Methods in Enzymology, Volume 2: Preparation and Assay of Enzymes

METHODS IN ENZYMOLOGY Edited by SIDNEY P. COLOWICK and NATHAN O. KAPLAN

McCoUum-Pratt Institute The Johns Hopkins University, Baltimore, Maryland

VOLUME

II

Advisory Board BRITTON CHANCE CARL F. CORI K. LINDERSTROM~ANG FRITZ LIPMANN

F. F. NORD SEVERO OCHOA JAMES B. SUMNER HUGO THEORELL

1955 ACADEMIC PRESS

New York

San Francisco

A Subsidiary of Harcourt Brace Jovanovich, Publishers

London

Contributors to Volume II Article numbers are s h o w n in parentheses following the n a m e s of contributors. Affiliations listed are current.

E. P. ABRAHAM (13), Sir William Dunn School of Pathology, Oxford University, Oxford, England N. J. BERRIDGE (7), National Institute for Dairy Research, University of Reading, Reading, England FRANCIS BINKLEY (40), Emory University, Atlanta, Georgia SANFORD M. BIRNBAUM (12, 55), National Institutes of Health, Bethesda, Maryland KOr~RAD BLOCH (45), Harvard University, Cambridge, Massachusetts ROGER K. BONNICHSEN (138), Nobel Medical Institute, Stockholm, Sweden ARNOLD F. BRODIE (121), Harvard Medical School, Boston, Massachusetts HARRr P. BROQUIST (104), Lederle Laboratories, Pearl River, New York KENNETH BURTON (23), Medical Research Council Unit for Cell Metabolism, Oxford, England G. C. B(:TLER (89), University of Toronto, Toronto, Canada P. S. CAMMARATA (6), Yale University, New Haven, Connecticut G. L. CANTONI (33, 34), National Institutes of Health, Bethesda, Maryland BRITTON CHANCE (136), University of Pennsylvania, Philadelphia, Pennsylvania H. CHANTRENNE (46), Facult$ des Sciences, Universit$ Libre de BruxeUes, Bruxelles, Belgium PHILIP P. COIIEN (19), University of Wisconsin, Madison, Wisconsin SIDNEY P. COLOWICK (68, 99), The Johns Hopkins University, Baltimore, Maryland BERNARD D. DAVIS (39), New York University, New York, N. Y. CHARLES R. DAWSON (145, 147), Columbia University, New York, N. Y.

ALEXANDER L. DOUNCE (137), University of Rochester, Rochester, New York S. W. EDWARDS(38, parts D,E,F), Harvard Medical School, Boston, Massachusetts NILS ELLFOLK (53), Biochemical Institute, Helsinki, Finland W. H. ELLIOTT (44), University of Oxford, Oxford, England HAROLD J. EVANS (59), North Carolina State College, Raleigh, N. C. CLAUDE FROMAGEOT (42), Universit$ de Paris. Paris, France J. S. FRUTON (6), Yale University, New Haven, Connecticut AKIJI FUJITA (103), Biochemical Institute, Kyoto Prefectural University, Kyoto, Japan COURTLAND C. GALEENER (31), University of Illinois, Urbana, Illinois ARTHUR W. GALSTON (140), California Institute of Technology, Pasadena, California DARCY GILMOUR (98), Commonwealth Scientific and Industrial Research Organization, Canberra, Australia CHARLES GILVARG (39), New York University, New York, N. Y. DAVID A. GOLDTHWAIT (78), Western Reserve University, Cleveland, Ohio ARDA A. GREEN (15, 151), The Johns Hopkins University, Baltimore, Maryland D. M. GREENBERG (5, 49), University of California, Berkeley, California a . ROBERT GREENBERG (78), Western Reserve University, Cleveland, Ohio JESSE P. GREENSTEIN (11), National Institutes of Health, Bethesda, Maryland SANTIAGO GRISOLIA (47), University of Kansas, Kansas City, Kansas I. C. GUNSALUS (18, 31, 109), University of Illinois, Urbana, Illinois

vi

CONTRIBUTORS TO VOLUME II

ERWIN HAAS (14, 122, 125), Mount Sinai Hospital, Cleveland, Ohio G. DE LA HADA (6), National Institutes of Health, Bethesda, Maryland OSAMU HAYAISHI (75), National Institutes of Health, Bethesda, Maryland LEON A. HEPPEL (57, 73, 80, 85, 90, 91), National Institutes of Health, Bethesda, Maryland DENIS HERBERT (139), Microbiology Research Department, Experimental Station, Porton, England ROGER M. HERRIOTT (1), The Johns Hopkins University, Baltimore, Maryland LEONARD A. HERZENBERG (17), California Institute of Technology, Pasadena, California R. J. HILMOE (85, 90), National Institutes of Health, Bethesda, Maryland B. L. HORECKER (73, 123), National Institutes of Health, Bethesda, Maryland f . EDMUND HUNTER, JR. (101), Washington University, St. Louis, Missouri MARGARET J. HUNTER (141), Harvard Medical School, Boston, Massachusetts R. M. JOHNSTONE (26), Montreal General Hospital, Montreal, Canada M. A. JOSLYN (149), University of California, Berkeley, California HERMAN M. KALCKAR (118), National Institutes of Health, Bethesda, Maryland NATHAN O. KAPLAN (69, 70, 86, 113, 114, 119, 135), The Johns Hopkins University, Baltimore, Maryland EDNA B. KEARNEY (108), Edsel B. Ford Institute for Medical Research, Detroit, Michigan W. WAYNE KIELLEY (95, 97), National Institutes of Health, Bethesda, Maryland DANIEL L. KLINE (16), Yale University, New Haven, Conn. W. E. KNox (32, 38), Harvard Medical School, Boston, Massachusetts SEYMOUR KORKES (129), Duke University, Durham, North Carolina ARTHUR KORNBERG (65, 76, 112, 116, 117), WasMngton University, St. Louis, Missouri

DANIEL E. KOSHLAND, JR. (87), Brookhaven National Laboratory, Upton, Long Island, N. Y. P. S. KRISHNAN (96), National Chemical Laboratory, Poona, India STEPHEN I~UBY (100), University of Wisconsin, Madison, Wisconsin HENRy LARDY (100), University of Wisconsin, Madison, Wisconsin M. LASKOWSKI (2, 3, 4), Marquette University, Milwaukee, Wisconsin HOWARD M. LEN~OFF (135), Loomis Laboratory, Greenwich, Connecticut ENzo LEONE (74), Istituto di Chimica Biologica dell' Universith, Naples, Italy AARON BUNSEN LERNER (146), University of Oregon, Portland, Oregon HENRY N. LITTLE (56), University of Massachusetts, Amherst, Massachusetts MARGARET R. MCDONALD (62, 63), Carnegie Institution, Cold Spring Harbor, New York WILLIAM D. MCELROr (150, 151), The Johns Hopkins University, Baltimore, Maryland R. W. McGILvERY (84), University of Wisconsin, Madison, Wisconsin WALTER S. McNuTT (67), Vanderbilt University, Nashville, Tennessee A. C. MAEHLY (136, 142, 143), University of Pennsylvania, Philadelphia, Pennsylvania RICHARD J. MAGEE (145, 147), Columbia University, New York, N. Y. HENRY R. MAHLER (120, 124), University of Indiana, Bloomington, Ind. INES MANDL (92, 93), Columbia University, New York, N. Y. A. H. MEHLER (29), National Institutes of Health, Bethesda, Maryland ALTON MEISTER (52), National Institutes of Health, Bethesda, Maryland HERSCHEL K. MITCHELL (17), California Institute of Technology, Pasadena, California SusuMu MITSUHASHI (39), New York University, New York, N. Y. ROBERT K. MORTON (81, 88), University, of Melbourne, Melbourne, Australia

vii

CONTRIBUTORS TO VOLUME II

AGNETE MUNCH-PETERSON (118), Uni-

versity of Copenhagen, Denmark

Copenhagen,

Institutes of Health, Bethesda, Maryland MURRAY SAFFRAN (77), McGiU Univer-

VICTOR A. NAJJAR (20, 61), The Johns

Hopkins University, Baltimore, Maryland ALVIN NASON (58, 59, 60, 128), 'The Johns Hopkins University, Baltimore, Maryland J. B. NEILANDS (134), University of California, Berkeley, California CARL NEUBERG (92, 93), New York Medical College, New York, N. Y. HANS NEURATH (8), University of Washington, Seattle, Washington GORDON NIKIFORUK (68), University of Toronto, Toronto, Canada LAFAYETTE NODA (100), University of Wisconsin, Madison, Wisconsin G. DAVID NOVELLI (102, 106,

SANFORD M. ROSENTHAL (54), National

115),

Western Reserve University, Cleveland, Ohio EVELYN L. OGINSKY (50), Merck Institute of Therapeutic Research, Rahway, New Jersey M. CLYDE OTEY (64), National Insgitutes of Health, Bethesda, Maryland A. M. PAPPENHEIMER, JR. (132), New York University, New York, N. Y. KARL-GusTAV PAUL (133), Nobel Medical Institute, Stockholm, Sweden S. V. PERRY (94), University of Cambridge, Cambridge, England PAUL PLESNER (64), University of Copenhagen, Copenhagen, Denmark B. DAVID POLIS (144), Naval Aviation Medical Research Laboratory, Johnsville, Pennsylvania VINCENT E. PRICE (64), National Institutes of Health, Bethesda, Maryland J. H. QUASTEL (26, 105), Montreal General Hospital, Montreal, Canada E. RACKER (127), Public Health Research Institute of the City of New York, New York, N. Y. S. RATNER (24, 28, 48), Public Health Research Institute of the City of New York, New York, N. Y. W. E. RAZZELL (109), University of Illinois, Urbana, Illinois

sity, Montreal, Canada ANTHONY SAN PIETRO (152), The Johns

Hopkins University, Baltimore, Maryland OTTO SCHALES (21, 22), Alton Ochsner

Medical Foundation, Louisiana

New

Orleans,

SELMA S. SCrIALES (21), Alton Ochsner

Medical Foundation, Louisiana

New

Orleans,

GERHARD SCHMIDT (79), Tufts Medical

School, Boston, Massachusetts H. W. SHMUKLER (144), Naval Aviation

Medical Research Laboratory, Johnsville, Pennsylvania Louis S~USTER (72, 86), National Institutes of Health, Bethesda, Maryland EMIL L. SMITH (9, 10), University of Utah, Salt Lake City, Utah LUCILE SMITH (130), University of Pennsylvania, Philadelphia, Pennsylvania C. V. SMYTHE (41), Rohm and Haas Company, Philadelphia, Pennsylvania JoHN E. SNOKE (45), University of Chicago, Chicago, Illinois B. H. SSRBO (43), Nobel Medical Institute, Stockholm, Sweden JOHN R. STAMER (18, 31), University of Illinois, Urbana, Illinois R. Y. STANIER (37), University of California, Berkeley, California ELMER STOTZ (131), University of Rochester, Rochester, New York HAROLD J. STRECKER(27), Columbia University, New York, N. Y. P. K. STUMPF (35), University of California, Berkeley, California JAMES B. SUMNER (51, 137), CorneU University, Ithaca, New York MARJORIE A. SWANSON (83), BowmanGray School of Medicine, WinstonSalem, North Carolina CELIA WHITE TABOR (54), National Institutes of Health, Bethesda, Maryland HERBERT TABOR (29, 54), National Institutes of Health, Bethesda, Maryland

viii

CONTRIBUTORS TO VOLUME I I

BIRGIT VENNESLAND (126), University of Chicago, Chicago, Illinois P. J. VIGNOS, JR. (34, part B), Western Reserve University, Cleveland, Ohio ARTTURI I. VIRTANEN (53), Biochemical Institute, Helsinki, Finland ELLIOT VO~KIN (82), Oak Ridge National Laboratories, Oak Ridge, Tennessee HEINRICH WAELSCH (36), Columbia University, New York, N. Y. T. P. WANG (66, 71, 110, 111), Institute of Physiology and Biochemistry, Shanghai, China

E. RoY WAYGOOD

(148), University of

Manitoba, Winnipeg, Canada H. G. K. WESTENBRINK (107), Laboratorture voor Physiologische Chemie, Utrecht, Holland W. A. WOOD (25), University of Illinois, Urbana, Illinois WALTER D. WOSILAIT (128), Western Reserve University, Cleveland, Ohio CHARLES YANOFSKY (30), Western Reserve University, Cleveland, Ohio MILTON ZVCEER (58, 60), Connecticut Agricultural Experiment Station, New Haven, Connecticut

Outline of Volumes I, HI, and IV VOLUME I P R E P A R A T I O N AND ASSAY OF E N Z Y M E S Section I. General Preparative Procedures A. Tissue Slice Technique. B. Tissue Homogenates. C. Fractionation of Cellular Components. D. Methods of Extraction of Enzymes. 1~. Protein Fractionation. F. Preparation of Buffers.

Section II. Enzymes of Carbohydrate Metabolism A. Polysaccharide Cleavage and Synthesis. B. Disaccharide, Hexoside and Glueuronide Metabolism. C. Metabolism of Hexoses. D. Metabolism of Pentoses. E. Metabolism of Three-Carbon Compounds. F. Reactions of Two-Carbon Compounds. G. Reactions of Formate.

Section III. Enzymes of Lipid Metabolism A. Fatty Acid Oxidation. B. Acyl Activation and Transfer. C. Lipases and Esterases. D. Phospholipid and Steroid Enzymes.

Section IV. Enzymes of Citric Acid Cycle

VOLUME

III

P R E P A R A T I O N AND ASSAY OF SUBSTRATES Section I. Carbohydrates A. Polysaccharide Analysis and Preparation. B. Substrates for Glucuronidases. C. Free Sugars. D. Sugar Phosphates and Related Compounds. E. Unphosphorylated Intermediates and Products of Fermentation and Respiration. F. Spectrophotometric Enzymatic Methods for Aldehydes and Ketoaldehydes.

Section II. Lipids and Steroids A. General Procedure for Separating Various Lipid Components of Tissues. B. Preparation and Determination of Higher Fatty Acids. C. Preparation and Analysis of Phospholipids. D. Phosphoric Monoester and Diester Derivatives of Phospholipids. E. Nitrogenous Phospholipid Constituents. F. Chromatographic Procedures for Deter... XVlll

OUTLINE OF VOLUMES I~ III~ AND IV

xi~x

mining Fatty Acids. G. General Procedures for Lower Fatty Acids. H. Molecular Distillation of Higher Fatty Acids and Other Lipids. I. Preparation and Assay of Cholesterol and Ergosterol.

Section I I I . Citric Acid Cycle Compounds A. Chromatographic Analyses of Organic Acids. B. Alpha-keto Acids. C. Beta-keto Acids. D. Tricarboxylic Acids. E. 4-Carbon Dicarboxylic Acids. F. Itaconic Acid and Related Compounds.

Section IV. Proteins and Derivatives A. Spectrophotometric Methods for Determination of Protein. B. Procedures for Measuring Amino Acids (Amino N). C. General Procedures for Preparation of Peptides. D. Resolution of DL Mixtures of Amino Acids. E. Determination and Preparation of Specific Amino Acids and Related Compounds.

Section V. Nucleic Acids and Derivatives A. Methods for Determination of Nucleic Acids in Tissues. B. Methods Nucleic Acids. C. Methods for Characterization of Nucleic Acids. D. Characterization and Isolation of Mono- and Oligonucleotides. E. Other Assay of Nucleotides and Nucleosides. F. Preparation and Assay of Cyclic G. Chemical Synthesis of Nucleosides and Nucleotides.

for Isolating Methods of Methods for Nucleotides.

Section VI. Coenzymes and Related Phosphate Compounds A. General Procedure for Isolating and Analyzing Tissue Organic Phosphates. B. Characterization of Phosphorus Compounds by Acid Lability. C. Determination and Preparation of N-Phosphates of Biological Origin. D. Methods for Preparation and Assay of ATP, ADP, and AMP. E. Methods of Assay and Preparation of Pyridine Nucleotides and Derivatives. Y. Assay and Preparation of Coenzyme A and Derivatives. G. Assay and Preparation of Lipoic Acid and Derivatives. H. Fluorometric Assay of Cocarboxylase and Derivatives. I. Assay and Preparation of Flavine Adenine Dinucleotide and Flavine Mononucleotide. J. Assay and Preparation of Pyridoxal Phosphate. K. Preparation and Analysis of UDPG and Related Compounds.

Section VII. Determination of Inorganic Compounds A. Nitrite and Nitrate. B. Total Nitrogen and Ammonia. C. Sulfur. D. Phosphorus. E. Metals.

XX

OUTLINE OF VOLUME I, III~ AND IV VOLUME IV

SPECIAL TECHNIQUES FOR THE ENZYMOLOGIST Section I. Techniques for Characterization of Proteins (Procedures and Interpretations) A. Electrophoresis; Macro and Micro. B. Ultracentrifugation and Related Techniques (Diffusion, Viscosity) for Molecular Size and Shape. C. Infra-red Spectrophotometry. D. X-ray Diffraction. E. Light Scattering Measurements. F. Flow Birefringence. G. Fluorescence Polarization and Other Fluorescence Techniques. H. The Solubility Method for Protein Purity. I. Determination of Amino Acid Sequence in Proteins. J. Determination of Essential Groups for Enzyme Activity.

Section II. Techniques for Metabolic Studies A. Measurement of Rapid Reaction Rates; Techniques and Applications, Including Determination of Spectra of Cytochromes and Other Electron Carriers in Respiring Cells. B. Use of Artificial Electron Aceeptors in the Study of Dehydrogenases. C. Use of Percolation Technique for the Study of the Metabolism of Soil Microorganisms. D. Methods for Study of the Hill Reaction. ~E. Methods for Measurement of Nitrogen Fixation. F. Cytochemistry.

Section III. Techniques for Isotope Studies A. The Measurement of Isotopes. B. The Synthesis and Degradation of Labeled Compounds (Including Application to Metabolic Studies).

Errata for Volume I Page xiii: for Milton F. Utter, read M e r t o n F. U t t e r Page xv: in Section I I B, for glucuronoside, read glucuronide. Page 63, 1. 7: for Ten Brock, read Tenbroeck Page 326: First sentence after Step 2. Acetone Precipitation should read: Dilute the yeast extract (170 ml.) with 940 ml. of cold water and adjust to p H 4.8 with about 20 ml. of 2 N acetic acid.

Outline of Organization VOLUME II PREPARATION

AND

ASSAY OF ENZYMES Article

Numbers

Pages

1-17

3-169

B. Enzymes in Amino Acid Metabolism (General)

18-26

170-220

C. Specific Amino Acid Enzymes

27-43

220-337

D. Peptide Bond Synthesis

44-46

337-350

E. Enzymes in Urea Synthesis

47-50

350-378

F. Ammonia Liberating Enzymes

51-55

378-400

G. Nitrate Metabolism

56-61

400-423

A. Nucleases

62,63

427-447

B. Nucleosidases

64-67

448-468

C. Deaminases

68-72

469-482

D. Oxidases

73-75

482-497

E. Nucleotide Synthesis

76-78

497-519

A. Phosphomonoesterases

79-88

523-561

B. Phosphodiesterases

89,90

561-570

C. Inorganic Pyro- and Poly-phosphatases

91-93

570-582

D. ATPases

94-98

582-598

1¢.. Phosphate-Transferring Systems

99-101

598-616

Section I. Enzymes of Protein Metabolism A. Protein Hydrolyzing Enzymes

Section II. Enzymes of Nucleic Acid Metabolism

Section III. Enzymes in Phosphate Metabolism

Section IV. Enzymes in Coenzyme and Vitamin Metabolism A. Synthesis and Degradation of Vitamins

102-105

619-632

B. Phosphorylation of Vitamins

166-109

633-649

110-118

649-677

C. Coenzyme Synthesis and Breakdown ix

x

OUTLINE OF ORGANIZATION Article Numbers

.Paoes

A. Pyridine Nucleotide-Linked, Including Flavoproteins

119-129

681-732

B." Iron-Porphyrins

130-144

732-817

C. Copper Enzymes

145-147

817-835

D. Unclassified

148-152

836-870

Section V. Respiratory Enzymes

Outlines of V o l u m e s I, III, and IV start on page xviii.

[1]

SWINE PEPSIN AND PEPSINOGEN

3

[1] Swine Pepsin and Pepsinogen By ROGER 71~/I. HERRIOTT Assay Method Pepsin Principle. This method, described by Anson, ~ involves digestion of denatured hemoglobin by dilute pepsin under standard conditions after which the undigested protein is precipitated with TCA and this is then removed by filtration. The extent of digestion which is the measure of protease action is determined on the filtrate with the aid of Folin's phenol reagent. A blue color is produced by a reaction with the tyrosine and tryptophan of the protein split products in the filtrate. Conversion of the color value to units of pepsin is readily made from a standard curve. Pepsinogen Principle. Swine pepsinogen may be determined in the presence of pepsin by destroying the latter at pH 8 where the precursor is stabile and then converting the pepsinogen into pepsin by acidification to pH 2. This pepsin is then assayed as described below. An evaluation of both components in a mixture of pepsin and pepsinogen entails two enzyme determinations, one after direct acidification which yields the sum of pepsin from pepsinogen and the initial pepsin, the other after first making the solution alkaline to pH 8 followed by acidification to pH 2 which measures the pepsinogen equivalent. The difference in the two values is the pepsin of the original mixture.

Reagents Substrate. The substrate solution contains 2% hemoglobin in 0.06 N HC1. An acidity of pH 1.8 is produced which denatures the hemoglobin. A satisfactory bovine hemoglobin may be purchased from Armour and Company or it may be prepared by plasmolysis of washed beef red cells followed by centrifugation of the stroma. Dry weight or colorimetry may be used to determine the concentration of hemoglobin. T C A - - 5 % (0.3 M). 0.5 N NaOH. Folin's phenol reagent 2 diluted 1:3 with water. Enzyme. Aqueous solutions containing 0.3 to 2 ~, of swine pepsin nitrogen per milliliter are optimal for this method. M. L. Anson, J. Ge~. Physiol. 22, 79 (1938). O. Folin and V. Ciocalt6u, J. Biol. Chem. 73, 627 (1927).

4

~NZYMES OF PROTEIN METABOLISM

[1]

Procedure. To 5 ml. of substrate in a test tube, equilibrated at 25 °, is added 1 ml. of enzyme solution. After 10 minutes' digestion, 10 ml. of 5% TCA is added and the precipitated protein removed by filtration. To 5 ml. of the filtrate in a 50-ml. Erlenmeyer flask is added 10 ml. of 0.5 N NaOH and 3 ml. of the diluted phenol reagent slowly with constant agitation. The blue color that develops is read after 5 minutes in a visible colorimeter against a standard or in a photoelectric colorimeter at 660 m~. The color value is expressed in milliequivalents of tyrosine. A color standard is readily produced with 8 X 10-4 meq. of tyrosine in 5 ml. of 0.2 M HC1 in place of the TCA filtrate. A hemoglobin enzyme blank is performed by adding the 1 ml. of enzyme to the TCA before the latter is added to the hemoglobin. Filtration and evaluation of the chromogenic equivalent of the filtrate completes this operation. An empirical standard curve in which the pepsin units plotted against the liberated tyrosine equivalents in 5 ml. of TCA filtrate permits a rapid conversion of color value to peptic units. Such a curve may be constructed from the following data taken from "Crystalline Enzymes": ~ 1, 2, 3, 4, 5, and 8 X 10-4 pepsin unit correspond to the release of 1.8, 3.5, 5.0, 6.3, 7.5, and 11 × 10-4 meq. of tyrosine, respectively, into 5 ml. of the TCA filtrate over and above the blank. Reproducibility. Duplicate assays agree within 5%, but analyses in different laboratories may not agree closer than 10%. Purification of Pepsin from Commercial Preparations Crystalline swine pepsin can be prepared readily from any of several crude products commercially available. A simple method which has considerable interest and merit employs ethanol fractionation and gives crystals low in nonprotein nitrogen. 4 The method described below uses salt fractionation and yields hexagonal bipyrimidal crystals which have been homogeneous in solubility studies. 4~ Step 1. One killogram of Cudahy U.S.P. 1:10,000 pepsin is dissolved in 2 1. of 0.5 M acetate buffer, pH 5.0. Step 2. To the above pepsin solution is added 4.5 1. of saturated magnesium sulfate and 50 g. of Hyflo Super-Cel (Johns Manville Co.), after which the suspension is filtered on large B~chner funnels. The residue is washed on the funnel with 1 1. of 0.6 saturated magnesium sulfate-0.2 M acetate, pH 5.0, hereafter termed "solvent." Step 3. The residue from step 2 is broken up in 14 1. of solvent and 3 j. H. Northrop, M. Kunitz, and R. M. Herriott, "Crystalline Enzymes," 2nd ed., Columbia University Press, New York, 1948. 4 j. H. Northrop, J. Gen. Physiol. 30~ 177 (1946). 4~ R. IV[. Herriott, V. Desreux, and J. H. Northrop, J. Gen. Physiol. 24, 213 (1940).

[1]

SWINE PEPSIN AND PEPSINOGEN

5

stirred for 20 hours at 20 ° , filtered, and the residue washed on the funnel with 1 1. of solvent. To the filtrate and washings is added 3 kg. of solid magnesium sulfate (crystalline, with 7 H20) and 100 g. of Filter-Cel. This is filtered and washed with 1 1. of solvent. Step 4. The residue from step 3 is stirred with 2.5 1. of solvent for 20 hours at 20 ° and filtered. Then 500 g. of magnesium sulfate is added to the filtrate, followed by 60 ml. of 5 N H2S04. The precipitate is filtered and washed with 100 ml. of cold N/50 H2S04. The cake is aspirated until no more mother-liquor can be withdrawn. Step 5. The filter cake is stirred with one-fourth its weight of water in a beaker. The beaker is placed in a 35 ° water bath, and the mixture is stirred to a smooth paste. 0.5 N NaOH is added very slowly through a capillary with continuous stirring until the precipitate just dissolves. The pH should be 3.9 to 4.0. The mixture is stirred slowly and allowed to cool. If hexagonal bipyramid crystals do not form, it should be reheated to 35 ° and the inside of the beaker scratched. Once the crystals form, the suspension should be stirred for 10 to 20 hours at 20 ° and then placed at 5° overnight. I t is then filtered. Purification of Pepsinogen from Swine Mucosa 3,5

Preparation of the Fundi. The stomachs from freshly killed swine are inverted and washed thoroughly with cold water. They can then be transported to the laboratory and the fundi cut out with heavy scissors. The fundus may be distinguished from the rest of the mucosa, since in general it appears slightly darker in color and usually occupies a 6- to 8-inch circular portion of the mucosa approximately midway between the entrance (cardiac) and exit (pyloric) of the stomach. Separation of the mucosa from the muscle tissue can best be performed with a scalpel or scissors. The separated mucosa should then have the slimy mucin removed by scraping gently with a straightedged piece of glass. The mucosa may now be used in step 1 or stored frozen. A fundus mucosa usually weighs about 100 g. and contains iust under a gram of pepsinogen. Step 1. One thousand grams of mucosa is minced twice through a meat grinder with 4-mm. holes and then mixed thoroughly with 4 1. of 0.45 saturated ammonium sulfate (SAS) in 0.1 M NaHC03 and stirred for an hour at room temperature. Step 2. F o r ty grams of Filter-Cel and 20 g. of Hyflo Super-Cel are added, and the suspension is filtered on 30-cm. B~tchner funnels. The filter cake is washed twice with 100 to 150 ml. of 0.42 SAS-0.1 M bicarbonate. The residue is discarded, and the filtrate and washings combined. 5 R. M. Herriott, J. Gen. Physiol. 21, 501 (1938).

6

ENZYMES OF PROTEIN METABOLISM

[1]

Step 3. The protein is precipitated from the above solution l~y the addition of 180 g. of solid ammonium sulfate per liter of solution. The addition of 15 to 30 g. of Hyflo Celite permits reasonably rapid filtration of this precipitate. The filtrate is discarded, and the residue suspended in water. The Celite is removed by filtration, but it should be washed thoroughly to recover all the pepsinogen. Step 4. The above pepsinogen solution is diluted to contain approximately 1 mg. of protein N per milliliter, titrated to pH 6.0 + 0.2 (yellow to methyl red and bromothymol blue) with 4 M acetate buffer, pH 4.7, and then mixed with an equal volume of " M / 1 " washed copper hydroxide. The mixture is stirred for 5 to 10 minutes, then filtered on large Bfichner funnels. If protein is found in the filtrate, more copper hydroxide may be added, filtered, and combined with the first residue. The combined residues are then broken up into a smooth creamy suspension in a volume of 0.1 M phosphate, pH 6.8, equal to the volume of the protein solution just prior to mixing with copper hydroxide. After being stirred for half an hour the suspension is filtered and the residue washed twice with 75 ml. of 0.1 M phosphate, pH 6.8. Step 5. The filtrate and washings from step 4 are combined and mixed with 50 g. of ~ilter-Cel per liter of solution. This is filtered with suction, and the cake washed twice with a volume of 0.1 M P Q , pH 6.8, equal to the weight of Filter-Ce] used, The filtrate is then brought to 0.7 saturation by the addition of 474 g. of solid ammonium sulfate per liter of filtrate. To this, 50 to 100 g. of Hyflo Super-Cel is added and the suspension is filtered on large Bfichner funnels, after which the protein in the residue is extracted as in step 3. Step 6. The protein solution is now diluted to contain about 1 mg. of PN per milliliter, after which steps 4 and 5 are repeated, except that no Hyflo Super-Cel is added to the 0.7 SAS suspension. The suspension is filtered slowly with suction on a hardened filter paper. Step 7. The residue from step 6 is stirred with 9 vol. of 0.4 saturated ammonium sulfate in 0.1 M P04, pH 6.25, filtered, and stirred slowly at 10° for a few hours. The solution~should become opalescent and a swirl of needle crystals develop. After two days the crystals may be filtered.

Properties Swine pepsin is unstable in solutions alkaline to pH 6, yet it is relatively stable to acid solutions, even to pH 1. The maximum protease action is observed at pH 1.8 to 2.0. Pepsinogen is unstable below pH 6, being autocatalytically converted to pepsin. In the absence of salts, pepsinogen is reversibly denatured at pH 7 above 50 °, or at room temperature and alkalinities of pH 8.5 to 11.

[1]

SWINE PEPSIN AND PEPSINOGEN

7

I n h i b i t o r s . A n i n h i b i t o r of p e p s i n h a s b e e n i s o l a t e d f r o m t h e a c t i v a t i o n m i x t u r e of p e p s i n o g e n 2 I t i n h i b i t s p e p t i c a c t i o n o n l y in t h e r e g i o n of p H 4 t o 6.

Other Species P u r i f i c a t i o n of p e p s i n a n d / o r p e p s i n o g e n f r o m species o t h e r t h a n swine h a v e b e e n d e s c r i b e d . T h u s , s a l m o n , 7 s h a r k , 8 b o v i n e , 9 a n d c h i c k e n 1° may be noted. TABLE I PREPARATION OF PEPSIN

Weight of cake

[PU]=~. ~b PN

500 g. (dry)

0.28 0.32 0.33-0.34 0.33-0.34

Crude pepsin Step 3 Step 4 Step 5

100 g. 40 g.

TABLE II PREPARATION OF PEPSINOGEN

Step

Volume, ml.

Protein N/ml.

Total protein

" [PU]ml" rfb,,,,

1 2 3 4 6 Crystals

4,700 3,800 1,000 1,470 135 37

0.67 0.37 1.3 0.33 2.1 3.5

3,150 1,400 1,300 485 280 130

0.042 0.048 0.15 0.075 0.43 0.74

1tl. . . . PN ,, [PU]mg.

0.06 0.13 0.12 0.23 b 0.21 0.21

Per cent of original activity 100 94 79 55 30 14

This is potential peptic activity, for it is determined only after active*ion of the pepsinogen. b This figure is probably not representative. In other preparations the activity per milligram of protein nitrogen was intermediate between this value and that of step 3. 6 R. M. Herriott, J. Gen. Physiol. 24, 325 (1941). 7 E. R. Norris and D. W. Elam, J. Biol. Chem. 134, 443 (1940). 8 G. P. Sprissler, "An Investigation of the Proteinase of the Gastric Mucosa of Shark," Dissertation, The Catholic University of America Press, Washington, D. C., 1942. 9 j. H. Northrop, J. Gen. Physiol. 16, 615 (1933). ~0R. M. Herriott, Q. M. Bartz, and J. H. Northrop, J. Gen. Physiol. 21, 575 (1938).

8

ENZYMES OF PROTEIN METABOLISM

[9.]

[2] Chymotrypsinogens and Chymotrypsins B y ~I. LASKOWSKI

Two chymotrypsinogens and several chymotrypsins are known at present. The first, a-chymotrypsinogen, was crystallized b y Kunitz and Northrop. 1 Several modifications of the original procedure were later contributed b y Kunitz. 2-4 Small a m o u n t s of trypsin (0.5 mg. per 10 g., 5 °, 48 hours) slowly activate a - c h y m o t r y p s i n o g e n into a-chymotrypsin. 1 The latter undergoes a slow transformation into ~- and ~-chymotrypsins, presumably as a result of limited autolysis. C h y m o t r y p s i n s f~ and ~, were obtained in crystalline form. a Jacobsen, 5 using a fast activation of a - c h y m o t r y p s i n o g e n (35 rag. of trypsin per gram of a-chymotrypsinogen, 0 °, 1 to 2 hours), described two new forms of chymotrypsin, a very unstable form ~, and a fairly stable form 5, which were more active t h a n a - c h y m o t r y p s i n . The preparation of amorphous ~-chymotrypsin, was repeated b y Schwert and Kaufman. 6 The crystalline diisopropyl phosphate, 8" DP-~-chymotrypsin, was recently obtained b y Desnuelle and co-workers. 7 Considerable progress in elucidation of the activation process has been recently achieved. The activation process depends on rupturing of the cyclic molecule of a - c h y m o t r y p s i n o g e n b y trypsin, and b y chymotrypsin itself (autolysis). Schemes of the activation process have been suggested b y Gladner and N e u r a t h s and b y Rovery, Poilroux, and Desnuelle. 7 The latter scheme is reproduced in Fig. I sa. The mechanism of transformations leading to f~ and ~/forms is not yet clear. The terminal groups of a-, ~-, and ~,-chymotrypsins are the same, 9,1° but the rate of release of the C terminal groups b y carboxypeptidase differs. 9 M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19, 991 (1936). M. Kunitz, J. Gen. Physiol. 33, 349 (1950). 3 M. Kunitz, J. Gen. Physiol. 2, 207 (1938). 4 M. Kunitz, J. Gen. Physiol. 32, 263 (1948). 5 C. F. Jacobsen, Compt. rend. tray. lab. Carlsberg, sdr. chim. 25, 325 (1947). 6 G. W. Schwert and S. Kaufman, J. Biol. Chem. 180, 517 (1949). s~ The following abbreviations are used: diisopropyl fluorophosphate is called DFP; the enzyme which reacted with DFP is called DFP-treated enzyme or DP enzyme. 7 M. Rovery, M. Poilroux, and P. Desnuelle, Biochim. et Biophys Acta 14, 145 (1954). s j. A. Gladner and H. Neurath, J. Biol. Chem. 205, 345 (1953). s, F. R. Bettelheim and H. Neurath, g. Biol. Chem. 212, 241 (1955) on the basis of analysis of terminal groups postulated a new intermediate between ~ and a chymotrypsins, and concluded that it probably resulted from the action of chymotrypsin and not trypsin. 9j. A. Gladner and H. Neurath. J. Biol. Chem. 206, 911 (1954). i0 M. Rovery, C. Fabre, and P. Desnuelle, Biochim. et Biophys. Acta 10, 481 (1953).

9

CHYMOTRYPSINOGENS AND CHYMOTRYPSINS

[9.]

A second crystalline chymotrypsinogen, named B, which on activation leads to a crystalline c h y m o t r y p s i n B was obtained b y Laskowski and his co-workers, n-14 Proteins of the B series differ from those of the a series in electrophoretic mobility 15 and in the ease with which t h e y are retained on resins, ~6 b u t not in molecular weight. 17 C h y m o t r y p s i n B shows the same specificity as a - c h y m o t r y p s i n when tested on several synthetic substrates, is However, the rates at which the two enzymes hydrolyze various substrates 1~,~,~9,2° differ. The analysis of terminal groups ~-Chymotrypsin

~r-Chymotrypsin

a-Chyrnot rypsinogen [ q I t Tyr, Ileu I

',

Ala L

I

Base

trypsin >

T ~ e u I i Ala I Base

LLeu- ]

/ #

'

I Tyr

i

I

,

I

Ileu

(~)

Ala ~

trypsin /

+ x --Base Leu ( ~ II autolysis J

trypsio, autolysis

• a-Chy mot ry psin - -

~

Q

Tyr,

Ileu @ l

Ala ,

L

+ x - - Base

eu©

FIG. 1. Scheme of activation of o~-ehymotrypsinogen! confirmed the nonidentity of the B proteins with the c~ protein% b u t did not elucidate the mechanism of activation. 9 Preparation of a-Chymotrypsinogen 1 Ten to twelve average-size beef pancreases, removed immediately after slaughter, are immersed at once in enough 0.25 N ice-cold H~S04 to cover the glands. After removal of fat and connective tissue the glands are minced in a meat chopper. Three liters of minced pancreas is suspended in 6 1. of 0.25 N H2SO4 at 5 °, and the suspension is allowed to stand at this temperature for 18 to 24 hours. The suspension is strained n M. Laskowski, J. Biol. Chem. 166, 555 (1946). •~ M. Laskowski and A. Kazenko, J. Biol. Chem. 167, 617 (1947). ~3C. K. Keith, A. Kazenko, and M. Laskowski, J. Biol. Chem. 170, 227 (1947). 14K. D. Brown, R. E. Shupe, and M. Laskowski, J. Biol. Chem. 173, 99 (1948). l~ V. Kubacki, K. D. Brown, and M. Laskowski, J. Biol. Chem. 180, 73 (1949). i~ C. H. W. Hirs, J. Biol. Chem. 205, 93 (1953). ~7E. L. Smith, D. M. Brown, and M. Laskowski, J. Biol. Chem. 191, 639 (1951). ~sj. S. Fruton, J. Biol. Chem. 173, 109 (1948). 19j. A. Ambrose and M. Laskowski, Science 115, 358 (1952). ~0F. C. Wu and M. Laskowski, Federation Proc. 13, 326 (1954); J. Biol. Chem. 213, 609 (1955).

10

ENZYMES OF PROTEIN METABOLISM

[2]

through two layers of gauze; the tissue is resuspended in 3 I. of cold 0.25 N H2S04 and immediately strained through gauze. The extracts are combined, and the residue is rejected. To each liter of extract 242 g. of solid ammonium sulfate is added to attain 0.4 saturation. The mixture is filtered through fluted paper (S. and S. No. 14501/~) in the cold room. ~ The precipitate is rejected. 22 The liquid is brought to 0.6 saturation with ammonium sulfate (205 g./1.). The copious precipitate which forms is allowed to settle at 5 ° and is filtered at that temperature on soft 22a filter paper on a large Bfichner funnel. ~3 The yield is about 100 g. of moist filter cake. The filtrate is saved for the preparation of ribonuclease (see Vol. II [62]). Each 100 g. of the precipitate is dissolved in 300 ml. of water, and 200 ml. of saturated solution of ammonium sulfate (20 to 25 °) is added. T M The precipitate which forms is filtered off with the aid of 5 g. of Celite and discarded. To each liter of filtrate 205 g. of solid ammonium sulfate is added slowly, and the precipitate which forms is filtered through a hardened paper. The yield is about 90 g. The filtrate is rejected. Crystallization of a-Chymotrypsinogen. Each 100 g. of the precipitate is dissolved in 150 ml. of water, treated with 50 ml. of saturated ammonium sulfate, and adjusted to pH 5.0 by dropwise addition of 5 N" NaOH (about 2 ml. per 100 g. of precipitate). The solution is allowed to stand for 2 days at 20 to 25 °. A heavy crop of chymotrypsinogen crystals (long needles) gradually forms. The suspension of crystals is filtered through hardened filter paper. The yield is about 25 g. The filtrate is saved for the preparation of trypsinogen (Vol. II [3]) or trypsin (Vol. II [3]), and afterward trypsin inhibitor (Vol. II [4]). 21 In this laboratory Sargent No. 500 filter paper is used and 10 g. per liter of SuperCel and 10 g. per liter of Celite No. 545 are added to facilitate filtration. 22 This precipitate contains deoxyribonuclease and chymotrypsinogen B. If the isolation of these two proteins from the same batch of pancreas is desired, it is recommended to follow the extraction procedure described for the preparation of deoxyribonuclease (see Vol. II [63]), using the less acid medium in order to protect deoxyribonuclease. The extract is then first brought to 0.2 saturation of ammonium sulfate (114 g./1.), and the precipitate is discarded. The liquid is brought to 0.4 saturation of ammonium sulfate (121 g./1.), and the precipitate (containing deoxyribonuclease and chymotrypsinogen B) is collected. The liquid is acidified by the addition of 6 ml. of concentrated sulfuric acid per liter of water used for the original extraction. From there on the procedure described in the text should be followed. 22a In this laboratory, for the soft paper Whatman No. 1 or 4 is used; for the hardened paper Whatman No. 50 or 52. 23 In this laboratory a stainless steel Btichner funnel, Model 503, is used, manufactured by American Biosynthetic Corp., Milwaukee, Wisconsin. ~ From there on all operations are carried at room temperature (20 to 25 °) unless specified otherwise.

[2]

CHYMOTRYPSINOGENS .A.ND CHYMOTRYPSINS

11

Recrystallization from A m m o n i u m Sulfate. 23bT h e crystalline filter cake is suspended in 3 vol. of water, and 5 N H2S04 is added from a b u r e t with stirring until the precipitate is dissolved. T h e solution is b r o u g h t to 0.25 saturation with a m m o n i u m sulfate b y addition of 1 vol. of s a t u r a t e d a m m o n i u m sulfate. An a m o u n t of 5 h r N a O H equivalent to the acid used is then added with stirring, and the solution is inoculated and allowed to stand at 20 ° . Crystallization should be practically complete in an hour. The yield is a b o u t 80%. Recrystallization from Alcohol. 4 After several (five or six) recrystallizations with a m m o n i u m sulfate, 10 g. of semidry filter cake of crystals of a - c h y m o t r y p s i n o g e n is stirred up with a b o u t 30 ml. of w a t e r are dissolved with the aid of several drops of 5 N H~SO~. T h e solution is dialyzed against slowly running distilled w a t e r for 24 hours at 5 °, preferably with stirring. T h e dialyzed solution of a - c h y m o t r y p s i n o g e n is filtered clear and then m a d e up with w a t e r to 50 ml. The p H of the solution is adiusted with dilute acid or alkali to a b o u t 4.0. T h e solution is cooled in an ice-salt b a t h to 1 to 3 °, and 12.5 ml. of ice-cold 95% ethanol is added slowly with stirring; the t e m p e r a t u r e of the solution is not allowed to rise a b o v e 5 ° during the addition of alcohol. T h e p H of the solution is then adjusted T M with 1 N N a O H to a b o u t 5.0 (0.01% m e t h y l red solution as an indicator on a test plate, and a 0.01 M acetate buffer as a standard). A h e a v y a m o r p h o u s precipitate forms at p H 5. T h e suspension is kept at 20 to 25 °. The precipitate gradually dissolves and is replaced within several hours b y a crop of large well-formed crystals. The crystallization is generally complete within 24 hours. T h e crystals are filtered with suction on hardened paper, washed with ice-cold acetone, and dried at room t e m p e r a t u r e for 24 hours. The yield is a b o u t 80%. T h e dried material is ground up in a m o r t a r and stored in refrigerator. 24 2~bIn the reviewer's laboratory, recrystallizations of a-chymotrypsinogen were repeatedly successful when the vriginal description of Kunitz was followed. Slight modifications of this method have been employed in other laboratories. E. F. Jansen (personal communication) suspends a-chymotrypsinogen in 5 vol. of water, adds H2S04 until pH 3.0 (glass electrode) is reached, then adds the required amount of ammonium sulfate and adjusts pH to 4.0 (instead to 5.0) with 5 N NaOH. Under these conditions recrystallization is essentially complete in about half an hour. G. W. Schwert (personal communication) similarly dissolves a-chymotrypsinogen at pH 3.0 (glass electrode), filters off the insoluble material, if any is present, but recrystallizes it at pH 5.0. 2acDuring the addition of alcohol the solution usually gels. On addition of NaOH the gel changes into a precipitate described below. ~4Neurath (personal communication) calls attention to a possible contamination of a-chymotrypsinogen with the active a-chymotrypsin. He noticed that when all necessary precautions in collecting glands were observed the first crystals of chymo-

12

ENZYMES OF PROTEIN METABOLISM

[2]

Crystallization of a - C h y m o t r y p s i n 1

T e n g r a m s of the crystalline a - c h y m o t r y p s i n o g e n filter cake containing a m m o n i u m sulfate, or 5 g. of salt-free crystalline a - c h y m o t r y p s i n o gen, is suspended in 30 ml. of w a t e r and dissolved b y the addition of a few drops of 5 N H2S04. T e n milliliters of M / 2 p h o s p h a t e buffer, p H 7.6, and a q u a n t i t y of N N a O H equivalent to the acid used are added. T h e solution is inoculated with 0.5 mg. of crystalline trypsin and left for 48 hours at 5 °. T h e p H of the solution is then adjusted to 4.0 b y addition of a b o u t 5 ml. of N H2SO4. T w e n t y - f i v e g r a m s of solid a m m o n i u m sulfate is added, and the precipitate is filtered with suction. T h e filter cake is dissolved in 0.75 vol. of 0.01 N H2SO~ and filtered if the solution is not clear. T h e clear solution is inoculated with crystalline (rhombohedral) a - c h y m o t r y p s i n and allowed to stand at 20 ° for 24 hours. A b o u t 5 g. of crystalline filter cake should form. Recrystallization. ~4~ T h e crystalline filter cake is dissolved in 1.5 vol. of 0.01 N H2S04, and a b o u t 1 vol. of s a t u r a t e d a m m o n i u m sulfate is added cautiously until crystallization commences. After 1 hour at room t e m p e r a t u r e the crystallization is almost complete. Recrystallization from Alcohol. 4 a - c h y m o t r y p s i n is first recrystallized two to three times f r o m a m m o n i u m sulfate. T e n g r a m s of crystalline filter cake is dissolved in 30 ml. of w a t e r and dialyzed against slowly running 0.005 2V H2SO4 at 5 ° for 24 hours with stirring. T h e dialyzed solution is filtered clear, then m a d e up with w a t e r to a volume of 50 ml., and cooled in an ice-salt b a t h to 2 to 3 °. T h e p H of the solution is adjusted to a b o u t 4.8 with the aid of several drops of 1 hr N a O H . Ten milliliters of ice-cold 95 % alcohol is added slowly with stirring, while the t e m p e r a t u r e of the solution is m a i n t a i n e d at a b o u t 5 ° . T h e solution is then trypsinogen contained 0.2 % of the active enzyme. The best preparation of Gladner and Neurath 8 contained only 0.03% of chymotrypsin, and no tryptic activity could be detected. This preparation was recrystallized seven times from ammonium sulfate and twice from alcohol. On the other hand, it the precautions in collecting glands were not observed, the first crystals may contain as much as 7 to 10% of active enzyme, and subsequent recrystallizations cannot reduce this amount to less than 1%. Hirs 18 described a chromatographic separation of a-chymotrypsinogen from crude acid extracts of pancreas using the resin IRC 50 (XE-64) (see Vol. I [13]). Good separation was obtained after a single passage through the column. As yet no attempts have beeen made to obtain a-chymotrypsinogen free from a-chymotrypsin. However, chromatography was found useful in detection of impurities in crystalline preparations of a-chymotrypsinogen. See also Vol I [12]. 24, E. F. Jansen (personal communication) recommends that all the ammonium sulfate be added rather rapidly to an Erlenmeyer flask containing the enzyme with adequate stirring. By this procedure, a clear supersaturated solution is obtained, which, when seeded, results in fairly large crystals.

[2]

13

CHYMOTRYPSINOGENS AND CHYMOTRYPSINS

titrated with 1 N H2S04 to pH 4.0 (light green to bromocresol green on a test plate). A heavy amorphous precipitate is formed. The suspension is kept at 5°. The amorphous precipitate slowly changes into a paste of very fine needles and rosettes. Seeding with a drop of a suspension of the crystals assures prompt crystallization within 24 hours. The paste of crystals is filtered with suction on hardened paper at 5 ° . The filtration generally takes several hours. The filter cake is dried on a watch glass placed near the cooling coil of the refrigerator. The dry material is ground to a fine powder and stored in the refrigerator.

The first preparation of cL-chymotrypsin diisopropyl fluorophosphate derivative was accomplished by Jansen et al. 2~ The mechanism of the reaction has been elucidated as follows. ~8,27 O a-Ch--H +

OC3Hv

!!/ F--P \

O

OC3H~

H/ --+ a - C h - - P \ OC~H7

+ HF OC3H7

One mole of phosphorus 28 and two isopropyl groups 27 are introduced per mole of enzyme with the liberation of H F and the formation of diisopropyl phosphate a-chymotrypsin (DP-a-chymotrypsin). Thirty per cent of the protein-bound phosphorus of the DP-a-chymotrypsin, after complete hydrolysis, has been recovered as phosphoserine, ~8 suggesting that the diisopropyl phosphate radical is bound to chymotrypsin through the hydroxyl groups of serine. 28~ Balls and Jansen 29 critically reviewed the method of preparation. The method consists essentially in dissolving the several-times-recrystallized and salt-free enzyme in 0.2 M phosphate buffer, pH 7.7; the concentration of enzyme in solution is not critical2 ° Diisopropyl fluorophosphate is added as a 1 M solution in isopropanol; this is recommended as a safety precaution, 29 since D F P is highly toxic. When working with a-chymotrypsin (molecular weight ca. 22,500) only a small excess of D F P is recommended 3° (1.2 millimoles of D F P per millimole of enzyme). ~5 E. F. Jansen, M. D. F. Nutting, R. Jang, and A. K. Balls, J. Biol. Chem. 179, 189, (1949). ~6 E. F. Jansen, M. D. F. Nutting, and A. K. Balls, J. Biol. Chem. 179, 201 (1949). 27 E. F. Jansen, M. D. F. Nutting, R. Jang, and A. K. Balls, J. Biol. Chem. 185, 209

(1950). ~s N. K. Schaffer, S. C. May, Jr., and W. H. Summerson, J. Biol. Chem. 202, 67 (1953). 2s, Caution in accepting this evidence as final is suggested by R. M. Herriott, The Mechanism of Enzyme Action (W. D. McElroy and B. Glass, eds.), Johns Hopkins Press, Baltimore, 1954, pp. 24-49, who discusses other possible sites of binding of D F P by chymotrypsin. 2~ A. K. Balls and E. F. Jansen, Advances in Enzymol. 13, 321 (1952). 30 E. F. Jansen, personal communication.

14

ENZYMES

OF PROTEIN METXBOLIS~

[2]

With trypsin and other forms of chymotrypsins 31 a greater excess (2 millimoles per millimole of enzyme) of D F P is recommended. Desnuell~ and co-workers7 used 5 millimoles of D F P per millimole of 5-chymotrypsin. The reaction is fast but not instantaneous. Usually it is allowed to proceed overnight in the cold room or for 1 to 2 hours at room temperature. The reaction is stopped by adjusting the pH to 4.0 with the aid of 2 N H2SO4. After this, the amorphous enzyme derivative is precipitated with ammonium sulfate at 0.8 saturation. The amorphous DP-a-chymotrypsin is then recrystallized according to the method of Kunitz and Northrop 1 described for the native a-chymotrypsin. Crystals of DP-achymotrypsin have the same appearance as those of the native a-chymotrypsin but are virtually inactive. After two recrystallizations only traces of activity can be detected. Crystallization of ~- and ~,-Chymotrypsins 3 As a starting material, either a-chymotrypsin or the mother liquor from a-chymotrypsin crystallizations can be used. With the latter the protein is first salted out in 0.7 saturated ammonium sulfate, and the precipitate is then used in the following operations in the same manner as the crystal cake of a-chymotrypsin. One hundred grams of crystal cake of a-chymotrypsin (containing ammonium sulfate) is suspended in 100 ml. of water. Fifty milliliters of 0.5 M phosphate buffer, pH 8.0, is added, and the clear solution is allowed to stand at 5 ° for three weeks. Then 120 ml. of saturated ammonium sulfate is added, the pH is adjusted to 5.6 by means of 5 N H2SO4 added drop by drop, and the mixture is allowed to stand at 20 ° for 3 days. Large bipyramidal crystals of ~/-chymotrypsin are filtered with suction. The yield is about 30 g. of filter cake. The filtrate (first ~, mother liquor) is stored at 5°. The ~, crystals are recrystallized by dissolving 10 g. in 30 ml. of water and adding 20 ml. of saturated ammonium sulfate. After 24 hours the crop of second crystals of ~,-chymotrypsin is filtered with suction. The precipitate is stored at 5° . The filtrate is combined with the first ~, mother liquor, adjusted to pH 4.2 with 5 N H2SO4, and the protein is salted out by addition of 21 g. of solid ammonium sulfate to each 100 ml. of solution. The precipitate is filtered with suction and is dissolved in 0.75 vol. of 0.01 N H2SO4. I t is allowed to stand for several days at 20 to 25 ° until a heavy precipitate of fine needle crystals of crude f~ is formed. The solution frequently turns into a thick fibrous gel of crystals, which are filtered with suction. The filtrate on standing may yield another crop of needle crystals. The 31E. F. Jansen and A. K. Balls, J. Biol. Chem. 194, 721 (1952).

[2]

CHYMOTRYPSINOGENS AND CHYMOTRYPSINS

15

total yield is about 50 g.31~ of crude/~ filter cake per 100 g. of original a-chymotrypsin filter cake. Recrystallization of Crude ~ Crystals. Ten grams of crystal cake is dissolved in 30 ml. of water, and 30 ml. of saturated ammonium sulfate is added. The p H is adjusted to 5.6 b y means of a few drops of 5 N NaOH, and after inoculation with ~, crystals the solution is allowed to stand for several days at 20 °. The crystals of 7-chymotrypsin are filtered off, and the p H of the filtrate is adjusted to 4.2. Crude ~ crystals gradually appear. T h e y are filtered after several days and subjected to a second recrystallization for the crude f~ crystals. Isolation of Pure ~ Crystals. Ten grams of three-times-recrystallized crude crystal cake is dissolved in 250 ml. of water, and 10 ml. of 0.4 M borate buffer, 31b pH 9.0, is added. The solution is heated to 37 ° and allowed to stand at this temperature for 1 hour. I t is then cooled to 20 ° and adjusted to pH 4.2 by means of 5 N H2SO,. Sixty-five grams of ammonium sulfate is added, and if a precipitate forms it is filtered off with the aid of 5 g. of Super-Cel through 9-cm. W h a t m a n No. 3 filter paper. T o each 100 ml. of filtrate 21 g. of ammonium sulfate is added, and the precipitated protein is filtered on hardened paper. The filtrate is rejected. Each gram of the precipitate is dissolved in 3 ml. of water, and 2 ml. of saturated ammonium sulfate is added. The solution is adjusted to pH 4.2 and allowed to stand at 20 °. An amorphous precipitate forms which gradually changes into very fine crystals. After several days the crystals are filtered through a hardened paper. The yield is about 2 g. of pure ~ crystal cake per 10 g. of crude ~ filter cake. The procedure for the recrystallization of the " p u r e " fl crystals is the same as for the crude ~ crystals.

Preparation of ~-Chymotrypsin ~-Chymotrypsin was first prepared b y J a c o b s e n ) The procedure described here is essentially t h a t of Schwert and Kaufman. 6 First 1.1 g. of lyophilized, essentially salt-free, a-chymotrypsinogen (prepared from chymotrypsinogen recrystallized eight times) 32 is dissolved in 50 ml. of water. A small trace of insoluble material is removed by filtration, and the pH of the solution is adjusted to 7.3 with 0.1 N NaOH. The solution is placed in a refrigerated bath at 0 °. After 20 minutes 70 mg. of crystalline trypsin (containing 50% MgS04) is added. ~1~ H. Neurath (personal communication) states that in his laboratory the usual yield

is about 25 g. 31bStock borate solution contains 49.6 g. of boric acid and 80 ml. of 5 N NaOH per 1000 ml. of solution. Borate buffers (0.4 M), pH 8.0 and 9.0, are mixtures of 100 parts of stock borate and 78.6 and 17.6 parts of 0.4 N HC1, respectively. ~2Chymotrypsinogen recrystallized from alcohol would be equally good.

16

ENZYMES OF PROTEIN METABOLISM

[2]

After a total of 98 minutes (half of this time interval would probably lead to the same result) the pH of the activation solution is rapidly adjusted to 4.2 with 2 N H~S04 and the solution is shell-frozen and lyophilized. The dry protein is stored at - 2 0 °. Over a period of two months no change in the activity of this preparation is observed. Crystalline DP-~-chymotrypsin was obtained 7 by following this activation procedure of Jacobsen, 5 and adding, at pH 7.6, 5 moles of D F P per mole of ~-chymotrypsin. The amorphous protein was then precipitated, and the needle-shaped crystals were obtained by following the procedure of Kunitz and Northrop 1 described for the crystallization of a-chymotrypsin.

Preparation of Chymotrypsinogen B The original extract from thirty average-size beef pancreases is prepared according to Kunitz and Northrop, 1 exactly as described for a-chymotrypsinogen. The whole operation is carried out in the cold room. The extract is brought to 0.2 saturation of ammonium sulfate (114 g. of solid salt per liter of extract). Ten grams of Celite No. 545 and 10 g. of Standard Super-Cel are added per liter, and the mixture is filtered through four large fluted filters (Sargent No. 500). The residue is rejected. The filtrate is brought to 0.4 saturation by addition of 121 g. of solid ammonium sulfate per liter. The precipitate which forms is filtered through a soft paper on a large Biichner funnel. The liquid may be used for the preparation of a-chymotrypsinogen, trypsinogen, trypsin inhibitor, and ribonuclease. The precipitate is dissolved in 5 vol. of water, and 20 ml. of a saturated solution of ammonium sulfate is added per 100 ml. of enzyme solution. A small precipitate is removed by filtration with the aid of 2 g. of Standard Super-Cel per 100 ml., using soft paper on a Btichner funnel, and is rejected. To each 100 ml. of filtrate 19 ml. of saturated ammonium sulfate is added. The precipitate which forms is collected on a hardened filter paper, and the filtrate is rejected. The precipitate is dissolved in 4 vol. of water plus 1 vol. of 1 M acetate buffer, pH 4.0, and the solution is adjusted to pH 4.0 (glass electrode). For each 100 ml. of solution 25 ml. of saturated ammonium sulfate is added. The precipitate is centrifuged off in the conical head of an International centrifuge at 5° and is washed twice, each time with one-half of the previous volume of 0.2 saturated solution of ammonium sulfate containing 20% of M acetate buffer, pH 4.0. The supernatant and washings are combined (the volume is measured), the pH is adjusted to 6.5 with 5 N NaOH (the volume of which is recorded), and a saturated solution of ammonium sulfate is added to at-

[2]

CHYMOTRYPSINOGENS AND CHYMOTRYPSINS

17

tain 0.4 saturation, a correction being made for the volume of NaOH used (33.3 ml. per 100 ml. of enzyme solution plus 0.67 ml. per each milliliter of NaOH). The precipitate is collected on a hardened filter paper on a Biichner funnel and is washed with 0.4 saturated ammonium sulfate containing 20% of M acetate buffer, pH 6.5. The washed precipitate is dissolved in a minimum amount of water kept at pH 4.0 by dropwise addition of 1 N HC1. The solution is centrifuged at high speed in a Servall SS-1 centrifuge in order to remove any of the extraneous material and is dialyzed in the cold, with stirring, against 0.01 M acetate buffer, pH 5.5, with frequent changes of buffer. Typical large plates of chymotrypsinogen B appear after several hours. The crystallization is usually complete after 2 to 3 days. Recrystallization is performed by dissolving crystals in a minimum amount of water which is kept at pH 4.0 by dropwise addition of 1 N HC1. After a complete solution is achieved, 0.5 N NaOH is added drop by drop until the first sign of "silkiness." The solution is allowed to stand for an hour at room temperature, after which it is transferred to a dialyzing bag and dialyzed for 24 hours against 0.01 M acetate buffer, pH 5.5, to complete crystallization. After three to five recrystallizations the crystals are centrifuged in a glass tube in a Servall centrifuge and are ]yophilized in the same tube. They may be kept in the refrigerator for several months without significant activation. If chymotrypsinogen B with a low content of chymotrypsin B is desired, this method should be followed. Even with this method partial activation occurs during the process of crystallization. 12 Fortunately, the activated enzyme remains in the mother liquor. In view of the lack of a better method, it is recommended that several (three to five) recrystallizations be made in rapid succession with a minimum exposure to room temperature. Preparations of chymotrypsinogen B containing only 0.15 % of the active chymotrypsin B have been occasionally obtained in the laboratory. An alternative method, which has the advantage of obtaining both crystalline deoxyribonuclease and chymotrypsinogen B, has been described. 17 This method, however, has two disadvantages: the yield is smaller, and considerable activation occurs during the process. This method can be used only when the ultimate goal is chymotrypsin B. Fresh pancreas is treated exactly as described by Kunitz 2 in the preparation of deoxyribonuclease (Vol. II [63]) up to the stage at which a precipitate with 0.7 saturation of ammonium sulfate was obtained. The 0.5 ammonium sulfate precipitate still containing Celite is suspended in 4 vol. of water, adjusted to pH 4.0 by addition of 5 N H2SO4, and stirred mechanically at low speed at 5° for 2 hours. The pH is checked several

18

ENZYMES OF PROTEIN METABOLISM

[2]

times and readjusted to 4.0 when necessary. One volume of cold saturated ammonium sulfate is added slowly with stirring to produce a 0.2 saturated solution. A small precipitate together with Celite is removed by filtration on a Btichner funnel through a soft paper. The filtrate is brought to pH 5.3 by addition of 5 iV NaOH, and the protein is salted out by addition of 20.9 g. of solid ammonium sulfate per 100 ml. of filtrate. The resulting precipitate is collected as dry as possible on a Bfichner funnel with Whatman No. 50 filter paper. The precipitate is redissolved in a minimum amount of water kept at pH 4.0 by dropwise addition of 1 N HC1 and is dialyzed against 0.01 M acetate buffer, pH 5.5, exactly as described above. Preparation of Chymotrypsin B 1~,14 Five grams of five-times-reerystallized chymotrypsinogen B is dissolved in 50 ml. of 0.2 M borate buffer, pH 7.8, and adjusted to pH 7.8. Then 2.5 mg. of crystalline trypsin is added, and the solution is allowed to stand for 4 days at 5 °. It is then transferred to a dialyzing bag and is dialyzed against 0.01 M acetate buffer, pH 5.0, at 5 ° (buffer is changed frequently). A heavy amorphous precipitate forms, which is centrifuged down at high speed (Servall SS-1 centrifuge). The precipitate is dissolved in a minimum amount of water, kept at pH 4.0 by the addition of 1 N HCI, and a small amount of gelatinous insoluble material is removed by centrifugation. The clear liquid is dialyzed against 0.01 M acetate buffer, pH 5.5. The precipitate which forms on dialysis is composed of poorly shaped needles and prisms. The crystalline form improves considerably on recrystallization. The crystals are collected by either centrifugation or filtration through a hardened paper. Recrystallization is achieved by dissolving the semidry crystal cake in a minimum amount of water kept at pH 4 by dropwise addition of 1 N HC1, and subsequent dialysis against 0.01 M acetate buffer, pH 5.5. It was found convenient to include at least one recrystallization in which crystals were first dissolved in a minimum amount of 0.4 M borate buffer, pH 8.0, and dialyzed against 0.01 M acetate buffer, pH 5.0. Four recrystallizations are recommended, after which the crystal cake is lyophilized and stored in the refrigerator. A convenient nomogram for calculating required amounts of ammonium sulfate has been published by Dixon 32~ (see Vol. I [10]). The nomogram is based on a solubility value of 760 g./1., corresponding to room temperature (ca. 25°). The values used by Northrop and Kunitz are calculated for the cold-room temperature (ca. 5°) and differ considerably from the values of Dixon. 82~ M. Dixon, Biochem. J. 54, 457 (1953).

[2]

CHYMOTRYPSINOGENS AND CHYMOTRYPSIN8

19

Determination of Activity Activity of chymotrypsins can be determined b y a variety of methods which m a y be conveniently subdivided into two groups: (1) methods using natural substrates; (2) methods using synthetic substrates. B o t h types of method require dilute solutions of enzymes of accurately determined concentrations. Kunitz 33 introduced a very convenient method of expressing the concentration of pure proteins in dilute solutions b y measuring specific absorption at 280 mu. Kunitz's method is now a common practice in m a n y laboratories. I t was found convenient to express the specific absorption as the optical factor. The optical factor is defined as the reciprocal of the optical density at 280 m~, in a cell 1 cm. wide, when the concentration of protein is 1 mg./ml. The variation in absorption is comparatively small in the range of pH values from 2 to 6, and the factors are generally useful for this range. In establishing the optical factor, the purity of the preparation is of highest importance. Beside being homogeneous in regard to electrophoresis and sedimentation the preparation should be free from ultravioletabsorbing contaminants and should be corrected for the moisture content. The optical factors for chymotrypsins have been recently reinvestigated 2° and the following values were obtained. a-Chymotrypsinogen a-Chymotrypsin Chymotrypsinogen B Chymotrypsin B

0. 484 34determined at wavelength 282 mg, 0.500 34~ 0. 500,330.495,200.465 34~ 0.55(0. 546)30 0.54(0. 538)20

The solution of a desired enzyme concentration is prepared b y first making a solution in the range from 0.1 to 0.35 mg./ml. The exact concentration of this solution is then determined by its absorption at 280 mg, and the solution of a desired concentration is prepared b y an appropriate dilution. The majority of methods using the natural substrates are based on determination of the rate of proteolysis, and often the same m e t h o d can be applied to several proteolytic enzymes. The representative m e t h o d of this t y p e is the spectrophotometric method of Kunitz, 33 in which casein serves as substrate. T h e method was originally devised for the determination of trypsin and has been later applied to the determination of trypsin 33 M. Kunitz, J. Gen. Physiol. 30, 291 (1947). a~ M. A. Eisenberg and G. W. Schwert, J. Gen. Physiol. 34, 583 (1951). 34~p. E. Wilcox (unpublished), quoted from H. Neurath, J. A. Gladner, and E. W. Davie, "The Mechanism of Enzyme Action" (W. D. McElroy and B. Glass, eds.), pp. 50-69, Johns Hopkins Press, Baltimore, 1954.

20

ENZYMES OF PROTEIN METABOLISM

[2]

inhibitors and chymotrypsins. The original method is described in detail in the next section on trypsin (see p. 32). This method is recommended for the purpose of following the purification of either an enzyme or an inhibitor. In respect to chymotrypsins, in the reviewer's laboratory 20 this method was modified in that 0.1 M borate buffer, pH 8.0, was substituted for the phosphate buffer, and CaC1236 was added to attain a final concentration of 0.005 M in the enzyme-substrate mixture. 1.2

1.0 O 0O

c~ 0.8

0.6 0.4 0.2

o

1'o

2'0

'

~o

'

45

'

50

Chymotrypsin, 3, per ml. 0.5% casein

FIG. 2. Standard activity curves for chymotrypsins ~ and B. ~°

Standard curves for chymotrypsins a and B obtained under the above conditions are illustrated in Fig. 2. Indicated concentrations of enzyme represent the final concentrations in the enzyme substrate mixture. Since the total volume in this procedure is 2 ml., the actual amounts of enzyme pipetted into the tube are twice the amounts indicated in the graph. Some of the methods utilizing the natural substrates are based on the ability of chymotrypsins to clot milk--the ability which was responsible for the original name of the enzyme and which distinguished it from trypsin. Several methods of the type have been described. Essentially the same methods are used for the determination of milk-clotting activity of rennin, pepsin, and chymotrypsin2 6 35 N. M. Green, J. A. Gladner, L. W. Cunningham, Jr., and H. Neurath, J. Am. Chem. Soc. 74~ 2122 (1952) found that calcium enhanced the activity of a-chymotrypsin. In unpublished work from this laboratory it was found that calcium accelerates also the rate of proteolysis by chymotrypsin B. With the casein method, a calcium concentration of 0.005 M is optimal for a-chymotrypsin and 0.0005 M is optimal for chymotrypsin B. With synthetic substrates or hemoglobin 0.1 M CaC12 is optimal for chymotrypsin B and 0.05 M for a-chymotrypsin. 8s See articles on pepsin and rennin, Vol. II [1, 7].

[2]

CHYMOTRYPSINOGENS AND CHYMOTRYPSINS

21

The introduction of simple synthetic substrates 37 for proteolytic enzymes must be credited to Bergmann and his co-workers2 8 The use of synthetic peptides made it possible to establish for each major proteolytic enzyme the peptide bonds susceptible to its action. F u r t h e r simplification of the substrate was achieved when it was found t h a t the amides 39 and esters t° of N-substituted aromatic amino acids are good substrates for chymotrypsins. The structural requirements for the specificity have been discussed in detail in the review of N e u r a t h and Schwert. 41 More recently Sprinson and Rittenberg 4~ have shown t h a t carbobenzoxy-L-phenylalanine, incubated in the presence of a-chymotrypsin in H20 Is, exchanges oxygen atoms of the carboxyl group with the medium, and that this reaction does not occur in the absence of a-chymotrypsin. D o h e r t y and T h o m a s 4~found t h a t a - c h y m o t r y p s i n is capable of hydrolyzing the C - - C bond in compounds of the type of ethyl 5-phenyl-3-ketovalerate with the formation of ethyl acetate and phenylpropionic acid. Only the methods employing synthetic substrates can be used when the specificity of the enzyme is to be determined. Similarly only these substrates can be used when the degree of contamination of c h y m o t r y p sin with trypsin is to be determined. 44 An additional advantage is t h a t zero-order kinetics persists for a longer period than with natural substrates. However, even with synthetic substrates the action of a-chymotrypsin m a y not be limited to the hydrolysis of a single susceptible bond, since transpeptidation (see Vol. I I [6]) has been shown to occur, a~-/ Numerous techniques, suggested for use with the synthetic substrates, were critically reviewed b y N e u r a t h and Schwert. 4° Two methods have 37 Methods of preparations of the typical substrates will be described by E. L. Smith in Vol. III [80]. 38See M. Bergmann, Advances in Enzymol. 2, 49 (1942). sq j. S. Fruton and M. Bergmann, J. Biol. Chem. 145, 253 (1942). 40G. W. Schwert, H. Neurath, S. Kaufman, and J. E. Snoke, J. Biol. Chem. 172, 221 (1948). 41 H. Neurath and G. W. Schwert~ Chem. Revs. 46, 69 (1950). 42D. B. Sprinson and D. Rittenberg, Nature 167, 484 (1951). 4a D. G. Doherty and L. Thomas, Federation Proc. 13, 200 (1954). a* Even with the synthetic substrates a careful selection must be made. Schwert et al.4O described a case of cross-reactivity with BAME (benzoyl-L-arginine methyl ester), a typical substrate for trypsin which was also susceptible to hydrolysis by a- and 7-chymotrypsins. See also J. A. Gladner and H. Neurath, Biochim. et Biophys. Acta 9, 335 (1952). ,4~ R. B. Johnston, M. J. Mycek, and J. S. Fruton, J. Biol. Chem. 187, 205 (1950). *4bj. S. Fruton, R. B. Johnston, and M. Fried, J. Biol. Chem. 190, 39 (1951). 44cM. Brenner, H. R. Mrtiler, and R. W. Pfister, Helv. Chim. Acta 33, 568 (1950). 44dM. Brenner, E. Sailer and K. Riifenacht, Helv. Chim. Acta 34, 2096 (1951). 44"H. Tauber, J. Am. Chem. Soc. 74, 847 (1952). 44! K. Blau and S. G. Waley, Biochem. J. 57, 538 (1954).

22

ENZYMES OF PROTEIN METABOLISM

[2]

been added since t h a t time. Parks and Plaut 4s utilize the manometric technique (described in detail below), and Ravin et al. ~6 a colorimetric technique. The latter m e t h o d is based on the use of N-benzoyl-DL-phenylalanine-~-naphthyl ester. T h e hydrolyzed naphthol is coupled with tetrazotized diorthoanisidine; the resulting azo dye is extracted in ethyl acetate and determined colorimetrically. M a n y of the suggested techniques are applicable to the determination of b o t h c h y m o t r y p s i n and trypsin, provided t h a t appropriate substrates are used. Only a few of these methods have been employed in the reviewer's laboratory. T h e methods described below were chosen rather arbitrarily, and it is realized t h a t m a n y valuable methods have been omitted. Determination of the Amidase Activity of Chymotrypsins. 4° T h e reaction mixtures for determining amidase activity are made by mixing equal volumes of 0.1 M solution (or suspension) of substrate in 0.1 M phosphate buffer and of enzyme solution in phosphate buffer. 47 A stop watch is started, and the mixture is placed at 25 ° and shaken mechanically. At intervals 0.2-ml. samples are withdrawn for analysis and are introduced into the outer chambers of Conway plates, 48 which contain in the inner chamber 0.75 ml. of 2 % boric acid. One milliliter of saturated solution of K2C03 is added to the outer chamber to volatilize the ammonia. T h e m o m e n t at which the K 2 C Q solution touches the sample is considered as the end of the time interval for each withdrawn sample. The plates are allowed to stand for at least ] hour before being titrated with approximately 0.01 N HC1 and 1 drop of Tashiro's indicator. Thus, 0.01 ml. of acid corresponds to approximately 1% hydrolysis. Since the indicator color varies with the volume of the system at the end point, water is added to the plates in which the extent of hydrolysis is small in order to bring the final volume for all titrations close to a constant volume. The horizontal burets used for these titrations were made b y drawing out the ungraduated portions of Kimble Exax 1-ml. measuring pipets and fitting the other ends with Clay-Adams pipet suction units. Blank determinations are made b y placing 0.1 ml. each of substrate 45 R. E. Parks, Jr., and G. W. E. Plaut, J. Biol. Chem. 203, 755 (1953). 46 H. A. Ravin, P. Bernstein, and A. M. Seligman, J. Biol. Chem. 208, 1 (1954).

47If calcium is used in the system, borate buffer should replace the phosphate buffer. Since chymotrypsins are not very stable in dilute solutions at the pH of their optimal activity it is recommended to prepare the enzyme solution immediately before use, and to include 0.1 M CaCl2 in the borate buffer to increase the stability of chymotrypsins (unpublished). 48E. J. Conway, "Micro-Diffusion Analysis and Volumetric Error," Crosby, Lockwood and Son, London, 1939.

[2]

CHYMOTRYPSINOGENS AND CHYMOTRYPSINS

23

and enzyme solutions a short distance apart on Conway plates and by tipping the plates so that the solutions are mixed with the saturated K2C03 solution before they are mixed with each other. Typical substrates are glycyl-L-phenylalaninamide and acetyl-Ltyrosinamide. Potentiometric Determination of Esterase Activity of Chymotrypsin. This method was first described by Schwert et al. 4° The principle of the method, applicable to both trypsin and chymotrypsin, is a continuous titration of liberated carboxyl groups from an appropriate ester, at 25 °, using a potentiometer as a null instrument. This method is widely used with small modifications introduced in different laboratories. The following procedure is recommended by Balls and Jansen. 29 The reaction mixture of 20 ml., at pH 6.25, consists of sufficient L-tyrosine ethyl ester (TEE) to make it 0.025 M, sufficient NaC1 to make it 0.25 M, and routinely an amount of enzyme which would cause the liberation of 0.005 meq. of carboxyl groups per minute. The pH is maintained at 6.25 by addition of 0.02 N NaOH, and readings of the added alkali are recorded periodically. Under these conditions the reaction is of zero order. The potency of a chymotrypsin preparation is expressed in milliequivalents of carboxyl groups per milligram of enzyme or per milligram of enzyme nitrogen. When N-acetyl-L-tyrosine ethyl ester (ATEE) is the substrate, the procedure is essentially the same except that the final concentration of ATEE is 0.018 M and hydrolysis is allowed to proceed at pH 7.8 in the presence of 0.01 M phosphate buffer in place of NaC1. Since the hydrolysis of ATEE is of apparent first order, the results are calculated on the basis of the initial slope. In Desnuelle's laboratory 49 the assay for chymotrypsin is performed exactly as described for trypsin (see Vol. II [3]) except that the substrate is 0.1 M ATEE, and the solution of enzyme contains approximately 2 mg. of salt-free a-chymotrypsin per 100 ml. Since the reaction is of apparent first order, the concentration of substrate is critical. After the experimental points are plotted, the initial slope is used for calculations of activity and potency. Other typical substrates used for potentiometric titrations are carbobenzoxy-L-tyrosine ethyl ester, L-phenylalanine ethyl ester, and acetylL-phenylalanine ethyl ester. Manometric Assay of Esterase Activity. 4s This method has an advantage in that a series of determinations may be performed simultaneously, and that a zero-order reaction persists for a considerable period of time. ~9 M. Rovery, C. Fabre, and P. Desnuelle, Biochim. et Biophys. Acta 12, 547 (1953).

24

a-Chymotrypsinogen

ENZYMES OF PROTEIN METABOLISM

[2]

TABLE I MOLECULAR WEIGHTS 36,000 Osmotic pressure a 22,000 Sedimentation-diffusion b 23,000 Sedimentation-diffusion c 22,500 Sedimentation-diffusion ~ 24,000 Osmotic pressure *

a-Chymotrypsin

40,000 32,000 17,500 21,500 22,500 27,000 23,000

Osmotic pressuref X-rayg Sedimentation-diffusion b Sedimentation-diffusion h Sedimentation-diffusion d Light scattering ~ Calculated from nitrophenol Released in preparation of E 600 derivativei

DP-a-Chymotrypsin

27,000 27,500 24,800

Light scattering k Osmotic pressure k From P content ~

DP-t~-Chymotrypsin DP-fl-Chymotrypsin

30,000 23,100

Osmotic pressures From P content ~

~-Chymotrypsin

DP-~,-Chymotrypsin

27,000 30,100 15,500 25,800

Osmotic pressurel X-rayo Sedimentation-diffusion b From P content k

Chymotrypsinogen B

22,500

Sedimentation-diffusion ~

Chymotrypsin B 22,500 Sedimentation-diffusion d a M. Kunitz and J. H. Northrop, J. Gen. Physiol. 18, 433 (1935). b G. W. Schwert, J. Biol. Chem. 179, 665 (1949). c G. W. Schwert, J. Biol. Chem. 190, 779 (1951). E. L. Smith, D. M. Brown, and M. Laskowski, J. Biol. Chem. 191, 639 (1951). e H. Gutfreund, Trans. Faraday Soc. 50, 624 (1954). s M. Kunitz, J. Gen. Physiol. 22, 207 (1938). a I. Fankuchen, in "Proteins, Amino Acids and Peptides as Ions and Dipolar I o n s " (E. J. Cohn and J. T. Edsall, eds.), Reinhold Publishing Corp., New York, 1943. h G. W. Schwert and S. Kaufman, J. Biol. Chem. 190, 807 (1951). K. J. Palmer, quoted from Balls and J a n s e n ) i B. S. Itartley and R. A. Kilby, Biochem. J. 56, 288 (1952). A. K. Balls and E. F. Jansen, Advances in Enzymol. 13, 321 (1952).

[2]

CHYMOTRYPSINOGENS AND CHYMOTRYPSINS

25

T h e a s s a y is p e r f o r m e d in 15-ml., 5° single side a r m W a r b u r g vesselh in a t o t a l fluid v o l u m e of 3 ml. a t 30 °. T h e e n z y m e s o l u t i o n is p l a c e d in t h e side a r m , a n d its final v o l u m e is b r o u g h t t o 0.3 ml. w i t h w a t e r . I t is a d v i s a b l e t o use c a r e f u l l y c a l i b r a t e d m i c r o p i p e t s t o d i s p e n s e t h e e n z y m e , since t h e v o l u m e is c r i t i c a l in o b t a i n i n g t h e m o s t a c c u r a t e results. I f t h e e n z y m e s o l u t i o n t o b e a s s a y e d c o n t a i n s a p p r e c i a b l e a m o u n t s of acid, i t is n e c e s s a r y t o b r i n g i t t o p H 6.5. TABLE II ISOELECTRIC POINTS

a-Chymotrypsinogen

5.0 6.3 9.5 9.1

Cataphoresis a Donnan equilibrium b Electrophoresis in 0.01-t` buffers ~ Electrophoresis in 0.1-# buffers d

a-Chymotrypsin

5.4 8.6 8.1 8.3 8.5

Cataphoresis a Electrophoresis in Electrophoresis in Electrophoresis in Electrophoresis in

~-Chymotrypsin ~,-Chymotrypsin

8.6 8.5

Electrophoresis in 0.05-t` buffers' Electrophoresis in 0.05-t~ buffers"

Chymotrypsinogen B

5.2

Electrophoresis in 0.1-• buffers d

0.01-t` buffers c 0. l-t` buffers c 0.l-t, buffers e 0.05-t` buffers'

Chymotrypsin B 4.7 Elcctrophoresis in 0.l-t` buffers d M. Kunitz and J. H. Northrop, J. Gen. Physiol. 18, 433 (1935). b V. M. Ingrain, Nature 170, 250 (1952). c A. E. Anderson and R. A. Alberty, J. Phys. and Colloid Chem. 52, 1345 (1948). V. Kubacki, K. D. Brown, and M. Laskowski, J. Biol. Chem. 180, 73 (1949). R. Egan, personal communication. The isoelectrie points of a- and ~-chymotrypsins are apparently identical, and that of fl form is very similar. The mobilities in more acid or more alkaline regions are different for each of these three chymotrypsins, a-Chymotrypsin on prolonged electrophoresis exhibits some heterogeneity. T h e m a i n c o m p a r t m e n t c o n t a i n s t h e s u b s t r a t e a n d b i c a r b o n a t e solution. L - P h e n y l a l a n i n e e t h y l e s t e r h y d r o c h l o r i d e ( P h E E ) , t h e m o s t useful of t h e s u b s t r a t e s t e s t e d , is a d d e d in 2.0 ml. of 0.0375 M s o l u t i o n (prep a r e d a n d a d j u s t e d t o p H 6.5). T h e n 0.7 ml. of a n 0.18 M s o l u t i o n of s o d i u m b i c a r b o n a t e is a d d e d . F i n a l c o n c e n t r a t i o n s of 0.025 M for P h E E a n d 0.042 M for s o d i u m b i c a r b o n a t e a r e t h u s o b t a i n e d . T h e p H of t h i s c o n c e n t r a t i o n of b i c a r b o n a t e w i t h an a t m o s p h e r e of 1 0 0 % c a r b o n d i o x i d e is 6.5. A c o n t r o l vessel c o n t a i n i n g no e n z y m e m u s t b e i n c l u d e d in t h e 50 In this laboratory 12-ml. vessels with a side arm blown up to contain 1 ml. of fluid are used.

26

ENZYMES OF PROTEIN METABOLISM

[3]

set, since the s u b s t r a t e alone shows a slight b u t measurable rate of hydrolysis. After a 10-minute period of gassing with 100% carbon dioxide followed b y a 5-minute period of t e m p e r a t u r e equilibration in the w a t e r bath, the e n z y m e is tipped into the m a i n c o m p a r t m e n t . I t has been found advisable to t a k e the zero readings after 2 or 3 minutes. T h e reaction is linear with t i m e until a b o u t 6 0 % of the ester is h y d r o lyzed. T h e rate of COs evolution is proportional to the concentration of e n z y m e over a range 10 to 100 ~,/ml. (best 20 to 70 ~,/ml.).

[3] Trypsinogen and Trypsin B y M. LASKOWSKI

T r y p s i n ~,~ and trypsinogen 3 were obtained in crystalline form from beef pancreas b y N o r t h r o p and Kunitz. F u r t h e r work 4 has i m p r o v e d m e t h o d s of p r e p a r a t i o n and established t h a t the t r a n s f o r m a t i o n of trypsinogen i n t o trypsin is a proteolytic process and m a y be accomplished 5 either b y autocatalysis or b y enterokinase, or b y the kinase f r o m a mold of the genus P e n i c i l l i u m . ~ T h e m e c h a n i s m of activation of trypsinogen has been studied in detail. 7-9 T h e t r a n s f o r m a t i o n is not quantitative, since in addition to trypsin an " i n e r t p r o t e i n " is formed. T h e relative a m o u n t of " i n e r t p r o t e i n " depends on the p H and the presence of other ions in the m e d i u m in which a c t i v a t i o n occurs. ~° The f o r m a t i o n of the " i n e r t prot e i n " is suppressed b y calcium ions. 1° T h e latter finding led to an improved m e t h o d of crystallization of trypsin. ~1 Evidence has been presented ~,~3 t h a t during the a c t i v a t i o n of t r y p sinogen a peptide is split off the N terminal end of the trypsinogen mole1j. H. Northrop and M. Kunltz, Science 73, 262 (1931). J. H. Northrop and M. Kunitz, J. Gen. Physiol. 16, 267 (1932). a j. H. Northrop and M. Kunitz, Science 80, 505 (1934). M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19, 991 (1936). 6 M. Kunitz and J. H. Northrop, Science 80, 190 (1934). 8 M. Kunitz, J. Gen. Physiol. 21, 601 (1938). 7 M. Kunitz, J. Gcn. Physiol. 22, 293 (1939). s M. Kunitz, J. Gen. Physiol. 22, 429 (1939). g M. Kunitz, Enzymologia 7, 1 (1939). 10 M. R. McDonald and M. Kunitz, J. Gen. Physiol. 26, 53 (1941). 11 M. R. McDonald and M. Kunitz, J. Gen. Physiol. 29, 155 (1946). ~ E. W. Davie and H. Neurath, Biochim. ct Biophys. Acta 11, 442 (1953). 1~ M. Rovery, C. Fabre, and P. Desnuelle, Biochim. et Biophys. Acta 12, 547 (1953).

[3]

TRYPSINOGEN AND TRYPSIN

27

cule. A tentatively suggested '2 composition of the peptide is H2N Val. (Asp) 5ore-Lys. COOH. 13~ Crystallization of Trypsinogen 4 Beef pancreas is treated exactly as described in Vol. II [2] up to the crystallization of a-chymotrypsinogen.13b The mother liquor and the washings from the crystallization of a-chymotrypsinogen are adjusted to pH 3.0 (pink with 0.01% methyl orange on a test plate) with about 1 ml. of 5 N H2S04 per 100 ml. of filtrate. Solid ammonium sulfate is then added (30.4 g. per 100 ml.), the precipitate which forms is collected on a hardened paper, and the filtrate is rejected. Each 10 g. of the precipitate is dissolved in 30 ml. of water and is treated with 20 ml. of saturated ammonium sulfate and 2 g. of Filter-Cel. The mixture is filtered with suction through a soft paper, and the precipitate is washed with 0.4 saturated ammonium sulfate and is discarded. The volume of the filtrate is measured, and an equal volume of saturated ammonium sulfate solution is added. The mixture is filtered with suction through a hardened paper on a large Bfichner funnel (18.5 cm.) until the precipitate is quite hard (the cracks which appear are worked out with a spatula). A saturated solution of magnesium sulfate in 0.02 N H2S04 is then poured over the precipitate to form a layer of about 5 mm. and is allowed to remain on the filter for 1 to 2 minutes. After this, the funnel is removed, and the magnesium sulfate solution is poured off by tipping the funnel. The filtration is continued until the rest of the washing solution is removed. This precipitate is referred to as "crude trypsinogen" and may be used for either the purification of trypsinogen or for the activation to trypsin, followed by the isolation of the trypsin inhibitor-trypsin complex. Crude trypsinogcn (30 g.) is dissolved in 30 ml. of 0.4 M borate 14 buffer, pH 9.0, at 2 to 5° (in an ice-water bath), and saturated solution of potassium carbonate is added dropwise until the pH is brought to 8.0. The volume of the solution is measured, and an equal volume of a saturated solution of magnesium sulfate is added. The mixture is allowed to stand in the cold room. Short triangular prisms of trypsinogen appear in the course of 2 to 3 days. If the solution is inoculated with crystals of 13~ I n a personal communication to the reviewer Dr. N e u r a t h stated t h a t E. W. Davie and H. N e u r a t h [J. Biol. Chem. 212, 515 (1955)] established the n u m b e r of aspartie acid residues as four. 13b Unless otherwise specified all operations are carried out a t room t e m p e r a t u r e (20 to 25°). 14 Stock borate solution contains 49.6 g. of boric acid a n d 80 ml. of 5 N sodium hydroxide per 1000 ml. of solution. Borate buffers, 0.4 M, p H 8.0 a n d 9.0, are mixtures of 100 p a r t s of stock borate a n d 78.6 a n d 17.6 parts of 0.4 M hydrochloric acid, respectively.

28

ENZYMES OF PROTEIN METABOLISM

[3]

trypsinogen, crystallization is much more rapid but the crystals are not so well formed. If the crystallization is delayed for more than 4 to 5 days, crystals of trypsin may appear. The crystals are filtered at 5° . The precipitate is washed on the funnel several times with cold 0.5 saturated magnesium sulfate made up in 0.1 M borate buffer and finally with saturated magnesium sulfate made up in 0.1 N H2S04 at room temperature. The crystals are then dried in an electric refrigerator at 5 ° and stored in the icebox. The dried material generally contains about 40% of trypsinogen protein and 60% of magnesium sulfate. No safe method for the recrystallization of trypsinogen can be recommended. Direct recrystallization leads to a mixture of trypsinogen and trypsin. Recrystallization has been accomplished in the presence of an excess of the pancreatic trypsin inhibitor. 4 This method is expensive owing to the high price (or low yield) of the pancreatic trypsin inhibitor (for the method of preparation, see Vol. II [4]). If this method is used the trypsin content of crystalline trypsinogen should be determined and an equal weight of the inhibitor (equivalent to a twofold excess) should be added. The recrystallized trypsinogen is then treated as follows, s Ten grams of trypsinogen crystals is dissolved in 200 ml. of N/400 HC1, and 200 ml. of 5 % trichloroacetic acid is added. The solution is left at 20° for 1 hour and then filtered with suction and washed several times with small amounts of 2.5% trichloroacetic acid and finally with water. The semidry precipitate is dissolved in twenty-five times its weight of N/50 HC1 and allowed to stand for about 30 minutes. Ammonium sulfate (242 g./1.) is added to attain 0.4 saturation. The precipitate is filtered off and rejected. The filtrate is brought to 0.7 saturation with solid ammonium sulfate (205 g./1.) and filtered with suction. Yield is about 30%. Tietze 15 recently described a method which led to a preparation of recrystallized trypsinogen, almost free from trypsin (0.016%). However, the author does not recommend his procedure for the routine preparations, since failures have been encountered. In Tietze's procedure 0.25 ml. of pure diisopropyl fluorophosphate (DFP) is added to a solution of 96 g. of "crude trypsinogen" in 96 ml. of 0.4 M borate buffer, pH 9, in order to inhibit any of the free trypsin present or forming. As in the procedure of Kunitz and Northrop 4 the pH is adjusted to 8.0 with saturated potassium carbonate, an equal volume of saturated magnesium sulfate is added, and the mixture is allowed to stand for 48 hours in the cold room. The crystalline trypsinogen (7.8 g.) is collected and then dissolved in 70 ml. of water, containing 0.003 ml. of DFP per milliliter. The pH is adjusted to 3.0, and a small amount of insoluble matter is removed by filtration through hardened filter paper. 15 F. Tietze, J. Biol. Chem. 204, 1 (1953).

[3]

TRYPSINOGEN AND TRYPSIN

29

The solution is dialyzed against 0.001 N HC1 and lyophilized. Trypsinogen is recrystallized in the following manner. Each gram of the lyophilized protein is dissolved in 4 ml. of ice-cold 0.4 M borate buffer, pH 9.3, and filtered in the cold. The pH is adjusted to 8.0 by a dropwise addition of 5/V H2S04, and an equal volume of saturated magnesium sulfate solution is added. The mixture is exposed to room temperature for a period of 24 hours, which results in formation of a copious crystalline precipitate. Crystallization of Trypsin In the method of Kunitz and Northrop 4 trypsin was obtained by activation of the crystalline trypsiuogen. The yield was small, however, owing to a side reaction leading to the formation of "inert protein." 1V[cDonald and Kunitz ~1 improved the yield by introducing calcium ions into the activating mixture. If trypsin, and not trypsinogen, is the desired product, the best yields are obtained in a minimum time by following the method of Kunitz and Northrop up to the stage of "crude trypsinogen" and activating the crude trypsinogen according to McDonald and Kunitz, as described below. Fifty grams of "crude trypsinogen" (or 30 g. of crystalline trypsinogen) is dissolved in 200 ml. of 0.005 N HC1. This solution is added to a previously prepared ice-cold mixture of 100 ml. of 1 M calcium chloride, 250 ml. of 0.4 borate buffer, ~4 pH 8, and 400 ml. of distilled water. 15~ The volume of the mixture is adjusted to 1000 ml. with cold water, and the mixture is allowed to stand for 24 hours in the cold room. The solution is treated with 2 g. of Standard Super-Cel and is filtered in the cold room through soft paper. The precipitate is rejected. The filtrate is adjusted to pH 3.0 (tested with methyl orange on a spot plate) with 5 N H2SO4 (about 4 ml.). Solid ammonium sulfate is added (242 g./1.) to 0.4 saturation, and the mixture is left in the cold room for 2 days. A heavy sediment of calcium sulfate crystals is formed. They are removed by filtration through a soft (Whatman No. 3) filter paper. The precipitate is rejected. The filtrate is brought to 0.7 saturation by addition of 205 g. of ammonium sulfate per liter, and the mixture is filtered with suction through the hardened filter paper. The filtrate is rejected. The filter cake (about 50 g.) is dissolved in 150 ml. of distilled water, and trypsin is reprecipils~ McDonald and Kunitz 11 used crystalline trypsinogen which always contains sufficient amounts of active trypsin to start the autocatalytic reaction. If the "crude trypsinogen" containing an excess of the trypsin inhibitor is the starting material, an activating agent (excess of trypsin, or enterokinase) should be added, otherwise trypsinogen, and not trypsin, would be crystallized. In the reviewer's laboratory 200 E.K.U. of enterokinase (5 mg. of the purified preparation) per 100 g. of crude trypsinogen is used.

30

ENZYMES OF PROTEIN METABOLISM

[3]

tated b y a slow addition of 350 ml. of saturated ammonium sulfate from a dropping funnel, while the mixture is mechanically stirred. The mixture is filtered with suction through a piece of hardened paper 18.5 cm. in diameter or larger, and the precipitate is washed as described for the "crude t r y p s i n o g e n " with saturated magnesium sulfate in 0.02 N H2S04 in order to remove the excess of ammonium sulfate. The semidry filter cake is dissolved in ice-cold 0.4 M borate buffer, p H 9.0 (10 ml. per 10 g. of filter cake), ~6 in an ice-water bath. Crystals of fine needles appear almost immediately. The mixture is left in the cold room for 24 hours and is filtered in the cold on hardened paper with suction. The crystals are washed with cold 0.5 saturated magnesium sulfate in 0.1 M borate buffer, pH 8.0, in the cold room and then at room temperature with saturated magnesium sulfate in 0.1 N H~S04. The yield is 15 to 20 g. The m o t h e r liquor (filtrate E) and the washings from the crystallization of trypsin are combined and saved for the preparation of pancreatic trypsin inhibitor-trypsin complex (see Vol. I I [4]). RecrystaUization of Trypsin. 4 T h e semidry filter cake is dissolved in 0.02 N H2S04 (10 ml. per 10 g. of filter cake) b y mixing it gradually into the acid to avoid foam. I t is filtered on a small W h a t m a n No. 3 fluted paper, which is washed with several milliliters of 0.02 N H2S04. The filtrate is cooled to a b o u t 5 ° and is adjusted to p H 8.0 (pink to 0.01% phenol red, b u t not to 0.01% cresol red on a spot plate) with cold 0.4 M borate buffer, p H 9.0 (about 8 ml. per 15 ml. of solution). Crystallization begins almost immediately. The mixture is left in the cold room for 24 hours. T h e precipitate is collected and is washed as described for the first crystallization. T h e yield is about 10 to 12 g. The filter cake is allowed to d r y in the refrigerator, after which it is ground to a fine powder and stored in the cold room. Purification of Trypsin by Trichloroacetic Acid. 4 When first crystallized, trypsin sometimes has a low specific activity, owing p a r t l y to the presence of some inhibitor; the activity m a y be raised to the m a x i m u m value b y repeated recrystallizations or b y precipitation with trichloroacetic acid followed b y crystallization. Ten grams of crystalline filter cake of trypsin is dissolved in 100 ml. of water, and 100 ml. of 5 % solution of trichloroacetic acid is added. T h e 16In this laboratory, occasionally, such proportions resulted in a pH of the final solution lower than 8.0, owing to the acid retained from the wash solution. If that occurs, saturated potassium carbonate is added until pH 8.0 is reached, followed by solid magnesium sulfate to start crystallization (not more than 0.36 g./ml, of solution). A preferred alternative is to thoroughly stir the precipitate with one-half of the required volume, adjust the pH to 8 with potassium carbonate, and bring up to volume with pH 8.0 buffer.

[3]

TRYPSINOGEN AND TRYPSIN

31

mixture is allowed to stand at room temperature for 30 minutes, until precipitation is about complete. It is filtered with suction; the precipitate contains trypsin, and the filtrate contains trypsin inhibitor. The precipitate is washed with water to remove the free acid. Each gram of the precipitate is dissolved in 20 ml. of 0.02 2V HC1 and allowed to stand at room temperature for 30 minutes, after which 5 g. of solid ammonium sulfate is added (per each 20 ml. of 0.02 ~V HC1 used). The mixture is filtered through Whatman No. 42 fluted filter paper until clear. The precipitate is rejected. An additional 5 g. of ammonium sulfate is dissolved (per each 20 ml. of solvent used), and the precipitate which forms is collected with suction on hardened filter paper. The filtrate is rejected. The precipitate is washed on the paper with saturated magnesium sulfate in 0.02 N H2SO4 (as described for the "crude trypsinogen"). Each gram of the precipitate is dissolved in 0.5 ml. of water, cooled to about 5°, and about 0.5 ml. of 0.4 M borate buffer, pH 9.0, is added to bring the pH of the solution to 8.0 (pink to 0.01% phenol red but not to 0.01% cresol red on a test plate). The volume is measured, and an equal volume of saturated solution of magnesium sulfate is added. The mixture is allowed to stand at 5° for 24 hours. Typical crystals of trypsin are collected and washed as previously described. The yield is low, about 0.3 g./g. o'f the original trypsin. Preparation of Purified Enterokinase 17

The contents of duodena of freshly killed swine are collected by gentle squeezing. Then 2.5 1. of duodenal contents is diluted with 7.5 1. of tap water at room temperature. Next 450 g. of Hyflo Super-Cel is added, and the whole mass is filtered with suction through filter cloth on a large Btichner funnel. The first extract is saved. The residue is resuspended in 3 1. of tap water and refiltered through cloth. The second extract is combined with the first, and the residue is discarded. The combined extracts are cooled to 5° and are adiusted with 5 iV H~SO4 to pH about 4.0 (tested with methyl orange). The precipitate formed is filtered off rapidly with suction with the aid of 20 g. of Standard Super-Cel per liter of solution. The filtrate is brought immediately to pH 8.0 with 5 iV NaOH. Solid ammonium sulfate (530 g./1.) is added to bring the filtrate to 0.8 saturation. The pH of the solution is again adiusted to pH 8.0 with 5 N NaOH. Four milliliters of 0.4 M borate buffer, pH 9.0, is then added to every liter of solution. The formed flocculant precipitate is allowed to rise to the surface and is then easily collected into a doughlike mass and removed from solution. The weight of the precipitate is about 20 g. 17M. Kunitz, J. Gen. Physiol. 22, 447 (1939).

32

ENZYMES OF PROTEIN METABOLISM

[3]

The precipitate is dissolved in about 5 vol. of cold water, and solid ammonium sulfate (24.2 g. per 100 ml.) is added to 0.4 saturation. The mixture is filtered with suction with the aid of 5 g. per 100 ml. of Standard Super-Cel. The residue is rejected. The filtrate from 0.4 saturated ammonium sulfate is brought to 0.8 saturation with solid ammonium sulfate and filtered with suction. The filtrate is discarded, and the precipitate is once more fractionated between 0.4 and 0.8 saturation as described above. The yield is about 10 g. Activity. The activity of enterokinase is determined as follows. 8 Into a 50-ml. volumetric flask are pipetted 5 ml. of 0.065% solution of trypsinogen in 0.005 N HC1 and 10 ml. of 0.1 M phosphate buffer, pH 5.8. The solution is left at 5 ° to equilibrate. One milliliter of enterokinase solution and precooled water to make up the volume are added. The flask is allowed to stand at 5 °. Aliquots are withdrawn at hourly intervals, and the amount of activated trypsin is determined by any suitable method. The reaction is of apparent first order. One enterokinase unit (1 E.K.U.) is defined as that amount of kinase that brings about the activation of 0.065 mg. of crystalline trypsinogen in 0.02 M S6rensen's phosphate buffer, pH 5.8, per hour at 5°.

Determination of Tryptic Activity Methods for the determination of trypsin are similar to those used for the determination of chymotrypsins (see Vol. II [2]), with the exception that synthetic substrates are amides and esters of basic amino acids. The range of optimal activity, similar to that of the chymotrypsins, lies between pH 7.0 and 8.0, but the activity can be measured over a wider range. At the range of the optimal activity trypsin is unstable. Gorini TM and Bier and Nord 19 independently found that the stability of trypsin is enhanced in the presence of calcium ions. These findings were extended to several other cations. ~°,21 The effect is attributed to the stabilization of the active form of trypsin. The enhanced stability is manifested as an increased activity in a majority of the systems investigated. However, no increase was found in the rate of proteolysis of protamine or in the rate of activation of a-ehymotrypsinogen.2° Recently the nitrogen content of trypsin was reported 2° as 15.0%. A correction for the optical factor (see section on chymotrypsins, Vol. II [2]), which was originally reported 22 as 0.585, was also introduced 2° as 18 L. Gorini, Biochim. el Biophys. Acta 7, 318 (1951). 19 M. Bier a n d F. F. Nord, Arch. Biochem. and Biophys. 33, 320 (1951). s0 N. M. Green a n d H. N e u r a t h , J. Biol. Chem. 204, 379 (1953). 2i W. G. Crewther, Australian J. Biol. Sci. 6, 597 (1953). ~2 M. :Kunitz, J. Gen. Physiol. 30, 291 (1947).

[3]

TRYPSINOGEN AND TRYPSIN

33

0.695. In this laboratory the optical factor was determined on a preparation of DP-trypsin, kindly sent to us b y Dr. E. F. Jansen, and was found to be 0.67, in fairly good agreement with the value of Green and Neurath. 20

Determination of Activity by the Casein Digestion Method (Kunitz22). A stock solution of casein is made b y suspending 1 g. of casein (preferably " H a m m a r s t e n " ) 23 in 100 ml. of 0.1 M S6rensen phosphate buffer, pH 7.6. 24 The suspension is heated for 15 minutes in boiling water, 25 thus bringing about a complete solution of the casein. This 1% casein solution IO-~(T.UJ.... 0.6 0

20

40

I

60

i

80 i

'

0.5 0

~ 0.4

~'0.3 o

~

~

-0.2

,y "

c,,s

0.25

-3

IlK, I I It

0

n

I

I

a

I

I

0.004 0.008 0.012 Trypsin protein, mg. per rnl. 0.5% casein

FIG. 1. Standard activity curve for trypsin according to Kunitz. 22

is stored in a refrigerator and is stable for at least a week. Prior to use, the casein solution is placed in a water bath at 35 ° for at least 5 minutes. A solution of crystalline trypsin (or a solution in which tryptic activity is to be determined, or a mixture of trypsin and inhibitor) is pipetted into 15-ml. pyrex centrifuge tubes. 2~ The volume of the enzyme solution in each tube is brought to 1 ml. with an appropriate buffer, and the tubes are placed in the water bath. One milliliter of casein solution is pipetted into the first tube, and a stop watch is started. Each subsequent tube receives 1 ml. of casein at 30-second intervals. Three milliliters of 5 % trichloroacetic acid (TCA) is added in the same order to each test tube ex~3 Casein prepared according to M. S. Dunn [Biochem. Preparations 1, 22 (1949)] is recommended. 24 If calcium is to be used in the system, 0.2 M borate buffer containing 0.005 M CaCl~ is recommended. 25 With casein prepared according to Dunn, 8-minute heating was found sufficient. ~8 I t was found convenient to use 15-ml. Lucite tubes for the Servall SS-1 centrifuge. The time of standing after addition of TCA can be reduced to 20 minutes, and the time of centrifugation to 7 minutes a t 8000 r.p.m.

34

ENZYMES OF PROTEIN METABOLISM

[3]

actly 20 minutes after the addition of casein. The content of the tubes is mixed well. The tubes are removed from the bath, allowed to stand for an hour, and centrifuged for 20 minutes. The optical density of the supernatants is read at 280 m~. The readings are corrected for the values of blanks. The blanks are prepared by first mixing 1 ml. of casein solution with 3 ml. of TCA solution, and then adding 1 ml. of the highest concentration of enzyme used, or 1 ml. of the buffer used in making up the trypsin dilutions. The corrections for blanks for the intermediate concentrations of trypsin are calculated by interpolation. The standard curve for trypsin is shown in Fig. 1. The abscissa is expressed in two scales: as concentration (milligrams per milliliter, calculated on the basis of the optical factor of 0.585) of the standard trypsin as prepared by Kunitz, 22 and in Kunitz's tryptic units. Determination of Trypsin by the Hemoglobin Digestion Method of Anson. ~ In this method denatured hemoglobin is the substrate. It is digested for 5 or 10 minutes (at 37 ° or 25°), the reaction is stopped by the addition of trichloroaeetic acid, and the nondigested hemoglobin is removed by filtration. The amount of split products remaining in solution is determined colorimetrically by means of the phenol reagent, or spectrophotometrically. Either a crystalline hemoglobin, or ~ hemoglobin prepared according to Anson (see Vol. II [1]), is used. First 2.2 g. of hemoglobin is placed in a 100-ml. volumetric flask, half filled with water, 36 g. of urea and 8 ml. of 1 ~V NaOH are added, and the solution is made up to volume with water. The alkaline solution is kept at room temperature for 30 to 60 minutes to denature the hemoglobin and is then mixed with 10 ml. of 1 M potassium dihydrogen phosphate ~8 and 4 g. of urea. The final pH is 7.5. Two milligrams of Merthiolate (Lilly) is added as a preservative, and the solution is stored at 5° . The procedure for the determination of trypsin is identical with that for pepsin (see Vol. II [1]), except that after addition of trichloroacetie acid the solution is allowed to stand for 30 minutes before filtration. Methods of determination of tryptic activity with synthetic substrates are based on determining either the amidase or the esterase activity. The amidase activity of trypsin is conveniently determined by the method of Schwert et al., 29 the details of which have been described in ~7 M. L. Anson, J. Gen. Physiol. 22, 79 (1938). ~8 If calcium is to be used in the system, borate buffer should be used plus enough HCI to bring p H of the mixture to 7.5. ~9 G. W. Schwert, H. Neurath, S. Kaufman, and J. E. Snoke, J. Biol. Chem. 172, 221 (1948).

[3]

TRYPSINOGEN AND TRYPSIN

35

TABLE I ISOELECTRIC POINT OF TRYPSIN

ca. 7.0 ca. 11.0 10.8

Cataphoresis a Electrophoresis b Electrophoresis in the presence of Ca, two components at p H range 3-7 c

M. Kunitz and J. H. Northrop, J. Gen. Physiol. 16, 295 (1935). b M. Bier and F. F. Nord, Arch. Biochem. and Biophys. 33, 320 (1951). c F. F. Nord and M. Bier, Biochim. et Biophys. Acta 12~ 56 (1953).

TABLE II MOLECULAR WEIGHT OF TRYPSINOGEN AND TRYPSIN

Trypsinogen

23,700

Sedimentation-diffusion a

Trypsin

36,500 35,000 41,000 (dimer) 15,100 30,600-34,000 24,000 17,000 24,000

Osmotic pressure b Spread monolayers c Spread monolayers d Sedimentation-diffusion, By deutron and electron bombardments Light scatteringg Osmotic pressure h Calculated from 1 : 1 ratio with soybean inhibitov

DP-trypsin

20,700 21,400-24,800 24,000 23,800

Calculated from P contentJ Calculated from P content ~ Sedimentation-diffusion ~.~ Sedimentation-diffusion"*

a F. Tietze, J. Biol. Chem. 204, 1 (1953). b M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19, 991 (1936). c H. B. Bull, J. Biol. Chem. 185, 27 (1950). d E. Mishuck and F. Eirich, J. Polymer Sci. 7, 341 (1951). • V. G. Bergold, Z. Naturforsch. 1, 100 (1946). I E. Pillard, A. Buzzell, C. Jeffreys, and F. Forro, Jr., Arch. Biochem. and Biophys. 33, 9 (1951). R. F. Steiner, Arch. Biochem. and Biophys. 49, 71 (1954). h H. Gutfreund, Trans. Faraday Soc. 50, 624 (1954). ' M. Kunitz, J. Gen. Physiol. 30~ 291 (1947). J E. F. Jansen and A. K. Balls, J. Biol. Chem. 194, 721 (1952). k L. W. Cunningham, Jr., F. Tietze, N. M. Green, and H. Neurath, Discussions Faraday Soc. 13, 58 (1953). z F. F. Nord and M. Bier, Biochim. et Biophys. Acta 12, 56 (1953). "~L. W. Cunningham, Jr., J. Biol. Chem. 211, 13 (1954).

36

ENZYMES OF PROTEIN METABOLISM

[4]

Vol. II [2]. For the determination of trypsin the authors used arginine derivatives: a-benzoylargininamide (BAA) and a-p-toluensulfonyl-L-argininamide (TSAA). Other substrates which have been used are a-benzoyl-L-lysinamide (BLA), a-p-toluensulfonyl-L-lysinamide (TSLA), and a-hippuryl-L-lysinamide (HLA). The potentiometric determination of esterase activity of trypsin is carried out by the method of Schwert et al. 2" according to Rovery et al. 13 The buffer solution of pH 7.9 is 0.005 M in respect to tris(hydroxymethyl)aminomethane, 0.04 M in respect to NaC1, and 0.02 M in respect to CaC12. Eight milliliters of this solution is placed in a small beaker, and 1 ml. of 0.1 M benzoyl-L-arginine ethyl ester (BAEE) is added. The beaker is placed in a 25 ° water bath, and the electrodes of a (Beckman Model G) potentiometer and a small mechanical stirrer are introduced into the liquid. With the aid of a horizontal buret, 30 0.1 N NaOH is added to adjust the pH to 8.0. One milliliter (100 7) of trypsin solution (approximately 10 mg. of trypsin containing 50 % of magnesium sulfate per 50 mh of 0.001 iV HC1) is added. The pH of the reaction mixture decreases. The stop watch is started when the pH of 7.9 is reached. At that time approximately 0.01 ml. of 0.1 N NaOH (free from carbonate) is added, and the time at which the pH 7.9 is again reached is recorded. After five or six repetitions the volume of NaOH added is plotted versus time. A straight line is obtained. The slope of this line represents the activity. The potency of the preparation of trypsin could be expressed by dividing the activity by either the micrograms of enzyme protein (which can be calculated from the optical density) or the micrograms of enzyme nitrogen. Other commonly used substrates are a-p-toluensulfonylL-arginine methyl ester (TSAME), a-benzoyl-L-arginine methyl ester (BAME), and L-lysine ethyl ester (LEE). 80See section on chymotrypsin, Vol. II [2].

[4] N a t u r a l l y O c c u r r i n g T r y p s i n I n h i b i t o r s B y M. LASKOWSKI

The following naturally occurring trypsin inhibitors will be discussed: (1) pancreatic inhibitor of Kunitz and Northrop, 1 (2) a second inhibitor from pancreas crystallized by Kazal, Spicer, and Brahinsky, 2 (3) soybean 1 M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19~ 991 (1936). L. A. Kazal, D. S. Spicer, and R. A. Brahinsky, J. Am. Chem. Soc. 70, 3034 (1948).

[4]

NATURALLY OCCURRING TRYPSIN INHIBITORS

37

inhibitor, 3,4 (4) c o l o s t r u m inhibitor, s,6 (5) lima b e a n inhibitor, 7,8 (6) ovomucoid, 9-11 (7) blood p l a s m a inhibitor, 12 and (8) inhibitor f r o m A s c a r i s 2 3 T h e n a t u r a l l y occurring t r y p s i n inhibitors h a v e been r e c e n t l y reviewed 14 in regard to their m o d e of action and properties. All inhibitors isolated so far a p p e a r to be proteins. ~5 M o s t of t h e m (Nos. 1, 2, 4, 5, 6, and 8) are r e m a r k a b l y stable t o w a r d acid and heat, whereas the others (Nos. 3 and 7) are relatively labile. T h e original conclusion of K u n i t z a n d N o r t h r o p ~ t h a t one molecule of the inhibitor reacts with one molecule of trypsin, and t h a t the complex is an addition c o m p o u n d , has been essentially confirmed b y the recent evidence. T h e first four of the a b o v e listed inhibitors are obtainable in crystalline form, a n d Nos. 1, 3, a n d 4 are also obtainable as crystalline complexes with trypsin. These complexes are v i r t u a l l y devoid of either t r y p t i c or t r y p s i n i n h i b i t o r y activity. W i t h i n a fairly wide p H range these complexes fulfill several criteria of h o m o g e n e i t y (electrophoresis, u l t r a c e n t r i f u g e ) ; outside this range complexes dissociate into their active c o m p o n e n t s . I t has been r e c e n t l y shown t h a t complexes of t r y p s i n with o v o m u c o i d ~s,~7 a n d with the K a z a l ' s inhibitor ~s are unstable, the inhibitor being digested with t h e s i m u l t a n e o u s liberation of the free trypsin. This p h e n o m e n o n was called t e m p o r a r y inhibition2 ~ T h e a c t i v i t y of the inhibitors is m e a s u r e d in t e r m s of the t r y p s i n inhibited, a n d therefore all m e t h o d s used for t h e d e t e r m i n a t i o n of t r y p t i e s 3~[. Kunitz, Science 101, 668 (1945). 4 M. Kunitz, J. Gen. Physiol. 29, 149 (1946). 5 M. Laskowski, Jr., and M. Laskowski, Federation Proc. 9, 194 (1950). 6 M. Laskowski, Jr., and M. Laskowski, J. Biol. Chem. 190, 563 (1951). 7 H. Tauber, B. B. Kreshaw, and R. D. Wright, J. Biol. Chem. 197, 1155 (1949). s H. L. Fraenkel-Conrat, R. C. Bean, E. D. Ducay, and H. S. Olcott, Arch. Biochem. and Biophys. 37, 393 (1952). 9 A. K. Balls and T. L. Swenson, J. Biol. Chem. 106, 409 (1934). 10 H. Lineweaver and C. W. Murray, J. Biol. Chem. 171, 565 (1947). 11 E. Fredericq and H. F. Deutsch, J. Biol. Chem. 181, 499 (1949). 12 R. J. Peanasky and M. Laskowski, J. Biol. Chem. 204, 153 (1953). is H. B. Collier, Can. J. Research 19B, 91 (1941). 14 M. Laskowski and M. Laskowski, Jr., Advances in Protein Chem. 9, 203 (1954). 15 No distinction is being made between proteins and large polypeptides. 16L. Gorini and L. Audrain, Biochim. et Biophys. Acta 8, 702 (1952). 17L. Gorini and L. Audrain, Biochim. et Biophys. Acta 10, 570 (1953). 18 M. Laskowski and F. C. Wu, J. Biol. Chem. 204, 797 (1953). is, In order to explain the mechanism of temporary inhibition it was postulated 18from the kinetic data that one molecule of inhibitor combines with 2 molecules of trypsin to form an intermediate trypsin-inhibitor-trypsin (TIT) complex, which subsequently breaks into products +2 T. Recently, J. Sri Ram, L. Terminiello, M. Bier, and F. F. Nord [Arch. Biochem. and Biophys. 52, 451 (1954)] supplied additional evidence for the existence of TIT.

38

ENZYMES OF PROTEIN METABOLISM

[4]

activity (see section on trypsin, Vol. II [3]) are applicable to the determination of inhibitors. The solution of inhibitor (in a buffer to be used) and the solution of trypsin (in 0.0025 N HC1) are mixed, allowed to react, and the remaining trypsin activity is determined. With the exception of the pancreatic trypsin inhibitor, which requires up to 5 minutes for the completion of the reaction with trypsin, all other inhibitors react almost instantaneously over the range of pH values from 6 to 8. In the majority of cases within a proper pH range the inhibition is a linear function of the inhibitor concentration and does not depend on the purity of the preparation used. Two exceptions are noted: inhibition by the crude blood plasma 19 and by the inhibitor from Ascaris. 13 The former exhibits the linear relationship only when the ionic strength of the medium is close to physiological range; the latter in many respects behaves abnormally. Since the activity of inhibitors is expressed in terms of the inhibited trypsin, it is important that the standard preparation of trypsin be used. In the reviewer's laboratory it was found convenient to accept the activity curve of trypsin published by Kunitz ~° as a standard (see section on trypsin, Vol. II [3], Fig. 1). Preparation of Pancreatic Inhibitor of Kunitz and Northrop 1 Fresh pancreas is treated by the procedure of Kunitz and Northrop, as described in the article on chymotrypsinogens and chymotrypsins (Vol. II [2]), through the stage of crystallization of the ~-chymotrypsinogem The mother liquor is then treated as described in the chapter on trypsinogen and trypsin through the stage of crystallization of trypsin (Vol. II [3]). The mother liquor from the crystallization of trypsin is referred to as filtrate E and is treated as follows. 2°" Crystallization of Inhibitor-Trypsin Compound. ~ Filtrate E is adiusted to pH 3.0 with 5 N H2S04, saturated with crystals of magnesium sulfate at 25 ° , and filtered with suction through hardened paper. The filtrate is rejected. The precipitate (10 g.) is dissolved in 50 ml. of N/16 HCI and poured with stirring into a large beaker containing 250 ml. of N / 1 6 HC1 at 90°. After 1 minute it is cooled in running cold water to 25 °. Then 24.2 g. of solid ammonium sulfate is dissolved in each 100 ml. of solution, and the suspension is filtered through fluted paper; next 20.5 g. of solid ammonium sulfate is dissolved in each 100 ml. of filtrate, and the suspension is refiltered with suction. The last filter cake (3 g.) is dissolved in 19 S. F. M c C a n n a n d M. Laskowski, J. Biol. Chem. 204, 147 (1953). 20 M. Kunitz, J. Gen. Physiol. 30, 291 (1947). 20~ All m a n i p u l a t i o n s are carried out a t room t e m p e r a t u r e (20 to 25 °) unless otherwise

specified.

[4]-

NATURALLY OCCURRING TRYPSIN INHIBITORS

39

12 ml. of water and cooled in ice water. About 3 ml. of 0.4 M borate, 21 pH 9.0, is added in order to bring the solution to pH 8.0, and the mixture is poured with stirring into a large beaker containing 75 ml. of boiling distilled water. A heavy precipitate forms. After 1 minute the suspension is cooled to 25 ° in running cold water, and 24.2 g. of solid ammonium sulfate per 100 ml. is added. The mixture is filtered with suction through hardened paper, and the precipitate is rejected. The filtrate is adjusted to pH 3.0 by addition of several drops of 5 N H2SO4, and then 20.5 g. of solid ammonium sulfate is dissolved in each 100 ml. of solution. The suspension is filtered with suction on a large funnel, the filtrate is rejected, and the precipitate is washed with saturated magnesium sulfate. The precipitate (1 g.) is dissolved in 5 ml. of M/IO acetate buffer, pH 5.5, and the pH is adjusted to 5.5 with about 1 ml. of'0.4 M borate buffer, pH 9.0. The suspension is filtered through Whatman No. 42 paper into a flask containing enough crystals of magnesium sulfate to saturate the solution. The filter paper is washed with 4 ml. of M/IO acetate buffer, pH 5.5. The solution is stirred after completion of filtration. Hexagonal crystals of inhibitor-trypsin compound rapidly appear. The suspension is allowed to stand for 1 day at 20 ° to complete crystallization. The yield is about 0.25 g. of filter cake. Recrystallization. The filter cake of crystals (accumulated from several preparations) is dissolved in 10 vol. of M/IO acetate buffer, pH 5.5, and filtered through Whatman No. 42 fluted paper. Crystals of magnesium sulfate are added to saturate the solution. 2~ Hexagonal crystals of the inhibitor-trypsin compound rapidly appear. After 1 day at 20 to 25 ° they are.filtered off; the yield is about 60%. Crystallization of the Free Trypsin Inhibitor. To a solution of 1 g. of crystalline filter cake of three-times-recrystallized inhibitor-trypsin compound in 10 ml. of water, 10 ml. of 5% trichloroacetic acid is added, and the mixture is allowed to stand at 20 ° for 30 minutes, until precipitation is about complete. The mixture is filtered with suction; the precipitate may be used for crystallization of trypsin. The filtrate is heated for 5 minutes at 80 °, cooled to 25 °, and filtered through fluted Whatman No. 42 paper. The precipitate is rejected. The pH of the filtrate is adjusted to 3.0 with 5 N NaOH. Solid ammonium sulfate (5.6 g. per 10 ml.) is added, the suspension is filtered with suc31 Stock borate solution contains 49.6 g. of boric acid a n d 80 ml. of 5 N sodium hydroxide per 1000 ml. of solution; 0.4 M borate buffers, p H 8.0 a n d 9.0, are mixtures of 100 parts of stock borate a n d 78.6 a n d 17.6 parts of 0.4 M hydrochloric acid, respectively. ~ Green a n d Work 24 recommend 0.7 s a t u r a t e d MgS04 for recrystallization. T h e reviewer has successfully used 0.75 saturated.

40

ENZYMES

OF PROTEIN

METABOLISM

[4]

tion, and the filtrate is rejected. The precipitate (0.25 g.) is dissolved in 2.5 ml. of water, and the pH is adjusted to 5.5 with 0.4 M borate buffer, pH 9.0. Saturated ammonium sulfate is added to slight turbidity, and the suspension is filtered through Whatman No. 42 filter paper. The paper is washed with 0.5 saturated ammonium sulfate. More saturated ammonium sulfate is added to the combined filtrate and washings until a slight precipitate forms. The amorphous precipitate gradually changes into long hexagonal prisms. The suspension is allowed to stand for 2 days at 20 ° and is then filtered with suction. The yield is 0.15 g. of inhibitor crystals. If it is desired to have crystals free from ammonium salt, the filter cake should be washed with saturated magnesium sulfate and then recrystallized from a magnesium sulfate solution. Recrystallization. The crystals (0.15 g.) are dissolved in 1.5 ml. of M/IO acetate buffer, pH 5.5. Then 7.5 ml. of saturated ammonium sulfate or 7.5 ml. of saturated magnesium sulfate plus a few crystals of solid magnesium sulfate are added. The mixture is allowed to stand at 20 ° for 1 day. Crystals of inhibitor gradually appear. The yield is about 0.1 g. of filter cake. 23 A different method for the preparation of the crystalline trypsintrypsin inhibitor complex and crystalline inhibitor has been recently described by Green and Work. 24 In this method the residue remaining after the extraction of pancreas for insulin serves as starting material. Preparation of the Second Crystalline Trypsin Inhibitor from Pancreas,

According to Kazal, Spicer, and Brahinsky ~ This inhibitor is extracted from pancreas together with crude insulin and remains in solution after insulin is precipitated from an acid 15% NaC1 brine. In order to obtain sufficient quantities of the starting material, several tons of pancreas must be extracted. Very few laboratories have such facilities; therefore the details of this preparation are omitted and the reader is referred to the original paper. 2

Preparation of the Crystalline Soybean Trypsin Inhibitor 4 Step 1. Washing with 80 % Alcohol. One thousand grams of cold-processed, defatted soybean meal 2s is added to a mixture of 2400 ml. of 95% 3a Green and Work 24 recommend the following procedure for recrystallization: 0.6 g. of the inhibitor is dissolved in 0.1 M acetate buffer, p H 6.5 (6 ml.), and the solution is saturated with MgS04. A little of the partially crystalline material which precipitates is filtered off. The filtrate is acidified to p H 3. A precipitate which forms rapidly changes into crystals. It is filtered off after standing overnight. Yield, 0.3 to 0.4 g. after two recrystallizations. 24 N. M. Green and E. Work, Biochem. J. 54, 257 (1953). 2s Soybean meal, Nutrisoy X X X , in the form of flakes, supplied by the ArcherDaniels-Midland Co., Chicago, Illinois.

[4]

NATURALLY OCCURRING TRYPSIN INHIBITORS

41

alcohol cooled to 5° and 450 ml. of distilled water. The suspension is stirred well and left at 20 to 25 ° for 30 minutes. It is then filtered with suction on a 32-cm. Biichner funnel through filter cloth. The filtrate is reiected. Step 2. Extraction in 0.25 N H2S04. The semidry meal is resuspended in 5000 ml. of 0.25 h r H2SO4 (7 ml. of concentrated H~SO4 per liter of water) at 20 to 25 ° and left for 1 hour at room temperature with occasional stirring. The suspension is refiltered with suction on the same filter cloth. The meal residue is rejected. Step 3. Removal of Inert Protein by Means of Bentonite. Twenty grams of stock mixture of 1 part by weight of Bentonite (U.S.P. powder, Amend Drug and Chemical Co., New York) and an equal part of Hyflo SuperCel (Johns-Manville Corp.) are added to the acid filtrate, and then it is stirred for 10 minutes. The suspension is filtered with suction on 32-cm. E-D No. 303 filter paper (The Eaton-Dikeman Co., Mr. Holly Springs, Pennsylvania). The residue on the paper is washed twice with portions of 125 ml. of water. The residue is rejected. Step 4. Adsorption of the Inhibitor on Bentonite. One hundred grams of the stock of Bentonite-Super-Cel mixture is added gradually to the combined filtrate and washings from step No. 3. The solution is stirred gently while the Bentonite mixture is added, and the stirring is continued for 10 minutes. The suspension is filtered and the residue is washed, as described in step 3. The filtrate and washings are rejected. Step 5. Elution with Pyridine and Dialysis. The Bentonite residue is stirred up with 270 ml. of water. At this stage the suspension can be stored in the refrigerator overnight. The suspension is warmed to 25 °, and 30 ml. of pyridine (Stock No. 214, Eastman Kodak Co.) is added with stirring. The thick suspension is filtered with suction on 24-cm. E-D No. 303 filter paper in a hood. The filtration generally requires several hours. The residue on the funnel is washed once with 200 ml. of 5 % pyridine in water. The combined filtrate and washing is dialyzed overnight in 12-inch-long cellophane tubings, 28 placed in a tall jar with running tap water, in the hood, if possible. Step 6. Removal of Inert Material at pH 5.3. The dialyzed solution, free of any gummy residue adhering to the dialysis tubes, is adjusted to pH 5.3 with the aid of about 2 ml. of 1 N HC1. (The pH is tested by the drop method on a plate with 0.1 M acetate buffers as standards and 0.01% methyl red as indicator.) Four grams of Bentonite-Super-Cel mixture is stirred into the solution which is then filtered with suction on 15-cm. E-D No. 303 filter paper, and the residue is washed several times NuJax Visking Cellulose Casing manufactured by the Visking Corporation, Chicago, Illinois.

2~ 27/~2_inch

42

ENZYMES OF PROTEIN METABOLISM

[4]

with 15-ml. portions of water. The washings, if not clear, are refiltered. The residue is rejected. Step 7. First Precipitation of the Inhibitor at pH 4.65. The combined filtrate and washings of step 6 is cooled to 5 ° and then titrated with 1 N HCI to pH 4.65 (tested carefully with 0.05% bromocresol green on a drop plate). A heavy precipitate is formed which is filtered off at 5 to 8 ° on 15-cm. E-D No. 303 filter paper on a Bfichner funnel without suction. The filtration is completed with a very light suction. The weight of filter cake is from 10 to 12 g. The filtrate is rejected. Step 8. Second Precipitation at pH 4.65. The filter cake is suspended in 100 ml. of water cooled to 5 °, the water being added gradually to the precipitate and incorporated thoroughly with a porcelain spatula. Then 1 N NaOH is added dropwise with stirring until the precipitate is dissolved. Care should be taken, however, not to raise the pH of the solution above 6.4. The clear solution is warmed to 25 ° and titrated slowly with 1 N HC1 until a slight permanent precipitate is formed. Two grams of Standard Super-Cel is stirred into the solution which is then filtered with suction on 7- to ll-cm. E-D No. 303 filter paper. The residue on the paper is washed with several milliliters of water. The combined filtrate and washings is cooled to 5°, titrated to pH 4.65, and then filtered with light suction on 15- to 18-cm. E-D No. 612 filter paper at 5 to 8 °. The yield is about 8 to 10 g. of filter cake which is stored in refrigerator. The filtrate is rejected. Step 9. Crystallization. 27 The filter cake of step 8 (about 10 g.) is ground up to a uniform suspension with 10 ml. of cold water and then warmed to about 35 °. Next 0.5 N NaOH is added dropwise, with careful stirring, until the precipitate is almost completely dissolved and the pH of the solution is about 5.2. The clear solution is decanted into a 50-ml. centrifuge tube. Any residue in the beaker is stirred with 1 to 2 ml. of cold water, dissolved with the aid of a dro p of 0.1 N NaOH, and added to the main bulk of the solution in the centrifuge tube which is then placed at 35 to 37 ° for crystallization. A heavy sediment of crystals is obtained within 5 to 6 hours. Inoculation with a few crystals greatly facilitates the process of crystallization. The suspension is centrifuged for 10 minutes at about 3000 r.p.m. The residue is stored in the refrigerator, whereas the supernatant liquid is either stored or, if time permits, titrated with a few drops of 0.2/V HC1 to pH 5.1 at 36 to 37 °, inoculated, and left at that temperature. Another crop of crystals is gradually formed, which is centrifuged off after several hours and added to the first crop of crystals. The supernatant liquid is rejected. ~ I t is advisable to begin the crystallization (step 9) with at least 50 g. of amorphous precipitate collected from several preparations.

[4]

NATURALLY OCCURRING TRYPSIN INHIBITORS

43

It is preferable to begin step 9 in the morning, so as to be able to centrifuge before the end of the day. The crystals, as well as the supernatant solution from the first crystallization, should be stored overnight in the refrigerator. Step 10. Recrystallization. The combined crystal residues of step 9 (about 7 ml.) are stirred up with twice the volume of cold water and titrated with 0.5 N NaOH to clearing, the final pH being about 6.0. The clear solution is warmed to 35 ° and titrated with 0.5 N HC1 to pH 5.1 when a slight permanent precipitate is formed. The solution is mixed with 2 g. of Standard Super-Cel and filtered clear with suction on a small E-D No. 303 filter paper. The filtrate is inoculated and left at 36 to 37 °. A heavy suspension of crystals forms gradually and is centrifuged after 5 to 6 hours. The residue is stored at 5°. The pH of the supernatant liquid is readiusted with 1 to 2 drops of 0.2 N HC1 to 5.1 and left for several hours longer at 36 to 37 °, when another crop of crystals is formed which is centrifuged off and added to the first crop. The final supernatant solution may yield still more crystals by cooling it to 5 ° and then adding one-quarter of its volume of cold 95 % alcohol as described in the following paragraph. The crystallization is repeated three times. Step 11. Crystallization in Dilute Alcohol. The centrifuged crystals are stirred up with five times the volume of cold water, and 0.5 N NaOH is added drop by drop until the crystals are all dissolved. The pH of the solution is not allowed, however, to rise above 6.6. The clear solution is titrated with 0.2 N HC1 to pH 5.2. Any precipitate formed is filtered off with suction on No. 303 filter paper with the aid of 4 g. of Standard Super-Cel per 100 ml. of solution. The residue on the funnel is washed with several milliliters of water. The volume of the filtrate and washings is measured, and the solution is then cooled in an ice-water bath to about 5° . One-quarter of its volume of 95% alcohol, cooled to 5 °, is added slowly to the cold solution. A heavy precipitate is formed. The pH of the mixture is adjusted with 0.2 N HC1 to 5.0, and the mixture is left at 30 °. The amorphous precipitate changes within 2 hours into well-formed hexagonal and rhomboid crystals and plates which settle rapidly to the bottom of the vessel. The supernatant solution is decanted every hour, adjusted with 0.2 N or more dilute HCI to pH 5.0, and returned to the original vessel containing the settled crystals. This is continued for several hours until no formation of precipitate is noticed when the pH of the supernatant solution is adjusted to 5.0. The crystallization mixture is allowed to stand at 30 ° for 30 minutes longer and then filtered with suction on hardened paper, washed on the funnel several times with cold acetone, and allowed to dry in the room for 24 hours. It is stored in the refrigerator.

44

ENZYMES OF PROTEIN METABOLISM

[4]

Step 12. Recrystallization in Alcohol. The dry crystals are suspended in thirty times their weight of cold water, allowed to soak for 5 to 10 minutes, and then treated exactly as in step 11. Preparation of Crystalline Trypsin-Soy Inhibitor Complex 28

Step 1. Preliminary Step. One gram of dry soy inhibitor crystals is suspended in 40 ml. of distilled water at 5°. The mixture is titrated with 0.2 iV NaOH to pH 7.5. This brings about complete solution of the inhibitor crystals. One gram of a preparation of dry crystalline trypsin (containing about 50% anhydrous MgS04) is then added slowly with stirring. The mixture thus contains an excess of inhibitor in order to avoid any proteolysis by trypsin. If necessary, the pH is readjusted to 7.5 with several drops of 0.1 M borate buffer, pH 9.0. The solution, if turbid, is filtered with suction with the aid of 1 g. of Super-Cel on a small Bflchner funnel. The residue on the funnel is washed with about 5 ml. of cold H20. The washings, if clear, are added to the main bulk of the filtrate which is titrated with 0.1 N HC1 to about pH 6.0 and dialyzed overnight against slowly running distilled water at 5 to 10°, preferably with stirring. A granular precipitate gradually forms in the dialysis bag. The dialyzed suspension is titrated with a few drops of 0.1 N HC1 to pH about 5.4 (tested on a drop plate with 0.01% solution of methyl red). The suspension is centrifuged. The residue yields the crystalline compound; the supernatant solution (designated as "first supernatant solution") contains the excess of soy inhibitor which can be partly recovered. Step 2. Crystallization of the Compound. The residue is suspended in about 20 ml. of cold H20 and recentrifuged. The washed residue is resuspended in 40 ml. of H20 at about 5° and titrated dropwise with 0.2 or 0.5 N NaOH to pH 9.0 (pink to 0.1% phenolphthalein on a test plate), when complete solution generally occurs. The solution is then titrated with a few drops of 0.2 N HC1 to very slight opalescence and stored at about 20 ° . Fine crystals in the form of small rosettes or bundles of needles and plates gradually appear. The suspension of crystals is centrifuged after a day or so; several drops of 0.1 N HC1 are added to the supernarant solution until a slight turbidity appears. The solution is stored for several hours at 20 °. A second crop of fine crystals generally appears, which is centrifuged on the top of the first crop of crystals. 5iore acid is added to the supernatant solution, and the process is repeated until pH 5.8 is reached or until the final acidified supernatant solution no longer yields crystals. It is rejected or is combined with the "first supernatant solution" to be worked up for soy :inhibitor, as described in step 7. 2a M. Kunitz, J. Gen. Physiol. 30, 311 (1947).

[~]

NATURALLY OCCURRING TRYPSIN INHIBITORS

45

Step 3. Recrystallization of the Compound. The crystals are suspended in about 20 vol. of cold water and titrated with several drops of 0.5 N NaOH to incipient clearing. The solution is allowed to stand for 5 to 10 minutes at 5 ° and then filtered, if turbid, on fluted W h a t m a n No. 3 paper moistened with cold water, pH 9.0. The filter paper is washed once with cold water. The combined clear filtrate and washing is titrated with several drops of 0.2 N HC1 to very slight opalescence, seeded, and left at 20 °. Crystallization is generally complete within 24 hours. The crystals are centrifuged. The supernatant solution is titrated with 0.2 N HC1 to slight turbidity and left at 20 ° for several hours. A second crop of crystals is obtained and collected by centrifugation in the same tube used to collect the first crop of crystals. The operation is repeated several times until no further yield of crystals is obtainable. The supernatant solution is treated as described in step 5. Step 4. Drying of Crystals. The combined crystals are resuspended in a small amount of distilled water and filtered with suction on hardened paper. The crystals are dried for 24 hours in a refrigerator at about 5 ° and then in a desiccator over anhydrous CaS04 (Drierite) at 20 °. The dried material is ground fine in a mortar and stored in a refrigerator. Step 5. Crystallization in Dilute Alcohol. The final supernatant solution in step 3 m a y further yield crystals if it is cooled to 5 °, one-fourth of its volume of cold 95 % alcohol is added, and the pH of the solution is adjusted with 0.2 N HC1 to 5.8. A precipitate forms which, when left at 20 ° , changes gradually into rosettes of fine plates. The crystals are filtered on hardened paper and dried first in a refrigerator and then in a desiccator over anhydrous CaSO4 (Drierite). Step 6. Recrystallization in Dilute Alcohol. The dry crystalline powder is suspended in about fifty times its weight of water. (The centrifuged residue of crystals, not dried, is suspended in twenty-five times its volume of water.) The suspension is titrated with several drops of 0.5 N N a O H to pH 9.0. The crystals gradually dissolve when left for about 10 minutes at 5° . The solution, if turbid, is filtered, and then one-fourth of its volume of cold 95% alcohol is added. The pH of the solution is adjusted to about 5.8. A heavy precipitate is formed which changes into crystals on storing for a day or two at 20 °. The crystallization in alcohol is more rapid and the yield of crystals is greater than in the absence of alcohol. There is also the advantage t h a t the alcohol keeps the solution sterile. There is, however, the possibility t h a t alcohol causes slight denaturation of the protein. Step 7. Partial Recovery of Excess of Inhibitor. The first supernatant solution of step 1 is titrated with 0.5 N HC1 to pH 4.65 at 5 ° and centrifuged at about the same temperature. The supernatant solution is re-

46

ENZYMES OF PROTEIN METABOLISM

[4]

jected. The residue is suspended in about 5 vol. of cold water and is titrated with 0.2 N NaOH to pH 5.2. Any precipitate left undissolved is centrifuged off and is rejected (or worked up for compound as described in step 2). The supernatant solution is cooled to 5 °, and one-fourth of its volume of 95% alcohol precooled to 5° is added. The solution is adjusted with several drops of 0.1 N HC1 to pH 5.0, seeded with soy inhibitor crystals, and left at 30 °. Crystals of inhibitor gradually form. The crystals are filtered after several hours, washed with cold acetone, and dried in the room. The yield is 0.1 to 0.2 g. of dry soy inhibitor crystals.

Preparation of the Crystalline Trypsin Inhibitor from Colostrum 6

Step 1. To each liter of bovine colostrum, 1 1. of water and 1 1. of 7.5% trichloroacetic acid are added (final concentration 2.5% with respect to trichloroacetic acid). The mixture is heated to 80 ° with constant stirring and allowed to stand at that temperature for 5 minutes. It is then cooled to 25 ° and filtered on a large stainless steel Biichner funnel 29 with Whatman filter paper No. 1. The heavy cheeselike precipitate is discarded. The filtrate is brought to 80% saturation by addition of solid ammonium sulfate (603 g./1.) a° and allowed to stand overnight at room temperature. The slight precipitate which floats on the surface is removed by filtration with suction through Whatman filter paper No. 4. The filtrate is discarded. Step 2. The precipitate (plus crude fractions from previous preparations) is dissolved in 7 vol. of water with the aid of a Waring blendor, and enough trichloroacetic acid is added to attain a final concentration of 2.5%. The mixture is heated to 80 ° for 5 minutes, cooled to 25 °, and filtered with suction through Whatman No. 4 filter paper. The precipitate is washed with 2.5% trichloroacetic acid and discarded. The combined filtrate and washings is brought to 80% saturation of ammonium sulfate, and the mixture is filtered with suction through Whatman filter paper No. 4. The filtrate is discarded. Step 3. After the precipitate has been dissolved in 5 vol. of water (Waring blendor), the solution is adjusted to pH 6.5 with 1 N NaOH (glass electrode) and brought to 30% saturation of ammonium sulfate (22.6 g. per 100 ml.). After addition of 5 g. of Celite No. 545 per 100 ml., the mixture is filtered with suction through Whatman filter paper No. 4. The filtration is slow. The dark precipitate is discarded. The filtrate is 20 I n this l a b o r a t o r y a stainless steel Biichner funnel, Model 503, is used, m a n u f a c t u r e d b y American Biosynthetic Corp., Milwaukee, Wisconsin. 30 These figures are higher t h a n t h e figures used b y K u n i t z a n d Northrop, 1 who refer t o a s a t u r a t e d solution a t 5 °, whereas these figures refer to 25 °. See Vol. I [10].

[4]

NATURALLY OCCURRING TRYPSIN INHIBITORS

47

brought to 70% saturation of ammonium sulfate (26.7 g. per 100 ml.), which results in the formation of a rubberlike precipitgte. The latter is filtered with suction through Whatman filter paper No. 4 and kept as precipitate 3. Some inhibitor can be saved by adjusting the filtrate to pH 2 and 80% saturation, filtering, and adding it to the next preparation (step 2). Step 4. Precipitate 3 is dissolved in 5 vol. of water, trichloroacetic acid is added to attain a concentration of 2.5%, and the solution is shaken thoroughly in a separatory funnel with an equal volume of ether. It is then allowed to stand for 2 hours. The water layer is separated and kept. The ether layer is washed with one-half of the previous amount of water, the washings are added to the next preparation (step 2), and the rest is discarded. The liquid, which is still saturated with ether, is brought to 30% saturation of ammonium sulfate (22.6 g. per 100 ml.). The rubberlike precipitate which forms is filtered through filter paper Whatman No. 4 and the filtrate is discarded. Step 5. The precipitate is dissolved by stirring in a minimum amount of water, and the solution is dialyzed against distilled water overnight in the cold room21 The dialyzed liquid is adjusted to pH 5.5 with 1 h r NaOH and is treated with an equal volume of methanol, precooled to - 1 8 °. The solution is allowed to stand in a deep-freeze ( - 1 8 °) for an hour and is centrifuged in the conical head of a refrigerated centrifuge at - 1 0 ° for 15 minutes at 3500 r.p.m. The precipitate may be used in the next preparation (step 2). The slightly cloudy supernatant is treated with four times the previous volume of methanol (a total of 5 vol.). It is set in a deep-freeze for an hour and is centrifuged at - 1 0 ° for 20 minutes at 3500 r.p.m. The supernatant is discarded. Crystallization of Trypsin-Colostrum Trypsin Inhibitor Complex. Precipitate 5 is dissolved in a minimum amount of water, and the solution is adjusted to pH 3. The amount of trypsin required to neutralize the inhibitor completely is determined on an aliquot. The calculated amount of recrystallized trypsin is added, the solution is adjusted to pH 5.5, and dialyzed in the cold against 0.01 M acetate buffer, pH 5.5, for a period of 10 to 14 days, ~2 with frequent (every 12 hours) changes of buffer. The small precipitate is centrifuged off and discarded. To the solution an equal volume of saturated ammonium sulfate is added, followed by dropwise addition until the first sign of turbidity. After seeding with crystals, the solution is allowed to stand for 3 to 4 days at room temperature. The ~ Sizable amounts of inhibitor are lost during dialysis, since the inhibitor slowly passes through the cellophane membrane. The dialysis is, however, necessary for

the succeeding precipitation with methanol[ 3~M. Laskowski, Jr., P. H. Mars, and M. Laskowski, J. Biol. Chem. 198t 745 (1952).

48

ENZYMES OF PROTEIN METABOLISM

[4]

mixture first becomes gelatinous; then needles of the crystalline complex slowly form. Crystallization of the Free T r y p s i n Inhibitor f r o m Colostrum. The twicerecrystallized trypsin-trypsin inhibitor complex is dissolved in a small a m o u n t of water. An equal volume of 5 % trichloroacetic acid is then added, and the solution is allowed to stand for an hour. The precipitated trypsin is centrifuged off and saved for recrystallization. The solution containing the inhibitor is heated at 80 ° for 5 minutes, cooled to 25 °, and filtered through a small fluted filter to remove a slight precipitate, which is discarded. T h e filtrate is brought to 80% saturation with ammonium sulfate and centrifuged in the high-speed head of a refrigerated International centrifuge at about 25,000 r.p.m. The precipitate is dissolved in a TABLE I BALANCE SHEET OF EXPERIMENT WITH

4

GALLONS OF COLOSTRUMa

Step

Total amount of trypsin inhibited,b g.

Potency, trypsin inhibited (~,) E~800 m"

1 2 3 4 5

1.7 1.1 1.0 0.6 0.4

43 130 220

550 730

M. Laskowski, Jr., and M. Laskowski, J. Biol. Chem. 190, 563 (1951). b Calculated on the basis of 0.585 as an optical factor for trypsin. minimum a m o u n t of 0.05 M acetate buffer, p H 5.5, an equal volume of saturated a m m o n i u m sulfate is added, and then, v e r y carefully, an excess of a few drops until the minute t h a t t u r b i d i t y appears. Crystals (long needles) form almost immediately. The solution is allowed to stand overnight, and the crystals are collected b y centrifugation in a highspeed a t t a c h m e n t . Recrystallization is carried out in the same m a n n e r as the first crystallization. Table I summarizes the purification procedure.

Preparation of the Lima Bean Trypsin Inhibitor T a u b e r et al. ~ described a m e t h o d for crystallization of the trypsin inhibitor from lima beans. Fraenkel-Conrat et al. s repeated the method of T a u b e r et al. 7 and introduced a modification of the recrystallization procedure which resulted in the removal of inhibitory activity from the crystals and concentrating it in the m o t h e r liquor. Fraenkel-Conrat el al. s described a m e t h o d for the preparation of the amorphous inhibitor, the reported potency of which is about four and one-half times t h a t of the crystalline material of T a u b e r et al. 7 and about two and one-half times

[4]

NATURALLY OCCURRING TRYPSIN INHIBITORS

49

that of the crystalline soybean trypsin inhibitor. 4 The procedure is similar to the procedure of Kunitz 4 for the crystallization of soybean trypsin inhibitor. Preparation of Ovomucoid The simplest and probably the most widely used method is that of Fredericq and Deutsch. 11 It leads to an amorphous product with ninefold higher potency than the original egg white. The two steps are: (1) precipitation of the inactive proteins of the egg white by addition of an equal volume of 10% trichloroacetic acid previously adjusted to pH 3.0, and readjustment of the pH of the mixture to 3.5; (2) precipitation of ovomucoid from the filtrate by addition of 2 vol. of 95% alcohol at pH 6.0, at - 8 °. This precipitate represents crude ovomucoid, which is electrophoretically heterogeneous. Further purification improves the electrophoretic pattern but does not improve the potency of the preparation. Preparation of Partially Purified Blood Plasma Inhibitor 12 Indirect evidence suggests the presence of more than one trypsin inhibitor in blood plasma. 14 The preparation presented here refers to the quantitatively predominant trypsin inhibitor. Partial purification of trypsin inhibitor from blood plasma with properties different from the inhibitor described here has been described, 3~ confirmed, 34 and denied. 35 Step 1. Bovine blood is collected in the slaughterhouse, oxalated, and centrifuged. Plasma, usually about 6 1., is diluted with an equal volume of physiological saline. The mixture is acidified with 5/V H2SO4 to pH 4.0 and cooled to 5 ° . It is brought up to 40% saturation (for that temperature) by a slow addition of solid ammonium sulfate (250 g./1.) and allowed to stand overnight. It is filtered in the cold with the aid of Celite No. 545 (20 g./l.) on a large stainless steel Biichner funnel 29 through one sheet of Whatman No. 1 filter paper (32 cm.) with gentle suction. The precipitate is reiected. The filtrate is brought to 90% saturation with ammonium sulfate (375 g./1.). The mixture is filtered through the same Bilchner funnel at room temperature with the aid of 5 g. of Celite per liter. The filtrate is reiected. Step 2. The precipitate is suspended in 20 vol. of water, and the Celite is filtered off. The clear filtrate is adjusted to pH 4.7 with 5 N NaOH and brought to 50% saturation with solid ammonium sulfate (377 g./1.). It is allowed to stand at room temperature overnight. The precipitate is filtered off with the aid of Celite (10 g./1.) and is discarded. The filtrate 33 A. Schmitz, Z. physiol. Chem. 255, 234 (1938). 84 D. Grob, J. Gen. Physiol. 26, 405 (1943). 35 E. S. Duthie and L. Lorenz, Biochem. J. 44, 167 (1949).

50

ENZYMES OF PROTEIN METABOLISM

[4]

is b r o u g h t t o 6 5 % s a t u r a t i o n w i t h a m m o n i u m s u l f a t e (100 g./1.) a n d a l l o w e d t o s t a n d for s e v e r a l h o u r s . T h e p r e c i p i t a t e is c o l l e c t e d on a 18.5-cm. B i i c h n e r f u n n e l w i t h t h e a i d of 2 g. of C e l i t e p e r liter. TABLE I I EXTENT OF THE PURIFICATION OF BLOOD PLASMA TRYPSIN INHIBITORa

Fraction

trypsin inhibited (~)b E~0m'

Potency,

Whole plasma Step 1 Step 2 Step 3 Step 4

12 65 160 300 500

(11-13) (60-70) (140-180) (280-330) (480-520)

Yield, % 100 45 20 10 5

" R. J. Peanasky and M. Laskowski, J. Biol. Chem. 204, 153 (1953). b Calculated on the basis of 0.585 as an optical factor for trypsin. TABLE I I I OPTICAL PROPERTIES

Substance Pancreatic inhibitor

Factor a

Remarks

Kazal's inhibitor Soybean inhibitor Colostrum inhibitor

1.26 1.22 1.54 1.10 2.00

Acid solutionb pH 7 buffer c,d pH 5.7 ° Acid solutionl Acid solutiong

Pancreatic complex Soybean complex Colostrum complex

0. 810 0. 765 0. 840

Acid solution b Calculated from Kunitz's datal Acid solution ~

a After multiplying the observed value E ~ " by this factor, the concentration of protein would be expressed in milligrams per milliliter. b M. Laskowski, Jr., P. H. Mars, and M. Laskowski, J. Biol. Chem. 198, 745 (1952). c N. M. Green and E. Work, Biochem. J. 64, 257 (1953). N. M. Green, J. Biol. Chem. 205, 535 (1953). L. A. Kazal, D. S. Spicer, and R. A. Brahinsky, J. Am. Chem. Soc. 70, 3034 (1948). f M. Kunitz, J. Gen. Physiol. 30, 291 (1947). g M. Laskowski, Jr., and M. Laskowski, J. Biol. Chem. 190, 563 (1951). Step 3. T h e p r e c i p i t a t e is s u s p e n d e d in 2 vol. of w a t e r , a n d t h e C e l i t e is f i l t e r e d off. A n a d d i t i o n a l v o l u m e of w a t e r is u s e d t o w a s h t h e C e l i t e . T h e f i l t r a t e a n d w a s h i n g a r e c o m b i n e d a n d a r e b r o u g h t t o 30 % s a t u r a t i o n w i t h solid a m m o n i u m s u l f a t e (22.6 g. p e r 100 ml.). T h e c l e a r s o l u t i o n is carefully a d j u s t e d t o p H 3.6 a n d is a l l o w e d t o s t a n d u n t i l a h e a v y p r e c i p i t a t e f o r m s . T h i s u s u a l l y r e q u i r e s n o t m o r e t h a n 15 m i n u t e s . L o n g e r

[4]

NATURALLY OCCURRING TRYPSIN INHIBITORS

51

exposure results in a partial loss of activity. The mixture is centrifuged at full speed in a Servall type SS-la centrifuge for 8 minutes. The centrifuge is stopped b y applying mechanical brake. The clear supernatant is decanted and quickly adjusted to p H 6.5. An equal volume of 30% TABLE IV ISOELECTRIC POINTS

Substance

pH

Remarks

Pancreatic inhibitor

ca. 10.0 > 8,7 10.1 4.8, 5.2, 5.9 4.5 5.0 4.2 7.2 Higher than 3.6 4.5 4.3 3.9

Electrodialysis a Electrophoresis b Electrophoresis b Three separate peaks, electrophoresis ~ Cataphoresis d Cataphoresis a Electrophoresis b Electrophoresis b Electrophoresis e Electrophoresis/ Electrophoresis~ In 0.1-u buffers; five peaks in 0.01-~ buffersh Five separate peaks in 0.01-~ buffers ~,i

Pancreatic complex Kazal's inhibitor Soybean inhibitor Soybean complex Colostrum inhibitor Colostrum complex Lima bean inhibitor Ovomucoid

4.41, 4.28, 4.17, 4.01, 3.83

N. NI. Green and E. Work, Biochem. J. 54, 257 (1953). b M. Laskowski, Jr., P. H. Mars, and M. Laskowski, J. Biol. Chem. 198, 745 (1952). c L. A. Kazal, D. S. Spicer, and R. A. Brahinsky, J. Am. Chem. Soc. 70, 3034 (1948). d M. Kunitz, J. Gen. Physiol. 30, 291 (1947). o H. L. Fraenkel-Conrat, R. C. Bean, E. D. Ducay, and H. S. Olcott, Arch. Biochem. and Biophys. 37, 393 (1952). f L. Hesselvik, Z. physiol. Chem. 254, 144 (1938). o L. G. Longsworth, R. K. Cannan, and D. A. McInnes, J. Am. Chem. Soc. 62i 2580 (1940). E. Fredericq and H. F. Deutsch, J. Biol. Chem. 181,499 (1949). M. Bier, A. J. Duke, R. J. Gibbs, and F. F. Nord, Arch. Biochem. and Biophys. 37, 491 (1952). i lV[. Bier, L. Terminiello, A. J. Duke, R. J. Gibbs, and F. F. Nord, Arch. Biochem. and Biophys. 47, 465 (1953). saturated a m m o n i u m sulfate is added, followed b y addition of 15.1 g. of solid a m m o n i u m sulfate per 100 ml. of mixture. The precipitate which forms is filtered off with the aid of Celite (1 g. per 100 ml.) and is rejected. To the clear filtrate 10 g. of a m m o n i u m sulfate per 100 ml. is added, and the precipitate containing most of the activity is collected on a small Btichner funnel with the aid of Celite. Step 4. The precipitate is suspended in a small volume of water; the Celite is removed and washed. The liquid is dialyzed against running t a p

52

ENZYMES

OF

PROTEIN

METABOLISM

.~

"~.~ ~

0

~~

[4]

°~

°r~

~

~

©

¢4

~ ~

O ~q C~

~2

~o o

0

0

O~q

I

Cq

~9

% o

°~

°~

O

¢D O

[4]

NATURALLY

0CCUR~ING

TRYPSIN

INHIBITORS

53

~.~ ~~

"~

e~

,.4

o~.

~

~

'~

~o

Lea v

h- ~" .,o~,'~

o

~

.~

~

~

O~

~0

oo

0

•- ~ •

~

"~'~ ~ " ~

.....

m

0

©

~'~

"~.~

h,

~. ~

•

~

•

~

~'~'~ ~ '~ ~0"~- ~

~

~

~

.

, ~. ' ~ ~~o ~. ~- ~ . ~ - ~ ~~. ~ ~- ~

-

m ~.~

.=~

I

~,~

~ ~

.~

~I~,i 0 .~

m~ ~

-~_~ ~.~-o.~o.~ ~ ...o o . ~ " ~ ~ ~ ~ ~- ~

~

.

~

~

.~

~..

,.~ ,

.

~

~'~ ~

~.,~ ~..~

~ ~..~ ~

~

=

=

~

=

.

~..~ . . . .

~

,.~

r/i~

~.~

.

.

o

~~~

. . .~ . . ' ~0 ~

~.~

~ ~

.

..~

~-~ ~ ~

.~ ~ . . ~~.; ~. ~. 0,~ •

" ~ 0 ~ ' ~

~

~

,"~.'~

~.~

• h-~

54

ENZYMES OF PROTEIN METABOLISM

[5]

water for 60 hours. It is diluted with distilled water to produce a solution having an optical density of 2.0 at 280 m~. The pH is carefully adjusted to 3.6 with 0.5 N H~SO~. Twenty milligrams of bentonite is added per each milliliter, and the mixture is allowed to stand overnight at 5 ° . The Bentonite is centrifuged off, and the supernatant, containing most of the activity, is lyophilized. Table II summarizes the purification procedure. Preparation of the Inhibitor from Ascaris 13 The worms are homogenized in 1% NaC1 in Waring blendor. Diastase is added, and the suspension is allowed to autolyze under toluene for several days. Trichloroacetic acid is added to attain a concentration of 2.5%. The mixture is heated to 80 ° for 5 minutes, cooled, and filtered. The precipitate is rejected. The filtrate is treated with charcoal to remove the color and odor, and after adjustment to pH 3 it is saturated with MgSO4. The precipitate which contains the activity is collected on hardened filter paper. The inhibitor from Ascaris appears to be the only trypsin inhibitor which forms with trypsin a dissociable complex.13 Furthermore, trypsin treated with the inhibitor was reportedly no longer precipitable with trichloroacetic acid. Some properties of the trypsin inhibitors are summarized in Tables III, IV, and V, taken from Advances in Protein Chemistry. 14 The potencies of different inhibitors and the substrate on which they were determined are specified in the last column of Table V.

[5] P l a n t Proteolytic E n z y m e s B y D. M. GREENBERG

Distribution and Properties Proteolytic enzymes have been found in both dicotyledonous and monocotyledonous plants. Representative of the former are the fig, papaya, milkweed, and euphorbia; of the latter, the pineapple and cereals. There appears to be two major groups of these proteinases, one requiring free SH groups for activity, having their optimum activity at pH ~ 7 for the digestion of hemoglobin, casein, egg albumin, etc., and strong milk-clotting activity; the other group lack active SH groups, have a more alkaline optimum pH, and an inferior milk-clotting power. Both groups of plant proteinases are unusually heat resistant, often maintaining their activity at temperatures of 60 to 70 ° .

[5]

PLANT PROTEOLYTIC ENZYMES

55

The physiological function of the proteinases in plants is unknown. Certain of the plant proteinases have a number of technological and medical usages. I Probably commercially the most important is the tenderizing of meat and other protein material by papain and bromelin. Other technological uses are in the preparation of leather and in the brewing industry. They are used medically to some extent in the treatment of digestive disturbances involving the proteolytic enzymes and in the treatment of sloughing wounds. Most of the plant proteinases are able to digest living parasitic worms, such as ascaris and trichuris, but it has not been found safe to employ them as vermifuges. The characteristic properties of these enzymes are summarized in Table I.

Determination of Activity The most reliable and convenient procedures are those which determine the split products, soluble in trichloroacetic acid (TCA), of a standard protein, and the clotting activity against milk. Phenol Color Method3 Five milliliters of a solution of denatured hemoglobin at pH 7.4 (or other well-defined protein) is digested by i ml. of a suitably diluted proteinase solution for 5 minutes at 25 °. The reaction is terminated by the addition of 10 ml. of 3 M TCA, the mixture is filtered through Whatman No. 3 paper, and the tyrosine and tryptophan in an aliquot of the filtrate is determined by the blue color given with the FolinCiocalteau phenol reagent 3 in alkaline solution. The phenol reagent is diluted with twice its volume of H20 before use. This color is compared against a standard tyrosine solution dissolved in 1 M HC1. To 5 ml. of the digestion filtrate in a 50-ml. Erlenmeyer flask are added 10 ml. of 0.5 M NaOH and 3 ml. of the phenol reagent. The solution is kept agitated during addition of the phenol reagent. The color is read against a standard prepared in the same manner after 2 to 10 minutes of standing. The standard tyrosine solution contains 8 X 10-4 meq. of tyrosine (0.0112 mg. of tyrosine N) in 5 ml. of 0.2 M HC1, with 0.5% formaldehyde as a preservative. The tyrosine standard can be prepared by accurately weighing out 289.7 mg. of pure, dry tyrosine, dissolving it in 1 1. of 0.2 M HC1, and diluting this solution ten-fold in 0.2 M HC1 for use. Alternatively, the tyrosine nitrogen can be determined by micro-Kjeldahl. A blank is to be run with each series of determinations. The SIt protein1 See H. Tauber, "The Chemistry and Technology of Enzymes," Johla Wiley & Sons, New York, 1949. M. L. Anson, J. Gen. Physiol. 20, 561 (1937); 22, 79 (1938), 30. Folin and V. Cioealt6u, J. Biol. Chem, 78~ 627 (1929),

56

ENZYMES

OF

PROTEIN

METABOLISM

[~]

ND

Nil

~ r~

~.~

=

N

m ©

m

~ o"~'~

"g o

<

N

N

~

~

~

~

o~ o

©

i

O

,-~

0

o~

O

[5]

PLANT PROTEOLYTIC ENZYMES

57

0 r~

v kO

CO o o

! 2, ~

®

~

t~ ~.t. ~

~.¢ "~

b0,1

~ b -

~

,~

~ . ~~, 4

-

•

~

.

.

.

~

~

~

~

o~ ~ - ~ ~ ~ , ~~

~

.

•

~

.

•

-

.

.

.

~-~ ~ ~ ~

.

.

•

~

~

©

@ ~ "

~r~.

.

•

.

58

ENZYMES OF PROTEIN METABOLISM

[5]

ases are activated before determination of activity by adding 5 drops of 2 M NaCN to 0.5 ml. of the enzyme solution. After standing for 3 minutes at 25 °, 9.25 ml. of H20 is added. One unit of proteinase is defined as the amount of enzyme which yields a color equivalent to 1 millimole of tyrosine per minute in the 6 ml. of digestion mixture under the standard conditions of pH and temperature employed. Nonprotein Nitrogen Method. 4 Ten-milliliter samples of the enzyme solution are added to a series of tubes containing 5 ml. of protein solution (casein) adjusted to the temperature of 35.5 °. A blank determination is made with each test series by adding 1 ml. of enzyme solution inactivated by heat or other suitable method. After incubation for successive time intervals, 5 ml. of 20 % TCA is added to each tube, the contents filtered, and the total N in 5 ml. of the filtrate determined by the micro-Kjeldahl method. The quantity of nonprotein nitrogen in the 6 ml. of the original digestion mixture is calculated, suitably corrected for the blank, and these values are plotted against the time of digestion. The activity is then determined as the initial slope of the curves obtained; the specific activity of the enzyme is the slope divided by the quantity of enzyme present. Milk-Clotting Method. 5 Twenty grams of whole powdered milk is ground to a smooth paste with a small amount of pH 4.6 acetate buffer (prepared by mixing 2 vol. of M CH3COOH with 1 vol. of M NaOH). Ten milliliters of this buffer is diluted to 85 ml. and added to the powdered milk to give a total volume of 100 ml. The reconstituted milk is filtered through cheesecloth for use. It is stable for several weeks in the refrigerator under toluene. One-milliliter portions of serial dilutions of the enzyme being tested are incubated in test tubes 15 mm. in diameter at 40 ° with 10 ml. of the milk preparation. When the tubes show a thickening on tilting, the reaction is considered complete. The SH proteinases are activated prior to test by diluting them with H2S water and bubbling H~S through the solution for 30 to 60 minutes at room temperature, or alternatively by treating the enzyme solution with an excess of NaCN or cysteine (at pH 5) at 40 °. Kinetics. In general, the relation between clotting time and enzyme concentration is a straight line that can be expressed by the equation E = K/t

where E is the weight of enzyme (in milligrams) and t the time (in minutes). J. H. Northrop, J. Gen. Physiol. 16~ 41 (1932). 5 A. K. Bails and S. R. Hoover, J. Biol. Chem. 121j 737 (1937).

[5]

PLANT PROTEOLYTIC ENZYMES

59

At low concentrations some of the enzyme is removed from "the sphere of activity and the equation found to hold is (E -- c)t = K '

In this expression c represents the amount of enzyme removed from action during the clotting. The value of c can be determined by solving a series of simultaneous equations for the observed points or by determining the intercept on the E axis when 1/t is plotted against E.

Preparation of SH Enzymes Five of these plant proteinases have been crystallized, namely, papain, chymopapain, ficin, asclepain, and mexicain. The others are impure products, P a p a i n . The procedure for crystallizing papain 6 from the fresh latex is given in Table II. The enzyme has a molecular weight of about 30,000. It is only slightly soluble in dilute salt solutions; but, like the prolamines, it is soluble in 70 % alcohol. In the native state it does not give a positive test for an SH group, with nitroprusside or porphyrindin. However, there is little doubt that an SH group not easily accessible for chemical reaction is essential for the activity of papain. The protein contains 15.5% nitrogen, 1.2% total sulfur, and 9.0% cystine sulfur. Smith and co-workers ~,b have devised a method of crystallizing papain from commercial dried papaya latex and also have obtained a crystalline mercury derivative of papain (mercuripapain). These investigators used a-benzyl-L-arginine-amide, which is deamidated by papain for assay of the enzyme. The carboxyl groups liberated were titrated. The hydrolysis is a first order reaction, and the specific activity of the papain is expressed as the log10 rate constant per mg. protein N per ml. reaction mixture. Success in crystallizing papain from dried latex depends upon grinding the latex to a fine powder in order to obtain a satisfactory extract. This can be done with washed sand or by agitating in a Waring blendor. The purification steps are the same as given in Table II with the modifications given below. The extraction of the enzyme is best carried out at pH 5.5, instead of at pH 6.7, as for fresh latex. The NaCN is replaced by cysteine for activation. In step 3 of Table II the 600 ml. of 0.02 M eysteine replacing the NaCN is maintained at pH 7 to 7.5. Mercuripapain. Crystalline mercuripapain is obtained by dissolving crystalline papain to a concentration of 1.5 to 2.0% in 70% ethanol cons A. K. Balls and H. Lineweaver, J. Biol. Chem. 130, 669 (1939). 6~ j. R. Kimmel and E. L. Smith, J. Biol. Chem. 207, 515 (1954). ~bE. L. Smith, J. R. Kimmel and D. M. Brown, J. Biol. Chem. 207, 533 (1954).

60

ENZYMES OF PROTEIN METABOLISM

[5]

TABLE II PREPARATION OF CRYSTALLINE PAPAIN a

Papain milkclotting units b

Material and manipulation 1 kg. wet papaya latex. 1 1.0.04 M N a C N c and 100 g. diatomaceous earth added to 1 kg. wet latex (180 g. dry weight) and stirred for 1 hr.; p H should be 6.5-7 d (green to bromothymol blue). After filtration residual liquid squeezed out of solid by supporting filter paper with cheese-cloth. Liquid fractions combined. Filtrate. Filtrate 1 brought to p H 9 (slightly greenish to thymol blue) with 2 M N a C N (25 ml.) and centrifuged. Supernatant liquid made 0.4 saturated with solid ammonium sulfate (250 g./1.), cooled, and filtered on a weighed layer of Hyflo filter aid. 73 g. precipitate. Precipitate 2 dissolved in 600 nil. 0.02 M N a C N (resulting pH, 8-8.5) and filtered to remove Hyflo. 60 g. NaCI added to filtrate. Suspension cooled and centrifuged while cold. 0.02 M cyanide solution, p H 6.4, added to centrifuged precipitate to make 400 ml., and p H of suspension adjusted to 6.5 (yellow-green to bromothymol blue) with 0.1 M HC1. Suspension. Suspension 3 cooled after standing about 30 min. at room temperature. The precipitate which formed was centrifuged at 5 ° after standing 18 hr. in cold. This crystalline precipitate dissolved in 300 ml. neutralized 0.02 M cyanide at room temperature; then 10 ml. saturated NaC1 solution added slowly to solution. Suspension of crystals. Recrystallizations made by repeating treatment of suspension 3. Suspension of second crystals.

Volume Fracof Per mg. tion fraction, protein no. ml. N 17 1

Total X 10-3 200

1540

2

15.5

39

3

420

16.5

9.7

4

310

22.5

4.5

5

155

26

4.0

A. K. Bails and H. Lineweaver, J. Biol. Chem. 180, 669 (1939). b One milk clotting unit is the amount of papain that will clot 5 ml. of standard milk preparation in 1 minute of 30 °. c If the preparation is to be made in the absence of added activator, 1 N N a O H is used to make the alkaline p H adjustments; otherwise the procedure is as given. Wherever p H is used in this table it refers to the apparent p H as given with Clark's indicators on a spot-plate.

[5]

PLANT PROTEOLYTIC ENZYMES

61

raining 0.001 M HgC12. The solution is filtered, if turbid, and stored at 4 ° C. Crystallization of the enzyme is complete in 3 to 4 days. The mercuripapain crystals are long rectangular plates, large enough to be seen with the naked eye. Mercuripapain is enzymatically inactive, but full activity can be restored by addition of cysteine and a metal-chelating agent (Versene). The highest papain specific activity was obtained with reactivated mercuripapain, namely, 1.2. The mercury content suggests that one atom of mercury combines with 2 molecules of papain. Chymopapain. 7 In the preparation of crystalline papain it was observed that the yield of this enzyme represented only a fraction of the total proteolytic activity of fresh papaya latex. Another crystalline proteinase, named chymopapain, because of its higher ratio of milk clotting to protein digestion activity in comparison to papain, was isolated from the latex. The isolation was aided by the stability of the enzyme in acid solution. The latex was acidified to pH 1.8 to 2.0, which precipitates papain together with considerable inert protein. More inert protein was precipitated from the filtrate by half-saturation with NaC1 at pH 3.5 to 4.0. The crystals develop on adjusting the pH to 2.0 and adding NaCI to full saturation. Crystalline chymopapain is stable at pH 2.0; it is many times more soluble in aqueous solution than is papain, and it gives a strong nitroprusside test for SH groups. Ficin. Ficin was crystallized s by adjusting the clarified latex to pH 5 and allowing the solution to stand for several weeks at 5° . Yellow hexagonal crystals form. Recrystallization was accomplished by redissolving the enzyme in 0.02 ill HC1, filtering, and readjusting the pH to 5.0. The recrystallized enzyme was colorless and ash-free. Crystalline Asclepain. This enzyme was crystallized from the pressed juice of the roots of Asclepias syriaca2 Three kilograms of cleaned milkweed roots was ground in a meat grinder, and the juice pressed out in a Carver laboratory press, yielding 1 1. of crude juice. This was centrifuged to remove cellular debris and then filtered through asbestos and paper pulp. The yield was 815 ml. of a clear filtrate with a pH of 6.5. This filtrate was saturated with solid (NH4)2SO4, added in small portions while being mechanically stirred, and then allowed to stand overnight at 5° . The precipitated material was centrifuged off, dissolved in about 300 ml. of H20, any insoiuble material removed, and the enzyme reprecipitated by half-saturation with solid (NH4)2SO4. The precipitate was isolated, redissolved in about 50 ml. of H20, filtered through asbestos and r ]:~. F. Jansen and A. K. Bails, J. Biol. Chem. 137, 459 (1941). 8 A. Walti, J. Am. Chem. Soc. 60, 493 (1938). 9 D. C. Carpenter and F. E. Love]ace, J. Am. Chem. Soc. 65, 2364 (1943).

62

ENZYMES OF PROTEIN METABOLISM

[5]

paper pulp and placed in cellophane dialyzing tubes, protected with toluene, and dialyzed against distilled H20 to remove the (NH4)2SO4. This precipitates the bulk of the enzyme protein. The precipitate (yield, 6 to 8 g.) was removed from the dialysis tubes, dissolved in phosphate buffer of pH 7.0, and dialyzed against saturated (NH4)2SO~, which induces crystallization. Amorphous asclepain from this milkweed was prepared from the leaves of the plant by H. Tauber and S. Laufer.1 The enzyme was inactivated on dialysis and strongly activated by sulfite. Highly active amorphous preparations of asclepain have been obtained from the latex of the milkweed species Asclepias speciosa 1° and A. mexicana 11 by filtering out suspended particles, adjusting the pH to 7.0 with NaOH, and then adding solid (NH4)2SO4 to 2/~ saturation. The salted-out enzyme was filtered on a Biichner, washed first with 0.5 saturated (NH4)2S04 and then with a small amount of water; lastly, the preparation was vacuum dried. One and one-half grams of enzyme was obtained from 100 ml. of latex. Mexicain. The presence of an active SH proteinase in the latex of the fruit and leaves of Pileus mexicanus, a plant known as " c u a g u a y o t e " in Mexico, was reported by Castafeda et al. 1~ Subsequently, this was crystallized by Castafieda and coworkers is as described below. Two volumes of H20 was added to 1 vol. of fresh latex, and the pH adjusted to 7.5 with 0.5 M NaOH with constant stirring. The mixture was then placed in a refrigerator at 5 ° for 24 hours. The solution was then filtered through Hyflo Super-Cel, and the pH adjusted to 5.5 with 0.05 M HC1. At this pH the solution becomes opalescent, and after 24 hours at 5° a fine precipitate of crystals appears in the form of lanceolate plaques. The crystals are four to five times as active as the dry latex in digestion of protein or milk clotting. The crystalline enzyme is very soluble and at pH 5.8 does not lose activity at room temperature and does not require activators, such as N a C N or cysteine, for enzymatic stability. It is not certain, therefore, that mexicain is an SH proteinase. Bromelin. This enzyme is present in the leaves and stalks, as well as in the fruit, of both green and ripe pineapple plants. The enzyme may be precipitated from the fresh fruit juice by 0.6 saturated (NH~)2S04 or by acetone or alcohol. ~4 The yield is about 2 to 3 g./1. of juice of impure enzyme with 25 to 100 % of the activity of commercial papain powder. 10T. Winnick, A. R. Davis, and D. M. Greenberg, J. Gen. Physiol. 23, 275 (1940). 11D. M. Greenberg and T. Winnick, J. Biol. Chem. 185, 761 (1940). 1~M. Castafieda, F. F. GavarrSn, and M. R. Balcazar, Science 96, 365 (1942). 18 M. Castafeda-Agull6, A. Hern~ndez, F. Loaeza, and W. Salazar, J. Biol. Chem. 159, 751 (1945). 14A. K. Balls, R. R. Thompson, and M. W. Kies, Ind. Eng. Chem. 38, 950 (1941).

[5]

PLANT PROTEOLYTIC ENZYMES

63

When bromelin was inactivated (denatured) by heating to 80 ° at pH 5 to 8, a partial recovery of activity occurred upon readjusting the pH to 3.2, although at this pH the enzyme is unstable. Pinguinain. The juice of the fruit of the " m a y a " plant of Puerto Rico (Bromelia pinguin) produces a burning sensation if applied to the hands and lips. This juice contains the proteinase named pinguinain. Five grams of crude enzyme may be obtained by precipitation with acetone from 100 ml. of juice, 15 which is about twenty times the yield of bromelin from a corresponding volume of pineapple juice. Tabernamontanain. This enzyme was prepared by Jaffe 1~ from the juice of the fruit of the Venezuelan shrub Tabernamontana grandiflora by acetone precipitation. The activity was ten times as great as that of crude papain. It was observed that juice collected in April was activated by cysteine or NaCN, whereas that obtained in July was not further activated, presumably because of the presence of a natural activator in the latter. Other plant sources of SH proteinases are wheat, 17 the lima bean, is and the soybean. 19 The enzyme of the latter has been named soyin. Preparation of Non-Sit Proteinases

Arachain. 2° This enzyme was prepared by extracting defatted peanut meal with alkalinized water and then with 0.5 M NaC1. Upon removal of the salt by dialysis, the enzyme activity was found to be present in the precipitated protein. The enzyme activity was approximately twenty times that of peanut meal. In contrast to most plant proteinases, it was rapidly destroyed on heating to 40 ° . Solanain. ~1 This proteinase was prepared from the fruit of Solanum elaeagnifolium by extracting the ground fresh fruit with dilute phosphate buffer (pH 7.5) and centrifuging the extract free of solids. The solution was made up to about 0.7 saturation with solid (NH4)2SO4, and the resulting precipitate collected on a Biichner funnel, washed with 0.7 saturated (NH4)2S04, and drained dry. The precipitate was dissolved in H20 and the enzyme reprecipitated with 4 vol. of acetone. The precipitate was washed with acetone and drained free of liquid. It was again dissolved in H20, precipitated by 0.7 saturation with (NH4)2S04, and col15 C. F. Asenjo and M. del Capella de Fernandez, Science 95, 148 (1942); J. Agr. Univ. Puerto Rico 29, 35 (1945). 16 W. G. Jaffe, Rev. brasil, biol. 3, 149 (1943). 17 A. K. Balls and W. S. Hale, Cereal Chem. 15, 622 (1938). 18 W. B. Davis, Food Research 4, 613 (1939). 19 H. Tauber and S. Laufer, Federation Proc. 2~ 72 (1943). 20 G. W. Irving, Jr., and T. D. Fontaine, Arch. Biochem. 6, 351 (1945).

64

ENZYMES OF PROTEIN METABOLISM

[6]

leered on a filter. Finally, the enzyme preparation was precipitated twice more from aqueous solution by 4 vol. of acetone, washed with acetone and ether, and dried in a v a c u u m desiccator. About 0.8 g. of white enzyme powder was obtained per 100 g. of fresh fruit. H u r a i n . 21 Hurain was isolated from the sap of the bark and roots of the tree of the family of Euphorbiaceae, H u r a crepitans (commonly known as " j a b i l l o " in Venezuela). Upon centrifugation insoluble material separated, and the supernatant liquid was clear. T o this was added 2 volumes of acetone which precipitated the enzyme. The precipitate was purified b y redissolving and reprecipitating with acetone. Hurain is precipitated b y saturated (NH4)2S04 or NaC1. Proteolytic activity in species of E u p h o r b i a was observed m a n y years agO. 22'23 E u p h o r b a i n . E n z y m e preparations named euphorbain have been prepared b y extracting acetone-precipitated latex of E . lathyris, 24 " C a p e r Spurge," and from E . cerifera. 25 P o m i f e r i n . This enzyme was discovered in the latex of the green fruit of the osage orange ( M a c l u r a p o m i f e r a Raf.) by H. T a u b e r and S. Laufer. 1 F r o m the latex and press juice of the fruit an enzyme powder was obtained b y precipitation with 95 % ethanol. T h e product is not as active as fresh p a p a y a latex. A non-SH proteinase has also been observed in the pumpkin. 26 ~1W. G. Jaffd, J. Biol. Chem. 149, 1 (1943). 2~C. Fermi, Ann. Inst. Bot. Roma 7, 99 (1898). ~ C. Gerber, Compt. rend. soc. biol. 73, 578 (1912). 24W. J. Ellis and F. G. Lennox, Australian J. Sci. 4, 187 (1942); Biochem. J. 39, 465 (1945). 25 M. Castafieda, M. R. Balcazar, and F. F. GavarrSn, Anales escuela nacl. cienc, biol. (Mex.) 3, 65 (1943). ~ R. Willsti~tter, W. Grassmann, and O. Ambros, Z. physiol. Chem. 151, 286 (1926).

[6] Cathepsin C from Beef Spleen B y G. DE LA HABA, P. S. CAMMARATA, and J. S. FRUTON

Assay Method Principle. Cathepsin C, a proteinase obtained from beef spleen and other tissues, 1 catalyzes the hydrolysis of the amide bond of glycyl-Lphenylalaninamide (GPA) to form glycyl-L-phenylalanine and ammonia, which m a y be determined b y the Conway microdiffusion technique. ~

1 H. R. Gutmann and J. S. Fruton, J. Biol. Chem. 174, 851 (1948). 2 R. B. Johnston, M. J. Mycek, and J. S. Fruton, J. Biol. Chem. 185, 629 (1950).

[6]

CATHEPSIN C FROM BEEF SPLEEN

65

This enzyme also catalyzes a transamidation reaction between GPA and hydroxylamine3 to yield the corresponding hydroxamic acid, which may be conveniently determined by the technique of Lipmann and Tuttle. 4 Either of these reactions may be employed to assay the activity of cathepsin C, and both have been found equally reliable. The method based on the determination of ammonia, although more accurate than the colorimetric determination of the hydroxamic acid formed in the transamidation reaction, is somewhat more cumbersome and time consuming. The latter method will be described in this chapter.

Reagents 0.05 M GPA acetate in 0.04 M Na veronal buffer; pH of mixture adjusted to 7.2. 2 M NH2OH hydrochloride adjusted to pH 7.1 with 10 N NaOH immediately before use. 0.25 M L-cysteine hydrochloride. 0.5 N NaOH. Distilled water. 20% TCA. 5% FeCI~-6H20 dissolved in 0.1 N HC1.

Procedure. The above solutions are added to test tubes in the following order: 0.5 ml. of GPA, 0.1 ml. of hydroxylamine, 0.1 ml. of L-cysteine HC1, 0.05 ml. of NaOH, the enzyme solution, and water to a final volume of 1.0 ml. A blank solution without enzyme is also prepared. Before the addition of enzyme, all tubes are incubated for 4 minutes at 38 °. The enzyme solution to be assayed is then added, and the reaction is allowed to proceed for exactly 10 minutes at which time 0.6 ml. is withdrawn from each tube and quickly pipetted into tubes containing 0.5 ml. of 20 % TCA and 0.9 ml. of water. (Except in the case of the crude tissue extract, no turbidity due to protein precipitation was observed; for the crude tissue extract, the mixture was centrifuged and the supernatant fluid decanted.) To each tube, 0.5 ml. of the FeC13 solution is then added, and the color is read immediately in a Bausch and Lomb colorimeter with a 550-mtL filter. The values for per cent transmission (T) obtained with this instrument are converted to density values (2 - log T). The density value obtained for the blank solution is subtracted from all assay values. In our procedure two enzyme concentrations are routinely assayed and the readings are averaged. Definition of Activity Unit and Specific Activity. One unit of enzyme is defined as that amount of enzyme which gives (after correction for the 3 M. E. Jones, W. R. Hearn, M. Fried, a n d J. S. Fruton, J. Biol. Chem. 195, 695 (1952). 4 F. L i p m a n n a n d L. C. Tuttle, J. Biol. Chem. 159, 21 (1945); see also Vol. I I I [39].

66

ENZYMES OF PROTEIN METABOLISM

[6]

blank.) a density value of 0.1. I n order to obtain satisfactory proportionality between a c t i v i t y and e n z y m e concentration, the e n z y m e should be assayed a t levels which give density values not exceeding 0.15 (uncorrected for e n z y m e blank). Specific a c t i v i t y is expressed in units of e n z y m e (H.U.) per milligram of protein. Protein concentration was determined b y m e a n s of the biuret procedure 5 or the micro-Kjeldahl technique, 8 assuming the nitrogen content of the total protein to be 16%. Preparation of Substrate. Glycyl-L-phenylalaninamide acetate is prep a r e d as described previouslyY T h e synthesis of this c o m p o u n d b y means of the mixed a n h y d r i d e m e t h o d of V a u g h a n and Osato 8 was also found to be satisfactory.

Purification of Beef Spleen Cathepsin C T h e following is a typical purification of cathepsin C; the procedure is s u m m a r i z e d in the a c c o m p a n y i n g table. PARTIAL PURIFICATION OF CATHEPSIN C FROM BEEF SPLEEN

Fraction Crude extract Acid extract 0-70% AS (dialyzed) 40-70% ASb (undialyzed) 40-70% ASb (dialyzed)

Acetone I Acetone I I Acetone I I I Acetone I, heated Acetone II, heated

Protein concenVolume, tration, Enzyme units, Total ml. mg./ml. H.U./ml. units 10,433 6,370

15.12" 0.69

18.6 19

Total protein, Specific mg. activity

190,053 121,030

157,796 4,395

1.2 27.5

140

16

795

111,300

2,240

49.7

36

50

2316

83,376

1,800

46

38

25.4

1490

56,620

965

58.6

12.1 22.4 13.6 18.8 14 19.5

1974 1474 0

23,885 20,046 0

271 255 273

88 78.4 0

10.8

6.6

1834

19,807

71.3 278

11.9

4.3

1492

17,754

51

347

Protein nitrogen determined by the micro-Kjeldahl procedure and multiplied by 6.25. b Only 23.7 ml. of 40 to 70% AS (or 54,889 units) was dialyzed. Extraction and Acid Treatment. F o u r beef spleens obtained immediately after slaughter (chilled in ice and b r o u g h t to the l a b o r a t o r y ) 6 H. W. Robinson and C. G. Hogden, J. Biol. Chem. 185, 727 (1940). 6L. Miller and J. A. Houghton, J. Biol. Chem. 159, 373 (1945). 7 j. S. Fruton and M. Bergmann, J. Biol. Chem. 145, 253 (1942). 8 j, Vaughan and R. L. Osato, J. Am, Chem, ,$oc. 75~ 5553 (1951).

[6]

CATHEPSIN C FROM B E E F SPLEEN

67

were cut into 2-inch cubes and frozen at - 2 0 °. Twenty hours later this material was thawed slightly at room temperature and passed twice through a chilled power meat grinder using a fine grinder blade (3477 g. of ground spleen was obtained). To each 1000 g. of ground spleen was added 2000 ml. of distilled water containing 0.9 mg. of Versene Na per milliliter (pH 7). The suspension was stirred mechanically until all the solid material was dispersed, a sample was taken for activity determination, and 100 ml. of 6.37 N sulfuric acid was then added slowly to a final pH of 3.5. (If the pH is allowed to become more acid, rapid inactivation of the enzyme occurs. If the extract is insufficiently acid, a lower degree of purification is obtained.) The acid suspension was placed in a water bath maintained at 38 ° and stirred mechanically. After 1 hour the pH was readjusted to 3.5 (10 ml. of 6.37 N sulfuric acid was used). Ten milliliters of toluene was added, the mixture shaken vigorously, and then incubated at 38 ° for 24 hours after reaching the temperature of the bath. The clear layer was then siphoned off, and the remainder was filtered through Whatman No. 1 filter paper by gravity at room temperature. To the filtrate enough solid ammonium sulfate was added slowly with mechanical stirring to give a final concentration of 70% saturation (47.4 g. to each 100 ml. of filtrate). The suspension was stirred mechanically for 30 minutes at room temperature and was then filtered through Whatman No. 1 filter paper by gravity in the cold room (5 °) overnight. The protein was then scraped from the filter paper and suspended in an acidic solution of 80 % saturated ammonium sulfate (the pH of a saturated solution of ammonium sulfate was adjusted to 4 with concentrated sulfuric acid, the pH being measured with a Beckman pH meter on a 1:10 dilution of this solution) and then centrifuged at 16,000 × g for 30 minutes. The supernatant fluid was discarded, and the precipitate (0 to 70% AS, see table) was dissolved in enough 0.9% NaC1 to give a final volume of approximately 100 ml., which was then dialyzed against 6 1. of 0.9% NaC1. The dialyzing medium was replaced by a fresh 6 1. of 0.9% NaC1 after 18 hours, and the dialysis was continued for an additional 8 hours. Refractionation with Ammonium Sulfate. To the dialyzed 0 to 70% AS fraction (the protein concentration should be 15 to 20 mg./ml.), saturated ammonium sulfate (pH 4.0) was added slowly to 40 % saturation (66.7 ml. to each 100 ml. of protein solution) in an ice bath with mechanical stirring. The suspension was centrifuged in the cold room at 16,000 X g for 15 to 30 minutes, and the precipitate was discarded. To the supernatant fluid enough saturated ammonium sulfate (pH 4.0) was added to a final saturation of 70% (100 ml. to each 100 ml. of supernatant fluid). The precipitate was collected by eentrifugation, as above, and

68

ENZYMES OF PROTEIN METABOLISM

[6]

dissolved in 30 to 40 ml. of 0.02% NaC1. This solution (40 to 70% AS) was dialyzed against 6 1. of 0.02% NaC1 in the cold room for 18 hours, the dialyzing medium being replaced by a fresh 6 1. of the same after 8 hours. (The undialyzed 40 to 70% AS fraction may be assayed if the concentration of ammonium ion in the assay system does not exceed 10 micromoles per milliliter.) Acetone Fractionation. The protein concentration of the dialyzed 40 to 70% AS fraction was adjusted to 11 mg./ml, with 0.02% NaC1 and the pH to 4.95 with 1 N acetic acid. To 81.6 ml. of this fraction acetone was added (20.4 ml.) at 0 to - 2 ° to a concentration of 20% (v/v) over a period of 10 minutes. The suspension was then centrifuged at this temperature for 15 minutes at 1000 X g, and the precipitate was dissolved in 11 ml. of 2% NaC1 (acetone I). To the supernatant fluid more acetone was added to a concentration of 33 % (20.4 ml.), as described above. The precipitate was collected by centrifugation and dissolved in 2% NaC1 (acetone II). Acetone was again added to the supernatant fluid to a final concentration of 50% (40.8 ml.). The precipitate was collected and dissolved in 2% NaC1 (acetone III). It should be pointed out that some variability in the quantitative distribution of cathepsin C between acetone fractions I and II has been observed; due probably to inadequate control of the ionic strength of the 40 to 70% AS fraction. Heat Inactivation. The relatively high heat stability of cathepsin C 9 may be employed to advantage in its purification. Acetone fractions I and II were immersed in a 65 ° bath and quickly brought to that temperature with stirring. The enzyme solutions were maintained at this temperature for 40 minutes with occasional stirring. At the end of this period the solutions were cooled quickly to ice bath temperature and centrifuged at 16,000 X g for 15 minutes. The supernatant fluid was stored at - 1 5 to --20 ° .

Properties and Specificity of Partially Purified Cathepsin C Cathepsin C purified in the manner described above is stable for weeks when kept frozen at - 1 5 to - 2 0 ° . For maximal activity, either in the hydrolysis of GPA or in the transamidation reaction with hydroxylamine, cysteine is required (glutathione or ~-mercaptoethanol will replace cysteine). Cathepsin C appears to be specific for the hydrolysis, at pH 5, of a dipeptide derivative such as glycyl-L-phenylalaninamide or glycyl-Ltyrosinamide (or their corresponding ethyl esters) and at more alkaline pH values (ca. 7.4) catalyzes successive transamidation reactions leading to the formation of insoluble polymeric peptides. 10 g H. H. Tallan, M. E. Jones, and J. S. Fruton, J. Biol. Chem. 194, 793 (1952). 10 j. S. Fruton, W. R. Heurn, V. M. Ingram, D. S. Wiggans, and M. Winitz, J. Biol. Chem. 204, 891 (1953).

[7]

PURIFICATION AND ASSAY OF RENNIN

69

[7] Purification and Assay of Rennin

By N. J. BERRIDGE Assay Method Principle. Rennin changes milk from a fluid to a gel in a time which, under suitable conditions, is inversely proportional to the concentration of the enzyme added to the milk. The moment of clotting may be detected by the appearance of macroscopic heterogeneity in a thin, flowing film of milk. The precautions to be taken to ensure that the conditions are suitable have previously been described in some detail.l An improved method of providing a visible end point 2 will be described below. The necessary precautions hinge on the variability of milk from sample to sample, and in one sample with time, and also on the double nature of the clotting process. The variability is partly overcome by using a moderate bulk of well-mixed skim milk powder, by making up in 0.01 M CaC12, and by treating the substrate in a constant way before use. The clotting process is said to have a double nature because two reactions, or groups of reactions, occur. It is the rate of the first one that is controlled by rennin. The second proceeds later and independently. Providing, however, that the milk powder is not too unsuitable, the 0.01 M CaC12 will make the second reaction proceed rapidly. If, then, the first reaction is made to go slowly by the use of a low concentration of enzyme, this reaction will control the rate of the whole process which may thus be used as a measure of the quantity of the enzyme. The details summarized in these simple statements have been discussed in previous reviews. 3.4 It is important to emphasize that the safe limits of the precautions for one batch or kind of milk powder may not apply to another batch or kind. They should preferably be redetermined whenever the use of a fresh batch is begun (see below), although the danger of being misled is not great if one works well within the limits.

Reagents CaC12 solution, diluted accurately to 0.01 M from a more concentrated stock solution of analytical reagent, which has been standardized, for example against silver nitrate. 1 ~. j. Berridge, Analyst T7, 57 (1952). 2 N. J. Berridge, J. Dairy Research 19~ 328 (1952). 3 N. J. Berridge in " T h e Enzymes" (J. B. Sumner and K. Myrb~ek, eds.), Vol. I, Part 2, p. 1079, Academic Press, New York, 1950. 4 N. J. Berridge, Advances in EnzymoL 15, 423 (1954).

70

ENZYMES OF PROTEIN METABOLISM

[6]

Spray-dried skim milk powder. The less heating or preheating this has suffered, the better. Although it is possible to use milk which has been preheated, it is worth going to some trouble to secure that which has not. It gives proportional clotting times over a wider range.

Special Apparatus A cylinder and plunger whisk is useful for dissolving the milk powder, because the upward stroke of the plunger creates a vacuum which pulls the milk granules apart. It is difficult to achieve reproducibility of the substrate without this apparatus. (Tissue blendors or homogenizers have not been tried.) A glass-fronted water bath at 30 ° _ 0.2. Suitable illumination. It is necessary to view the milk by reflected light. A 100-watt electric lamp should be fixed above and a little to one side of the operator so that neither direct light nor that reflected from the glass can dazzle him, but the milk is brilliantly lit. The background should be dark. Pyrex or other high-silica glassware cleaned with chromic-sulfuric acid is desirable. It appears, although it has not been proved, that ions from soft glass, or imperfections in the surface, may influence the clotting time. Device for forming reproducible films. If the tube containing milk is sloped at an angle of 30 ° to the horizontal and rotated around its long axis, a thin film of milk is formed on the glass vertically above the surface of the liquid. The thickness of the film depends only on the physical constants of the apparatus and of the milk. A suitable device can be constructed very quickly from Meccano parts and an electric clock motor. 2 A speed of 3 r.p.m, is satisfactory, although some workers may prefer the thicker film that more rapid rotation would give. Boiling tubes of about 2.2-cm. internal diameter are convenient; they may be fitted on to the rotating spindle by means of a rubber bung fixed permanently thereon. A free-wheel or other simple device allowing limited forward rotation facilitates manipulation. Several other methods of determining the clotting point have been described.

Procedure. When the work is first begun certain preliminary experiments are necessary, but as they will be better understood after the procedure has been read they will be described later. Reconstitute the milk powder at the rate of 12.0 g. in 100 ml. of 0.01 M CaCI~, and dispense in 10-ml. quantities into boiling tubes. This can be done very rapidly by

[7]

PURIFICATION AND ASSAY OF RENNIN

71

means of a large hypodermic syringe fitted with a stop on the stem to limit the volume delivered to 10 ml. Set aside until solution is complete; with some milk powder this may require an hour or two. Other kinds will dissolve at once. When solution is complete allow a further 30 to 45 minutes in the water bath for the attainment of equilibrium. Tubes which have inadvertently been left in longer ought to be discarded. Dilute the rennin preparation with distilled water so that when 1 ml. is added to 10 ml. of milk clotting will take place in about 5 minutes (see preliminary experiments below). When the milk has aged sufficiently allow 1 ml. of diluted rennin to run on to the surface of the milk. Shake gently but thoroughly to mix, avoiding froth formation, and set a stop watch going simultaneously. Fix the tube onto the rotating shaft so that the surface of glass on which the film is formed is below the surface of the water in the thermostat, and determine, to the nearest second, the time for the appearance of casein particles. In order to obtain a consistency of +__1% it is essential to measure clotting times at least in triplicate. This can be done with little extra trouble by adding the rennin to each of three tubes at intervals of 1 minute, mixing at exactly 0, 60, and 120 seconds. The first two clotting times are then noted without stopping the watch. If it is held or fixed just out of the observer's sight, he can take a rapid glance at it at the moment of clotting and so observe the momentary position of the second hand, make a note of the figure for seconds only, and proceed to the next tube. It is fatal to attempt the continual observation of both the tube and the watch. For the final result the watch is stopped in the usual way. The three clotting times should not differ by more than a few seconds, so that the proper values for minutes and seconds can be deduced from the final reading of the stop watch. Preliminary Experiments. These are carried out by the same general procedure and are necessary firstly to determine the amount of dilution required by an unknown sample to give a clotting time near 5 minutes, and secondly to decide how far from 5 minutes the times may deviate without loss of accuracy. The second experiment is normally required only once for each kind of milk powder. If in a series of experiments with a wide range of rennin concentrations the reciprocal of the clotting time is plotted graphically against the rennin concentration, a curve approximating to a straight line through the origin will be obtah~ed. The wider the range of clotting times, the poorer the approximation will be. From this graph, therefore, it is possible to decide over what range the law of proportionality holds within the desired limits. Calculation, Units. There is no absolute unit. The activity of a preparation toward a certain milk under the given conditions may be described as x units per milliliter or per gram, where

72

ENZYMES OF PROTEIN METABOLISM

X

~

[7]

D t --

D is the dilution (number of milliliters containing i ml. or 1 g.), and t is the clotting time in seconds. When changing from one batch of milk powder to another it is necessary to compare the two thoroughly before the old is used up so that new units may be related to old if necessary. An additional check may be made by keeping a standard rennet in the form of carefully preserved dry powder. Application to Various Kinds of Material. In practice rennin is obtained in admixture with its precursor prorennin. When only preformed rennin is to be measured the dilution should be reasonably near a neutral pH, and it should not be allowed to stand too long. Activation of the prorennin may thus be avoided, but clotting times should be determined at different intervals after dilution to confirm that activation is not occurring. When the total potential activity is required the original solution must be activated by keeping it under acid conditions and assaying from time to time until a maximum is reached. At pH 3.6 and 2 ° activation proceeds smoothly, and hourly determinations, with later values overnight and after 24 hours, are worth while. Some preparations of rennin are unstable when diluted. Here, again, clotting times may need to be repeated at intervals, and it may be necessary to use a buffer for dilution. The effect of the buffer on the clotting time of the milk should be determined with a stable preparation. Crystalline rennin is slow to dissolve in distilled water. For assay it is therefore first dissolved in a little 0.2 M phosphate buffer at pH 6.8 and diluted immediately with distilled water. The determination of residues of low activity is important in works control. Here, acid, sodium chloride, or other materials may affect the substrate, and adequate controls must be run, at least in the early stages. By this test other proteolytic enzymes will appear as rennin. It is important always to bear this in mind when interpreting results. Purification Procedure--Preparation of Crude Extract

It is usual to bypass the early steps by using a commercial extract. If necessary, however, the crude extract may be made from the fourth stomachs of calves as follows. The method is based on details given by Leitch ~ and by Thornley and Hilton. s 5 R. H. Leitch, Compt. rend. 5th congr, intern, tech. et chim. inds. agr. 2, 307 (1937). 6 B. D. Thornley and S. Hilton (Benger's Food, Ltd.), British Patent 532,458 (1941); U.S. Patent 2,337,947 (1943).

[7]

PURIFICATION AND ASSAY OF RENNIN

73

Step 1. Preparation of Dried Stomachs. The stomachs are emptied and cleaned b y rinsing momentarily in cold water and divested of f a t t y tissue. T h e y are then dried, either b y inflating, tying off, and hanging the bladders in a dry room, or by salting them thoroughly and hanging them up to dry without inflating. I t is also possible to prepare crude extracts without drying the stomachs. 7,s The dried material m a y be stored for m a n y months. Step 2. Preparation of the Extract. To prepare an extract the dried yells are torn or cut into shreds of any convenient size (e.g., 0.5 to 3 cm. wide) and extracted for several days at room temperature with enough 10% sodium chloride solution to make a suspension which can conveniently be stirred. The actual quantities are unimportant, but less than a dozen yells ~vill give quantities too small for convenient handling at later stages. F r e q u e n t stirring hastens the extraction, and most of the enzyme is in solution after 2 or 3 days. The salt is normally sufficiently bacteriostatic for extractions lasting up to 3 days, but preservatives such as thymol or sodium benzoate m a y be required for longer periods. It is worth while to determine the activity of the extract daily and to terminate it by straining off as soon as the activity ceases to climb steadily. Step 3. Activation. At this stage much prorennin m a y be present, and this m a y be activated b y t r e a t m e n t with acid at p H 1.6 to 2.0 and at room temperature for several hours when the most rapid activation is required; but for the maximum yield p H 3.6 and 0 to 5 ° are better conditions, and these should be maintained until there is no further significant rise in activity. One or two days are required. The p H is then raised to 5.4 to produce a stable, highly active, but viscous and cloudy solution. Step 4. Clarification. This m a y be achieved b y the procedure of van der Burg and van der Scheer2 A concentrated solution containing 0.8 to 0.9 g. of KAl(SO4)212H20 for every 100 ml. of rennet extract is added rapidly to the gently stirred solution, by which procedure a flocculent precipitate of aluminium hydroxide with all the rennin adsorbed upon it and all the protein particles entrained within it is formed. The rennin, but not the solid particles, is then released by adding enough Na2HPO4 solution to raise the p H to between 5.3 and 6.3. The mixture is stirred for a few minutes and filtered. This is the first filtration in the process; it is easy and it gives a clear solution containing the activity in good yield. Step 5. Precipitation. The clarified solution m a y well serve as a starting 7 H. L. Keil and B. K. Stout, U.S. Patent 2,145,796 (1939); British Patent 503,730 (1939). 8 H. Tauber and I. S. Kleiner, J. Biol. Chem. 96, 745 (1932). 9 B. -can der Burg and A. F. van der Scheer, Compt. rend. 5th congr, intern, tech. et chim. inds. agr. 2, 321 (1937).

74

ENZYMES OF PROTEIN METABOLISM

[7]

point for preparing the crystalline enzyme, but in fact all the preparations made by Berridge 1°,11 have begun with the commercial solution prepared by Benger's Ltd. (Holmes Chapel, Cheshire, England), and it would therefore be advisable to continue with the method of preparation patented for them by Thornley and Hilton2 In this method the rennin is precipitated by the addition of sodium chloride to 26 % and of hydrochloric acid to an apparent pH (glass electrode) of 1.5. The resulting copious precipitate contains a high proportion of the activity. It may be stored as such or redissolved in distilled water by adding enough alkali to give a pH of 5.4.

Preparation of Crystalline Rennin Step 1. Fractionation with NaCl. Four liters of commercial rennet, or of a solution prepared as described above, is saturated with sodium chloride and filtered through large fluted Whatman No. 3 papers strengthened at the tip with small No. 54 papers. The filtration is very slow (3 to 7 days), the filtrate opalescent, although the first runnings are normally returned to the filters, and the yield of precipitate small. Typical yields of activity (as opposed to visible material) at various stages are given in the table taken from Berridge and Woodward.18 At the end of the filtration the No. 3 papers contain much solid NaC1 and but little protein. This therefore must be dissolved out. The papers are pulped with enough distilled water to make a thick slurry and the imbibed liquid is then pressed out by squeezing in the hand. Crude as this method is, it is quite effective. The squeezed lumps are re-pulped with more water and the whole process carried out four times in all, yielding about 1 I. of extract. All solutions after this point are kept in contact with solid thymol. Step 2. Preparation of Coarse Precipitate. After filtration (Whatman No. 1) and adjustment to pH 5.4 the solution which has been expressed from the No. 3 papers is again saturated with sodium chloride, added this time, however, by diffusion through a rotating semipermeable membrane according to the method of McMeekin. 1~Visking tubing is suitable, and so is cast collodion. The semipermeable tubing is about 3 cm. in diameter, it is doubled, and the closed end of the resulting U dips into the liquid to a depth of about 4 cm. The tube hangs from an electric stirrer which rotates at 60 to 120 r.p.m, being governed by the air resistance of a large card fixed to the spindle. This gives more reliable operation than reducing the input voltage. This technique brings about saturation in lo N. J. Berridge and C. Woodward, J. Dairy Research 20, 255 (1953). 11 N. J. Berrldge, Biochem. J. $9, 179 (1945). ~ T. L. McMeekin, J. Am. Chem. Sac. 61~ 2884 (1939),

[7]

PURIFICATION AND ASSAY OF RENNIN

75

2 or 3 days. Fresh solid NaC1 has to be added to the membrane from time to time. Because of the slow precipitation the protein appears in a relatively coarse form which is easily separated by centrifuging. Step 3. Crystallization. The precipitate is redissolved in 50 ml. of distilled water, a small piece of thymol added, and the solution set aside in the refrigerator for at least 24 hours, preferably several days. A heavy deposit of crystals is usually formed. If crystals do not appear, the solution needs further purification. This is achieved by repeating the saltingout process using a membrane of 7-mm. diameter to correspond to the smaller volume of solution. The precipitate from this is dissolved in only a little less water than formerly and again given the opportunity to crystallize by being left in the refrigerator. Solutions which fail to crystallize are not discarded before about a fortnight. All such failures have been preparations from other brands of commercial extracts. These seem to require further fractionation, and by partial precipitation with sodium chloride it is possible to obtain from them low yields of crystalline material. The solution decanted from crystals is retained for a month or two as it frequently produces further crops. Step 4. Recrystallization. The crystals from 4 1. of commercial rennet, if present in the yield given in the table, are washed with a few milliliters of 10% sodium chloride and dissolved in 100 ml. of 0.2 M potassium phosphate buffer at pH 6.8 + 0.1. Half an hour with occasional stirring is required. The solution is filtered if necessary and brought to pH 5.4. Often crystals will appear from this solution, but it is better to precipitate the rennin by saturating with NaC1. The precipitate is filtered off with Whatman No. 54 paper and then redissolved in 100 ml. of iced distilled water. The filtration is quite rapid, and the soft creamy precipitate is easily scraped off the paper. Undissolved denatured protein is removed by filtration through No. 42 paper, and the clear filtrate is set aside to crystallize. If the crystals which appear in step 3 are irregular, some additional fractionation is practiced in step 4 by adding NaC1 to the phosphate solution at pH 5.4 a little at a time until the solution becomes turbid. The turbidity, consisting of less pure rennin, is filtered off before proceeding to the saturation. If step 4 is interrupted at any stage, crystallization may occur. If iced distilled water is not used for redissolving the amorphous rennin, losses of 50% or so may occur. Berridge and Woodward 1° used water at room temperature and experienced high losses. Additional Remarks. The simple process described has yielded crystals each of the six or eight times it has been used. Failures have occurred with other types of commercial rennet, and it may be surmised that the processes here described for the preparation of the crude solution are

76

ENZYMES

OF PROTEIN

METABOLISM

[7]

SUMMARY OF CRYSTALLIZA.TION PROCEDURE

Fraction

Total volume, Recovery, ml. %

Commercial rennet Extract of filter papers

4000

100

1000

55

Solution of coarse precipitate Suspension of crystals Solution of crystals

50

43

--

18

Suspension of recrystallized rennin

100

--

100

9-17

Operation Saturate with NaC1. Filter, extract filters 4 times with distilled water. Saturate with NaC1 via semipermeable membrane. Centrifuge, redissolve precipitate. Set aside to crystallize. Decant supernatant. Decant and dissolve residue in phosphate buffer at pH 6.8. Adjust to pH 5.4, saturate with NaC1, filter, and redissolve in H20 at 0 °. Set aside to crystallize.

effective in removing substances which interfere with crystallization. Failures with the longer m e t h o d " h a v e been mentioned privately, b u t success with a slight modification of it has been reported. '3 P r e p a r a t i o n s said to be in the form of needles h a v e been described. '4,15 T h e solubility of crystals at p H 5.4 or lower is v e r y low, and it seems an a d v a n t a g e not to h a v e too highly s u p e r s a t u r a t e d solutions at the early stages. T h e first precipitation with sodium chloride separates m u c h impurity. No successful alternative m e t h o d of removing the precipitate has been found. G r e a t e r yields of activity m a y be expected to indicate less purification. On one occasion (not published) a large yield of precipitate was isolated from a solution which filtered easily. Crystals were eventually obtained from this material, b u t t h e y were atypical. Several recrystallizations were needed to secure crystals of good shape, and at e v e r y stage losses were high. Properties P u r i t y . Crystals p r e p a r e d b y the earlier long m e t h o d 11 a p p e a r e d reasonably pure b y the constant solubility test. The short m e t h o d gives

la E. Cherbuliez and P. Baudet, Helv. Chim. Acta 33, 1673 (1950). 14C. L. Hankinson, J. Dairy Sci. 26, 53 (1943). 1~R. Mo De Baun, W. M. Connors, and R. A. Sullivan, Arch. Biochem. and Biophys. 43, 324 (1953).

[8]

CARBOXYPEPTIDASE AND PROCARBOXYPEPTIDA_SE

77

crystals of the same appearance, but they have not been tested for purity. Activity. One part of crystals will clot about 107 parts of normal milk in 10 minutes at 37 °. The enzyme is proteolytic, acting optimally on hemoglobin at p H 3.7 11.15 with relatively insignificant activity above p H 4.9. When acting in large excess at p H 6.8 it liberates a total of 0.28 meq. of acidic groups and 0.22 meq. of basic groups per gram of casein. ~Sa F r o m the a-casein in milk it liberates peptones not precipitable b y trichloroacetic acid. 16 The q u a n t i t y liberated approaches a maximum well before clotting occurs. A marked phosphoamidase activity was found in crystalline rennin by Holier and Si-Oh Li. I~ Mattenheimer et al. ~s found phosphatase activity in crystalline rennin when it was allowed to act on phosphopeptone in the presence of a thermostable activator from an ultrafiltrate of milk or of Hansen's rennet. Physical and Chemical Properties. Schwander el al. 19 determined the amino acid make-up of crystalline rennin. T h e y also carried out a number of electrophoretic examinations on filter paper, concluding that the isoelectric point lay near p H 4.5. The molecular weight was found to be about 40,000, the diffusion coefficient, which depended on the concentration being 9.5 • 10-7 sq. cm./sec, on extrapolation to infinite dilution. ~5,~H. Nitschmann and R. Varia, Helv. Chim. Acta 34, 1421 (1951). 1GC. Alais, G. Mocquot, H. Nitschmann, and P. Zahler, Helv. Chim. Acta 36, 1955 (1953). 1TH. Holter and S.-O. Li, Acta Chem. Scan& 41, 1321 (1951). 1~It. Mattenheimer, H. Nitschmann, and P. Zahler, Helv. Chim. Acta 35, 1970 (1952). ~9H. Schwander, P. Zahler, and H. Nitschmann, Helv. Chim. Acta 35, 553 (1952).

[8] Carboxypeptidase and Procarboxypeptidase

By HANS NEURATH Properties and Assay of Carboxypeptidase Chemical Characteristics. Pancreatic carboxypeptidase is usually obtained from the p a r t l y autolyzed exudate of freshly collected beef pancreas glands. 1 Of the four electrophoretic components 2 seen in the crude exudate at p H 8.5, carboxypeptidase represents the one of next-to-lowest 1 M. L. Anson, J. Gen. Physiol. 20, 663 (1937).

F. W. Putnam and H. Neurath, J. Biol. Chem. 166, 603 (1946).

78

ENZYMES

OF PROTEIN

METABOLISM

[8]

mobility. Preparations of the enzyme recrystallized seven times are electrophoretically monodisperse at pH 8.3, but at pH 9.3 20% of the protein migrates with a faster mobility than the main fraction. The cause for this behavior remains to be elucidated. The isoelectric point of the enzyme (moving boundary electrophoresis at 0.2 ionic strength) is at pH 6. The sedimentation constant, S, of the purified enzyme is 3.07; the diffusion constant (D20.~) is 8.68 X 10-7, corresponding to a molecular weight of 34,300, which is also the minimum molecular weight as determined from amino acid analyses2 The protein shows no unusual properties in amino acid composition, but it is distinguished chemically by being a metalloprotein, containing 1 mole of firmly bound zinc per mole of protein. ~ The molecule contains a" single polypeptide chain with a N-terminal threonine group, the C-terminal group being unidentified. The molar extinction coefficient at 278 m~ is 8.6 × 104. Crystalline preparations of the enzyme are practically insoluble in water and only difficultly soluble in dilute salt solutions. The enzyme is relatively soluble in 10% LiC1, particularly in the cold. It is stable within the pH range of 7.0 to 10.0, but for enzymatic analyses freshly prepared solutions should be used. Unlike some of the other proteolytic enzymes, carboxypeptidase is not inactivated by diisopropylphosphofluoridate (DFP) 2 Specificity. 3,5,6 Specific substrates for carboxypeptidase may be represented by the symbol R1.R2.CO-R~.COOH, hydrolysis occurring at the CO-R bond which may be either a peptide or an ester bond. The presence of a carboxyl group on the a position is an absolute requirement for the hydrolysis of peptides, esters, or proteins. The most susceptible substrates contain in position R3 side chains of aromatic amino (or hydroxy) acids, the rate decreasing in the order of phenylalanine, tyrosine, tryptophan, leucine, methionine, and isoleucine. The Michaelis constants (25 °) are of the order of 10-3 to 10-3 M, and the specific rate constants (k3) of highly susceptible substrates of the order of 1 (mole/1./min./mg. enzyme N/ml.). The substrates usually employed for enzymatic assay are carbobenzoxyglycyl-L-phenylalanine (CGP), carbobenzoxyglycyl-L-tryptophan (CGTr), and carbobenzoxyglycyl-L-leucine (CGL). Certain acylamino acids, such as chloroacetyl-L-phenylalanine, are also hydrolyzed, and, 8 References to these and other properties of the enzyme may be found in Chapter 25 by N. M. Green and H. Neurath, in "The Proteins" (Neurath and Bailey, eds.), Vol. II, Part B, p. 1057, Academic Press, New York, 1954. 4B. L. Vallee and H. Neurath, J. Am. Chem. Soc. 76, 5006 (1954). 5 H. Neurath and G. W. Schwert, Chem. Revs. 46, 69 (1950). 6E. L. Smith, Advances in Enzymol. 12, 191 (1951).

[8]

CARBOXYPEPTIDASE AND PROCARBOXYPEPTIDASE

79

because of the relative ease of synthesis, they have been often used in earlier work. Carboxypeptidase is inhibited by structural analogs of specific substrates, including, notably, aromatic D-amino acids and certain aromatic and heterocyclic carboxylic acids. The most effective inhibitor is fl-phenylpropionic acid [K~(25°) = 6 × 10-6]. Enzymatic Assay. The most reliable enzymatic assay is based on the estimation of liberated amino acids by the colorimetric ninhydrin method of Moore and SteinJ Monovalent buffers with relatively low affinity for zinc (Veronal, Tris) are preferable to phosphate buffer. The usual assay system contains 0.02 or 0.05 M substrate (CGP) in 0.02 M Veronal buffer, pH 7.5, containing 0.1 M NaC1 and 2 to 5 X 10-~ mg. of enzyme N per milliliter. Under these conditions, approximately 20% hydrolysis occurs within 30 minutes. Aliquots of 0.2 ml. are removed from the incubation mixture at 5- or 10-minute intervals and subjected to colorimetrie ninhydrin analysis, the enzymatic reaction being halted during the boiling process. The zero-time sample serves as blank, and under these conditions the method is equally applicable to pure and to crude enzyme preparations. With pure carboxypeptidase, the "proteolytic coefficient" is approximately 14 when the initial substrate concentration is 0.05 M, and 20 at an initial substrate concentration of 0.02 M CGP (provided that initial reaction velocities are being measured). Procarboxypeptidase The existence of this zymogen was inferred by Anson s from observations that the carboxypeptidase activity of fresh pancreatic extracts increased spontaneously at 37 ° or on the addition of trypsin. Procarboxypeptidase was partially purified by fractional precipitation with 0.2 to 0.35 saturated ammonium sulfate, but little else was known concerning its properties as a protein. More recently, 9 the zymogen was obtained in 80 % purity by a process which includes the following steps: (1) extraction of freshly collected, defatted pancreas glands with 2% LiC1, in the presence of DFP; (2) fractional precipitation of the clarified extract at pH 6.5 with acetone, the bulk of the zymogen being precipitated between 30 and 50% acetone at - 1 7 ° ; (3) fractional precipitation of the preceding material with 0.43 saturated ammonium sulfate at pH 6.7, 4 °, followed by (4) refractionation within the limits of 0.3 to 0.4 saturated ammonium sulfate. Because this work remains to be published, no details of isolation will be given herein. The main component of the purified zymogen had an electrophoretic mobility of 5.9 × 10-6 cm.2/volt/sec, at 7 S. Moore and W. H. Stein, J. Biol. Chem. 176, 367 (1948). 8 M. L. Anson, J. Gen. Physiol. 20, 777 (1937). 9 G. S. Albrecht, Thesis, University of Washington, 1954.

80

ENZYMES OF PROTEIN METABOLISM

[8]

p H 7.56 (phosphate buffer, ionic strength 0.22). Purified procarboxypeptidase is specifically activated by trypsin; chymotrypsin, thrombin, carboxypeptidase, and the protease from B. subtilis are completely ineffective in this respect. The rate and extent of tryptic activation depend on the purity of the zymogen preparation and are greatly affected by the presence of other proteins.

Purification of Carboxypeptidase Although earboxypeptidase can be isolated from pancreatic exudate as well as from minced pancreatic tissue, only the former will be considered here, since the exudate is easier to handle and is free of the structural protein components of the glands. The isolation procedure consists in the following principal steps: (1) collection of the pancreatic exudate; (2) full activation of procarboxypeptidase; (3) precipitation of the euglobulin precipitate; (4) extraction of the euglobulin precipitate; (5) crystallization of the enzyme from the soluble extract of the euglobulin precipitate; (6) recrystallization. Several of these steps are as yet illcontrolled, and hence highly variable yields of the enzyme may be expected from batch to batch. The least controlled steps are 1 and 2. Thus the amount of proteins collected in the exudate and the extent of activation of procarboxypeptidase will depend on the history of the glands and on the method and time period of collection of the exudate. Since, moreover, the time curve of tryptic activation depends both on the concentration of active trypsin (which is highly variable in the collected juice) and on the concentration of proteins other than procarboxypeptidase (vide supra), it is clear that the yield of carboxypeptidase can vary within very wide limits. Although the method described below has been used with success in this laboratory and elsewhere, it cannot be recommended as the one of ultimate choice. In fact, because of its ill-controlled aspects, a new method is being developed in this laboratory based on the isolation of purified procarboxypeptidase, which is subsequently activated under carefully controlled conditions. This work will be described at a later time. The following method of isolation is based on Anson's original description, 1 modified as described in the literature 2,9-11 and by practical experience in this laboratory. Step 1. Collection of Pancreatic Exudate. Fifty pounds of freshly collected, frozen pancreas glands 12 are cut on a band saw, while frozen, into 10 H. N e u r a t h , E. Elkins, a n d S. K a u f m a n , J. Biol. Chem. 170, 221 (1947). 11 H. N e u r a t h a n d G. De Maria, u n p u b l i s h e d experiments quoted in ref. 5. 12 T h e glands can be obtained from commercial slaughterhouses; it is i m p o r t a n t t h a t t h e y be frozen p r o m p t l y after collection a n d shipped in dry ice to the laboratory.

[8]

CARBOXYPEPTIDASE AND PROCARBOXYPEPTIDA~E

81

longitudinal sections, approximately 1 inch wide, and spread evenly on racks 13 in the cold room at 4 °. The drip juice is allowed to collect in glass or enamel trays, approximately 3 1. of exudate accumulating over a period of 60 hours. Step 2. Activation. The juice is adjusted to pH 7.8 (glass electrode) by the addition of 1 N NaOH and heated in 1-1. Erlenmeyer flasks in a 37 ° bath for 1 hour. During this process, the color of the juice turns from the original reddish-brown to chocolate-brown. Step 3. Precipitation of the Euglobulin Precipitate. Acetic acid (5 N) is added to the activated juice, with stirring, until pH 4.6 (glass electrode) is reached. According to the original procedure of Anson, I the acidified solution is then kept at 37 ° for 2 hours, resulting in the formation of a copious precipitate which is discarded. This latter step has been omitted in this laboratory, H since carboxypeptidase is unstable below pH 6. Instead, after adjustment to pH 4.6, the solution is immediately diluted tenfold with cold distilled water, the pH readjusted to 4.6 if necessary, and the suspension allowed to stand in the cold room until the euglobulin precipitate has settled, requiring 2 to 5 hours. The clear, supernatant solution is then syphoned off and the euglobulin precipitate collected by centrifugation in a refrigerated centrifuge. Approximately 15 to 20% of the proteins of the pancreatic juice is found in the euglobulin precipitate. Step 4. Extraction of the Euglobulin Precipitate. The euglobulin precipitate is suspended in distilled water to a volume approximately onefifth that of the pancreatic juice. Freshly prepared 0.2 M Ba(OH)2 (nearly saturated at room temperature) is added dropwise, under stirring, to pH 6 (glass electrode). This operation is carried out in the cold room or in a cold bath at 0 to 4 °. In this and in all subsequent extractions, the pH tends to drop as protein goes into solution, and hence time should be allowed for the pH to become nearly constant as the solution becomes saturated. After the pH becomes stabilized, the suspension is centrifuged and the supernatant solution, containing little, if any, enzymatic activity, is discarded. The precipitate is suspended in enough water to yield a volume approximately one-tenth that of the pancreatic juice, and 0.2 M Ba(OH)2 is added, with stirring, until a stable pH of approximately 9.5 is reached. The suspension is then centrifuged in the cold, and the clear supernatant solution is subjected to crystallization, as described below. The insoluble precipitate is subjected to a second extraction by suspending it in cold water to yield a volume approximately one-twentieth that of the pancreatic juice. Ad~3 Paraffin-coated wire mesh m a y be used for this purpose; plastic m a t t i n g , such as Neotex, appears to be b e t t e r suited, since c o n t a m i n a t i o n b y metal ions is eliminated.

82

ENZYMES OF PROTEIN METABOLISM

[8]

ditional 0.2 M Ba(OH)2 is added dropwise, in the cold, until a stable pH of approximately 10 is reached. The pH may be raised to 10.4, but at no time should this upper limit be exceeded. The protein dissolves only slowly, and several hours of extraction in the cold may be required. After centrifugation, the residual precipitate may be subjected to additional extractions, although little activity remains after the fifth extraction. During these extractions, the color of the precipitate changes from an initial gray to olive-green. Step 5. Crystallization. The supernatant solution resulting from each extraction is subjected immediately to crystallization. This is done by the dropwise addition of 1 N acetic acid, under stirring, additions of acid being withheld until the amorphous precipitate initially formed has dissolved. When the solution assumes a permanent, faint opalescence, as seen against a light placed behind the beaker, it is seeded and allowed to stand in the cold until crystallization ensues (12 to 24 hours). When microscopically visible crystals have appeared, more 1 N acetic acid is added, with stirring in the cold, until pH 7.2 is reached. Photomicrographs of crystals have been published. Step 6. Recrystallization. This is usually effected by suspending the crystals in a small amount of water, and adding, instead of Ba(OH)2, 0.1 N Li(OH) in the cold, under stirring, until pH 10 is reached. Dissolution occurs slowly, and it is sometimes necessary to raise the pH to 10.4. Crystallization of the protein is achieved by the addition of 0.1 N acetic acid, under conditions otherwise identical to those described in the preceding section. The supernatant solutions remaining after recrystallization are essentially free of carboxypeptidase activity. Usually, the crystals obtained from the first four extractions of the euglobulin precipitate are pooled for purposes of recrystallization. Five recrystallizations are usually required before the enzyme reaches maximum specific carboxypeptidase activity and becomes free of tryptic and chymotryptic activity. The yield is approximately 500 to 700 mg./1, of pancreatic juice. Alternate Methods of Recrystallization. It is sometimes found that, even after several recrystallizations by the above procedure, pigments remain associated with the enzyme. Under these conditions, the method of crystallization by graded dialysis has been found to be effective in achieving purity. The disadvantage of the method is that it seems to cause undue losses of enzyme. The crystals are dissolved in cold 10 % LiC1, and the solution is then successively dialyzed against 5% LiC1, 2 % NaC1, and distilled water. The change from one solvent to the next should be gradual and is best achieved by dropwise replacement in the feeding bottle. The crystals collecting in the dialysis bag have the habits of thin, square plates.

[9]

AMINOPEPTIDASES

83

It has also been reported 14 that, because the contaminating proteins are even less soluble in 0.2 M NaC1 than is crystalline carboxypeptidase, extraction of the crystals with 0.2 N NaC1 followed by dialysis against water is an expedient method of purification; however, no yields were given. 14 E. L. Smith, D. M. Brown, and H. T. Hanson, J. Biol. Chem. 180, 33 (1949).

[9] Aminopeptidases By EMIL L. SMITH A. Aminotripeptidase (Tripeptidase) Assay Method Principle. This enzyme hydrolyzes tripeptides at the bond adjacent to the essential free amino group to yield a free amino acid and a dipeptide as in the following typical reactions: Glycylglycylglycine-~ Glycine -~ glycylglycine L-Leucylglycylglycine--~ L-Leucine -~ glycylglycine L-Prolylglycylglycine --~ L-Proline + glycylglycine Glycylglycyl-L-leucine--* Glycine + glycyl-L-leucine

(1) (2) (3) (4)

These and a large number of other substrates can be used for the assay of the enzyme. The substrate of choice is glycylglycylglycine (triglycine) for a number of reasons, among them being its availability from commercial sources and the simplicity of its synthetic preparation (see Vol. III). The leucine compouud, in reaction 2, has been widely used; however, with crude extracts ambiguous data may be obtained, since it is rapidly hydrolyzed by leucine aminopeptidase. Hydrolysis of the tripeptide is readily measured by the Grassmann and Heyde 1 procedure for the estimation of liberated carboxyl groups.

Reagents Glycylglycylglycine solution at 0.125 M. Dissolve 236 mg. of the peptide in 0.4 ml. of 1 N NaOH and dilute to 10 ml. with water. Tris buffer (0.1 M) at pH 7.9. 0.01 M KOH in 90% ethanol. 100% ethanol. 0.1% thymolphthalein in 95 % ethanol. Enzyme. Dilute to obtain 5 to 15% hydrolysis in 15 minutes. 1 W. Grassmann and W. Heyde, Z. physiol. Chem. 183, 32 (1929).

84

ENZYMES OF PROTEIN METABOLISM

[9]

Procedure. The assay is conducted at 40 ° as described for other peptidases (see Vol. II [10]) in 2.5-ml. flasks containing 1.0 ml. of substrate, 1.0 ml. of Tris buffer, and enzyme. Definition of Unit and Specific Activity. The hydrolysis of triglycine and other tripeptides at the initial concentration of 0.05 M has been found with some sources of enzyme to follow first-order kinetics, and with others zero-order kinetics. The specific activity can then be defined as C1 when a first-order rate constant (K1) is obtained and calculated in terms of minutes and decimal logarithms. C1 = K I / E , where E is the protein nitrogen in milligrams per milliliter. When zero-order kinetics are found Co = K o / E , and K0 is calculated in per cent hydrolysis per minute. Units of activity are calculated by multiplying C1 by total E (or Co by total E), where total E is given in milligrams of protein N. Hence, 1 unit is the amount of enzyme which will give a K0 of 1%/min. or a K~ of 1.0.

Purification Procedures The aminotripeptidase has been extensively purified from calf thymus by Fruton and his associates, 2,3 and to about half the specific activity from equine erythrocytes by Adams et al. 4 Both procedures are given below, since the latter method is exceedingly simple and yields a preparation which appears to be as free of other proteolytic enzymes as the enzyme from thymus. Purification of Erythrocyte Enzyme. This method is based on the relative insolubility of horse hemoglobin in 0.4 saturated solution of ammonium sulfate and on the observations of Smith and Bergmann 5 and of Fruton et al. ~ that the aminotripeptidases of swine intestinal mucosa and calf thymus are soluble in 0.4 saturated and insoluble at 0.6 to 0.7 saturated ammonium sulfate. Freshly collected, citrated horse blood (5 to 15 1.) is permitted to stand overnight at 5 ° to permit sedimentation of the cells, and the plasma and buffy coat are removed by aspiration. The sedimented erythrocytes are washed in the centrifuge cups once or twice with cold isotonic saline and are then hemolyzed with an equal volume of cold distilled water. The hemolyzate is brought to 0.4 saturation with solid ammonium sulfate (242 g./1.) and allowed to stand overnight at 5°. The heavy precipitate of hemoglobin is removed first by centrifugation and then by filtration on a Biichner funnel with the aid of Hyflo Super-Cel. The paler filtrate is brought to 0.7 saturation by addition of solid ammonium sulfate 2 j. S. Fruton, V. A. Smith, and P. E. Driscoll, J. Biol. Chem. 173, 457 (1948). 8 D. Ellis and J. S. Fruton, J. Biol. Chem. 191~ 153 (1951). 4 E. Adams, N. C. Davis, and E. L. Smith, J. Biol. Chem. 199~ 845 (1952). E. L. Smith and M. Bergmann, J. Biol. Chem. 158, 627 (1944).

[9]

AMINOPEPTIDASES

85

(205 g./1.), and the precipitate is collected by centrifugation. The precipitate is resuspended in a solution of ammonium sulfate at 0.4 saturation of approximately half the volume of the original hemolyzate. This procedure is repeated two or three times, with successive collection of the fraction precipitating between 0.4 and 0.7 saturation and resuspension of this precipitate in progressively smaller volumes of 0.4 saturated ammonium sulfate solution. These repeated fractionations precipitate the bulk of the hemoglobin at 0.4 saturation in sulfate, whereas the tripeptidase activity precipitates at 0.7 saturation. The final precipitate, pale orange-brown in color, is dialyzed against cold distilled water until free of sulfate and is then lyophilized. Once dried, preparations of tripeptidase have retained their activity for over a year. However, preparations of the enzyme in solution lose approximately half their original activity on lyophilization. The C1 for the hydrolysis of triglycine averages 0.0009 in crude hemolyzates, the range being 0.0008 to 0.0013. Individual preparations of tripeptidase, purified as described, vary in activity from a C~ggof 0.1 to 0.6 with yields from 10 to 30% that of the total original activity of crude hemolyzates. The most active preparations thus represent a 500 to 750-fold purification of the enzyme. Purification of Thymus Enzyme. The method of isolation is that of Ellis and Fruton2 The procedure originally described is based on starting material of finely comminuted frozen thymus but can be applied to finely ground frozen thymus. Step 1. Extraction. Comminuted or finely ground thymus (850 g.) is stirred with 1700 ml. of a 2% sodium chloride solution at 1° for 21 hours. The insoluble material is removed by centrifugation followed by filtration with the aid of Hyflo Super-Cel. Yield, 1810 ml. of filtrate. Step 2. Ethanol Precipitation. To 1800 ml. of filtrate, 5.4 ml. of 1.0 M sodium acetate buffer at pH 4.0 is added to bring the pH to 5.0. There is then slowly added through a fine capillary tube, at - 6 ° with stirring and cooling, a mixture (precooled to - 6 °) containing 569 ml. of 95% ethanol, 21.6 ml. of 1.0 M sodium acetate buffer at pH 5.0, and 304 ml. of water. (The final conditions are pH 5.0, 0.23 M NaC1, 0.01 M sodium acetate, 20 % ethanol, and 1.3 % protein.) One hour after the addition is completed, the clear supernatant fluid is collected by centrifugation at - 6 °. A sample of the solution is dialyzed against 1% NaC1 to remove ethanol and acetate for assay of activity. Step 3. First Zinc-Ethanol Precipitation. The solution from step 2 (2100 ml.) is kept at - 6 °, and the following mixture at the same temperature is added in the manner-prescribed for the previous step: 180 ml. of 95% ethanol, 63 ml. of 4 M sodium acetate, 210 ml. of 0.25 M zinc acetate buffer (pH 5.8), and 72.6 ml. of water. (The final conditions are pH 5.8,

86

ENZYMES OF PROTEIN METABOLISM

[9]

0.18 M NaC1, 0.1 M sodium acetate, 0.02 M zinc acetate, 22.5% ethanol, and 0.1% protein.) The precipitate is collected by centrifugation at - 6 ° and is stirred with 75 ml. of ice-cold 0.1 M sodium citrate. This mixture is dialyzed against several changes of distilled water at 1°. The insoluble residue is removed by centrifugation and the clear solution (260 ml.) is saved. Step 4. Second Zinc-Ethanol Precipitation. The following mixture is added at 0 ° to the supernatant of step 3:23 ml. of 0.25 M zinc acetate buffer (pH 5.8) and 5.8 ml. of 1.0 M sodium acetate buffer at pH 5.8. After 20 minutes at 0 °, the following mixture is added at - 5 ° in the manner described: 68.5 ml. of 95% ethanol, 7.7 ml. of the 0.25 M zinc acetate buffer, 2.0 ml. of the 1.0 M sodium acetate buffer, and 20.7 ml. of water. (The final conditions are pH 5.8, 0.02 M sodium acetate, 0.02 M zinc acetate, 17% ethanol, and 0.36% protein.) After 75 minutes at - 5 °, the precipitate is removed by centrifugation, leaving a clear supernatant (357 ml.). To 350 ml. of this solution, the following mixture is added at - 6 ° : 31.9 ml. of 95% ethanol, 3.2 ml. of the 0.25 M zinc acetate buffer, 0.8 ml. of the M sodium acetate buffer and 4.6 ml. of water. (The final conditions are pH 5.8, 0.02 M sodium acetate, 0.02 M zinc acetate, 23% ethanol, and 0.15% protein.) After 1 hour at - 6 °, the precipitate is collected by centrifugation, dissolved in a few milliliters of 0.1 M sodium citrate, and dialyzed against 1% NaC1 for 36 hours and then against distilled water for 24 hours. This solution is used immediately for the next step, since the enzyme gradually loses activity in salt-free solution. Step 5. Ammonium Sulfate Precipitation. To 15 ml. of solution, 3.71 g. of ammonium sulfate is added at 0 °. After 3 hours, the precipitate is removed by centrifugation, and to the supernatant solution (16.6 ml.) 2.08 g. more of the salt is added. The precipitate is collected by centrifugation, dissolved in a few milliliters of water, and dialyzed against 1% NaC1 for 96 hours. The activities and yields are summarized in Table I. TABLE I PURIFICATION OF AMINOTRIPEPTIDASE FROM CALF THYMUS

(The m e t h o d a n d the d a t a are from Ellis a n d F r u t o n 2 ) Step 1. 2. 3. 4. 5.

Extraction E t h a n o l precipitation First Zn-ethanol precipitation Second Zn-ethanol precipitation A m m o n i u m sulfate precipitation

Co

Total units

Yield in per cent a

2.0 17.5 22.5 117 219

7780 6930 4890 1500 1030

100 89 63 19 13

Yield is uncorrected for samples removed for assay.

[9]

AMINOPEPTIDASES

87

Properties S p e c i f i c i t y . The enzyme hydrolyzes a variety of tripeptides at the N-terminal peptide bond. A s u m m a r y of some of the studies with the erythrocyte and t h y m u s enzymes is given in Table II. The purified enzyme has no action on dipeptides, dipeptide amides, acyl tripeptides, tripeptide amides, or tetrapeptides, hence its classification as an aminotripeptidase. I t appears that free amino and carboxyl groups in the substrate must be at a definite distance from one another. A c t i v a t o r s a n d I n h i b i t o r s . No activators are required for this enzyme and there is, as yet, no information as to the nature of essential groupings in the enzyme. The t h y m u s enzyme 3 is inhibited 75% b y 0.01 M cysteine, 95% b y 0.001 M Cd ++, 80% by 0.001 M Hg ++, and more weakly by a few other divalent cations. Similar, though not identical, results are found with the erythrocyte enzyme. 4 P u r i t y . No evidence is available as to the absolute purity of presently available preparations. The enzyme m a y prove to be useful for the identification of the N-terminal residue of tripeptides. If the preparation contains detectable traces of leucine aminopeptidase or metal-activated dipeptidases, these m a y be inhibited by the addition of ethylenediaminetetracetate (Versene) at 0.005 M which has only a small inhibitory effect (10 to 15%) on the tripeptidase.

TABLE II SPECIFICITY OF AMINOTRIPEPTIDASE

Relative activity~ Substrate Glycylglycylglycine L-Leucylglycylglycine D-Leucylglycylglycine L-Alanylglycylglycine Glycylglycyl-L-proline L-Prolylglycylglycine Hydroxy-L-prolylglycylglycine Glycyl-L-prolylglycine Glycyl-Lqeucylglycine Glycyl-D-leucylglycine Glycylglycyl-L-leucine Glycylglycyl-D-leucine

Thymus enzyme Erythroeyte enzyme 100 88 0 108 71 ---98 0 40 9

100 80 0 175 55 235 40 0 -----

a The data for the thymus enzyme are calculated from Co values given by Fruton et al. 2 and Ellis and Fruton, 3 those for the erythrocyte enzyme from C~ values of Adams et al. 4 Further data on the specificity may be found in the aforementioned publications.

88

ENZYMES OF PROTEIN METABOLISM

[9]

B. Leucine Aminopeptidase Assay Method Principle. The most convenient and sensitive substrate for this enzyme is L-leucinamide which may be used as the hydrochloride; its synthesis is given in Vol. III. The assay depends on the measurement of carboxyl groups liberated during the course of hydrolysis. These may be estimated by titration in 90% ethanol by the method of Grassmann and Heyde,' or, alternatively, by any of the methods which estimate free ammonia. Reagents Leucinamide HC1 solution (0.125 M). Dissolve 208 mg. of the compound in 8.0 ml. of 0.1 N NaOH and bring to a volume of 10 ml. in a volumetric flask. The compound is completely stable in the cold as long as contamination is avoided. 0.2 M Tris buffer, pH 8.0. 0.04 M MnS04 or 0.04 M MnCl~. 0.01 M KOH in 90% ethanol which must be standardized daily. 100 % ethanol. 0.1% thymolphthalein in 95% ethanol. Enzyme. The stock solution should be diluted to yield about 5 to 15 % hydrolysis of the substrate in 15 minutes.

Procedure. Crude and partially purified enzyme preparations require a prior incubation with Mn ++ (or Mg ++) to obtain maximal activation. To a 2.5-ml. volumetric flask, add 1 ml. of Tris buffer, 0.5 ml. of 0.04 M MnSO4, 0.2 to 1.0 ml. of enzyme, and water to volume. Mix the contents of the flask and allow to incubate at 40 ° for 10 to 120 minutes; with crude extracts the longer time is needed, with purified extracts the shorter time is sufficient. For the assay, add to a 2.5-ml. flask 1.0 ml. of 0.125 M substrafe solution, 1.0 ml. of Tris buffer, 0.1 to 0.5 ml. of preincubated enzyme, and water to volume, as needed. Mix the contents rapidly and remove 0.2-ml. samples at zero time and subsequently; titrate each sample immediately to a blue end point (thymolphthalein) with the standardized alcoholic KOH which is near 0.01 M. (See the procedure under prolidase, Vol. II [10].) The difference in titer of the initial sample and those removed later gives the amount of liberated carboxyl. With 0.01 M KOH, 100% hydrolysis corresponds to an increase in titer of 1.00 ml. Specific Activity and Units. Hydrolysis of L-leucinamide at 0.05 M (concentration in the test solution) is first order. The first-order constant (K1) is calculated in minutes and decimal logarithms. Specific activity is given by the proteolytic coefficient C1, where CI = K~/E and E is the

[9]

AMINOPEPTIDASES

89

protein N in milligrams per milliliter. Units are calculated by the product C1 × total E in milligrams. Assay of Enzyme in Crude Extracts. The aminopeptidase appears to be freely soluble in water. Assay with leucinamide is desirable. Other and more specific peptidases which are present in crude tissue extracts hydrolyze leucylglycine and leucylglycylglycine,the peptides used originally by LinderstrCm-Lang and by Johnson and co-workers in their studies of this enzyme. 6,v Purification Procedure

The enzyme has been obtained in partially purified form from a number of sources. The best purification has been obtained from swine kidney rather than the less convenient swine intestinal mucosa used earlier. The procedure given below is that of Spackman, Smith and Brown s which incorporates features used earlier by Smith and co-workers 5,9 for the purification of the intestinal enzyme. An aqueous clarified extract of swine kidney has a C~ = 0.06. Step 1. Preparation of Acetone Powder. Whole swine kidney (2400 g.) is coarsely ground in the frozen state and then blended for 1 minute with an equal amount (2400 ml.) of 53.3 % ethanol at - 5 °. One-fifth of the volume of the mixture or 960 ml. of 95% ethanol at - 2 0 ° is then added, and the mixture is allowed to stand for 30 minutes before centrifuging in the cold to collect the precipitate. (The supernatant contains prolidase and may be saved for the preparation of this enzyme.) The precipitate is extracted with an equal volume of absolute ethanol for 30 minutes, the mixture is centrifuged, and the supernatant is discarded. The insoluble fraction is rinsed into a beaker and mixed with 2 vol. of cold acetone. After 2 hours, the material is collected on a Btiehner funnel, sucked dry, and the procedure repeated. Finally, the precipitate is again collected on a funnel, washed successively with more acetone (0.5 vol.), with 50% acetone-ether (0.25 vol.) and finally with ether. The fibrous material is first rapidly air-dried at room temperature, and then in vacuo over sulfuric acid. The yield is 360 g. of powder. A clarified aqueous extract of this powder made at pH 8 has a C1 = 0.25. Step 2. First Ammonium Sulfate Fractionation. The acetone powder is extracted by treatment in a Waring blendor for 1 minute with 7 vol. of water. After 30 minutes, the mixture is centrifuged. The precipitate is re-extracted for 30 minutes with an equal volume of water. The insoluble 6 M. J. Johnson and J. Berger, Advances in Enzymol. 2, 69 (1942). 7 E. L. Smith, Advances in Enzymol. 12, 191 (1951). 8 D. H. Spackman, E. L. Smith, and D. M. Brown, J. Biol. Chem. 212,255 (1955). 9 E. L. Smith and N. B. Slonim, J. Biol. Chem. 176, 835 (1948).

90

ENZYMES OF PROTEIN METABOLISM

[9]

residue is removed by centrifugation and discarded. The two extracts are combined, and the turbid solution is adjusted with 1 N NaOH (approximately 10 to 12 ml./1.) to pH 8.0 to 8.1. This solution is brought to 0.4 saturation with solid ammonium sulfate (242 g./1.), and after 30 minutes the precipitate (which is discarded) is removed by filtration on fluted paper or with weak vacuum on a Btichner funnel (Hyflo Super-Cel is recommended). To the supernatant more solid ammonium sulfate is added (280 g./1.) to bring to 0.8 saturation. The precipitate may be collected in a small refrigerated Sharples centrifuge at 40,000 to 50,000 r.p.m, or in a refrigerated bucket centrifuge at 4700 r.p.m. The supernatant is discarded. The precipitate is dialyzed against 0.005 M Tris buffer at pH 8.0. The solution should have a C1 = 0.9 to 1.2 and contain about 2.5 to 3.0% protein. Step 3. Second Ammonium Sulfate Fractionation. The solution from step 2 (at pH 8.0 to 8.1) is brought to 0.5 saturation with solid ammonium sulfate (312 g./1.), and the precipitate is removed by centrifugation and discarded. The supernatant is brought to 0.7 saturation with solid ammonium sulfate (135 g./1.), and the precipitate is collected in the manner described for the 0.8 precipitate in step 2. The precipitate is dialyzed against the Tris buffer as described in step 2. C1 = 1.5 to 2.4. Step 4. Precipitation with MgCl2. The solution from step 3 is brought to pH 7.0 by addition of N HC1 and to 0.01 M MgC12 by addition of the solid salt. After 2 hours, the inactive precipitate is removed by centrifugation. The supernatant is brought to pH 8.0 with N NaOH. C1 = 2.5 to 3.5. Step 5. Heating. The solution is transferred to a stainless steel beaker and heated in an 80 ° water bath with mechanical stirring until the temperature of the solution reaches 70 °, and it is held there for a total time of 10 minutes, including the 6 to 7 minutes required to reach 70 °. The preparation is then quickly cooled in an ice-water bath. The inactive precipitate is removed by filtration or centrifugation. C1 = 3.8 to 5.8. Step 6. Acetone Fractionation. The solution from step 5 is brought to pH 7.0 with N HC1, and acetone precooled to - 6 0 ° is added to make the concentration 20 %. The inactive precipitate is removed by centrifugation in the cold. The supernatant is brought to 30% acetone, and the precipitate collected by centrifugation. The precipitate is dissolved in water containing 0.005 M MgC12 and is dialyzed for 16 to 18 hours against three changes of a solution containing 0.005 M MgC12 and 0.005 M Tris buffer at pH 8.0 to remove acetone. C1 = 16 to 43. (Note on step 6. In most cases preparations have been obtained with C~ values of 30 to 43. In those instances where the purity was low (C1 = 16 to 18) the fractionation was repeated between 18 and 25% acetone to bring the C1 to

[9]

AMINOPEPTIDASES

91

a b o u t 30. With preparations of CI = 32 to 43, repetition of the acetone fractionation does not increase the purity significantly.) Step 7. Aging. When preparations from step 6 are diluted to a concentration of about 2 rag. of protein per milliliter with a solution containing 0.005 M MgC12 and 0.005 M Tris at pH 8 and kept under toluene in the refrigerator for six to ten weeks, some precipitation occurs, with a slow decrease in pH. The content of aminopeptidase remains constant, and a twofold purification occurs. F u r t h e r storage has no effect. A preparation of CI = 43 went to C1 = 86 after 10 weeks. Purity. The procedure is summarized in Table I I I . F r o m electrophoretic analysis and identification of the active component, the preparation with CI = 86 is estimated to be essentially homogeneous. TABLE III PURIFICATION OF ]JEUCINE AMINOPEPTIDASE FROM SWINE KIDNEY

(A representative run for 2400 g. of kidney. The activity of the crude clarified extract is calculated from average data obtained on aliquots; such extracts are not prepared for the purification procedure given.) Step Crude extract 1. Acetone powder extract ~ 2. First sulfate precipitation 3. Second sulfate precipitation 4. Precipitation with MgCI~ 5. Heating to 70° 6. Acetone fractionation 7. Aging

C1

Total protein, rag.

0.06 0.25 1.1 2.4 3.6 5.8 43 86

293,000 45,300 11,600 4,170 2,640 1,320 110 55

Total units Yield~ % 2450 1700 1900~ 1500 1430 1120 720 360

100 69 78b 61 58 46 29 29

a From a yield of 360 g. of powder. b The high recovery in this step is presumably due to the removal of inhibitors. I n most cases, the above procedure has given material of C1 = 40 to 60. Purification b y electrophoresis on filter paper has given preparations of C1 = 85 to 88. 8

Properties Specificity.

The aminopeptidase is relatively nonspecific. I t hydrolyzes at different velocities amino acid amides, di- and tripeptides, and, very likely, longer polypeptides as well. A free carboxyl group is not required, but a free amino group is essential. The enzyme is optically specific for the residue bearing the free amino group, i.e., L-leucinamide and L-leucylglycine are substrates, the corresponding D isomers are not. I n dipeptides, the residue bearing the carboxyl m a y in some instances be of the D

92

ENZYMES OF PROTEIN METABOLISM

[9]

configuration, e.g., L-leucyl-L-alanine is hydrolyzed twenty-five times as rapidly as the diastereoisomer, L-leucyl-D-alanine. In dipeptides in which the C-terminal residue is large, the presence of a D residue hinders the action of the enzyme (e.g., L-leucyl-D-isoleucine, L-leucyl-D-valine). ~°,1~ The name leucyl peptidase (or leucine aminopeptidase) was originally suggested b y L i n d e r s t r t m - L a n g to distinguish it from other peptidases and to describe its rapid action on leucyl peptides. I t is now evident that, although the action on leucyl compounds is most rapid, the peptidase also attacks amides and peptides containing other N-terminal amino acids. The relative rate of hydrolysis of a number of amino acid amides is given in Table IV. 12 I n no case is the D isomer susceptible to hydrolysis or inhibitory. TABLE IV HYDROLYSIS OF AMINO ACID AMIDES BY LEUCINE AMINOPEPTIDASE

(The rates were determined at a substrate concentration of 0.05 M for the L component at pH 8.0 and 40° in the presence of 0.002 M Mn ++. The value of L-leucinamide is given as 100 and for the other substrates as the relative rate. The data are from the work of Smith and Spackman. 13) Substrate L-Leucinamide DL-Norleucinamide DL-Norvalinamide DL-a-Amino-n-butyramide L-Alaninamide Glycinamide L-Valinamide I~Isoleucinamide L-Alloisoleucinamide

Rate 100 101 84 36 3.5 0.1 17 20 7

Substrate

Rate

L-Serinamide L-Phenylalaninamide L-Tryptophanamide L-Tyrosinamide L-Isoglutamine L-Aspartic diamide L-Lysinamide L-Prolinamide Hydroxy-L-prolinamide

1 26 24 16 2 3 7 0.7 0.6

The peptidase also hydrolyzes glycyl-L-leucinamide and other dipeptide amides as well. The action on glycyl-L-leucinamide rapidly liberates a m m o n i a and is followed b y very slow hydrolysis of glycyl-L-leucine2 Thus, the enzyme can hydrolyze substrates at a susceptible bond one residue removed from t h a t adjacent to the free amino group2 ,I° F u r t h e r detailed discussion of the specificity and mode of action of this enzyme can be found elsewhere. 12,18 lo E. L. Smith and W. J. Polglase, J. Biol. Chem. 180, 1209 (1949). 1~E. L. Smith, D. H. Spackman, and W. J. Polglase, J. Biol. Chem. 199, 801 (1952). 12E. L. Smith, N. C. Davis, E. Adams, and D. H. Spackman, in "The Mechanism of Enzyme Action" (W. D. MeElroy and B. Glass, eds.), Johns Hopkins Press, Baltimore, 1954. la E. L. Smith and D. It. Spackman, J. Biol. Chem. 212, 271 (1955). This paper gives details of specificity, activation and mode of action of the kidney enzyme.

[10]

DIPEPTIDASES

93

Activators and Inhibitors. The enzyme is maximally activated by 0.001 M Mn ++ or Mg++. 5 With crude preparations the activation is slow, 2 to 3 hours being required. With preparations at the level of C1 = 5 to 85 which already have been heated with Mg ++, the activation is optimal in a few (2 to 5) minutes2 The enzyme is more stable in the presence of Mg ++ than Mn ++. With Mg ++ present, the enzyme, protected from contamination with toluene or thymol, may be kept in solution in the refrigerator for 6 months or longer. No other metal ions will activate, and many are inhibitory, e.g., Zn ++, Pb ++, Hg ++, and Fe ++. The enzyme is strongly inhibited by anions which bind with Mg ++ and Mn ++, e.g., citrate, Versene, and pyrophosphate. Sulfhydryl reagents, such as iodoacetamide and p-chloromercuribenzoate have no effect, nor does glutathione. Diisopropylfluorophosphate has no inhibitory effect. Effect of pH. The pH activity curve shows an optimum at pH 9.1 to 9.3 depending on the activator and the substrate. 13 The most suitable buffers to use with the aminopeptidase are Tris, Veronal, and cacodylate; these show no specific ion effects. Distribution. The enzyme has been found in all animal tissues examined thus far and in many plants and microorganisms as well. 6,7

[10] D i p e p t i d a s e s

By EMIL L. SMITH A. Carnosinase Assay Method

Principle. Presently available preparations of this enzyme hydrolyze carnosine and several other dipeptides containing L-histidine as the C-terminal residue. Carnosine is the preferred substrate, partly because of its presence in large amounts in muscle,1 but also because its isolation and synthesis have been carefully studied. A method for the synthesis of carnosine is given in Vol. III. The assay of the enzyme depends on the measurement of carboxyl groups liberated during the course of hydrolysis by the microtitration method of Grassmann and Heyde 2 or, alternatively, by any of the other methods which estimate carboxyl or amino groups. 1The literature on carnosine up to 1939 is discussed in an excellent review by V. du Vigneaud and 0. K. Behrens, Ergeb. Physiol. 41, 917 (1939). W. Grassmann and W. Heyde, Z. physiol. Chem. 183, 32 (1929).

94

ENZYMES OF PROTEIN METABOLISM

[10]

Reagents

Carnosine nitrate solution (0.125 M). Dissolve 362 rag. of the salt in 1.25 ml. of 1 N NaOH and bring to a volume of 10 ml. The compound is stable in the cold. 0.2 M Tris buffer at pH 8.0 or 0.1 M Veronal buffer at pH 8.0. 0.01 M 1V[nCl~ or MnS04. 0.01 M K O H in 90 % ethanol (standardized daily). 100% ethanol. 0.1% thymolphthalein in 95 % ethanol. Enzyme. Assayed most conveniently when diluted to obtain 5 to 15% hydrolysis in 15 minutes. Procedure. Extracts of spleen, kidney, and liver contain carnosinase, ~ but the preferred source is swine kidney 4,5 and most of the information has been obtained from the enzyme of this tissue. The enzyme is activated by both Zn ++ and Mn ++, but the stability of the enzyme is much greater with Mn ++. For optimal activation of crude extracts a preincubation with iVIn++ for 1 hour is necessary. To a 2.5-ml. volumetric flask, add 1 ml. of Tris (or Veronal) buffer, 0.1 ml. of MnC12, enzyme, and water to volume. After I hour at 40 °, add an aliquot of the preincubated enzyme solution (0.1 to 0.5 ml.) to a 2.5-ml. flask containing 1 ml. of substrate and 1 ml. of Tris (or Veronal) buffer; then add water to volume. After the contents are mixed, titrate 0.2-ml. aliquots for carboxyl groups with 0.01 M alcoholic K O H as will be described for prolidase. One hundred per cent hydrolysis corresponds to an increase in titer of 1.00 ml. of 0.01 M KOH. Specific Activity and Units. The hydrolysis of carnosine at 0.05 M follows the kinetics of a zero-order reaction to at least 80% of completion. The zero-order constant, K0, is calculated as per cent hydrolysis per minute. The proteolytic coefficient, Co, is given by K o / E , where E is given in milligrams of protein N per milliliter. One unit of activity is the amount of enzyme (in milligrams of protein N per milliliter) which will give a Ko of 1. Total units = Co X total E.

Purification Procedure Partial purification of carnosinase has been accomplished from swine kidney by Hanson and Smith. 5 s p. G. Garkovi, Bull. Biol. et todd. exptl. U.R.S.S. 4, 57 (1937); Chem. Abstr. 32, 8514 (1938). 4 H. T. Hanson and E. L. Smith, Federation Proc. 8, 204 (1949). 5 H. T. Hanson and E. L. Smith, J. Biol. Chem. 179, 789 (1949).

[10]

DIPEPTIDASES

95

Step 1. Extraction. Fresh frozen swine kidney (400 g.) is ground in a mechanical meat grinder before the tissue is completely thawed. The grinder is rinsed with 4 vol. of cold distilled water (1600 ml.) which is used to extract the enzyme. After 10 to 15 minutes, the mixture is centrifuged for 45 minutes at 2500 r.p.m. (This and subsequent steps are done in a cold room at 2 to 5° unless otherwise specified.) The upper fatty layer is then aspirated and discarded; the turbid enzyme solution is decanted from the insoluble residue which is discarded. Step 2. Heating and Ammonium Sulfate Precipitation. To the turbid solution (1300 ml.) from step 1 there is added 130 ml. of toluene, and the mixture is heated in a water bath at 55 ° for 15 minutes. The mixture is cooled to 0 °, and the solution is brought to 0.25 saturation with solid ammonium sulfate (144 g./1.). The precipitate is removed on fluted filter paper and discarded. The clear red filtrate (1100 ml.) is brought to 0.55 saturation with ammonium sulfate (192 g./1.), and the precipitate collected on fluted filter paper. The red filtrate is discarded. After thorough dialysis of the precipitate against distilled water, the fine suspension containing the enzyme is centrifuged. About a third of the activity is in the supernatant, and the remainder is in the precipitate. The residue is extracted with 20 ml. of 0.15 M sodium chloride and centrifuged; the insoluble, inactive material is discarded. The saline extract (solution A) is used for the next step. Step 3. Incubation with MnCl~. To solution A there are added sufficient solid MnC12 to make the solution 0.01 M with respect to Mn ++, 0.1 vol. of 0.1 M Veronal buffer at pH 8.0, and finally 0.1 M NaOH (added dropwise with stirring) to bring the pH to 7.9 to 8.1. The mixture is incubated at 40 ° for 1 hour, and the inactive precipitate is removed by centrifugation. The clear and almost colorless solution (solution B) contains all the activity. Comments on Purification. A summary of the purification is given in Table I. The absolute purification reached in solution B cannot be estimated because of the activation produced by Mn ++, but the specific activity is 14.7 times that of the initial extract assayed without Mn ++. TABLE I PURIFICATION OF CARNOSINASE (The values given are from H a n s o n a n d Smith 5 for a 400-g. b a t c h of swine kidney.) Step 1. Extraction 2. Heating and salt precipitation (solution A) 3. I n c u b a t i o n with MnCI~

Co

Total units

Yield, %

0.38 0.86 5.6

640 67 149

13

96

ENZYMES OF P R O T E I N METABOLISM

[10]

Solution A loses about 20 % of its activity in 8 days at 2 °. Under the same conditions, solution B is completely stable for m a n y weeks.

Properties

Specificity and Purity. Solution B contains no kidney carboxypeptidase (acylase), a trace of leucine aminopeptidase, and only small quantities of certain other peptidases, e.g., those which hydrolyze glycylglycine, L-prolylglycine, glycyl-L-proline, and triglycine, all at m u c h lower levels than carnosinase; none of these purified enzymes has a n y action on carnosine and certain other histidine-containing peptides (Table II). T h e TABLE II SPECIFICITY OF C•RNOSINASE a

Substrate ~-Alanyl-L-histidine #-Alanyl-D-histidine I,-Alanyl-L-histidine D-Alanyl-L-histidine Glycyl-L-histidine L-a-Aminobutyryl-L-histidine Glycyl-L-histidinamide ~-L-Aspartyl-L-histidine

Relative activity, % 100 1 150 89 115 13 19 1

Data of Hanson and Smith.~ enzyme has no action on the acyl peptide carbobenzoxy carnosine; hence a free amino group is essential. Hydrolysis of glycyl-L-histidinamide yields glycine and L-histidinamide. Because the enzyme is so much more active on dipeptides t h a n on glycylhistidinamide, it m a y be t e n t a t i v e l y classified as a dipeptidase rather than as an aminopeptidase. Activation, pH Optimum, Inhibition. The enzyme is activated b y M n ++ and b y Zn ++. The p H o p t i m u m with M n ++ is at 8.0 to 8.4, with Zn ++ a t p H 7.8 to 7.9, and without added metal at 7.4 to 7.5. The stability is greatest with M n ++ and much less so with Zn ++ or without added metal. The unactivated and Zn ++ enzymes are not inhibited b y citrate or phosphate, whereas with M n ++ 0.08 M citrate inhibits 80 % and 0.08 M phosphate more than 90%. The unactivated material is inhibited 80% b y 0.001 M sulfide, 50% b y 0.001 M cysteine, and 80% b y 0.01 M cyanide. Iodoacetate at 0.001 M has no effect. The enzyme appears to be a metalenzyme in the " u n a c t i v a t e d " state, and its behavior resembles the Zn ++ preparation rather than the Mn++-activated system.

[10]

DIPEPTIDASES

97

B. Iminodipeptidase (Prolinase) 6 Assay Method P r i n c i p l e . This enzyme hydrolyzes a v a r i e t y of dipeptides which contain N - t e r m i n a l L-proline or hydroxy-L-proline, typical substrates being L-prolylglycine and hydroxy-L-prolylglycine; the synthesis of the latter is given in Vol. I I I . L-Prolylglycine m a y be synthesized b y the procedure of Abderhalden and Nienburg. 11 T h e assay is based on the titration of liberated carboxyl groups with alcoholic K O H b y the m e t h o d of Grassm a n n and Heyde.-' Reagents

Hydroxy-L-prolylglycine (HG) or L-prolylglycine solution (PG) (0.125 M). Dissolve 235 mg. of H G or 215 mg. of P G in 4.2 ml. of 0.10 N N a O H , and bring to a volume of 10 ml. in a volumetric flask b y addition of water. These stock solutions m a y be k e p t for only a few days at 2 °, since diketopiperazine f o r m a t i o n will occur spontaneously in aqueous solutions near neutrality. 0.2 M Tris buffer at p H 8.0. 0.2 M Tris buffer at p H 8.5. 0.01 M MnC12 or MnS04. 0.01 M K O H in 90% ethanol, which m u s t be standardized daily. 100% ethanol. 0.1% t h y m o l p h t h a l e i n in 9 5 % ethanol. E n z y m e . T h e stock solution should be diluted to achieve 10 to 15% hydrolysis in 15 minutes. Procedure. I n crude extracts of kidney or other tissues, this enzyme is optimally active at a b o u t p H 8.0. Partially purified preparations show negligible activity at p H 6 and steeply increasing activity with increasing p H up to at least p H 9.2. Since the enzyme is extremely unstable a t the

6 Grassmann el a!J ascribed the hydrolysis of prolylglycine and prolylglycylglycine to the same enzyme which they called prolinase. It was later shown that the tripeptide is hydrolyzed by aminotripeptidase 8,9 and that purified preparations of the enzyme which hydrolyzes prolylglycine have no activity toward the tripeptide. 10. The general name, iminodipeptidase, suggests the known specificity more correctly. 7 W. Grassmann, H. Dyckerhoff, and O. yon Schoenebeck, Bet. 62~ 1307 (1929); W. Grassmann, O. yon Schoenebeck, and G. Auerbach, Z. physiol. Chem. 210, 1 (1932). 8 M. J. Johnson, J. Biol. Chem. 122, 89 (1937-38). 9 E. Adams, N. C. Davis, and E. L. Smith, J. Biol. Chem. 199, 845 (1952). 10N. C. Davis and E. L. Smith, J. Biol. Chem. 200, 373 (1953). 11E. Abderhalden and H. Nienburg, Fermentforsehung 13, 573 (1933).

98

ENZYMES OF PROTEIN METABOLISM

[10]

higher pH values, however, pH 8.5 has been chosen as a suitable value at which to conduct assays of the purified samples. To a 2.5-ml. volumetric flask, add 1.0 ml. of Tris buffer, 1.0 ml. of substrate solution, 0.25 ml. of 0.01 M MnC12 or MnS04, 0.1 to 0.25 ml. of enzyme solution, and water, if needed, to bring to volume. Mix the contents of the flask, and remove 0.2-ml. aliquots at zero time and subsequently. Titrate each sample immediately with the alcoholic KOH as descrlbed for prolidase (pp. 100-101). The difference in titer between the initial sample and those removed later gives the quantity of liberated carboxyl. Specific Activity and Units. The hydrolysis of the substrates given above is zero order up to 60 to 80% of completion. 1°,1~ The proteolytic coefficient (Co) is defined as the per cent hydrolysis per minute per milligram of protein nitrogen per milliliter. The number of units of activity is given by the Co multiplied by the total milligrams of protein N in the fraction. Purification Procedure The method is that of Davis and Smith, 1° and its success depends on the stabilization of the enzyme by Mn ++. It should be noted that the same initial procedure is used for the isolation from swine kidney of prolidase and leucine aminopeptidase, as well as the iminodipeptidase. Step 1. Preparation of Acetone Powder. Fresh frozen swine kidney (2.5 kg.) is coarsely ground in the cold in a meat grinder. (An assay of the water-soluble fraction at this stage gives Co = 14.) This material is blended in portions in a Waring blendor for 1 minute with an equal volume of 53.3% ethanol at - 5 ° . The alcohol concentration is then raised to approximately 40 % by the addition of 1 I. of 95 % ethanol at - 5 °. The mixture is centrifuged for 1 hour at 2500 r.p.m., and the soluble fraction is discarded or saved for the isolation of prolidase. The insoluble fraction is then washed at --5 ° successively with absolute ethanol, acetone, and ether; the powder is then air-dried in the usual manner. The yield is 500 g. of a tan-colored powder which retains its activity for weeks at 5 °. An extract of the powder is made by adding 20 vol. of water at room temperature and adjusting the pH to 7.8 with 0.1 M sodium hydroxide, and centrifuged or filtered through Hyflo Super-Cel. It will be noted (Table III) that no increase in specific activity is accomplished by this step; however, it is essential for the subsequent steps which do not work on crude aqueous extracts of kidney. 12R. E. Neuman and E. L. Smith, J. Biol. Chem. 193, 97 (1951).

[10]

DIPEPTIDASES

99

TABLE III PURIFICATION OF IMINODIPEPTIDASE

Step Crude extract a 1. Acetone powder 2. First alcohol step 3. Second alcohol step 4. Third alcohol step

Co

Weight of powder, g.

Yield, units

Yield, %

14b 14b 59b 108b 394c

500 10 2.5 0.63

175,000 91,000 25,000 17,000 14,000

52 14 9.7 8.0

Total units are based on a batch of 2.5 kg. b Assayed at pH 8.0 with L-prolylglycine or hydroxy-L-prolylglycine. c Assayed at pH 8.5 with i,-prolylglycine; with hydroxy-L-prolylglycine, Co ~ 570 at this pH.

Step 2. First Alcohol Precipitation. The clear filtrate is cooled to 2 ° and adjusted to a concentration of 50% ethanol b y the slow addition of an equal volume of absolute ethanol at - 1 0 °, and to 0.02 M MnC12 b y the addition of the solid salt. After standing overnight in the cold, the resulting precipitate is collected b y centrifugation, dissolved in water, frozen, and dried in vacuo. The powder is extracted with 60 vol. of 0.02 M MnC12 at p H 7.8 for 30 minutes and the preparation filtered through Hyflo Super-Cel. Step 3. Second Alcohol Precipitation. The clear filtrate is cooled in an alcohol-dry ice b a t h while absolute ethanol is slowly added at - 3 0 ° to bring the alcohol concentration to 30%. After 1 hour, the precipitate is removed b y centrifugation in the cold and discarded. The clear a m b e r fluid is frozen and lyophilized to yield a powder which is extracted with 10 vol. of 0.02 M MnCl~ a t 0 ° and at p H 7.8 (adjusted with N a O H ) and is centrifuged. Step 4. Third Alcohol Precipitation. T h e solution is chilled, and an equal volume of 95 % ethanol precooled to - 2 0 ° is added. After 30 minutes, the precipitate is collected b y centrifugation and is washed once in the centrifuge bottle with cold 48 % ethanol. T h e precipitate is dissolved in water, frozen, and lyophilized. Properties Purity. T h e absolute p u r i t y of the iminodipeptidase is unknown. T h e final product of the purification scheme described is free of tripeptidase and of leucine aminopeptidase b u t still contains some prolidase and a highly active glycylglycine dipeptidase (see pp. 107-109). Specificity. The enzyme appears to be a specific dipeptidase. I° T h e r e is no action on compounds in which the terminal imino group is acylated

100

ENZYMES OF PROTEIN METABOLISM

[10]

or on the following tripeptides: L-prolylglycylglycine, hydroxy-L-prolylglycylglycine, and glycyl-L-prolylglycine. The amides of L-proline and hydroxy-L-proline are not hydrolyzed. All the known substrates are dipeptides which contain N-terminal i-proline or hydroxy-L-proline. The nature of the C-terminal residue has only a minor influence on rate with the exception of compounds which contain terminal glutamic or aspartic acids; these are completely resistant. Activators. In crude extracts of kidney, the hydrolysis of specific substrafes is activated by Mn ++ and by Cd++. 12 Because the enzyme is specifically stabilized by Mn ++, this ion is added during the purification. 1° Inhibition of the enzyme in crude extracts is produced by the usual agents which bind Mn++: pyrophosphate, citrate, phosphate, and fluoride. 12 C. Prolidase (Imidodipeptidase)

Assay Method Principle. The most useful substrate for the assay of this peptidase is glycyl-L-proline,13 and the assay is based on the measurement of carboxyl groups liberated during the hydrolysis of the peptide by titration with KOH in 90% ethanol by the method of Grassmann and Heyde. 2 The synthesis of glycyl-L-proline is given in Vol. III.

Reagents Glycyl-L-proline stock solution (0.125 M). A stock solution of glycyl-L-proline'l/6H~O may be made up and kept for only a few days at refrigerator temperature (5°), since cyclization to form the diketopiperazine will occur slowly in aqueous solution. 14Transfer the substrate (226 rag.) into a 10-ml. volumetric flask, add 4.2 ml. of 0.1 N NaOH, and dilute to volume with water. 0.2 M Tris buffer at pH 8.0. 0.1 M N[nSO4 or 0.1 M MnC12. 0.01 M KOH in 90% ethanol which must be standardized daily against 0.02 N potassium biniodate with phenolphthalein as indicator. 100 % ethanol. 0.1% thymolphthalein in 95% ethanol. Enzyme. Dilute the enzyme preparation to achieve about 5 to 15 % hydrolysis in 15 minutes.

Procedure. Crude and partially purified enzyme preparations require a prior incubation with 1V[n++ to obtain maximal activation. To a 2.5-ml. la M. B e r g m a n n a n d J. S. Fruton, J. Biol. Chem. 117, 189 (1937). 14 E. L. S m i t h a n d M. Bergmann, J. Biol. Chem. 158~ 627 (1944).

[10]

DIPEPTIDASES

101

volumetric flask add 1 ml. of Tris buffer, 0.5 ml. of 0.1 M MnS04, and 0.2 to 1.0 ml. of enzyme solution; add water to bring to volume. Mix the contents of the flask and allow to incubate in a water bath at 40 ° for 30 to 60 minutes. For the assay, to a 2.5-ml. volumetric flask add 1.0 ml. of glycylproline solution (0.125 M), 1.0 ml. of Tris buffer, and incubate to equilibrate at 40 °. Add 0.1 to 0.5 ml. of preincubated enzyme solution, and water to bring to volume, if necessary. Mix the contents rapidly, and remove samples (0.2 ml.) at zero time and subsequently; titrate each sample immediately with standardized alcoholic KOH which is near 0.01 M to the first blue end point with thymolphthalein (2 drops); add 1.8 ml. of 100% ethanol, and complete the titration with the alcoholic KOH. The alkali may be conveniently added from a 2 ml. microburet. The difference in titer of the initial sample and those measured later gives the amount of liberated carboxyl. Specific Activity and Units. The hydrolysis of glycylproline at the given concentration in the test solution (0.05 M) is first order. TM The first-order rate constant (K1) is calculated in minutes and decimal logarithms. The specific activity is expressed by the proteolytic coefficient C1, where C1 = K~/E. E is the protein N in milligrams per milliliter. Units are calculated by multiplying C~ by total E in milligrams; hence one unit of enzyme is the amount of protein N necessary to give a K~ of 1.0. Assay of Enzyme in Tissue Extracts. Prolidase appears to be freely soluble in water and stable in the Waring blendor. As far as is known, no other enzyme of animal tissues appears to be capable of hydrolyzing glycylproline; hence, this substrate may be used for assay of the enzyme in crude extracts of cells and tissues. It is usually desirable to remove water-insoluble material and to perform the assay after preincubation with Mn ++ as described above. Purification Procedure This enzyme has now been obtained free of detectable amounts of peptidases which attack peptide bonds of normal character in which the linkage is - - C O N H - - . The two sources from which such preparations have been obtained are equine erythrocytes15 and, in a higher state of absolute purity, swine kidney. I6,17 Since the method of preparation from erythrocytes is exceedingly simple and yields material which is adequate for many purposes, the methods are described for both preparations. The is E. Adams and E. L. Smith, J. Biol. Chem. 198, 671 (1952). 16N. C. Davis and E. L. Smith, FederationProc. 12, 193 (1953). i7 N. C. Davis and E. L. Smith, unpublished studies.

102

ENZYMES OF PROTEIN METABOLISM

[10]

enzyme has also been partially purified from swine intestinal mucbsa by Smith and Bergmann. 14 From Equine ]~rythrocytes. The procedure is that of Adams and Smith ~ and depends on the observation that the Tsuchihashi procedure for the denaturation of hemoglobin, as described by Keilin and Mann, is does not destroy prolidase but inactivates or precipitates all the other peptidases in the hemolyzate. The method has usually been applied to batches of about 10 1. of whole blood and should be performed in a cold room at 2 to 5° . Step 1. Blood is collected from freshly killed animals, and clotting is prevented by the addition of 5 g. of sodium citrate per liter of blood. After the blood is stored in a cold room or refrigerator for 12 to 24 hours to permit sedimentation of the cells, the citrated plasma and buffy coat are siphoned off and discarded. The packed erythrocytes are then hemolyzed by the addition of an equal volume of cold distilled water. Step 2 should be started early in the morning and should be carried through in one day. Step 2. To each liter of hemolyzate are added 900 ml. of 90 % ethanol and 65 ml. of chloroform, both solvents precooled to - 3 0 °. The mixture is rapidly stirred with a glass or stainless steel paddle or with heavy rods in order to produce prompt coagulation of hemoglobin into a spongy mass from which the residual liquid can be first decanted and then expressed. After filtration through Celite, the clear faintly yellowish solution is adjusted from pH 6.9 to pH 6.0 by the addition of 1 M HC1; the moderately heavy precipitate contains negligible activity. (The pH is determined in a glass electrode after dilution of the samples with 9 vol. of water.) This precipitate is removed by filtration on a Btichner funnel precoated with Celite. To the filtrate an equal volume of acetone precooled to - 3 0 ° is added, and the heavy flocculent precipitate is allowed to settle. After most of the supernatant fluid is siphoned off, the precipitate is collected on a filter pad precoated with Celite, washed successively with acetone and ether, and rapidly air-dried. The dried powder is stable for many weeks. Step 3. The acetone-dried powder containing Celite is dissolved in water and clarified by filtration. The protein concentration should be 0.5 to 1% as estimated turbidimetrically or by refractive index. Solid ammonium sulfate (39 g. per 100 ml. of solution) is added to 0.6 saturation. The precipitate is collected by centrifugation and is dialyzed against cold distilled water. The solution may be lyophilized and kept for months in the refrigerator with no loss in activity. A representative run is given in Table IV. Yields have varied between 18 D. Keilin a n d T. M a n n , Biochem. J . 34, 1163 (1940).

[10]

DIPEPTIDASES

103

TABLE IV PURIFICATION OF ERYTHROCYTEENZYME Step 1. Crude hemolyzate 2. Acetone powder 3. Ammonium sulfate precipitate

C1

Total units

Yield~ %

0.0012 0.18 0.39

336 55 13

100 16 4

8 and 13 % of the initial activity. The C1 has varied between 0.1 and 0.5. An occasional failure may result when the coagulation of hemoglobin is incomplete, as indicated by a finely dispersed coagulum and turbid highly colored filtrates instead of large spongy masses of denatured hemoglobin and clear filtrates. From Swine Kidney. This procedure is that of Davis and Smith 1~,~7 and incorporates the chloroform treatment used for the erythrocyte enzyme followed by fractionation with acetone and ammonium sulfate. For the steps given, the over-all purification is 2000 to 3000 times and yields a preparation with a C~ = 20 to 30. The enzyme from this source has been obtained with CI = 120 to 130, but the steps cannot always be performed in the same manner and, therefore, are not given here. An aqueous extract of swine kidney gives a CI = 0.01. Step I. Preparation of Acetone Powder. Fresh frozen swine kidney (10 kg.) is ground in a meat grinder. The ground tissue is treated in portions with an equal volume (10 1.) of 53.3% ethanol at - 5 ° in a Waring blendor. Ethanol (4 1., 95%) precooled to - 2 0 ° is added to make the final ethanol concentration approximately 40%, and chloroform (65 ml./ kg. of tissue) is added. The precipitate is removed in portions by centrifugation at 0 to 5° and may be saved for the preparation of other peptidases (see leucine aminopeptidase and iminodipeptidase). To the clarified mother liquor, acetone at --20 ° (250 ml./1.) is added. The precipitate is allowed to form for 1 hour and is then removed by centrifugation. To the clear, yellow centrifugate more acetone at - 2 0 ° is added (750 ml./1.), and the mixture is allowed to settle overnight. The clear supernatant is siphoned off and discarded; the precipitate is collected by centrifugation, washed on to a Biichner funnel with acetone, washed with several portions of acetone, with ether, and air-dried. The yield is about 30 to 40 g. of a light tan powder. Step 2. Treatment with Mn ++, Ammonium Sulfate, and Acetone. The dried powder from step 1 is extracted with 0.02 M MnC12 (25 ml./g, of powder) at 40 ° for 1 hour. The mixture is cooled in an ice bath, and the precipitate is removed by centrifugation. Solid ammonium sulfate is added to bring the solution to 0.5 saturation (31.2 g. per 100 ml.). After

104

ENZYMES OF PROTEIN METABOLISM

[10]

1 hour at 2 °, the precipitate is collected b y centrifugation, dissolved "in the minimal a m o u n t of water and dialyzed free of sulfate. T o the dialyzed solution of the enzyme, solid !V[nC12 is added to give a 0.02 M concentration which lowers the p H to 5.4 to 5.6. T h e solution is incubated at 40 ° for 20 minutes. T h e inactive precipitate is r e m o v e d b y centrifugation and discarded. T h e solution is cooled to 0 ° in an alcohol-dry ice bath, and the t e m p e r a t u r e is gradually lowered to a b o u t - 1 0 ° as acetone precooled to - 2 0 ° is added to the solution in a ratio of 30 ml. of acetone for each 70 ml. After 1 hour the precipitate is collected b y centrifugation, suspended in a small a m o u n t of water, frozen in an alcohol-dry ice bath, and lyophilized. T h e purification is s u m m a r i z e d in T a b l e V. TABLE V PURIFICATION OF KIDNEY PROLIDASE

(Figures are given for 10 kg. of fresh frozen kidney.) Step Crude aqueous extract 1. Acetone powder ~ 2. Lyophilized powder

C1

Weight, g.

Yield, %

0.01 2.0-3.0 20-30

30-40 1.0-1.3

60-70 ~'

An aqueous extract of this powder containing 0.02 M MnC12 is assayed. b From the acetone powder. Approximately 50 to 80% of the prolidase originally present in the kidney is obtained in the acetone powder. Properties

Specificity. Prolidase is an imidodipeptidase; i.e., it hydrolyzes only dipeptides in which the susceptible linkage lacks a peptide hydrogen. 15.17,19 Typical substrates are glycyl-L-proline, L-prolyl-L-proline, glycylhydroxyL-proline, glycylallohydroxy-L-proline, L-phenylalanylhydroxy-L-proline, glycylsarcosine, etc. I n general, the action is on dipeptides of the following kinds : NH2

C H 2 - - C H [OH]

I

HC--C--N

t

R

H

O

/ \

NH2 and

CH---CH2

J

I

HC--C--N--CH~COOH

I

R

II

O

I

CH~

COOH The e n z y m e has no action on dipeptides which h a v e the normal peptide bond - - C O N H - - or on acyl dipeptides such as carbobenzoxyglycyl19E. L. Smith, N. C. Davis, E. Adams, and D. H. Spackman, in "The Mechanism of Enzyme Action" (W. D. McElvoy and B. Glass, eds.), Johns Hopkins Press, Baltimore, 1954.

[10]

DIPEPTIDASES

105

L-proline, on the tripeptides glycylglycyl-L-proline and glycyl-L-prolylglycine, and on the dehydropeptide dehydrophenylalanyl-L-proline. pH Optimum. The optimal action of the enzyme from muscle, erythrocytes, or kidney is at pH 7.8 to 8.0. The activity falls smoothly on both sides of the optimum, being about 20% as active at pH 6.5 and 9.0. Activators and Inhibitors. Mn ++ is the only known metal activator and should be present in the final assay solution at a level of 0.001 to 0.005 M. Other metal ions, Mg ++, Co++, Fe ++, are inactive or are inhibitory, e.g., Zn++, Hg ++, Pb ++, Cd ++, etc. Metal-binding agents are inhibitory. The erythrocyte enzyme is inhilJited more than 99 % by 0.001 M pyrophosphate and by 0.01 M Versene; citrate (0.01 M) and fluoride (0.01 M) cause 30 to 50% inhibition. In the absence of Mn ++, iodoacetamide and p-chloromercuribenzoate are strong inhibitors. 16,17 Mn ++ protects against the iodoacetamide inhibition, which suggests that Mn ++ may be bound to an SH group of the protein. 19 Mechanism of Action. It has been suggested that the metal (Mu ++) forms chelate compounds with both protein and substrate. A detailed discussion is given by Smith et al. 19 Turnover Number. For the best preparation thus far obtained, C = 130; the estimated turnover number is approximately 100,000 moles of substrate per minute per 100,000 g. of enzyme. Distribution. Prolidase has been found in all animal tissues examined, e.g., intestinal mucosa, skeletal and smooth muscle, erythrocytes, serum, pituitary, lung, and kidney.

D. Glycyl-L-leucine Dipeptidase Assay Method Principle. Enzymes from certain animal tissues which hydrolyze glycyl-L-leucine show properties which appear to distinguish this activity from other known peptidases. 2° Assays are conducted by the GrassmannHeyde 2 microtitration method for liberated carbo×yl groups. The sinthesis of glycyl-L-leucine is described in Vol. III. Reagents Glycyl-L-leucine solution (0.125 M). Dissolve 235 mg. of finely pulverized substrate in 4.2 ml. of 0.1 M NaOH and dilute to 10 ml. with water. 0.01 M ZnC12 for the uterine enzyme. 0.25 M MnC12 or 0.25 M MnS04 for the intestinal enzyme. 0.3 M phosphate buffer at pH 7.8. 2°E. L. Smith, J. Biol. Chem. 176, 9 (1948).

106

ENZYMES OF PROTEIN METABOLISM

[10]

0.01 M KOH in 90 % ethanol. 100 % ethanol. 0.1% thymolphthalein in 95 % ethanol. Enzyme solution. May be assayed at levels which give as little as 5% hydrolysis per hour or as high as 15% hydrolysis in 10 minutes. The two sources of the enzyme considered below are human uterus and swine intestinal mucosa.

Procedure. For assay of the uterine enzyme, to a 2.5-ml. volumetric flask add 1.0 ml. of glycyl-L-leucine stock solution, 1.0 ml. of buffer, and 0.25 ml. ZnC12, and incubate at 40 ° for l0 minutes. Then add 0.1 to 0.25 ml. of enzyme solution and water to volume, if needed. Samples of 0.2 ml. are titrated by the technique of Grassmann and Heyde described for prolidase (pp. 100-101). For assay of the intestinal enzyme, preincubation of 1V[n++ and enzyme are necessary to obtain full activation. To a 2.5-mi. volumetric flask add 1.0 ml. of buffer, 0.5 ml. of 0.25 M h/[nCl~, an aliquot of enzyme, and water to volume. Incubate at 40 ° for 1 to 2 hours. Then to a prepared flask (2.5 ml.) equilibrated at 40 ° containing 1.0 ml. of substrate and 1.0 ml. buffer add 0.5 ml. of preincubated enzyme. This gives a final concentration of 0.01 M Mn ++ which is optimal for this enzyme. Aliquots are titrated as given for prolidase (pp. 100-101). Specific Activity. For both enzymes, the hydrolysis follows first-order kinetics over a wide range of enzyme concentration. The first order constant (K1) is calculated in minutes and decimal logarithms. C1, the specific activity, is given by C1 = K1/E, where E is the protein N in milligrams per milliliter. Units may be defined as given for prolidase (p. 101).

Purification Procedure Neither of the two enzymes has been purified to any extent. A stable preparation of the intestinal enzyme may be obtained from a crude aqueous extract of the mucosa by precipitation between 0.4 and 0.8 saturation with ammonium sulfate, followed by dialysis. A stable preparation of the uterine enzyme may be obtained from a crude aqueous extract of the tissue by precipitation with 2 vol. of cold acetone and the preparation of an acetone-dried powder. A clarified aqueous extract of this powder at pH 7.5 to 8.0 is stable and highly active.

Properties Although the enzymes from either source given have not been extensively purified, there is evidence that they differ from the other known

[10]

DIPEPTIDASES

107

peptidases as judged by stability, activation behavior, and other properties. Nevertheless, it should be noted that Robinson et al. ~1 (see Vol. II [11]) have obtained from swine kidney an enzyme which hydrolyzes glycyl-L-leucine and many other dipeptides, so that not all activities toward this particular substrate necessarily possess the same specificity. One specific feature may be mentioned here. The enzyme obtained by Robinson et al. 21 possesses almost equal activity toward glycyl-L-leucine and its D-isomer. The enzymes of swine intestinal mucosa and human uterine tissue are optically specific; there is no action on the D-isomer (E. L. Smith, unpublished observations). Activators and Inhibitors. The uterine enzyme manifests maximal activity with Zn ++ and phosphate. In the absence of phosphate, Ca ++ is inhibitory, and Zn++ may be also. A crude intestinal extract shows no activation effect. After precipitation by ammonium sulfate and dialysis, the enzymatic activity is increased more than 25-fold by Mn ++. The course of activation follows the mass law equation with an apparent dissociation constant of 1.8 X 10-4 M for the metal-protein complex. Specificity. Neither enzyme is able to attack carbobenzoxy glycylL-leucine, carbobenzoxyglycyl-L-leucinamide, or glycyl-L-leucinamide, hence the classification as a dipeptidase. Sarcosyl-L-leucine is slowly hydrolyzed by the dipeptidase. 2°

E. Glycylglycine Dipeptidase Assay Method Principle. Extracts of various animal tissues hydrolyze the simple dipeptide glycylglycine. In some tissues indirect evidence of several kinds indicates the presence of a specific enzyme with an action only on the aforementioned dipeptide and on sarcosylglycine. The activity is strongly enhanced by Co ++ and more weakly by Mn ++. The enzyme is assayed by titration of liberated carboxyl groups by the method of Grassmann and Heyde 2 or by other micromethods for estimation of an increase in amino groups such as a colorimetric ninhydrin method. Glycylglycine and its hydrochloride are commercially available. Reagents

Glycylglycine solution (0.125 M). Dissolve 165 mg. in 4.2 ml. of 0.1 M NaOH and dilute to 10 ml. with water. 0.1 M Veronal or 0.2 M Tris buffer at pH 7.6. 0.01 M COC12. 0.01 M KOH in 90% ethanol. ~ D. S. Robinson, S. M. Birnbaum, and J. P. Greenstein, J. Biol. Chem. 202, 1 (1953).

108

ENZYMES OF PROTEIN METABOLISM

[10]

100 % ethanol. 0.1% thymolphthalein in 95 % ethanol. Enzyme. Dilute to obtain 5 to 15 % hydrolysis in 15 minutes.

Procedure. To a 2.5-ml. volumetric flask add 1.0 ml. of substrate solution, 1.0 ml. of buffer, and 0.25 ml. of 0.01 M CoCI~, and incubate at 40 ° for 10 minutes. Then add 0.1 to 0.25 ml. of enzyme solution and water to volume, if necessary. Titrate samples of 0.2 ml. by the Grassmann-Heyde technique as described for prolidase (pp. 100-101). Specific Activity and Units. With glycylglycine at 0.05 M, the hydrolysis up to 70 to 80% of completion follows zero-order kinetics. 22 The rate constant, K0, is calculated in per cent hydrolysis per minute. The specific activity is given as Co, where Co = Ko/E; E is the enzyme concentration as protein N in milligrams per milliliter. Sources of Enzyme and Purification. All animal tissues which have been investigated thus far contain activities which hydrolyze glycylglycine and show strong activation by Co++. 22-~4 This is also shown by some plants and microorganisms. 25 It has not been established, however, that all these activities are due to the same enzyme. For the enzymes of rat muscle and human uterus, the enzyme appears to be highly specific as judged by differential inactivation and by the effect of Co ++ and other divalent metals, i.e., the activity toward glycylglycine does not parallel those toward other simple optically active L-peptides: alanylglycine, glycylalanine, leucylglycine, glycylleucine, and others. 22,23 It should be noted, however, that Robinson et al. 21 have obtained a purified preparation from the particulate fraction of swine kidney (see Vol. II [11]) which exhibits high activity toward glycylglycine, many other dipeptides, and dehydropeptides, and weaker activity toward tripeptides and amino acid amides; this enzyme appears to differ in many respects from the glycylglycine-hydrolyzingenzymes of muscle. Rat Muscle Enzyme. This enzyme may be obtained in solution by dispersing the muscle tissue at 0 ° with 5 vol. of cold water in a Waring blendor. The extracts are centrifuged and adjusted to pH 7.5 with 0.1 N Na0H. The preparation must be used immediately; even within a few hours some activity is lost. Human Uterine Enzyme. This enzyme is extracted in the same manner as the rat muscle enzyme, and the clarified extract is treated with 2 vol. of cold acetone. The precipitate is collected and washed successively with acetone and ether and air-dried in the usual manner. The enzyme ~ E. L. 2~ E. L. 24 E. L. 25 ~YI. J.

Smith, J. Biol. Chem. 173,571 (1948). Smith, J. Biol. Chem. 176, 21 (1948). Smith, Advances in Enzymol. 12, 191 (1951). J o h n s o n a n d J. Berger, Advances in Enzymol. 2, 69 (1942).

[11]

DEHYDROPEPTIDASES FROM KIDNEY

I09

is stable for months in the dried powder. An aqueous extract of the powder is adjusted to pH 7.5 and used immediately. Swine Kidney Enzyme. Crude preparations of the first alcohol-precipitated fraction of iminodipeptidase (see pp. 97-100) have a high activity toward glycylglycine; however, it is unknown whether this is the same enzyme obtained by Robinson et al. ~ from particulate fractions.

Properties of the Muscle and Uterine Enzymes The rat muscle enzyme is exceedingly unstable, and most of the activity is lost in 24 hours at 2 ° . The uterine enzyme is more stable. The pH optimum for both enzymes is at pH 7.6. The kinetics at 0.05 M substrate are zero order. Optimal activation is obtained at 10-3 M Co++; for the rat muscle enzyme, 10- to 50-fold activation m a y be found. Ka, the dissociation constant of the hypothetical metal-enzyme complex, is 2.8 × 10-5 M at 40 °, as determined by the effect of Co ++ concentration on activity. Mn ++ shows a weaker activation. Mg ++ has no effect on the enzyme, and Zn ++ is a strong inhibitor. Specificity. These enzymes hydrolyze sarcosylglycine, as well as glycylglycine, but not N-dimethylglycylglycine, diglycylglycine, N-acyl substituted glycylglycine, glycylglycinamide, or fl-alanyl dipeptides. The classification as a dipeptidase is based on these findings. 22 It has been postulated t h a t Co ++ acts as a bridge in forming chelate complexes between the specific protein and the substrate. 22-24

[11] Dehydropeptidases from K i d n e y Soluble Acylase I and Solubilized Aminopeptidase X C H R C O N H C ( = C H R ' ) C O O H -{- 2H~O --~ X C H R C O O H ~- NH3 -t- R ' C H s C ( = O ) C O O H (where X = H, C1, or NH2, R = H or alkyl, R ' = H, alkyl, or aryl)

By JESSE P. GREENSTEIN Assay Method Principle. Dehydropeptidase II (soluble acylase I) acts on acetyl, chloroacetyl, and glycyldehydroalanine, whereas dehydropeptidase I (insoluble aminopeptidase) acts on all dehydropeptides containing a free NH2 group on the acyl residue. 1,2 Chloroacetyldehydroalanine is there1 K. R. Rao, S. M. Birnbaum, and J. P. Greenstein, J. Biol. Chem. 203, 1 (1953). D. S. Robinson, S. M. Birnbaum, and J. P. Greenstein, J. Biol. Chem. 202, 1 (1953).

110

ENZYMES OF PROTEIN METABOLISM

[11]

fore the specific dehydropeptide substrate of choice for the former enzyme, glycyldehydroleucine for the latter. Although peptides of dehydroalanine possess a specific absorption band at 240 m~ and their hydrolysis can be followed by the rate of decrease in the density of this band, 8 the more general method of measuring ammonia is preferable, since it is applicable to all dehydropeptide substrates. 3

Reagents Preparation of Substrates. (a) Chloroacetyldehydroalanine:2 7.6 g. of redistilled chloroacetonitrile is mixed with 10.6 g. of freshly distilled pyruvic acid, the mixture chilled, and saturated with dry HC1 gas. After standing for 1 hour at 5° the mixture solidifies to a mass of crystals. After 12 hours of further standing, the mass is suspended in dry ether, filtered, washed with dry ether, and recrystallized from acetone. The yield is 14 g. or 86% of the theoretical. (b) Glycyldehydroleucine:4 7.6 g. of redistilled chloroacetonitrile is mixed with 22 g. of freshly prepared a-ketoisocaproic acid (Vol. I I I [65]), the mixture chilled, and saturated with dry HC1 gas. After standing for 3 to 5 days at 5 °, the mixture solidifies to a yellow-brown, crystalline mass. It is washed several times with petroleum ether and once with ether. On solution in the minimum amount of warm acetone followed by addition of petroleum ether, the product crystallizes in long prisms. There is generally considerable contamination by chloroacetamide, and the chloroacetyldehydroleucine is therefore directly aminated by dissolving in the twentyfold amount of ammonia water which had been saturated at 0 °. After standing for 4 days at 25 °, the solvent is removed, the residual mass washed several times with 95% alcohol, dissolved in the minimum amount of water, and crystallized by addition of alcohol to 80 %. The glycyldehydroleucine is recrystallized in the same way several times or until free of NH4 +. The yield is 20 to 30% of the theoretical. Substrate stock solutions (0.025 M). Dissolve 102.5 rag. of chloroacetyldehydroalanine in 12 ml. of H20, and add a drop of phenol red indicator, followed by 0.1 N NaOH to pH 7.0. Water is then added to 25 ml. final volume. Dissolve 116.3 rag. of glycyldehydroleucine in 20 ml. of water, followed by added water to 25 ml. final volume. The solutions are stored at 5 ° when not in use, and are made up freshly every week. 0.1 M sodium borate buffer at pH 8.0. 0.1 M phosphate buffer at pH 7.0. Saturated K2C03 solution. 3 V. E. Price and J. P. Greenstein, J. Biol. Chem. 171, 477 (1947). 4 A. Meister and J. P. Greenstein, J. Biol. Chem. 195, 849 (1952).

[11]

DEHYDROPEPTIDASES FROM KIDNEY

111

2% H2SO4 Nessler's reagent. Enzyme. Samples of the lyophilized preparations are weighed and dissolved in water.

Procedure. One milliliter of the substrate solution is mixed with I ml. of buffer and 1 ml. of either water or enzyme solution. Phosphate buffer at pH 7.0 is used with chloroacet:~ldehydroalanine, borate buffer at pH 8.0 with glycyldehydroleucine. The mixtures are incubated at 38 ° for varying lengths of time, removed, and treated with 1 ml. of saturated K2C03 solution. The ammonia evolved is absorbed into tubes containing 5 ml. of 2% H2S04 by attachment of the latter to a vacuum line. ~ The air drawn through the train is rendered ammonia-free by prepassing it through a sulfuric acid trap. The period of aeration is 45 minutes, at the expiration of which time the inlet tubes into the acid traps are washed, the combined acid and washings are brought to 20 ml., and 5 ml. of Nessler's reagent is added. The color developed is compared at 500 mtt with standards prepared from a known amount of ammonium sulfate solution in the spectrophotometer. Complete hydrolysis of the substrates yields 350 ~, of NH3-N. Experimental conditions are so chosen that one-tenth to onethird of the substrate is hydrolyzed, i.e., where the hydrolysis is linear with time of incubation. Definition of Unit and Specific Activity. One unit of enzyme is defined as the amount which will hydrolyze 1 micromole of substrate per hour under the above conditions. Specific activity is expressed as units per milligram of protein N. The latter is determined by prolonged digestion in concentrated H2SO~ using 30% H202 as catalyst, nesslerizing the ammonia produced, and determining the intensity of the Nessler color as above. Application of Assay Method to Crude Tissue Preparations. In order to reduce the ammonia blank in crude homogenates or extracts of tissues it is advisable to subject them to a 10- to 15-hour period of dialysis at 5° against frequent changes of distilled water. The procedure employed is similar to that with the purified enzymes except that in the ammonia aeration step a few drops of caprylic alcohol are added to the alkalinized protein solutions in order to reduce the possibility of foaming. Traces of caprylic alcohol carried over into the acid traps interfere with the development of Nessler color and are therefore removed either by boiling the acid solutions for a few minutes, or by letting them stand at room temperature for 20 hours. In any event, an extra control is invariably run on the ammonia content of the tissue preparation in the absence of substrate, 5 C. E. Carter and J. P. Greenstein, J. Natl. Cancer Inst. 7, 51 (1946).

112

ENZYMES OF PROTEIN METABOLISM

[11]

and the test run corrected for this value. In carefully prepared materials, and with freshly prepared substrates, both tissue and substrate spontaneous evolution of ammonia is quite negligible. Purification Procedure The preparation of the soluble renal acylase I by Birnbaum is described elsewhere (Vol. II [12]). The following preparation of renal aminopeptidase, solubilized from the particulate fraction of hog kidney by the method of Morton, 8 is taken from the report of Robinson et al. 2 Step 1. Four kilograms of fresh, frozen hog kidneys is thawed, the fat cut away, and homogenized in a Waring blendor with 2 vol. of ice water. The homogenate is strained through cheesecloth and centrifuged at 1200 X g for 20 minutes. Step 2. The supernatant fluid is chilled to 0 °, brought to pH 5, and the resulting thick suspension immediately centrifuged at 0 ° and 3000 X g for 30 minutes. The supernatant fluid is discarded, and the sediment taken up in an equal volume of 0.06 M phosphate buffer at pH 7. The lumpy suspension is briefly homogenized in the blendor and frozen solid at - 10°. After thawing, the suspension is brought to pH 5, and the centrifugation and resuspension in phosphate buffer repeated. Step 3. Cold acetone is added to the suspension and stirred at 0 ° to a final concentration of 70%. The suspension is slowly added to several Biichner funnels layered thinly with Hyflo Super-Cel on No. 4 Whatman paper, and suction applied to yield a clear filtrate. The residue on the filter is carefully washed with cold water, the entire filtration taking place at 5 ° . The filtration procedure must exhaustively remove all traces of soluble protein, as tested by addition of 10 % trichloroacetic acid to the filtrate, and the procedure may require several days and several relayerings on the filter. Step 4. The filter cake is washed off the funnel and homogenized with 8 to 10 1. of water, n-Butanol, chilled to 0 °, is added with rapid stirring to 20 %. The suspension is mechanically shaken for 1 hour at 5 ° and dialyzed for 18 hours against cold running tap water to remove the butanol. 2 N HC1 is added at 0 ° to pH 5, and the mixture centrifuged at 3000 × g for 30 minutes at 0 °. The sediment is discarded, and the supernatant fluid taken to pH 7 with 2 N NaOH. Step 5. The volume is reduced at this stage from about 10 1. to 500 ml. by placing the solution in cellophane bags before a powerful fan. A small amount of insoluble material is centrifuged off and discarded. 6R. K. Morton, Nature 166, 1092 (1950).

[11]

113

DEHYDROPEPTIDASES FROM KIDNEY

Step 6. Solid ammonium sulfate is added at pH 7 to 50% saturation (35.3 g. per 100 ml. at 0°), and after standing 1 hour the precipitate is discarded after centrifugation at 3000 X g for 45 minutes at 0 °. To the supernatant fluid, solid ammonium sulfate is added to 75% saturation, and after standing for several hours, the suspension is centrifuged at 3000 X g for 1 hour. The sediment is taken up in a small volume of water and dialyzed for several hours. Lyophilization yields a white powder, weighing about 200 rag., with a nitrogen content of 10.8%, and a lipid content of approximately 30 %. The dried protein is completely soluble in water. When freshly prepared, the specific activity is about 600 times that of the homogenate. Properties

Specificity. Soluble renal acylase I is specific for dehydropeptides containing dehydroalanyl residues, regardless of the nature of the acyl residue. It also acts on a variety of acylated L-amino acids but does not act on acylated D-amino acids. Renal aminopeptidase (solubilized from the particulate fraction of kidney) acts equally well on all varieties of dipeptides containing dehydro-, L-, and D-terminal amino acid residues but requires the presence of a free amino group on the acyl residue. These relations are illustrated in the table. SPECIFICITY OF THE DEHYDROPEPTIDASES (UNITS OF SPECIFIC ACTIVITY)

Substrates Chloroacetyl-L-aminoacids Chloroacetyl-D-aminoacids Chloroacetyldehydroalanine Chloroacetyldehydroaminoacids other than dehydroalanine Glycyl-~amino acids Glycyl-D-aminoacids Glycyldehydroalanine Glycyldehydroaminoacids other than dehydroalanine L-Leucinamide

Dehydropeptidase II DehydropeptidaseI Acylase I (soluble) Aminopeptidase (solubilized) 9-100,000 Negligible 740

Negligible Negligible Negligible

Negligible 54-15,000 Negligible 70

Negligible 4300-350,000 7100-300,000 300,000

Negligible Negligible

69,000-300,000 Negligible

Stability. Lyophilized preparations of renal aminopeptidase rise in specific activity on storage at 5° . The increase in activity is equal for the L-, D-, and dehydropeptides, and approximately doubles for each two months of storage for the first six months after preparation of the enzyme.

114

ENZYMES OF PROTEIN METABOLISM

[11]

Electrophoresis and ultracentrifugal studies on the freshly prepared enzyme indicate the presence of a monodisperse system, and these characteristics do not apparently alter during the period of storage and increase in specific activity. ~ Activators and Inhibitors. Dehydropeptidase activity is moderately enhanced by added Zn ++ and Co ++ and is inhibited by cyanide and sulfhydryl.l.3.7.s Historical and Terminological Reference. Dehydropeptidase is a name applied by Bergmann and Schleich to a renal preparation which hydrolyzed glycyldehydrophenylalanine9 and which they believed to be specific for the class of dehydropeptides. The possibility that more than one dehydropeptidase exists in kidney was raised by Greenstein, Price, and Leuthardt; the designation of dehydropeptidase I was given to the particulate enzyme which rapidly hydrolyzes glycyldehydroalanine in kidney, and the designation of dehydropeptidase II to the soluble kidney enzyme which rapidly hydrolyzes chloroacetyldehydroalanine.TM Since the soluble acylase I of kidney has been found to hydrolyze all peptides of dehydroalanine, whereas the solubilized aminopeptidase of this tissue hydrolyzes not only glycyldehydroalanine but all dehydropeptides which possess a free s-amino group, the terminology of these two systems has been retained as above (cf. the table). In view of the fact that dehydropeptidase II (acylase I) is also effective toward a wide variety of Nacylated L-amino acids, and that dehydropeptidase I (solubilized aminopeptidase) is also effective toward a wide variety of glycyl-L- and D-amino acids, the designation of these enzymes as dehydropeptidases has apparent validity only when dehydropeptides are employed as substrates. Dehydropeplidases of Liver. Hepatic tissue supernatants contain three separate activities. That toward glycyldehydroalanine is destroyed by heating at 48 °, that toward chloroacetyl-L-alanine is destroyed by heating at 60 °, and that toward chloroacetyldehydroalanine is nearly completely stable at this latter temperature. 11 The last-mentioned activity, namely that of dehydropeptidase II, disappears when the hepatic tissue becomes cancerous, the second-mentioned remains nearly the same, whereas the first-mentioned (dehydropeptidase I) markedly increases.12 7 W. H. Yudkin and J. S. Fruton, J. Biol. Chem. 169, 521 (1947). 8 V. E. Price, A. Meister, J. B. Gilbert, and J. P. Greenstein,J. Biol. Chem. 181, 535 (1949). M. Bergmann and H. Schleich, Z. physiol. Chem. 205, 65 (1932); 207, 235 (1932). i0 j. p. Greenstein, V. E. Price, and F. M. Leuthardt, J. Biol. Chem. 175, 953 (1948). 11S. M. Birnbaum, and J. P. Greenstein, Bull. Israeli Acad. Sci. 4, 6 (1954). 12j. p. Greenstein, P. J. Fodor, and F. M. Leuthardt, J. Natl. Cancer Inst. 10, 271 (1949).

[12]

AMINOACYLASE

115

[12] A m i n o a c y l a s e Amino Acid Acylases I and II from Hog Kidney 1,2 O R2

H H

L

R 1 C H 2 - - C - - N - - C H - - C O O H --~ R1CH2COOH + N H 2 R 2 C H C O O H {R 1 = Acylase I R2 R = Acylase II R~

C1, H, NH2, etc. L-Aminoacid side chain other than L-aspartic C1 or H Aspartic acid

B y SANFORD M. BIRNBAUM

Assay Method The method of choice for the measurement of the hydrolysis of N-acyl derivatives of naturally occurring a-amino acids is the Van Slyke ninhydrin-CO2 analysis 3 of suitably prepared digests. Other well-known procedures are u n d o u b t e d l y applicable. 4-6 Reagents

0.1 M phosphate buffer at p H 7. Acyl amino acid solution, 0.025 M with respect to the L-form, and at p H 7.0. Saturated picric acid solution. E n z y m e suitably diluted in 0.01 M phosphate buffer. Procedure. Two tubes, a digest.and a blank, are employed for each assay. Each contains 1 ml. of 0.1 M phosphate buffer at p H 7 and I ml. of the enzyme solution. At zero time, 1 ml. of substrate is added to the

S. M. Birnbaum, L. Levintow, R. B. Kingsley, and J. P. Greenstein, J. Biol. Chem. 194, 445 (1952). 2 Aeylase I is probably the enzyme previously referred to as hippuricase or histozyme [cf. F. Leuthardt in "The Enzymes" (J. B. Sumner and K. Myrb~ck, eds.), Vol. I, Part 2, Academic Press, New York, 1951]. Hippuric acid is hydrolyzcd at a rate of 50 micromoles per hour per milligram N by this preparation, and the degree of concentration of this activity over the crude homogenate is of the same order as for the other acyl amino acids. ' D. D. Van Slyke, R. T. Dillon, D. A. MacFadyen, and P. Hamilton, J. Biol. Chem. 141, 627 (1951). 4 D. D. Van Slyke, J. Biol. Chem. 83, 425 (1929). 5 W. Grassmann and W. Heyde, Z. physiol. Chem. 183, 32 (1929). 6 K. LinderstrCm-Lang, Z. physiol. Chem. 173, 32 (1928).

116

ENZYMES OF PROTEIN METABOLISM

[12]

digest tube and both tubes are incubated at 37 °. At t, 3 ml. of saturated picric acid is added to each of the tubes, and 1 ml. of substrate is added to the blank. Suitable aliquots (usually 3 ml.) are taken from each for ninhydrin-CO2 analysis. Where the possibility of crystallization of the formed amino acid exists, one-half the above amounts of reagents can be added to and incubated directly in the Van Slyke tube. Estimation of Specific Activity. The pC02 of the digest minus the pCO~ of the blank after suitable aliquot corrections can be directly converted to micromoles of acyl amino acid hydrolyzedJ The hydrolytic reaction catalyzed by acylases I and II follows zero-order kinetics up to at least 40% splitting of the total available substrate. Thus, from any value obtained below 40 %, the number of micromoles of substrate hydrolyzed per milligram of protein nitrogen per hour can be calculated. Preparation of Acylase I

Two and one-half kilograms of fresh frozen hog kidneys was thawed, defatted, and homogenized in a Waring blendor with 2 vol. of ice water. The homogenate was strained through cheesecloth and centrifuged at 2500 r.p.m, for 20 minutes to remove cellular debris. The activity of this preparation tested against acetyl-DL-methionine (1 ml. of enzyme solution, 1 ml. of phosphate buffer at pH 7.0, and 1 ml. of 0.05 M neutralized substrate) was 800 micromoles hydrolyzed per hour per milligram of N. The total activity was 18 moles per hour. The preparation was chilled to 0 ° in a cold bath and brought to pH 4.7 by the careful addition of 2 N HC1. The resulting thick suspension was immediately centrifuged at 0 ° and 4000 r.p.m, for 20 minutes in the refrigerated centrifuge. The sediment was discarded, and the clear red supernatant quickly adjusted to pH 6.5 by addition of 2 N NaOH. The activity at this stage was 2300 micromoles of substrate hydrolyzed per hour per milligram of N; the total activity was 15 moles per hour. The ~upernatant was treated with 266 g. of solid ammonium sulfate per liter of solution, whereupon the pH decreased to 6.0 to 6.2. The resulting precipitate, which contained most of the activity, was separated either in the Sharples supercentrifuge or in the International refrigerated centrifuge. When the latter instrument was used, the suspension was allowed to settle overnight at 5 °, and as much of the supernatant solution as possible was siphoned off the following morning prior to centrifuging. The clear red supernatant was set aside for the preparation of acylase II. The sediment was suspended in about 40 ml. of ice water and dialyzed against running tap water until 7 I t must, of course, be kept in mind that dipeptides give 2 moles of CO~ per mole hydrolyzed and that aspartic acid gives 2 moles of C02 per mole formed in the digest.

[19.]

AMINOACYLASE

117

completely free of ammonium sulfate. The contents of the dialysis sack were centrifuged to remove inactive protein which separated during the dialysis. The activity value of the clear, almost black supernatant fluid was 12,200; total activity was 11 moles per hour. This solution (150 to 200 ml.) was adjusted to pH 5.9 to 6.0 with dilute acetic acid and treated with 0.4 vol. of chilled acetone in a cold bath at - 10 to - 15°. The precipitate was separated at - 8 ° in the refrigerated centrifuge and discarded. The clear, straw-colored supernatant solution was chilled again in the cold bath and treated with an additional 0.6 vol. of chilled acetone (based on the original volume). The precipitate was centrifuged at - 8 °, taken up in cold distilled water, and centrifuged again. The light pink-colored supernatant was quickly frozen and lyophilized. The activity value was 29,000; total activity was 9 moles per hour, or close to one-half the original activity of the crude homogenate. The weight of the dried preparation was generally about 2 g.; N content, 16.0%.

Preparation of Acylase II (Aspartic Acid Acylase) Most of the activity against the N-acylated aspartic acids remained in the supernatant fluid after the initial treatment with solid ammonium sulfate. The active acylase II fraction could be quantitatively precipitated from this supernatant by the further addition of 150 g. of solid ammonium sulfate per liter. The resulting precipitate was centrifuged in the Sharples centrifuge, the supernatant solution discarded, and the sediment dissolved in water and dialyzed free of salt. The dialyzed solution was then frozen and lyophilized, yielding about 14 g. of a fluffy, easily soluble preparation.

Properties Acylase I. The lyophylized preparation of acylase I is extremely stable and can be kept for long periods of time without loss in activity. In solution at pH 7, the enzyme can be heated to 70 ° for 60 minutes without change of activity. Below pH 5, however, it is rapidly and irreversibly destroyed at ordinary temperatures. The specificity of acylase I has been studied extensively.I, 8-11 With a given acyl moiety (chloroacetyl) the rates of hydrolysis vary from 5 micromoles per hour per milligram of N (chloroacetyl-L-proline) to 100,000 (chloroacetyl-L-methionine). The acyl derivatives of amino acids 8 K. R. Rao, S. M. Birnbaum, and J. P. Greenstein, J. Biol. Chem. 203, 1 (1953). 9 K. R. Rao, S. M. Birnbaum, R. B. Kingsley, and J. P. Greenstein, J. Biol. Chem. 198, 507 (1952). 19 S. C. J. Fu and S. M. Birnbaum, J. Am. Chem. Soc. 75, 918 (1953). 11 W. S. Fones and M. Lee, J. Biol. Chem. 201, 847 (1953).

118

ENZYMES OF PROTEIN METABOLISM

[19. ]

with aliphatic side chains are strongly favored b y this enzyme, in sharp contrast to pancreatic carboxypeptidase. 1,12,13 With the exception of methionine derivatives, the acyl compounds of the simple straight-chain aliphatic L-a-amino acids are the most rapidly attacked, increasing regularly in rate up through the norvaline compounds and then decreasing. Branching of the amino acid side chain inhibits the reaction somewhat to the point where chloroacetyl-L-tert-leucine, which possesses a completely substituted S-carbon, is not attacked a t all. 14 The action of aeylase I on dehydropeptides is described elsewhere (Vol. I I [11]). The effect of variation of the acyl derivative on two amino acids, glycine and L-alanine, is illustrated in Table I. Fones and Lee I~ have further studied the result of variation of the acyl moiety of the substrate on the rates of hydrolysis by acylase I. T h e y confirm a rough correlation between the rate of hydrolysis and the electronegativity of the acyl residue. 1° The most susceptible derivative studied was the triftuoroacetyl. Following this in descending order were fluoroacetyl, chloroacetyl, propionyl, acetyl, bromoacetyl, formyl, and iodoacetyl. TABLE I EFFECT OF N~AcYL RADICALON RATES OF HYDROLYSISWITH ACYLASE I a Rates of hydrolysis of N-acylated amino acid residue N-Acyl radical Acetyl Chloroacetyl Glycyl L-Alanyl Propionyl d/-Chloropropionyl

L-Alanine

Glycine

2,740 14,800 645 1,160 3,840 59

520 2640 88 170 930 23

a Initial reaction rates in terms of micromoles of substrate hydrolyzed per hour per milligram of protein N. 1~E. L. Smith in "The Enzymes" (J. B. Sumner and K. Myrb~ck, eds.), Vol. I, Part 2, Academic Press, New York, 1951. 13Acylase I hydrolyzes the N-chloroacetyl and N-acetyl derivatives of most of the L-amino acids about thirty times as fast as does the kidney homogenate. The exceptions to this observation, in addition to the N-acylated derivatives of L-aspartie acid, are those of the aromatic amino acids. Part of the activity of the homogenate against these substrates is due to a third fraction of hog kidney with a specificity probably similar to that of pancreatic carboxypeptidase. We can tentatively designate this fraction as acylase III. 14N. Izumiya, S. C. S. Fu, S. M. Birnbaum, and J. P. Greenstein, J. Biol. Chem. 205, 221 (1953).

[19.]

AMINOACYLASE

119

Added Co ++ accelerates generally the hydrolytic rates b y acylase I of those chloroacetyl and glycyl derivatives of the amino acids which are ordinarily less susceptible to the action of this enzyme, and, even in very low concentrations, Co ++ inhibits the hydrolysis of the very susceptible derivatives of other amino acids. Of all the compounds whose hydrolysis by acylase I is activated b y added Co *+, the ehloroacetyl and glycyl derivatives of aspartic acid are those most markedly affected2 Acylase II. This fraction has an almost absolute specificity for acyl aspartic acids, excluding those bearing a free amino group, e.g., glycyl-Laspartic acid. This is an unusually high degree of specificity for a peptidase. Table II gives the hydrolytic values against several representative compounds and contrasts this fraction with acylase I. TABLE II INITIAL HYDROLYTIC RATES WITH HOG KIDNEY ACYLASES I AND II ~

Rates with Substrate Chloroacetyl-L-glutamic acid Glycyl-L-glutamic acid Chloroacetyl-L-aspartic acid Glycyl-L-aspartic acid Chloroacetyl-I,-asparagine Glycyl-I~-asparagine Acetyl-DL-aspartic acid Chloroacetyl-DL-serine Chloroacetyl-DL-leucine

Homogenate 480 310 32 45 8 1120 11 455 630

Acylase I

Acylase II

12,700 490 4 4 129 54 5 11,600 16,500

6 3 142 2 0.3 19 27 3 7

a In terms of micromoles of substrate hydrolyzed at 38° per hour per milligram of protein N. Attempts to further purify acylase II b y alcohol or acetone fractionation or b y specific adsorbents have resulted only in a wide smearing of the activity over several fractions with little purification. Resolution of a-Amino Acids Because of their high degree of optical specificity, these enzymes have been extensively employed in the resolution of racemic a-amino acids (Vol. I I I [82]).

120

ENZYMES OF PROTEIN METABOLISM

[13]

[13] P e n i c i l l i n a s e Penicillin -}- H20--~ Penicilloic acid B y E. P. ABnAHAM

Assay Methods P r i n c i p l e . The enzyme has been assayed by measuring either the rate of disappearance of penicillin or the rate of formation of penicilloic acid. The disappearance of penicillin has usually been estimated from the loss of antibacterial activity, and the formation of penicilloic acid from the increase in acidity. However, other methods for estimating penicillin and penicilloic acid might be used. 1-4 M a n y of the early procedures for assaying penicillinase were unsatisfactory. Low concentrations of penicillin were often used, and the rate of reaction decreased as hydrolysis proceeded. The remaining antibacterial activity was sometimes measured in solutions which still contained the active enzyme. More recent procedures have used penicillin in concentrations t h a t are sufficient to maintain a reaction of zero order over most of its course; concentrations varying from 200 units/ml, to 3500 units/ml. have been stated to be satisfactory by different investigators. Under these conditions the rate of reaction is proportional to the concentration of penicillinase. If the reaction is carried out at a pH near 7 and at 25 to 30 ° the nonenzymic destruction of penicillin is negligible. Three procedures for assaying penicillinase are described below. In the first, as by Levy, 5 the remaining penicillin is measured by its antibacterial activity. In the second, as by Wise and Twigg, 8 formation of penicilloic acid is measured by titration with alkali. In the third, as originally performed by Henry and Housewright/ penicilloic acid is determined manometrically under the conditions described by Pollock. 8 Procedure. (1) To samples of 20 ml. of a solution of benzylpenicillin in 0.1 M phosphate buffer, pH 7, containing about 3500 units/ml., are added 1-ml. portions of a solution of penicillinase, of suitable activity, in 0.1 M

1j. V. Scudi, J. Biol. Chem. 164, 183 (1946). J. V. Scudi and V. C. Jelinek, J. Biol. Chem. 164, 195 (1946). 8j. H. Ford, Ind. Eng. Chem. Anal. Ed. 19, 1004 (1947). 4j. F. Alicino, Ind. Eng. Chem. Anal. Ed. 18, 619 (1946). 6 G. B. Levy, Nature 166, 740 (1950). 6 W. S. Wise and G. H. Twigg, Analyst 75, 106 (1950). R. J. Henry and R. D. Housewright, J. Biol. Chem. 167, 559 (1947). s M. R. Pollock, Brit. J. Expil. Pathol. 31, 739 (1950).

[13]

PENICILLINASE

i21

phosphate at p H 7.0. The solutions are kept at 25 °. After predetermined intervals of time the remaining penicillin is extracted as follows: The sample is rapidly brought to p H 2.3 with phosphoric acid (50 vol. %) and shaken twice with 20-ml. portions of ice-cold amyl acetate. The combined amyl acetate extracts are shaken twice with 40 ml. of 0.2 M phosphate, p H 7.0, and the resulting aqueous solution of penicillin made up to 100 ml. Controls are prepared in a similar manner from solutions to which penicillin was added immediately before extraction. The penicillin in all the extracts is assayed against Staph. aureus b y a turbidimetric or diffusion method2 The assay values for the penicillin which survives contact with the enzyme are subtracted from the values for the corresponding controls. The plot of the resulting values against time is a straight line, whose slope, over a wide range, is proportional to the a m o u n t of enzyme. The accuracy of this procedure is probably limited b y the precision of the method of assay. (2) F i f t y milliliters of a solution of benzylpenicillin containing 200 to 300 units/ml, is stirred with a stream of carbon dioxide-free air in a vessel kept at 25 °. The p H of the solution, measured by a glass electrode, is adjusted to 7.0 with 0.01 N NaOH, and a known a m o u n t of a penicillinase solution, also at p H 7.0, is added. 0.01 N N a O H is then run in from a buret at such a rate t h a t t h e p H remains constant, and buret readings are taken every minute. The slope of the graph of buret readings plotted against time is proportional to the a m o u n t of enzyme. (3) The C02 liberated when penicilloic acid is formed in a C Q - b i c a r bonate buffer at p i t 7 is measured in constant-volume Warburg respirometers at 30 °. Samples of 1 to 2 ml. of the enzyme solution are added to 0.5 ml. of 0.043 M sodium bicarbonate in the main c o m p a r t m e n t and the volume made up to 2.5 ml., if necessary, with water. In the side bulb are placed 0.1 ml. of a solution containing 100,000 units//ml, of benzyl penicillin, 0.1 ml. of 0.043 M sodium bicarbonate, and 0.3 ml. of water. The manometers are gassed with 5 % CO~ in N2. After equilibration for 20 minutes, the contents of the side bulb are tipped into the main flask. A constant rate of gas evolution is established within 2 to 5 minutes and persists until about 75% of the penicillin is destroyed. This rate is a measure of the penicillinase activity.

Application of the Manometric Assay Method to Crude Preparations. When the enzyme solution contains substances such as amino acids and 9 The assay of penicillin by microbiological methods is discussed by H. W. Florey, E. Chain, N. G. Heatley, M. A. Jennings, A. G. Sanders, E. P. Abraham, and M. E. Florey in "Antibiotics" Vol. 1, p. 110, Oxford University Press, New York, 1949.

122

ENZYMES OF PROTEIN METABOLISM

[18]

phosphate, the COs retention is calculated from the difference between the amounts of gas evolved when tartaric acid is added to the solution and the bicarbonate buffer together, and to the bicarbonate buffer alone. For the assay of penicillinase in bacterial suspensions the production of further enzyme is stopped by the addition of 0.6 ml. of M/200 oxine to 3-ml. samples of the culture. 8 Definition of Unit and Specific Activity. No unit of penicillinase has yet come into general use. Levy ~ has suggested that a unit be defined as that amount of enzyme which hydrolyzes 0.1 micromole of penicillin (35.6 ~, or 59.3 units of sodium benzylpenicillin) per hour at 25 ° and at pH 7.0, the rate of inactivation being measured in a concentration of benzylpenicillin sufficient to maintain a zero-order reaction rate. This has been called the Schenley unit. Pollock and Torriani 1° define a unit as that amount of enzyme which hydrolyzes 1.0 micromole of penicillin per hour at 30 ° and at pH 7.0. They express specific activity as units per milligram of nitrogen. Production and Purification Procedure Certain strains of aerobic spore-forming bacteria are convenient sources of extracellular penicillinase. The enzyme is adaptive: its production is greatly increased in the presence of penicillin. 8,1~ The following procedure for the production and purification of penicillinase from Bacillus cereus is that described by Pollock and Torriani.S° Production. A strain of B. cereus (NRRL 569) is grown in the following medium: 1% Difco proteose-peptone, 0.1 M potassium phosphate buffer at pH 7.0, Mg ++ (1 ml.fl, of 20% MgS04), Fe ++ (0.5 ml./1, of 0.1% FeSO4.7H20). Fifty liters of medium, inoculated with a culture in the logarithmic phase of growth, is stirred and aerated at 35 ° . When the bacterial density reaches 0.06 mg./ml. 5 X 104 units of penicillin is added, and a further 5 X 10 ~ units when it reaches 0.18 mg./ml. After about 4.5 hours, when the bacterial density reaches 1 mg./ml., the culture is harvested, cooled at once, and the cells separated in a Sharples centrifuge. The supernatant fluid contains 1000 units/ml, of penicillinase (860 units/ mg. N), the unit being that already defined. 1° Purification. Precipitations with acetone and ethanol are carried out at a temperature between - 3 and - 0 . 5 °, and precipitations with ammonium sulfate are done between 0 and -}-2°. The enzyme is precipitated by the addition of 1.5 vol. of acetone to the solution, and the precipitate is redissolved in 5 1. of water. Impurities are precipitated from the resulting solution by the addition of 2.5 1. of acetone, and the enzyme precipitated from the supernatant by the addition of a further 2.5 1. of acetone. The 10 M. R. Pollock a n d A.-M. Torriani, Compt. rend. 257, 276 (1953). 11 E. S. Duthie, Brit. J. Exptl. Pathol. 26, 96 (1944).

[13]

PENICILLINASE

123

precipitate is dissolved in 250 ml. of water, and the solution dialyzed against repeated changes of 0.001 M phosphate buffer until most of the phosphate precipitated by the acetone has been removed. The enzyme is then precipitated by 0.9 saturation with ammonium sulfate. At this stage the yield is approximately 60%. Further considerable purification can be brought about by precipitation with ammonium sulfate between 0.60 and 0.67 saturation. The yield in this step is about 50 %. The product has been found to have an activity of 6.7 × 105 units/mg. N. This preparation has been purified further by two precipitations with ethanol (final concentration 43 %) from a solution in 0.1 M phosphate buffer, pH 7. The yield at this stage was 25%. The specific activity of the final precipitate was 8.7 × 105 units/mg. N, representing approximately a 1000-fold purification from the medium nitrogen. However, on a TCA-precipitable nitrogen basis, the purification is only tenfold.

Properties Penicillinase is produced by a variety of microorganisms, and it is not yet known how closely enzymes from different sources resemble each other. The following properties refer mainly to the enzyme from B. cereus. Specificity. The enzyme appears to be specific for members of the penicillin family. Crude preparations of penicillinase, however, may contain an enzyme which inactivates the antibiotics helvolic acid and cephalosporin p.~2 Stability. Concentrated solutions of the purified enzyme are stable at 0 °, but dilute solutions rapidly lose their activity. The crude enzyme is usually more stable than the purified enzyme in dilute solution and sometimes retains a high proportion of its activity after heating for several minutes at 100 °. This appears to be due to the protection afforded by compounds of high molecular weight in crude preparations. Infusion broth and 1% gelatin have a striking protective action. 13 The enzyme is inactivated by incubation at pH 5 with papain activated by cysteine. 7 Inhibitors. The activity of the enzyme is inhibited almost completely by Fe +++ in concentrations of 50 ~//ml., and to the extent of 65% by Ca ++ in concentrations of 25 ~,/ml. 7 It is also inhibited by 2-benzylglyoxaline and by penicillamine (~-mercaptovaline). 14 Antipenicillinase serum, obtained from rabbits which have been immunized with penicillinase, inhibits the activity of the enzyme. 15 12H. S. Burton and E. P. Abraham, Biochem. J. 50, 168 (1951). 18E. E. D. Manson and M. R. Pollock, J. Gen. Microbiol. 8, 163 (1953). 14O. K. Behrens and L. Garrison, Arch. Biochem. 27, 94 (1950). i, R. D. Housewrightand 1%.J. Henry, J. Bacteriol. ~8, 241 (1947).

124

ENZYMES OF PROTEIN METABOLISM

[14]

Penicillinase activity is not affected by the following enzyme in'hibitors: 7 0.15 M sodium azide, 0.01 M potassium cyanide, 6 X 10-3 M diethyl dithiocarbamate, 0.2 M sodium fluoride, 0.5 M formaldehyde, 4.4 × 10-3 M sodium thioglycolate, 10-3 M sodium-p-chloromercuribenzoate, 0.05 M iodoacetamide. It has been concluded from these results that penicillinase is not an iron- or copper-containing enzyme and that free thiol groups or amino groups are not essential for its activity. Effect of pH. In bicarbonate buffer at 36 ° the activity of penicillinase rises to a rather flat plateau between pH 6.9 and 7.6; the optimum pH is probably about 7.2. 7

[14] R e n i n f r o m K i d n e y Hypertensinogen

[Renin] -~ Hypertensin

By ERWlN HAAS Assay Method Principle. Renin may be assayed directly 1,2 by its pressor effect on the test dog or indirectly ~,3 by the determination of the hypertensin formed during the incubation of hypertensinogen with renin in vitro. In both instances hypertensinogen is the substrate and it is transformed into hypertensin by the enzymatic action of renin. Hypertensin, the vasoconstrictor and pressor substance, induces the elevation of the blood pressure by acting on the peripheral vascular bed. Reagents. In the direct assay of renin no other reagents are required, since a sufficient amount of hypertensinogen is present in the serum of the normal, healthy test dog. Procedure. For quantitative determinations by the direct method renin is injected intravenously into normal, trained, unanesthetized dogs and the elevation of the mean femoral blood pressure is measured. For this purpose a 20-gage needle is inserted into the femoral artery; it is connected through a rubber tube with a U-tube mercury manometer and the system is filled with a 0.9% sodium chloride solution. Because of the enzymatic nature of the reaction there is a short (about 1H. Goldblatt, Y. J. Katz, H. A. Lewis, and E. Richardson, J. Exptl. Med. 77, 309 (1943). H. Goldblatt, H. Lamfrom, and E. Haas, Am. J. Physiol. 175, 75 (1953). 3j. M. Mun5z, E. Braun-Men~ndez, J. C. Fasciolo, and L. F. Leloir, Am. J. Med. Sci. 200, 608 (1940).

[14]

RENIN FROM KIDNEY

125

15 seconds) induction period, and the blood pressure elevation reaches its maximum about 2 minutes after the injection of renin. This specific time-pressure relation permits the distinction of renin from other pressor agents, such as hypertensin or pressor amines which might be present in crude renal extracts and which would react instantaneously. This test procedure, furthermore, allows the detection of depressor effects. Definition of Unit and Specific Activity. TM The amount of renin required to elevate the direct mean blood pressure 30 mm. Hg has been designated as one dog unit. In this range, the blood pressure elevation is directly proportional to the renin concentration. The purity of a renin preparation is expressed in a quantitative way by the term Units of renin Specific activity = Milligrams dry substance First Purification Procedure A simple method is available 5 for the isolation of relatively crude renin, which can be generally applied to the kidneys of various animal species. The kidneys are kept for 1 day at - 2 4 ° in a deep-freeze cabinet and then thawed at room temperature. This procedure is repeated three more times, and the kidneys are then ground up. Experimental conditions will be described for 100 g. of renal tissue, but the identical procedure has been applied to a few grams of rat kidney and to 44 kg. of rabbit kidney. One hundred grams of ground kidney tissue is extracted twice at room temperature by stirring each time with 75 ml. of water, followed by centrifugation for 30 minutes at 2000 r.p.m. The combined extracts, measuring about 160 ml., are filtered through a sieve, cooled to 0 °, and acidified to pH 1.6 by dropwise addition of about 7.6 ml. of 4 N sulfuric acid. After 10 minutes at 0 °, the acid-denatured insoluble proteins are precipitated by neutralization of the solution by the slow addition of about 5.6 ml. of a 5 N KOH solution, and these inert proteins are then separated by centrifugation. The clear supernatant solution contains the renin. No difficulties have been encountered as tile result of the intravenous injection into dogs of this type of renin prepared from the kidneys of various animals, although, in some instances, an amount of extract equivalent to 6 g. of heterologous kidney tissue was administered. The only exception was dog renin, which required dialysis prior to testing in order to remove accessory pressor substances the nature of which has not yet been determined. The acid treatment of the first crude tissue extract removes about 85 % of the inert proteins, resulting in a sixfold purification of the renin. 4 E. Haas, H. Lamfrom, and H. Goldhlatt, Arch. Biochem. and Biophys. 42,368 (1953). s E. Haas, H. Lamfrom, and H. Golbdlatt, Arch. Biochem. and Biophys. 48, 256 (1954)

126

ENZYMES

OF PROTEIN

METABOLISM

[14]

Of the remaining inert proteins, about 80% can be removed easily by a simple salt fractionation, resulting in an over-all thirtyfold purification of the renin. Acidification of the renin solution to pH 4.3 and fractionation with ammonium sulfate between 1.0 and 1.7 M, followed by dialysis, will separate renin with a yield of 64% and a specific activity of 2.0 dog units per milligram of dry substance. The method described has the advantage of being simple and uniformly applicable; it removes sensitizing anaphylactic depressor and accessory pressor substances, but it achieves only a thirtyfold purification. Second Purification Procedure

The second procedure, to be described here in greater detail, has been designed specifically for the isolation of highly purified renin from hog kidneys. 4 It consists of about fourteen individual steps and results in a 56,000-fold purification. In each step, the main fraction, which contains the bulk of the renin and which has the highest specific activity, is carried through the subsequent steps of the purification procedure, as shown in the summary. There is usually no loss, and the side fractions containing the remaining renin in a lower state of purity are collected, channeled back into the appropriate step of the isolation procedure according to their specific activity, and worked up, again yielding renin of high specific activity. Starting Material. For large-scale preparative work, and to ensure a high yield, it is important to select the proper time of the year (October to May) for the isolation of renin from hog kidneys of slaughterhouse animals. Seasonal fluctuations of the renin content can result in onequarter of the yield from kidneys of summer hogs compared with the yield from winter and spring hogs. A detailed description of the experimental procedure will be given here for a batch of 67 kg. of hog kidney. Fresh kidneys are stored for 1 day at 2 ° before the extraction is undertaken. If storage at this temperature is omitted, difficulties may be encountered in one of the subsequent steps, namely, the precipitation of renin by tungstate in step 3. The kidneys thereafter may be stored in the frozen state or worked up immediately. Step 1. Autolysis and Extraction. The kidneys, without being dissected or defatted, are put through a meat grinder, a motor-driven l~-horsepower commercial model. The autolysis of the kidney tissue is permitted to proceed in a concentrated suspension and at an elevated temperature to achieve effective autodigestion by the tissue enzymes. Ten liters of a 0.18 N ammonium hydroxide solution is heated to 52 ° and slowly added to the ground kidney tissue, followed by 5 1. of benzene.

[14]

RENIN FROM KIDNEY

127

The tissue suspension is mixed slowly with wooden ladles, immersed in a water bath of 55 ° , and warmed to 37 ° . After incubation without stirring for about 4 hours at 37 ° the suspension is cooled to 25 ° and stored overnight in the cold room. The subsequent extraction likewise is conducted with a minimum of liquid to keep the volume of solution to be processed at a minimum. Eleven liters of distilled water is added to the suspension, and this is thoroughly mixed at room temperature with 9 kg. of a filter aid (Johns Manville Celite, No. 503). The extract containing the renin is separated from the insoluble residue by filtration through heavy cloth in a hydraulic press at a total pressure of 15 tons which results in the recovery of 50 1. of solution. The residual material is broken up into smaller pieces, reextracted with 20 1. of water, and filtered again with pressure, which yields a second extract of about 26 1. The combined solutions in a volume of 76 1. contain the renin in addition to about 5700 g. of other proteins. The average yield in twenty-five batches was found to be 250,000 units of renin or 3.7 units/g, of kidney after the first step of the isolation procedure. The following steps of this isolation procedure are performed at about 1° because of the instability of purified renin. Steps 5 and 8 are exceptions, and specific conditions will be outlined. Step 2. Fractionation with Ethanol. The renin solution (76 1.) is cooled to 1°, and approximately 3 1. of 4 N cold sulfuric acid is added slowly and with efficient stirring to adjust the solution to pH 2.15. Stirring and cooling are continued, and 9.3 1. of 95% ethanol, previously cooled to - 2 4 °, is added at a rate slow enough to avoid an increase of temperature. Centrifugation results in the separation of a heavy precipitate of inert protein, and 73 1. of a clear, yellow solution is obtained which contains the renin. Step 3. Precipitation of Renin with Tungstate. The selection of the correct concentration of tungstate is essential. It varies from one batch to the next, and it is determined in serial pilot experiments in which the tungstate concentration is varied in small increments. The minimum amount required to induce the precipitation of all the protein in 30 minutes at 2° was found also to be the correct amount of tungstate for the precipitation and recovery of renin. The absence of turbidity after the addition of tungstate serves as a convenient end point in these titrations of protein content. With too small amounts the precipitation of renin is incomplete. With excess tungstate the recovery of renin is unsatisfactory, since the renin-tungstate complex cannot be dissociated effectively. Furthermore, the renin-tungstate complex is redissolved by an excess of tungstate, and for this reason a local excess has to be avoided by slow addition and efficient stirring. As a rule, about 9 ml. of M sodium

128

ENZYMES OF PROTEIN METABOLISM

[14]

tungstate was required per liter of renin solution No. 2 with variations ranging between 7 and 15 ml. To 73 1. of the acid renin-ethanol solution obtained by step 2 is added 660 ml. of M sodium tungstate, with rapid stirring. A heavy precipitate containing the renin-tungstate complex settles out overnight, and the supernatant clear solution can be siphoned off and discarded. The precipitate is further concentrated by centrifugation, suspended in 5.3 1. of water, dissolved by the addition of about 280 ml. of 2 N KOH, and adjusted to pH 7.3. In the resulting solution (7.3 1.) renin has been concentrated tenfold. Renin tungstate in this form has been stored for several months at - 2 4 ° and frozen and thawed repeatedly without loss of activity.

Step 4. Separation of Tungstate and Fractionation with Ammonium Sulfate. For the precipitation of tungstate 1.0 1. of a 3 M calcium chloride solution is added to the renin solution, and it is stirred slowly for 60 minutes and centrifuged. An additional amount of purified renin can be obtained by re-extraction of the calcium tungstate precipitate. For this purpose it is stirred for 30 minutes with 4.5 1. of water and separated by centrifugation. The supernatant solutions obtained after the two centrifugations contain the renin. They are combined, resulting in a volume of 12 I. An excess of calcium is removed by addition of 1.70 kg. of solid ammonium sulfate, stirring for 10 minutes, decanting, and filtering. The clear renin solution is acidified to pH 4.3 with approximately 34 ml. of 2 N sulfuric acid, and a small amount of protein settles out overnight; this is removed by filtration. Renin is precipitated from the supernatant solution by the addition of 1.60 kg. of ammonium sulfate, corresponding to a concentration of 1.7 M. After stirring for 10 minutes, the precipitate is collected by centrifugation and dissolved at pH 5.6 by the addition of 900 ml. of water and 7 ml. of N KOH. Clarification by centrifugation finally results in 1000 ml. of renin solution No. 4. Step 5. Fractionation with Acetone. An ammonium sulfate concentration of 0.4 M and a buffer concentration of 0.1 M are established by the addition of 38 g. of solid (NH4)2S04 and 113 ml. of M acetate buffer of pH 4.8 to 1000 ml. of renin solution No. 4. Acetone (560 ml.), previously cooled to - 2 4 °, is added to the renin solution slowly and with stirring, corresponding to a final concentration of 33 %. The suspension is stirred in stainless steel containers for 5 minutes at a temperature of - 5 ° and is centrifuged for 8 minutes in aluminum cups. The precipitate is washed with 560 ml. of a solution containing 33 % acetone and 0.1 M ammonium sulfate, separated by centrifugation, and discarded. To the combined supernatant solution (2160 ml.) containing the renin, 850 ml. of cold acetone is added to a final concentration of 52%, and the suspension is

[14]

RENIN FROM KIDNEY

129

stirred for 5 minutes at 0 °. After centrifugation for 30 minutes, the supernatant solution consisting of two layers of liquid is poured off and discarded. The precipitate containing the renin is placed on filter paper for a few minutes and then dissolved in about 220 ml. of water, resulting in 250 ml. of renin solution No. 5. No loss of activity has been observed during storage of this solution for 71 days at - 2 4 ° and pH 5.1.

Step 6. Fractionation with Ammonium Sulfate at pH 5.6, Precipitation by Dialysis and Acidification. Renin in purified form is rather labile, especially in a salt-free solution, but it can be stabilized by pyrophosphate, and for this purpose 90 ml. of a 0.19 M pyrophosphate buffer of pH 5.3 is added to 250 ml. of renin solution No. 5. Acetone is removed by dialysis for 15 hours at 2 ° against distilled water, which increases the volume of the solution to 450 ml. For the following fractionation, 26 ml. of molar acetate, pH 5.5, is added to establish a buffer concentration of 0.05 M, the pH is adjusted to 5.58 with about 2 ml. of 2 N KOH, and inert proteins are precipitated by the addition of 82 g. of solid ammonium sulfate (1.20 M). After being stirred for 10 minutes, the precipitate is removed by centrifugation and discarded. From the supernatant solution (500 ml.) renin is precipitated by the addition of 61 g. of ammonium sulfate (2.0 M) with stirring for 10 minutes, and high-speed centrifugation for 10 minutes at 20,000 × g. The precipitate is dissolved in 300 ml. of 0.05 ill pyrophosphate buffer, pH 5.3, and dialyzed for 13 hours against distilled water for the removal of salt which interferes with the quantitative precipitation of renin. The main fraction of renin is precipitated, with good purification and with a yield of about 80%, by acidification of the dialyzed solution to pH 3.80 with about 24 ml. of 0.1 N HC1 and centrifugation. After the separation, a side fraction of lower specific activity is recovered from the supernatant solution by precipitation with 2.3 M ammonium sulfate. The precipitate containing the main fraction of renin is suspended in 235 ml. of saline and dissolved at pH 6.0 by the addition of about 2 ml. of N KOH, resulting in 250 ml. of renin solution No. 6. Storage of this renin preparation in a neutral saline solution in the frozen state at - 2 4 ° and thawing results in an appreciable loss of activity. Various methods of stabilization can be employed, such as acidification to pH 4.7, or the addition of 9 % NaC1 or of 0.015 M pyrophosphate, pH 5.3. Renin obtained by step 6 has been purified about 2000-fold. Sterilized by Seitz filtration, it has been injected subcutaneously and intramuscularly for periods up to 33 weeks into human hypertensive patients for the production of antirenin. No untoward effects such as pyrogenic, anaphylactic, or depressor reactions were observed, which

130

ENZYMES

OF P R O T E I N

METABOLISM

[14]

frequently have been reported after the repeated injection of foreign proteins. Step 7. Fractionation with Ammonium Sulfate at pH 7.5. Renin of greater purity than in renin solution No. 6 is adversely affected by stainless steel, especially in an alkaline solution. Therefore, glass containers and glass or plastic centrifuge tubes are used in all the subsequent steps of the isolation procedure. The solubility of renin is a function of both the renin and the ammonium sulfate concentration. In the fractionation procedures of the following steps it is essential therefore to adhere to the concentrations specified for renin. To renin solution No. 6, which contains 100,000 units of renin, is added 190 ml. of water and 100 g. of ammonium sulfate, followed by 10 ml. of N ammonium hydroxide, to establish the following conditions: renin concentration, 200 units/ml. ; (NH4)~SO~, 1.50 M; NH4OH, 0.02 M; total volume, 500 ml. ; pH 7.55. Stirring for 5 minutes and centrifugation for 7 minutes at 6000 X g result in the precipitation of the side fraction I. Addition of 38 g. of ammonium sulfate (2.0 M) to the supernatant solution, stirring, and precipitation by centrifugation yield fraction II, the main fraction of renin. A further increase to 2.5 M by the addition of 38 g. of ammonium sulfate to the supernatant solution results in the precipitation of another side fraction III, accounting for the complete recovery of renin. For further purification, fraction II was dissolved in 670 ml. of water, and 150 g. of ammonium sulfate (1.5 M) and 17 ml. of a N ammonium hydroxide solution (0.022 N) were added. Renin concentration, 100 units/ml.; volume of the solution, 780 ml. ; pH 7.9. Renin was precipitated from this solution and was recovered quantitatively by the addition of 60 g. of (NH4)~SO4 (2.0 M), stirring, and centrifugation. After solution of the precipitate in water, 400 ml. of renin solution No. 7, which contains 0.1 M ammonium sulfate, is obtained. To improve the stability of renin, acidification with acetic acid to pH 3.8 is recommended. Spectrophotometric investigation of the renin preparations obtained in step 7 indicates that they are free of pigments such as hemoglobin, myoglobin, or flavoproteins. Step 8. Fractionation with Ammonium Sulfate at pH 3.85, Variation of Temperature. Renin has a somewhat unusual negative temperature characteristic, indicated by a decrease of its solubility with increasing temperature. Renin dissolved at 0 ° can be precipitated by warming the ammonium sulfate solution to room temperature, and this allows fractional purification of renin. Renin solution No. 7, which contains 78,000 units of renin in 400 ml.,

[14]

RENIN FROM KIDNEY

131

is cooled to 0 ° and diluted with 2500 ml. of water to a final renin concentration of 25 units/ml. Ammonium sulfate was added (530 g. = 1.25 M), and the pH adjusted to 3.85 with about 40 ml. of N acetic acid. This solution in aliquots of 320 ml. was placed in a water bath at 40 °, warmed in about 1 minute to 16°, and separated immediately by centrifugation for 7 minutes at room temperature. A precipitate designated as fraction I is separated by this procedure. The supernatant solution (3200 ml.) is cooled to 0°, 142 g. of ammonium sulfate is added (1.55 M), and fraction II, the main fraction of renin, is precipitated by the warming of the solution to 25 ° and centrifugation at the same temperature. Again, warming and centrifugation were performed in smaller batches, to avoid inactivation of renin by prolonged exposure to the elevated temperature. From the supernatant solution all the remaining renin is precipitated at 0 ° by the addition of 375 g. of ammonium sulfate (2.3 M) and centrifugation, resulting in fraction III. The two side fractions I and III, stabilized by 0.01 M pyrophosphate buffer, pH 5.3, are stored at - 2 4 °. The main fraction II, dissolved in water and clarified by centrifugation, represents renin solution No. 8, with a volume of 424 ml. and an ammonium sulfate concentration of 0.05 M. Step 9. Fractionation with Ammonium Sulfate and Acidification to pH 2.5. Two side fractions of lower specific activity are separated here by fractionation with ammonium sulfate. At a specific activity of about 100 units/mg., renin can be acidified to pH 2.5 without inactivation, if the temperature is low, if the reaction time is short, and if it is protected by high concentrations of ammonium sulfate. This acidification and the fractionation with acetone in the following step modify the solubility characteristics of renin in such a way that it will precipitate out in a highly purified form during the dialysis in the final step of this isolation procedure. The modification of renin induced by acid treatment appears to be a reversible process, and spontaneous reconversion to the pre-acid state of renin has been observed during storage. For this reason, storage of renin after step 9 for longer than 4 weeks at - 2 4 ° should be avoided. Otherwise renin fails to precipitate out during the subsequent dialysis, and a repetition of the acid treatment becomes necessary, resulting in considerable losses. Renin solution No. 8, with a volume of 424 ml. and a renin content of 43,000 units, is cooled to 0 °, 74 g. of ammonium sulfate (1.20 M) is added, and the acid concentration is adiusted to pH 2.50, which requires the dropwise addition of about 19 ml. of N sulfuric acid. Fraction I, which is precipitated under these conditions, is separated by stirring for 5 minutes and centrifugation for 6 minutes at 6100 X g.

132

ENZYMES OF PROTEIN METABOLISM

[14]

To the supernatant solution (480 ml.), 28 g. of ammonium sulfate (1.60 M) is added for the precipitation of fraction II, and the suspension is stirred and centrifuged as described before. Finally, all the remaining renin is precipitated in fraction I I I by the addition of 54 g. of ammonium sulfato to the supernatant solution (2.3 M), stirring, and centrifugation. The precipitate obtained in the range 1.2 to 1.6 M ammonium sulfate (fraction II) contains the main fraction of renin. Because of the high acidity it is transferred without delay into the buffered solution. Solution in 197 ml. of 0.04 M acetate buffer, pH 4.5, and clarification by centrifugation result in 200 ml. of renin solution No. 9. Step 10. Fractionation with Acetone. The relative solubility of renin and of the other biologically inactive protein in diluted acetone can be reversed by the addition of ammonium sulfate. As a practical consequence, renin can be "salted i n " by ammonium sulfate, and it becomes thereby relatively more soluble in a 45 % acetone solution than the inert protein, which precipitates out under these conditions, and which can be separated in a fraction of lower specific activity. After an increase of the acetone concentration to 60%, all the renin can be recovered in fractions I and II, indicating that high acetone concentrations will be tolerated at this state of purity without impairment of renin activity. Renin solution No. 9, which contains 33,000 units of renin in 200 ml. of 0.04 M acetate buffer, pH 3.5, is cooled to 0 °, and 16 ml. of a 2 M ammonium sulfate solution is added (0.15 M), followed by 180 ml. of cold ( - 2 4 °) acetone to a final acetone concentration of 45%. Stirring for 10 minutes and centrifugation for 15 minutes in glass tubes results in the precipitation of fraction I. Addition of 145 ml. of cold acetone to the supernatant solution (acetone concentration = 60%), stirring, and centrifugation result in the precipitation of fraction II, which is dissolved in 100 ml. of 0.05 M acetate buffer, pH 3.8. This is referred to as renin solution No. 10. Step 11. Precipitation by Dialysis. Dialysis of renin solution No. 10 results in the precipitation of most of the renin in a highly active form; no loss of activity is encountered. Specifically, renin solution No. 10 is dialyzed for a period of 15 to 40 hours at 0 ° against the distilled water of the laboratory (pH 4.2 to 4.8). During that time a fine white precipitate is formed which is separated by centrifugation and which represents the renin preparation of the highest specific activity obtained so far. The precipitate is suspended in water and dissolved at pH 6.6 by the addition of about 0.1 ml. of 0.1 N KOH, resulting in the final renin solution No. 11 with a volume of 50 ml. containing about 20 mg. of protein. The over-all purification with respect to renin is 56,000-fold. The

[14]

R E N I N FROM KINNEY

133

over-all yield in this m a i n fraction represents a b o u t 6 % of the t o t a l renin obtained in the starting solution (step 1), b u t additional a m o u n t s of the highly active renin can be recovered from the side fractions. T h e highly purified renin obtained b y step 11 is unstable in neutral solution, b u t it can be stabilized b y the addition of a b o u t 0.01 M pyrophosphate buffer, p H 5.3. No loss of activity has been observed during storage of the frozen renin p y r o p h o s p h a t e solution for 70 days at - 2 4 °, or as a result of lyophilization of such a solution, or during the storage of renin as a d r y powder for more t h a n 3 months at room t e m p e r a t u r e or at 0 °. SUMMARY OF PURIFICATION PROCEDURE

Fraction First crude extract 1. l Autolysis at 37° 2. Fractionation 10% ethanol, pH 2.15 3. Precipitation with Na2WO4 Dissociation with CaC12 4. ~Fraetionation with (NH4)2S04, ( 1.0-1.7 M; pH 4.3 5. Fractionation with acetone 33-52%; pH 4.8 fFractionation with (NH4).~S04, 6. ~ 1.2-2.0 M; pH 5.5 (Precipitation after dialysis pH 3.8 7. Fractionation with (NH4):S04, 1.5-2.0 M; pH 7.5 fFractionation with (NH4)2SO4, 8. ~ 1.25-1.55 M; pH 3.85 (Temperature, 25 ° 9. Fractionation with (NH4)2SO4, 1.2-1.6 M; pH 2.50 10. Fractionation with acetone, 45-60%; pH 3.5 11. Precipitation by dialysis, pit 4.3

Total units

Specific activity, Recovery, units/mg. %

80,000 250,000 205,000 195,000

0. 014 0.044 0.92

137,000

3.6

55

130,000

9.4

52

-82 78

100,000

25

40

78,000

50

31

43,000

110

17

33,000

182

13

22,000 15,000

240 780

9 6

Properties Physiological, 2 biochemical, and physicochemical properties of renin, 6 its ultraviolet spectroscopy, 7 and its antigenic properties 8 h a v e been investigated recently with highly purified preparations. 6 E. Haas, H. Lamfrom, and H. Goldblatt, Arch. Biochem. and Biophys. 44, 63 (1953). 7 E. Haas, H. Lamfrom, and H. Goldblatt, Arch. Biochem. and Biophys. 44, 79 (1953). s H. Lamfrom, H. Goldblatt, and E. Haas, Am. J. Physiol. 177, 55 (1954).

134

ENZYMES OF PROTEIN METABOLISM

[14]

Intravenous injection of a single dose of renin into a normal dog results in a temporary blood pressure elevation which is directly proportional to the amount of renin up to a rise of 35 mm. Hg. Under anesthesia (ether, morphine, Nembutal, chloralosane) the blood pressure response to renin is reduced greatly. A constant elevation of blood pressure can be maintained by means of a slow, constant infusion of renin at a rate of 0.5 units per minute. Repeated subcutaneous or intramuscular injection of heterologous renin for weeks or months into an animal 8,9-H or into human, hypertensive subjects 8,~2 is followed by the development of antirenin. The pressor effect of renin is neutralized by antirenin, and this reaction is unaffected by variations in the specific activity of the renin preparation between 3 and 470 units/mg, dry substance. Renin can be dissolved readily in a neutral, salt-free solution but, purified beyond step 6 of the isolation procedure, it is insoluble, in the dialyzed form, at a reaction more acid than pH 4.8. Renin does not seem to require an easily dissociable prosthetic group or metal ion for its enzymatic activity. This is indicated by the fact that highly purified renin can be dialyzed for 2 days, under certain conditions, without any decrease of activity. Purified renin is very susceptible to inactivation, but stabilization could be accomplished by pyrophosphate, by glycine, or merely by the presence of inert proteins. Dialysis without inactivation is possible with renin of relatively low purity, but almost complete destruction takes place when highly purified renin is dialyzed at 2 ° for 16 hours against distilled water. Dialysis of the purest renin without loss of activity is possible, however, against pyrophosphate buffer of pH 5.2. The protective action of pyrophosphate seems to suggest that metal tracer might be the cause of the inactivation of renin. Complete inactivation of renin was observed by 10-3 M solutions of ferric, auric, and platinic salts, partial inactivation by silver and cupric ions. Renin is protected against metal poisoning in the presence of proteins of kidney tissue or serum. Renin is unusually stable toward acid reaction, especially in a state of low purity and at a low temperature, and no inactivation was found after acidification of hog renin to pH 1.6. This property has been utilized in the simplified method described for the isolation of semipurified renin. ~ 9 C. A. Johnson and G. E. Wakerlin, Proc. Soc. Exptl. Biol. Med. 44, 277 (1940). a0 H. Goldblatt, Y. J. Katz, H. A. Lewis, E. Richardson, A. Guevara-Rojas, and F. Gollan. Proc. Cen. Soc. Clin. Research lfi, 31 (1942). x, C. A. Johnson, G. E. Wakerlin, and E. L. Smith, J. Immunol. 48, 79 (1944). 12 O. M. IIelmer, R. E. Shipley, J. D. Peirce, and K. G. Kohlstaedt, J. Lab. Clin. Med. 33, 1484 (1948).

[15]

RENIN SUBSTRATE (HYPERTENSINOGEN) FROM HOG SERUM

135

Ultraviolet spectroscopy indicates that the renin-protein complex can be transformed reversibly into three distinct configurations. Identical transformation reactions have not been observed in other proteolytic enzymes such as pepsin, trypsin, and chymotrypsin, and they cannot be induced in other proteins extracted from kidney tissue. The light absorption coefficients for the three forms of renin at the wavelength 275 m~ are : a-renin = 1.0 cm.2/mg. f3-renin = 6.0 cm.2/mg. ~/-renin = 7.2 cm.2/mg.

[15] R e n i n

Substrate (Hypertensinogen) from Hog Serum Renin Renin substrate--* Angiotonin (hypertensinogen) (hypertensin)

By ARDA A. GREEN Assay Method

Principle. Renin substrate, or hypertensinogen, can be properly assayed only in terms of the angiotonin, or hypertensin, formed from it on incubation with renin. The angiotonin so formed is assayed in terms of the rise in blood pressure in an animal on intravenous injection of the angiotonin. Preparation of Angiotonin. Incubate for 20 minutes at pH 7.2 at 38 ° in the presence of 0.03 M NaC1, a 1.5% dialyzed substrate solution and 1~0 vol. of 1% hog renin solution I with an activity of 2 Goldblatt 2 units/mg. Both the substrate and the renin should be free of angiotonase. After incubation deproteinize; bring the pH to 5.6 to 5.7, and add 1.5 vol. of 95% ethyl-alcohol. Allow to stand for 10 minutes; then centrifuge. Wash the precipitate with two times its volume of alcohol of the same concentration, and centrifuge. Concentrate the combined supernatants under nitrogen with reduced pressure at 45 ° to about one-tenth the incubation volume. Impurities interfering with the assay are removed by another alcohol precipitation. Add 5 voh of 95% ethyl alcohol to the above concentrate, and allow to stand overnight at 5 ° . Centrifuge, and wash the precipitate with alcohol of the same concentration. ConcertThe renin used here was only partially purified by an unpublished method. Consult E. Haas, Vol. II [14], for better methods. 2 H. Goldblatt, Y. J. Katz, H. A. Lewis, and E. Richardson, J. Exptl. Med. 77, 309 (1943).

136

ENZYMES OF PROTEIN METABOLISM

[1~]

trate the supernatants under reduced pressure as before. If the incubation volume is small enough, 5 vol. of alcohol can be added immediately and one concentration step omitted. Heating in an acid solution can also be used to deproteinize.8 Bioassay. The unit of angiotonin is a r b i t r a r y and is defined here as the a m o u n t with presser activity equal to t h a t of 1 rag. of a crude preparation kept for a standard. This crude material is about 1/~000 as active as the best material obtained b y E d m a n 3 as determined b y his m e t h o d of assay against t y r a m i n e phosphate. The best material we obtained 4 contained 15,000 units/rag, of peptide N. Assays are performed on dogs. Dogs pretreated b y section of the sine-aortic buffer nerves and injection of t e t r a e t h y l ammonium chloride are more responsive to angiotonin. 5 In anesthetized dogs so treated, 4 units of the reference standard, intravenously injected, causes an arterial blood pressure rise of 40 to 60 mm. of Hg. Activity of the renin substrate is reported as units of angiotonin formed per gram of substrate. The purification is also followed b y ultraviolet absorption of the angiotonin formed. This is calculated as absorption at 275 m~ in a Beckman D U spectrophotometer in a 1-cm. cell in a concentration of 10 anI0 u n i t s giotonin units/ml, and reported in the tables as D275 m,. T h e purer the substrate, the lower this factor becomes. I t starts at 5.0, and the best obtained after extensive purification of the angiotonin itself is 0.01. Purification Procedure

A n u m b e r of purification procedures yielding about tenfold increase in activity have been reported. 6-9 T h e m e t h o d described here was reported b y Green and Bumpus. 1° I t is designed primarily for the preparation of large amounts of renin substrate to be used for the subsequent preparation of angiotonin. A m m o n i u m sulfate fractionations at different acidities are employed, but the most essential step in the procedure is selective acid denaturation. This step removes most of the inactive protein and, also, the angiotonase which destroys angiotonin. Thus both the yield and the p u r i t y are greatly increased b y this one step. 8 p. Edman, Arkiv Kemi, Mineral. Geol. 22, 1 (1945). 4 F. M. Bumpus, A. A. Green, and I. H. Page, J. Biol. Chem. 210, 287 (1954). 5 j. W. MeCubbin and I. H. Page, Am. J. Physiol. 170, 309 (1952). 6 I. H. Page and O. M. Helmer, J. Exptl. Med. 71, 29 (1940). 7 A. A. Plantl, I. H. Page, and W. W. Davis, J. Biol. Chem. 147, 143 (1943). 80. Schales, M. Holden, and S. S. Schales, Arch. Biochem. 2, 67 (1943). 9 E. Braun-Men6ndez, J. C. Fasciolo, L. F. Leloir, J. M. Mufioz, and A. C. Taquini, in "Renal Hypertension" (Dexter, ed.), Charles C Thomas, Springfield, 1946. ~0A. A. Green and F. M. Bumpus, J. Biol. Chem. 210, 281 (1954).

[15]

RENIN SUBSTRATE

(HYPERTENSINOIGEN)

FROM HOG SERUM

137

J

TABLE I ] PURIFICATION OF SUBSTRATE BY SER~ES OF I~EPRECIPITATION8

Series

pH

I II ~ III

6.5 4.0 6.0

10 unite (NH4)~S04,moles/i. Original protein, % Units/g. protein D27~m~ 1.35-2.25 1.2 - 2 . 2 5 1.64-2.1

20 2 1

(10) 2000 4000

5.0 O. 1 O. 05

The protein stood at pH 2.5 in 0.56 M (NH4)~S04for 1 hour at 25° before series II was started. The initial procedure consists of three series of ammonium sulfate precipitations as outlined in Table I. Series I. Uncentrifuged " s e r u m " from 60 1. of hog blood is processed at one time. The serum is obtained by collecting the clots in cheesecloth, leaving most of the serum behind, and then allowing the clots to drain overnight in a large container with a mesh bottom. Although the "ser u m " is uncentrifuged, it is better to decant the relatively clear material after the " s e r u m " has settled overnight and treat it separately from that containing some red cells. Convenient quantities are diluted with 3/~ vol. of 2% saline, and the pH adjusted to 6.5 with 1 M acetate buffer, pH 4. The precipitate formed on the addition of 200 g. of (NH4)2SO4 per liter (1.35 M) is filtered by gravity with the aid of Hyflo Super-Cel and discarded. The filtrate is brought to 2.25 M (NH4)~SO4 by the addition of 142 g./1., and the precipitate is collected. The 2.25 M precipitate is dissolved in a known amount of water and reprecipitated at the same concentration of (NH4)~S04. This is repeated until a blue-green precipitate is obtained. From one to four reprecipitations are required. Series [I. The precipitate from series I is dissolved in 3 vol. of water, brought to pH 2.5 with 1 N HC1, and allowed to stand at room temperature for 1 hour. The solution is then brought to pH 4 with 2 N NaOH and adjusted to 1.2 M (NH4)~SO4 concentration. In calculating the amount of salt to be added, the protein precipitate is assumed to have the same concentration of ammonium sulfate as the solution in which it was precipitated. Also, if a protein precipitate is dissolved in a measured amount of water, the increase in volume is assumed to be equal to the volume of the precipitate. Obviously these assumptions are not exactly correct, but they are adequate and extremely convenient. A thick gummy precipitate is formed which is filtered on eight or ten 381/~-cm. Eaton-Dikeman No. 193 filters in the cold room overnight or over the week-end if preferred. The precipitate is suspended in 6 to 8 1. of water with stirring, again brought to pH 4 and 1.2 M (NH4)~SO4, and

138

ENZYMES OF P R O T E I N METABOLISM

[15]

refiltered. This extraction is carried out five times in all. The fltrate is brought to 2.25 M (NH~)2S04, and the precipitate collected on a 50-cm. Whatman No. 50 filter paper from which it can be scraped cleanly. Series I I I . The precipitate from series II is dissolved in 3 vol. of water, the pH adjusted to 6.0, and (NH4)2SO4 added to 1.64 M. The solution is filtered overnight and the precipitate redissolved in a measured amount of water, usually 1 1. (to approximately the original volume), and (NH4)2SO4 added again to 1.64 M. This process is repeated until five precipitations have been carried out. The filtrates are brought to 2.1 M (NH4):SO4 at pH 6, allowed to settle, decanted as much as possible, and the precipitate collected in a 50-cm. No. 50 Whatman filter. Representative results of the first three series of extractions and precipitations are given in Table I. Series I reduces the amount of protein by 80% of that in the original serum and yields angiotonin equal in purity to our crude standard preparation. The quantity is low if the material is incubated at this stage because much of it is destroyed by angiotonase. Series II removes 90% of the remaining protein, and the ultraviolet absorption at 275 m~ of the angiotonin is reduced fifty times. Series I I I removes half of the protein, doubles the activity per gram of protein, and halves the ultraviolet absorption. The above description deals with a routine method of producing relatively purer substrate and therefore purer angiotonin in large amounts. All steps were finally adopted only if there was relatively little loss of substrate. Electrophoretic analysis of the most active fractions obtained showed two components, one in the a and one in the ~, area. This finding was independent of the pH of fractionation from pH 4 to 7. This suggested that the proteins were precipitating as a complex and that they might be separated if a higher or a lower pH were employed. The acid range proved more practicable; the results of series IV at pH 2.5 are presented in Table II. Series IV. The protein from series III is dissolved in 3 vol. of H~O, most of the (NH~)2S04 necessary to bring the concentration to 1.44 M is added, and the pH is brought to 2.5 with 1 N HC1 after which the reTABLE II SEPARATION BY FRACTIONATION AT PH 2.5

Fraction (NH4)2SO4,1noles~. A B C

1.44 1.64 2.05

Protein, %

Total units

45 47 8

16,000 37,000 1,600

10 u n i t s Units/g. protein D276m~

2800 6500 1600

0.06 0.023 0.1

[16]

ENZYMES IN BLOOD CLOTTING

139

maining (NH4)2SO4 required for the acidified solution is added. The precipitate is removed on an Eaton-Dikeman No. 193 filter paper, redissolved in H20 to one-half the original volume, and reprecipitated at pH 2.5 and 1.44 M. The precipitate is again separated. The combined filtrates are brought to 1.64 M (NH4)2SO4 without pH adjustment, the precipitate, B, is removed on a No. 50 filter, and the filtrate is brought to 2.05 M (NH4)2S04, forming the third precipitate, C. As a result of this fraetionation the 3,-globulin decreases and the a increases with increasing (NH4)2SO4 concentration. The middle fraction, B, is the most active, and the third fraction, which is almost wholly a-globulin, is the least active. This shows only that much of the a-globulin is not renin substrate. Evidence for the inactivity of the 3"-globulin comes from the incubation of fractions removed by syringe and long needle from the Tiselius cell after electrophoresis. The slow 3,-fraction was inactive as was a fast a-fraction, and activity was found in between. Thus, renin substrate is apparently a very small part of the a2-fraction of hog serum. The most active fractions so far obtained yield 30,000 units of angiotonin per gram of protein substrate. They were obtained by repeated precipitations at various acidities and (NH4)2SO~ concentrations, incubating and assaying each fraction and always combining the most active fractions. Since these fractions are, on the basis of electrophoretic analysis, not more than half pure, it would seem that renin substrate is not more than 0.1 to 0.2% of the total protein of hog serum.

[16] Enzymes in Blood Clotting By DANIEL L. KLINE The following hypothesis of the clotting mechanism has been used in order to present the enzymes in blood clotting in an orderly manner. Prothrombin is converted to thrombin by thromboplastin (thrombokinase). The latter is a complex of a stable and a heat-labile factor. The full complex is found in brain, lung, platelets, and other tissues. The heat-labile factor occurs in plasma and has also been designated as the antihemophilic factor (AHF) and plasma thromboplastin component (PTC). A precursor, plasma thromboplastin antecedent (PTA), has also been described. The conversion of prothrombin is accelerated by serum Ac-globulin (accelerin), which exists in plasma as a precursor, plasma Ac-globulin (proaccelerin), and is also found in platelets. Another enzyme, convertin (serum prothrombin conversion accelerator or SPCA,

140

ENZYMES OF PROTEIN METABOLISM

[16]

factor VII), which is activated from a plasma precursor, proconvertin, shortens the prothrombin time. Proconvertin appears to be the chief substance whose synthesis is interfered with by dieoumarol. Thrombin reacts with fibrinogen to form a fibrin clot. The physical qualities of the clot are modified by the presence of a plasma protein. The anticlotting substances are as yet poorly defined. The clot-dissolving system consists of profibrinolysin (plasminogen) which is converted to active fibrinolysin (plasmin) by a number of activators. Two reviews la,b attempt to orient the investigator in the complex subject of the names and functions of the enzymes in blood clotting.

I. Prothrombin Assay Methods Principle. The estimation is based on the time required for the conversion of prothrombin to thrombin and the interaction of the latter with fibrinogen to form a clot. The one-stage method introduced by Quick lc or the two-stage procedure originated by Warner et al., 2 in which the thrombin formed is reacted with fibrinogen in a second tube, may be used. There is no agreement as to the merits of each under various circumstances2 One-Stage: Modification of Owren 4 Reagents Thromboplastin. Human brain is extracted with 1.5 I. of saline per brain at 37 °. The solution is stored at - 2 0 °. Diluting solution. To 800 ml. of 0.9% NaC1 solution add 200 ml. of veronal buffer, pH 7.35 (1.175 g. of sodium diethylbarbiturate to 43 ml. of 0.1 N HC1), and 700 mg. of potassium oxalate monohydrate. CaC12. Usually 0.030 M; an optimal concentration is determined. Prothrombin-free ox plasma. After an initial clarifying filtration, pass oxalated plasma once through 50% asbestos filter paper (400 ml. for 15-cm. diameter). 1~ C.-B. Laurell, Blood 7, 555 (1952). lb j. H. Milstone, Medicine 31, 411 (1952). lc A. J. Quick, M. Stanley-Brown, and F. W. Bancroft, Am. J. Med. Sci. 190, 501 (1935). 2 E. D. Warner, K. M. Brinkhous, and H. P. Smith, Am. J. Physiol. 114, 667 (1936). a Trans. 3rd Conf. on Blood Clotting and Allied Problems (J. E. Flynn, ed.), pp. 135-224, Josiah Macy, Jr., Foundation, New York, 1948. 4 p. A. Owren and K. Aas, Scand. J. Clin. & Lab. Invest. 3, 201 (1951).

[16]

ENZYMES IN BLOOD CLOTTING

141

Stored serum. Add 3 ml. of thromboplastin solution to 100 ml. of blood. Stir. Centrifuge, and store at 4 ° for 10 days. Mix the serum obtained with an equal volume of prothrombin-free ox plasma, and store at - 2 0 ° in quantities suitable for 1 day's use.

Procedure. Mix 4.5 ml. of blood with 0.5 ml. of 0.2% potassium oxalate. Centrifuge. Dilute 0.20 ml. of the plasma with 1.8 ml. of diluting solution. In a small serology tube mix 0.40 ml. of stored serum-prothrombin-free plasma solution + 0.20 ml. of plasma dilution + 0.20 ml. of thromboplastin solution. Place in a water bath at 37 ° for 5 minutes, and then add 0.20 ml. of CaC12 solution of optimal concentration and determine the clotting time. Calculation of Activity. Normal pooled plasma diluted 1:10 is taken as 100%, and dilutions to 10% are made. When the percentage of normal prothrombin is plotted against clotting time on log-log paper, a straight line is obtained. Unknowns are expressed as percentage of normal. Two-Stage: Modification of Ware and Seegers 5 Reagents Thrombin. Dissolve 5000 units of thrombin topical (Parke, Davis and Co.) in 25 ml. of water. Add an equal volume of glycerol, C.P. This solution is stable for months in the refrigerator. Acacia solution. Dissolve 15 g. of powdered U.S.P. acacia in 100 ml. of 0.9% NaC1 solution. Centrifuge to clarify. The Ca content of this preparation is 0.023 M. Imidazole buffer. Dissolve 1.72 g. of imidazole in 90 ml. of 0.1 N HC1, and dilute with water to 100 ml., pH 7.2 to 7.4. Thromboplastin. See preparation from lung or brain. Commercial thromboplastin may be used after resuspension in saline (0.15 g. per 10 ml.). Dilute four to six times with saline for use. The undiluted solution is stable for one week in the refrigerator. Bovine serum. Permit beef blood to clot. Centrifuge after 1 to 2 hours at 2500 r.p.m, for 20 minutes, and remove the serum. Add 200 mg. of powdered BaCOa to a small amount of serum; then add the remainder. Agitate for 10 minutes at room temperature, and centrifuge. The Ac-globulin activity of this prothrombin-free serum is stable for 12 hours at room temperature, for two to three weeks at 5 °, and for at least a year at - 2 0 °. Dilute 600 times for use.

Fibrinogen. Fraction I (Armour and Co., Chicago, Illinois) is suitable. The solution is in 0.9% NaC1 and should contaia 1% clotA. G. Ware a n d W. H. Seegers, Am. J. Clin. Pathol. 19, 471 (1949).

142

ENZYMES OF PROTEIN METABOLISM

[16]

table protein and 5% imidazole buffer by volume. It is stable at - 2 0 ° for months. Reaction mixture. Three parts of thromboplastin in saline, 2 parts of 15% acacia in saline (contains 0.023 M Ca), 0.5 parts of imidazole buffer, 3.5 parts of saline. This mixture can be lyophilized. It can be purchased from the Difco Laboratories, Detroit, Michigan. Three parts of reaction mixture to 1 part of fibrinogen solution should remain clear for at least 3 minutes.

Procedure. After discarding the first 3 ml., draw 9 ml. of blood into a syringe containing 1 ml. of 3.2% sodium citrate (oxalate may be used if Ac-globulin is not to be determined also). Mix the contents, and transfer to a clean, dry test tube. Determine the hematocrit. Keep the blood cold, and centrifuge at 2000 r.p.m, for 20 to 30 minutes. Remove the plasma, and keep at 5 ° or less (use within 2 days at 5°; indefinitely stable at -20°). If stored in dry ice, place the plasma in a sealed container. To 0.5 ml. of plasma in a test tube, add 0.4 ml. of saline and 0.1 ml. of thrombin (100 units/ml.) at room temperature. Normal plasma clots within 15 to 20 seconds. Shortly after the clot forms, remove it by winding it around a glass rod or steel wire. Allow the defibrinated plasma to stand for 10 minutes to permit destruction of the added thrombin. Mix 0.1 ml. of the defibrinated plasma with 2.4 ml. of saline-beef serum diluent. To make diluent, add 0.1 ml. of beef serum to 15 ml. of saline. Use shortly after making. Add 1 ml. of diluted defibrinated plasma to 3 ml. of the reaction mixture, and mix well. Time the beginning of the addition. At approximately 2-minute intervals, add 0.4 ml. of the above mixture to 0.1 ml. of fibrinogen solution in a clean 10 X 75-mm. test tube. Mix immediately, and note the first definite signs of fibrin formation. Tilt the tube gently back and forth and view it against a light source. A cloudiness appears a second or two before the end point. Obtain two or three like readings. The conversion of prothrombin is usually complete in 4 to 6 minutes. If the clotting times are outside of 13 to 17 seconds, make a new dilution. New dilutions are predicted as follows: 10 seconds, dilute twice as much; 22 seconds, one-half as much; 30 seconds, one-third as much. Calculation of Activity. Multiply the dilution (usually 250) by the unit conversion factor (see Table I). For additional accuracy, correct for dilution with the anticoagulant. If the hematocrit is 40%, of 10 ml., 4 ml. is cells, 5 ml. is plasma, and 1 ml. is anticoagulant. Therefore, multiply prothrombin value by 6/g. Definition of Unit and Specific Activity. The prothrombin unit corresponds to the National Institutes of Health thrombin unit, i.e., the

[16]

ENZYMES IN BLOOD CLOTTING

143

TABLE I Clotting time, sec.

Conversion factor (thrombin conc.), units/ml,

13.0 13.2 13.4 13.6 13.8 14.0 14.2 14.4 14.6 14.8 15.0

1.20 1.17 1.16 1.13 1.12 1.10 1.07 1.05 1.03 1.02 1.00

Clotting time, sec.

Conversion factor (thrombin conc.), units/ml.

15.2 15.4 15.6 15.8 16.0 16.2 16.4 16.6 16.8 17.0

0.97 0.96 0.95 0.94 0.92 0.91 O. 89 0.88 0.86 0.85

a m o u n t of enzyme which will clot a s t a n d a r d fibrinogen solution in 15 seconds. Specific activity is expressed as units per milligram of nitrogen, dry weight, or of tryosine. Purification P r o c e d u r e s

Principle. P r o t h r o m b i n is concentrated from dilute plasma b y isoelectric precipitation and adsorption on Mg(OH)2. T h e enzyme is eluted b y decomposing the Mg(OH)~, and the eluate is fractionated with a m m o n i u m sulfate and isoelectric precipitation. Materials

Imidazole buffer. See two-stage assay procedure. Oxalated saline. Mix 0.075 g. of K~C20~.H20 with 0.85 g. of NaC1, and dissolve in 100 ml. of water. Mg(OH)2 cream. Slowly, and with stirring, add 5 1. of concentrated N H ~ O H to 20 1. of 2 0 % MgC12. Wash the settled precipitate several times with water. Suspend 500 g. of centrifuge-packed Mg(OH)2 in 1 1. of water. Procedure. Mix 4.5 gallons of blood with 1 1. of special anticoagulant (1.85% K 2 C 2 Q ' H 2 0 + 0.5% H2C~O4.2H20). Centrifuge, and remove the p l a s m a as soon as possible. If storage is necessary, place at - 2 0 ° . T o 3 1. of oxalated plasma, add 42 1. of cold water, and mix with 1% acetic acid until acidified to p H 5.1. Allow to settle for 4 hours. Decant. Collect

6 W. H. Seegers, E. C. Loomis, and J. M. Vandenbelt, Arch. Biochem. 6, 85 (1945); A. G. Ware and W. H. Seegers, J. Biol. Chem. 174, 565 (1948).

144

ENZYMES OF PROTEIN METABOLISM

[16]

the precipitate by centrifugation in the cold. Add 500 ml. of cold oxalated saline, and disperse with a Waring blendor. Bring the pH to 6.4 with 0.1 N N a O H (approximately 30 ml.). Centrifuge in the cold to remove insoluble proteins. Proceed with the next step at once. Adsorption by and Elution from Mg(OH)2. With mechanical stirring, add 140 ml. of a cold Mg(OH)~ suspension. Centrifuge cold in an angle centrifuge. Wash the precipitate with 200 ml. of cold water. Centrifuge. Repeat the washing and centrifugation. Suspend the Mg(OH)2 in 250 ml. of water. Place in a glass pressure bottle, and decompose the Mg(OH)~ by shaking with CO2 at 2 to 2.5 arm. Cool immediately to 0 °. This product may be stored for 15 hours, or the purification may be continued immediately. Ammonium Sulfate Fractionation. Clarify the above eluate by straining through washed gauze. If the clarified eluate is less than 65% of the starting activity, it is not suitable for fractionation. Approximately 300 ml. of eluate is placed in a 1500-ml. beaker, set in a salt ice bath, and stirred. When 0 ° is reached, saturated ammonium sulfate solution warmed to 26 ° is added dropwise. The temperature is kept at 0 °. When an equal volume of ammonium sulfate has been added, centrifuge for 10 minutes in the cold at 3500 r.p.m. Cool the clear supernatant solution to 0 °, and again add saturated ammonium sulfate solution until the concentration is 65% of saturation. After the addition is complete, permit the temperature to drop to - 5 °, and set in the cold room so that 1 hour elapses from the end of the addition. Decant the prothrombin suspension from the inorganic crystals at the bottom, and centrifuge at 3500 r.p.m. Collect the precipitate in a single centrifuge tube, and dissolve in 10 ml. of cold water. The next step must be carried out at once. Isoelectric Precipitation. Dialyze the above solution against water at pH 7.0 until the specific resistance is from 2000 to 3000 ohms at 5 °. This can be accomplished in 1}~ hours by dialyzing in half-filled casing with a vertical pull once each second. Bring the solution to pH 5.4 by adding 0.1 N HC1 with constant stirring. Centrifuge off a small precipitate. Precipitate the prothrombin by adjusting the pH to 4.6. Centrifuge, and dissolve in 10 ml. of water by cautiously adding 0.1 N NaOH to pH 7 to 7.5. Dry from the frozen state. To remove the remaining Ac-globulin activity, hold the solution at 53 ° for 2 hours in neutral distilled water. Prothrombin is rapidly destroyed at 53 ° in saline solution. If the resulting prothrombin is 23,000 units/mg, tyrosine or less, further purification is accomplished as follows. 7 Twenty milliliters of prothrombin in aqueous solution (0.5% protein, pH 7.2) is mixed with 1 g. of BaCOn. The BaCO~ is removed by centrifugation. Further adsorpW. H. Seegers and N. Alkjaersig, Am. J. Physiol. 172~ 731 (1953).

[16]

ENZYMES IN BLOOD CLOTTING

145

tions of impurities are without effect. When dried from the frozen state, these preparations were stable for 1 m o n t h at room temperature. The yield which has been obtained with this m e t h o d is exceedingly unpredictable, as has been the purity of the product. For a simpler preparation of a less pure material which is free of Ac-globulin, thromboplastin, fibrinogen and antithrombin, see the method of Owren. 8 TABLE II SUMMARY OF PURIFICATION PROCEDURE

Fraction Precipitate at 5.1 Adsorbed and eluted Ammonium sulfate fractionation Isoelectric precipitation BaC03 treatment

Total units 6.4 X 4.1X 1.73 X 1.02 X

105 105 105 105

Specific activity 215/mg. N 1900/mg. N 13,500/mg. tyrosine 23,000-28,000/rag. tyrosine Depends on starting material

Properties Specificity. Prothrombin is the precursor of thrombin which reacts with fibrinogen to form a clot. Activators and Inhibitors. The physiological activators are thromboplastin and Ca. Purified prothrombin is also activated by 25% sodium citrate, ammonium, Mg and N a sulfates, potassium citrate, potassium oxalate, and sodium dihydrogen phosphate. Agents which alter the S-S linkage of proteins inhibit prothrombin, whereas the blocking of SH groups has no effect2 3-Chloro-4,4'-diaminodiphenyl sulfone stimulates the activation with citrate, whereas 2,4,4'-triaminodiphenyl sulfone inhibits. When prothrombin is activated with citrate, 3,4,4'-triaminodiphenyl sulfone inhibits the conversion to thrombin but does not block the disappearance of prothrombin. 1° Stability. Thrombin-free preparations in neutral aqueous solution are stable for months in the deep-freeze. T h e y m a y be dried from the frozen state without loss of activity, but activity is lost in a desiccator at room temperature. Preparations m a y be obtained in a dry and stable form b y precipitating and drying with acetone.

8 p. A. Owren, Acta Med. Scand. Suppl. 194, 5 (1947). 9 j. R. Carter and E. D. Warner, Am. J. Physiol. 173, 109 (1953). 10L. Lorand, N. Alkjaersig, and W. H. Seegers, Arch. Biochem. and Biophys. 45, 312 (1953).

146

ENZYMES OF PROTEIN METABOLISM

[16]

Physicochemical Properties. I~ COMPOSITION. Carbon 44.12, hydrogen 6.51, nitrogen 13.57, sulfur 0.96, ash 0.36, phosphorus 0.00, tyrosine 4.58, tryptophan 3.33, carbohydrate (orcinol) 4.3, glucosamine + . ELECTROPHORETIC MOBILITY.At pH 5.0, 3 × 10-5 cm.2/v./sec., which decreases with pH to 6.2 X 10-5 at 8.0. The mobility places prothrombin between albumin and the a-globulins. About 5 % impurity is present in the best preparations. ISOELECTRIC POINT. 4.2 (by extrapolation). PHYSICAL CONSTANTS. The specific extinction coefficient at 280 m~, 0.75; refractive index at 486 m~, 25 °, increment was 1.89 X 10-3; partial specific volume, 0.70; sedimentation constant, 4.83; diffusion constant 6.24 X 10-7 cm.2/sec. ; viscosity, 0.041; molecular weight by light scattering, 80,000; length, 119 A.; width 34 A.; prolate spheroid. Species Specificity. Human and bovine prothrombin appear to be identical. Nature of Activation. When purified prothrombin is converted to thrombin in the presence of citrate, there is an increase in the nonprotein nitrogen and carbohydrate of the TCA supernatant. There is a lag between the disappearance of prothrombin and the appearance of thrombin. II. Thromboplastin (Thrombokinase) Assay Method

Principle. In an otherwise complete system, the log of the clotting time is inversely proportional to the log of the thromboplastin concentration. As in the case of prothrombin, both one-stage and two-stage procedures are widely employed. One-Stage: Method of Owren 12 Reagents. See prothrombin assay. Procedure. To 0.20 ml. of oxalated plasma (1 ml. of K2C204, 2%, to 9 ml. of blood), add 0.20 ml. of veronal buffer, pH 7.3, and 0.20 ml. of the thromboplastin solution. After heating to 37 °, add 0.20 ml. of CaCI~ solution for optimal recalcification, and determine the clotting time. A plot of the clotting time against thromboplastin concentration gives a straight line on log-log paper in the middle range. At low levels, the thromboplastin present in the oxalated plasma used introduces a large error. 11 F. Lamy and D. F. Waugh, J. Biol. Chem. 205, 489 (1953). ~2 p. A. Owren, Acta Med. Scand. Suppl. 194, 106 (1947).

[16]

ENZYMES IN BLOOD CLOTTING

147

Two-Stage: Method of McClaughry and Seegers 18 Reagents Standard prothrombin solution. A solution of prothrombin containing 6.7 units/ml., which gives a 12-second clotting time with a standardized fibrinogen solution. Ac-globulin (factor V). See method of preparation. Acacia. See prothrombin assay. CaC12. A 1% solution of anhydrous CaCI~ in saline. Imidazole buffer. See prothrombin assay. Fibrinogen. A 1% solution of 95 % clottable fibrinogen.

Procedure. Mix the reagents in the following proportions: standard prothrombin solution, 3 parts; thromboplastin solution, 3 parts; Ac-globulin, 2 parts; CaCI~ solution, 1 part; acacia, 2 parts; imidazole buffer, 1 part. Place this reaction mixture in a water bath at 28 °. At various times, add 0.4 ml. of the mixture to 0.1 ml. of fibrinogen solution, and determine the clotting time. These data show how much thrombiu is formed and how rapidly. The curve obtained with varying quantities of thromboplastin is compared with a standard curve using purified material or may be expressed as a percentage of the maximal thrombin yield. The assay of thromboplastin is not very satisfactory. Schmid TM has reported a method for the inactivation of inhibitors which are found in thromboplastin preparations. The use of platelet-poor plasma as a reagent minimizes the variability of the one-stage test. Assay of Plasma Thromboplastin Component (Antihemophilic Factor) and Stable Platelet Factor: Method of Soulier and Larrieu 14 Reagents Horse plasma (free of all thromboplastins except antihemophilic factor). Centrifuge horse blood for 30 minutes at 2000 r.p.m. Centrifuge the oxalated plasma three times at 24-hour intervals for 1 hour at 4000 r.p.m. Maintain 1° temperature during and between centrifugations. The resulting plasma is incoagulable on recalcification in silicone or glass at 37 °. If clotting should occur, recalcify in the cold with one-fourth the plasma volume of CaC12, 1.11 g. % (0.10 M). The precipitate of Ca oxalate adsorbs the residual thromboplastin. Let settle for 12 hours. Centrifuge. Before freezing, add i part of sodium citrate (3.8%) to 9 parts of plasma. This solution contains thromboplastin 0, prothrombin 50 %, proconvertin 78 %, proaccelerin 100%, fibrinogen 100%, antihemophilic factor 60%. 13 R. I. McClaughry a n d W. H. Seegers, Blood 5~ 303 (1950). 13~ j . Schmid, Acta Haematol. 4, 265 (1950). ~ J. P. Soulier and M. J. Larrieu, J. Lab. Clin. Med. 41, 849 (1953).

148

ENZYMES OF PROTEIN METABOLISM

[16]

Hemophilic plasma reagent. Collect 200 ml. of hemophilic blood, using siliconized apparatus in chilled silicone tubes which contain 0.3 g. of sodium oxalate per 200 ml. of blood. Treat the same as horse blood reagent, and store at - 2 0 °. Traces of AHF are still present, but the solution is not coagulable in the presence of calcium. Platelet reagent. Heat concentrated platelets at 64 ° for 30 minutes. Resuspend with violent stirring for 15 minutes, freeze, and thaw twice. Then centrifuge for 5 minutes at 2000 r.p.m. CaC12, M/40. Procedure. AssAY OF AHF ACTIVITY. TO 0.1 ml. of hemophilic reagent 0.1 ml. of platelet factor ~- 0.1 ml. of test plasma (1:20) add 0.1 ml. of CaCl: solution, and determine the clotting time. The activity is determined from a curve established with various dilutions of normal plasma (1 : 20). AssAY OF PLATELETFACTOR. TO 0.1 ml. of horse plasma -}- 0.1 ml. of test plasma (1:20) add 0.1 ml. of CaC12 solution, and determine the clotting time. Activity is computed from a normal curve as in the antihemophilic factor assay. Definition of Unit and Specific Activity. Thromboplastin activity is usually expressed as a percentage of normal pooled plasma. In the twostage assay, it may be expressed as a percentage of the total possible thrombin yield. Specific activities, in terms of dry weight or nitrogen content. Purification Procedure

Preparation of Thromboplastin .from Lung. Method of Chargaff. 15 One kilogram of finely minced fresh beef lung is extracted with 600 ml. of icecold saline for 3 hours. The mixture is pressed through a canvas bag, and the filtrate is subjected to three centrifugations of 45 minutes each at 0 ° and 4200 r.p.m. The turbid solution is centrifuged at 42,000 r.p.m, in a vacuum ultracentrifuge for 25 minutes. Before each run, the rotor is chilled to 4 °. The pellets obtained are then dissolved in one-third the original volume of borate buffer, pH 8.6 ionic strength 0.15, and again centrifuged at 42,000 r.p.m, for 25 minutes. The sediment, dissolved in the same amount of buffer as the original volume, is centrifuged at 7500 r.p.m, for 20 minutes, and the supernatant obtained is centrifuged at high and low speeds alternately for three to five times. Sediments obtained at low speeds are discarded. Finally, the white pellet obtained is dissolved in borate buffer, pH 8.6, and the colorless, turbid solution is used. The yield is 65 rag. per 100 g. of tissue. 1~E. Chargaff, D. H. Moore, and A. Bendich, J. Biol. Chem. 145, 593 (1942).

[161

ENZYMES IN BLOOD CLOTTING

149

If a continuous supereentrifuge separator is available, the method described by Chargaff et al. ~6 may be used. Preparation of Brain Thromboplaslin (Rabbit). Method of Quick. '7 Rabbit brain may be stored for 24 hours at 4 °. Completely remove the pia and other blood vessels. Triturate with acetone in a glass mortar. Pour off the acetone, and replace with fresh acetone until a granular, nonadhesive product is obtained. Complete the dehydration within 10 minutes. Dry the product thoroughly. If suction is used, care must be taken to avoid condensation of moisture caused by the rapid evaporation of acetone. A final drying at 37 ° for 1 hour is recommended. Store in the cold in sealed, evacuated ampules. Preparation of Thromboplastin Emulsion. Place 0.2 g. of dry material in a small Pyrex tube. Cover with 5 ml. of saline. Mix by blowing through the suspension. Do not triturate. Incubate for 15 to 20 minutes at 50°; then place in a water bath at 37}~°. Agitate occasionally by blowing through with a pipet. Permit the coarse particles to settle, and use 0.1 ml. of this suspension in the analysis by the one-stage method. Purification of Plasma Component of Thromboplastin (Antihemophilic Factor). Is Collect blood with the silicone technique, using one-tenth of the volume of 0.2 M citrate. Centrifuge at 1000 r.p.m, for 15 minutes at 4 °, and transfer the plasma to glass test tubes. Keep at 37 ° for 30 minutes, and centrifuge at 4000 r.p.m, for 30 minutes at 4 °. Treat the supernatant with 50 mg./ml, of BaS04. Wash the B a S Q precipitate with water at 4 °, and then elute with saline or 0.2 M sodium citrate. The total eluate is one-tenth the original volume. The optimal pH for elution is 6.3, the temperature 37 °. Repeat the adsorption and elution. Acidify the eluate to pH 5.0 with CO~ at 4 °, and centrifuge down the precipitate. Add ammonium sulfate to 25 % of saturation, and collect the precipitate. Redissolve to the original volume of eluate in saline, and dialyze against saline for 24 hours at 4 °. Contains 20 units/rag. N. UNIT. The unit is the amount of material which, when added in the volume of 0.1 to 0.9 ml. of platelet-poor native human plasma, will halve the prothrombin activity of the resulting serum 1 hour after completion of clotting as measured by the one-stage prothrombin consumption test. Other Methods. Fraction IV (Armour and Co., Chicago, Illinois) is a potent source of plasma thromboplastin component. Additional purification methods from plasma are available. ~9,2° It has been reported that '~ E. Chargaff, A. Bendich, and S. S. Cohen, J. Biol. Chem. 156, 161 (1944). ~7A. J. Quick, Am. J. Clin. Pathol. 15, 560 (1945). is E. W. Campbell and M. Stefanini, Proc. Soc. Exptl. Biol. Med. 83, 105 (1953). 1~S. G. White, P. M. Aggeler, and M. B. Glendening, Blood 8, 101 (1953). ~-~S. A. Johnson, W. M. Smathers, and C. L. Schneider, Am. J. Physiol. 170, 631 (1952).

150

ENZYMES OF PROTEIN METABOLISM

[16]

a precursor of plasma thromboplastin component (PTC) is present in plas/na and serum ~1 and is adsorbed by BaSO4. It has been called plasma thromboplastin antecedent (PTA). Purification of Stable Thromboplastin Activator from Platelets. Van Crevetd and Paulsen, ~2 using Chargaff's centrifugation technique, described above, on the residue of platelets after water extraction, found the stable factor in the phosphatide fraction.

Properties Specificity. The complete enzyme in the presence of calcium catalyzes the conversion of prothrombin to thrombin. Lung thromboplastin has phosphatase activity. Thromboplastin prepared from one species will be effective in other species but at varying rates. Activators and Inhibitors. Calcium is necessary. The presence of Acglobulin (proaccelerin) and serum prothrombin conversion accelerator (convertin, factor VII) accelerates the conversion of prothrombin to thrombin. Stability. Both the complete and the protein component of thromboplastin are stable at - 2 0 ° for weeks. The plasma component (antihemophilie factor) lost 50% of its activity in one week at 4 °. There was no loss of activity at 56 ° for 30 minutes. A pH of 6.8 is optimal for stability, and phenol stabilizes. Chemical Properties. Complete thromboplastin is a lipoprotein. The lung preparation gives a positive Molisch test, a positive fuchsin test, and a negative D N A reaction. Lipids: one-half of the weight is lost on hot alcohol-ether extraction. The extract contains free cholesterol ]9%, fat 18%, phosphatides 63 %, acetal phosphatides 1.3 %. The residue contains protein, carbohydrate, and ribonucleic acid (1.8 % of the total complex). Both protein and lipid moieties are essential for activity. Plasma thromboplastin is a protein which is nondialyzable and does not settle at 40,000 r.p.m, for 1 hour. It is adsorbed by filter paper with an asbestos content above 20%. Physical Properties. Complete lung thromboplastin has the following constants: partial specific volume at 27 °, 0.87; sedimentation constant S, 330; diffusion constant at 20 °, 0.38 X 10-7, from which the particle size is 167 X 10 e. The molecule is a sphere with a diameter of 80 to 120 m~. The mobility at pH 7.5 is 8.0 X 10-5 cm.2/v./sec.; at pH 8.6, 8.4 X 10-5. 21 R. L. Rosenthal, O. H. Dreskin, and N. Rosenthal~ Proc. Soc. Exptl. Biol. Med. 82, 171 (1953). ~2 S. van Creveld and M. M. P. Paulsen, Lancet 262, 23 (1952).

[16]

ENZYMES I N BLOOD CLOTTING

151

Thromboplastin Complexes Flynn and Coon 23 report that thromboplastin ~- convertin (SPCA, factor VII), thromboplastin-~ Ac-globulin (proaccelerin), or thromboplastin -[- convertin -}- Ac-globulin can be isolated as complexes by centrifugation of plasma at 4 ° for 45 minutes at 40,000 r.p.m. III. Plasma Ac-Globulin (Proaccelerin, Factor V) Assay Method Principle. In an otherwise complete system, the clotting time is inversely related to the Ac-globulin concentration. Either the one- or twostage procedure may be used. The two-stage method is preferable to control SPCA variations. One-Stage Method 24 Procedure. Draw 5 ml. of blood into a syringe containing 1.046% potassium oxalate in a quantity equal to the plasma volume of the blood drawn. Centrifuge. Place the following cold reagents in small tubes: 0.20 ml. of prothrombin (200 prothrombin nnits/ml.), 0.20 ml. of fibrinogen solution (0.4 to 0.5%), 0.2 ml. of plasma diluted to (a) 5%, (b) 2.5%, and (c) 1.25%, and allow to stand at 37 ° for 13 to 4 minutes. Add 0.20 ml. of CaC12 solution (25 mM.) and record the coagulation time. Calculation of Activity. By the use of different dilutions of standard plasma, the relation between the concentration of factor V (Ac-globulin) and clotting time is determined. The concentration of factor V corresponding to the concentration of the test plasma is read off, and the total concentration is calculated on the basis of the dilution used. Three determinations are averaged. The concentration is usually expressed as a percentage of the concentration in standard plasma. Two-Stage Method 25

Ac-globulin may be estimated by a comparison of prothrombin levels with and without Ac-globulin. The test appears to be reliable down to prothrombin levels of - 3 0 %. This test does not detect abnormally high levels. See the two-stage determination of prothrombin for details. Purification Procedure 26 Ox blood is collected in oxalate, quickly cooled, and centrifuged to prevent platelet disintegration. Siliconed apparatus is used. After 2 to 3 ~3j. E. Flynn and R. W. Coon, Am. J. Physiol. 175, 289 (1953). 24p. A. Owren, Acta Scand. Med. Suppl. 194, 260 (1947). 2sA. G. Ware and W. H. Seegers, Am. J. Clin. Pathol. 19, 471 (1949). as p. A. Owren, Acta Scand. Med. Suppl. 194:, 88 (1947).

152

ENZYMES OF PROTEIN METABOLISM

[16]

days at 4 °, the ox plasma is filtered through Seitz Filter (EK), 6 cm. in diameter. The first 10 ml. is discarded whenever a new filter is used. After five filtrations, the plasma is entirely prothrombin free and is low in thromboplastin. One hundred milliliters of prothrombin-free filtrate is diluted with 100 ml. of water and precipitated with 100 ml. of saturated ammonium sulfate solution, pH 7.3 at 20 °. After removal of the precipitate, the supernatant solution is precipitated at 50% ammonium sulfate saturation by the addition of 100 ml. of saturated ammonium sulfate. The precipitate obtained is dissolved in 25 ml. of saline and dialyzed against running water for 24 hours. The sediment is centrifuged off. The solution is adjusted to pH 5.3 with dilute acetic acid and left in the refrigerator for a few hours. The solution is centrifuged, and the sediment is dissolved in distilled water plus sodium bicarbonate to pH 7. Dilute acetic acid is added to pH 6, and after 3 hours in the refrigerator the solution is centrifuged and the sediment discarded. Dilute acetic acid is added to pH 5.2. After several hours the solution is centrifuged The precipitate is dried in vacuo over CaCl~ or dried from the frozen state.

Properties Specificity• The conversion of prothrombin to thrombin by thromboplastin + calcium is accelerated by Ac-globulin. It is not certain whether Ac-globulin is necessary for prothrombin conversion. No marked species specificity has been described. Activators and Inhibilors. Thrombin activates plasma Ac-globulin (proaccelerin) to serum Ac-globulin (accelerin). Citrate either inhibits or destroys plasma Ac-globulin activity. Effect of pH. The enzyme is inactivated by precipitation below pH 4 and is irreversibly destroyed above pH 10.5. Stability. Aqueous solutions lose activity rapidly. In oxalated plasma at 2 °, 30 to 40% of the activity is lost in a week; at room temperature, 50% in 3 days. The enzyme is rapidly inactivated at 56 °, in 20 to 30 minutes at 50 °, but there is no loss in 30 minutes at 37 °. Chemical Properties. Proaccelerin has many properties in common with prothrombin but is more sensitive to alkali and, therefore, cannot be eluted with phosphate or citrate buffers from Mg(OH)2 but can be eluted from Al(OH)3. It is less soluble in ammonium sulfate solutions than prothrombin and is less completely adsorbed by asbestos filters• The purified preparations are water soluble but precipitate at pH 5.4. Plasma Ac-globulin totals 0.4 to 0.5% of the plasma proteins of bovine plasma but is present in smaller concentration in human plasma. Electrophoretic studies at pH 7.4 and 0.2 lomc strength show a mobility of - 4.73 X 10-~

154

ENZYMES OF PROTEIN METABOLISM

[16]

Procedure. Oxalate blood as described for the prothrombin assay. If the proconvertin-free ox plasma contains less than 25 % of normal human prothrombin, dilute the test plasma 1:10 (i.e., no further dilution); if 25% or above, dilute the test plasma to a final dilution of 1:20 by adding an equal volume of diluting solution containing 100 mg. of potassium oxalate per 100 ml. To 0.2 ml. of proconvertin-free ox plasma, add 0.2 ml. of thromboplastin solution, 0.2 ml. of plasma dilution, and recalcify with 0.2 ml. of CaC12 solution at 37 °. Determine the clotting time using various dilutions of normal pooled plasma (1:20 = 100), and plot on log-log paper. The concentration of proconvertin in the unknown may be read directly from this graph as a percentage of normal. P u r i f i c a t i o n P r o c e d u r e 80

To 100 ml. of oxalated human plasma, add 10 g. of BaSO4. Mix for 10 minutes at 25 °, and centrifuge for 15 minutes at 5000 r.p.m. Wash the BaS04 precipitate with saline three times and once with water. Elute proconvertin and prothrombin with 10 ml. of 0.2 M sodium citrate at 25 °. Repeat the elution, and combine eluates. Dialyze the eluate against 0.0005 M phosphate buffer, pH 7.3, then adjust the pH to 5.5 with 1% acetic acid. Centrifuge for 15 minutes at 5000 r.p.m. The precipitate contains prothrombin; the supernatant, proeonvertin. Concentrate the supernatant to 10 ml. by evaporation from a dialysis bag, evaporation in vacuo, or lyophilizatiou. Adjust the salt concentration to 0.85% NaC1 and the pH to 7.3. Comparative data with other methods are not available. The method of Owren 31 should also be consulted. Properties

Specificity. Proconvertin is the precursor of eonvertin. Activators and Inhibitors. Proconvertin is activated in the presence of thromboplastin and calcium. This is one of the earliest detectable events in blood clotting. No specific inhibitors of this conversion have been described. Dicoumarol interferes with its synthesis in the liver. Stability. Proconvertin is relatively stable in stored serum, even at room temperature. It may be preserved indefinitely at - 2 0 °. Chemical Properties. The enzyme is absorbed by BaSO4, BaCOn, and asbestos. I t may be eluted by citrate, but prothrombin is always a contaminant.

n0j. H. Lewis and J. H. Ferguson, J. Clin. Invest. 32, 915 (1953). 81p. A. Owren, Rev. h~natol. 7, 145 (1952).

[16]

ENZYMES IN BLOOD CLOTTING

155

VI. Convertin (SPCA, Factor VII, Cothromboplastin) Assay Method 32 In an otherwise complete system, the clotting time is inversely proportional to the enzyme concentration. Convertin affects the rate of prothrombin conversion but not the amount.

Reagents SPCA-free plasma. Filter the plasma slowly through a Seitz asbestos filter pad (50 to 60 drops per minute at room temperature). Freeze in small portions, and keep in a deep-freeze. Thromboplastin. To produce clotting in 11 seconds. See assay of prothrombin and preparation of thromboplastin. Veronal-acetate buffer, pH 7.35. Dissolve 9.714 g. of sodium acetate trihydrate -}- 14.714 g. of sodium veronal in 500 ml. of water. The complete buffer comprises 250 ml. of veronal-acetate solution -~ 200 ml. of 4.25% NaC1 solution ~ 217 ml. of 0.1 N HC1 -t- 683 ml. of water. CaCI:. 3.675 g. of CaCl~'2H20 per liter (M/40).

Procedure. Place 9 ml. of blood and 1 ml. of sodium oxalate (1.34%) in a siliconed vessel. Centrifuge, and dilute the plasma 1:10 with buffer. At 37 ° mix 0.1 ml. of diluted plasma, 0.1 ml. of SPCA-free plasma, and 0.1 ml. of thromboplastin solution. After 20 seconds, add 0.1 ml. of CaC12 solution. Determine the clotting time. Unit and Specific Activity. Prepare a standard curve of normal human plasma diluted 1 : 10 as 100 % down to 10 %. A plot of clotting time versus percentage of normal gives a straight line on double log paper. Normal is 20 to 25 seconds. Purification Procedure TM

From Plasma. Ten grams of BaSO4 is suspended in 200 ml. of oxalated plasma at 2 °, stirred for 40 minutes, and the suspension centrifuged. The supernatant solution is discarded. The BaS04 is washed twice with 30 ml. of saline at 2 °. Chromatography: At room temperature, a homogeneous mixture of B a S Q (10 g.) containing adsorbed SPCA and Hyflo SuperCel (5 g.) suspended in saline is introduced into a chromatography tube. Any excess saline is discarded. The prothrombin is eluted by means of 150 ml. of 0.14 M trisodium citrate-citric acid solution, pH 5.8. Elution is continued with a 0.14 M trisodium solution, pH 7.8. Prothrombin comes off the column first, then prothrombin and SPCA, and finally, ~2 F. Koller, A. Loeliger, and F. Duckert, Rev. h$matol. 7, 156 (1952). 3 a ~ F. Duckert, F. Koller, and M. Matter, Proc. Soc. Exptl. Biol. Med. 82, 259 (1953).

156

ENZYMES OF PROTEIN METABOLISM

[16]

after 200 ml. of pH 7.8 citrate, SPCA comes off free of other factors. The rate of flow is 1 drop in 12 seconds. From Serum. Twenty grams of BaSO4 is suspended in 200 ml. of diluted serum (9 parts of serum ~- 1 part of 0.1 M sodium oxalate). The suspension is stirred for 45 minutes and centrifuged. The BaSO4 is washed twice with saline and once with 30 ml. of 0.006 M sodium citrate. SPCA is eluted by suspending the washed BaSO4 in 30 ml. of 0.14 M citrate, pH 7.8, stirring for 30 minutes, and centrifuging. SPCA is in the supernatant. The yield is 90%. It is noteworthy that this procedure is almost identical with that described earlier by Milstone 3~bfor the preparation of thrombokinase. Properties

Specificity. SPCA is not species specific. It shortens the prothrombin time but does not affect prothrombin consumption (thrombin yield). Activators and Inhibitors. See SPCA precursor. Effect of pH. Optimal activity is shown at pH 7.3 to 7.4. Stability. At room temperature, SPCA loses 80 % of activity in a week; at 37 ° full activity is retained for 5 hours; is heat stable at 45 °. The enzyme may be preserved at 5 ° for at least six weeks, indefinitely at - 2 0 °. It is pH stable between 5 and 8. Chemical Properties. SPCA is quantitatively adsorbed from plasma or serum by BaSO4, BaCOn, and 20% asbestos filter paper. It is eluted by citrate. The enzyme is not precipitated at pH 5.1 or 5.7 but is at 5.3. It is not dialyzable. Physiological Properties. SPCA is inactive in the absence of Acglobulin. VII. Thrombin Assay Method 34 The clotting time of a fibrinogen solution is inversely related to the concentration of thrombin added. The dilutions used minimize interference by antithrombin.

Reagent Thrombin titration mixture. 6.5 parts of saline, 2 parts of 15 % acacia in saline (commercial acacia contains Ca), 0.5 part of imidazole buffer.

Procedure. Add 0.1 ml. of thrombin solution (100 units/ml.) to 1.9 ml. of saline. Transfer 0.5 ml. of this diluted thrombin to 1.5 ml. of titration j. H. Milstone, J. Gen. Physiol. 35, 67 (1951). ~4 A. G. V~Tare and ~r. H. Seegers, Am. J. Clin. Pathol. 19, 471 (1949). 3Sb

[16]

ENZYMES ZN BLOOD CLOTTZNG

157

mixture. Add 0.4 ml. of the second solution to 0.1 ml. of fibrinogen contained in a 10 X 75-mm. tube. (See prothrombin assay for fibrinogen solution.) Mix immediately, and note the clotting time. If the clotting time is outside of 13 to 17 seconds, use a new dilution. Definition of Unit and Specific Activity. In this assay, the dilution, which is usually 100, is multiplied by the unit conversion factor (see the table for calculation of prothrombin units) to obtain units per milliliter of test solution. These units should be equal to and may be compared with a standard thrombin preparation which can be obtained from the National Institutes of Health, Bethesda, Maryland. The N.I.H. standard is that amount of thrombin which will produce a clot in 15 seconds in a standard fibrinogen solution. Specific activity is usually expressed in terms of units per milliliter of solution, although dry weight or nitrogen content of purified material may be used. Purification Procedure 35,36

Thrombin may be prepared from plasma, s7 but the purest preparations are those obtained from purified prothrombin in the presence of citrate. A small amount of 3-methyl-4,6,4'-triaminodiphenyl sulfone is added to 15 ml. of 1% prothrombin solution (1400 units/mg, dry weight) in 25% sodium citrate solution. This is allowed to stand at room temperature, and at the end of 24 hours the yield of thrombin is complete. The thrombin in citrate solution is diluted to 100 ml. with water and precipitated by slowly adding 200 ml. of saturated ammonium sulfate solution. The temperature of the mixture is kept at 0 °. The precipitate is then dissolved in 10 to 15 ml. of water and dialyzed against cold water. When, in about 11/~ hours, the specifc resistance is near 6000 ohms, the thrombin is dried from the frozen state. There is no loss of activity during drying, and the dry material is stable. The specific activity is usually 17,000 to 19,000 units/mg, of tyrosine. Properties Spec~city. The enzyme converts fibrinogen to fibrin enzymatically. Thrombin has also been reported to activate plasma Ac-globulin and to lyse platelets2 s Thrombin activation of plasma thromboplastin (antihemophilic factor) has also been claimed. 39 35w. H. Seegers, R. I. McClaughry, and J. L. Fahey, Blood 5, 421 (1950). 36p. D. Klein and W. H. Seegers, Blood 6, 742 (1950). 37W. H. Seegers and H. P. Smith, Am. J. Physiol. 137, 348 (1942); W. H. Seegers and D. A. McGinty, J. Biol. Chem. 146, 511 (1942). 3sE. de Robertis, P. Paseyro, and M. Reissig, Blood 8, 587 (1953). 39A. J. Quick, C. V. Hussey, and E. Epstein, Am. J. Physiol. 174, 123 (1953).

158

ENZYMES OF PROTEIN METABOLISM

[16]

Activators and Inhibitors. No specific activator is needed, but calci~um and a plasma factor (see Fibrinogen) are needed to form a urearesistant fibrin clot. Thrombin is inhibited by heparin in the presence of a cofactor which is present in plasma and mast cells and by antithrombin. 4° Effect of pH. The pH for optimal activity is about 7.3 to 7.4. Stability. Thrombin is stable in the dry form and retains its activity for long periods at 4 °. There is some inactivation at 40 ° in 30 minutes, rapid inactivation at 60 °. Below pH 4.1 and above 10, thrombin is inactivated. Glycerol can stabilize thrombin. Chemical Properties. There are two active electrophoretic components. Solubility curves were inhomogeneous, but the specific activity of dissolved and undissolved material was equal. There appeared to be two proteins with equal thrombin activity. Thrombin precipitates from pH 5.1 to 3.4. Sulfur and carbohydrate (5 to 6%) are present in the molecule. During clot formation, thrombin is adsorbed to fibrinogen and may be recoyered by lysis of the clot. Thrombin also adsorbs strongly to glass. Purified thrombin had no fibrinolytic activity. VIII. Fibrinogen Assay Method Fibrinogen, in the presence of purified thrombin, is quantitatively converted to fibrin. The precipitation of fibrinogen from solution does not appear to be specific enough for its determination. Procedure. 4~ Mix 1 ml. of a 0.2 to 0.8% fibrinogen solution with 30 ml. of saline containing 1 ml. of M / 5 phosphate buffer, pH 6.4, and 10.5 ml. of 1% CaC12. To the diluted fibrinogen, add 1 ml. of a 50% glycerol solution containing about 100 units of thrombin (commercial) in a 30 X 120-mm. tube. Mix immediately by inversion, and allow to s~and for 30 minutes at room temperature. Remove the fibrin clot by means of a stainless steel wire, and express the remaining fluid mechanically with the aid of filter paper. Wash the clot with tap water, then saline. Definition of Unit and Specific Activity. The quantity of fibrin may he determined by nitrogen or tyrosine analysis or by dry weight. Specific activity of fibrinogen is usually expressed as clottability which is determined by analyses of the fibrinogen solution before and after clotting.

Purification Procedure

From Blood. 41 Twenty liters of bovine blood is mixed with 1 1. of special anticoagulant (1.85 % potassium citrate dihydrate ~ 0.5 % citric acid 40 T. Astrup and S. Darling, Acta Physiol. Scan& 6~ 13 (1943). 41 A. G. Ware, M. M. Guest, and W. H. Seegers, Arch. Biochem. 18, 231 (1947).

[16]

ENZYMES IN BLOOD CLOTTING

159

dihydrate). The oxalated blood is centrifuged, and the plasma is poured into a 1-gallon metal tin can and frozen solid at - 30 °. This is then placed in a refrigerator at 5 °. Two days later, the plasma contains no ice. Two clean trunnion cups (21/~ inches i.d. X 41/~ inches deep) are cooled in an ice bath. Part of the cold plasma is poured into these and centrifuged at room temperature at 2500 r.p.m, for 1 minute. After decanting, this is repeated with cold plasma until all the fibrinogen has been collected. The centrifuge cups are again placed in an ice bath. A total of 110 ml. of ice-cold saline is then poured over the fibrinogen. Thorough mixing is achieved by using a strong glass rod fitted at one end with a rubber stopper. Mechanical stirring denatures fibrinogen. This is again centrifuged for 1 minute. After decantation, the cups are returned to the ice bath. Again 110 ml. of ice-cold saline is added and mixed with the fibrinogen. The solution is centrifuged for 1 minute at 2500 r.p.m. Washing is repeated for a total of five more times with 100, 90, 80, 70, and 50 ml. of ice-cold saline. The final centrifuging is for 3 minutes. The fibrinogen is then suspended in 200 ml. of saline and set in a bath at 35 °. The mixture is kept in constant motion by means of the special stirrer until the temperature is 33 °. The fibrinogen solution is then placed in one metal centrifuge cup and is centrifuged for 2 hours at room temperature at 2500 r.p.m. The clear fibrinogen solution is then decanted. The fibrinogen is 97.5 % clottable and represents about a 2.6 % solution. The clot formed with thrombin has a high tensile strength and is free of lytic factors. From Fraction I (Armour and Co.). 42 Two grams of Armour bovine fibrinogen, 60 to 70% clottable, is dissolved in about 200 ml. of borate buffer (11.25 g. of boric acid + 4 g. of sodium borate. 10H20 + 2.25 g. of NaC1) pH 7.75, and the solution is filtered. Prothrombin is removed by BaSO4 adsorption (50 mg./ml.), and the fibrinogen is then precipitated three times, respectively, with 25, 20, and 20% saturated ammonium sulfate solution. After dialysis against borate buffer, the solution is adjusted to final desired strength by nitrogen analysis or thrombinclottable fibrinogen assay. This method yields 97 % clottable protein. The simpler method of Laki 43 may also be used.

Properties Fibrinogen is not an enzyme, strictly speaking, but is quantitatively converted to fibrin by thrombin. Activators and Inhibitors. Substances which accelerate the thrombinfibrinogen reaction are known as fibrinoplastie. Acacia is an example of 4~j. H. Ferguson in "Blood Cells and Plasma Proteins" (J. L. Tullis, ed.), p. 85, Academic Press, New York, 1953. 4~ K. Laki, Arch. Biochem. and Biophys. 32j 317 (1951).

160

ENZYMES OF P R O T E I N METABOLISM

[16]

this nonspecific type of action. Calcium enters into the p r i m a r y polymerization process and is necessary for the formation of a urea-insoluble clot. 44 In addition to calcium a thermolabile serum factor is needed for the formation of a urea-resistant clot. 45 Thioglycollate and glutathione act like the serum factor. High calcium concentrations are inhibitory (decrease the velocity), and thrombin activity is slowed at high ionic strength. Effect of pH. Bovine and h u m a n fibrinogen show maximal activity at p H 6.4 to 6.6. The optimal p H for other species is different. Stability. Fibrinogen is stable in the d r y state at 4 ° or below. Storage of solutions in 2 % saline has been recommended but is of doubtful value. On thawing, an insoluble material appears and must be centrifuged off. This is probably profibrin which can be completely removed b y the addition to the cold solution of 5 % kaolin or 2 % aluminum oxide. 46 Chemical Properties. The physicochemical processes involved in fibrin formation have been intensively studied by Morrison and Seiler. 47 Fibrinogen is a rod-like particle, 700 to 800 A. X 40 A., molecular weight 350,000. Mannose, galactose, and glucosamine are part of the fibrinogen molecule. T h e y are also found in fibrin in which the hexose is decreased and glucosamine is unchanged. 4s Fibrinogen, on conversion to fibrin, shows a change from tyrosine and glutamic acid end groups to tyrosine and glycine. Two peptides are split off. Both contain m a n y amino acids in common. Peptide A contains serine as well; peptide B contains alanine and tyrosine which are not present in peptide A. 49

IX. hntithromboplastins Assay Method Principle. T h e delay in coagulation of recalcified, citrated plasma activated by strong thromboplastin solution is proportional to the concentration of inhibitor enzyme present. Procedure? ° Varying amounts of inhibitor are added to the reagents of the one-stage prothrombin assay, and the clotting time is recorded. Definition of Units and Specific Activity. The delay in clotting time is compared to the effect of standard heparin, and a corresponding unit

44S. Katz, S. Shuhnan, I. Tinoco, Jr.. I. H. Bullock, K. Gutfreund, and J. D. Ferry, Arch. Biochem. and Biophys. 47, 165 (1953). 45S. Shulman, A.ature 171, 606 (1953). 46p. A. Owren, Acta Med. Scan& Suppl. 194, 133 (1947). 47p. R. Morrison and A. Seiler, Am. J. Physiol. 168, 421 (1952). 48 S. Sz~ra and D. D. Bagdy, Biochirn. et Biophys. Acta 11, 313 (1953). ~9F. R. Bettelheim and K. Bailey, Biochim. el Biophys. Acta 9, 578 (1952). 50L. M. Tocantins, R. T. Carroll, and T. J. McBride, Proc. Soc. Expil. Biol. Med. {}8, 110 (1948).

[16]

ENZYMES IN BLOOD CLOTTING

161

is determined. The specific activity is expressed as units per milliliter or milligram.

Purification ProcedureS°-Lipid Antithromboplastin Thirty grams of acetone-dried brain powder is extracted for 5 to 6 days at 5° with 600 ml. of absolute methanol. The supernatant is filtered, and the filtrate is distilled in vacuo in a flask immersed in a water bath at 38 to 40 °. The residue remaining in the flask after distillation is complete is removed with absolute ethyl ether. The ether solution is kept at 5 ° overnight, during which time a white precipitate settles out. The supernatant is decanted off, and the precipitate washed with cold ether. The combined ether extracts are evaporated off in vaeuo, leaving a creamy, white waxy powder. A 10% suspension is made in saline, put through a homogenizer seven to ten times, and the pH adjusted to 7.3.

Properties Specificity. The inhibitor has no effect on thrombin, does not bind calcium, and does not alter the fibrinogen to fibrin rate. Some species specificity is shown. Stability. In saline solution at 65 to 70 ° for 10 minutes, almost complete inactivation occurred. The material is sensitive to acid and alkali. Chemical Properties. The enzyme is soluble in most lipid solvents and warm ethanol, is insoluble in acetone. The Molisch test is negative; biuret, xanthoproteic, and Acree-Rosenheim tests are positive. Fraction IV of Cohn is a source.

Purification Procedure-Protein Anfithromboplastin sl Rabbit muscle is ground and extracted twice with 1 vol. of water, then filtered through gauze. The combined extracts are filtered through cotton, then through Whatman No. 1 filter paper. The extract is adjusted to pH 5.7 with 0.15 M acetate, pH 4.6, and centrifuged for 15 minutes at 10,000 r.p.m. The precipitate is dissolved in a minimum of 0.02 N NaOH (usually 15 ml. per 500 g. of muscle), with the pH kept between 7 and 9. The solution is clarified by centrifugation at 10,000 r.p.m, for 15 minutes. The solution is then fractionated with ammonium sulfate, and the precipitate between 45 and 70% saturation is collected and dissolved in saline. The solution is then adsorbed on 0.2 vol. of alumina gel (Cv). The enzyme is eluted with 5 vol. of a solution of 30 % saturated ammonium sulfate + 0.10 M disodium phosphate. The eluate is fractionated with ammonium sulfate between 45 and 70% saturation. All 51J.-C. Dreyfuss, Biochim. et Biophys. Acta 11, 313 (1953).

162

ENZYMES OF ]PROTEIN METABOLISM

[16]

procedures are carried out in the cold. The purified material contained no accelerators. Twenty milligrams was obtained per 500 g. of muscle. This inhibitor may be similar to that reported by Fiale 5~ and Lanchantin and Ware. 5a

Properties

Specificity. There is no effect on the the conversion of purified prothrombin stage test prolonged. Chemical Properties. The enzyme is a inhibited by protamine. There is no lipid

conversion of thrombin or on to thrombin but is the oneheat-labile protein and is not component.

X. Antithrombin Assay Method ~ Introduce 7 parts of blood to 1 part of 1.85% potassium oxalate. Mix, and centrifuge at 3000 to 4,000 r.p.m, for 10 to 15 minutes. Remove the plasma. Defibrinate the plasma by heating at 56 ° for 3 to 5 minutes. Centrifuge. Mix 0.5 ml. of each standard thrombin solution (for human, 1100 units) with 0.5 ml. of defibrinated plasma in paraffin-lined tubes. Allow the reaction to proceed for 2 hours at 28 °. Place the tubes in ice water until final thrombin assays are completed by a method which measures thrombin quantitatively. The method of Astrup and Darling 5~ may also be used. Calculations. The percentage of thrombin destroyed per milliliter of oxalated plasma equals T --T2(t) X 100, and the thrombin units destroyed by 1 ml. of plasma equals T -- 2(t), where T is the concentration of thrombin added (units/ml.) and t is the concentration of thrombin at equilibrium (units/0.5 ml.).

Purification Procedure No method is available for the purification of antithrombin.

Properties

Stability. Activity was retained without loss for four weeks in the refrigerator; by the fifteenth week all activity was lost. Freeze-drying and thawing had no effect. The enzyme is destroyed above 56 °. Chemical Properties. Antithrombin does not dialyze from serum. 52 S. Fiale, Arch. intern. Physiol. 68, 386 (1951). 6s G. F. Lanchantin and A. G. Ware, J. Clin. Invest. 32, 381 (1953). 54 W. H. Seegers, K. D. Miller, E. B. Andrews, and R. C. Murphy, Am. J. Physiol. 169, 700 (1952). 65 T. Astrup and S. Darling, Acta Physiol. Scan& 6, 13 (1943).

[16]

ENZYMES IN BLOOD CLOTTING

163

XI. Cofactor of Heparin Assay Method Antithrombin activity is determined in the presence and absence of heparin. The difference in antithrombic activity is a measure of the heparin cofactor.

Purification Procedure from Mast Cells 56 Fresh liver capsules are stripped off and mechanically freed of liver cells. The sheaths are frozen in solid COs and minced three times in a handmill. The resulting material is very gently ground in a mortar for 5 minutes. Small amounts of isotonic phosphate buffer, pH 7.0, are added, and a reddish, gelatinous mass is obtained. More solvent is added gradually to a final volume of four times the original pulp. The following differential centrifugation at 4 ° is carried out to obtain fraction $3: centrifuge for 6.5 minutes at 800 )< g, discard the sediment; centrifuge for 15 minutes at 40,000 X g, discard the sediment; centrifuge for 6 hours at 50,000 × g. The final supernatant is $3. To freshly prepared $3 in phosphate buffer at pH 7.0 at 4 ° add phosphate to complete saturation whereupon the protein and lipid material precipitate and polysaccharides remain in solution. Shake the precipitate with 2 to 3 vol. of a mixture of 1 : 1 ethanol and ether at 4 ° for 48 hours. The dissolved lipids are then recovered by drying in vacuo. Five hundred grams of capsules yielded a phosphate precipitate of 150 g. After separation, protein and salts totaled 50 g., lipid 10 g., heparin 2 mg.

Properties Chemical Properties. Electrophoretic studies show a mobility of 5.9 × 10-s cm.2/v./sec, for the active component of $3. This can be salted out with phosphate buffer, pH 7.2, from 65 to 80% saturation at 20 °. This fraction is homogeneous in the ultracentrifuge ($20, 4.4). After protamine treatment to remove heparin, the mobility approached that of albumin. Nucleic acid is absent. The protein portion is a polypeptide containing six amino acids, mol. wt. 8000. The lipid portion contains lecithin, cholesterol, and neutral fat. Both portions are needed for activity. Thioglycollate, dithionite, and thiobenzoic acid reduce the antithrombin effect.

XII. Profibrinolysin (Plasminogen)

Assay Method Principle. Any standard technique for measuring proteolysis may be used. More sensitive assays involve the action of the enzyme on a fibrin clot. 56 O. Snellman, B. Sylv6n, and C. Jul6n, Biochim. et Biophys. Aa a 7, 98 (1951).

164

ENZYMES OF PROTEIN METABOLISM

[16]

Clot Method 5~

To a small tube add 0.5 ml. of 1% bovine fraction I (fibrinogen) and 0.5 ml. of borate buffer, pH 7.75. To a second tube add 0.5 ml. of enzyme solution (activated), 0.5 ml. of borate buffer, 0.2 ml. of thrombin (20 units/ml.). The activator is usually in the borate buffer. Mix the contents of the tubes by pouring back and forth and then incubate at 37 °. The lysis time is that required for the complete disappearance of the gelatinous clot. Units. One hundred units is that amount of fibrinolysin which will lyse the clot in 5 minutes (unit = 500/lysis time). Plate Method 5s Reagents

Fibrinogen. 40 to 50% clottable or more, 0.2%. Thrombin. 0.20 ml. (100 units/ml.). Buffer. M / 1 5 phosphate or diethylbarbiturate, 0.1 M, pH 7.15. Petri dishes. 10-cm. diameter with even thickness bottoms. Cleaned and sterilized before use. Procedure. Buffered fibrinogen solutions are poured into petri dishes and clotted with thrombin. On the surface of the fibrin, 0.030 ml. of the enzyme solution is placed as small drops (3 drops on each plate), and the dishes are incubated at 32 ° for about 18 hours. The lysed zones are measured (in square millimeters), and the product of two perpendicular diameters is taken as a measure of the lysis, based on the average of three zones. Units. The results are plotted with the concentration of enzyme as abscissa and the product of the diameters as ordinate. A dilution curve of the unknown solution is compared to a standard dilution curve. Purification P r o c e d u r e 59

The starting material is fraction III. 6° After an initial extraction with 0.05 N H~S04 (1 g. per 20 ml.) for 10 minutes at room temperature, the resulting suspension is centrifuged at 2500 r.p.m, for 10 minutes. After removal, by means of a policeman, of floating lipids which occasionally appear, the supernatant solution is decanted and adjusted to pH 11 with N NaOH. The pH is then immediately brought to 5.3 with N HC1, and the preparation is stored at 4 ° for a minimum of 3 hours. 57 j . It. Lewis a n d J. H. Ferguson, Am. J. Physiol. 170, 636 (1952). 58 S. Miillertz, Ac~a Physiol. Scand. 25, 93 (1952). .~9 D. L. Kline, J. Biol. C'hem. 204, 949 (1953). 6o j. T. Edsall, Advances in Protein Chem. S, 383 (1947).

[16]

ENZYMES IN BLOOD CLOTTING

165

The pH of the solution is adjusted to 2.0 with N HC1 and centrifuged for 1 hour at 2700 r.p.m, or more. The supernantant solution containing profibrinolysin is carefully decanted. The solution is adjusted to pH 8.6. If a visible precipitate remains undissolved, the solution is adjusted to pH 9.0 and centrifuged. Slowly, with stirring, 1 ml. of 0.02 M phosphate buffer, pH 6.0, is added per 100 ml. of solution, and the solution is stored in the refrigerator for at least 1 hour, preferably overnight. It is then centrifuged, and the profibrinolysin dissolved in distilled water with the aid of a few drops of N HC1. Properties Specificity. The activated enzyme dissolves clots in vitro and in vivo. Activators and Inhibitors. Many activators have been described. The

most important appear to be chloroform, protamine, streptokinase, staphylokinase, and tissue activators. The bacterial activators show species specificity. Chemical Properties. The enzyme is a nondialyzable protein which coprecipitates readily. It is relatively heat-, acid-, and alkali-stable, although it is inactivated above pH 10. XIII. Antifibrinolysin Assay Method Proteolytic or fibrinolytic assays are carried out in the presence of varying concentrations of inhibitor. Purification Procedure 6~ Grind ox lung finely five times in a meat grinder. Then suspend the pulp in saline with an excess of xylol, and centrifuge. Repeat twice. Leave suspension in the refrigerator overnight. Centrifuge. Repeat the saline-xylol wash twice, and follow by three washes with water. After dehydration with acetone (three times), wash with ether and dry in air. An almost white powder is obtained. The starting material must be ox lung. Properties The spontaneous activity of bovine plasmin and of streptokinase-activated human plasmin is inhibited. The material is a potent trypsin inhibitor. It is not heparin. ~ T. Astrup, Acta Physiol. Scan& 26, 243 (1952).

166

ENZYMES OF PROTEIN METABOLISM

[16]

XIV. Fibrinolytic Activators from Tissues Methods of Preparation Fibrinokinase. 6~,63 Pig heart is ground with sand and washed with water. The residue is extracted for 1 hour with M K S C N and then filtered through gauze. The filtrate is dialyzed against diethylbarbiturate buffer, pH 7.8, ionic strength 0.05. I t is then precipitated with acetone and dried with acetone and ether. Fibrinolysokinase. 64 Perfuse lung with cold saline-phosphate buffer (0.85 % NaC1 buffered at pH 7.3 with 0.0025 M phosphate). Macerate in a Waring blendor, strain, and centrifuge the supernatant at 20,000 X g for 90 minutes. Wash the sediment with saline buffer, and suspend in a volume of buffer equal to half the original lung weight with the aid of a Waring blendor. Urine Activator. e5 Adjust fresh urine to pH 9.5. Centrifuge, and neutralize to litmus. Cool, and precipitate the enzyme with 3 vol. of icecold 95% ethanol. After 15 minutes, centrifuge. Dissolve in a small volume of saline, and dialyze overnight against saline. Centrifuge, and lyophilize the supernatant solution. Yield, 500 rag./1. M e a s u r e m e n t of Activity. Methods for the assay of the activators 6~ and inhibitors 67,6s of fibrinolysin have been described, but the desired precision has not yet been obtained.

62p. M. Pertain, Nature 160, 571 (1947). 6aT. Astrup and A. Stage, Nature 170, 929 (1952). 64j. H. Lewis and J. H. Ferguson, J. Clin. Invest. 29, 486, 1059 (1950). 65T. Astrup and I. Sterndorff, Proc. Soc. Exptl. Biol. Med. 81, 675 (1952). ,s A. E. Wasserman, J. L. Ciminera, L. Hayfliek, and W. F. Verwey, J. Lab. Clin. Med. 41, 812 (1953). 67D. G. C. Clark, E. E. Cliffton, and B. L. Newton, Proc. Soc. Exptl. Biol. Med. 69, 276 (1948). 6s A. Bertoye and P. Detolle, Ann. inst. Pasteur 83, 248 (1952).

[17]

ENZYMATIC DEGRADATION OF CYTOCHROME C

167

[17] E n z y m a t i c D e g r a d a t i o n of C y t o c h r o m e C Cytochrome c (Fe +++) ~- H20--~ Hematin peptide (Fe +++) + Amino acids (1) Succinate -t- Hematin peptide (Fe +++) -* Fumarate -t- Hematin peptide (Fe ++) (2) O2 ~- Hematin peptide (Fe ++) -~ Verdohemochrome peptide

(3) By HERSCHEL K. MITCHELL and LEONARD A. I~ERZENBERG Assay Method

Principle. The over-all reaction produces a visible color change from red to green. 1 The green compound has absorption bands at 655 mt~, 530 m~, and 410 m#. The course of the primary reaction which hydrolyzes about 80% of the cytochrome peptide bonds can be observed by means of quantitative ninhydrin analyses. 2 The second, oxidative step, which requires reduction of the iron (in this case by means of succinic dehydrogenase and succinate) can be followed spectrophotometrically, fluorometrically, or manometrically. The first method, based on the large decrease of the optical density at 410 m~ during the course of the reaction, is described below. Reagents

Buffers. 0.05 M phosphate, pH 6.9, containing 75 g./1. of mannitol. 0.1 M Tris-HC1, pH 8.7. Sodium succinate, 2.5 × 10-2 M in buffer. Cytochrome c (Sigma), 5 mg./ml, in buffer (2.5 × 10-4 M). Enzyme. Mitoehondria from young cultures of the poky strain of Neurospora as described below. Procedure. Flask A containing 0.8 ml. each of cytochrome, succinate, and enzyme plus 2.0 ml. of pH 6.9 buffer, and flask B containing 0.8 ml. each of cytochrome and enzyme plus 2.8 ml. of pH 6.9 buffer are incubated with shaking at 35 °. At the time of mixing and at 10-minute intervals, 0.5 ml. samples are removed and pipetted into a mixture of 1.0 ml. of pH 6.9 buffer and 1.5 ml. of pH 8.7 buffer. The pH of the resulting mixture is 8.3. The optical density at 410 m# of the A series is measured in a

F. A. Haskins, A. Tissieres, H. K. Mitchell, and M. B. Mitchell, J. Biol. Chem. 200, 819 (1953). W. Troll and R. K. Cannan, J. Biol. Chem. 200, 803 (1953).

168

ENZYMES OF PROTEIN METABOLISM

[17]

Beckman DU spectrophotometer using the B series as blanks for zeroing the instrument.

Purification Procedure The cytochrome-destroying system described mitochondria of the poky strain of Neurospora but wild-type strain. The mitochondria are prepared essentially the same way as described for animal

here is found in the not in those from the by centrifugation in tissues by Hogeboom

et al. 3 Growth of the Mold. Inoculate 7 1. of Fries minimal medium 4 contained in a 9 1. bottle equipped with a tube and cotton filter for aeration with sterile air with the conidia from a 6 to 12 day old, 25 ml. agar-minimal culture of the poky strain of Neurospora. Aerate at 25 ° for 2 to 3 days to give about 15 g. wet weight of mold after filtering through paper or cheesecloth. Wash the mold twice with 50 parts of distilled water. Grind the mold in an ice-cold mortar with 0.5 part of sand and 3 parts of cold phosphate-mannitol buffer, pH 6.9. Centrifuge at 2000 X g for 3 minutes, and repeat the grinding and centrifugation with the residue. Centrifuge the combined supernatant solutions in a refrigerated centrifuge for 20 minutes at 30,000 X g. Resuspend, and wash the red pellet twice with 100 vol. of buffer. Suspend the particles in a volume of buffer equal to half the weight of the original moist mold. Aging the Particles and Storage of the Preparation. With freshly prepared particles there is a lag of 2 to 4 hours in the cytochrome destruction reactions, and this is reduced to 5 to 30 minutes by storing the particle suspension at 8 ° for 2 to 4 days. The suspension is then frozen quickly in dry ice and Cellosolve and stored at - 2 0 ° for use over a period of at least 30 days. Thawed particles can be used directly or after centrifugation and resuspension in fresh buffer.

Properties

Specificity. Aged mitochondria from poky cause the release of amino acids from cytochrome c (beef or Neurospora) and from protamine or casein but not appreciably from egg albumin or hemoglobin. The aging process itself involves an extensive self-proteolysis which does not occur with mitochondria from wild-type Neurospora. It is not known whether more than one particle-attached proteolytic enzyme is involved, but the action on cytochrome c does produce a definite residual fragment which retains the absorption spectrum of the undigested cytochrome. At neutral 3 G. H. Hogeboom, W. C. Schneider, a n d G. E. Palade, J. Biol. Chem. 172, 619 (1948); see also Vol. I [3]. 4 G. W. Beadle a n d E. L. T a t u m , Am. J. Botany 82, 678 (1945).

[17]

ENZYMATIC DEGRADATION OF CYTOCHROME C

169

pH values this residual fragment is either insoluble or is adsorbed by the particles. The second stage of the reaction, the oxidation of the hematin peptide after reduction of the iron through succinic dehydrogenase, does not occur with hematin, hemoglobin, or catalase, although indirect evidence suggests that it does occur with cytochromes a and b. The isolated hematin peptide decomposes spontaneously in the presence of hydrosulfite and oxygen, and it is quite possible that its breakdown in the presence of suecinate, succinic dehydrogenase, and oxygen is also spontaneous after reduction, analogous to the reactions described for pyridine hemochromogen. 5 However, ascorbate or glutathione do not serve as satisfactory reducing agents. Inhibitors. The proteolytic action on cytochrome c is inhibited by an unknown water-soluble substance(s) present in extracts of Neurospora. It is also inhibited by protamine (0.05 mg./ml.). The oxidative step of the reaction is inhibited by malonate and fumarate when succinate is used for reduction, and by cyanide. Effect of pH. The over-all reaction has a pH optimum between pH 6.8 and 7.0, and it does not occur appreciably below pH 6 or above pH 7.3. 5 R. Lemberg and J. W. Legge, "Hematin Compounds and Bile Pigments," p. 499, Interscience PublishErs, New York, 1949.

170

ENZYMES OF PROTEIN METABOLISM

[18]

[18] Transaminases in Bacteria H

Be al P

R--C--NH~

I

COOH

~- R I - - C ~ O

I

COOH

~

H

~ R--C~---O ~ R 1 - - C - - N H 2

J

C00H

I

COOH

B y I. C. GUNSALUS and JOHN 1~. STAMER

T r a n s a m i n a t i o n in bacteria was, until recently, restricted to surveys for the presence of the t r a n s a m i n a s e systems previously d e m o n s t r a t e d in animal tissue s and to the d e m o n s t r a t i o n of pyridoxal p h o s p h a t e (B6 al P) as the coenzyme. 1" E a c h t r a n s a m i n a s e is considered so far to involve two amino a c i d s - - o n e m a y be an a m i n o acid amide2.~"--and their respective a - k e t o acid analogs, with the n o m e n c l a t u r e indicated b y the amino acid pair, t h a t is, glutamic-aspartic t r a n s a m i n a s e for the reaction A s p a r t a t e + a - k e t o g l u t a r a t e ~ Oxalacetate ~ g l u t a m a t e T h e progress of the t r a n s a m i n a s e reaction can be followed b y m e a s u r ing the f o r m a t i o n or disappearance of a n y one or more of the components, provided t h a t side reactions which remove c o m p o n e n t s are eliminated. T h e rate has characteristically been followed b y measuring the f o r m a tion of glutamic acid, using a specific bacterial decarboxylase, 3,4 the form a t i o n of oxalacetate, measured either s p e c t r o p h o t o m e t r i c a l l y 5 or b y decarboxylation with aniline-citrate, 6 or, more recently, b y measuring pyruvic acid after oxalaceta~e decarboxylation. 7 T h e a - k e t o g l u t a r a t e can be measured, although less conveniently, with succinoxidase after acid p e r m a n g a n a t e oxidation to succinate s or as the 2,4-dinitrophenylhydrozone after correction for oxalacetate and p y r u v a t e . 2,9 A s p a r t a t e accumulation, or disappearance, has been followed, using the c h l o r a m i n e - T m e t h o d 1° in which a s p a r t a t e yields two moles of CO2 whereas the all1IL C. Lichstein and P. P. Cohen, J. Biol. Chem. 157, 85 (1945). la H. C. Liehstein, I. C. Gunsalus, and W. W. Umbreit, J. Biol. Chem. 161, 311 (1945). 2 A. Meister and S. V. Tice, J. Biol. Chem. 187, 173 (1950). ~ A. Meister and P. E. Fraser, J. Biol. Chem. 210, 37 (1954). 3 E. F. Gale, Biochem. J. 89, 46 (1945); See also Vol. II [20]. 4 E. F. Gale, Nature 157, 265 (1946). s D. E. Green, L. F. Leloir, and V. Nocito, J. Biol. Chem. 161, 559 (1945). 6 N. L. Edson, Biochem. J. 29, 2082 (1935). N. E. Tonhazy, N. G. White, and W. W. Umbreit, Arch. Biochem. 28, 36 (1950). s H. A. Krebs, Biochem. J. $2, 108 (1938). 9 T. E. Friedemann and G. E. Haugen, J. Biol. Chem. 147, 415 (1943). ~0p. p. Cohen, J. Biol. Chem. 186, 565 (1940).

[18]

TRANSAMINASES IN BACTERIA

171

phatie amino acids and glutamate yield but one. This method has the drawback of being slightly cumbersome and of measuring transamination as the difference between two large values of CO~ evolution. More recently, microorganisms have been used to explore the participation of a wide variety of amino acids in the transaminase reaction, and the distribution of these enzymes. 11.12Paper chromatography was employed to demonstrate the qualitative formation of glutamie acid from a-ketoglutarate and its dependence on individual amino acids as donors. The disappearance of the donor amino acid and its dependence on the presence of a-ketoglutarate was also shown. The quantitative methods applied to the glutamic-aspartic transaminase--particularly the glutamic decarboxylase~ean be applied to these reactions as well. The deearboxylase measurement can also be extended to polyfunctional amino acid systems for which specific decarboxylases are known; these include arginine, ornithine, histidine, tyrosine and lysine, 13 and aspartate. 14 Transamination between aliphatie amino acids, for example, valine and isoleucine, or among aromatic amino acids without the obligatory participation of glutamate, has been reported in animal tissue and in microorganisms. 12,~5 The qualitative paper chromatographic procedures are applicable to these systems, but suitable quantitative methods for their measurement have not been fully explored. Transamination occurs among D-amino acids ~e and may be studied by the same procedures used with the L-amino acid transaminases, with the additional provision that the decarboxylases are specific for the L-amino acids which can thus be differentiated from the D-amino acids which accumulate, ~8 and D-amino acids, especially D-alanine, can be measured with D-amino acid oxidase from pig kidney. ~7 Transamination between the dicarboxylic amino acid amides, glutamine and asparagine, and several aliphatic keto acids has been demonstrated in animal tissue, although not so far in bacterial cells. 2 The progress of these transaminases can be measured by the same methods employed for the amino acid transaminases. In addition, ammonia formation can be followed if interfering side reactions are controlled or eliminated; in most eases, however, it is preferable to follow amino acid formation. 11L. I. Feldman and I. C. Gunsalus, J. Biol. Chem. 187, 821 (1950). 12 D. Rudman and A. Meister, J. Biol. Chem. 200, 591 (1953). 13 E. F. Gale, Advances in Enzymol. 6, 1 (1946). 14 A. Meister, H. A. Sober, and S. V. Tice, J. Biol. Chem. 189, 577 (1951). is E. B. Kearney and T. P. Singer, Biochim. et Biophys. Acta 11, 276 (1953). le C. B. Thorne, C. G. Gomez, and R. D. Housewright, J. Bacteriol. 69, 357 (1955). ~7 W. A. Wood and I. C. Gunsalus, J. Biol. Chem. 190, 403 (1951), modified from E. Negelein and H. BrSmel.

172

ENZYMES OF PROTEIN METABOLISM

[18]

Unit of Transaminase. A unit of bacterial transaminase for several donors to form glutamate was defined by Feldman and Gunsalus ~1 as that amount of enzyme which will form 1 micromole of glutamate per hour. The assay was run according to the protocol outlined l~elow, for 10 minutes at 37 °. Rudman and Meister ~ have used a similar unit and expressed specific activity as units per milligram of protein nitrogen. Green et al. 6 took as a unit of glutamic-aspartic transaminase the amount of enzyme which forms oxalacetate equivalent to 100 ~l. of COs in 10 minutes at 38°; the protocol is described in their paper.

Assay Methods 1. Qualitative Assay. Principle. The amino donor activity of any amino acid, in the presence of a suitable a-keto acid acceptor or of amino acceptor activity of its keto acid analog, is detected by paper chromatography, using a solvent which separates the amino acids in question, is Semiquantitative estimation of the appearance of an amino acid, its dependence on a particular amino donor, and the disappearance of that amino acid can be obtained. This method has been employed for the glutamic-linked transaminases, H,19 for the systems interlinking aliphatic amino acids, 12,2° and more recently for the cysteine sulfinate-glutamate transaminase. 15 The glutamic-forming systems with a-ketoglutarate as amino acceptor will be used as an example. I~,12

Reagents Amino acid in question, M/8, pH 8. a-Ketoglutarate, M/2, pH 8. Pyridoxal phosphate, ca. 100 ~,/ml. M phosphate buffer, pH 8.3. Trichloroacetic acid, 100%. For paper chromatographs: (a) Whatman No. 3 filter paper, 15 × 18-inch sheets, for ascending method. (b) Ammonia vapor cabinet. (c) Solvent for chromatography: water-saturated phenol. (d) Ninhydrin, 25 rag. per 100 ml. in n-butanol. Enzyme source. Dried cells or extracts: Escherichia coli/1,12,21 Pseu18 R. 19 p. 20 E. 21 H.

Consden, A. H. Gordon, and A. J. P. Martin, Biochem. J. 88, 224 (1944). S. Cammarata and P. P. Cohen, J. Biol. Chem. 187, 439 (1950). V. Rowsell, Nature 168, 104 (1951). E. Umbarger and B. Magasanik, J. Am. Chem. Soc. 74, 4253 (1952).

[18]

TRANSAMINASES

IN BACTERIA

173

domonas fluorescens, n Bacillus subtilis, 16 Aerobacter aerogenes, ~2 etc. Procedure. Pipet the reactants in the order listed into 13 X 100-mm. test tubes: 0.1 ml. of M phosphate, pH 8.3; 0.2 ml. of a suspension containing 10 mg. of dried cells; 0.1 ml. (10 7) of pyridoxal phosphate; 0.2 ml. (25 ~M.) of 0.125 M L-amino acid; 0.1 ml. (50 ~M.) of 0.5 M A-ketoglutarate; and water to 1 ml. Incubate for 60 minutes at 37°; stop the reaction by adding 0.1 ml. of 100% TCA. Transfer 0.02 ml. or other suitable amount, to Whatman No. 3 filter paper sheet with a micro- or a 0.1-ml. pipette, spacing at approximately 1-inch intervals along a base line approximately 1 inch from the bottom of the paper; the chromatograms are to be run in the direction of the 15-inch dimension. Allow the spots to dry, suspend the paper in a chamber containing ammonia vapor to neutralize the acid salts, and free the amino acids. Transfer to solvent chambers (6 or 12 X 18 inch Pyrex battery jars) containing 200 ml. of water-saturated phenol, allow to equilibrate, place the paper in the solvent, and allow to ascend at 25 ° until the solvent front is within 2 inches of the top of the paper. Remove the chromatogram, dry at 90 ° for 10 minutes, spray with ninhydrin reagent, and heat again at 90 ° for 5 minutes. Read the chromatograms visually, comparing the Rs and intensity to known quantities of the amino acids in question. For examples using a pyridoxamine phosphate-alanine transaminase of C. wclchii, see Meister et al. u 2. Quantitative Assay. Principle. As indicated in the introduction, the method of choice for quantitative measurement of the transaminase reaction depends on the amino acid pairs involved. As examples, the following are given as characteristic. Others may be found by reference to the initial papers. A. GLUTAMIC ACID AS ONE OF THE PRODUCTS--USE OF GLUTAMIC DECARBOXYLASE FOR ASSAY.3'4'14 This principle is applicable to the assay of formation of any amino acids for which specific decarboxylases have been observed. ~3,~4 Reagents1 Amino acid in question, M / 8 , pH 8. a-Ketoglutarate, M / 2 , pH 8. Pyridoxal phosphate, ca. 100 7/ml. M phosphate buffer, pH 8.3. 0.075 M phthalate buffer, pH 5.0. Enzyme source. Dri'ed cells or extracts: E. coli, A. aerogenes sp., Proteus sp.

174

ENZYMES OF PROTEIN METABOLISM

[18]

Procedure. For the manometric determination of transaminat]on, incubate 1 ml. of the reaction mixture (prepared according to the method described above) for 60 minutes at 37 °. Stop the reaction by placing the tube in boiling water for 5 minutes, adjust the pH to 5.0, and transfer 1 ml. to the main cup of a Warburg vessel containing 1 ml. of 0.075 M phthalate buffer, pH 5.0. Add 0.5 ml. (10 rag.) of a suspension of glutamic decarboxylase 3,4,14 (dried cells) to the side arm. After 10 minutes of equilibration at 37 °, tip the contents from the side arm and follow CO2 evolution until decarboxylation is completed (15 to 30 minutes). B.

ASPARTIC ACID AS ONE REACTANT---DETECTION OF OXALACETATE

W I T H A N I L I N E CITRATE. 5

Reagents Aspartic acid, M/2, pH 7.3. a-Ketoglutarate, M/2, pH 7.3. Pyridoxal phosphate, ca. 100 ~/ml. Phosphate buffer, M/5, pH 7.3. Aniline citrate reagent. Mix equal volumes of aniline and citric acid solution (50 g. of citric acid in 50 ml. of water). Enzyme source (same as above.)

Procedure. Pipet the following solutions into the main compartment of a Warburg cup: 0.5 ml. of M/5 phosphate, pH 7.3; 0.2 ml. (5 mg.) of dried cells; 0.1 ml. (10 -~) of pyridoxal phosphate; and water to required volume. After 10 minutes of incubation at 37 °, tip from the side arm 0.2 ml. each of aspartic acid and a-ketoglutarate. After a 10-minute reaction period, tip 0.5 ml. of aniline citrate from the second side arm, and read the gas evolved within 10 minutes. A control containing all the reagents except the enzyme is run simultaneously. C.

OXALACETATE MEASUREMENT BY DECARBOXYLATION TO PYRUVATE. 7

Reagents Aspartic acid, M/5, pH 7.4. (Dissolve 1.33 g. of DL-aspartic acid and 0.86 g. of K2HPO4 in water, adjust to pH 7.4 with KOH, and dilute to 50 ml.) a-Ketoglutarate, M/IO, pH 7.4. Phosphate buffer, M/IO, pH 7.4. Aniline citrate, see above. Trichloroacetie acid, 100%. Dinitrophenylhydrazine. (Dissolve 100 rag. of 2,4-dinitrophenyl-

[18]

TRANSAMINASES IN BACTERIA

175

hydrazine in 20 ml. of concentrated HC1 and add 80 ml. of water.) Toluene, reagent grade, water-saturated. Alcoholic KOH (2.5 g. of K 0 t I made up to 100 ml. with 95% alcohol).

Procedure. To 0.2 ml. containing the enzyme, coenzyme and buffer add 0.5 ml. aspartate. After warming to 37 ° add 0.2 ml. of a-ketoglutarate. Mix and incubate at 37 ° for 10 minutes. Add 0.1 ml. of trichloroacetic acid, shake and add 0.1 ml. of aniline citrate. Shake and incubate for 10 minutes or more. Add 1 ml. of the dinitrophenylhydrazine reagent and incubate at 37 ° for 5 minutes. Add 2 ml. of the toluene and shake vigorously. After 5 minutes remove i ml. of the toluene layer to a colorimeter tube containing 5 ml. of alcoholic KOH. Mix and allow to stand 5 to 10 minutes. Add 1 ml. distilled water and read in a photoelectric colorimeter with a green filter (540 mu), using appropriately prepared reagent blanks and pyruvate standards. The amount of pyruvate which may be determined lies in the range 10 to 80 micrograms. D.

OXALACETATE MEASUREMENT BY SPECTROPHOTOMETRIC METHOD.

Reagents Aspartic acid, M/IO, pH 7.3. a-Ketoglutarate, M/IO, pH 7.3. Phosphate buffer, M/IO, pH 7.3. Enzyme (0.02 to 0.10 Green unit~), equivalent to 0.5 to 2.5 micromoles of oxalacetate formed per hour. n

Procedure. Pipet the following reagents into a 1-cm. 3 ml. quartz absorption cell, and observe the increase in optical density at 280 rag: 0.3 ml. of M/IO phosphate buffer; 10 ~ of pyridoxal phosphate; enzyme (0.5 to 2.5 units n) ; 0.3 ml. of M/IO amino acid (10 gM./ml.) ; 0.3 ml. of M/IO a-ketoglutarate; and water to 3.0 ml. The reaction is started by addition of a-ketoglutarate at zero time: U . M E A S U R E M E N T OF D-AMINO ACID TRANSAMINASES. 16

Reagents. See above. Procedure. The reaction mixture is made up to contain, in 1 ml., 0.1 ml. of enzyme, 10 to 20 ~/ of pyridoxal phosphate, and 100 micromoles of the appropriate substrates in 0.05 M phosphate buffer, pH 8.0. The reaction is run at 37 ° for the desired time and stopped by boiling for 5 minutes, and, after centrifugation, the supernat~nt is used for analysis. The total glutamic, ~spartic, and alanine are determined quantitatively by elution from paper chromatograms and reading the ninhydrin color

176

ENZYMES OF PROTEIN METABOLISM

[18]

in a Coleman colorimeter. :~ L-Glutamic acid is determined with decarboxylase, 3,4 D-glutamic by difference between total and L-glutamic and D-alanine with D-amino acid oxidase. 17

Preparation of Bacterial Transaminases Growth of various bacteria with high transaminase content can be accomplished as outlined in the original papers: S. faecalis, ~,'I E. coli, 1',~2,2' C. welchii, 2 B. subtilis, 1',IG among others. ',11,~ The bacterial transaminases have been purified only to a limited extent, and separation has been largely restricted to the report of two "group-specific" transaminases from E. coll. ~2 It is evident from preliminary studies, however, that the bacterial transaminases are relatively stable enzymes which could be separated and purified by the means employed for fractionation of other enzymes from bacterial extracts. For the preparation of extracts, see Vol. I [7]; for the removal of nucleic acids and protein separation, see Vol. I [77], etc. As a specific example, Rudman and Meister ~2 have prepared from E. coli, largely by gel absorption and elution, a " t r a n s a m i n a s e A," which catalyzes the transfer of amino groups among the dicarboxylic and aromatic amino acids including glutamic, aspartie, tryptophan, tryosine, and phenylalanine, and "transaminase B," which transfers amino groups among the aliphatic amino acids, isoleucine, valine, leucine, norleucine and norvaline, and from each of these to glutamate. Some overlapping in activities is exhibited, presumably due to incomplete separation. A third "transaminase," relatively specific for valine-alanine and valine-a-aminobutyric acid, was obtained from an E. coli mutant. Bacterial extracts were prepared by alumina grinding (see also Vol. I [7]; 1 part dry cells, 3 parts alumina), followed by addition of 20 parts of water and by centrifugation and dialysis. Trausaminase A . The bacterial extract was adjusted to pH 5.3 with M sodium acetate buffer and mixed with an equal quantity of calcium phosphate gel (40 rag. dry weight per milliliter). After occasional shaking for 10 minutes at 5 °, the mixture was centrifuged, the pellet resuspended in the original volume of 0.1 M phosphate buffer, pH 4.5, and allowed to stand for 10 minutes at 5 °. After centrifugation, the supernatant was dialyzed for 10 hours vs. distilled water, and M acetate buffer of pH 5.3 added to a final concentration of 0.1 M. Saturated ammonium sulfate was added at 0 ° to 0.45 saturation, allowed to stand i hour, centrifuged, and brought to 0.6 saturation with saturated ammonium sulfate. After 1 hour, the precipitate was collected and dissolved in the original volume of 0.1 M phosphate buffer, pH 8.3, and used as a source of transaminase A. 2~ R. D. ttousewrigh¢ and C. B. Thorne, J. Bacteriol. 60, 89 (1950).

[18]

TRANSAMINASES 21,,1BACTERIA

177

Transaminase B. The bacterial extract was diluted with 0.1 vol. of M pH 5.3 acetate buffer and, after cooling to 0 °, cold acetone was added slowly with continued cooling to - 5 ° and to 20% concentration. Mter 15 minutes, and centrifugation, the supernatant at - 5 ° was brought to 40 % acetone. After 15 minutes at - 5 °, the precipitate was collected, dissolved in one-half the initial volume of 0.1 M potassium phosphate, pH 8.3, and dialyzed for 10 hours against distilled water. Then 0.1 vol. of M acetate buffer, pH 5.3, was added. An equal volume of alumina A gel (30 mg. dry weight per milliliter) was added, and, after occasional shaking for 10 minutes at 5 °, the gel was collected by centrifugation and the pellet washed with five 15-ml. fractions of 0.1 M phosphate buffer, pH 6.2. The gel was suspended in 15 ml. of 0.1 M phosphate buffer, pit 8.3, and the supernatant collected and dialyzed against water. The absorption and elution was repeated, and the pH 8.3 eluate dialyzed for 10 hours against water. After dialysis, 0.1 vol. of M acetate buffer, pH 5.3, was added and saturated ammonium sulfate added to 0.4 saturation. After 1 hour at 0 °, the precipitate was removed. The supernatant was brought to 0.6 saturation, and allowed to stand for 1 hour at 0 °. The precipitate was collected and dissolved in one-fifth the initial volume of 0.1 M phosphate buffer~ pH 8.3. This fraction is "transaminase B."

Properties p H Activity. The optimum pH for the bacterial transaminases studied to date is around 8.0.1,11,12 The equilibrium for the transaminases is dependent on the amino acid pairs and not on the specific enzyme. The most pertinent equilibrium data are for the glutamic-aspartic la and the glutamic-glycine11 systems, and particularly with the heat-activated transaminations, studied by Snell and co-workers. 23 Activators and Inhibitors. Pyridoxal phosphate has been shown to be an activator of all the transaminases so far resolved. Carbonyl reagents-for example, hydroxylamine and hydrazine--inhibit B~-eatalyzed reactions, 13,24 presumably by reaction with the coenzyme and, in the case of the transaminases, more slowly with the keto acid substrates. The specificity of the substrates for the transaminases and precise data on the substrate analogs as inhibitors must await the separation and purification of the enzymes, and very probably a clarification of the transaminase mechanism, since a system involving one protein, two coenzymes, and four substrates does not lend itself readily to kinetic studies. The present state of knowledge of bacterial transaminases is fragmentary; more specific data on purification, properties, equilibria and specificity needed. 53D. E. Metzler, J. Olivard, and E. E. Snell, J. Am. Chem. Soc. 76, 644 (1954). *~H. Blaschko, Advances in Enzymol. 5, 67 (1945).

178

ENZYMES OF PROTEIN METABOLISM

[19]

[19] Estimation of Animal Transaminases B y PHILIP P. COHEN

A variety of analytical methods suitable for estimating transaminase activity has appeared in the recent literature. Since each transamination system consists of four components, a substrate amino acid and a-keto acid, and an end product amino acid and a-keto acid, measurement of the disappearance of one or the other of the former or formation of one or the other of the latter will serve as a m e t h o d of estimating transaminase activity. As a general rule, it is of course more desirable to measure the formation of a compound t h a n disappearance of an added substance to a system. I n dealing with crude enzyme preparations of relatively u n k n o w n composition it is essential t h a t at least two components of the system be estimated and preferably all four in establishing t h a t a transamination reaction has taken place. M e t h o d s for estimating transaminase activity have been published involving the following procedures: 1. 2. 3. 4.

Quantitative filter paper chromatography.l-3 Spectrophotometric. 1,4-8 M a n o m e t r i c amino acid decarboxylase. 9-14 Other. 14-1e

The methods to be presented in detail here are: I. Spectrophotometric measurement of oxalacetic acid formation. I I . M a n o m e t r i c estimation of glutamic acid b y means of glutamic acid decarboxylase. 1j. Awapara and B. Seale, J. Biol. Chem. 194, 497 (1952). 2L. Fowden, Biochem. J. 48, 327 (1951). 3 p. G. Tulpule and V. N. Patwardhan, Nature 169, 671 (1952). 4 p. S. Cammarata and P. P. Cohen, J. Biol. Chem. 193, 45 (1951). 5 A. Nisonoff, S. S. Henry, and F. W. Barnes, Jr., J. Biol. Chem. 199, 699 (1952). 6 A. Nisonoff and F. W. Barnes, Jr., J. Biol. Chem. 199, 713 (1952). 7 p. S. Cammarata and P. P. Cohen, Biochim. et Biophys. Acta 10, 117 (1953). 8 H. Brandenberger and P. P. Cohen, Helv. Chim. Acta 36, 549 (1953). 9 p. S. Cammarata and P. P. Cohen, J. Biol. Chem. 187, 439 (1950). 1oH. A. Krebs, Biochem. J. 47, 605 (1950). 11L. I. Feldman and I. C. Gunsalus, J. Biol. Chem. 187, 821 (1950). 12A. F. Miiller andF. Leuthardt, Helv. Chim. Acta 33, 268 (1950). 13A. Meister, H. A. Sober, and S. V. Tiee, J. Biol. Chem. 189, 591 (1951). 14H. A. Krebs, Biochem. J. 54, 82 (1953). 15N. E. Tonhazy, N. G. White, and W. W. Umbreit, Arch. Biochem. 28, 36 (1950). 16p. p. Cohen, J. Biol. Chem. 136, 565 (1940).

[19]

ESTIMATION OF ANIMAL TRANSAMINASES

179

Spectrophotometric Measurement of Oxalacetic Acid Formation 4 This method may be used with any transamination system in which one of the components, either added as substrate or formed as a reaction product, has a measurable characteristic absorption at a suitable wavelength. In principle the method requires that the other components do not have an interfering absorption at the wavelength used for a given compound which interferes. It thus is necessary to have data on the absorption properties of all the components in any given system. Although the spectrophotometric method is limited to only a few transaminating systems, its usefulness as a microprocedure and its suitability especially for kinetic studies make it the method of choice in some instances. The procedure for measuring oxalacetic acid formation as shown in reaction 1 will be presented.

Reagents Enzyme preparation. In using crude tissue extracts, dialysis of the preparation is desirable in order to minimize tissue blanks and side reactions requiring coenzymes. Substrates 1. 1. a-Ketoglutaric acid. Made up to contain 100 micromoles per milliliter and adiusted to pH 7.4. Made up to final volume with 0.05 M phosphate buffer, pH 7.4. 2. L-Aspartic acid. Made up to contain 20 micromoles per milliliter and adjusted to pH 7.4. Made up to final volume with 0.05 M phosphate buffer, pH 7.4. 3. Pyridoxal phosphate (obtained from Dr. W. W. Umbreit, Merck Institute for Therapeutic Research, Rahway, New Jersey). M~ade up to contain 30 ~ of calcium salt per milliliter and up to final volume with 0.05 M phosphate buffer, pH 7.4. The above solutions are stable indefinitely if stored in a deep freeze box.

Assay Conditions. To the quartz cell of a Beckman spectrophotometer is added 1 ml. of tissue extract and 1 ml. of pyridoxal phosphate solution. The mixture is incubated for 20 minutes at 38 °, and then 1 ml. of aspartic acid solution is added. This mixture is further incubated for 10 minutes at 38 °, after which 0.2 ml. of a-ketoglutaric acid solution is added. As blanks, complete systems in which aspartic acid and a-ketoglutaric acid, respectively, are omitted are routinely employed. It is important that these blanks be run simultaneously with the test system in order to detect any change in absorption due to the presence of an L-amino acid oxidase,

180

ENZYMES OF PROTEIN METABOLISM

[19]

or a change in absorption due to protein denaturation or precipitation, and consequent opacity. (In studying the reverse reaction an additional blank containing coenzyme, amino acid, boiled enzyme, and oxalacetic acid must be run in order to determine the rate of nonenzymatic decarboxylation of the keto acid.) It will be found, in most cases, that these blank corrections are not significant. Assays are made at 280 m#. Absorption of the other components at this wavelength is minimal? For this assay, the Beckman model D U spectrophotometer is equipped with a water jacket through which water from a constant-temperature bath is pumped. Calculagons. Density readings are taken every 3 minutes for 30 minutes and plotted against time. The best straight line is drawn through the points, and the value of dD/dt, the rate of change of optical density with time, is determined from the slope of the curve. In a generalized transamination system the following reaction will occur: (1) COOH COOH COOH COOH

I

HCNH2

r

CH~

I

COOH

I

C~O

r

~- CH2

!

CH2

I

I

C=0

f

-~ CH2

HCNH2 +

I

COOH

I

COOH Aspartic a-Ketoglutaric Oxalacetic acid acid acid (Cl) (C~) (C,)

i

CH2

I

CH~

I

COOH Glutamic acid

(C4)

If we start with (C1) = (C2), then at any time (C.) = (C,)

(2)

and also

(c,) =

(Co--C3) = ( c , )

(3)

where (Co) is the original concentration of (C1). The optical density of such a generalized transamination system can be represented by equation 4. D = kl(C1) + k~(C2) + k3(C3) ~- k4(C4) -~ K(C)

(4)

where kl, k2, k3, and k4 are the extinction coefficients of the substances whose concentrations are given by (C1), (C2), (C3), and (C4), (C) represents the sum of concentrations of all constant components such as protein, coenzyme, buffer ions, etc., and K represents the sum of the extinction coefficients of these substances. It is to be noted that the appearance

[19]

ESTIMATION OF ANIMAL TRANSAMINASES

181

of a transitory enzyme-substrate complex with a characteristic extinction coefficient would not interfere with this treatment, since kinetic data are taken only during the steady-state period when the concentration of the complex is a constant. Because the concentration of this complex is constant during this period, it can be grouped with the constant term K ( C ) .

0.70 >..

I.-Z bJ

0.60 -J 0

ha. 0

0.50

5

I0 15 20 TIME(Minutes)

25

FIG. 1. T i m e - - D e n s i t y Curve. Enzyme preparation, 1 ml. of dialyzed aqueous

extract of pig heart; aspartic acid, 20 tLM.; a-ketoglutarie acid, 20 t~M.; pyridoxal phosphate, 30 ~; phosphate buffer, 0.05 M, pH 7.4. Final volume, 3.2 ml. Temperature, 38° (corrected for blanks). If in equation 4 we substitute the values of (C1), (C~), and (C4) obtained from equations 2 and 3, an equation is obtained which on grouping and transposition of terms simplifies to: (C~) =

D

(Co)(kl + k~) K(C)

--(k3+k,--k~-k~)

(5)

On taking the derivatives of this equation with respect to time, we obtain d(C3) dD 1 dt - dt X (k3 + Ic4 -- k~ -- k2)

(6)

The values of the extinction coefficients are taken from the absorption curve of the substance concerned, and the iTalue of d D / d t is obtained as described above. Oxalacetic acid has a millimolar extinction coefficient (micromoles per milliliter) of 0.53 in 0.05 M phosphate buffer, pH 7.4.

182

ENZYMES OF PROTEIN METABOLISM

[19]

The resulting rate of formation of (C3) when multiplied by 22.4, divided by the protein concentration, and placed on an hour basis yields the Q transamination value. A typical time-density curve for transaminase assay of the aspartic acid -~ ~-ketoglutaric acid system is shown in Fig. 1.

Manometric Assay Using Glutamic Acid Decarboxylase The principle of this method is that of using a specific amino acid decarboxylase and estimating C02 formation manometrically. The formation or disappearance of any given amino acid participating in a transamination reaction can be measured provided that a specific and reasonably active enzyme preparation is available. Since glutamic acid formation and disappearance are so commonly estimated to measure transaminase activity and since a highly specific and active enzyme preparation is available for this purpose, this discussion will be limited to the use of glutamic acid decarboxylase. The reactions to be studied may be expressed in the general form Amino acid ~ a-ketoglutaric acid ~-~ a-keto acid -~ glutamic acid

(7)

The assay involves the estimation of glutamic acid manometrically in an aliquot of an incubation mixture by measuring COs formation in accordance with the following: glutamic acid Glutamic acid

~ C02 ~ ~-aminobutyric acid

(8)

deearboxylase

Preparation of Glutamic Acid Decarboxylase. Three active glutamic acid decarboxylases are available from ready sources, one from plants 17 and two from microorganisms, E. coli is and Cl. welchii. 14,19,2° The need for an enzyme which is readily prepared and free of other interfering systems limits the choice in the author's experience to the E. coli preparation. The plant enzyme preparation (from squash) contains highly active transaminases and keto acid decarboxylases 21 and thus is not suitable without going through extensive purification procedures which have not as yet been worked out. The glutamic acid decarboxylase preparation from C1. welchii is satisfactory for many transamination systems, but it contains an active aspartic acid decarboxylase 9,~2,22 and transaminases of its own active at pH 5. 9 E. coli glutamie acid decarboxyl17 O. Schales and S. S. Schales, Arch. Biochem. 11, 445 (1946). 18 W. W. Umbreit and I. C. Gunsalus, J. Biol. Chem. 159, 333 (1945). x9 E. F. Gale, Biochem. J . 39, 46 (1945). ~0 H. A. Krebs, Biochem. J. 45, 51 (1948). ~1 O. Schales and S. S. Schales, Federation Proc. 12, 264 (1953). ~2 A. Meister, H. A. Sober, and S. V. Tice, J. Biol. Chem. 189, 577 (1951).

[19]

ESTIMATION OF ANIMAL TRANSAMINASES

183

ase is prepared with minor modifications according to the method of Umbreit and Gunsalus. 18 Medium: NaCI Trypticase (Difco) Yeast extract (Difco) Cerelose K2HP04 Distilled H20

100 g. 100 g. 100 g. 100 g. 25 g. 10 1.

E. coli (American Type Culture, strain 4157) is transferred from a nutrient agar slant into 100 ml. of medium and incubated at 30 ° for 24 hours. Sixteen liters of medium is then inoculated with the 100 ml. of culture and held at 30 ° for 5 hours and continued at room temperature for 24 hours. Occasional shaking is desirable for maximum yields. The organisms are then harvested in a Sharples centrifuge at 20,000 r.p.m. The collected paste is washed twice with 10 vol. of 0.85% saline and then spread out on petri dishes and dried in vacuo over CaC12. The resulting dried powder is finely triturated and stored in a desiccator. The yield is between 3 to 4 g. of dry powder. Reagents. ~-Ketoglutaric acid (or other keto acids) and amino acids are prepared in suitable concentration according to the experimental conditions desired. A concentration of 20 to 40 micromoles per milliliter has been employed in this laboratory in most instances. The preparation is adjusted to pH 7.4 and made up to final volume with 0.05 M phosphate buffer, pH 7.4. Pyridoxal phosphate is prepared as previously stated for the spectrophotometric assay. 3 M Acetate buffer, pH 4.8, is prepared by mixing 27.2 g. of sodium acetate (NaC~H3023H20) with 6 g. of glacial acetic acid and diluting up to 100 ml. Transaminase Assay Procedure. The conditions and volume of the incubation system will of course depend to some extent on the amount of material available for assay, the nature of the material, etc. Since with this method it is necessary to measure COs production, conditions must be such that the gas volume can be accurately estimated with the usual manometer. If conditions do not permit dialysis beforehand to remove coenzymes and oxidizable substrates, an anaerobic gas phase must be employed unless the tissue can be diluted sufficiently so as to minimize oxidative reactions. In the case of weak transaminating systems, it has been found helpful to prepare a lyophilized homogenate of the tissue which is then suspended in a suitable volume of buffer so as to have a higher concentration of tissue per unit volume. Such a preparation must be dialyzed before use or incubated anaerobically. The following assay conditions have been used successfully in assaying a large number of different animal tissues.

184

ENZYMES OF PROTEIN METABOLISM

[19]

To the main compartment of a Warburg flask is added 1 ml. of an enzyme solution which has been dialyzed for 24 hours, immediately prior to the assay, against 0.01 M phosphate buffer, pH 7.4. One-half milliliter of pyridoxal phosphate solution (30 ~/ml.) is then added, and the mixture is allowed to equilibrate for 20 minutes at room temperature. Amino acid solution, 1 ml., is added to the flask which is then mounted in a Warburg bath by means of special rack. After equilibration at 38 °, 0.5 ml. of a-ketoglutaric acid solution is added, the flask stoppered, and shaking started. The reaction is stopped by detaching the flask from the rack and immersing it in a boiling water bath, ~ inch deep, for a period of 5 minutes. The solution in the flask is then brought to pH 5.0 by the addition of 0.2 ml. of 3 M acetate buffer, pH 4.8, and 0.5 ml. of the E. coli suspension (20 mg. of dried organisms per milliliter in 0.1 M acetate buffer, pH 5.0) is added to the side arm. A blank containing all components except amino acid is routinely employed. CO~ evolution is measured until no more gas evolves. This usually requires 30 minutes under the condiq tions employed. Air is used as the gas phase. Calculations. One micromole of glutamic acid will form 22.4 td. of C02. Transaminase Activity of Different Cell Preparations. Representative transaminase activity values for different microorganisms and plant and animal tissues are shown in Table I. TABLE I GLUTAMIC-OXALACETIC TRANSAMINASE ACTIVITY OF ANIMAL AND PLANT TISSUES AND MICROORGANISMS

Micromoles of substrate transaminated per milligram nitrogen of enzyme preparation per hour E. coli A. vinelandii Cl. welchii Oat seedlings (96 hours) Potato root Potato stem Potato leaf Brain (rat) Liver (rat) Kidney (rat) Heart (rat)

39.7 70.5 52.3 252.0 146.0 102.0 29.0 125.0 98.2 78.2 148.5

[20]

AMINO ACID DECARBOXYLASES OF BACTERIA

185

[20] Amino Acid Decarboxylases of Bacteria RCH COOH--~ RCH~ + CO2

L

NH2

t

NH2

By VICTOR A. NAJJAR

Assay Method Principle. The enzymatic decarboxylation of amino acids is essentially not reversible. The carbon dioxide formed is trapped as bicarbonate or liberated as gas, depending on the pH. Below pH 5.0 the amount of CO2 in solution is negligible, and above that value the amount of CO2 retained in solution increases with pH. Enzyme activity can therefore be assayed manometrically in the Warburg apparatus by measuring the C02 liberated as gas, provided that the pH is about 5.0 or below at the time the reading is made. Fortunately a great many decarboxylases have an optimum pH around 5.0 or below, and direct measurement of C02 can be made. In instances where the optimum pH is over 5.0, a quantity of acid is tipped in at the desired time, which should lower the pH sufficiently to cause a complete cessation of enzyme activity and a complete evolution of C02. For that purpose 0.2 to 0.3 ml. of 5 N H2SO4 is amply sufficient. Reagents Amino acid, 2 to 10 micromoles per milliliter, adjusted to the pH desired. Buffer, 0.2 M. The type of buffer chosen depends on the optimum pH of the enzyme; phosphate, acetate, and phosphate-citrate are used. 5 N H2SO4. Enzyme solution.

Procedure. The preparation of the enzyme differs with the type of microorganism used and the particular enzyme desired (see below). However, the following method of assay applies to all bacterial decarboxylases in general. The conventional Warburg technique is used, and the rate of COs evolution per unit of time (5 to 10 minutes) is taken as a measure of enzyme activity. The temperature of the bath is fixed at a convenient level (30 to 37°). The gas phase is usually room air. The main compartment contains the substrate and the buffer. One side arm contains the enzyme, and the other contains the acid if an acid tip is necessary. A

186

ENZYMES OF PROTEIN METABOLISM

[20]

blank manometer containing no substrate is run simultaneously. After temperature equilibration (10 to 15 minutes) the enzyme is tipped in and readings are taken at a convenient time. The amount of substrate should be in excess of that expected to be decarboxylated at the end of the experiment. Usually 4 to 10 micromoles is sufficient. The final molarity of the buffer is usually 0.1 to 0.2 M, and the final volume is 1 to 2.5 ml.

Purification Procedures The preparation of some amino acid decarboxylases will be discussed in more detail than others. Only those studies made with cell-free extracts will be included in this discussion.1 T.-Glutamic Acid Decarboxylase 2 from E . c o l i a - - H O O C ( C H ~ ) 2 CHNH2COOH --~ HOOC(CH2) 3NH2 (~,-Aminobutyric). The organism is grown in 6 1. of 3% trypticase soy broth 4 for 18 hours at 37 °. The cells are harvested by centrifugation and ground with alumina 5 at 6 to 8 °. The material is then extracted with 50 ml. of cold water, centrifuged, and the supernatant separated. The extraction is repeated with 30 ml. of cold water. To the combined extracts ammonium sulfate solution saturated at 6 ° is then added stepwise to 0.50 saturation and the precipitate collected by centrifugation. The precipitate is dissolved in 10 ml. of cold water and dialyzed against distilled water for 4 hours at 6 °. The turbidity is centrifuged down, and the supernatant is refractionated with ammonium sulfate. The fraction collected at 0.4 to 0.5 saturation contains 7% of the activity with over eightfold purification. The preparation so obtained shows some lysine and arginine decarboxylase activity. However, acetone powders can be prepared containing only glutamic acid activity.l.~ A maximum of 20 % of this activity can be extracted in water and can be subjected to the fractionation steps as above. The yield, however, is small. Neither the acetone powder nor its aqueous extract has any glutaminase activity. During growth of the organism the enzyme activity is near maximal after 4 hours and that is maintained for 24 hours. The enzyme is specific for L-glutamic acid and attacks the a-carboxyl to yield stoichiometric amounts of COs and ~-aminobutyric acid. It does not attack glutamic acid derivatives such as ~-methyl amide, ~-ethyl ester, or acetyl or 1 Method of bacterial growth and mode of preparation of decarboxylases studied mainly in cell suspension or acetone dried cells are described in Vol. III [75A]. V. A. Najjar and J. Fisher, J. Biol. Chem. 206, 215 (1954). a American Type Culture Collection (No. 11246), 2029 M. Street, N. W., Washington, D.C. 4Baltimore BiologicalLaboratory, Baltimore, Maryland. 5 1557 AB Buehler, Ltd., Chicago, Illinois.

[20]

AMINO ACID DECARBOXYLASES OF BACTERIA

187

carbamyl glutamic acid. The activity under the conditions described above is proportional to the enzyme concentration when the enzyme is saturated with substrate. The pH optimum is 5.0. The enzyme shows some activity below pH 4.0 but none above 5.9. The effect of pH on enzyme activity was shown to be due to an effect on the enzyme surface rather than on the substrate. The Miehaelis constants at pH 4.5 and 5.6 are 16.6, 4.5, and 14.3 (X10-~), respectively.

Arginine Decarboxylase-HN

/

CNH(CH~)3CH NH2COOH --*

H2N HN

/

CNH(CH2)4NH2 (Agmatine)

H2N E. coli 6 (N.C.T.C. 86) is grown in 3% casein digest and 2% glucose at 25 ° for 24 hours. Cell-free preparations 1 are obtained by acetone treatment of the cells followed by extraction in M / 4 5 borate buffer, pH 8.5. The optimum pH is at 5.25, and the Miehaelis constant is 7.5 × 10-4.

Histidine Decarboxylase-HC CCH2CHNH~COOH--~ HC

I

L

HN

N C H

C(CH~)2NH2 (Histamine)

!

I

HN

N C H

Cell-free preparations 1 are obtained from Cl. welchii BW 21 (N.C.T.C. 6785). 7 Cells are grown in 3% casein digest and 2% glucose with heart muscle at 37 ° for 16 hours. Acetone-dried cells are extracted with 0.05 M borate buffer, pH 8.5, at 37 °. Purification is obtained by adsorbing the enzyme at pH 5.0 on neutral C~. The washed alumina is then twice eluted with ammonium sulfate, 0.50 saturation, at room temperature. The enzyme is precipitated by adding 1.5 vol. of the saturated salt, and the precipitate dissolved in water. The enzyme is then fractionated with saturated ammonium sulfate solution. The fraction between 0.50 and 0.75 saturation shows about fiftyfold purification. The Michaelis constant is 7.5 X 10-~ M with an optimum pH at 4.5. 6 E. S. Taylor and E. F. Gale, Biochem. J. 39, 52 (1945). 7 H. M. R. Epps, Biochem. J . 89, 42 (1945).

188

ENZYMES OF PROTEIN METABOLISM

[20]

Tyrosine Decarboxylase-HO~CH2CHNH2COOH

~ HO~(CH2)2NH2

(Tyramine)

S. faecalis s (N. C.T.C. 6783) is grown in 3 % casein digest and 2 % glucose. Cell-free extracts I are obtained by extracting acetone-dried cells of S. faecalis with M / 4 5 phosphate buffer, pH 5.5, at 37 ° for 21 hours. The pH is then adjusted to 5.0 and adsorbed on neutral Ca~(POt)2 suspension, 3 ml. per 10 ml. of extract. The washed suspension is then twice eluted with 0.5 saturated ammonium sulfate solution, 1 ml. per 20 rag. To the combined eluates an equal volume of 0.5 saturated ammonium sulfate solution is added. The enzyme is then precipitated with 40 g. of the solid salt per 100 ml. of enzyme solution. The precipitate is then dissolved in water and fraetionated with saturated (NH4)2SO4 solution. The fraction precipitated at 0.50 to 0.65 saturation is dissolved in water and similarly refractionated. The fraction collected at 0.52 to 0.58 is dissolved in water and the pH adjusted to 5.0 with acetate buffer and again adsorbed on the calcium phosphate and eluted with (NHt)2SOt solution 0.25 saturation (1 ml. per 15 mg. of the calcium salt). The purification of the final product is approximately 100-fold. Aspartic Acid Decarboxylase--HOOCCH2CHNH2COOH--~ HOOC(CH2)2NH~ (~-Alanine). Cell-free extracts 1 have been obtained from a number of organisms. 9 ttowever, a more detailed study has been made on the enzyme found in C1. welchii SR 12 (N.C.T.C. 6784). 1° The organisms are grown in 0.5% Difco yeast extract, 1.5% casein digest (N-Z-Case Sheffield), 0.25% NaC1, 0.005% L-cysteine, and 2% glucose for 12 to 18 hours. Washed cells are then lyophilized and ground with ten times their weight of alumina (Aluminum Co. of America No. A-301); 20 ml. of water is then added for every gram of dry cells and centrifuged. The clear supernatant contains the enzyme. The enzyme catalyzes the decarboxylation of the a-carboxyl group of L-aspartic acid to yield f~-alanine. The enzyme activity is accelerated by low concentrations of a-keto acids. However, semicarbazide and cetyltrimethylammonium bromide in concentrations of 6 X 10-8 M completely inhibit the enzyme. The inhibition due to the latter is partially reversed by pyruvate. The pH optimum for enzyme activity is 5.5. Lysine Decarboxylase--H2N(CH~) 4CHNH2COOH--~ H2N(CH2) 5NH2 (Cadaverine). Bact. cadaveris or E. coli (N.C.T.C. 6758 and 86, respec8 H. M. R. Epps, Biochem. J. 38, 242 (1944). 90. Schales, in " T h e E n z y m e s " (J. B. Sumner and K. Myrb~ck, eds.), Vol. II, Part 1, p. 216, Academic Press, New York, 1951. 10 A. Meister, H. A. Sober, and S. V. Tice, J. Biol. Chem. 189, 577 (1951).

[20]

AMINO ACID DECARBOXYLASES OF BACTERIA

189

tively) 11is grown on 3 % casein digest and 2% glucose. Cell-free preparations 1are made by extracting acetone-dried cells with M/45 borate buffer, pH 8.5, for 2 hours at 37 °. To the extract ethanol is added to 20% concentration and the pH adjusted to 5.5 to 6.0 with acetic acid. The enzyme is then adsorbed on C~, 8 to l0 mg./ml, of the original extract. Elution is then performed twice with 0.2 M phosphate buffer, pH 7.0, each with 0.5 ml./ml, of the original extract. Solid ammonium sulfate is then added, 50 g. per 100 ml. of eluate. The protein precipitate is centrifuged down and dissolved in water. This is then fractionated with saturated ammonium sulfate solution, and the fraction 0.4 to 0.56 is collected, dissolved in water, and refractionated. The 0.4 to 0.47 fraction is collected, and the refractionation repeated as before. The fraction collected at 0.41 to 0.47 represents a purification of about 15-fold with cadaveris and about 25-fold with coli as compared to the crude acetone powder extract. The pH optimum for the enzyme from either source is 6.0, and the 1V[ichaelis constant is approximately 1.5 X 10-3 M. Ornithine Decarboxylase--ii~N(CH~)3CHNH2COOH ~ H2N(CH2),NH2 (Putrescine). Cell-free preparations 1 are prepared from Cl. septicum PIII (N.C.T.C. 547). ~ The organisms are grown in 3 % casein digest and 3 % glucose with heart muscle at 37 ° for 16 hours. Extracts are made by shaking washed cell suspensions with glass beads. The enzyme is quite unstable and deteriorates in 1 to 2 days. It has an optimum pH at 5.25 and a Michaelis constant of 4 × 10-3 M. Properties Speci~city. All decarboxylases so far studied are specific for L-amino acids and for the particular amino acid. Activators and Inhibitors. Aspartic acid decarboxylase obtained from Cl. welchii 1°is activated by a-keto acids as well as by pyridoxal phosphate. L-Lysine, L-tyrosine, L-arginine, and T.-ornithine decarboxylases have been resolved into apoenzyme and coenzyme, n,12 Addition of pyridoxal phosphate stimulates and restores the activity of the enzyme. It has not been possible to demonstrate any codecarboxylase content in or a requirement for L-glutamic acid and L-histidine decarboxylases. The enzymes that are capable of resolution are markedly inhibited by Fe ++ and hydrazine, whereas the glutamic acid and histidine enzymes show little or no inhibition. 11 E. F. Gale and tI. M. R. Epps, Biochem. J. 38, 232 (1944). 1~ E. S. Taylor and E. F. Gale, Biochem. J. 39, 52 (1945).

190

ENZYMES OF PROTEIN METABOLISM

[21]

[21] A m i n o Acid D e c a r b o x y l a s e s of Plants

By OTTO SCHALES and SELMA S. SCHALES

Glutamic Acid Decarhoxylase of Higher Plants I-3 HOOCCHsCH2CI-t(NHs)COOH--~ HOOCCH2CH2CH2NH2 -~- CO2

Assay Method Principle. The glutamic acid decarboxylase activity of an enzyme preparation is determined by measuring manometrically the amount of CO2 liberated during a period of 10 minutes on anaerobic incubation of the enzyme (at pH 5.75 and 37 °) with an excess of substrate. Reagents L-Glutamic acid solution (0.068 M). To 1.000 g. of L-glutamic acid (6.8 millimoles) in a 100-ml. volumetric flask are added 6.8 ml. of carbonate-free N NaOH (6.8 millimoles) and sufficient water to bring to a final volume of 100 ml. This solution has a pH near 7.0. 1.2 N sulfuric acid (concentrated H~SO4 diluted 1:30 with water). M / 1 5 (0.067 M) phosphate buffer, pH 5.75.

Procedure. Warburg flasks with two side arms are used for the determination of glutamic acid decarboxylase activity. Into the main compartment of each flask is pipetted 4 ml. of enzyme solution in M / 1 5 phosphate buffer, pH 5.75. The first side vessel receives 0.5 ml. of L-glutamic acid solution, containing 5 mg. (34 micromoles) of L-glutamic acid with 762 ~1. of CO~ available for enzymatic release. The second side vessel receives 0.5 ml. of 1.2 N H2SQ. A blank vessel, in which water takes the place of substrate, must be used to permit correction for the small amounts of COs which may be contained in the buffer or may be formed by the enzyme preparation in absence of L-glutamic acid. The air in all vessels is replaced with nitrogen by passing a stream of the gas through the vessels for 3 minutes in the usual manner. The reason for using anaerobic conditions is to eliminate oxygen uptake by the enzyme preparation which, in air, in a few experiments, was found to amount to 5 to 15% of the COs produced anaerobically and would result in erroneously low activity values. 10. Schales, V. Mims, and S. S. Schales, Arch. Biochem. 10, 455 (1946). O. Schales and S. S. Schales, Arch. Biochem. 11, 155 (1946). 3 0. Schales and S. S. Schales, Arch. Biochem. 11, 455 (1946).

[9.1]

AMINO ACID DECARBOXYLASES OF PLANTS

191

The reaction is started after temperature equilibrium has been reached by adding the substrate from side vessel I to the main compartment. Manometer readings are taken immediately before the addition of substrate (zero minute) and again after the reaction has proceeded for 10 minutes. The enzymatic decarboxylation is then terminated by adding the content of side vessel II to the enzyme substrate mixture. This addition of acid not only inactivates the enzyme but also liberates that portion of the COs which had been retained in solution at pH 5.75. After an additional 10 minutes of shaking the final reading is taken. The volume of COs formed by the reaction of enzyme with substrate is calculated in the usual manner under consideration of the various corrections necessary. Unless carbonate-free NaOH is used in the preparation of the substrate solution, a second blank becomes necessary. The value of this blank is determined by measuring the amount of COs liberated on mixing 0.5 ml. of substrate solution with 0.5 ml. of 1.2 N H2SO4. N"2 Calculation of Activity. Activities are expressed as Qco2 values (microliters of CO2 per hour per milligram of fresh plant material). If dried enzyme preparations are used instead of plant suspensions or plant extracts, microliters of CO2 per hour per milligram of dry material is used N2 as an index of activity. The Qco, values are obtained by multiplying microliters of CO2 formed in 10 minutes by 6 and dividing by the milligram of material used in the assay. Ideal Q values should be based on initial reaction velocities. In the calculation used here they must be considered as minimal values, since the velocity of decarboxylation decreases during the course of 10 minutes. As this process of inactivation continues, one finds that the actual amount of COs liberated per milligram of plant material per hour is smaller than would be expected from the Q value. Although Qco~ calculated from the 10-minute readings is, therefore, a somewhat artificial concept--the multiplication with 6 is carried out to conform with the customary definition of Q values--it is, nevertheless, convenient for comparisons, particularly as values calculated in a similar way have been used in activity data for amino acid decarboxylases of mammalian tissues. 4 Preparation of Extracts and Dry Powders with Glutamic Acid Deearboxylase Activity Glutamic acid decarboxylase occurs in a wide variety of plants. 1 Squash is particularly rich in this enzyme and may be used conveniently for the preparation of extracts and stable dry powders according to the following procedure. 3 In a Waring blendor 90 g. of squash is mixed with 4 H. Blaschko, Advances in Enzymol. 5, 67 (1945).

192

[21]

ENZYMES OF PROTEIN METABOLISM

300 ml. of water for 4 minutes at room temperature. The material is placed in a refrigerator for 1 hour and then centrifuged for 15 minutes at 3000 X g (size 2 centrifuge, conical head, 4000 r.p.m.). The slightly turbid supernatant fluid (about 330 to 350 ml.) is rapidly frozen and dried i n vacuo from the frozen state. Yellow squash yields 3.5 to 4.5 g. of dry powder, whereas white squash gives between 2.1 and 2.6 g. of residue. The dried material is very hygroscopic. It should be stored i n ~'acuo over CaC12, and under such storage conditions, even at room temperature, it will not lose activity for at least a year• Samples stored in the same manner, but at 4 °, were found to be fully active after 5 years of storage. Suspensions of fresh squash in M/15 phosphate buffer, pH 5.75, give Qc~2 values of 1.2 to 3•0, depending on the type of squash used. 1 Dry powders, prepared as described above, show a Qc~2 of 20 to 50, and this activity increases to 30 to 60 when pyridoxal-5-phosphate is added to the incubation mixture. The dry powders may be purified by precipitation with ammonium sulfate• Example : 60 g. of dry powder was dissolved in 250 ml. of water, centrifuged, and filtered. To the filtrate (265 to 270 ml.) was added 2 vol. of saturated ammonium sulfate solution, and the mixture was stored at 4 ° for 20 hours• The precipitate was then isolated by centrifugation, dissolved in water, and dialyzed at 4 ° until free of ammonium sulfate (30 hours)• This solution was then lyophilized and yielded 5.4, 7.0, and 7.7 g. of a white, nonhygroscopic powder in three different experiments• In the presence of pyndoxal phosphate the Qco2was found to be 177 to 188, whereas under the same conditions the original dry powders gave Q values of 32 to 58. On the average, a four- to fivefold purification is achieved by this treatment with a yield of about 50% in terms of original activity. •

N 2

Properties Specificity. Of the naturally occurring amino acids, only L-glutamic acid is decarboxylated by squash extract. Slow decarboxylation takes place when synthetic fl-hydroxy glutamic acid 5 is used as substrate. 6 However, since, contrary to earlier claims, the hydroxy acid is not found in protein hydrolyzates, 7 its reaction with the enzyme is only of academic interest. Crude, lyophilized squash extracts constitute a conveniently available reagent for the quantitative determination of L-glutamic acid in protein hydrolyzates. ~ Despite the presence of a-keto acid carboxylase in crude squash dry powders, it is possible to determine glutamic acid selectively in mixtures 5 W. J. Leanza a n d K. Pfister, J. Biol. Chem. 9.01, 377 (1953). s O. Schales a n d S. S. Schales, u n p u b l i s h e d observations. 7 C. E. D e n t a n d D. I. Fowler, Biochem. J. 56~ 54 (1954)•

[9.1]

A M I N O ACID D E C A R B O X Y L A S E S

OF P L A N T S

193

containing pyruvic acid or a-ketobutyric acid, since the action of carboxylase is quantitatively inhibited by 2 rag. of phenol per milliliter of incubation mixture, s,9 Phenol has no effect in this concentration on L-glutamic acid decarboxylase. Erroneous results are obtained, however, when a-ketoglutaric acid is incorporated into the incubation mixtures. On addition of this keto acid crude squash powder releases C02 even in the absence of glutamic acid, since this amino acid is formed by transamination. Removal of the amino group donors (aspartic acid, alanine) from squash powders by short dialysis abolishes the formation of glutamic acid from a-ketoglutaric acid) It should be mentioned here that, if transamination occurs, the reaction products pyruvic acid (from alanine) and oxalacetic acid (from aspartic acid) supply an additional 1 or 2 moles of CO2, respectively, as a result of their reaction with carboxylase. Activators and Inhibitors. Glutamic acid decarboxylase on prolonged dialysis loses its prosthetic group (pyridoxal-5-phosphate) with a corresponding decrease in activity. Loss of some of the coenzyme is also observed during purification processes involving dialysis. Activity is immediately restored in these instances on addition of pyridoxal phosphate. The aldehyde group of pyridoxal is essential for enzymatic activity, and substances reacting with aldehydes are, therefore, inhibitors for glutamic acid decarboxylase. Hydroxylamine, for example, reduces the initial reaction velocity by about 50% when its concentration in the i~cubation mixture is 3 × 10-5 M, but a concentration of 8 X 10-5 M is required to lower the total C02 output over a period of 60 minutes to about 50% of that obtained in absence of the inhibitor. 2 Effect of pH. Optimal activity of the enzyme takes place between pH 5.5 and 5.8, and no activity is found beyond pH 4.0 and 7.5, respectively. 2 The C02 output during a period of 60 minutes is lowered to onehalf of that observed under optimal conditions when the incubation is carried out at pH 4.6 or 6.85. Reaction Kinetics. 2,~° The time-activity curves for the action of glutamic acid decarboxylase do not represent curves for a monomolecular reaction, and there is a rapid decrease of k ~ while the experiment is in progress. When the activity of dialyzed extracts is restored by the addition of pyridoxal-5-phosphate, or when an excess of the coenzyme is O. Schales, S. S. Schales, and G. M. Schwarzenbach, Federation Proc. 9, 223 (1950). 9 0. Schales and S. S. Schales, Federation Proc. 12, 264 (1953). ~oO. Schales, in " T h e Enzymes" (J. B. Sumner and K. ~¢Iyrb/ick, eds.), Vol. II, Part 1, p. 216, Academic Press, New York, 1951. ,1 k = l l o g a a

194

ENZYMES OF P R O T E I N MET.~BOLISM

[21]

added to fresh extracts, one obtains reaction curves which fit very well the equation for a first-order reaction. It was found that the reaction velocity decreased as a linear function of the extent of glutamic acid decar.boxylation, or, in other words, straight lines are obtained when x / t (microliters of COs per minute) is plotted against x (total microliters of COs formed at time t). Extending these lines to the ordinate (t = 0 minute) permits an easy determination of initial reaction velocities. These initial reaction velocities may be used, instead of Q values based on 10-minute readings, to express the activity of an enzyme preparation. The equation for the straight lines just discussed may be written as x/t =mx + b

and this can be rearranged to read 1/t = b . 1 / x + m

This, again, is an equation for a straight line. The slope of this line, obtained when l i t is plotted against l / x , is, then, b or the initial velocity. The Michaelis-Menten constant for glutamic acid decarboxylase (from carrots) was determined as 3.6 _ 0.4 × 10-3 M.

Other Amino Acid Decarboxylases of Higher Plants Some evidence has been presented 12 that certain plants form histamine from histidine and hydroxytyramine from 3,4-dihydroxyphenylalanine. Decarboxylation of these amino acids was observed only with intact plants, and attempts to prepare cell-free, enzymatically active extracts were unsuccessful. 1~E. Werle and A. Raub, Biochem. Z. 318, 538 (1948).

[22]

195

AMINO ACID DECARBOXYLASES OF ANIMALS

[22] A m i n o Acid D e c a r b o x y l a s e s of A n i m a l s

By OTTO SCHALES 3,4-Dihydroxyphenylalanine Decarboxylase from Animal Tissues H0--~~CH~CH(NH~)C00H

--~

H0 HO--~--CH~CH2NH2

~- CO2

H0

Assay Method

Principle. Manometric determination of the CO~ liberated during a period of 10 minutes on anaerobic incubation of the enzyme preparation at pH 6.80 and 37 ° in the presence of an excess of substrate serves as a measure of enzyme activity ~,2 and is the procedure to be described. However, other methods have been published also. Since the reaction product hydroxytyramine is physiologically active and raises blood pressure, the biological determination of the amount of this amine formed under specified conditions may also be used for the assay of this enzyme. ~,3 Furthermore, it has been suggested recently 4 to determine hydroxytyramine colorimetrically by reaction with Folin and Ciocalt6u's phenol reagent, after the amine has been separated from the remaining substrate by cation exchange adsorption on activated Permutit. For each microliter of CO2 liberated, there are formed 6.83 -y of hydroxytyramine. Reagents L-Dihydroxyphenylalanine solution (0.0223 M). Dissolve 22 mg. of L-dihydroxyphenylalanine in 5 ml. of water and store in the cold. The colorless solution turns brown and darkens on standing for a few days and should then be replaced by a fresh one. 1.2 N sulfuric acid (concentrated H~S04 diluted 1:30 with water). M/15 (0.067 M) phosphate buffer, pH 6.80.

Procedure. Warburg flasks are set up containing 4 ml. of enzyme solution in M/15 phosphate buffer, pH 6.80, in the main compartment, 0.5 ml. O. P. a E. 4 L.

Schales and S. S. Schales, Arch. Biochem. 24, 83 (1949). Holtz, R. Heise, and K. Lfidtke, Arch. exptI. Pathol. Pharmakol. 191, 87 (1938). W. Page, Arch. Biochem. 8, 145 (1945). S. Dietrich, J. Biol. Chem. 204, 587 (1953).

196

ENZYMES OT PROTEIN METABOLISM

[22]

of L-dihydroxyphenylalanine solution (2.2 mg. of L-DOPA, capable of releasing 250 ul. of C02, DOPA concentration in incubation mixture 0.0025 M) in side vessel I, and 0.5 ml. of 1.2 N H2S04 in side vessel II. A blank vessel in which water takes the place of substrate must be used to permit correction for the small amounts of COs contained in the buffer or formed by the tissue extract in the absence of DOPA. Air is replaced by nitrogen to avoid confusing side reactions, such as action of amine oxidase on hydroxytyramine. The reaction is started, after temperature equilibrium has been reached, by adding the substrate from side vessel I to the main compartment. Manometer readings are taken at zero (before addition of substrate) and 10 minutes, and the reaction is then terminated by adding the content of side vessel II to the enzyme-substrate mixture; this addition of acid not only inactivates the enzyme but also liberates that portion of the COs which has been retained in solution at pH 6.80. After an additional 5 to 10 minutes of shaking the final reading is taken. The volume of CO~ formed by the reaction of enzyme with substrate is calculated in the usual manner under consideration of the various corrections necessary. Nz Calculation of Activity. Activities are expressed as Qco, values (microliters of C02 per hour per milligram of fresh tissue). If dried enzyme preparations are used instead of tissues or tissue extracts, microliters of COs per hour per milligram of dry material is used as an index of activity. Finally, the nitrogen content of the tissue or dry powder instead of its weight can be used to calculate Qco,, which then reads microliter of CO~ per hour per milligram of protein nitrogen. It is customary 5 to use initial velocities (10-minute values) for the calculation of activities rather than the actual measured amount of COs formed in 1 hour. The 10-minute values are multiplied by 6 to give the hypothetical CO~ production per hour. This value is, as a rule, considerably higher than actual measurements after 60-minute reaction time.

Preparation of a Stable Dry Powder Dihydroxyphenylalanine decarboxylase occurs in a variety of tissues of numerous species, ~,6 but guinea pig kidneys are particularly rich in this enzyme. A stable dry powder may be prepared from guinea pig or rabbit kidneys as follows. 1 In a Waring blendor, fresh kidneys are mixed with 3 to 4 vol. of ice-cold water for 4 minutes at room temperature. The material is then placed in a refrigerator for 1 hour and later centrifuged for 15 minutes at 3500 X g (size 2 centrifuge, conical head No. 845, 5 H. Blaschko, Advances in Enzymol. 5, 67 (1945).

60. Sehales, in "The Enzymes" (J. B. Sumner and K. Myrb~.ck, eds.), Vol. II, Part 1, p. 216, Academic Press, New York, 1951.

[22]

AMINO ACID DECARBOXYLASES OF ANIMALS

197

4400 r.p.m.). The pink and turbid supernatant fluid is decanted, frozen rapidly, and dried i n vacuo from the frozen state. The weight of dry powder obtained from guinea pig kidneys is quite regularly 10% of that of the fresh kidneys; the yield from rabbit kidneys varies from l0 to 14 g. per 100 g. of fresh kidneys. The dried material is stored in vacuo over CaC12 at room temperature (23 to 25°). A comparison between low-speed eentrifugation (2000 X g) and high-speed centrifugation (20,000 X g) showed 7 that the higher speed removed (in three out of five experiments) additional inactive ballast material from the kidney extract. The decarboxylase activities of the dry powders (dissolved in M / 1 5 phosphate buffer, pH 6.80) is about 75% (rabbit) and 45 to 60% (guinea pig) of those observed with phosphate buffer extracts from fresh kidneys of the same batch. An extract from fresh rabbit kidneys, for example, gave a Qco, of 0.91 and yielded a dry powder with a Qco, (per milligram of fresh tissue) of 0.68. Calculated per milligram of dry powder instead of fresh kidneys, the activities of several preparations from rabbits varied between 3.7 and 4.9, but decreased on standing over CaC12 i n vacuo. During a storage period of 6 weeks, for example, the activity of one of these preparations dropped from 3.72 to 1.98 (47% loss). Several extracts from guinea pig kidneys had a Qco2 of 2.0 to 2.5, and the corresponding dry powders gave a Qco, of 1.0 to 1.2 (per milligram of fresh kidney) or 10.3 to 12.3 (per milligram of dry powder). The material from guinea pig kidneys is relatively stable. A product with a Qco, of 11.1 on the day of preparation retained its activity for 4 months, giving a Qco, averaging 11.0 in nine determinations performed after 100 to 120 days of storage. Longer storage over CaC12 i n vacuo led to a decrease in activity, and the Qco, values after 6.5 and 8 months of storage were 7.7 and 5.4, respectively. The amounts of dry powders used in these stability studies (and suitable for assay studies as outlined above) were 25 to 60 mg. (guinea pigs) and 90 to 120 rag. (rabbits) per Warburg flask and liberated between 35 and 85 ~1. of CO2 during the first 10 minutes after addition of substrate. Properties Specificity. The enzyme is not absolutely specific for 5-3,4-dihydroxyphenylalanine but also decarboxylates other substituted phenylalanines with at least one hydroxy group in ortho or meta position, such as 2-hydroxy-, 3-hydroxy-, 2,5-dihydroxy-, s and 3,5-dihydroxyphenylalanine 7 at rates somewhat slower than those observed with 3,4-dihydroxyphenylalanine. It is interesting that the introduction of a second hydroxy 7 T. Sourkes, P. tteneage, and Y. Trano, Arch. Biochem. and Biophys. 40, 185 (1952). H. Blaschko and G. H. Sloane-Stanley, Biochem. J. 42, iii (a,b) (1948); 48, xxxvi (1948); H. Blaschko, ibid. 44, 268 (1949).

198

ENZYMES OF PROTEIN METABOLISM

[22]

group in meta position into 3-hydroxyphenylalanine, leading to 3,5-dihydroxyphenylalanine, considerably reduces the rate of decarboxylation. Activators and Inhibitors. The activity of tissue extracts or of dry powders prepared from such extracts is nearly doubled on addition of pyridoxal phosphate, 1 and, although absolute proof through isolation is still lacking, a number of observations suggest that pyridoxal-5-phosphate is the prosthetic group of this enzyme, as it is for other amino acid decarboxylases (for a discussion see Schales and Schalesl). In accordance with this role of pyridoxal phosphate, the enzyme is inhibited by aldehyde reagents, such as hydroxylamine, which shows appreciable effect at concentrations of 5 X 10-~ M.~ A variety of other inhibitors, acting through unknown mechanisms, has been reported, as summarized elsewhere. 6 Inhibition by an excess of substrate has also been described. 9 Effect of pH. Optimal activity of the enzyme from guinea pig and rabbit kidneys was found ~ to occur at pH 6.80, with the activity falling to one-half of optimal at about pH 6.2 and 7.8, respectively. Reaction Kinetics. The time-activity curve for the enzymic decarboxylation of 3,4-dihydroxyphenylalanine does not follow the course of a firstorder reaction. In an evaluation of experimental data using the equation k = l i t log (a/a - x), it was found I that the numerical value for k at 60 minutes was only about one-half of that calculated for the initial 10-minute period. Similar observations had been made already in studies with glutamic acid decarboxylase, ~° and it was reported that the addition of pyridoxal phosphate resulted in a first-order reaction curve for this enzyme. With DOPA decarboxylase, however, this change to a firstorder reaction does not occur, although the addition of pyridoxal phosphate lessened the extent of the decrease in k during a 60-minute reaction period. Inhibition by an excess of substrate seems to play a role in this connection. 9 Examination of the data obtained with DOPA decarboxylase showed (as was observed with glutamic acid decarboxylase) that the reaction velocity decreased as a linear function of the extent of substrate decomposition. As illustrated elsewhere, 1 this relationship results in the fact that straight lines are obtained when 1Ix (x = microliters of COs liberated at time t) is plotted against l i t . For any given observation period, there is a linear relationship between enzyme concentration (milligrams of dry powder per 4.5 ml. of incubation mixture) and amount of COs produced in the presence of 2.2 mg. of DOPA. This permits the correlation of the activities of unknown preparations with those of standard preparations of this enzyme. The Michaelis constant of 3,4-dihydroxyphenylalanine decarboxylase 9 H. F. Schott a n d W. G. Clark, J. Biol. Chem. 196, 449 (1952). 10 O. Schales a n d S. S. Schales, Arch. Biochem. llp 155 (1946).

[23]

D-AMINO ACID OXIDASE FROM KIDNEY

199

in the presence of pyridoxal phosphate was estimated to be 5.4 × 10-4 mole per liter at pH 6.80. 9

Other Amino Acid Decarboxylases of Mammalian Tissues Various mammalian tissues, especially kidney and liver, are able to decarboxylate, in addition to 3,4-dihydroxyphenylalanine, one or several of the following amino acids: histidine, tyrosine, tryptophan, eysteic acid, hydroxyphenylserine, and phenylalanine. Glutamic acid was found to be decarboxylated by mouse brain. A detailed description of these enzymes together with references to the original literature has been presented elsewhere. 6 Suffice it to say that the activities of extracts containing these enzymes are usually well below those of DOPA decarboxylase preparations. In some instances (histidine, tyrosine, tryptophan) the decarboxylation is far too slow for manometric assays, and the pharmacological effect of the reaction product must be used as an index of the progress of the enzymic reaction.

[23] D - A m i n o A c i d O x i d a s e f r o m K i d n e y R.CHNH2COOH W 02 + H20 --~ R.CO.COOH + NH3 + H~O~

By KENNETH BURTON Assay Method

Principle. The activity is measured by the rate of oxygen consumption. Excess catalase is added to ensure complete destruction of the H202 formed in the oxidase reaction; no interference is then introduced by the varying levels of catalase activity in the samples being assayed. With excess catalase, the consumption of 1 mole of oxygen corresponds to the oxidation of 2 moles of D-amino acid. In order to ensure that all the D-amino acid oxidase protein is in the enzymically active conjugated form, an excess of the prosthetic group (FAD) is added to the assay system. DL-Alanine is frequently used as the source of the substrate if, as with aqueous extracts of acetone-dried mammalian tissues, L-alanine is known not to be appreciably oxidized. Procedure. The main compartment of each Warburg vessel contains 1 ml. of 0.1 M pyrophosphate buffer, pH 8.3 (8 ml. of N HC1 and 100 ml. of 0.2 M Na4P207 diluted to 200 ml.), catalase (equivalent to about 0.5 ~, of pure catalase; 0.1 ml. of red blood cells diluted 1:100 in water

200

ENZYMES OF PROTEIN METABOLISM

[23]

may be used), 0.1 ml. of 10-4 M FAD, ~and D-amino acid oxidase in a total volume of 2.3 ml. The side bulb contains 0.2 ml. of 5% DL-alanine (or 2.5% D-alanine), and the center well contains 0.2 ml. of 2 N NaOH and filter paper for COs absorption. The assembled manometers are shaken at 37°; after 2 minutes the glass joints are ground in and the contents of the main compartment and the side bulb are mixed. After a further 5 minutes of shaking, temperature equilibrium of the flasks and their contents is attained, and the first reading is made; subsequent readings are taken at 5-minute intervals. The rate of reaction should be constant until at least 100 ~1. of 02 has been consumed. One unit of activity corresponds to the consumption of 1 ttl. of 02 per minute. The range of the assay method is 1 to 5 units of activity. Purification Procedure The procedure described is that of Negelein and Bromel; 2 it has been followed successfully in many laboratories up to step 3, where all the catalasc activity and almost all the FAD are separated from the oxidase protein. The enzyme is usually purified to obtain a FAD-free preparation for the assay and identification of FAD; no procedure has been described to obtain a highly purified preparation containing FAD. The removal of the FAD results in considerable instability of the enzyme, and in the present author's experience losses may be heavy at steps 4 and 5. Negelein and Bromel report a variable loss on drying at step 7. There is considerable variation (reported to be seasonable 3) in the activity of the sheep kidneys used as starting material. Pig kidneys are often as suitable as sheep kidneys, and the identical purification procedure may be followed at least to step 3. Step 1. Acetone Drying and Extraction of the Powder. 4 Stir freshly minced sheep kidney cortex into ice-cold acetone (400 ml. per kidney) and allow to stand at room temperature for 1 hour. Decant off the supernarant, and stir the residue with more cold acetone (125 ml. per kidney). Filter after 30 minutes and dry in vacuo over P205. Yield: 10.7 g. of powder per kidney. Stir the powder with 20 times its weight of 0.017 M pyrophosphate buffer (pH 8.3) at 38 ° for 45 minutes. Filter through cloth, and squeeze the residue by hand. 1 See footnote 5, and step 3 of the enzyme purification procedure. E. Negelein and H. Br6mel, Biochem. Z. 300, 225 (1939). s A. E. Bender and H. A. Krebs, Biochem. J . 46, 210 (1950). 4 As an alternative to mincing, the tissue m a y be treated with acetone in a Waring blendor. No activity is lost b y drying the filter cake in a gentle current of air at 15 ° instead of drying it in vacuo.

[23]

D-AMINO ACID OXIDASE FROM KIDNEY

201

Step 2. Heating at pH 5.1 and Ammonium Sulfate Precipitation. A d d , w i t h c o n t i n u o u s stirring, 21.6 ml. of 2 N acetic acid per liter of t h e extract. K e e p a t 38 ° for 5 m i n u t e s , cool to 25 °, centrifuge, a n d d i s c a r d t h e p r e c i p i t a t e . Dissolve (NH4)2S04 (236 g./1.) i n t h e s u p e r n a t a n t , a n d ~UMMARY OF ~Y)UI~IFICATION PROCEDURE a

Fraction I. ]~xtract of acetone powder 2. Redissolved (NH4)2SO~ precipitate 3. HCl precipitate 4. Heating at pH 6.0 5. Redissolved (NH4)2S04 precipitate 6. Dried (NH4)2S04 precipitate 7. Lyophilized powder

Total Total units, Specific volume, thou- Protein, activity, Recovery, 1. Units/ml. sands mg./ml, units/mg. %

12.0

103

1240

11.2

9.0

--

6.0 0.6 1.76

140 735 202

840 435 355

2.6 16.5 2.55

54 44 79

68 35 29

1.00

250

250

0.97

259

20

---

---

86 52

---

740 935

6.9 4.2

'~ The data in this table are from E. Negelein and H. Br6met, Biochem. Z. 800, 225 (1939). Since these workers did not use catalase in their activity test, the data given here are calculated assuming that no catalase activity was present in fraction 3, and that fraction 2 contained sufficient catalase to destroy the H~O~ as it was formed in the activity test. Negelein and Bromel used 700 g. of acetone powder corresponding to about 65 sheep kidneys. c e n t r i f u g e a f t e r 30 m i n u t e s a t 0 °. R e s u s p e n d t h e p r e c i p i t a t e in cold 3.3 × 10 -3 M p y r o p h o s p h a t e buffer, p i t 8.3 (one-half t h e v o l u m e of t h e original extract).

Step 3. Precipitation by Hydrochloric Acid from 20% Saturated Ammonium Sulfate. T h i s step m u s t be p e r f o r m e d e n t i r e l y a t 0 °. P e r liter of fract i o n 2, a d d 260 ml. of s a t u r a t e d (NH4)2SO4, a n d t h e n slowly a d d 30 ml. of N HC1 w i t h c o n t i n u o u s stirring. T h e p H is r o u g h l y 2.8 a t t h i s p o i n t . C e n t r i f u g e . 5 W a s h t h e p r e c i p i t a t e w i t h 1 1. of s a t u r a t e d (NH~)2SO~, 5 If sheep kidney is used as the starting material, the supernatant at this stage is a convenient source of FAD, almost completely free from other flavins. It may be concentrated by extraction into a small volume of phenol, adding 5 vol. of ether to the phenol extract, followed by extraction with a few milliliters of water. The FAD may be used in the assay after removing the phenol by washing with ether and subsequent aeration to remove the ether.

202

ENZYMES OF PROTEIN METABOLISM

[23]

centrifuge, and resuspend in 0.016 M pyrophosphate buffer, pH 8.3 (100 ml./1, of fraction 2). Adjust the pH to 6.5. Step 4. Heating at p H 6.0. Dilute fraction 3 with 2 vol. of water, and adjust to pH 6.0 using 2 N acetic acid (approximately 2.2 ml./h of fraction 3). Heat for 10 minutes at 38 °, cool to 20 °, centrifuge, and discard the precipitate. Step 5. Cool to 0 ° and adjust to pH 5.0, using approximately 2.3 ml. of 2 N acetic acid per liter. Dissolve 118 g. of (NH4)~SO4 per liter in the solution and centrifuge after 30 minutes at 0 °. Redissolve the precipitate in ice-cold 1.3 × 10-3 M pyrophosphate buffer, pH 8.3 (570 ml./1, of fraction 4). Step 6. Adjust to pH 5.1, using 2 N acetic acid (approximately 1.4 ml./1.). Warm to 38 °, and after 10 minutes cool to 20 °, centrifuge, and discard the precipitate. Dissolve 119 g. of (NH4)~SO4 per liter in the supernatant, centrifuge after 30 minutes at 0 °, and dry the precipitate at 0 ° over P205 and KOH in vacuo. Negelein and Bromel's material contained 50% protein and 50% (NH4)2SO4 at this stage. Step 7. Dissolve 250 mg. of the powder from step 6 in 2.5 ml. of 0.1 M NaHCO3. Dilute with 17.5 ml. of water, clarify by centrifuging, and dilute the clear solution to 375 ml. Add 100 ml. of saturated (NH4)~SO4 at 0 °, followed by 25 ml. of M acetate buffer, pH 4.92. Centrifuge immediately at 0 °, and suspend the precipitate in 10 ml. of 0.01 M NaHC03. Clarify by centrifuging, and lyophilize the clear solution. Negelein and Bromel's dried material contained 43% protein and 57% (NH4)2SO4.

Properties Specificity. The enzyme oxidizes a large variety of D-a-amino acids 3 and N-substituted D-a-amino acids, 6,~ including D-proline. One hydrogen atom on the a-carbon atom and one on the a-amino group must be unsubstituted. Glycine, 8 D-glutamic acid, 8 and the naturally occurring (? meso-) diaminopimelic acid 9 are not attacked. No peptides or acyl amino acids are known to be attacked. Activators and Inhibitors. The enzyme is sensitive to traces of heavy metals; the activations by amino acids, by pyrophosphate buffer, and by other metal-chelating substances TM are probably due to the removal of inhibitors rather than to direct activation. The enzyme is inhibited by

s D. Keilin and E. F. Hartree, Proc. Roy. Soc. (London) Bl19, 114 (1936). 7p. Handler, F. Bernheim, and J. R. Klein, J. Biol. Chem. 130, 203 (1941). s H. A. Krebs, Biochem. J. 29, 1620 (1935). 9 E. Work, Biochem. J. 49, 17 (1951). 10S. Edlbacher, O. Wiss, and A. Walser, Helv. Chim. Acta 29, 162 (1946).

[23]

D-AMINO ACID OXIDASE FROM KIDNEY

203

certain " s u l f h y d r y l " reagents H and by H2S ~ but is not affected by cyanide or by sec-octanol. 8 Azide is inhibitory at pH 6.8 but not at pH 7.8. ~ Many compounds, including L-leucine, L-phenylalanine,12 fatty acids, 13 kojic acid, 14 benzoic acid, 15 and substituted benzoic acids 1~ inhibit by competing with the D-amino acid substrate. Other compounds (ADP, 5'-AMP) inhibit by competing with the FAD, and quinine appears to inhibit by forming a complex with the FAD and thus inducing dissociation of the prosthetic group from the enzyme.17 The inhibitions by atebrin (mepacrine) and some other antimalarial drugs is seem to be more complex. Stability. FAD-free preparations lose activity at an appreciable rate (10% per week), even when stored frozen at - 1 5 ° or when lyophilized and kept dry over CaC12 at 0 °. They are stabilized by adding 0.03 M D-alanine or 2.5 uM FAD. ~ The pH of optimum stability is about 6.5. pH. The pH of optimum activity is about 8.8. 8 Dissociation Constants. The values reported for the Michaelis constants of D-alanine at pH 8.3 and 37 ° vary between 2 and 9 mM ~7-~9 for the sheep kidney enzyme saturated with FAD. The cause of this variation is unknown, although it should be noted that independent workers 1~-~7 agree that this Michaelis constant is approximately 100 times the K, value for benzoic acid. Values of 2 mM and 8 mM, respectively, have been reported for the Michaelis constants of D-valine 8 and D-methioaine.13 The values reported for the apparent dissociation constant of FAD 2° also vary and lie between 0.13 and 0.25 #M 17,2~-~3 at pH 8.3 and 37 °. The data of Hellerman et al. ~s give different values (about 0.19 and 0.27 ~M, respectively) for two different enzyme preparations studied under similar conditions; these authors also noted that at 30 ° the enzyme is saturated with much lower concentrations of FAD. 11 T. P. Singer, J. Biol. Chem. 174, 11 (1948). 12 S. Edlbacher a n d O. Wiss, Helv. Chim. Acta 27, 1831 (1944). ~a W. T. Brown a n d P. G. Scholefield, Proc. Soc. Exptl. Biol. Med. 82, 34 (1953). 14 j. R. Klein a n d N. S. Olsen, J. Biol. Chem. 170, 151 (1947). 1~ j. R. Klein a n d H. Kamin, J. Biol. Chem. 138, 507 (1941). 1~ G. R. Bartlett, J. Am. Chem. Soc. 70, 1010 (1948). 1~ K. Burton, Biochem. J. 48, 458 (1951). 18 L. Hellerman, A. Lindsay, a n d M. R. Bovarnick, J. Biol. Chem. 163, 553 (1945). 1~ W. C. Stadie a n d J. A. Zapp, J. Biol. Chem. 160, 165 (1943). ~ The concentration of F A D which gives one-half of the m a x i m u m velocity obtained with higher concentrations of FAD. ~ O. W a r b u r g a n d W. Christian, Biochem. Z. 298, 150 (1938). 2~ T. P. Singer a n d E. B. Kearney, Arch. Biochem. 27, 348 (1950). 2~ L. G. Whitby, Biochem. J . '54, 437 (1953).

204

ENZYMES OF PROTEIN METABOLISM

[24]

Enzyme-inhibitor dissociation constants of 1 mM and 1.4 mM, respectively, have been reported for 5-AMP and for ADP.17 Hydrogen Acceptors. Certain basic dyes (e.g., methylene blue) 8 react sluggishly as hydrogen acceptors in place of molecular Oz. Equilibrium. It may be calculated, from available free energy data, that in dilute aqueous solution the equilibrium constant (CHaCO'COO-). (H202)" (NH4 +) (CH3CHNH2+COO -) is approximately 10 la M s in the presence of oxygen at 1 atmosphere. The oxidation by molecular oxygen is therefore practically irreversible. Turnover Number. Two thousand molecules of D-alanine are oxidized per minute per mole of protein-bound FAD at pH 8.3 and 370. 5

[24] L-Amino Acid Oxidases (Mammalian Tissues and Snake Venom) RCHNH2COOH + ~ 0 2 ~ RCOCOOH + IXTH3 (catalase present) R C H N H 2 C O O H W H20 -}- O2--* RCOCOOH ~ NH3 ~- H202 (catalase absent) RCOCOOH + H~O2-~ RCOOH -[- COs % H20 RCHNH~COOH + 02--~ RCOOH + NH3 + CO2 (sum of lb + lc) By S. t~ATNER

(la) ilb) (lc) (ld)

Assay Method Principle. The assay, originally introduced by Krebs, ~ consists in measuring the rate of oxygen uptake by means of the Warburg manometer. In the presence of catalase (reaction la) 1/~ mole of 02 is utilized for the oxidation of 1 mole of amino acid. In the absence of catalase, oxygen consumption is doubled (reaction lb or ld). Activity may also be followed, but far less conveniently, by measuring NH3 or a-keto acid formation. The latter cannot be used in the absence of catalase, since H202 will oxidize the a-keto acid in accordance with reaction lc. With insufficient catalase present, oxygen uptake will tend to exceed the requirement for reaction la, and some of the a-keto acid will disappear. Application of Assay Procedure to Crude Preparations. The procedure is applicable to crude tissue extracts, but low activity is usually masked 1 H. A. Krebs, Z. physiol. Chem. 217, 191 (1933).

[24]

L-AMINO ACID OXIDASES

205

by the oxygen uptake of the blank. Small differences may become manifest after prolonged incubation. Most crude snake venoms are highly active. A. L-Amino Acid Oxidase of Snake Venom

Reagents 0.1 M L4eucine adiusted to pH 7.2 with a few drops of 0.1 N NaOH. 0.2 M Tris buffer, pH 7.2 (tris (hydroxymethyl) aminomethane). 0.1 M KC1.

Procedure. In the side arm is placed 0.21 ml. of the leucine solution. In the main space are placed 1.0 ml. of buffer and an amount of enzyme, suitably diluted with the KC1 solution, to give 100 to 200 ~1. of oxygen uptake in 30 minutes. Water is added to a final volume of 3.0 ml. The center well contains alkali. Measurements are made at 38 ° with air in the gas phase. Definition of Unit and Specific Activity. In the paper of Singer and Kearney, 2 whose purification procedure is given below, the unit of activity is not defined, but the yields and purity are stated. Specific activity is given as the activity ratio: microliters of oxygen uptake per 30 minutes per milliliter of enzyme/optical density at 280 m~ in a 1-cm. cell. The values are also expressed as Qo2: microliters, per hour per milligram of protein. From their data it may be calculated that 1 mg. of protein per milliliter has an optical density of 1.64. Under the assay conditions, activity is proportional to enzyme concentration. All assays are carried out in the absence of catalase, since it is not present in the crude venom. Purification Procedure

The purification steps were developed with moccasin venom (Agkistrodon piscivorus), which is the least expensive source in this country, s The procedure is applicable to small quantities (from 200 rag. to 5 g.). The first two steps, which can be carried out in a day, bring the enzyme from 2 % to 80 % purity. The enzyme is unstable to freezing or lyophilization and is best stored at 0 to 5° as an (NH4)~S04 precipitate, or as a concentrated solution in 0.5 saturated (NH4)2S04 at pH 5.5. Advantage is taken of the relative stability to heat in the presence of substrate, and of the stability and minimum solubility at the isoelectric point. The procedure has been reproduced in the author's laboratory with excellent results. T. P. Singer and E. B. Kearney, Arch. Biochem. 29, 190 (1950). 3 Snake venoms may be purchased from Ross Allen's Reptile Institute, Silver Springs, Florida.

206

ENZYMES OF PROTEIN METABOLISM

[24]

Step 1. Heat Treatment. One gram of dried moccasin venom is dissolved in 100 ml. of water, and 10 ml. of 0.1 M L-leucine is added. The solution is immediately heated to 73 ° as rapidly as possible, and it is kept at 73 ° for 5 minutes. After chilling in ice and a brief centrifugation at 5000 r.p.m., the denatured proteins contained in the precipitate are discarded, and the clear, yellowish supernatant, containing all the enzyme, is cooled to 0 to 3 ° in an ice bath. The rest of the procedure is carried out at this temperature range. Step 2. Adsorption on Ca3(P04)2 Gel and Fractionation with (NH4)2S04. To each 100 ml. of supernatant solution, neutral Ca3(PO4)2 gel 4 (dry weight 350 mg.) is added dropwise, and the resulting suspension is stirred for 15 minutes, after which it is briefly centrifuged. The precipitate is discarded, and the nearly colorless supernatant, pH 6.5 to 6.6, containing all the enzyme, is treated with 20 ml. of 0.1 M acetate buffer, pH 4.61, whereupon the enzyme immediately turns yellow. (The various acetate buffers are prepared from sodium acetate by neutralization with HC1.) It is then treated dropwise with 400 rag. of Ca3(PO4)~ gel for each 100 ml. of supernatant obtained in step 1. After 15 minutes of stirring the suspension is centrifuged for 5 minutes at 5000 r.p.m. The colorless supernatant (pH 5.57 to 5.61) is discarded, and the gel, containing 95% or more of the enzyme, is thoroughly drained. The gel is resuspended in 100 ml. of acetate buffer, pH 5.5 to 5.7, 0.06 to 0.1 M, by careful homogenization by hand and subsequent stirring for 15 minutes. The suspension is centrifuged, and the colorless supernatant solution, pH 5.7 to 5.8, is discarded; this contains less than 10% of the enzyme and large amounts of inert protein. For best yields, the concentration and pH of the buffer used for washing the gel should be selected in a preliminary experiment, within the limits indicated for each batch of gel. The acetate wash should elute no more than 5 to 10% of the enzyme present (determined by assay) and an amount of protein which gives an optical density of at least 0.380 at 280 m~ in a 1-cm. cell. The oxidase is eluted by keeping the pH in its isoelectric range and raising the ionic strength. This is accomplished by resuspending the gel in 0.25 ml. of 0.65 saturated (NH4)2SO4 in acetate buffer, pH 4.6, with the aid of a homogenizer, and then stirring for 15 minutes. The eluting solution is made by diluting 65 ml. of cold saturated (NH~)~SO4 to 100 ml. with 0.2 M acetate buffer, pH 4.6. The clear yellow eluate obtained on brief centrifugation, which is approximately at pH 6 and is about 0.58 saturated with respect to (NH4)~SO4, is cautiously adjusted to pH 5.5 to 5.6 with a small amount of 1 N acetic acid, and 1.35 g. of (NH,)~SO4 is added to each 10 ml. of solution. The slight precipitate formed on 15 4 F o r p r e p a r a t i o n of calcium p h o s p h a t e gel, see Vol. I [11].

[24]

L-AMINO ACID OXIDASES

207

minutes of stirring is the active enzyme. I t is separated b y centrifugation at 5000 r.p.m, for 30 minutes. Step 3. (NH4)~S04 Fractionation at pH 5.5. T h e light yellow precipitate obtained in step 2 is redissolved in 3 ml. of 0.1 M acetate buffer, p H 5.5, and it is then dialyzed for 12 hours with continuous stirring against 200 ml. of 0.626 saturated (NH4)2S04. This raises the saturation to 0.62. The light yellow precipitate formed is centrifuged off and discarded. The (NH4)~S04 saturation is now raised to 0.73 by dropwise addition of saturated (NH4)2S04 in acetate buffer, p H 5.5, 4.07 ml. being required for 10 ml. of enzyme solution. After 15 minutes of stirring, the heavy yellow precipitate, containing most of the enzyme, is centrifuged off. In this step, the pH is given at 0 °. Step 3. Electrophoresis. T h e precipitate is redissolved in a small volume of 0.05 M Tris buffer, p H 7.2, and then it is dialyzed against the same buffer overnight. Any slight precipitate formed at this stage is removed by centrifugation. The yellow solution is electrophorized in a Tiselius apparatus using the separatory cell, at 1.5 °, and 0.012 ampere; the enzyme migrates to the positive pole. The yield, of course, depends on the length of electrophoresis. In practice, the enzyme m a y be obtained in homogeneous form after 24 hours. T h e recovery in this step is 50 %. F u r t h e r (NH4)2S04 fractionation m a y be substituted 2 for this step. SUMMARY OF PURIFICATION PROCEDURE2

Fraction 1. Crude extract 1. Heat step 2. Gel elution and (NH~) ~SO4 pptn. 3. (NH4)2SO4 fractionation 4. Electrophoresis

Total Total volume, protein, Activity ml. mg. ~ ratio 110 108 3

730 400

180-200 385

Qo~ 440-530

100

18-20 5000-5500 16,440-18,100 12 6

5900 6300

Recovery, %

19,300 20,810

Purity 0.02 0.06

60-70

0.79-0.87

40-50 25

0.93 1.0

Protein values are approximate.

Properties The enzyme was found in snake venoms b y Zeller, 5 who investigated the properties in venoms from a great m a n y species. Since there are some minor variations among them, only the more general properties are given 5 E. A. Zeller and A. Maritz, Helv. Chim. Acta 27, 1888 (1944) ; 28, 365 (1945).

208

ENZYMES OF PROTEIN METABOLISM

[24]

here along with some properties of the purified enzyme from moccasin venom.

Prosthetic Group and Physical Constants. 2,6,The oxidase is a flavoprotein having a molecular weight of 62,000 with 1 mole of FAD per mole of enzyme. Resolution does not occur during purification. Reoxidation of the reduced flavoprotein appears to be the rate-limiting step, for the enzyme is bleached by substrate in the presence of air, and oxygen consumption is tripled when 02 replaces air in the gas phase. The turnover number in O2 is 3100 moles of leucine oxidized per minute per mole of protein at 38 °, pH 7.2, in the absence of catalase. The enzyme is homogeneous by phase-solubility criteria and by electrophoresis and sedimentation patterns. Diffusion constant, D20 = 11.0 X 10-~ cm.2/sec. electrophoretic mobility, g = 2.02 )< 10-a cm.2/sec./volt at pH 6.0, succinate buffer, F/2 = 0.08; sedimentation velocity, S:0 = 6.93. Specificity. ~,7 D-Amino acids are not attacked. The highest activity is found among monoaminomonocarboxylic acids. The relative rates vary among different species; leucine, methionine, phenylalanine, norvaline, norleucine, and frequently cysteine, tyrosine, tryptophan, citrulline are among the most rapid; glycine, alanine, serine, threonine, and valine are usually inactive or among the slowest. Histidine, arginine, and ornithine are slowly oxidized; glutamic acid, aspartic acid, proline, N-methylamino acids, and peptides are not oxidized. Dependence on pH and Substrate. 2,5.~ The Km for leucine is about 1 X 10-3 M. The affinity for the other substrates has not been investigated. In general, substrate concentrations above 0.01 M are inhibitory. The region of optimum pH extends from 7.0 to 7.5; activity falls off above and below this range. Inhibitors and Activators. Benzoic, mandelic, salicylic, and iodoacetic acids, sulfonamides, aromatic sulfonic acids, aliphatic a-aminosulfonic acids, and the carbonyl reagents all inhibit in a competitive manner. 5,7 Riboflavin above 1 )< 10-~ M and many of its analogs above 1 X 10-3 M exert an inhibition which is not reversed by FAD. e Cyanide is not inhibitory. The enzyme in many but not all venoms rapidly undergoes a spontaneous, reversible inactivation in water or in the presence of phosphate and other bi- and trivalent anions. Protection and reactivation occur in the presence of chloride, substrate, or flavins. The papers of Kearney 6 T. P. Singer a n d t~. B. Kearney, Arch. Biochem. 9.7, 348 (1950). 7 E. A. Zeller, Advances in Enzymol. 8, 459 (1948).

[24]

L-AMINO ACID OXIDASES

209

and Singer 8 should be consulted for the influence of pH and temperature on the rate and extent of inactivation and reactivation. B. Mammalian L-Amino Acid Oxidase 9

Reagents 0.1 M L-leucine in 0.5 M phosphate buffer, pH 8.8. 0.1 M sodium phosphate buffer, pH 8.8. Catalase as a partially purified preparation or as washed erythrocytes.

Procedure. The side arm contains 0.5 ml. of leucine. In addition to water, the main space contains 0.5 ml. of buffer, 1.0 ml. of enzyme, and 0.3 ml. of erythrocytes. Other conditions are the same as for the snake venom enzyme. Catalase is present in the crude extract but is destroyed during purification. Catalase supplementation is necessary after step 3. Definition of Unit and Specific Activity. One unit catalyzes the uptake of 60 ul. of 02 per hour at 38 ° and pH 8.8, in the presence of excess catalase. Specific activity is not defined as such, but a purity index is employed, which is defined as the ratio of the optical density at 280 m~ in a l-cm. cell to the number of units per milliliter of enzyme. Purification Procedure

Rat kidney and, to a lesser extent, rat liver have proved to be the only sources sufficiently active for isolation purposes. Large quantities are required. The tissue retains activity when stored frozen on dry ice. Step I. Preparation of Extract. Rat kidneys (about 1.5 kg.) are minced in a Waring blendor to a fine cream and poured into 4 vol. of acetone, cooled to - 5 ° . The suspension is rapidly filtered with suction and the residue washed several times with cold acetone. The residue is then allowed to dry in air at room temperature while being rapidly broken up to a powder. It is then resuspended in 10 vol. of acetone to remove residual fat, filtered, and again allowed to dry in air. 318 g. of dry powder are obtained. The powder is extracted with 10 vol. of water for 30 minutes with constant stirring. Insoluble material is filtered off and washed with water. Step 2. Fractionation with Na2S04. Anhydrous sodium sulfate is added to the filtrate (15 g. per 100 ml. of extract) and then glacial acetic acid to bring the pH to 5.1, as measured with the glass electrode. The precipitate may be separated by filtration on fluted paper, left to stand overnight if necessary. The enzyme is quite stable at this stage. E. B. Kearney and T. P. Singer, Arch. Biochem and Biophys. 33, 377, 397, 414 (1951). 9 M. Blanchard, D. E. Green, V. Nocito, and S. Rather, J. Biol. Chem. 161,583 (1945).

210

ENZYMES OF P R O T E I N METABOLISM

[24]

Step 3. Heat Treatment and Second Na2S04 Precipitation. The precipitate is suspended in about 350 ml. of water, and 6 N NaOH is carefully added to bring the pH to 8.5. The suspension is heated with swirling in a 57 ° bath for 5 minutes, and the bulky precipitate removed by filtration. The filtrate is again treated with sodium sulfate (15 g. per 100 ml.), and the pH is brought to 5.6 with 10% acetic acid. The adjustment of pH is critical and should be carried out electrometrically. The precipitate is centrifuged off, suspended in 50 ml. of water, and brought to pH 8.7 with 6 N NaOH, and then clarified by centrifugation. Step 4. Successive Fractionations with (NH4) ~S04. The enzyme solution from the above step is dialyzed for 18 hours at 0 ° against 3 1. of 0.025 M phosphate buffer, pH 7.3. The pH of the cold solution is cautiously brought to 4.9 with 10 % acetic acid. The precipitate, containing the bulk of the hematin impurities, is discarded. The supernatant fluid, which from this step on has the greenish yellow appearance of flavin solutions, is neutralized with 6 N NaOH. The solution is then subjected to a series of stepwise fractionations with (NH4)2S04 between 30 and 60% saturation. The two middle fractions (36 to 43 and 43 to 50) are combined and refractionated. The two middle fractions thus obtained are again combined and refractionated. The 43 to 50 fraction finally obtained is homogeneous on electrophoresis. SUMMARY OF PURIFICATION PROCEDURE 9

Fraction l. 2. 3. 3. 4.

Crude extract First Na:SO~ precipitation Heat treatment Second Na~S04 precipitation Three (NH4)2SO4 fractionations

Total volume, ml.

Total units

Purity index

3900 360 310 56 2

347 222 85 48 4.8

347

13.9 1.7 ~

a Corresponds to a Qo2 of 52.

Properties Prosthetic Group and Physical Constants. 9 The enzyme is a flavoprotein and occurs in two molecular species, one being four times the size of the other. The lighter species has a molecular weight of about 120,000 and contains 2 moles of F M N per mole of enzyme (0.66% FNIN). The turnover number, 6 moles of amino acid oxidized per minute per mole of enzyme at 38 °, is unusually low for flavoproteins of this class. Under anaerobic conditions, the enzyme is bleached by substrate.

[24]

L-AMINO ACID OXIDASES

211

T h e electrophoretic p a t t e r n shows a single p e a k at p H 7.4 in 0.02 M sodium phosphate buffer, 0.15 M NaC1. I n the ultracentrifuge, two components of equal activity appear, 6 0 % as a h e a v y fraction ($20 = 13.5), and a light fraction ($20 = 5.0). T h e diffusion constant of the light component D20 = 4.0 X 10 -7 cm.2/sec. Specificity2 -11 Practically all monoaminomonocarboxylic acids of the L-series are oxidized. I n descending rate order, these include N - m e t h y l leucine, leucine, aminocaproic acid, methionine, proline, isoleucine, aminovaleric acid, t r y p t o p h a n , valine, and tyrosine. Serine, glycine, arginine, ornithine, and aspartic and glutamic acids are not attacked. T h e e n z y m e also oxidizes a n u m b e r of a - h y d r o x y acids of the L series; a m o n g the most rapid are the ~-hydroxy derivatives of isovaleric, butyric, isocaproic, and caproic acids and phenylglycolic, phenyllactic, and lactic acids. The last is oxidized at 2.7 times the r a t e of leucine. Inhibitors. I n contrast to the snake v e n o m enzyme, the m a m m a l i a n enzyme is inhibited b y iodoacetate at 10 -3 M and a m m o n i u m ions at 10 -2 M but is not appreciably inhibited b y benzoate at 10 -3 M. I n contrast to the L-amino acid oxidase activity of slices or brei, there is no inhibition b y cyanide at 10 -2 M or b y capryl alcohol.

C. Other L-Amino Acid Oxidases I n specificity, response to inhibitors, and lower activity of crude preparations, the isolated enzyme differs from the behavior of tissue slices and brei. 1.~2Some of the differences can be a t t r i b u t e d to an oxidative deamination in slices and brei which is catalyzed b y t r a n s a m i n a s e in conjunction with glutamic dehydrogenase and the cytochrome system. An L-amino acid oxidase is present in the mycelium and m e d i u m of Neurospora crassa, grown on low biotin. ~3-15 T h e activity of crude preparations is m u c h higher and the specificity broader t h a n the m a m m a l i a n enzyme. T h e enzyme from the m e d i u m has been partially purified and shown to be a flavoprotein with F A D as prosthetic group. ~5 F o r L-amino acid oxidase activity in other sources see Krebs. 16 10 M. Blanchard, D. E. Green, V. Nocito, and S. Rather, J. Biol. Chem. 155, 421 (1944). 11 M. Blanchard, D. E. Green, V. Nocito-Carroll, and S. Ratner, J. Biol. Chem. 163, 137 (1946). 12H. A. Krebs, Biochem. J. 29, 1620 (1935). is A. E. Bender and H. A. Krebs, Biochem. J. 46, 210 (1950). 14p. S. Thayer and N. H. Horowitz, J. Biol. Chem. 192, 755 (1951). 15K. Burton, Bioehem. J. 50, 258 (1951-52). 16H. A. Krebs in "The Enzymes" (J. B. Sumner and K. Myrb~ck, eds.), Vol. II, Part 1, p. 499, Academic Press, New York, 1951.

212

ENZYMES OF PROTEIN METABOLISM

[25]

[25] A m i n o Acid R a c e m a s e s

By W. A. WOOD A. Alanine Racemase from Streptococcus faecalis L-Alanine --* DL-Alanine *-- D-Alanine

Assay Method

Principle. All assay procedures are based on the measurement of one stereoisomer of alanine, since the substrate and end product differ only in configuration. A manometric measurement of the rate of racemization may be made with L-alanine as the substrate and an excess of D-amino acid oxidase as described by Wood and Gunsalus. 1 Under these conditions the rate of racemization limits the rate of oxygen uptake. In addition, the removal of the reaction product prevents the formation of a racemic mixture the approach toward which is accompanied by a decrease in reaction velocity. The stoichiometry of the reaction is dependent upon the presence of catalase. In its presence 1 micromole of D-alanine is equivalent to 11.2 ul. of oxygen, whereas in its absence 1 mieromole of D-alanine is equivalent to 22.4 ul. of oxygen. Reagents 0.2 M L-alanine. 1.0 M K2HPO4-KH~P04 buffer, pH 8.1. Calcium pyridoxal phosphate, ~ 100 ~//ml. 20 % potassium hydroxide. Enzymes 1. Alanine racemase preparation is diluted to obtain 1 to 8 units/ml. (see definition below). 2. D-amino acid oxidase preparation. Purification of n-amino acid oxidase from acetone-dried pig kidney, by the procedure of Negelein and BrSmel, 3 was carried through the acid precipitation, heat treatment, and first ammonium sulfate precipitation steps. The oxidase activity in the assay system of Negelein and BrSmel, 3 with air as the gas phase, varied between 280 and 330 #l. of oxygen consumed per milliliter of enzyme in 5 minutes at 37 °. The enzyme is stored at - 1 5 ° until used and is 1 W. A. Wood and I. 2 Calcium pyridoxal New Jersey. Values a E. Negelein and H.

C. Gunsalus, J. Biol. Chem. 190, 403 (1951). phosphate is obtained from Merck and Company, Rahway, are expressed as pure calcium salt. BrSmel, Biochem. Z. 300, 225 (1938-39); see Vol. 2 [23].

[25]

AMINO ACID RACEMASES

213

stable for several months. Such preparations contain catalase and do not require added flavin adenine dinucleotide for activity. The pyridoxal phosphate content is approximately 3 m-y/ml.

Procedure. Place in the main compartment of a 15-ml. double side arm Warburg flask 0.3 ml. of phosphate buffer, 0.1 ml. of pyridoxal phosphate, 0.2 ml. of glutathione, 1.0 ml. of enzyme, and water to a volume of 1.6 ml. Add 0.4 ml. (80 micromoles) of L-alanine to one side arm, 1.0 ml. of D-amino acid oxidase to the second side arm, 0.15 ml. of 20% KOH and a strip of filter paper to the center well. After shaking at 37 ° for 5 minutes, tip the contents of both side arms, and allow the reaction to proceed for 10 minutes. Then close the stopcocks, and measure the rate of oxygen uptake over the next 30-minute period. Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount of enzyme which causes the uptake of 1 ~1. of oxygen per minute in the above assay system. Specific activity is units per milligram of protein. When the ratio of optical densities at 280 and 260 m~ is below 0.7, protein is determined by the biuret method of Robinson and Hogden. 4 After the removal, ,of nucleic acid by protamine (280/260 ratio >_- 0.7) protein is determined spectrophotometrically by the method of Warburg and Christian. 5 see Vol. III [73]. Application of the Assay Method to Crude Preparations. Dried cells, bacterial spores, 6 and crude bacterial extracts may be assayed by this procedure. To obtain the rate of racemization in such preparations, it is necessary to measure the endogenous rate of oxygen uptake and subtract this rate from that obtained in the presence of substrate. The presence of an active L-amino acid oxidase which oxidizes alanine such as is found in rabbit liver interferes with the racemase assay. Purification Procedure This procedure was perfected by Dofin 7 and has been carried out successfully several times. Step 1. Preparation of Crude Extract. Sixteen grams of acetone-dried Streptococcus faecalis, strain 10C1,s and 16 g. of alumina (A301-325 mesh 4 It. W. Robinson and C. G. Hogden, J. Biol. Chem. 135, 707 (1940). 50. Warburg and W. Christian, Biochem. Z. 310, 384 (1942). 6 B. T. Stewart and H. O. Halvorson, J. Bacteriol. 65, 160 (1953). 7 M. I. Dolin, Ph.D. Thesis, Indiana University, 1950. 8 Large batches of S. faecalis ceils are grown in 10-1. cultures containing 1% yeast extract, 1% tryptone, 0.5 % K2HPO4, and 0.3 % glucose. Growth is initiated with 0.1% of a 10- to 12-hour inoculum, and the culture is incubated for 10 to 12 hours at 37 ° (final pH 5.6 to 5.8). Dried cells racemized 2 to 6 micromoles of alanine per hour per milligram dry weight.

214

ENZYMES

OF P R O T E I N

METABOLISM

[9.5]

Alcoa) are suspended in 320 ml. of M/50 phosphate buffer, pH 8.0, and 20-ml. aliquots oscillated for 30 minutes at 9 kilocycles in a 50-watt magnetostriction oscillator. 9 The cell debris is removed by centrifugation (18,000 × g) at 0 °. The total volume of the extract was 318 ml. In all subsequent steps the preparation is kept at 0 °. Centrifugations are carried out at 18,000 × G. Step 2. Fractionation with Ammonium Sulfate. Solid ammonium sulfate is added to 28% saturation (69 g.). The precipitate is removed by centrifugation and discarded. Solid ammonium sulfate is added to the supernatant fluid to 60% saturation (77.5 g.). The precipitate is obtained by centrifugation and redissolved in 160 ml. of M/50 phosphate buffer, pH 8.0. At this point, if necessary, water is added to adjust the protein concentration to approximately 10 mg./ml. Step 8. Protamine Precipitation of Nucleic Acids. The nucleic acids are removed by adding 50 ml. of a 2% protamine solution 1° over a 1-hour period. The precipitate which forms is removed by centrifugation. The 280/260 ratio of the supernatant fluid equals 0.81. Phosphate buffer, M/50, pH 8.0, is added to bring the volume to 318 ml. Step 4. Fractionation with Ammonium Sulfate. Solid ammonium sulfate is added to 37% saturation (88 g.), and the precipitate removed by centrifugation and discarded. Thirty grams of solid ammonium sulfate is added to the supernatant fluid to 48% saturation. The precipitate is separated by centrifugation and redissolved in 160 ml. of M/50 phosphate buffer, pH 8.0 (280/260 ratio = 1.23). The supernatant fluid is discarded. Step 5. Adsorption on Calcium Phosphate Gel. 2.9 ml. of 2 M acetate buffer, pH 4.5, is added to adjust the pH to 5.6. One-tenth volume (16 ml.) of calcium phosphate gel 11is added dropwise with stirring. The stirring is continued an additional 15 minutes, and the gel then removed by centrifugation. Thirty-four milliliters of calcium phosphate gel is added to the supernatant fluid in the same manner and collected by centrifugation. The sedimented gel is treated with 80 ml. of M/50 phosphate buffer, pH 5.7, for 15 minutes at 0°. The gel is separated by centrifugation, and the supernatant fluid discarded. The racemase activity is eluted by treating the gel with 80 ml. of M/IO phosphate buffer, pH 5.7, for 15 minutes at 0 °. 9 The 200-watt 10-kilocycle oscillator also is satisfactory for obtaining a cell-free enzyme. Grinding with alumina, however, was unsuccessful. 10 Two grams of powdered protamine is dissolved in 90 ml. of water, adjusted to p H 5

with dilute acetic acid, and diluted to 100 ml. 11One hundred and fifty milliliters of calcium chloride solution (88.6 g. CaC12.2H20 per liter) is added to 160 ml. of distilled water. This is shaken with 150 ml. of trisodium phosphate (152 g. Na3PO4.12H20 per liter). The pH is adjusted to 7.4 with 1 M acetic acid. The precipitate is washed by decantation five times, centrifuged, and suspended in 300 ml. of water.

[25]

AMINO ACID RACEMASES

215

SUMMARY Of PURIFICATION PI~OCEDURE

Total volume, ml. Units/ml. 1. 2. 3. 4. 5.

Sonic extract (NH4)2SO4fraction 0.28-0.60 Supernatant from protamine (NH4)~SO4fraction 0.37-0.48 Eluate from Ca3(PO4)~gel

318 160 195 160 80

18.5 38 36.7 35.1 38

Specific Total Protein, activity, units mg./ml, units/rag. 5883 6080 7159 5616 3040

8.75 9.25 7.6 2.46 0.28

2.1 4.1 4.83 14.3 136

Properties Specificity. The purified enzyme is specific for alanine. The other amino acids which are attacked b y D-amino acid oxidase, including a-aminobutyrate, are not racemized. In another assay system containing L-glutamic acid decarboxylase, D-glutamate is not converted to L-glutamate. i2 Activators. Pyridoxal phosphate is required as a coenzyme. The purified preparation is approximately 90% resolved with respect to pyridoxal phosphate. Full activity is obtained with 2 to 3 - / o f pyridoxal phosphate per milliliter. About 0.9 ~/ml. is required for one-half maxim u m velocity (Kin = 2 X 10 -6 M). Glutathione also is required for maximum activity. The enzyme has a low affinity for the substrate, since the maximum velocity is not attained with 27 micromoles of L-alanine per milliliter. Approximately 8 micromoles of alanine per milliliter is required for one-half maximum rate (K,, = 8.5 X 10 -3 M). T h e p H for optimum activity is approximately 8.5. With conditions under which neither isomer of alanine is removed, either D- or L-alanine is converted to the racemic mixture. Alanine racemase has been demonstrated in a wide variety of bacteria. I A remarkably high concentration is found in spores of the genus Bacillus. 6 In the spores virtually no inactivation occurs after 2 hours at 80o. 6

B. Glutamic Acid Racemase from Laclobacillus arabinosus L-Glutamate --* DL-Glutamate *-- D-Glutamate

Assay Method Principle. The rate of racemization is measured b y the rate of T,glutamate formation from D-glutamate. l~,Ia Aliquots of the reaction mixture are assayed manometrically at desired intervals for L-glutamate with L-glutamic acid decarboxylase. A continuous assay utilizing plant 13 S. A. Narrod and W. A. Wood, Arch. Biochem. and Biophys. 35, 462 (1952).

is p. Ayengar and E. Roberts, J. Biol. Chem. 197, 453 (1952).

216

ENZYMES OF PROTEIN METABOLISM

[25]

or bacterial glutamic decarboxylase or glutamic dehydrogenase has not been devised.

Reagents 0.1 M K2HPO~-KH2P04 buffer, pH 6.8. Calcium pyridoxal phosphate, ~ 100 ~,/ml. 0.1 M 1)-glutamic acid (neutralized). 0.07 M potassium acid phthalate-NaOH buffer, pH 5.5. Enzymes 1. Dried L. arabinosus. ~4 2. Glutamic decarboxylase. Vacuum-dried cells of Escherichia coli, Crookes strain, ATCC 8739, prepared essentially as described by Umbreit and Gunsalus, ~5 were used as a source of decarboxylase. Such preparation had an activity (Qco,) of 550 to 600.

Procedure. The assays are carried out in test tubes at 37 °. The reaction mixture contains 0.8 ml. of pyridoxal phosphate, 0.8 ml. of D-glutamate, 1.0 ml. of phosphate buffer, dried cells or enzyme, and water to 4 ml. After equilibration at 37 ° racemization is initiated by adding the D-glutamate. One-milliliter aliquots are removed at 0, 1, and 2 hours and boiled for 5 minutes to stop the reaction. The pH is adjusted to 5.5 with dilute HC1, and the volume increased to 2 ml. with water. After removal of the coagulated protein by centrifugation, the L-glutamate content is determined manometrically. One milliliter of the acidified and diluted sample is placed in the side arm of a Warburg vessel. One milliliter of phthalate buffer, 5 mg. of glutamic decarboxylase preparation, and water to a 2-ml. volume are added to the main compartment. After 10 minutes' equilibration at 37 ° , the side arm contents are tipped and the evolved carbon dioxide measured. 22.4 ~l. of carbon dioxide is equivalent to 1 micromole of L-glutamate. Applicability of the Method. This method is suitable for the assay of acetone- or vacuum-dried bacteria and sonic extracts. Controls must be run to correct for the L-glutamate content of crude preparations. Purification Procedure A usable purification scheme has not been developed for this racemase. 14 The cells are grown in a medium containing 1% tryptone, 1% yeast extract, 0.5 % K2HP04, 0.5 % glucose, and Salts B. The medium is inoculated with 0.1% of an 18-hour culture and incubated for 18 hours at 37 °. The cells are either dried in vacuo or subjected to sonic vibration to obtain a cell-free enzyme. 1~ W. W. Umbreit and I. C. Gunsalus, J . Biol. Chem. 159, 333 (1945).

[26]

AMINO ACID REDUCTASES

217

Properties The optimum pH for racemization has been reported at pH 6.81~ and pH 8.0.13 Pyridoxal phosphate stimulates the rate of racemization by dried cells threefold. ~ Glutamine does not stimulate the rate of racemization. ~3 A purified alanine racemase from Streptococcus faecalis does not racemize glutamate. 1~

[26] A m i n o Acid R e d u c t a s e s By R. M. JOHNSTONE and J. H. QUASTEL Stickland, ~ in his studies of amino acid interactions, described the existence of an enzyme in Cl. sporogenes concerned with the reduction of a-amino acids. He demonstrated that glycine, proline, and hydroxyproline are capable of oxidizing reduced benzylviologen in the presence of washed suspensions of the organism. In amino acid interactions, glycine is reduced to acetic acid and ammonia, and proline to 8-aminovaleric acid. Woods 2 found, in addition, with Cl. sporogenes, that ornithine and arginine are both activated as hydrogen acceptors, ornithine giving rise to 8-aminovaleric acid.

Assay Methods Manometric. (1) When an amino acid is used as hydrogen donor, an interaction occurs between the donor and acceptor amino acids, with the evolution of carbon dioxide from the donor. The rate of carbon dioxide evolution is a measure of the reductase activity. 1,3 The following is a typical reaction: CH3CHNH~COOH nu 2CH2NH~.COOH + 2H20 = 3CHsCOOH + 3NH3 ~- CO~ (2) Molecular hydrogen is absorbed by Cl. sporogenes in the presence of amino acid hydrogen acceptors but not of amino acid hydrogen donators. 3,4 The rate of hydrogen absorption may be made a measure of reductase activity. Colorimetric. The amino acid hydrogen acceptors readily oxidize a variety of leuco dyes. The rate of oxidation of reduced benzylviologen 1L. H. Stickland, Biochem. J. 28, 1746 (1934); 29, 288,889 (1935). D. D. Woods, Biochem. J. 30, 1934 (1936). 3 R. NIamelak and J. H. Quastel, Biochim. el Biophys. Acta 12, 103 (1953). 4 j. C. Hoogerheide and W. Kocholaty, Biochem. J. 32, 949 (1938).

218

ENZYMES OF PROTEIN METABOLISM

[26]

has been used successfully by several workers as a measure of amino acid reductase activity. 1,2 Stickland ~ found that glycine, in the presence of Cl. sporogenes, oxidizes leucomethylviologen completely, leucobenzylviologen partly, and leucophenosafranine not at all. Proline oxidizes a variety of leuco dyes including leucophenosafranine. Ammonia Formation. When glycine, ornithine, or arginine is reduced by a hydrogen donor in the presence of Cl. sporogenes ammonia is formed. The rate of ammonia production gives a measure of reductase activity. It should be noted that, when ornithine, arginine, or glycine is used as a hydrogen acceptor for coupled anaerobic amino acid interactions, more ammonia is produced than when proline is used as the hydrogen acceptor. This is due to the fact that proline is not deaminated on reduction in contrast to the other amino acids. Occurrence. Apart from Cl. sporogenes, the following organisms are known to bring about amino acid interactions: 5 C1. butyricum, C1. flabelliferum, Cl. histolyticum, C1. saproticum, Cl. sordelii, C1. bifermentans, Cl. acetobutylicum, Cl. indolicus, and Cl. botulinum.

Preparations Amino acid reductase may be found in resting cell suspensions, lyophilized cells, and cell-free extracts of Cl. sporogenes. Cells grown on a variety of media show good amino acid reductase activities. ~-4,8 Resting Cell Suspensions. The organisms are grown on a suitable medium 2-4,6 for 12 to 16 hours at 37 °. The cells are then centrifuged and washed twice in an isotonic sodium or potassium chloride solution. The packed, washed cells from a liter of medium are suspended in 16 ml. of the same salt solution. One milliliter of this suspension will evolve 300 to 400 ~1. of carbon dioxide per hour at 37 ° from a mixture of 0.02 M alanine and 0.02 M proline in a 0.028 M bicarbonate medium in an atmosphere of 93% N2 and 7% C02. The optimum pH for amino acid reductases is 7.2 to 7.4. When molecular hydrogen is used as hydrogen donor, it is found advisable to boil salines and buffer solution preparatory to use 4 in order to remove oxygen, as hydrogenase is easily inactivated by air. Johnstone and Quastel (unpublished results) find that the presence of 0.1% neutral cysteine (or sodium pyruvate) in the wash saline will prevent loss of hydrogenase activity during the preparation of the resting cells. Care must be taken to avoid unnecessary exposure to air in the course of preparation. B. Nisman, M. Raynaud, and G. H. Cohen, Ann. inst. Pasteur 74, 323 (1948). e B. Nisman and G. Vinet, Ann. inst. Pasteur 78, 115 (1950).

[26]

AMINO ACID REDUCTASES

219

Preparation of Lyophilized Cells. 8 Packed cells of C1. sporogenes, prepared as above, are washed twice with 1% sodium thioglycoUate solution and suspended in 20 ml. of 0.15 M KC1. The cell suspension is frozen in a mixture of dry ice and acetone and lyophilized for 5 hours. The dried cells are stored in a vacuum desiccator at 0 to 10 °. For assay, 18 to 20 mg. of the dried cells is used per milliliter. Such a preparation shows considerable prolinc and ornithine reductase activities, b u t the activity for glycine is absent. Preparation of Bacterial Extracts. A bacterial press described by Hughes 7 was used b y Mamelak and Quastel ~ for preparation of cell-free extracts of Cl. sporogenes. The cells from 2 1. of medium, grown as described earlier, are harvested, washed once with 200 ml. of 1% sodium thioglycollate solution, and a second time with 10 ml. of this solution. T h e packed, washed cells are frozen for 5 to 10 minutes in dry ice and inserted into the press which has previously been cooled to - 2 0 ° . The crushed cells are suspended in 7 to 9 ml. of 1% sodium thioglycollate solution and centrifuged for 2 minutes at 20,000 X g at 0 °. T h e amber-colored supernatant fluid reduces proline or ornithine in the presence of suitable hydrogen donors 3 but will not reduce glycine (unpublished result). Properties

Stability. Exposure of a resting cell suspension of Cl. sporogenes to air for 3 or 4 hours causes a marked fall in the rates of the anaerobic amino acid interactions. 3 This fall is due to the presence of oxygen, as no diminution is experienced if the suspension is exposed to nitrogen for the same length of time. Exposure to small concentrations of hydrogen peroxide (1/50,000) for a few minutes is equally inhibitory. Reductase activity may be maintained for more than 2 weeks if the packed cells are stored at - 2 0 ° (unpublished result). If the cells are kept in suspension at 0 to 20 °, the activity is completely lost in less than 6 hours. Specificity. Woods 2 showed that either optical isomer of proline or ornithine is reduced b y a suitable hydrogen donor in the presence of C1. sporogenes. He ~ also demonstrated t h a t the amino group of glycine is essential for reductase activity, since replacement of the amino group b y a hydroxyl group results in a complete loss of the ability of the molecule to act as a hydrogen acceptor. N - M e t h y l glycine, N-acetyl glycine, and ethanolamine are not attacked b y the enzyme, nor do they inhibit glycine reductase (Johnstone and Quastel, unpublished results). D. E. Hughes, Brit. J. Exptl. Pathol. 32, 97 (1951).

220

EIgZYMES OF PROTEIN METABOLISM

[27]

Little is known at present of the specificity of amino acid reductases. The fact that the ability of Cl. sporogenes extracts to reduce glycine is lost under conditions where proline and ornithine reductions may still take place indicates the likelihood that separate enzymes are involved in the reduction of these amino acids. Necessity for DPN. Recent work by Mamelak and Quastel ~ has shown that the coenzyme involved in the reduction of proline and ornithine is DPN. It would appear, therefore, that amino acid reductase of C1. sporogenes is DPN-linked. Inhibitors. Stickland ~ demonstrated that arsenite inhibits amino acid interactions in C1. sporogenes. Nisman and Vinet Bshowed, however, that the oxidation of the amino acid donor is unaffected by arsenite (M/225), whereas the reductions of proline and glycine are completely suppressed. h/Iamelak and Quastel 3 demonstrated that organic arsenoxides, particularly m-amino-p-hydroxyphenylarsenoxide, are inhibitory to amino acid interactions in C1. sporogenes. They showed that the reduction of proline is affected more than the oxidation of alanine. For example, 8 "/ of the above organic arsenoxide per milliliter caused a 50% inhibition of the amino acid interaction but had no effect on the aerobic oxidation of alanine. ~ Pentavalent arsenic compounds have little or no toxicity. The inhibitions caused by organic arsenoxides can be largely reversed by the presence of thiol compounds such as glutathione2 The evidence indicates that the amino acid reductases are labile thiol enzymes.

[27] L - G l u t a m i c D e h y d r o g e n a s e

from Liver

L-Glutamate- ~- D P N + ~ H:O ~ ~-Ketoglutarate--~ D P N H ~ NH4 + ~- H +

By HAROLD J. STRECKER Assay Method Principle. The oxidation of glutamate is measured by the increase of optical density at 340 m~ caused by the reduction of DPN. Alternately the reductive amination of a-ketoglutarate may be measured by the decrease in optical density arising from the oxidation of D P N H . Reagents Potassium glutamate (0.5 M). 918 mg. of glutamie acid hydrochloride is suspended in 5 ml. of H~O. Sufficient K O H solution is

[27]

L-GLUTAMIC DEHYDROGENASE FROM LIVER

221

added to neutralize the glutamic acid and bring it into solution. The final volume is made to 10 ml. DPN solution. Three micromoles per milliliter in water. 0.05 M potassium phosphate buffer, pH 7.6. Enzyme. The enzyme solution is diluted with distilled water so as to obtain a concentration of 500 to 1000 units of enzyme per milliliter (see definition below).

Procedure. 2.6 ml. of buffer, 0.1 ml. of the DPN solution, and 0.1 ml. of the enzyme solution are mixed in a quartz cuvette with a 1.0-cm. light path. The same solution plus 0.2 ml. of H20 in another cuvette serves as the blank. 0.2 ml. of the potassium glutamate solution is added to the experimental cuvette. The solution is rapidly stirred with a small glass rod, and the optical density at 340 m/~ is recorded at 15-second intervals. Definition of Unit and Specific Activity. The increment in optical density (aE~40) between the readings at 15 and 30 seconds after making the last addition, multiplied by 4, is taken as the enzyme activity per minute. One enzyme unit is defined as that amount which causes a change in optical density of 0.001 per minute under the above conditions. Specific activity is expressed as units per milligram of protein. Protein is determined by a modified biuret reaction. 1 The values reported in the table are for activities determined in potassium phosphate buffer at pH 7.6. The activity increases with pH up to 8.5 to 8.6 and depending on the buffer system used (see section on properties of enzyme). Other Methods of Assay 2

Enzyme activity has been determined by measuring the rate of oxidation of DPNH, by the reduction of methylene blue, and by the uptake of oxygen. In general, reduction of DPN is the simplest and most economical method provided that initial rates are measured. Purification Procedure

Steps 1 and 2 are based on the observations of von Euler et al. 2 and Dewan a on the presence of glutamic dehydrogenase activity in extracts of acetone powders of liver and on the stability of the activity to acid pH. Step 1. Fresh beef or calf liver obtained from the slaughterhouse is homogenized for 1 to 2 minutes in a Waring blendor with an approximately equal volume of acetone at - 3 ° in 500-g. amounts. The acetone 1 j. W. Mehl, J. Biol. Chem. 157, 173 (1945). 2 H. y o n Euler, E. Adler, G. Gthather, a n d N. B. Das, Z. physiol. Chem. 254, 61 (1938). s j . G. Dewan, Biochem. J. 32, 1378 (1938).

222

ENZYMES OF PROTEIN METABOLISM

[27]

suspension is poured into 10 vol. of acetone at 0 to - 3 °, stirred thoroughly, and filtered by suction on a large Biichner funnel. The cake of acetone powder thus formed is again homogenized in a Waring blendor and stirred into 10 vol. of cold acetone and filtered as before. The cake is sucked as dry as possible. These operations are performed in a cold room at 0 to 7 °. The acetone powder cake is then removed to room temperature, broken up, and dried rapidly by rubbing between the palms of the hands. The dry liver powder, varying from 250 to 1000 g. in weight, is stirred for 30 minutes with 10 vol. of potassium phosphate buffer, pH 7.6, at room temperature. After allowing most of the insoluble material to settle (5 to 10 minutes), the supernatant is decanted and filtered through four layers of cheesecloth. The residue is again stirred with 10 vol. of water for the same length of time, and the suspension filtered through cheesecloth as before. The solution at this stage contains fine suspended material. Step 2. Acid Precipitation. The phosphate extract is chilled to 0 to 3 ° and the pH brought to 6.4 by slow addition of 10% acetic acid with stirring. The solution is centrifuged and the precipitate discarded. With large-scale preparations where centrifugation is difficult, this first acid precipitation may be omitted. Ten per cent acetic acid is further added until a pH of 4.8 is attained. At this pH a slow precipitation of active protein occurs which is complete in 5 hours or less. To determine the completeness of precipitation, aliquots of the supernatant fluid are tested at intervals. When no more than 10% of the total activity remains in the supernatant fluid, the solution is centrifuged in the cold (Laval cream separator or Sharples centrifuge for large volumes). The supernarant fluid is discarded. The precipitate is broken up and extracted with one-tenth of the original volume of cold 0.05 N potassium phosphate buffer, pH 7.6, with stirring. The pH is adjusted to 7.6 by addition of dilute alkali. The extraction can be carried out for periods of time ranging from 1 to 2 hours to overnight without much difference in activity recovered. The suspension is centrifuged and the insoluble residue discarded. Yield of enzyme activity: 50 to 80% of the initial phosphate extract. From this stage on, the temperature of the solution, where not specifically noted, is never allowed to rise above A-3°. The quantities of ethanol referred to are final concentrations on a volume basis. Step 3. First Alcohol Fractionation. The solution is adjusted with 10 % acetic acid to pH 6.4 and centrifuged if necessary. Ethanol is added to the supernatant to a concentration of 10%. After standing for about 1 hour at --1 to 0 ° the suspension is centrifuged and the precipitate discarded. Ethanol is added to the supernatant fluid to a final concentra-

[9.7]

223

L-GLUTAMIC DEHYDROGENASE FROM LIVER

tion of 35%, the temperature being maintained at - 5 °. The suspefision is kept overnight at - 1 5 to - 1 8 ° and centrifuged. The precipitate is dissolved in 0.05 M potassium phosphate buffer, pH 7.6, in a volume approximately one-tenth of the previous solution. Yield of enzyme activity: 30 to 50 %. Step 4. Second Alcohol Fractionation. This fractionation is carried out at pH 6.4 between the limits of 10 to 30% alcohol. The pH of the precipitation appears to be fairly critical. The precipitate from this step is dissolved in sufficient 0.05 M potassium phosphate buffer, pH 7.6, to attain a protein concentration of 20 to 25 mg./ml. The volume required for this is generally about one-half of the preceding volume. Yield of enzyme activity: 30 to 50 %. Step 5. Adsorption of Calcium Phosphate Gel. The solution is adjusted to pH 7.6 with dilute alkali and stirred with calcium phosphate gel (1.5 g. of Ca3(PO4)2 per 100 ml. of enzyme solution). In order to maintain the enzyme concentration, the gel is first centrifuged, the supernatant fluid discarded, and the enzyme solution added to the sedimented gel. After being stirred for 20 to 30 minutes, the suspension is centrifuged and the gel discarded. Step 6. Crystallization. The supernatant fluid is chilled to 0 °, and 1 M acetate buffer, pH 4.7, is added until a pH of 6.4 is reached. Alcohol is then slowly added. In the best preparations, addition of alcohol dropwise over a period of 1 to 2 hours results in the initiation of crystallization at a 6 to 8% ethanol concentration. Crystallization proceeds fairly rapidly without any further addition of alcohol. These crystals appear as long, colorless needles when examined as a dense smear under the microscope. The specific activity of the crystalline material is about 6000 units/rag., a fivefold increase over the previous stage. In some preparaPURIFICATION OF OX LIVER GLUTAMIC DEtIYDROGENASE (1000 g. of acetone powder)

Step 1. 2. 3. 4. 5.

Aqueous extract Acid precipitate First alcohol precipitate Second alcohol precipitate Calcium phosphate gel treatment 6. First crop crystallization

Specific activity, Protein, units/mg. Yield, mg. protein %

Volume of solution, ml.

Units

22,000 2,000 200 120

10,000,000 5,000,000 3,600,000 3,000,000

436,000 80,000 13,200 2,800

23 62 272 1,070

120 --

2,160,000 1,200,000

1,850 200

1,170 6,000

100 50 36 30 21.6 12

224

ENZYMES OF PROTEIN METABOLISM

[27]

tions, however, addition of alcohol to 8 % precipitates only a small portion of the total activity, and it is necessary to go up to 10 to 12% to obtain crystallization. In these cases the final product has a specific activity of 3000 units/mg, and the crystals are much smaller. Recrystallization does not result in any increase of specific activity. It is probable that coprecipitation of impurities is the cause of these variations. P r o p e r t i e s 2 , 4-6'

Specificity. In the forward direction the purified enzyme appears to be absolutely specific for L-glutamic acid, having been tested on the following substrates with no activity-~,-ethyl glutamate, diethyl glutamate, D-glutamate, N-acetyl glutamate, N-carbamyl glutamate, N-phthalyl glutamate, glutamine, v-ethyl amide, and ~,-methyl amide of glutamic acid, L-aspartate, L-a-amino adipic acid, pyrrolidone carboxylic acid, and the following L-amino acids and peptides composed of L-amino acids: ~,-glutamylglutamate, ~,-glutamylglycine, ~,-glutamylglycylglycine, ~,-glutamyl-L-leucine, ~/-glutamylalanine, a-glutamylglutamate, ~/-glutamylcysteine, ~,-glutamohydroxamic acid, methionine, cysteic acid, DL-homocysteine, i-methionine sulfoxide, asparaginylglycine, glutaminylglycine, aspartylglycine, and glyeylaspartic acid. T P N and deamino D P N can be substituted for DPN, although the activity is less. In the reverse direction methylamine, ethylamine, and diethylamine are inactive. Activators and Inhibitors. No activators have been reported. In the presence of equimolar concentrations of L-glutamate the following per cent inhibitions were noted: aspartate, 10, glutamine, 20, D-glutamate, 50. p-Mercurichlorobenzoate inhibits 50% at a concentration of 1.1 X 10-4 M. Silver, mercury~ zinc and ferric iron, and high salt concentration are also inhibitory. In the reverse direction in the presence of equimolar concentrations of NH4C1, hydroxylamine inhibits 30 %. Effect of pH. The optimum pH for enzymatic activity for the oxidation of glutamate is 8.5 to 8.6. The activity at any particular pH appears to depend somewhat on the buffer used. For example, at pH 7.6 the activity in Tris is about one-third of that obtained in a buffer mixture consisting of phosphate, Tris, and 2-amino-2-methyl-l,3-propanediol, whereas at pH 8.5 the activity in the former buffer is slightly higher than in the latter.

4j. A. Olson and C. B. Anfinsen, J. Biol. Chem. 197, 67 (1952). 6H. J. Strecker, Arch. Biochem. and Biophys. 46, 128 (1953). 6j. A. Olson and C. B. Anfinsen, J. Biol. Chem. 202~~41 (1953).

[28]

GLYCINE OXIDASE

225

Enzyme Substrate Dissociation Constants. The values of Km obtained for the various reactants at pH 7.6 in 0.5 M potassium phosphate buffer by application of the usual Lineweaver and Burk methods are: glutamate, 1.92 X 10-3; DPN, 2.47 × 10-5; a-ketoglutarate, 1.23 X 10-~; NH4 +, 0.057; D P N H , 1.8 X 10-5. The values for K~ for glutamate, however, have been found to change markedly according to the pH and buffer used. It is probable that the other substrates also behave in this way (cf. also ref. 6). Equilibrium Constant. The average value for the equilibrium constant for the equation Glutamate- + D P N + + H~O ~ a-Ketoglutarate-+ D P N H + NH4 + + H + and using the convention of 1 M water is 1.8 × 10-~3. For this value AF ° at 27 ° = +17,400 calories and Eo' for the reaction Glutamate- + H20 ~ a-Ketoglutarate-- + NH4 + + 2H + + 2 e - is - 0.106 volt. Other Physical .Constants. Sedimentation constant, 26.6 X 10-~3 second; diffusion constant, 2.54 X 10-7 sq. cm./see.; molecular weight, 1,000,000 g.

[28] G l y c i n e O x i d a s e CH~NH2COOH + ~0~--~ CHOCOOH + NH3 CH2NHCHsCOOH + 1/~0~--. CHOCOOtt + NH2CH3

By S. RATNER

Assay Method Principle. Oxygen consumption is measured manometrically by the Warburg technique. Catalase is present at all stages of the enzyme preparation, and therefore 1/~ mole of O2 is consumed per mole of glycine or N-monomethylglycine (sarcosine) oxidized? The glyoxylie acid is not oxidized further. Less conveniently, the reaction can also be followed by formation of NH3 or carbonyl group. Should the catalase be removed, the 02 consumption will double and the glyoxylic acid will be oxidized further by the H202 formed. : S. Ratner, V. Nocito, and D. E. Green, J. Biol. Chem. 182, 119 (1944).

226

ENZYMES OF PROTEIN METABOLISM

[9.8]

Reagents 1.0 M glycine. 0.5 M dimethylglycine buffer, pH 8.2 (sodium dimethylglycineHC1). ~ 0.01% FAD.

Procedure. One milliliter of enzyme, 0.5 ml. each of the glycine and buffer solutions, 0.2 ml. of the FAD solution, and water to a final volume of 3.0 ml. are placed in the main space; alkali is placed in the center well. Oxygen consumption is measured at 38 ° with air in the gas phase. The taps are closed after 10 minutes of temperature equilibration. The assay time is 15 or 30 minutes. Definition of Enzyme Activity. Specific activity is expressed here as Qo2 (microliters of O3 consumed per hour per milligram of protein). Application of Assay to Crude Tissue Preparations. The activity of crude extracts of acetone-dried tissue is usually masked by the oxygen uptake of the blank. Purification Procedure The procedure given below is taken from the original publication. 1 It is supplemented with Qo, values obtained recently (unpublished data of the author). Step 1. Preparation of Crude Extract. Fresh pig kidneys are divested of fat and minced with ~ vol. of water in a Waring blendor to a fine paste and poured into 5 vol. of acetone cooled to - 1 0 ° with dry ice. The mixture is rapidly filtered with suction on large Biichner funnels. The cake is washed several times with small portions of cold acetone and is then removed from the filter paper and broken up finely until dry. The powder is resuspended in 5 vol. of acetone, and, after 10 minutes of stirring at 0 °, the mixture is again filtered with suction. The fat-free cake is broken up finely at room temperature until dry. Then 250 g. of acetone powder is mixed thoroughly with 2.5 1. of water at 0 °. After being stirred for 30 minutes, the mixture is filtered or centrifuged. Step 2. Precipitations with Ammonium Sulfate. (NH~)~S04 (30 g. per 100 ml.) is slowly added to the cold filtrate (about 2 1.) with mechanical stirring. The precipitate is filtered off or collected by centrifugation at 13,000 r.p.m, in the Servall centrifuge and then redissolved in 800 ml. of Dimethylglycine can be readily prepared as the sodium salt by the procedure of L. Michaelis and M. P. Schubert, J. Biol. Chem. 115, 221 (1936). Buffer composition data are included.

[28]

GLYCINE OXIDASE

227

water. The preparation can be interrupted at this stage and stored at - 2 0 ° in half the volume. Step 3. Precipitation with Potassium Acid Phosphate. 192 g. of finely pulverized KH2PO4 (24 g. per 100 ml.) is slowly added to the 800 ml. of solution at room temperature with mechanical stirring. The precipitate is centrifuged off at room temperature and resuspended in 300 ml. of water. Sodium carbonate (10%) is added dropwise until the mixture is brought to pH 8.2. The insoluble material is centrifuged off and discarded. The precipitation procedure with KH2P04 is repeated as before, including adjustment to pH 8.2. The final enzyme solution (about 150 ml.) is pale greenish yellow in appearance but water clear. Activity is retained for about a week when stored at 0 ° and longer at - 2 0 °. Summary of Enzyme Activity. The activity of crude extracts and also of the fraction obtained after (NH4)2SO4 precipitation is uncertain. As the procedure is given, the solutions obtained after the first and second precipitation with KH2POa have from 25 to 30 mg. of protein per milliliter. Under the assay conditions, the Qo2 is about 7 after the first and about 9 after the second. Activity is about 30% higher in the absence of buffer.

Properties Prosthetic Group. Removal of the prosthetic group occurs during the (NH4)~S04 step, and enzyme activity is restored by FAD. The 20 ~/ called for in the assay is sufficient to saturate the enzyme present in 1 ml. Specificity. The enzyme acts only on glycine and N-monomethylglycine, the latter at about half the rate of the former. N-Dimethylglycine, glycylglycine, and other glycylpeptides tried are not oxidized, nor is creatine or hippuric acid. The preparation is quite high in D-amino acid oxidase activity which is quite distinct from glycine oxidase. As it occurs in the preparation, the D-amino acid oxidase activity is almost completely dependent on FAD supplementation. Dependence on pH and Substrate Concentration. The pH optimum is a narrow region at 8.3; activity declines sharply to either side. Veronal buffer is strongly inhibitory. Substrate concentrations greater than 0.16 M are required for maximum rates, and the Km is about 0.04 M. Inhibitors. The enzyme is sensitive to Cu++; 0.0002 M CuSO4 inhibits 64%. No inhibition is produced by capryl alcohol-saturated water, 0.2 M NaF, 0.01 M CN-, 0.001 M IAA, or 0.001 M ZnSO4.

228

[29]

ENZYMES OF PROTEIN METABOLISM

[29] Hisfidase and Urocanase

By H. TABOR and A. H. MEttLER A. Histidase

(Pseudomonas)

H C

H C

N

i

HC

NH

!

NH~

f

CCH2CHC00H L-Histidine

N

]

HC--

NH

+ NH,

i

CCH---~CHCOOH Urocanic acid

Assay Method Principle. Previous methods for the assay of histidase have depended on chemical methods such as the isolation of urocanic acid, determination of ammonia, or the combined use of diazotization and bromination reactions. A more sensitive and rapid method is a spectrophotometric assay based on the appearance of urocanic acid. Between pH 7.2 and 11 the molar extinction coefficient of urocanic acid at 277 m~ is 18,800, whereas histidine has essentially no absorption at this wavelength.1

Reagents 0.1 M histidine solution. Dissolve 209 mg. of commercial L-histidine monohydroehloride monohydrate in water, neutralize to pH 8.5 to 9.5 with approximately 1 ml. of N KOH, and make up to 10 mh with water. 0.1 M sodium pyrophosphate buffer, pH 9.2. 0.1 M glutathione. Dissolve 305 mg. of commercial glutathione in 10 ml. of water. Enzyme. Dilute the enzyme with sufficient 0.1 M glutathione to obtain approximately 200 to 500 units/mh (See definition below.)

Procedure. Into each of two silica cuvettes place 0.3 ml. of pyrophosphate buffer, 0.05 mh of glutathione, and a suitable aliquot of enzyme. Add water to give a total volume of 3.0 ml. in the reference cuvette and 2.9 ml. in the experimental. At zero time add 0.1 ml. of histidine solution to the experimental euvette and measure the rate of change at 277 m~ at 15- to 30-second intervals. 2 1 A. H. Mehler and H. Tabor, J. Biol. Chem. 201, 775 (1953). 2 With some preparations the rate m a y increase during the initial period, associated with a reactivation b y the added glutathione. In these cases the readings are taken until a m a x i m u m rate is attained.

[29]

HISTIDASE AND UROCANhSE

229

Definition of Unit and Specific Activity. One unit of enzyme is defined as t h a t a m o u n t which causes an increase in optical density at 277 m~ of 0.001 per minute at 25 °. Specific activity is expressed as units per millig r a m of protein. Protein concentration can be determined from the optical density at 280 m~, using the absorption at 260 m~ to correct for nucleic acid. Application of Assay Method to Crude Tissue Preparations. 3 T h e assay method described is suitable for use with crude as well as with purified preparations. The presence of urocanase in the crude extracts does not interfere with the histidase assay, since urocanase is relatively inactive at the p H of the assay. Purification P r o c e d u r e

Pseudomonas fluorescens cells, grown on histidine, possess high histidase and urocanase activity. These activities can be extracted into w a t e r by grinding with alumina. 4 A histidase preparation free from urocanase can be obtained f r o m this extract b y a heating step. This p r e p a r a t i o n is sufficient for m a n y studies on histidase as well as for the large-scale preparation of urocanic acid. 5 I f desired, however, further purification can be accomplished b y a m m o n i u m sulfate fraetionation and adsorptionelution steps with C~ and calcium phosphate gels. Culture and Harvesting. 4 Pseudomonas fluorescens (American T y p e Culture Collection No. 11,299) is grown on a m e d i u m containing 0.15% K2HPO4, 0.05% KH:PO4, 0.02% 5/IgSO4, 0.2% L-histidine m o n o h y d r o chloride monohydrate, and 0.1% Bacto yeast extract in tap water (usually 15 1.). After inoculation the culture is incubated at 25 °, with vigorous aeration, until m a x i m u m growth is obtained. ~ (The optical 3 Extracts of livers of guinea pigs and various other species also contain histidase, although the activities are considerably less than these found in the Pseudomonas preparations. The same spectrophotometric assay procedure is applicable to these preparations. Purification of the enzyme from liver has been reported by various authors, including iV[. Takeuchi [J. Biochem. (Japan) 34, 1 (1941)] and Y. Sera [Med. J. Osaka Univ. 4, 1 (1951)]. The purification procedure used for guinea pig liver in this laboratory has involved extraction by blending with 1% KC1, heating to 55° for 15 minutes, isoelectric precipitation of inert proteins at pH 5.0, adsorption on alumina C~, elution with pH 7.2 phosphate buffer, and precipitation with ammonium sulfate. This series of steps has given an over-all purification of about 25 times. Acetone fractionation has also been found useful for obtaining liver histidase free from urocanase. 4 H. Tabor and O. Hayaishi, J. Biol. Chem. 194, 171 (1952). 5 A. H. Mehler, H. Tabor, and O. Hayaishi, Biochem. Preparations in press. 6 The cells should be harvested at this time, since further incubation leads to a gradual decrease in the histidase activity.

230

ENZYMES OF PROTEIN METABOLISM

[29]

density at 650 mt~ in a cell with a 1-cm. light path is approximately ~.7, usually after 16 to 20 hours.) The cells are then harvested in a Sharples centrifuge, washed with a 0.25% NaC1-0.25% KC1 mixture, and stored at - 15°. Preparation of Extract. To prepare the cell-free extract, 7 g. of frozen cells is ground with 14 g. of alumina (Alcoa A-301) in a chilled mortar until pasty (about 5 minutes). The mixture is then triturated with 70 ml. of water and centrifuged at 22,000 X g in an angle centrifuge for 10 minutes (usually at 0°). Heating Step. The supernatant solution is rapidly heated (2 to 4 minutes) to 78 to 83 ° in a water bath and maintained at this temperature for 15 minutes. The solution is then cooled to 0 °, and any insoluble material is removed by centrifugation. Ammonium Sulfate Fractionation. The fractionation obtained with ammonium sulfate varies considerably with each extract. It is therefore necessary to assay each fraction and select the fraction showing the best specific activities. The heated extract (60 ml.) is treated with 1.0 vol. of saturated (4 M) ammonium sulfate (saturated at room temperature); the precipitate is collected by centrifugation and dissolved in approximately 9 ml. of 0.05 M sodium pyrophosphate buffer (pH 9.3). A second precipitate is obtained by the addition of another vol. of ammonium sulfate to the supernatant solution. The solutions are assayed, and the fraction with the higher specific activity selected. If the specific activity is less than 10,000, the material is refractionated by successive additions of 0.2 vol. of saturated ammonium sulfate. The precipitates are dissolved in 3 ml. or more of water and assayed. The fractions with the highest specific activities are combined and dialyzed overnight against 200 vol. of distilled water at 0 to 4 °. Alumina C~ Adsorption. The dialyzed solution is treated with 6 ml. of alumina C~ gel (11 rag. of solids per milliliter). The precipitate is centrifuged down and washed with 5 ml. of cold distilled water. The enzyme is eluted from the gel with approximately 5 ml. of 0.1 M phosphate buffer, pH 7.4. These steps give 15 to 40% of the original activity with a specific activity about 80 to 100 times that of the crude extract. Properties

Stability. The enzyme can be stored for several months at - 15° without significant loss of activity (when tested with added glutathione). Specificity. Although a thorough study of substrate specificity has not been carried out, the enzyme appears to be specific for L-histidine.

[29]

HISTIDASE AN'D UROCANASE

231

Activators and Inhibitors. On purification, thorough dialysis, or aging, the enzyme becomes completely inactivated. Complete reactivation can be attained by the addition of glutathione or of sodium thioglycolate. The enzyme is inhibited by ethylenediaminetetraacetate (90 % inhibition by 10-~ M), and less effectively by cyanide, glycine, and ethylenediamine. Effect of pH. The optimum pH is approximately 9.5, but the enzyme is active over a wide pH range. TABLE I SUMMARY OF HISTIDAS]~, PURIFICATION PROCEDURE

Fraction Pseudomonas extract Heating First ammonium sulfate (0-0.5 sat.) Second ammonium sulfate (0.25-0.5 sat.) C~ eluate

Total Protein Specific volume, Total conc., activity, Recovery, ml. Units/ml. units mg./ml, units/mg. % 60 60

3,300 198,000 8.6 3,300 198,000 7.5

380 440

-100

9.8

20,000 196,000 4.7

4,260

99

3.2 5.8

37,000 118,000 2.3 13,600 78,000 0.42

16,100 32,400

60 40

B. Urocanase (Pseudomonas)

Assay Method Principle. Previous methods for the assay of urocanase have depended on the disappearance of urocanic acid, as measured by an alkaline diazotization procedure, or by the appearance of alkali-labile ammonia. In the method used in this laboratory the degradation of urocanic acid was followed by measuring the disappearance of the absorption at 277 m~ (see p. 228). In addition to its sensitivity and convenience, this method is preferred because the nature of the reaction catalyzed is still unknown, and the number of enzymes involved in the production of the products which accumulate is uncertain.

Reagents 0.001 M sodium urocanate. Dissolve 17.4 mg. of urocanic acid dihydrate 5 in 100 ml. of 0.001 N NaOH. 0.2 M potassium phosphate buffer, pH 7.4. Enzyme. Dilute the enzyme with water to obtain 200 to 2000 units/ml.

Procedure. Into each of two silica cuvettes with 1-cm. light paths place 0.3 ml. of phosphate buffer, a suitable aliquot of enzyme, and water

232

ENZYMES OF PROTEIN METABOLISM

[29]

to a final volume of 3.0 ml. in the reference cuvette and to 2.9 ml. in the experimental. At zero t i m e add 0.1 ml. of urocanic acid solution to the experimental cuvette, and record the decrease in optical density at 277 m~ at 15- or 30-second intervals. T h e initial rate of decrease is proportional to the e n z y m e added. Definition of Unit and Specific Activity. These are the same as those listed under "Histidase." Application of Assay Method to Crude Tissue Preparations. N o interference with this m e t h o d has been found when crude extracts are used. Purification P r o c e d u r e T h e cell-free extract of Pseudomonas fluorescens, which was described under " H i s t i d a s e , " is used as the starting material. 7 Some additional purification can be accomplished b y removing nucleic acids with protamine, followed b y a C~ gel adsorption-elution step, and an a m m o n i u m sulfate fractionation. These steps are carried out at 0 °. Protamine Precipitation. T o 365 ml. of fresh extract is added, with stirring, 5 g. of p r o t a m i n e sulfate dissolved in 50 ml. of water. T h e gelatinous precipitate is then removed b y centrifugation and discarded. TABLE II SUMMARY OF UROCANASE PURIFICATION PBOCEDURE

Fraction

Pseudomonas extract

Total Specific volume, Total Protein, activity, Recovery, ml. Units/ml. units mg./ml, units/mg. %

365 Protamine supernatant 395 C~ eluate 200 Ammonium sulfate ppt. 1 8.8 Ammonium sulfate ppt. 2 9.3

76 78 120 1360 750

27,700 30,800 24,000 12,000 6,975

11.1 4.9 2.1 16.9 6.1

6.8 16 57 80 123

100 111 87 43 25

C~ Gel Adsorption and Elution. T o the s u p e r n a t a n t solution 90 ml. of alumina C~ gel (11.1 m g . / m l . ) is added. T h e gel is centrifuged down, and the urocanase activity is eluted with 200 ml. of 0.2 M p o t a s s i u m phosp h a t e buffer, p H 7.8. Ammonium Sulfate Fractionation. T o the C~ eluate is added 217 ml. of a m m o n i u m sulfate solution (prepared b y adding 7.5 ml. of 2 8 % a m m o n i a to each 100 ml. of a s a t u r a t e d (25 °) a m m o n i u m sulfate solution). T h e Urocanase is also present in liver extracts, and can be assayed in the same manner as described for Pseudomonas preparations. Purification procedures for liver urocanase have been described by M. Takeuchi [J. Biochem. (Japan) 34, 1(1941)] and Y. Sera and D. Aihara [J. Osaka Med. Soc. 41, 795 (1942)].

[30]

TRYPTOPHAN SYNTHETASE FROM NEUROSPORA

233

precipitate is collected by centrifugation and dissolved in water (first precipitate). To the supernatant solution 160 ml. of ammoniacal ammonium sulfate is added. The precipitate is collected by centrifugation and dissolved in water (second precipitate).

Properties The enzyme is stable in frozen cells; essentially no activity is lost over a period of several months at - 1 5 °. The extracts and partially purified preparations, on the other hand, are rather unstable and lose their urocanase activity rather rapidly. The pH optimum is near 7.

[30] T r y p t o p h a n S y n t h e t a s e I f r o m Neurospora Indole % L-Serine --~ L-Tryptophan

By

CHARLES YANOFSKY

Assay Method Assays of tryptophan synthetase activity are based on colorimetric determinations of indole and/or tryptophan. The author has found the colorimetric method for indole described below (a modification of the method of Stanley and Spray 2) excellent for the assay of this enzyme. In some instances it is desirable to check activity values which are based on indole disappearance. In such cases tryptophan should also be determined. The tryptophan assay procedure described by Nason et al. 3 is satisfactory for this purpose.

Reagents 0.5 M phosphate buffer, pH 7.8. 0.1 M phosphate buffer, pH 7.8. 0.2 M DL-serine. 0.005 M indole. 0.05 M glutathione (stored at --15°). 200 ~ ammonium pyridoxal phosphate 4 per milliliter. 1This enzyme has also been called tryptophan desmolase [M. Gordon and H. K. Mitchell, Genetics 35, 110 (1950)] and tryptophan desmasc [J. Monod and G. CohenBazire, Compt. rend. 236, 530 (1953)]. The name tryptophan synthetase was suggested by the editors of this volume. 2 A. R. Stanley and R. S. Spray, J. Bacteriol. 41, 251 (1941). 3 A. Nason, N. O. Kaplan, and S. P. Colowick, J. Biol. Chem. 188, 397 (1951). 4 Ammonium pyridoxal phosphate can be purchased from the California Foundation for Biochemical Research, Los Angeles, California.

234

ENZYMES OF PROTEIN METABOLISM

[30]

5 % NaOH. Color reagent. Dissolve 36 g. of p-dimethylaminobenzaldehyde (Eastman) in 500 ml. of ethanol. Add 180 ml. of concentrated HC1. When cool, bring the volume to 1 1. with ethanol. Enzyme. Dilute enzyme preparation with 0.1 M phosphate buffer to obtain 5 to 30 enzyme units/ml. (See definition below.) Procedure. Place 0.08 ml. of indole solution, 0.4 ml. of serine solution, 0.1 ml. of pyridoxal phosphate solution, 0.02 ml. of glutathione solution, and 0.12 ml. of 0.5 M phosphate buffer in a test tube. Add water and enzyme to bring the final volume in the tube to 1 ml. Prepare a similar tube containing all supplements except enzyme (for a standard curve). Incubate both tubes at 37 °. After 40 minutes add 0.2 ml. of 5% NaOH to each tube. Dispense 4 ml. of toluene into each tube, and shake vigorously for a few seconds. If desired, the tubes may be centrifuged for a few minutes to hasten separation of the two layers. Pipet aliquots of the toluene layer (up to 1 ml.) into separate test tubes, and add 4 ml. of ethanol and 2 ml. of color reagent to each. Allow the color to develop for 30 to 60 minutes, then read the tubes in a Klett-Summerson colorimeter (No. 54 filter). The indole standard curve is linear at low and intermediate indole levels and deviates slightly from linearity at high indole levels. Definition of Unit and Specific Activity. One unit of enzyme is defined as the amount of enzyme which will convert 0.1 micromole of indole to tryptophan under the conditions given above. Specific activity is expressed in terms of the number of enzyme units per milligram of protein. Protein is determined by the method of Lowry et al. 5 Application of Assay Method to Crude Homogenates and to Extracts from Other Organisms. The assay method described above works equally well with clarified extracts and crude mycelial homogenates of Neurospora. It has also been used successfully to determine tryptophan synthetase activity in enzyme preparations from Glomerella cingulata and Escherichia coll. When examining crude homogenates or extracts from other organisms for tryptophan synthetase it is advisable to perform both indole and tryptophan determinations on each sample. Purification Procedure

All wild-type strains of Neurospora crassa examined to date are good sources of tryptophan synthetase. Wild-type strain Em-5297a has been used by the author as a source of this enzyme, and the procedure described below was carried out with mycelium of this strain. 60. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

[30]

TRYPTOPHAN SYNTHETASE FROM NEUROSPORA

235

Step 1. Preparation of Crude Extract. Five-liter bottles, each containing 3 1. of Neurospora minimal medium, ~ are inoculated with a heavy conidial suspension of strain 5297. The bottles are incubated in a constant temperature room at 30 ° . Air, filtered through sterile cotton, is continuously bubbled through the medium. After 48 to 72 hours the contents of each bottle are filtered through cheesecloth, and the mycelium obtained is washed twice in distilled water. The washed mycelium is then frozen and lyophilized. The lyophilized mycelium is ground to a fine powder in a mortar or a Wiley mill, and 5-g. quantities are placed in 4-ounce screwcap vials with several dozen small glass beads and 80 ml. of cold 0.1 M phosphate buffer (10-3 M with respect to glutathione) at pH 7.8. The vials are shaken at maximum speed (approximately 300 strokes per minute) on an Eberbach shaker (in a cold room) for 2 to 3 hours. At the end of this period the contents of the vials are filtered through cheesecloth on a Biichner funnel to remove the glass beads. The filtered suspension is centrifuged in a Servall centrifuge (in a cold room) at 12,000 r.p.m, for approximately 30 minutes. The faintly turbid supernatant solutions are carefully poured off and stored at - 1 5 °. Step 2. Treatment with Protamine SulfateJ Add cold 1 N acetic acid to the thawed extract until the pH is lowered to 6.6. s Then add 1 ml. of protamine sulfate solution (15 mg./ml.) for every 100 rag. of extract protein. Stir, and let stand for 15 minutes. Remove the precipitate by centrifugation. Step 3. Ammonium Sulfate Fractionation. Adjust the pH of the supernatant solution to 5.8 with 1 N acetic acid. Add 15 g. of ammonium sulfate, with constant stirring, for each 100 ml. of solution. After the ammonium sulfate has dissolved, stir the suspension occasionally for 20 minutes. Collect the precipitate by centrifugation, and take it up in 0.1 M phosphate buffer (10-3 M with respect to glutathione) at pH 7.8. Use 15 ml. of buffer for every 100 ml. of extract treated with protamine sulfate. Step 4. Calcium Phosphate Gel Treatment. For every 10 mg. of protein in the final solution from step 3 add 6 mg. of calcium phosphate geP (30 rag. dry weight per milliliter), and stir for 5 minutes. Remove the gel by centrifugation, and repeat the treatment, using 10 mg. of gel for every 10 mg. of protein initially present. Remove the gel by centrifugation. Step 5. Ammonium Sulfate Fractionation. For every 50 ml. of supernatant solution from the previous step add 7.2 g. of ammonium sulfate. s G. W. Beadle a n d E. L. T a t u m , Am. J. Botany 32~ 678 (1941).

7This step and all the followingsteps in the purification procedure should be carried out at reduced temperatures. The initial pH of the extract is usually between 7.3 and 7.5. 9D. Keilin and E. F. Hartree, Proc. Roy. Sot., (London) B124, 397 (1937-38); see Vol. I [11].

236

ENZYMES OF PROTEIN METABOLISM

[30]

After 20 minutes remove the precipitate b y eentrifugation, and add an additional 4.3 g. of a m m o n i u m sulfate. After 20 minutes collect the precipitate by centrifugation, and take it up in 0.01 phosphate buffer at pH 6.8 (13 ml. for every 50 ml. of supernatant solution treated). Add glutathione (to a final concentration of 10 -3 M) and pyridoxal phosphate (to 20 ~//ml.). Dialyze the solution against the same buffer (5 X 10-4 M with respect to glutathione and containing 2 ~, of pyridoxal phosphate per milliliter) for 2 hours with mechanical stirring of the contents of the dialysis bag. If a precipitate forms, remove it b y centrifugation. Step 6. Calcium Phosphate Gel Treatment. T r e a t the solution from step 5 twice ~ i t h calcium phosphate gel (1 rag. of gel per milligram of protein, set step 4). Store the s u p e r n a t a n t solution at - 1 5 °. SUMMARY OF PURIFICATION PROCEDURE

Fraction 1. Crude extract 2. Protamine sulfate supernatant 3. Ammonium sulfate fraction 4. Supernatant from gel 5. Ammonium sulfate fraction 6. Supernatant from gel

Specific Total Total Protein, activity, Recovery. volume Units/ml. units mg./ml, units/rag. % 180

15

2700

17

0.88

210

12

2500

11

1.1

93

29 50

48 24

1400 1200

15 3.3

3.2 7.3

52 44

13 14

58 36

750 500

2.6 0.58

22 62

28 19

Properties Specificity. The purified enzyme has a requirement for L-serine which cannot be satisfied by any of the following compounds; D-serine, glycine, I)L-alanine, L-cysteine, L-cystine, DL-threonine, DL-methionine, L-aspartare, L-glutamate, acetate, pyruvate, suceinate, and malate. TM T h e L-serine concentration which gives half m a x i m u m velocity is 3.4 X 10 -3 M. The enzyme is not specific for indole; 6-methylindole and 7-hydroxyindole are also attacked. However, neither of these indole derivatives is taken up as rapidly as is indole when tested at equimolar concentrations. The indole concentration which gives half maximum velocity is 5.6 N 10-~ M. Coenzyme. T h e activity of crude synthetase preparations is stimulated b y pyridoxal phosphate. 11 Storing mycelium in the frozen state for lo C. Yanofsky, J. Biol. Chem. 194, 279 (1952). 11W. W. Umbreit, W. A. Wood, and I. C. Gunsalus, J. Biol. Chem. 165, 731 (1946).

[30]

TRYPTOPHAN SYNTHETASE FROM NEUROSPORA

237

several weeks before extraction increases the pyridoxal phosphate dependence of the enzyme preparations obtained. 11 Extracts which have low activity in the absence of pyridoxal phosphate may also be obtained from mycelium of a pyridoxine-requiring mutant grown on a limiting pyridoxine supplement. Fractionation with ammonium sulfate is also effective in aiding in the resolution of the enzyme. The pyridoxal phosphate concentration which is required to restore half maximum velocity to a resolved preparation is approximately 1 X 10-6 M. 12 Pyridoxal phosphate cannot be replaced by pyridoxamine phosphate. Stability. Crude synthetase preparations prepared without the addition of glutathione are relatively unstable; they lose half their activity in 24 hours when stored at 2 °. When stored at - 15°, however, full activity is retained for several months. Synthetase preparations to which reduced glutathione and pyridoxal phosphate have been added are stable at 2 ° for periods up to a week. Although either pyridoxal phosphate or reduced glutathione will partially stabilize the enzyme, both must be present for full protection (at concentrations of 20 ~,/ml. and 10-3 M, respectively). Cysteine will not replace reduced glutathione. Inhibitors. Tryptophan synthetase, like other pyridoxal phosphaterequiring enzymes, is inhibited by hydroxylamine and cyanide. It is also inhibited by cysteine and L-tryptophan. The metal-binding agents 8-hydroxyquinoline and etbylenediaminetetraacetic acid are ineffective as inhibitors except at unusually high concentrations. 13 Co ++, Zn ++, Cu++, 13 and p-chloromercuribenzoate also inhibit the enzyme. The inhibition by the latter compound is completely reversed by glutathione. D-Serine, at a concentration of 6 X 10-2 M, does not inhibit. Effect of pH. The enzyme exhibits maximum activity at about pH 7.8 (in phosphate buffer) although in the range 7.5 to 8.4 there is only slight variation in activity. At pH values below 7.0 enzyme activity decreases rapidly. Enzyme Formation. Neurospora appears to require zinc for the synthesis of tryptophan synthetase. 3,~3 Deficiencies of magnesium, manganese, and iron, on the other hand, do not appear to affect tryptophan synthetase formation. Tryptophan synthetase formation (in Aerobacter aerogenes) is inhibited by indole and tryptophan and stimulated by phenylalanine, methionine, and several other amino acids. TM Tryptophan synthetase formation in 12Calcium pyrido×al phosphate provided by W. W. Umbreit and assaying 28 % pyridoxal phosphate was used in this determination. 1~A. Nason, Science 112, 111 (1950). 14j. Monod and G. Cohen-Bazire, Compt. rend. 236, 530 (1953).

238

ENZYMES OF PROTEIN METABOLISM

[31]

Neurospora is also inhibited by tryptophan, but the inhibition is not as marked. Genetic control of tryptophan synthetase formation has been demonstrated in Neurospora. 15,16 15H. K. Mitchell and J. Lein, J. Biol. Chem. 175, 481 (1948). is C. Yanofsky, Proc. Natl. Acad. Sci. U.S. 38, 215 (1952).

[31] T r y p t o p h a n C l e a v a g e

Tryptophanase from E. colt Pyridoxal phosphate [

-)

CH2CH--COOH

~~N~

-$- NH3 q- CH3C COOH

(1)

0 By I. C. GUNSALUS, COURTLAND C. GALEENER, and JOHN R. STAMER

Assay Method Principle. The tryptophanase reaction, long recognized as the source of indole in growing cultures of E. colt, has been considered at the enzymatic and mechanism level principally by two groups of workers: (1) Happold and co-workers, data to 1950 summarized by Happold, 1 (2) Wood, Umbreit, and Gunsalus, 2 who obtained the enzyme in extracts, free of serine and alanine deaminase, defined the reaction as an anaerobic cleavage of tryptophan to indole, ammonia, and pyruvate (reaction 1 above), and demonstrated pyridoxal phosphate as the coenzyme. These workers also suggested as mechanism a cleavage of tryptophan into indole plus an intermediate--probably aminoacrylic acid--which decomposed spontaneously to ammonia plus pyruvate. The tryptophanase reaction can be followed most conveniently by measuring the indole accumulation with the specific p-dimethylaminobenzaldehyde method of Ehrlich. 2 Reagents M /20 L-tryptophan. Barium pyridoxal phosphate, 100 ~//ml. 1 F. C. Happold, Advances in Enzymol. 10, 51 (1950). W. A. Wood, I. C. Gunsalus, and W. W. Umbreit, J. Biol. Chem. 170, 313 (1947).

[31]

TRYPTOPHAN CLEAVAGE

239

M/1 phosphate buffer, pH 8.3. Trichloroacetic acid (TCA), 100% w/v. Toluene, reagent grade. Indole standard, 50 ~/ml. in M/IO phosphate buffer, pH 8.3. p-Dimethylaminobenzaldehyde, 5% in 95% ethanol. Acid alcohol, 1 1. of ethanol ~ 80 ml. of concentrated H2S04, added slowly with cooling. E. coli, dried cells or extracts prepared as indicated below.

Procedure. Tryptophanase activity was determined by measuring indole formation. The usual assay was run in 2 ml. containing the following reagents: 0.2 ml. of phosphate, 0.2 ml. (20 -y) of barium pyridoxal phosphate, enzyme or dried cells containing 1 to 20 units of tryptophanase, plus water to 1.8 ml. Incubate at 37 ° for 10 minutes, add 0.2 ml. of L-tryptophan, and allow to incubate for an additional 10 minutes as a reaction period. Stop the reaction with 0.2 ml. of TCA, add 2 ml. of toluene, and shake gently to extract the indole. The tryptophan cleaved, as measured by indole formation, is not a linear function of the amount of enzyme above 8 to 10 ~, of indole formed per 2 ml. reaction volume owing to the inhibition of the forward reaction by indole. A suitable calibration curve for enzyme quantity can be constructed by using an acetone powder or a cell-free coli extract as source of tryptophanase. 2 Indole is measured on a suitable aliquot of the toluene layer containing 1 to 15 ~, of indole as follows: Transfer the toluene, usually 0.2 to 1 ml., to an 18 X 150-ram. colorimeter tube, add 1 ml. of 5% p-dimethylaminobenzaldehyde reagent, and dilute to 10 ml. with acid alcohol. After 10 minutes, read in an Evelyn colorimeter with a 540-m~ filter, and calculate the indole from a standard curve prepared by toluene extraction of the indole standard in phosphate buffer. Definition of Units. The tryptophanase unit is defined as that amount of enzyme required to form 1 ~, of indole per 10-minute assay, protocol as listed above. The specific activity is the units of enzyme per milligram of protein or, with cell powder, per milligram of dry weight. Purification Procedure

1. Dried cell preparations of E. coli, Crookes strain, may be obtained as follows: Inoculate a medium composed of 1% tryptone, 1% yeast extract, 0.5% K~HPO4, and 0.1% glucose dispensed in 200-ml. amounts in 500-ml. Erlenmeyers with 5 % inoculum. Incubate for 4 to 6 hours at 30 °, then for an additional 18 to 20 hours on a mechanical shaker at 30 °.

240

ENZYMES OF PROTEIN METABOLISM

[31]

Harvest the cells by Sharpies centrifuge, resuspend in one-twentieth of the growth volume of distilled water, and dry in vacuo over Drierite, or pipet into 10 vol. of acetone at - 2 0 °. Collect the cells from the acetone on a Bfichner funnel, and wash with cold acetone, followed with cold ether. Work the filter cake from the funnel on a piece of wrapping paper until solvent free, as determined by absence of odor. The yield is about 2 g. of dry cells per liter of medium. The tryptophanase is stable in either vacuum- or acetone-dried cells maintained in an anhydrous state. 2. Cell-free tryptophanase was prepared by Wood et al. ~ (before more convenient means of extraction were available) by alternately freezing and thawing 10 g. of acetone-dried cells per 500 ml. of water. On thawing, the suspension was brought to 37 ° and allowed to autolyze for 3 hours. After three repetitions of this procedure, the debris was removed by centrifugation and discarded. The cell-free extract, which contains 60 to 80 % of the tryptophanase present in the dried cells, was fractionated by ammonium sulfate precipitation and phosphate gel adsorption. A similar procedure was used by Dawes et al. (see ref. 1). Tryptophanase can be extracted more conveniently by sonication or alumina grinding (see Vol. I [7]) as follows: Suspend 100 mg. of dried ceils per milliliter in M / 5 0 phosphate, pH 7.0, and sonicate for 20 minutes in a 200-watt, 10-kc. Raytheon oscillator. After removal of debris by centrifugatiorL, the extract contains about 90% of the tryptophanase units present in the dried cells. 3. Purification of tryptophanase can be accomplished by precipitation from the extracts with ammonium sulfate. The 0.3 to 0.6 saturated precipitate contains the enzyme at twofold increase in specific activity, i.e., extract S.A. = 6; 0.3 to 0.6 saturated ammonium sulfate precipitate, S.A. = 14. Redissolve the precipitated tryptophanase in M / 5 0 phosphate buffer, pH 7.0, containing M/1000 glutathione. Dilute to 10 mg. of protein per milliliter, adjust to pH 6.0 with 1 M acetic acid, and treat with protamine (20 ~ of protamine sulfate per milliliter, pH 5.0) until a test sample after centrifugation shows a 280/260-m~ absorption ratio of about 0.8 (requires about 0.1 vol.). Centrifuge to remove the protamine nucleate. Dialyze for 12 hours at 0 ° against distilled water, and remove the copious precipitate which forms by centrifugation. The supernatant, after protamine sulfate treatment and dialysis, contained 70% of the tryptophanase present in the initial extract at a specific activity of 13. A second ammonium sulfate fraetionation yielded tryptophanase in the 0.4 to 0.55 saturated fraction equivalent to 70% of the initial units at a specific activity of 25, and an additional 15,000 units (50% of the initial units) in the 0.55 to 0.65 saturated precipitate at a specific ae-

[31]

TRYPTOPHAN CLEAVAGE

241

tivity of 54--t0 give an over-all increase in units of tryptophanase measured. The combined 0.4 to 0.65 precipitate in M / 5 0 phosphate glutathione buffer adjusted to 10 rag. of protein per milliliter was subjected to calcium phosphate gel treatment. One volume of gel (20% solids) at pH 6 removed more than 50% of the protein without adsorbing the tryptophanase, and an additional volume of gel adsorbed the tryptophanase which can be eluted at a specific activity of approximately 150 by M / I O phosphate buffer pH 7. The effectiveness of the gel procedure is improved by using a trial sample of each preparation to determine the best gel proportion for both the negative and positive gel steps. Since the original definition of the tryptophanase reaction with extracts of E. coli, 2 no extensive attempt to purify this enzyme has been undertaken. The above data are taken from the research report of Mr. Courtland Galeener who also obtained the inhibitor data for the tryptophan analogs indicated below.

Properties The optimal pH of tryptophanase is around 7.5 (Wood, Galeener, unpublished experiments); the substrate Kin, about 2.5 × 10-5 M per liter; and the Km of the pyridoxal phosphate, about 2 × 10-8 M per liter. Activators and Inhibitors. The resolution for pyridoxal phosphate occurs spontaneously during the above purification procedure or more abruptly if the preparation is treated with 10-4 M cyanide before one of the ammonium sulfate precipitation steps. After calcium phosphate gel treatment, the tryptophanase can be stabilized by the addition of mercapto compounds, e.g., glutathione or cysteine; after spontaneous loss of activity, it can be reactivated by mercapto compounds. Inhibitors. Carbonyl reagents are inhibitory, presumably through combination with the cofactor (see Gale 3 for decarboxylases). Among these, KCN at 10-3 M inhibits the reaction 95 %; at 10-5 M, 35 %. Hydrazine and hydroxylamine at 10-3 M inhibit completely. Indole, at 10-3 M, inhibits the reaction better than 90%; at 3 X 10-5 M, approximately

50%. DL-Tryptophan is 90% as active as L-tryptophan, thus indicating a slight inhibition by the D isomer. Homotryptophan is inactive as substrate and inhibits the tryptophanase reaction 70% at equimolar concentration with substrate. Tryptazan, 4 is inactive as substrate, but inhibits the reaction 40% at equimolar concentration with the substrate. The 3E. F. Gale, Advances in Enzymol. 6, 18 (1946). 4H. R. Snyder, C. B. Thompson, and R. L. Hinman, J. Am. Chem. Soc. 74, 2009 (1952).

242

ENZYMES OF PROTEIN METABOLISM

[32]

6-methyl- and 4,6-dimethyltryptophans are active as substrates, approximately 25 and 15%, respectively, of the tryptophan rates; with tryptophan in equimolar amount, intermediate rates, not readily interpretable, are obtained. Activation of tryptophanase by potassium, ammonium, and rubidium ions after dialysis against 0.25 M NaCl has recently been reported by Happold and Struyvenberg. 5 5 F. C. Happold and A. Struyvenberg, Biochem. J. 58, 379 (1954).

[32] T r y p t o p h a n O x i d a t i o n

By W. E. KNOX A. L-Tryptophan Peroxidase from Liver +05

L-Tryptophan -}- H~O~-+ [

]

~ Formylkynurenine ~- H20~

Assay Method Principle. Formylkynurenine is hydrolyzed to kynurenine by excess kynurenine formamidase normally present in liver and determined by its absorption at 365 m~. This method was first used by Knox and Mehler. I The simple conditions given are suitable for rat liver homogenates, but the same method can be used with other preparations if they are reinforced as necessary with enzyme-generated peroxide, formamidase, and catalase. Reagents 0.14 M KC1, containing 2.5 millimoles of NaOH per liter. 0.03 M L-tryptophan. Dissolve.153 mg. in about 20 ml. of water by the addition of several drops of 6 N N a 0 H , immediately neutralize to pH 7, and dilute to 25 ml. Store at 5°, and renew weekly. 0.2 M NaH2PO4-Na2HPO4 buffer, pH 7.0. 1.0 N NaOH. 15% (w/v) metaphosphoric acid. One milliliter of this acid should be neutralized to pH 7.0 to 7.5 by 1 ml. of the 1.0 N NaOH. Store at 5° , and renew monthly. Enzyme. An amount of enzyme is used which will form 0.5 to 5.0 micromoles of kynurenine per hour, which is usually present in 0.5 to 2.0 ml. of 12.5 % liver homogenate. Maximum activities are i w. E. Knox and A. H. Mehler, J. Biol. Chem. 187, 419 (1950).

[32]

TRYPTOPHAN OXIDATION

243

obtained only if the enzyme is prepared as given below and assayed within an hour after death of the animal.

Procedure. The animals are exsanguinated and the livers immediately removed, cooled in ice water, and weighed to 0.1 g. All solutions, equipment, and the enzyme until it is assayed are kept cold (2 to 5°). The livers are homogenized for 2 minutes in 7 vol. of the alkaline KC1 solution. The final pH should be 7 to 7.5 (phenol red). Four cups for measuring two concentrations of the enzyme, each with a no-tryptophan blank, are equilibrated at 37 ° in a Dubnoff shaker or similar device in an atmosphere of oxygen. One of each pair of cups contains 0.3 ml. of L-tryptophan, and all cups contain 1.0 ml. of phosphate buffer, pH 7.0, and water to make a total volume of 4.0 ml. after additions of the enzyme. The refrigerated homogenate, usually 1 and 2 ml., is added to each pair of cups, which are then shaken at 120 oscillations per minute for 60 minutes. The reactions are stopped, in the order that enzyme was added, by 2.0 ml. of the metaphosphoric acid added while slower shaking continues. The cups are removed from the bath and, after 5 minutes, filtered. A 3.0-ml. aliquot of each filtrate is neutralized with 1.0 ml. of 1 N NaOH. The final pH should be between 6.5 and 7.5. The solutions should not be exposed to direct sunlight and must be read within an hour after neutralization, against water at 365 m~ in a Beckman spectrophotometer. Appropriate dilutions with water are made of any solutions reading more than 1.200. The density difference between the blank and the experimental is a measure of the kynurenine formed; it is proportional to the enzyme concentration and increases linearly with time for 2 or 3 hours. The dry weight is taken as that of the fresh homogenate (usually 2 ml.), dried for 2 to 3 hours at 100°, minus the weight of the contained KC1. Calculation of Activity. Enzyme activity is expressed in terms of the micromoles of kynurenine formed per milliliter of enzyme per hour, and the specific activity in terms of the grams of dry weight. The molar extinction coefficient of kynurenine at 365 mtL and pH 6.5 to 7.5 is 4.53 X 103. The micromoles of kynurenine formed by 1 ml. of homogenate per hour under the above conditions would be the difference in optical densities of the 1-ml. enzyme blank and that plus tryptophan determined in 1-cm. cells, divided by 0.567: Application of Assay Method to Other Types of Preparations. Formamidase, catalase, and enzyme-generated peroxide are present in the homogenates described but must be added to certain other types of preparations. The formamidase is normally present in a 600-fold excess in liver, and if separated on purification it can easily be added back as an aged soluble liver extract containing no tryptophan peroxidase activity, or as

244

ENZYMES OF PROTEIN METABOLISM

[32]

the purified enzyme (which see). Rat liver homogenates, and the supernatants from them, generate sufficient peroxide during the first 2 to 3 hours after preparation, mostly from xanthine oxidation. After storage for 24 hours at 2 ° the apparent tryptophan peroxidase activity decreases 40 to 50%, largely because of peroxidase lack. The routine addition of 15 micromoles of glucose and sufficient glucose oxidase (notatin) 2 to cause the uptake of 20 ~1. of O~ per 10 minutes will maintain optimal peroxide production in the system when assaying the aged or purified preparations, or enzymes from other species. Catalase in the system protects the tryptophan peroxidase from inhibition by excess peroxide, and 0.5 mg. should be added whenever glucose oxidase is used. When the purified system is catalase-free no exogenous peroxide source is necessary, since the coupled oxidation reaction of the tryptophan peroxidase supplies its own peroxide very efficiently. Although homogenates contain kynureninase and kynurenine transaminase, very little kynurenine reacts further under these assay conditions. Speetrophotometric, chemical, and chromatographic analyses show that only negligible amounts of anthranilic acid and little or no kynurenic and hydroxyanthranilie acids are formed during tryptophan oxidation. The tryptophan disappearance and oxygen uptake, however, are in excess of that accounted for by the kynurenine accumulated. Purification Procedure This procedure has been used successfully in several laboratories, but only on livers from rabbits and rats. Preferably, the enzyme should first be adaptively increased by the administration of tryptophan 6 hours before killing the animal. 3 The goal of the purification is the functional separation of catalase, formamidase, and the peroxide-generating systems from the tryptophan peroxidase. All manipulations must be done at 2 to 5°. Step 1. Preparation of Soluble Fraction. Fresh, chilled liver is homogenized briefly in 3 vol. of alkaline 0.14 M KC1. The particulate elements are centrifuged off at 11,000 X g for 20 minutes. Step 2. pH 5.4 Precipitation. This step should be completed in less than 45 minutes. One-tenth volume of 0.5 M NaH2P04, then 0.05 vol. of the same solution containing 1% (v/v) glacial acetic acid, are slowly stirred into the soluble fraction from step 1. A drop of the mixture should produce a just perceptible green in a drop of bromocresol green. After 2 A suitable preparation can be obtained commercially as Deoxygenase from Takamine Laboratory, Clifton, New Jersey; see also Vol. I [45]. W. E. Knox and A. H. 1VIehler, Science 113, 237 (1951); W. E. Knox, Brit. J. Exptl. Pathol. 32, 462 (1951).

[32]

T R Y P T O P H A N OXIDATION

245

rapid centrifugation, the s u p e r n a t a n t containing the catalase and formamidase is carefully drained off, and the precipitate washed b y resuspension and centrifugation in at least the original volume of 0.1 M acetate buffer, p H 5.4, and then with water in the same way. T h e precipitate after washing is i m m e d i a t e l y emulsified with 0.2 M Na2HPO4 (0.05 of the original volume), adjusted to p H 7 with 1 N N a O H , and diluted with water to a b o u t one-fifth the original volume. T h e enzyme remains active a b o u t one week at - 1 0 °. TABLE I SUMMARY OF PURIFICATION PROCEDURE a

Fraction

Specific activity Total Activity, Dry (dry wt.), vohlme, micromoles/ weight, micromoles/Recovery, ml. ml./hr. Total mg./ml, hr. %

1. 25% holnogenate 2. Soluble supernatant 3. pH 5.4 precipitate b

60

2.3

138

67.8

34.0

--

48 8

2.2 7.6

106 61

58.6 80

37.6 94.8

77 44

slivers from two rats given 2 millimoles of DL-tryptophan intraperitoneally 6 hours previously. b Substantially free of catalase and formamidase, as shown by the accumulation of formylkynurenine and the high activity without added peroxide or catalase. Assayed with added formamidase, catalase, and the glucose oxidase system in the amounts given in the method.

Properties Distribution. T r y p t o p h a n peroxidase has been found in the liver of the mouse, rat, guinea pig, rabbit, dog, pig, and pigeon b u t not in other tissues of these animals. I t is clearly differentiated from other peroxidases known in animals (milk and white blood cells). An enzyme with similar properties and catalyzing the same reaction occurs in t r y p t o p h a n a d a p t e d Pseudomonas. 4 Specificity. T h e enzyme has no action on D-tryptophan, DL-acetylt r y p t o p h a n , N l - m e t h y l t r y p t o p h a n , indolepropionic acid, or t r y p t a m i n e , and no discernible action on the common substrates used with peroxidases. Effects of p H and Substrate Concentration. T h e activity is detectable between p H 6.5 and 8.5 and is optimal near 7.0. T h e Km in the assay described is 4 X 10 -4 M, and 10 -3 M L - t r y p t o p h a n saturates the enzyme. 4 R. Y. Stanier and O. Hayaishi, J. Bacteriol. 62, 691 (1951).

246

ENZYMES OF PROTEIN METABOLISM

[32]

Activators and Inhibitors. Peroxide is required in the reaction, and it is consequently inhibited by catalase. The catalase inhibition may be specifically reversed by addition of extra peroxide generated enzymically. This means is used in the assay of aged preparations to overcome the effect of the high catalase concentration of crude liver preparations when the endogenous peroxide production is low. An unusual feature of the tryptophan peroxidase reaction is its inhibition by Cu ++ and its light reversible inhibition by CO, in addition to its inhibition by the usual ferric reagents such as azide, cyanide, and sulfide, which inhibit all peroxidases. Mechanism of the Reaction. The tryptophan peroxidase catalyzes a physiologically occurring coupled oxidation reaction with the uptake of molecular oxygen. There is no evidence suggesting that more than one enzyme is involved in the two-step oxidation. The same enzyme is thought to successively catalyze the peroxidase and oxidase steps, using oxygen and forming its own peroxide, and at the same time alternating between the ferric and ferrous states. Substrate Induction and Hormonal Control. The activity of the tryptophan peroxidase is increased in a few hours in vivo by administration of tryptophan and by cortisone2

B. Kynurenine Formamidase (Formylase) Formylkynurenine--* HCOOH ~- Kynurenine

Assay Method Principle. The method depends on the shift in the ultraviolet absorption spectrum which accompanies hydrolysis of aromatic formamido compounds. Formyl kynurenine is used as the substrate instead of formylanthranilic acid as was originally used by Mehler and Knox. 5 This makes the method more sensitive and avoids the inhibition due to anthranilie acid accumulation. Reagents

0.01 M formylkynurenine. Dissolve 12 mg. of the free acid 6 in 5 ml. of water by quickly adjusting to pH 7 with 0.1 N NaOH to avoid spontaneous hydrolysis. The presence of some kynurenine, causing absorption at 365 m~, is inconvenient but does not affect the results. The solution should be kept cold and neutral and renewed every day or two. A. H. Mehler and W. E. Knox, J. Biol. Chem. 187, 431 (1950). For preparation of formylkynurenine,see Vol. III [89].

[32]

TRYPTOPttAN OXIDATION

247

0.2 M phosphate buffer, pH 7.5. Enzyme. Dilute the well-centrifuged enzyme in 0.14 M KC1 to give a concentration producing a density change of 0.050 to 0.250 per minute per milliliter. This activity is present in 0.05 ml. or less of a 25 % homogenate of rat liver.

Procedure. To a 1-cm. cell is added 0.2 ml. of the formylkynurenine solution, 1.0 ml. of phosphate buffer, and water to make a total volume of 3.0 ml. after addition of 1 ml. of the enzyme. The enzyme is added last, and 365-m~ readings are taken at 30-second intervals thereafter for 3 minutes. Activity. Activity is expressed as the density change per minute which would be produced by 1 ml, of the enzyme in the above system. Specific activity is expressed as the activity per milligram of protein, determined from its absorption at 260 and 280 mt~ by the method of Warburg and Christian. 7 Since formylkynurenine does not absorb appreciably at 365 mt~, the micromoles of kynurenine formed can be calculated directly from the density change and the molar extinction coefficient of kynurenine under these conditions (4.53 X 103). Application to Crude Tissue Preparations. Use of the method is limited only by lack of clarity of the crude enzyme solutions. Centrifugation of an homogenate is usually sufficient to permit assay, since the enzyme is soluble and is present in high activity in liver. If deproteinization after an appropriate interval should be necessary for readings to be made, this must be done with a neutral precipitant such as Zn acetate-NaOH, to avoid the ready hydrolysis of formylkynurenine in acid solution. Purification Procedure The purification follows that of Mehler and Knox 5 and uses rat liver. The enzyme of horse and guinea pig livers will not withstand the heat treatment step. The numbered steps have been used repeatedly in several laboratories, with results at least as good as those given in the table. Steps 1 and 2. Preparation of Soluble Extract and Precipitation at pH 5.4. The rat livers are homogenized, centrifuged, and acidified in the cold exactly as described in the preparation of tryptophan peroxidase. 8 The supernatant after removal of the p H 5.4 precipitate contains the enzyme, and it is promptly brought to pH 7 with approximately one-tenth the original volume of 1 N NaOH. Step 3. Heat Treatment. The neutralized supernatant from step 2 is placed in a large Erlenmeyer flask, the liquid set swirling, and the whole 70. Warburg and W. Christian, Biochem. Z. 810, 384 (1941) ; see Vol. I I I [73]. s See p. 244 (tryptophan peroxidase preparation).

248

ENZYMES OF PROTEIN METABOLISM

[32]

immersed in a large water b a t h at about 90 °. The t e m p e r a t u r e is raised within 2 minutes to 60 °, as determined b y a t h e r m o m e t e r kept in the solution, and held accurately at t h a t t e m p e r a t u r e for 5 minutes with constant swirling, then quickly cooled below 20 ° in an ice bath. T h e coagulated protein is filtered off on fluted paper. Step 4. Ammonium Sulfate Fractionation. T h e enzyme is precipitated between 45 and 68% saturation with a m m o n i u m sulfate at 5 ° . T o the filtrate of step 3, 0.82 vol. of saturated a m m o n i u m sulfate is added and the precipitate centrifuged off and discarded. More saturated a m m o n i u m sulfate, equal to 0.7 vol. of the new supernatant, is added to bring it to 68 % saturation. This precipitate contains the enzyme, and it is dissolved in a small volume of water. The enzyme at this stage can be dialyzed against water, if desired, and will keep at 0 ° for several months. F u r t h e r purification can be achieved by t r e a t m e n t of the dialyzed enzyme with alumina C~. 9 Some inactive protein is preferentially absorbed b y the first addition of alumina gel and is centrifuged off and discarded. F u r t h e r addition of relatively large amounts of the gel absorbs the enzyme, which can then be eluted by two successive washings with 0.2 M phosphate buffer, p H 7.5. The amounts of alumina required and the success of the procedure differ with each preparation. Only by repeated assays of the activity and the total protein during stepwise additions of the gel can the considerable purification potential of this step be realized and the last traces of catalase removed. The enzyme after this t r e a t m e n t is also quite stable. TABLE II SUMMARY OF PURIFICATION PROCEDURE

Fraction 1. 2. 3. 4.

Soluble Supernatant, pH 5.4 Heated at 60° Ammonium sulfate, 45-68 %

Activity, Total A/rain. Specific volume, Protein, activity, Recovery, ml. Per ml. Total mg./ml. A/mg. protein % 235 246 214

3.8 3.3 2.9

890 810 620

64 36 16

0.059 0.092 0. 181

-91 70

17

12.2

207

27

0. 452

23

Properties Distribution. T h e livers of all species examined contain similar activities of the enzyme. Kidney, spleen, and intestine show less t h a n one-tenth the activity of liver, and other tissues none. 9 For preparation of alumina C~ gel, see Vol. I [11].

[39.]

TRYPTOPHAN OXIDATION

249

Specificity. The enzyme hydrolyzes a large series of aromatic formamido compounds. Formylkynurenine is hydrolyzed six times as fast as the second most rapid compound, formylanthranilic acid. The latter has also been used as the substrate for assay of the enzyme by a similar method to the one given. 5 Aromatic acetamido and aliphatic formamido compounds are hydrolyzed only very slowly. Inhibitors. Anthranilic acid inhibits the enzyme about 20% in 10-4 M concentration, but kynurenine does not. It is not inhibited by cyanide, sulfide, fluoride, or other metal-binding reagents. Effect of pH. The enzyme shows a broad pH optimum between pH 6 and 8. C. Liver and Bacterial Kynureninases L-Kynurenine--~ Anthranilic acid -t- L-Alanine

Assay Method

Principle. Colorimetric determinations of the kynurenine disappearing 1° and of alanine formed 11 were originally used to detect the kynureninase reaction. The simplest method, however, depends on the disappearance of the kynurenine absorption at 360 to 365 m~, as was used by Dalgliesh et al. z~ The change in extinction is measured with the slower liver enzyme after deproteinization, 13 but the reaction can be followed directly with the more active bacterial enzyme. 14 Procedures are given for each of these enzymes, because of their different activities and properties. Reagents for the Bacterial Enzyme 0.001 M L-kynurenine. 15 0.2 M tris(hydroxymethyl)aminomethane buffer, pH 8.5.

Procedure. One milliliter each of L-kynurenine and Tris buffer are placed in a 1-cm. cell with enough water to make 3.0 ml. total volume after addition of the enzyme. Sufficient enzyme is added to cause a density change of 0.05 to 0.20 per minute at 360 m~, and readings are taken at 30-second intervals for several minutes. The resulting curve remains linear while about two-thirds of the substrate is removed, and from this portion the rate is calculated. 10 y . Kotake and T. Nokayama, Z. physiol. Chem. 270, 76 (1941). 11 O. Wiss, Helv. Chim. Acta 32, 1694 (1949). 12 C. E. Dalgliesh, W. E. Knox, and A. Neuberger, Nature 168, 25 (1951). 13 W. E. Knox, Biochem. J. 53, 379 (1953). 14 O. Hayaishi and R. Y. Stanier, J. Biol. Chem. 196, 735 (1952). 1~ For preparation~ see Vol. I I I [89].

250

ENZYMES OF PROTEIN METABOLISM

[32]

Reagents for the Liver Enzyme 0.0075 M L-kynurenine. 15 20 mg. % pyridoxal phosphate. 0.2 M phosphate, pH 8.0. 1% boric acid in ethanol.

Procedure. An amount of enzyme which will split less than 0.5 micromole of kynurenine per hour is first incubated for 15 minutes at 37 ° with 0.1 ml. of pyridoxal phosphate and 0.5 ml. of phosphate buffer in a total volume of 1.0 ml. Then 0.2 ml. of water is added to one such tube and 0.2 ml. of kynurenine to a second, and both are immediately deproteinized by addition of 5 ml. of boric acid-ethanol. An identical pair are incubated for 40 minutes after the addition and then deproteinized. The initial and final kynurenine is determined by reading the enzyme filtrates against their blanks at 365 m~. Activity. The molar extinction coefficients of kynurenine under the conditions used with the bacterial enzyme and the animal enzyme are 4530 and 5150, respectively. The unit of bacterial enzyme activity used by Hayaishi and Stanier 14 was the amount causing 0.1 density change per minute in the 3.0-ml. assay system. This corresponds to 3.96 micromoles of kynurenine split per hour. For comparison between the enzymes, the activities of both are here expressed as micromoles of kynurenine split per hour under the given reaction conditions. The specific activities are expressed as the activities per milligram of dry weight of the bacterial enzyme and per milligram of protein for the animal enzyme. Application of Assay Method to Crude Tissue Preparations. The method used for the animal enzyme does not require optically clear solutions and can be used with homogenates. Addition of pyridoxal phosphate increases the activity of a liver homogenate about 25 %. A similar procedure can be used, if necessary, with the bacterial extracts. With crude animal and bacterial enzymes, a small proportion ( < 20 %) of the kynurenine disappearing does so through the kynurenine transaminase reaction, with the formation of kynurenic acid. ~3,18 This side reaction can be minimized by preliminary dialysis of the enzyme to remove a-keto acids and can be measured by the kynurenic acid formed, calculated from readings at 310 and 330 m~, the maxima of anthranilic and kynurenic acids, respectively23 Miller et al. ~7 and Wiss TM have demonstrated the separate existence of the bacterial and animal kynurenine transaminases. 16 I. L. Miller and E. A. Adelberg, J. Biol. Chem. 208) 691 (1953). 17 I. L. Miller, M. Tsuehida, and E. A. Adelberg, J. Biol. Chem. 203, 205 (1953). ~80. Wiss, Z. Naturforsch. 7b, 1933 (1952).

[32]

TRYPTOPHAN OXIDATION

251

Purification Procedures Bacterial Enzyme. The procedure is that of Hayaishi and Stanier, 14 and it has been checked by V. H. Auerbach. Pseudomonas fluorescens, strain Tr-23,19 is grown for 20 hours at 30 ° from a heavy inoculum in aerated flasks containing 8 1. of the following medium: 0.2% DL-tryptophan, 0.1% K2HPO4, 0.1% KH2PO4, 0.02% MgSO4"10H20, and 0.1% Difco yeast extract. The pH is adjusted to 7.0 before autoclaving. The cells are harvested by centrifugation in the cold and washed into a single cup with 0.02 M phosphate buffer, pH 7. They are washed and recentrifuged twice with this buffer. The yield of wet ceils is 7 to 11 g. Step 1. Preparation of Crude Extract. The paste of wet cells is ground diligently with three times its weight of dry alumina ~° in a chilled mortar for 7 to 10 minutes. The mixture takes on a moist, gel-like appearance as disintegration occurs. Five milliliters of 0.02 M phosphate, pH 7.0, is added per gram of wet cells, and the whole mixture is centrifuged hard to remove intact cells, debris, and alumina. Step 2. First Ammonium Sulfate Fractionation. To 100 ml. of the crude extract is added 21.0 g. of ammonium sulfate. The precipitate centrifuges off easily and is discarded. To the supernatant is added another 21.0 g. of ammonium sulfate. The precipitate which forms contains the enzyme and must be centrifuged hard. If the supernatant remains cloudy, it must be recentrifuged, and, if necessary, an additional 3.0 g. of ammonium sulfate can be added to cause a clean precipitation. The clear supernatant is discarded, and the precipitate is dissolved in 50 ml. of 0.02 M phosphate, pH 7.0. This solution can be kept frozen at - 10° for several weeks. Step 3. Acid Treatment. The solution of the enzyme at 0 ° is acidified to pH 5.4 with 3% (w/v) acetic acid added dropwise (ca. 7 ml.). The precipitate is discarded. Step 4. Reprecipitation with Ammonium Sulfate. To 50 ml. of the acid supernatant of step 3 is added 16.0 g. of ammonium sulfate, and the precipitate is discarded. The enzyme is then precipitated from the supernatant by an additional 3.5 g. of ammonium sulfate. The precipitate is dissolved in about 10 ml. of 0.02 M phosphate, pH 7.0. It can be dialyzed at 3 ° against water overnight, but it is generally less stable than the crude fractions. The enzyme at this stage is free of tryptophan peroxidase and pyrocatecase and does not oxidize anthranilic acid. It is not resolved from pyridoxal phosphate. 19 Available from R. Y. Stanier, Department of Bacteriology, University of California, Berkeley, California. so Levigated Alumina, Norton Company, Worcester, Massachusetts.

252

ENZYMES OF PROTEIN METABOLISM

[32]

Animal Enzyme. All procedures are carried out at 4 ° in a cold ~oom, following the m e t h o d of Knox.13 Step 1. Soluble Liver Fraction. R a t livers are disintegrated in 2 vol. of 0.14 M KC1 in a Waring blendor and centrifuged hard for 20 minutes. Step 2. pH 4.8 Supernatant. The soluble liver fraction is b r o u g h t to p H 4.8 at 0 ° with ca. 8 ml. of 0.5 M acetic acid per 100 ml. The precipitate is p r o m p t l y centrifuged off, and the supernatant neutralized to p H 6.5. Step 3. 45% Ethanol Precipitation. W i t h the enzyme in an effective cold bath, ethanol is added to a final concentration of 45% (v/v), keeping the t e m p e r a t u r e at - 5 ° , and the resulting precipitate dissolved in half the original volume of 0.14 M KC1. Step 4. Ammonium Sulfate Fractionation. One-half volume of saturated a m m o n i u m sulfate is added, and the precipitate is discarded. The supern a t a n t is made to 0.6 saturation with additional saturated a m m o n i u m sulfate and centrifuged. The precipitate is dissolved in a small a m o u n t of water and dialyzed against running water for 12 hours. The enzyme at this stage usually still contains some kynurenine transaminase but is almost completely free of acylpyruvase and has been resolved b y the a m m o n i u m sulfate t r e a t m e n t with respect to pyridoxal phosphate. The activity lasts only a few days when stored at 2 °. TABLE III SUMMARY OF PURIFICATION PROCEDURE

Liver kynureninase b

Bacterial kynureninase~

Step Soluble extract First ammonium sulfate fractionation Acid treatment Second ammonium sulfate fractionation

Total activity, micromoles/hr,

Specific activity, per mg. Yield dry wt. %

1378

0. 309

--

1180

0.748

86

840

1. 148

783

2. 140

Step Soluble extract

Acid precipiration Alcohol frac61 tionation Ammonium sulfate fractiona57 tion

Total activity, micromoles/hr,

Specific activity, per mg. Yield, protein %

258

0. 015

175

0.030

68

153

0. 074

59

126

0. 303

49

a O. Hayaishi and R. Y. Stanier, J. Biol. Chem. 195, 735 (1952). b W. E. Knox, Biochem. J. 53, 379 (1953).

[32]

TRYPTOPHAN OXIDATION

253

Properties Distribution. Dog, cat, pig, and beef, as well as rat, livers contain kynureninase activity. Strains of Pseudomonas with high and low kynurinase activities after adaptation to tryptophan are known. Both kynureninase ~I and kynurenine formamidase 2: have been isolated from Neurosl:ora crassa. Specificity. The animal enzyme splits L- but not D-kynurenine and presumably the L-form of DL-hydroxykynurenine and DL-(~-benzoyl)alanine, ~3 but not N"-acetylkynurenine. The bacterial enzyme has been tested only on L-kynurenine. Coenzyme. Both enzymes require pyridoxal phosphate. The animal enzyme is readily resolved and is best reactivated by preliminary incubations with the coenzyme. Inhibitors. Cyanide, hydroxylamine, semicarbazide, and other compounds reacting with the carbonyl group of the coenzyme inhibit both kynureninases. Experiments on kynureninase of Neurospora by W. B. Jakoby indicate that amino acids in general inhibit the enzyme. 21 p H and Substrate Concentrations. The bacterial and animal enzymes appear to have different pH optima (8.5 and 8.0, respectively) and different substrate affinities (K, = 3.9 × 10-5 and 4.0 X 10-~ M, respectively). However, the different conditions of the assays would tend to cause differences in this same direction. Biological Function. The bacterial kynureninase is adaptive. The liver enzyme does not adapt, although the enzyme preceding it metabolically, the tryptophan peroxidase, does adapt. In pyridoxine deficiency the liver enzyme activity decreases, although the protein moiety is preserved in nearly normal concentration. The reaction does not require the participation of a second, diketo acid-splitting enzyme,13 nor does it involve intermediate formation of free pyruvic acid. 1~ The mechanism of the reaction is still unknown but can be compared with the direct fission occurring in the tryptophanase reaction, which also requires pyridoxal phosphate.

21W. B. Jakoby and D. M. Bonner, J. Biol. Chem. 205, 699 (1953); Ibid. 205, 709 (1953). 22W. B. Jakoby, J. Biol. Chem. 207, 657 (1954). ~30. Wiss and H. Fuchs, Experientia 6, 472 (1950).

254

ENZYMES OF PROTEIN METABOLISM

[33]

[33] Methionine-Activating Enzyme, L i v e r B y G. L. CANTONI

Assay Method Principle. The reaction catalyzed by the methionine-activating enzyme 1 is described by the equation

GSH L-Methionine -t- A T P -

) S-Adenosylmethionine -~ 3 IP Mg++

The activity of the MAE, therefore, can be determined by measuring the amount of orthophosphate liberated from ATP in the presence and absence of methionine. ~,3 The exact mechanism underlying the formation of S-adenosylmethionine 1 is not known, and the equation is only a description of the over-all reaction. Clearly the fact that all three phosphates in ATP appear as orthophosphate concomitantly with the formation of one molecule of AMe must reflect the occurrence of hydrolytic breakdown of phosphorylated derivatives formed during the course of the activation reaction. The nature of the phosphorylated intermediates involved is not yet known. The method used for the determination of orthophosphate ~ does not differentiate between orthophosphate and labile phosphate compounds. However, even by the use of more sensitive techniques no evidence has as yet been found to suggest accumulation of acid-labile phosphate compounds. Reagents

L-Methionine, 0.1 M. Na~K~-ATP, 0.06 M (obtained by neutralization of the disodium salt with KOH). Tris(hydroxymethyl)aminomethanebuffer, 0.5 M, pH 7.4. (THAM was recrystallized from ethanol and Norit.) MgCl~, 1.0 M. The followingabbreviations will be used: MAE--methionine-activating enzyme; AMe--S-adenosylmethionine; THAM--tris(hydroxymethyl)aminomethane; ATP --adenosinetriphosphate; IP--orthophosphate, GSH--reduced glutathione; T CA-trichloroacetie acid. G. L. Cantoni, J. Biol. Chem. 189, 745 (1951). a G. L. Cantoni, J. Biol. Chem. 204, 403 (1953). K. Lohmannand L. Jendrassik, Biochem. Z. 178, 419 (1926).

[33]

METHIONINE-ACTIVATING ENZYME, LIVER

255

GSH, 25 mg./ml., neutralized immediately before use. Enzyme. Dilute with cold THAM buffer or KC1 (0.05 M) immediately before testing. Reaction mixture. ATP solution, 0.15 ml.; L-methionine solution or H~O, 0.1 ml.; buffer, 0.2 ml.; MgCl~, 0.25 ml.; GSH, 0.1 ml.; enzyme, 0.2 ml.

Procedure. The reaction was started by the addition of the cold enzyme dilution to the reaction mixture at 37°; after 30 minutes of incubation, 1.0 ml. of 10% TCA was added and orthophosphate determined 3 on a suitable aliquot of the protein-free filtrate (0.1 to 0.5 ml.). Units and Specific Activity. One unit of enzyme was defined as that amount which in the presence of methionine caused an increased formation of 3 micromoles of orthophosphate from ATP in 30 minutes. Specific activity was expressed as units per milligram of protein. Protein was determined by the spectrophotometric procedure of Warburg ~ and by the Weichselbaum method. ~ Purification Procedure Rabbits of medium size and of various breeds, most frequently albino, were used, in most cases some 15 to 20 hours after the last feeding. Two rabbits can be used most conveniently for one preparation. The animals were anesthetized by intravenous injection of a 10% solution of Na seconal (0.4 to 0.5 ml./kg, of body weight). The animals were bled by cutting the carotids, and the livers were removed and placed in ice. Step 1. Extraction and Ammonium Sulfate Fractionation. The livers were cut into small pieces and homogenized in a Waring blendor with 2.5 vol. of cold 0.01 N acetic acid. This and subsequent operations were carried out in a cold room maintained at 2°. The suspension was centrifuged for 30 minutes in a Servall at top speed (11,000 to 12,000 r.p.m.). The supernatant was decanted into a graduated cylinder through a funnel containing a small glass wool plug designed to retain fat particles. The volume of the extract was noted, and the preparation transferred to a beaker packed in ice and gently stirred mechanically until the temperature had fallen to 2 t o 4 °. (The temperature may rise to 12 to 15° during centrifugation.) When the desired temperature had been reached, a solution of ammonium sulfate saturated at 2 ° (56 ml. per 100 ml. of enzyme solution) was added slowly with continuous mechanical stirring, the mixture was allowed to stand with gentle 50. Warburg and W. Christian, Biochem. Z. 310, 384 (1941-42). 6 T. E. Weichselbaum, Am. J. Clin. Path., Tech. Sect. 10, 40 (1946); see also Vol. I H [731.

256

ENZYMES OF PROTEIN METABOLISM

[33]

agitation for 20 minutes, and then centrifuged in a Servall for 10 minutes at 10,000 r.p.m. The supernatant was decanted into a clean beaker, and an additional 36 ml. of the ammonium sulfate solution was added for each 100 ml. of the enzyme preparation (initial volume). After an interval as above, the precipitate was collected by centrifugation. The supernatant was discarded, and the tubes were allowed to drain for a few minutes in the cold room. When stored at - 2 0 °, the ammonium sulfate paste is stable for several weeks, or even months, and may be used after dialysis against 0.05 M phosphate buffer, pH 6.4. The specific activity of the dialyzed ammonium sulfate fraction varied somewhat in different preparations but was generally around 0.3.

Further Purification Studies on the purification of the methionine-activating enzyme beyond step 1 are in progress in this laboratory, but they have not advanced sufficiently to permit description of a standardized purification procedure. However, it may be stated that the methionine-activating enzyme has been purified further by isoelectrie precipitation at pH 5.2, adsorption on and elution from calcium phosphate gel, and a second ammonium sulfate fractionation. The highest specific activity reached so far has been in the neighborhood of 3, representing a purification of at least thirtyfold over the initial extract. The isoelectric precipitation step resulted in the separation of the methionine-aetivating enzyme into two protein fractions, one of which, the supernatant, was devoid of activity by itself but was capable of increasing the activity of the other fraction which presumably contains the methionine-aetivating enzyme proper. The activity of the supernatant fraction could be replaced by crystalline yeast pyrophosphatase. It has not yet been definitely established whether pyrophosphatase activity represents the only enzymatic factor contributing to the activating effect of the supernatant fraction for the methionineactivating enzyme.

Properties The methionine-activating enzyme was found to require -~SH groups for optimal activity. 2 Attention might be drawn to the exceptionally high requirement for Mg ++. The significance of this is not clear, and perhaps it will be clarified in the course of studies currently in progress on the mechanisms of the activation reaction. The requirement for Mg ++ was absolute, and NIn ++ would not replace Mg ++ as activator. The pH optimum of the reaction was around 7.4. In addition to compounds capable of oxidizing or binding - - S H groups, the enzyme was inhibited by fluoride.

[34]

METHYL ACCEPTOR SYSTEMS

[34] M e t h y l

257

Acceptor Systems

A. Nicotinamide M e t h y l p h e r a s e , Liver B y G. L. CANTONI

Assay M e t h o d Principle. Nicotinamide methylpherase activity was d e t e r m i n e d b y m e a s u r e m e n t of the rate of N~-methylnicotinamide 1 synthesis f r o m S-adenosylmethionine 1 and nicotinamide. F o r the determination of N M e N the sensitive fluorimetric m e t h o d of Huff 2 was used, with minor modifi cations. 3 Reagents

A M e (0.01 M). The preparation of A M e is described in Vol. I I I of this treatise. 4 Nicotinamide, 0.05 M. N a acetate buffer, p H 5.0, 0.25 M. Enzyme. Dilute with cold buffer (0.05 M) before use. Reaction mixture. 0.1 ml. of AMe, 0.1 ml. of nicotinamide, 0.1 ml. of buffer, and 0.2 ml. of enzyme dilution. Procedure. T h e chilled enzyme preparation was added to the reaction mixture at room temperature. T h e tubes were mixed and immersed in a water b a t h at 38 °. T h e reaction was t e r m i n a t e d after a 60-minute incubation p e r o d b y addition of 5 vol. of T C A and N M e N determined on an aliquot of the protein-free filtrate after suitable dilution. Units and Specific Activity. One unit of the enzyme was defined as t h a t a m o u n t which causes the synthesis of 1 ~, of N M e N in 60 minutes. Specific activity was expressed as units per milligram of protein. Protein was determined b y the spectrophotometric procedure of W a r b u r g 5 and b y the Weichselbaum method.6

1 The following abbreviations will be used: NMeN--Nl-methylnicotinamide; AMe-S-adenosylmethionine; TCA--trichloroacetic acid; GA--guanidinoacetic acid or guanidinoacetate; GSH--reduced glutathione. 2 j. W. Huff, J. Biol. Chem. 167, 151 (1947). 8 G. L. Cantoni, J. Biol. Chem. 169, 203 (1951). 4 G. L. Cantoni, Vol. III [85]. 50. Warburg and W. Christian, Biochem. Z. al0, 384 (1941-42). 6 T. E. Weichselbaum, Am. J. Clin. Pathol. 16 (1946); Tech. Bull. 10, 40 (1946); see also Vol. III [73].

258

ENZYMES OF PROTEIN METABOLISM

[34]

Purification Procedure Adult albino rats were killed by decapitation, the liver quickly removed, rinsed free of excess blood, and chilled in ice. It was found convenient to use 12 to 18 rats for one preparation. Step 1. Extraction and Heat Treatment. For extraction of the enzyme the livers were weighed and homogenized in the cold room in a Waring blendor, with 3.0 vol. of cold buffer (sodium acetate, 0.1 M, pH 5.0). Without delay the homogenate was centrifuged for 15 to 20 minutes at 11,000 r.p.m, in the cold. The clear supernatant was collected, and with the aid of the glass electrode the solution was adjusted to pH 5.0 by means of dropwise addition of cold acetic acid (1.0 M). A small precipitate which formed on acidification was disregarded, and the solution was transferred to a '.arge stainless steel beaker. The preparation was then rapidly heated by swirling in a 52 ° water bath until the temperature rose to 48 to 50° , but not higher. With suitable equipment this temperature may be reached within 2 minutes. The preparation was kept at 48 to 50 ° for 5 minutes, then chilled in an ice bath and centrifuged in the cold for 10 minutes at 10,000 r.p.m, to remove the denatured protein precipitate. The supernatant was used for the next step. Step ~. Ammonium Sulfate Fractionation and Dialysis. To the supernatant obtained above, solid ammonium sulfate (24 g. per 100 ml.) was added slowly with stirring while the solution was kept in an ice bath. The mixture was kept at 0 ° for 15 minutes after the addition of ammonium sulfate, and the precipitate removed by centrifugation in the cold. To the supernatant, which contained most of the enzyme activity, ammonium sulfate (15.5 g. per 100 ml.) starting volume was added as above. The precipitate was collected by centrifugation, and the supernatant discarded. The precipitate was then dissolved in 0.05 M Na acetate and dialyzed against large volumes of 0.05 M Na acetate buffer, pH 5.0, until addition of BaC12 to the dialyzate did not result in formation of a precipitate of BaSO4. The preparation may be interrupted either before or after dialysis; for storage it is best to keep the ammonium sulfate precipitate at - 2 0 ° ; on the other hand the enzyme preparation after dialysis can be kept in the cold room. Step 3. Absorption on and Elution from Ca Phosphate Gel. The dialyzed preparation was diluted with an equal volume of distilled water, and the protein concentration adjusted to 8 to 9 mg./ml, by further dilution with 0.025 M Na acetate buffer, pH 5.0. Next, calcium phosphate gel was added (7.0 rag. of dry weight per 9 rag. of protein), and the suspension centrifuged. The supernatant was discarded, and the gel eluted with 0.02 M phosphate buffer, pH 6.8, using a volume of buffer equal to onehalf the volume of the enzyme preparation before addition of the gel. The

[34]

METHYL ACCEPTOR SYSTEMS

259

elution was r e p e a t e d once w i t h t h e s a m e buffer a n d twice m o r e w i t h 0.04 M p h o s p h a t e buffer of t h e same pH. T h e a c t i v i t y of t h e eluates was determined, a n d those h a v i n g the highest specific a c t i v i t y (usually t h e first three) were pooled a n d were used for the next step. T h e eluates were stable for several d a y s w h e n stored at 2 ° in t h e cold room. Step 4. A m m o n i u m Sulfate Fractionation. 7 This step did not cause a n y substantial increase in specific a c t i v i t y b u t was useful for c o n c e n t r a t i n g the e n z y m e . T h e p H of the eluate pool was a d j u s t e d to 5.0, a n d solid a m m o n i u m sulfate was a d d e d with stirring in t h e cold as in step 2. A first f r a c t i o n h a v i n g specific a c t i v i t y lower t h a n the s t a r t i n g m a t e r i a l was collected after addition of 28 g. of a m m o n i u m sulfate per 100 ml. A second fraction was collected after addition of 18 g. of a m m o n i u m sulfate to the s u p e r n a t a n t . T h e s u p e r n a t a n t , which still contained some activity, was discarded. A r e p r e s e n t a t i v e r u n is described in T a b l e I. TABLE I PREPARATION OF NICOTINAMIDE METHYLPHERASE FROM

R.aT LIVER

Specific activity, units/mg. Yield, Unitsa/ml. protein % Acetate buffer extract After heat treatment b Ammonium sulfate ppt. (33-55 % saturated) c Ca phosphate gel treatment supernatant Eluate 1 Eluate 2 Eluate 3 Ammonium sulfate ppt. of eluates 1, 2, and 3 (40-66 % saturated) b

96 102 748 21.8 98 49 31 600

2.8 4.16 15.7

100 85 68

9.2 71 43.5 36.6

14 24 20 8

60

25

1 unit -- 1 -y of NMeN formed in 60 minutes at 38 °. b 5 minutes at 49 °. c Percentages are on the basis that addition of 70 g. of AS to 100 ml. will result in a saturated solution; see also Vol. I [10]. Properties T h e curve relating nicotinamide m e t h y l p h e r a s e a c t i v i t y to p H s h o w e d a p l a t e a u with a m a x i m u m at 5.1 and a slow decrease to 7.5. M g ++ a n d - - S H c o m p o u n d s were n o t required for o p t i m a l activity, a n d no a c t i v a tors h a v e been f o u n d so far. On theoretical g r o u n d s 8 it h a d been expected 7 This step is still under investigation since it has not been uniformly satisfactory. 8 G. L. Cantoni, in "Phosphorus Metabolism" (McElroy and Glass, eds.), Vol. 2, p. 129, Johns Hopkins Press, Baltimore, 1952.

260

[34]

ENZYMES OF PROTEIN METABOLISM

that the methylation of nicotinamide by AMe would show reversibility. However, efforts to obtain experimental evidence to support this postulate have so far been unsuccessful. Experiments on this point and other properties of the enzyme are continuing in this laboratory.

B. Guanidinoacetate Methylpherase, Liver

By G. L. CANTONI and P. J.

VIGNOS,

JR.

Assay Method

Principle. The enzyme activity was determined by measurement of the rate of creatine synthesis from S-adenosylmethionine1 and guanidinoacetic acid 1in the presence of GSH or other - - S H compounds. The determination of creatine was based on its conversion to creatinine and measurement of the latter by Borsook's modification9 of the alkaline picrate method of Folin. 1° Reagents AMe, 0.025 M. The preparation of AMe is described in Vol. I I I of this treatise. ~ GA (recrystallized from hot water), 0.015 M. GSH (25 mg./ml.), neutralized immediately before use. Tris(hydroxymethyl)aminomethane buffer, pH 7.4, 0.25 M. Enzyme. Dilute with cold buffer (0.05 M) immediately before use. Reaction mixture. 0.1 ml. of AMe solution, 0.2 ml. of GA solution, 0.1 ml. of GSH solution, 0.2 ml. of buffer solution, and 0.4 ml. of enzyme, properly diluted.

Procedure. The reaction was started by addition of the cold enzyme preparation to the reaction mixture at room temperature. After mixing, incubation was continued for 120 minutes at 37 °. Suitable controls (no AN[e, no GA, no enzyme) were run simultaneously with each assay. The reaction was terminated by the addition of 1.0 ml. of 10% TCA, and for creatine determination 1.0 ml. of the protein-free filtrate was used. Units and Specific Activity. One unit of the enzyme was defined as that amount which causes the synthesis of 1 micromole of creatine in 120 minutes. Specific activity was expressed as units per milligram of protein. Protein was determined by the spectrophotometric procedure of Warburg 5 and by the Weichselbaum method. 6 9 H. Borsook, J. Biol. Chem. 110, 481 (1935). 10 O. Folin, J. Biol. Chem. 17, 475 (1914).

[3~

METHYL ACCEPTOR SYSTEMS

261

Purification Procedure Pig liver was obtained at the slaughterhouse and brought to the laboratory packed in ice. All manipulations were carried out in a cold room maintained at 2° . Step 1. Extraction and Collection of Ammonium Sulfate Fraction. The liver was diced, rinsed free of excess blood with a buffer solution (sodium acetate 0.075 M, pH 5.0), weighed, and homogenized in a Waring blendor with 2.5 vol. of the same buffer solution. Next the homogenate was centrifuged at 9000 r.p.m, for 30 minutes. The supernatant, which was slightly opalescent, was packed in ice, and solid ammonium sulfate was added slowly with mechanical stirring (19.5 g. per 100 ml.). The precipitate was removed by centrifugation in a Servall high-speed centrifuge and discarded, and ammonium sulfate (10.5 g. per 100 ml.) was added to the supernatant. The precipitate collected as above contained essentially all the activity. Step 2. Dialysis, Absorption, and Elution from Alumina C~. The ammonium sulfate paste was dissolved in a small volume of 0.10 M Na acetate and dialyzed for 3 hours against running 0.05 M acetate buffer, pH 5.6. At the end of the dialysis an inactive precipitate was removed by centrifugation. The protein content of the supernatant material was then determined, and the protein concentration was adjusted by dilution with the same acetate buffer to 20 mg./ml. ; 0.33 vol. of alumina C~ (dry weight, 35 mg./ml.) 11 was added, with good mechanical stirring, the suspension was centrifuged at 3000 r.p.m., and the supernatant discarded. The residue was eluted four times with 0.0125 M phosphate buffer, pH 6.35, using each time a volume of buffer equal to the volume of the alumina C~ suspension. The eluates are not stable and whenever possible were fractionated on the same day as described in step 3. Step 3. Ammonium Sulfate Fractionation. The eluates having the highest specific activity, usually the first two, were pooled, the pH of the solution was adjusted to 7.2 with dilute NaOH, and then buffered at this pH by addition of 0.05 vol. of 2 M phosphate buffer, pH 7.2. Next saturated ammonium sulfate, pH 7.2, was added to 47.5% saturation, and the inert precipitate removed at high-speed centrifugation as above. Solid ammonium sulfate was added slowly with stirring to the supernatant (1 g. for each 10 ml.), and after 30 minutes the precipitate was collected by centrifugation. The precipitate was dissolved in dilute phosphate buffer (pH 7.4) and used directly, or, if more convenient, dialyzed against 0.05 M KC1 or 0.025 M phosphate buffer, pH 7.4, for ~1 See Vol. I [11].

262

ENZYMES OF PROTEIN METABOLISM

[34]

TABLE I112 PREPARATION" OF GUANIDINOACETATE METHYLPHERASE FROM PIG

LIVER

Specific activity, units/mg. Yield, Units"/ml. protein % Acetate buffer extract Ammonium sulfate ppt. I (25-40 % saturated) Treatment with alumina C~ Supernatant Eluate 1 Eluate 2 Eluate 3 Ammonium sulfate ppt. II (48-65 % saturated)

1.1 3.2

0. 034 0.13

0.56 2.76 2.38 0.83 15.5

0.14 1.6 1.7 1.66 2.6

100 90 19.5 28.7 25.6 9 34.5

a 1 unit = 1 micromole of creatine formed in 120 minutes at 37°. 3 hours. T h e precipitate was stable when k e p t at - 20 °. T h e results f r o m a representative run are presented in T a b l e I I . 12 T o prepare larger a m o u n t s of the enzyme, step 1 can be replaced b y the following procedure 13 whereby as m u c h as 4 pounds of liver can be processed conveniently in 1 day. T h e liver was homogenized with 4 vol. of sodium acetate buffer (0.075 M, p H 5.6). N e x t an a m o u n t of Celite equivalent to the a m o u n t of liver used was stirred gradually into the homogenate, and the suspension was filtered b y means of a pressure filter, 14 using filter p a d size 0. Solid a m m o n i u m sulfate (17.5 g. per 100 ml.) was added to the clear filtrate as above, and, after 20 minutes at 2 °, the suspension was filtered with the aid of 5 g. of Celite per 100 ml. (filter p a d size 2). T h e concentration of a m m o n i u m sulfate was raised b y the addition of 10.5 g. per 100 ml., and, after suitable incubation at 2 ° as above, the suspension was filtered with the aid of Celite as above, through a sheet of W h a t m a n p a p e r (No. 50) backed b y a filter p a d size 0. T h e filter cake was dissolved in 0.1 M N a acetate, and the Celite r e m o v e d b y centrifugation in a Servall centrifuge; the enzyme was then precipitated f r o m the clear s u p e r n a t a n t b y m e a n s of a m m o n i u m sulfate (32 g. per 100 ml.). T h e precipitate collected b y centrifugation was then stored in the deep-freeze and purified further as described in steps 2 and 3. Properties

T h e p H o p t i m u m for the reaction was found to be around 7.5; phos~ phate, bicarbonate/CO2, and t r i s ( h y d r o x y m e t h y l ) - a m i n o m e t h a n e can be 1~Reprinted from G. L. Cantoni and P. J. Vignos, Jr., J. Biol. Chem. in press. ~8This step was developed in collaboration with Dr. Eduardo Scarano. 14F. R. Hormann & Co., Newark, N. J.

[35]

GLUTAMYLTRANSFERASE (PLANTS)

263

used to buffer the reaction mixture. Adeninethiomethylpentose and methionine methylsufonium iodide were tested for their ability to function as methyl donor in this system, since they are chemically related to AMe; only AMe was active as a methyl donor. In the presence of an excess of the acceptor, GA, the transfer of the methyl group of S-adenosylmethionine appears to go to completion as indicated by the stoichiometric relationship between the amount of substrate furnished and the amount of creatine formed. Likewise in the presence of an excess of the methyl donor, all the guanidinoacetate supplied can be methylated to creatine. Reducing compounds such as glutathione, cysteine, BAL, and cyanide increased the activity of the enzyme. The activation by - - S H and other reducing compounds became more pronounced as the degree of purification of enzyme increased. Thus, the activity of crude liver homogenates was not increased by the addition of - - S H reagents; there was a moderate activation of the initial ammonium sulfate fraction and a very marked activation of the alumina C~ eluates, or the ammonium sulfate fractions obtained from them. Ca ++ or Mg ++ is not required in the reaction catalyzed by GA methylpherase. Sodium arsenate, sodium fluoride, folic acid, and citrovorum factor have no effect on the activity of the enzyme.

[35] ~,-Glutamyltransferase (Plants) Glutamine + ~H2OH--~ Glutamylhydroxamic Acid + NH3

By P. K. STUMPF Assay Method Reagents 0.01 M phosphate buffer, pH 6.5. 0.01 M manganese sulfate. 0.01 M ATP, pH 6.5; or 0.001 M ADP, pH 6.5. 0.1 M glutamine made up freshly. M NH2OH.HC1 made up freshly to pH 6.5.

Procedure. Each test tube contains 0.25 ml. of 0.01 M phosphate buffer, 0.1 ml. of 0.01 M manganese sulfate, 0.1 ml. of 0.01 M ATP, 1.0 ml. of L-glutamine, 0.1 ml. of 1 M NH2OH, enzyme diluted to a concentration which will form not more than 3 micromoles of hydroxamic acid under conditions of test, and water to a final volume of 2.25 ml. After incubation for 20 minutes at 30 °, 1 ml. of 10% TCA and 1 ml. of 0.5% alcoholic ferric chloride in 0.1 N HC1 are added to the reaction mixture.

264

ENZYMES OF PROTEIN METABOLISM

[35]

After 5 minutes the suspension is diluted to 10 ml., filtered, and the color intensity measured with the Klett-Summerson colorimeter employing the 540-mg filter. Definition of Unit. Elliott 1 has defined a unit of glutamyl transferase activity as that amount of enzyme required to produce under standard conditions 1.15 micromoles of glutamylhydroxamic acid in 20 minutes at 30 °. Stumpf et al. 2 have shown that when arsenate replaces phosphate in the reaction mixture, a three- to fivefold increase in activity is consistently observed. Thus the sensitivity of any test system would be greatly increased by substituting arsenate buffer for phosphate. Application of Assay Method to Crude Tissue Preparations. For distribution studies, acetone powders of plant tissue prepared by conventional techniques 2 are suspended in 0.01 M arsenate buffer, pH 6.5, in a test tube, placed in a vacuum desiccator, and evacuated in order to displace air pockets which interfere with complete contact of solvent with acetone powder. After 15 minutes the vacuum is released and the suspension is tested directly as described in Procedure. No attempt is made to separate the soluble from the insoluble material, since in some instances the enzyme is, in part, associated with insoluble material. Purification Procedure

A simplified two-step purification procedure for pumpkin glutamyl transferase has been described by Stumpf et al. ~ This preparation has low glutamine synthetase activity. The procedure described here is essentially that developed by Elliott, 1 who achieved a 2000-fold purification of the transferase, the starting material being pea meal. The final preparation appears to have a dual function, possessing both glutamine synthetase and glutamyltransferase which cannot be separated. Step 1. Extraction. Dry green pea seeds (dwarf Blue Bantam variety) were purchased locally and pulverized to a fine powder in a powerdriven hammer mill. Eighteen kilograms of the powder was stirred for 30 minutes with 144 1. of cold 0.1 M NaHCO3. Next, 3.7 1. (0.05 vol.) of 2 M MgSO4 was stirred in, and the precipitate was allowed to settle at 0 ° overnight. The supernatant fluid was poured off as cleanly as possible, and the remaining suspension was centrifuged on a centrifugal separator. The two supernatant fluids were combined. To avoid the centrifuging of large volumes, the suspension remaining after decantation can be rejected with a loss of only about 25% of the enzyme. Volume of extract, 109 1.; protein, 26 mg./ml. ; transferase, 2.0 units/ml. l W. H. Elliott, J. Biol. Chem. 201, 661 (1953). 2 p. K. Stumpf, W. D. Loomis, and C. Michelson, Arch. Biochem. 30, 126 (1951).

[35]

GLUTAMYLTRANSFERASE (PLANTS)

265

The treatment with MgS04 removes gummy material which otherwise prevents satisfactory fractionation of the enzyme. Step 2. Fractionation with Ammonium Sulfate. The extract was adjusted to pH 6.5 by the addition of 2 M KH~PO4, and 300 g. of solid ammonium sulfate was added per liter of extract. The precipitate was allowed to settle overnight at 0 °, and the supernatant fluid poured off and discarded. The precipitate was collected from the remaining suspension by centrifuging and resuspended in about 6 1. of cold distilled water. The thick suspension was brought to pH 7.2 with K2HPO4, put into cellophane tubes, and dialyzed against two changes of 40 1. of cold distilled water for about 36 hours. A small sample of the cleudy dialyzate was centrifuged for enzyme assay; the main bulk was carried onto the next stage without centrifuging. Volume of extract, 8.7 1.; protein, 47 mg./ml.; transferase, 11.0 units/ml. Step 3. Treatment with Protamine. The cloudy dialyzed extract was treated with a solution of 2% protamine sulfate until a small sample, after centrifugation, gave no further precipitate on addition of a drop of protamine solution. About 2 1. was required. The bulky inactive precipitate was centrifuged, and the supernatant fluid retained. Volume of extract, 10 1.; protein, 23 mg./ml.; transferase, 9.1 units/ml. The protamine treatment apparently removes an inhibitor from the extract, for an increase in total activity is usually obtained by this procedure. Step ~. Nucleic Acid Precipitation. The enzyme is completely precipitated around pH 5 by small amounts of yeast nucleic acid. One-liter aliquots of the extract were treated at 0 ° with 10 ml. of M acetic acid (to pH 5.1), followed by 60 ml. of 2% potassium nucleate solution (pH 5.5). The precipitate was centrifuged at 0 °, suspended in a minimal volume of cold distilled water, neutralized to pH 7.3 of M K2HP04, extracted for 15 minutes, and the suspension centrifuged. The precipitate was washed with cold dilute K2HPO4, and, after centrifugation, the two milky supernatant fluids were combined. Trial fractionation is performed for each batch of enzyme to determine the amount of nucleic acid required. Volume of extract, 400 ml.; protein, 40 mg./ml.; transferase, 187 units/ml. Step 5. Second Ammonium Sulfate Fractionation. Four hundred milliliters of the enzyme solution was mixed with 16 ml. of M phosphate buffer, pH 7.4, followed by 288 ml. of saturated ammonium sulfate solution. The enzyme and ammonium sulfate solutions had been previously cooled to 0 °. Mter standing for 15 minutes, the bulky inactive precipitate was removed by centrifugation, and a further 240 ml. of the saturated ammonium sulfate solution was added to the supernatant solution. After 20 minutes the precipitate was centrifuged and redissolved in cold water,

266

ENZYMES OF PROTEIN METABOLISM

[35]

with the addition of a few drops of M K2HP04 to pH 7.3. Volume of extract, 64 ml. ; protein, 18 mg./ml. ; transferase, 700 units/ml. Step 6. Dialysis. The solution was dialyzed with stirring against three changes of 4 1. of cold distilled water. The white precipitate was removed by centrifugation, and the supernatant fluid was collected. Volume of extract, 75 ml. ;protein, 6.1 mg./ml. ; transferase, 453 units/ml. Step 7. Second Nucleic Acid Precipitation. Seventy milliliters of the enzyme solution was treated with 6.7 ml. of 1% nucleic acid solution, followed by 1.3 ml. of 0.2 M acetic acid. The precipitate was collected and redissolved in dilute phosphate buffer, pH 7.3. As in the previous nucleic acid fractionation, a trial fractionation was carried out for each batch of enzyme by treating a small sample of enzyme with nucleic acid and adding graded amounts of acetic acid. At each stage the precipitate was collected by centrifugation and assayed for enzyme content. Volume of extract, 20 ml. ; protein, 8 mg./ml.; transferase, 1200 units/ml. The purified enzyme contains more than a single protein component. Electrophoretic studies on the enzyme solution at pH 8.3 in Veronal buffer showed the presence of one main peak and two small subsidiary ones. In the ultracentrifuge at pH 7.3 in phosphate buffer, two approximately equal peaks were present. By appropriate reduction in reagent volume, a smaller quantity of enzyme may be prepared by this procedure. SUMMARY OF PURIFICATION PROCEDURE

Step 1. 2. 3. 4. 5. 6. 7.

Extraction First ammonium sulfate precipitation Protamine First nucleic acid precipitation Second ammonium sulfate precipitation Dialysis Second nucleic acid precipitation

Purification, units

Yield, %

1 2.3 4.1 45 380 750 1570

(100) 30 37 27 16 13 11

Properties Specificity. Purified pumpkin glutamyltransferase (PGT) will react with only L-glutamine. A large number of amides, including asparagine, have been tested with negative results. 2 Activators and Inhibitors. Unlike the pea enzyme, PGT is activated only by Mn ++. Mg ++ is inert in the system. ADP in a final concentration of 10-s M will fully activate the system. AMP and ATP free of ADP are

[36]

GLUTAMOTRANSFERASE

267

inert. Arsenate will increase the rate of enzyme action some three- to fivefold above that of the phosphate-activated system. Fluoride at a concentration of 10-5 M gives complete inhibition. Iodoacetate, cyanide, diisopropylfluorophosphate, azide, dinitrophenol, and malonate at 5 × 10-3 M are ineffective. The enzyme is remarkably stable. PGT preparations have been stored at - 5 ° for a period exceeding two years with no appreciable loss in activity. Exposure to 55 ° for 5 minutes at pH 7 will cause complete and irreversible inactivation. Preparation of Glutamine (CONISH~). Since plant glutamyltransferase catalyzes the transfer of the glutamyl moiety to NH~ or to NH~OH, the enzyme can be employed for the preparation of glutamine (CON15H~). The conditions for the transfer reaction are described by Delwiche et al. 3 Labeled glutamine may be isolated from the reaction mixture by conventional procedures and purified by repeated crystallization from ethanolwater solvent. 4 3 C. C. Delwiche, W. D. Loomis,and P. K. Stumpf, Arch. Biochem. and Biophys. 33, 333 (1951). 4 H. B. Vickery and G. W. Pucher, Biochem. Preparations 1, 44 (1949).

[36] G l u t a m o t r a n s f e r a s e

(7-Glutamyltransferase) ~

B y HEINRICH WAELSCH

Two groups of glutamotransferases are known. Both groups catalyze the reaction Glutamine + hydroxylamine --~ Glutamohydroxamic acid + ammonia One group of transferases (GTF) has been found as yet only in microorganisms. These enzymes are highly active without the addition of metals or nucleotides, and they are not associated with glutamine synthetase. The other group of transferases (GTF~n) has been found not only in plants and animals but also in microorganisms (Proteus vulgaris). These transferases are active only in the presence of metals, phosphate (or arsenate), and nucleotides. They appear to be associated closely with glutamine synthetase activity.

Assay Method Principle. The method is based on the color reaction of enzymatically formed glutamohydroxamic (GHA) acid with ferric chloride as used by ~N. Grossowicz, E. Wainfan, E. Borek, and H. Waelsch, J. Biol. Chem. 187, 111 (1950).

268

ENZYMES OF PROTEIN METABOLISM

[36]

Lipmann and Tuttle ~ for estimation of hydroxamic acids. Synthetic GHA 1,8 serves as standard. Definition of Unit and Specific Activity. One micromole of GHA formed per hour equals one unit of enzyme activity. Specific activity is defined as number of micromoles of GHA formed per hour per milligram of protein. Bacterial GTF

Procedure. Two milliliters contain 20 micromoles each of glutamine and hydroxylamine (hydroxylamine buffers, adjusted to pH 8), and enzyme solution. After incubation at 35 °, 1.5 ml. of a solution containing 0.5 ml. of 15% TCA, 0.5 ml. of 2.5 N HC1, and 0.5 ml. of 5% ferric chloride in 0.1 N HC1 is added. After centrifugation the optical density of the supernatant solution is measured at 500 m~ against a reagent blank. Preparation of a Cell-Free Enzyme Extract from P. vulgaris. 1 In order to obtain large quantities of P. vulgaris X-19, liquid media are employed. The medium contains, per liter, 7.5 g. of Difco Bacto-casamino acid, 0.5 g. of ammonium sulfate, 0.5 g. of ammonium chloride, 1 g. of potassium nitrate, 3 mg. of ferric citrate, 5 g. of K H 2 P Q , 50 mg. of M g S Q . 7H20, i mg. of nicotinamide, 2.5 ml. of a 0.02% solution of phenol red, and 0.1 ml. of a mixture of equal parts of tributyl citrate and methyl benzoate. The pH is adjusted to 6.8 with 10 N NaOH. Usually 15 1. of medium is employed in one run. The medium is sterilized by autoclaving. A small inoculum of the organism grown on agar is transferred to the medium, and after 18 hours of incubation at 35 °, while growth is still limited, 25 ml. of a sterile solution of 50% glucose is added per liter of medium and a filtered stream of oxygen, containing 5% of CO2, is bubbled through the solution. The pH is kept constant by the repeated addition of 10 N NaOH. After 6 to 8 hours of additional incubation the cells are collected on a refrigerated Sharples centrifuge. The yield of dry cells is approximately 2 g./1. of medium. Because of the short period of incubation during which active multiplication takes place, the crop consists mainly of young cells. Preparation of Cell-Free Extracts. The harvest of wet cells is lyophilized, and the dry material is shaken with an equal volume of glass beads at a rate of 440 excursions per minute for 1 hour. The powder is suspended in water to yield a 5% suspension which is dialyzed for 48 hours against 40 vol. of distilled water at 4 °. The suspension is centriF. Lipmann and L. C. Tuttle, J. Biol. Chem. 159, 21 (1945). 3 The described method for chemical synthesis of GHA is laborious and gives the pure product in poor yield. A simplified method of synthesis has been developed (A. Neidle and H. Waelsch, in preparation).

[36]

GLUTAMOTRANSFERASE

269

fuged in a refrigerated centrifuge at 13,000 r.p.m. The slightly opalescent supernatant solution serves as the source of the enzyme. The total nitrogen content (Kjeldahl) of these extracts is 1 to 1.2 mg./ml, for a 5% suspension of the original bacterial powder. By repeated fractionation of the crude extract with ammonium sulfate a 250-fold purification with a loss of about 60 % of total activity is obtained. 4 The active fraction is found mainly between the limits of 65 to 80% ammonium sulfate saturation. The purified extract contains, in addition to GTF, aspartotransferase and asparaginase, but no glutaminase or glutamine synthetase. 4 Properties

Specificity. The enzyme does not act on acetamide, benzamide, glycinamide, or nicotinamide. ~ In place of hydroxylamine, hydrazine 5 or ammonia 6 may act as acceptor of the ~,-glutamyl radical. Inhibitors and Activators. 5 Amino acids and some amines inhibit to a varying degree enzymatic GHA formation, the former by competition with glutamine, the latter by competition with hydroxylamine. A free a-amino group is essential for the inhibition by amino acids. Cu ++ in concentration of M X 10-3 approximately doubles the activity of GTF. Ethanol, methanol, glycerol, and glucose also increase GTF activity considerably. Mn is not required for this enzyme. Effect of pH. ~ The enzyme exhibits optimal activity at pH 8 with a decrease to 50% activity at pH 5.8 and 9.3, respectively. K85 determined with crude extracts from P. vulgaris was found to be 5 to 7 )< 10-3 for glutamine, and 2.3 to 2.4 X 10-3 for hydroxylamine. Occurrence of GTF. The enzyme was found in Proteus vulgaris X-19, Aerobacter aerogenes, and Escherichia coli, but was not found in Shigella flexneri, Pseudomonas aeruginosa, Staphylococcus aureus, and Lactobacillus arabinosus. M n - D e p e n d e n t Glutamotransferase (GTFMn) 7

Procedure. Two milliliters contains 100 micromoles of acetate (pH 5.5), 40 micromoles of glutamine, 20 micromoles of hydroxylamine (pH 5.5), 10 micromoles each of manganous chloride and potassium chloride, 0.1 micromole of ATP, and enzyme solution. After incubation at 37 °, assay as for GTF. 4 N. Grossowicz and A. Miller, unpublished. M. Schou, N. Grossowicz, and H. Waelsch, J. Biol. Chem. 192, 187 (1951). s H. Waelsch, P. Owades, E. Borek, N. Grossowicz, and M. Schou, Arch. Biochem. 27, 237 (1950). 7 A. Lajtha, P. Mela, and H. Waelsch, J. Biol. Chem. 205~ 2 (1953).

270

ENZYMES OF PROTEIN METABOLISM

[86]

Purification of GTF,,o from Brain Cortex (Sheep). 7 One kilogram of brain cortex is homogenized in portions in a Waring blendor with a total of 3 1. of 0.06 M phosphate buffer (pH 7.5) for 30 seconds. The homogenate is stirred in the cold for 3 hours and centrifuged for 30 minutes. The precipitate is stirred with 1.5 1. of phosphate buffer for 3 hours and centrifuged. The pH of the combined extracts is adjusted to pH 6 with acetic acid and fractionated with ammonium sulfate at 20 % (FRo), 50% (Fs0), and 60% (Fe0) saturation. After each addition of the ammonium sulfate the mixture is kept in the cold for 3 hours and then centrifuged. The three protein precipitates are dissolved in small volumes of water and dialyzed against 0.02 M phosphate buffer (pH 7) for 18 hours. Fractions F20 and F6o are reprecipitated with ammonium sulfate, and the fractions obtained between 20 and 50% saturation combined with Fs0 after dialysis (F6o). F60 is adjusted to pH 6, fractionated at 20% (F~02), 25% (F~5~), 40% (F402), and 50% (Fs02) ammonium sulfate saturation and freed from salt by dialysis. F~02 is discarded; F~52 and Fs02 are combined and reprecipitated at 25 and 40% saturation, and the fraction obtained at 40% ammonium sulfate saturation is combined with F402 after dialysis (F~02). After adjustment to pH 6, F402 is fractionated at 28 and 36% (F368) ammonium sulfate saturation and freed from salt as described above. After adjustment to pH 6, F863 is treated with 24 ml. of calcium phosphate gel, the mixture kept for 1 hour and then centrifuged, and the supernatant solution again treated with the gel in the same manner. The procedure is repeated once more. The three portions of calcium phosphate gel are combined and stirred for 15 minutes with 80 ml. of 0.2 M phosphate buffer (pH 5.75), the suspension is centrifuged, and the supernatant fluid discarded. The washing of the gel is repeated twice in the same manner. The gel is suspended in 30 ml. of 0.2 M phosphate buffer (pH TABLE I SUMMARY OF PURIFICATION PROCEDURE

Fraction Homogenate Supernatant Ammonium sulfate, Ammonium sulfate, Ammonium sulfate, Calcium phosphate, Calcium phosphate,

F60 F4o2 F3aa Ca1 Ca~

Total volume, ml.

Protein, g.

GTFMn activity, units Total

Specific

2100 760 214 122 156 83 66

126 27 5.45 1.83 0.7 0.108 0.046

315,000 238,000 109,000 76,000 53,000 28,000 20,000

2.5 8.8 20.0 41.6 75.5 260.0 436.0

[36]

GLUTAMOTRANSFERASE

271

7.2), and the mixture is kept for 1 hour and centrifuged. The procedure is repeated twice, and the supernatant fluids are combined and adjusted to p H 6 (Ca1). Ca, is treated with calcium phosphate gel in the m a n n e r described above, care being taken to keep the temperature during all operations between 0 and -5°1 since the enzyme is rather unstable at this degree of purity (Ca2). Purification of GTF,~, from Pigeon Liver. 7 Acetone-dried powder (10 g.) is extracted with 100 ml. of 0.02 M phosphate buffer (pH 7.5), and after centrifugation of the suspension the supernatant fluid is used for purification. The purification is carried out as described for brain cortex, with the difference t h a t the refractionation of the side fractions is unnecessary, since a much greater portion of GTFMo is found in the main fraction (F36) than in the case of brain cortex. TABLE I[ SUMMARY OF PURIFICATION PROCEDURE

Fraction Supernatant Ammonium sulfate, Ammonium sulfate, Ammonium sulfate, Calcium phosphate, Calcium phosphate,

Fs0 F40~ F~6a Ca1 Ca2

Total volume, ml.

Protein, g.

GTFMn activity, units Total

Specific

100 36 31 10 5.7 15.1

3.4 1.07 0.24 0.087 0. 033 0. 013

44,000 30,000 19,000 11,000 8,000 5,000

13.0 28.0 79.0 126.0 242.0 388.0

Purification of GTF,~n from Proteus vulgaris. 7 A lyophilized powder (see above) (10 g.) of Proteus vulgaris is extracted with 200 ml. of phosphate buffer (0.01 M, p H 7) for 6 hours with dialysis against the same buffer. After centrifugation the supernatant fluid is adjusted to p H 6.5 TABLE III SUMMARY OF PURIFICATION PROCEDURE

Fraction Supernatant Ammonium sulfate, F40 Ammonium sulfate, F~ 2 Calcium phosphate, Ca1 Calcium phosphate, Ca~

Total volume, ml.

Protein, g.

GTFMn activity, units Total

Specific

200 50 2.7 3.3 1.3

2.0 0.89 0. 062 0.0105 0.0033

56 46 19 9 6.5

0. 028 0.052 0. 308 0. 855 1.96

272

E~ZVMES OF PROTEIN METABOLISM

[36]

and fractionated with ammonium sulfate between 20 and 40% (F40), and this fraction is refractionated between 20 and 36% ammonium sulfate saturation. F36 is subiected to two consecutive adsorptions on calcium phosphate gel. GTF is separated from GTFMn during the purification procedure.

Properties GTF~n from brain cortex has been most extensively studied, and most of the data refer to this enzyme system if not otherwise noted. In most respects GTF~n from pigeon liver and Proteus vulgaris behave in the same manner. Activators and Inhibitors. GTFMn has an absolute requirement for Mn ~+ salts (optical concentration 10-2 M). The optimal concentration of Mg ++ is in the same range, but this metal activates GTFMn much less than Mn ++. Co++ is more effective than Mg ++ but less so than Mn ++. The addition of phosphate (optimal concentration about 5 X 10-8 M) enhances the activity of GTFMn, arsenate being still more effective. The addition of ATP or ADP (optimal concentration 10-5 M) increases the activity of the enzyme, depending on its state of purification, two- to sevenfold. AMP is ineffective. GTF~n is strongly inhibited by ammonia and a-amino acids, an inhibition which is not overcome by hydroxylamine or ghtamine. Salts such as KC1 in high concentration inhibit GTF~,n activity. Pyrophosphate is a strong inhibitor, whereas hexa-, tri-, and metaphosphate are weak inhibitors of enzyme activity. pH Dependence. GTFM~ from brain, pigeon liver, and Proteus exhibits a pH optimum at 5.5. K~. The substrate enzyme dissociation constant for glutamine is 2.3 X 10-2 for GTFMn from brain. Increasing hydroxylamine concentrations inhibit enzyme activity. Purity of Enzyme Preparatien. The outstanding feature of the GTFM~ preparation from all sources is the association with glutamine synthetase which accompanies GTFMn from brain or pigeon liver at an approximately constant ratio of activity through all stages of purification.

[37]

CLEAVAGE OF AROMATIC RINGS

273

[371 Cleavage of Aromatic Rings with Eventual Formation of /~-Ketoadipic Acid

By R. Y. STANIER Introduction

Figure 1 shows the reaction sequences catalyzed b y the varied enzymes treated in this section. These reaction sequences occur, so far as is known, only in aerobic bacteria belonging to the genera Pseudomonas, ~ Vibrio, ~ and Mycobacterium2 Few of the enzymes have been studied in detail; some have not y e t been isolated. Those a b o u t which enough is known to merit specific discussion are shown in boldface in Fig. 1.

D(-)-Mandelic acid ~- i(+)-Mandelic acid Racemase

~ L-Mandelic dehydrogenase

Benzoylformic acid

Benzoylformic carboxylase

Tryptophan (via intermediates) L_~ Anthranilic acid

Benzaldehyde ~ Benzaldehyde Dehydrogenases Benzoic acid Phenol 1 I ~Catechol¢I Pyrocatechase cis,cis-Muconic acid Lactonizing enzyme

~,-Carboxymethyl-A"-butenolide J. Lactone-splitting enzyme "C2" + Succinic acid~------~-Ketoadipic acid (

p-Hydroxybenzoic acid ] Protocatechuic acid | Protocatechuic ~ oxidase /~-Carboxymuconic acid [

FIG. 1. Reaction sequences for aromatic oxidations which converge with formation of ~-ketoadipic acid. The enzymes discussed in this section are shown in boldface. The choice and t r e a t m e n t of the biological material used as a source of these enzymes requires special mention. Different strains of a given bacterial species m a y v a r y considerably in their ability to a t t a c k aromatic substrates, and hence careful strain selection is an essential preliminary to enzymatic studies. For example, most strains of Pseudomonas fluorescens can oxidize benzoic and p-hydroxybenzoic acids, but the ability to oxidize mandelic acid or phenol is relatively rare. 4 Furthermore, all the enzymes shown in Fig. 1 are more or less strictly inducible and are formed in appreciable concentrations only b y cells which have been exposed to the specific substrates. Hence the composition of the growth m e d i u m is of R. Y. Stanier, Bacteriol. Revs. 14, 179 (1950). W. C. Evans, Biochem. J. 41, 373 (1947). s W. H. Wagner, Biochem. Z. 322, 121 (1951). 4 R. Y. Stanier, J. Bacteriol. 55, 477 (1948).

274

ENZYMES OF PROTEIN METABOLISM

[37]

paramount importance in obtaining active starting material. 5 The growth medium should contain as principal carbon source either the substrate for the enzyme one wishes to isolate or a metabolic precursor of this substrate. Thus ceils of P. fluorescens with high mandelic racemase and L-mandelic dehydrogenase activities can be obtained only by growth on a medium containing mandelic acid, but cells with a high pyrocatechase activity can be obtained by growth on a medium containing any one of the following compounds: mandelic acid, benzoic acid, phenol, tryptophan, or anthranilic acid. These compounds are all metabolic precursors of catechol and thus cause indirect (sequential) induction of the catecholoxidizing enzyme, pyrocatechase. From the metabolic interrelationships shown in Fig. 1 one can infer the patterns of induction. All the isolation procedures described below employ suitably induced cells of Pseudomonas fluorescens as starting material. In order to conserve space, the common initial operations will be described here. Unless otherwise specified, they are conducted at 0 to 3 °. Cell Breakage. The methods of choice are: (1) grinding with alumina and (2) sonic oscillation. In method 1, wet, packed cells are ground by hand in a chilled mortar with twice their weight of levigated alumina s until cell breakage occurs (vigorous grinding for 3 to 4 minutes generally suffices). The mixture is slurried with cold phosphate buffer (0.02 M, pH 7.0), a suitable dilution being approximately 5 ml. of buffer per gram of wet cells. Residual intact cells and the bulk of the alumina are then removed by centrifugation for 15 minutes at 6000 r.p.m., yielding a crude extract. Such extracts are very viscous, owing to their content of highly polymerized DNA, and subsequent operations (particularly high-speed centrifugation) are greatly facilitated by treatment at this stage with DNAse. The extract is warmed to 30 ° and incubated for 10 minutes with crystalline DNAse (1 mg. per 100 ml. of extract) and MgS04 (at a final concentration of 0.01 M). The mixture is then immediately chilled again to 0 °. In method 2, wet, packed cells are resuspended in 0.02 M phosphate buffer at a concentration of 10% (w/v) and subjected to sonic oscillation until cell breakage is just complete, as determined by the absence of further appreciable decrement in optical density. With a Raytheon 9-kc. sonic oscillator operated at a plate voltage of 110 and a minimum output voltage, treatment for about 10 minutes is required when the cup contains 25 ml. of cell suspension. Coarse debris and any residual cells are removed by centrifugation for 15 minutes at 7000 X g, yielding a crude extract. Since sonic treatment partly depolymerizes DNA, such extracts are much 5 B. P. Sleeper, M. Tsuchida, a n d R. Y. Stanier, J. Bacteriol. 59, 129 (1950). 6 Purchased from N o r t o n Abrasives, Worcester 6, Massachusetts.

[37]

CLEAVAGE OF AROMATIC RINGS

275

less viscous than those prepared by grinding with alumina, and hence DNAse treatment can usually be omitted. Treatment of Crude Extracts. The enzymes in a crude extract are of two kinds: soluble and particulateY The particulate component carries the entire electron transport system of the bacterial cell together with certain substrate dehydrogenases, among them one of present concern, the L-mandelic dehydrogenase. All the other enzymes treated in this section are soluble. It is therefore in general desirable to separate the particulate and soluble fractions as a first step in purification. The bulk of the particulate fraction can be sedimented by centrifugation at high gravitational fields (20,000 × g or above) ; an exact specification cannot be given, since the rate of sedimentation of the particles is conditioned by the viscosity of the extract, as well as by the average particle size, which is smaller in sonic extract than in alumina extracts. The supernatant liquid after such high-speed centrifugation contains all the soluble enzymes of the extract, together with a small amount of very finely divided particulate material. This residual particulate material is completely removed from the soluble enzymes by addition of 0.5 vol. of saturated neutral ammonium sulfate, followed by ccntrifugation. The clear, straw-colored supernatant liquid is then saturated with solid ammonium sulfate, which precipitates all the soluble enzymes. This precipitate is resuspended in 0.02 M phosphate buffer (pH 7.0) and dialyzed overnight against the same buffer mixture. The resulting solution constitutes the crude soluble fraction. Protamine Treatment. Before further purification of the enzymes in the crude soluble fraction can be undertaken, it is necessary to remove as much of the nucleic acids as possible. The crude soluble fraction (which should contain 10 to 15 mg. of protein per milliliter) is adjusted to pH 6.0 by the addition of 1 N acetic acid, and protamine sulfate (20 mg./ml., pH 5 to 6) is added in a fine stream with vigorous agitation. The precipitate is removed periodically by centrifugation, and the ratio of optical densities at 280 m~/260 m~ is determined on a suitable dilution of the supernatant with the Beckman spectrophotometer. In our experience different samples of commercial protamine sulfate vary greatly in their effectiveness as selective nucleic acid precipitants, and this makes it impossible to specify exactly the amount which should be added. The crude soluble fraction has a 280/260 ratio of about 0.55, and protamine addition should be continued until this reaches at least 0.75. With some batches of protamine sulfate, the ratio can be increased to 1.2 before appreciable protein is lost, but with others attempts to increase the ratio above 0.75 have led to severe losses of material. 7 I. C. Gunsalus, C. F. Gunsalus, and R. Y. Stanier, J. Bacteriol. 66, 538 (1953).

276

ENZYMES OF PROTEIN METABOLISM

[37]

I. Mandeli¢ Acid R a c e m a s e 8

D(-)-C6HsCHOHCOOH ~-

L(+)-C~HsCHOHCOOH

Assay M e t h o d

Principle. The racemization of the D-isomer is coupled to an oxidation of the L-isomer by washed particles, which contain L-mandelie dehydrogenase together with the electron transport system to oxygen. Provided that the rate of formation of L-mandelic acid by the racemase is insufficient to saturate the dehydrogenase, the rate of oxygen uptake at the expense of the D-isomer is a function of racemase concentration. Reagents 0.1 M phosphate buffer, pH 7.0. 0.1 M Na D-mandclate in above buffer solution. A preparation of L-mandelate-oxidizing particles (see p. 277) on which the Qo, with the L-isomer has been previously determined. 10% KOH. Racemase, approximately diluted with 0.01 M phosphate buffer.

Procedure: To the side arm of a Warburg vessel add 0.3 ml. of Na o-mandelate. To the main compartment add an amount of particles sufficient to oxidize 30 #M. of L-mandelic acid per hour under the test conditions; racemase; and buffer to bring the total volume to 2.5 ml. If the racemase contains any benzoylformic carboxylase (the case with crude extracts), 0.2 ml. of KOH should be added to the center well. Equilibrate in a bath at 30 °, tip the substrate, and determine the rate of oxygen consumption between 5 and 25 minutes after substrate addition. Oxygen uptake increases in rate during the first 5 minutes, while the concentration of oxidizable isomer is reaching a steady state, and must be excluded from the measurement. Definition of Unit. One unit of racemase is that amount which causes the oxidation of D-mandelic acid at a rate of 15 tLM. per hour under the specified conditions. Since the rate of oxygen consumption is not a linear function of the concentration of racemase except at very low racemase levels, a standard curve should be constructed by determining the rate of oxygen uptake with a series of dilutions of the racemase. Purification Procedure

No systematic attempts to purify the enzyme have been made. 8 C. F. Gunsalus, R. Y. Stanier, and I. C. Gunsalus, J. Bacteriol. 66, 548 (1953).

[37]

CLEAVAGE OF AROMATIC RINGS

277

Properties The properties described have been determined only with crude soluble fractions. The enzyme is active at pH 7.0, but the pH optimum has not been determined. Lengthy dialysis against dilute buffer does not appreciably reduce its activity. Starting with either isomer of mandelic acid, the enzyme catalyzes the formation of a racemie mixture. The racemization of mandelic acid--C14OOH in the presence of inactive benzoylformic acid causes negligible incorporation of radioactivity in the benzoylformic acid: therefore, racemization does not appear to involve an interconversion of the isomers via the keto acid. II. L(+)-Mandelic Acid Dehydrogenase 9 C6HsCHOHCOOH + 0.5 02 ~ C6HsCOCOOH + H20 Assay Method

Principle. Measurement of the rate of oxygen consumption at the expense of substrate. Reagents 0.1 M phosphate buffer, pH 7.0. 0.l M Na L(+)-mandelatein the same buffer. Particle fraction from mandelate-grown cells of P. fluorescens.

Procedure. Ad~d0.5 ml. of Na L-mandelate to the side arm of a Warburg vessel. To the main compartment add washed particles and sufficient buffer to bring the total volume to 2.5 ml. Equilibrate at 30 °, tip, and determine the rate of oxygen uptake. For assay of crude fractions which still contain benzoylformic carboxylase, 0.2 ml. of 10% KOH should be added to the center well. Crude fractions can often also consume oxygen at the expense of benzaldehyde (formed by decarboxylation of benzoylformic acid), but the rate is relatively low, and the error thereby introduced will seldom exceed 10%. Removal of pyridine nucleotides from such crude preparations by dialysis will eliminate benzaldehyde oxidation completely. Alternative Assays. It should be noted that the assay method described above is dependent on the fact that the particle fraction in extracts, to which the L-mandelic dehydrogenase is bound, also contains an intact, functional cytochrome system through which substrate dehydrogenation is connected to oxygen. Solubilization of the dehydrogenase has not yet been attempted; but when this is done, an assay by determinaL 9 R. Y. Stanier, C. F. Gunsalus, and I. C. Gunsalus, J. Bacteriol. 66, 543 (1953).

278

ENZYMES OF PROTEIN METABOLISM

[37]

tion of oxygen uptake will no longer be possible, and other methocis will have to be devised. Preliminary experiments show that the dehydrogenation of L-mandelate can be connected to a reduction of 2,6-dichlorophenolindophenol, which can be followed spectrophotometrically by measuring the decrement of optical density at 605 m~. Definition of Unit. This has not yet been done and had best await the development of an alternative assay procedure. Purification Procedure

L-Mandelic dehydrogenase is one of the complex of enzymes which is physically bound to the particulate fraction of Pseudomonas fluorescens. Disaggregation of this insoluble complex has not yet been accomplished, and the purification so far achieved involves a simple removal of all soluble proteins and cofactors initially also present in a crude extract by repeated washing of the particles with 0.02 M phosphate buffer (pH 7.0). In this way, the L-mandelic dehydrogenase can be freed completely from mandelic acid racemase and benzoylformic acid carboxylase, both of which are soluble enzymes. Sedimentation of the particles requires highspeed centrifugation (preferably at 20,000 X g or higher). The particles are sedimented in the form of a translucent, gelatinous, light-brown pellet, which is difficult to redisperse evenly. Redispersion is most easily achieved by repeated ejection from a fine hypodermic syringe. Precipitation with ammonium sulfate is of no value in isolating the particles, since small amounts of soluble proteins tend to be occluded in the precipitate. III. Benzoylformic Carboxylase s

C~HsCOCOOH L~ CsHsCHO -}- CO2 Assay Method

Principle. Measurement of the rate of COs formation from the substrate.

Reagents 0.1 M phosphate buffer, pH 6.0. 0.1 M Na benzoylformate in the above buffer, pH adjusted to 6.0. TPP, 1 mg./ml., in the above buffer. Enzyme, appropriately diluted in the above buffer.

Procedure. To the side arm of a Warburg vessel add 0.5 ml. of Na benzoylformate. To the main compartment add 0.1 ml. of TPP, enzyme, and sufficient buffer to bring the total volume to 2.5 ml. Equilibrate at 30 °, tip the substrate, and determine the rate of C02 evolution. Note that at 30 ° and pH 6.0 the effective value of a¢o, is 1.44 times the true value,

[37]

CLEAVAGE OF AROMATIC RINGS

279

and vessel constants must be recalculated accordingly. Measurements can be made in air, except when assaying crude extracts or whole cells still capable of consuming oxygen at the expense of benzaldehyde. An atmosphere of nitrogen must be used in such cases. The rate of C02 evolution is a linear function of enzyme concentration over the measurable range. Definition of Unit and Specific Activity. One unit of enzyme is t h a t a m o u n t which decarboxylates 1 ~M. of benzoylformic acid per hour under the specified conditions. Specific activity is expressed as units per milligram of protein, the latter being measured b y the Folin-Ciocalteu method. Purification P r o c e d u r e The steps through protamine t r e a t m e n t have been outlined in the int r o d u c t o r y section. Starting with a protamine-treatcd crude soluble fraction (step 3), we proceed to steps 4 and 5. Step 4. Further Fractionation with Ammonium Sulfate. A saturated ammonium sulfate solution, whose p H has been adjusted to 8.0 with ammonia, is added to 0.4 saturation. The precipitate is removed b y centrifugation and discarded. The supernatant is brought to 0.7 saturation b y further addition of saturated ammonium sulfate, and the precipitate is collected, dissolved in 0.01 M phosphate buffer (pH 7.0), and dialyzed overnight against the same buffer. It is then refractionated in a similar manner, and the fraction precipitating between 0.5 and 0.7 saturation is collected, redissolved, and dialyzed against buffer to remove ammonium sulfate. Step 5. Heat Treatment. The material obtained in step 4, dissolved in 0.01 M phosphate buffer (pH 7.0) at a concentration of about 10 mg. of protein per milliliter is heated with stirring in a water bath to 50 ° and held at this temperature for 45 seconds. I t is then immediately cooled in ice, after which the precipitate is removed b y centrifugation and discarded. This constitutes the fraction of highest specific activity so far obtained. I t is about 20% resolved with respect to T P P . Complete resolution with negligible loss of activity can be obtained b y dialysis for TABLE I SUMMARY OF PURIFICATION PROCEDURES

1. 2. 3. 4. 5.

Fraction

Total units, thousands

Total protein, mg.

Crude sonic extract Crude soluble fraction Protamine-treated fraction 2 0.5-0.7 (NH4)2SO4fraction Supernatant after heating

117 110 84 20 19

4270 2650 1190 80 70

Specific Recovery, activity % 27 41 71 252 273

94 72 17 16

280

ENZYMES OF PROTEIN METABOLISM

[37]

24 hours against 0.02 M sodium pyrophosphate (pH 8.5), followed by dialysis for 12 hours against distilled water. Properties The purified enzyme shows very slight decarboxylative activity with pyruvic, ~-ketobutyric, and ~-ketoglutaric acids. In no case is the rate as much as 5% of that with benzoylformic acid. There is indirect evidence that p-hydroxybenzoylformic acid can also be decarboxylated at a high rate, but the pure compound has not been tested. The Km value for benzoylformic acid is 9 X 10-4 mole/1. The reaction is essentially irreversible. The optimal pH lies between 6.0 and 6.5. TPP is required, the K~ value being approximately 4 × 10-6 mole/1. A metal requirement has not been shown, but by analogy with other simple decarboxylases it may well exist. IV. TPN- and DPN-Linked Benzaldehyde Dehydrogenases 8 C6HsCHO + TPN--~ C6HsCOOH -F T P N H C6HsCHO + DPN--* C6HsCOOH + D P N H Assay Method

Principle. Both enzymes are assayed by the measurement of the rate of pyridine nucleotide reduction, as determined by the increase of optical density at 340 mu. Reagents 0.01 M benzaldehyde, freshly distilled. 1 M phosphate buffer, pH 8.5. DPN or TPN solution, 1 mg./ml, in water. Enzyme, appropriately diluted.

Procedure. To a cuvette (1-cm. light path) add 0.2 ml. of benzaldehyde, 0.3 ml. of DPN or TPN, 0.3 ml. of buffer, enzyme, and water to 3 ml. Determine the increase of optical density at 340 m~. The rate of increase is a linear function of enzyme concentration over the measurable range. Definition of Unit. The unit of activity is defined as the amount of enzyme which causes a change of 0.01 density unit per minute under the specified conditions. Purification Procedure No satisfactory method of purification has been developed. The two dehydrogenases have been separated from one another by the procedure outlined in Table II.

[3~

CLEAVAGE OF AROMATIC RINGS

281

TABLE II SEPARATION OF DPN- AND TPN-LINKED BENZALI)EHYDE DEHYDROGENASES

DPN-linked enzyme

TPN-linl~ed enzyme

Ratio Protein, Total Specific Total Specific activity, rag. units activity units activity T P N / D P N

Fraction 1. Protamine-treated soluble fraction 2. Ammonium sulfate fraction of fraction 1 (a) 0.33-0.5 (b) 0.5-0.7 3. Fraction 2a refractionated between 0.4 and 0.5 saturated ammonium sulfate (a) Aged in cold 6 days 4. Fraction 2b refractionated between 0.5 and 0.6 saturated ammonium sulfate (a) Heated to 50° for 45 seconds

243

8250

34

3540

15

0.43

82 77

2260 4600

28 60

1630 306

20 4

0.7 0.07

15 15

216 0

14 0

450 260

30 17

2.0 ~¢

15

396

26

43

3

0.12

15

130

9

0

0

0

Properties T h e D P N - l i n k e d e n z y m e has a s h a r p a n d n a r r o w p H o p t i m u m a t 8.5; t h e p H o p t i m u m of t h e T P N - l i n k e d e n z y m e lies a t 9.0 or above. A t these p H values, t h e r e a c t i o n s are e s s e n t i a l l y irreversible. N e i t h e r e n z y m e c a n oxidize a c e t a l d e h y d e , g l y c e r a l d e h y d e , or m e t h y l glyoxal. V. P y r o c a t e c h a s e ~°,H

COOH

OH

I

OH +

II

CH

t

CH

II

CH

I

C00H Catechol

cis,cis-Muconic Acid

10O. Hayaishi and K. Hashimoto, Med. J. Osaka Univ. 21 33 (1950). 11 M. Suda, K. Hashimoto, H. Matsuoka, and T. Kamehora, J. Biochem. (Japan) $81 289 (1951).

282

[37]

ENZYMES OF PROTEIN METABOLISM

Assay Method Principle. Manometric measurement of oxygen consumption. Procedure. A reliable procedure has not yet been worked out. As shown by Suda et al., 11 a requirement for ferrous iron develops during purification, and it has been found in our laboratory that glutathione also stimulates activity. However, the optimal concentrations are not established. Definition of Unit. None. Purification Procedure No reliable data on purification are available. The enzyme has'been obtained completely dependent on Fe ++ ions for activity by the following procedure. 11 One gram of acetone-dried cells is suspended in 50 ml. of 0.067 M phosphate buffer (pH 8.0), stored in an ice chest for 24 hours, and centrifuged at 17,000 r.p.m, for 20 minutes. The supernatant is fractionated with saturated ammonium sulfate, and the material precipitating between 0.3 and 0.6 saturation is collected. This is redissolved in buffer and dialyzed against distilled water for 20 hours.

VI. Lactonizing and Lactone-Splitting Enzymes 12 COOH

COOH I

I en2

CH

/H

II

CH

~

CH H

~ CH lactonizingII

I

CH

COOH I

enzyme

I

C

i

CH

CH.I

I I + H20'

o

I

C00H

C .

lactone-splitting C~O

enzyme

I

) CH2

r

CH2

I

C00H

IL

0 cis,cis-Muconic Acid

~-Carboxymethyl-A'butenolide

fl-Ketoadipic Acid

Assay Method Principle./~-Ketoadipic acid is virtually transparent in the ultraviolet region, whereas cis, cis-muconic acid (I) and 7-carboxymethyl-A~-butenolide (II) both show strong absorption. I has a well-marked peak with a maximum at 259 m~, whereas II shows only end absorption. Hence the activity of the lactonizing enzyme can be determined by measurement of 1~W. R. Sistrom and R. Y. Stanier, J. Biol. Chem. 210, 821 (1954).

[37]

CLEAVAGE OF AROMATIC RINGS

283

optical density change at 259 m~ irrespective of the product, since both II and ~-ketoadipic acid show negligible light absorption in this region. The activity of the lactone-splitting enzyme can be determined from measurement of optical density change at the short end of the ultraviolet range, the point selected being 230 m~.

Reagents for Lactonizing Enzyme 0.01 M MnC12. 0.1 M Tris buffer, pH 8.0. 0.001 M cis, cis-muconic acid in buffer. Enzyme, appropriately diluted.

Reagents for Lactone-Splitting Enzyme 0.1 M phosphate buffer, pH 6.8. 0.01 M -~-carboxymethyl-h"-butenolide in buffer. Enzyme, appropriately diluted.

Procedure for Lactonizing Enzyme. In a cuvette of 1-cm. light path, mix 0.3 ml. of MnC12, 0.3 ml. of Tris buffer, 0.3 ml. of I, enzyme, and water to 3 ml. Read the optical density change at 259 m~ in a spectrophotometer equipped with temperature control (30 ° + 1°). The change in optical density is a linear function of enzyme concentration at rates between 0.050 and 0.150 unit/min. Procedure for Lactone-Splitting Enzyme. In a cuvette of 1-cm. light path, mix 0.3 ml. of phosphate buffer, 0.3 ml. of II, enzyme, and water to 3 ml. Read the optical density change at 230 m~ at appropriate intervals in a temperature-controlled spectrophotometer ( 3 0 ° + 1°). The change in optical density is a linear function of enzyme concentration between 0.030 and 0.080 unit/rain. Definition of Unit. For both enzymes, the unit is defined as the amount of enzyme which causes the disappearance of substrate at a rate of 1 ~M./min. under the specified conditions. For the laet~izing enzyme, this is equivalent to a change in optical density at 259 mu of 5.750/min. ; for the lactone-splitting enzyme, to a change at 230 m~ of 0.476/min. Specific activity is recorded as units per milligram of protein, the latter being determined by the quantitative biuret method. Purification Procedure

The steps through protamine treatment have been outlined in the introduction. Starting with a crude soluble protamine-treated fraction (step 3), we proceed to step 4. Step 4. Further Fractionation with Ammonium Sulfate. The supernatant after protamine treatment is adjusted to pH 7.0 with 0.1 M

284

ENZYMES OF P R O T E I N METABOLISM

[37]

Na~HPO4 and t r e a t e d with neutral s a t u r a t e d a m m o n i u m sulfate. "The fractions precipitating between 0 and 0.35 saturation and 0.45 and 0.70 s a t u r a t i o n are collected and redissolved in 0.01 M Tris buffer. T h e former contains nearly all the lactonizing enzyme, free of lactone-splitring enzyme, and the latter nearly all the lactone-splitting enzyme, free of lactonizing enzyme. TABLE III SEPARATION OF THE LACTONIZING AND LACTONE-SPLITTING ENZYMES

Lactonizing enzyme

Protein, mg.

Fraction Crude extract After protamine (}-0.35 ammonium sulfate fraction 0.45-0.70 ammonium sulfate fraction

Volume, ml. Total Per ml.

Total units

Specific activity

Lactone-splitting enzyme Total Specific units activity

30 24

615 264

21 11

-318

-1.2

450 281

4

54

13

344

6.4

0

4

96

21

0

0

146

0.7 1.0 0 1.7

Properties T h e lactonizing e n z y m e requires either M g or M n for activity, whereas the lactone-splitting e n z y m e does not. T h e lactonizing e n z y m e catalyzes a freely reversible reaction; at p H 8, the equilibrium mixture contains app r o x i m a t e l y 9 5 % of I I and 5 % of I. T h e lactone-splitting step is essentially irreversible T h e p H o p t i m u m for the lactonizing e n z y m e is a b o u t p H 8.0, and for the lactone-splitting e n z y m e a b o u t p H 7.0. VII. Protocatechuic Acid Oxidase 13 COOH

COOH

r

+ OH

OH

II CCOOH

!

CH

II J

CH COOH la R. Y. Stanier and J. L. Ingraham, Or. Biol. Chem. 2111~799 (1954).

[37]

CLEAVAGE OF AROMATIC RINGS

285

Assay Method

Principle. The method is based on the fact that substrate and product have markedly different ultraviolet absorption spectra, permitting the enzymatic conversion to be followed by the change in optical density at a suitable wavelength. Reagents Protocatcchuic acid stock solution (0.01 M). Dissolve 172 mg. of protocatechuic acid in 100 ml. of distilled water. This may be stored for several months. 0.1 M phosphate buffer, pH 7.0. Enzyme, appropriately diluted.

Procedure. Dilute the stock solution of protocatechuic acid 1:10 with buffer. To a cuvette (1-cm. light path) add 2.0 ml. of phosphate buffer, 0.5 ml. of diluted protocatechuic acid, enzyme, and water to a total of 3 ml. Using a blank which contains all reagents except the substrate, determine the change in optical density at 290 mt~ at appropriate intervals. Definition of Unit. One unit of enzyme is defined as the amount that causes an initial change in optical density of 0.056 unit/rain, at 290 mt~ when ~-carbo×ymuconic acid is the product of the reaction. This is equivalent to the disappearance of 0.075 ~M. of protocatechuic acid per minute. With crude extracts, the ~-carboxymuconic acid undergoes further decomposition to ~-ketoadipic acid, which is transparent throughout the ultraviolet region. A correct measure of substrate disappearance under these conditions requires determination of optical density at two wavelengths (270 and 290 mt~) from which, after the curves for optical density change have been plotted, the amount of protocatechuic acid at any given time can be calculated by simultaneous equations. The molar extinction coefficients at pH 7.0 for protocatechuic acid are 2560 at 270 mt~ and 3800 at 290 m~; those for ~-carboxymuconic acid are 6300 at 270 m~ and 1500 at 290 m~. Purification Procedure The steps through protamine treatment have already been outlined in the introductory section. Starting with the crude protamine-treated soluble fraction, we proceed to steps 4 and 5. Step 4. Further Fractionatwn with Ammonium Sulfate and Heat Treatment. The supernatant after protamine treatment is fractionated with neutral saturated ammonium sulfate, and the fraction, precipitating between 0.5 and 0.7 saturation is collected, redissolved in 0.02 M phosphate

286

[37]

ENZYMES OF PROTEIN METABOLISM

buffer (pH 7), and dialyzed overnight against the same buffer. I t is then heated to 50 ° and held at this t e m p e r a t u r e for 3 minutes, after which it is immediately cooled in an ice bath. I t is then refractionated with ammonium sulfate, the material precipitating between 0.5 and 0.6 saturation being collected, redissolved in phosphate buffer, and dialyzed overnight against distilled water. Step 5. Gel Treatment. After eentrifugation of the dialyzed extract to remove precipitated material, the supernatant is treated with alumina C~ gel. One-tenth volume of gel (dry weight 20 mg./ml.) is sufficient with a solution of the enzyme containing 5 mg. of protein per milliliter. The mixture is immediately centrifuged, and the supernatant liquid collected. This constitutes the purest material so far obtained. I t should be noted t h a t the decarboxylase which converts ~-carboxymuconic acid to ~-ketoadipie acid accompanies the protocatechuic acid oxidase through all steps up to gel treatment, b y which it is quantitatively removed from solution with the appropriate a m o u n t of gel. TABLE IV PARTIAL PURIFICATION OF PROTOCATECHUIC ACID 0XIDASE

Protein Fraction 1. Protamine-treated crude soluble fraction 2. 0.5-0.7 ammonium sulfate fraction of fraction 1 3. Fraction heated 4. 0.33-0.6 ammonium sulfate fraction of fraction 3 5. Fraction 4 after dialysis 6. Fraction 5supernatant after gel treatment

Enzyme

Volume, Mg./ Units/ ml. ml. Total ml.

28

9.0 252 88 --

Specific Recovery, Total activity %

610 17,100

68

100

1,180 10,150 1,070 9,200

116 --

60 54

8.6 8.6

10.2 --

4.3

8.0

34.4 1,500

6,450

187

38

5.0

5.0

25.0 1,230

6,150

246

36

5.0

1.4

3,125

446

18

7.0

625

Properties Protocatechuic acid oxidase catalyzes an essentially irreversible reaction. I t shows extreme substrate specificity; other compounds tested as substrates with negative results include catechol, h y d r o x y h y d r o q u i none, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, all three monohydroxybenzoic acids, and the ethyl, isopropyl and isobutyl esters

[38]

CONVERSION OF TYROSINE TO ACETOACETATE

287

of protocatechuic acid. The oxidation cannot be connected to dyes, and oxygen appears to be essential for the reaction. The pH optimum is high (probably about 9.0) but cannot be determined with accuracy, since protocatechuic acid undergoes spontaneous oxidation in alkaline solution. No cofactor requirements have been shown. The enzyme is not inhibited by KCN or by Versene at a concentration of 10-2 M.

[38] Enzymes Involved in Conversion of Tyrosine to Acetoacetate A. L-Tyrosine-Oxidizing System of Liver L-Tyrosine ~ 202 -~ ~-Ketoglutarate--~ Acetoacetate ~- Fumarate ~ COs ~- L-Glutamate

By W. E. KNOX

Assay Method Principle. The several enzymes catalyzing the oxidation of L-tyrosine to acetoacetate are present in the soluble fraction of rat liver. The overall system can be most easily demonstrated with the preparation of Knox and LeMay-Knox, 1 using oxygen uptake as the index of activity. Partial oxidation with accumulation of intermediate compounds often results in other types of preparations, and this can be detected only by determination of the total oxygen uptake with tyrosine or by measurement of the final products. Reagents 0.2 M phosphate, pH 7.2. Six micromoles of L-tyrosine per milliliter of 0.2 M phosphate, pH 7.2; 10.9 mg. of L-tyrosine is dissolved by warming in 10 ml. of the phosphate buffer and used before crystallization occurs. 0.1 M a-ketoglutarate. The acid is dissolved by neutralization and adjusted to pH 7. It is kept in the refrigerator and is usable for several weeks. 0.05 M ascorbate. Sixteen milligrams of ascorbic acid is dissolved in 1.1 ml. of water and 0.9 ml. of 0.1 N NaOH (to pH 7) immediately before use. W. E. Knox and M. LeMay-Knox, Biochem. J. 49~ 686 (1951).

288

E N Z Y M E S OF PROTEIN M E T A B O L I S M

[38]

Procedure. Oxygen uptake is measured in Warburg vessels at 37 °, in air, and with NaOH papers in the center wells. The vessels contain 3 ml. total volume, including 1 ml. of enzyme, 6 micromoles of L-tyrosine, 1 ml. of 0.2 M phosphate, pH 7.2, 0.1 ml. of 0.05 M ascorbate, and 0.1 ml. of 0.1 M a-ketoglutarate in the side arm. The blank is identical but without tyrosine. The reaction is started by tipping in the a-ketoglutarate. The blank has an insignificant oxygen uptake. L-Tyrosine is oxidized with the uptake of about 100 #l. of 02 per 10 minutes until a total of 2 moles of 02 is consumed and 1 mole of acetoacetate is formed per mole of L-tyrosine. Activity. Within narrow limits (0.3 to 1.0 ml.) the rate of oxygen uptake is proportional to the enzyme concentration. Dilution effects and weakening of the component reactions by any purification result in variable completenesses of tyrosine oxidation. Activity and specific activity measured solely by oxygen uptake consequently have little meaning. Application of Assay Method to Crude Tissue Preparations. All the tyrosine-oxidizing activity of liver homogenates is measured in the soluble, dialyzed fraction. Undialyzed but centrifuged preparations often require 5 micromoles of ascorbate (that added in the assay) for maximal activity, whereas a dialyzed preparation may require only 0.1 micromole. Homogenates require more ascorbate, have a high blank oxygen uptake, and cause further reactions of the a-ketog!utarate , glutamate, acetoacetate, and fumarate. These complications are avoided by removal of the mitochondrial elements in the preparation described. Preparation. Rat livers are homogenized for 2 minutes with 2 vol. of ice-cold 0.14 M KC1 in a Waring blendor, then centrifuged in the cold for 20 minutes at 11,000 X g. The supernatant is adjusted to pH 7.5 and dialyzed in the cold for 12 hours against 0.14 M KC1. After 48 hours at 4 ° successive failures of the component reactions occur. Although the over-all system itself cannot be further purified, a number of enzymes believed to participate in the system have been identified in the above preparation. The L-tyrosine-glutamic acid transaminase, homogentisate oxidase, maleylacetoacetate isomerase, and fumarylacetoacetate hydrolase are described in subsequent sections. Properties

Activators and Inhibitors. There is virtually no reaction of L-tyrosine in the system without added a-ketoglutarate and catalytic amounts of ascorbate. Ascorbate, but not a-ketoglutarate, is required for the oxidation of p-hydroxyphenylpyruvate (and more ascorbate than is required for oxidation of tyrosine by the same preparation). Neither compound is required for the oxidation of the 2,5-dihydroxyphenylpyruvate and homogentisate. Only the latter is a proved intermediate.

[38]

CON~rERSION OF TYROSINE TO ACETOACETATE

289

D-Isoascorbic and glucoascorbic will replace ascorbic acid in the oxidation of p-hydroxyphenylpyruvate, but glutathione will not. Other compounds have increased tyrosine oxidation in different types of preparation. 2.3 A great variety of inhibitors affect the over-all reaction at one or another locus. These are best discussed in connection with the particular enzymes on which they act. Distribution. Rat, rabbit, guinea pig, horse, ox, and pig livers have active tyrosine-oxidizing systems.

B. Tyrosine-Glutamic Acid Transaminase L-Tyrosine nc a-Ketoglutarate--~ p-Hydroxyphenylpyruvate d- L-Glutamate

By W. E. KNOX This reaction has been detected by means of the glutamate formed, determined chromatographically,4 and measured with the bacterial decarboxylase. 5 It has been assayed in the soluble liver fraction by the latter method and also by determination of the p-hydroxyphenylpyruvate formed, both chemically and by its ultraviolet spectrum. 1 It has not been studied in sufficient detail to describe a fully satisfactory assay, but the tyrosine or glutamic acid decarboxylases appear to offer the specificity such a method would need. The enzyme can be precipitated from the soluble fraction of liver with 50% saturated ammonium sulfate and then dialyzed. With this treatment it is resolved from its coenzyme, pyridoxal phosphate. Its substrate specificity is not known. As in other transaminase reactions 6 the product is believed to be the keto, and not the enol, form of p-hydroxyphenylpyruvate.

C. p-tIydroxyphenylpyruvate Enol-Keto Tautomerase O OH

H

II

H

I

H0--/~_~--C--C--C00H~ H0--~--C=C--C00H By W. E. KNox Assay Method

Principle. The enol of p-hydroxyphenylpyruvate absorbs intensely at 300 m~, whereas the keto form does not absorb significantly in the 2 B. N. La Du, Jr., and D. M. Greenberg, Science 117, 111 (1953). J. N. Williams, Jr., and A. Sreenivasan, J. Biol. Chem. 203, 605 (1953). 4 F. J. R. Hird and E. V. Rowsell, Nature 166, 517 (1950). s p. S. Cammarata and P. P. Cohen, J. Biol. Chem, 187, 439 (1950). 6 A, Meister, J. Biol. Chem. 195, 813 (1952).

290

ENZYMES OF PROTEIN METABOLISM

[38]

near-ultraviolet region. 7 The equilibrium mixture is predominantly the keto form, but the enol can be stabilized in borate, presumably by the formation of a complex similar to those formed by other a-hydroxy acids. 8 Equilibrium mixtures in borate consist of nearly equal amounts of both forms, and the enzyme can be assayed in borate spectrophotometrically by following the approach to this equilibrium, starting with either form. The keto form is usually used, since its solutions are relatively stable. Reagents 0.5 M boric acid, adjusted to pH 6.2 with NaOH. 0.005 M p-hydroxyphenylpyruvate (keto). Eighteen milligrams of the crystalline free acid 9 (enol) is dissolved in 20 ml. of 0.05 M acetate, pH 6, and left for 24 hours to tautomerize. It is then in the keto form and can be kept in the icebox for use for two weeks. Procedure. 2.8 ml. of boric acid solution, 0.02 to 0.2 unit of enzyme, and sufficient 0.1 N NaOH to adjust the pH to 6.2 are added to each of two cells of l-em. light path. Water is added to make the total volumes 3.3 ml. (blank) and 3.2 ml. 0.1 ml. of 0.005 M p-hydroxyphenylpyruvate (keto) is added to the second cell, and readings of its optical density at 330 m~ are taken at l-minute intervals (ODt) for 5 minutes. Excess enzyme (ca. 0.3 unit) is then added to each cell, and the optical density at equilibrium read 10 minutes later and corrected for the dilution (ODeq). Activity. The reaction follows first-order kinetics. The constant k = log (ODeq -- ODt,) -- log (ODeq - ODt~) t2,-- t~ is proportional to the enzyme concentration. Time is measured in minutes. The spontaneous rate of enolization under these conditions is very slow (k < 0.002), so that no blank correction is necessary. The unit of activity is that amount of enzyme giving k = 1.0, and this amount is usually present in about 1 ml. of 33 % liver homogenate. The specific activity is expressed as k per milligram of protein. Protein has been determined by the method of Warburg and Christian.1° Application of Assay Method to Crude Tissue Preparations. The mixture of boric acid and enzyme need only be optically clear to use this 7 T. Bficher and E. Kirberger, Biochim. et Biophys. Acta 8, 401 (1952). 8 j. Boeseken, Advances Carbohydrate Chem. 4, 195 (1949). 9 Prepared by the method for phenylpyruvic acid, R. NI. Herbst and D. Shemin, Org. Syntheses 19, 77 (1939). 10 O. Warburg and W. Christian, Biochem. Z. 310, 384 (1941); see Vol. I I I [73].

[38]

CONVERSION OF TYROSINE TO ACETOACETATE

291

method. Carbohydrates combining with boric acid m a y lower the pH, which m u s t be avoided by early addition of NaOH. T h e equilibrium values obtained with crude liver extracts are not stable and should be read at their maximum.

Purification Procedure

Step i. Fresh pig liver is homogenized and centrifuged exactly as described for preparation of the soluble tyrosine-oxidizing system, 1~ but it is not dialyzed. Step 2. Heat Treatment. The soluble pig liver fraction is brought to 57 ° within 2 minutes of heating by swirling it in a hot water bath. The swirling solution is held at that temperature for 4 minutes, then rapidly cooled in an ice bath and the coagulum centrifuged off. Step 3. Ammonium Sulfate Precipitation. Saturated ammonium sulfate is added to the enzyme at 4 °. The precipitate forming at 40 % saturation is discarded, and t h a t forming after 15 minutes at 60% saturation is centrifuged down, dissolved in water, and adjusted to p H 7.5. T h e enzyme can be dialyzed against distilled water with the loss of about 20% of its activity. It is stable at - 1 0 ° for several months. TABLE I PURIFICATION OF TAUTOMERASE FROM PIG LIVER

Activity

Fraction Soluble, step 1 57°, heated 40-60 % ammonium sulfate

Specific Recovery, Protein, activity, Volume k/ml. Total % mg./ml, k/mg. 208 151 25

0.97 0.94 3.71

202 142 93

-70 46

156 47 66

0.006 0.020 0. 056

Properties

Distribution. The enzyme has been found in rabbit, pig, and beef livers and kidneys, but not in rabbit heart or skeletal muscle. Specificity. The tautomerase also catalyzes the enol-keto change in phenylpyruvate and m-hydroxyphenylpyruvate. No reaction of p y r u v a t e itself can be observed with this method. Effect of pH. The enzyme has been tested between p H 5.0 and 7.5. I t is somewhat more active at p H 6.2. The spontaneous rate of the reaction increases with pH, especially above p H 7. Activators and Inhibitors. Fe ++ and Cu ++ at 5 X 10-~ M slightly increase the spontaneous reaction r a t e / a n d they increase the rate of the 11Preparation of soluble tyrosine-oxidizing system, p. 288.

292

ENZYMES OF PROTEIN METABOLISM

[38]

enzyme-catalyzed reaction about 20%. However, H2S, HCN, EDTA, and ~,a-dipyridyl at 3 × 10-3 M do not significantly inhibit the reaction. The reaction is faster in phosphate buffer. An essential thiol group on the enzyme is suggested by the inhibition with p-chloromercuribenzoate (80% with 10-4 M) and with Hg ++ (65% with 3 X 10-~ M). The latter inhibition can be reversed with cysteine. Cysteine or ascorbate generally activates the enzyme a small amount. Iodoacetate inhibits only at 0.01 M concentration.

D. Homogentisate Oxidase from Rat Liver O

OH \

II

/

~ /

C--CH2

\

--CH2--COOH H O2--) HC"

C--CH~--COOH

OH

O

II

II

HC

\ COOH

By S. W. EDWARDSlla and W. E: KNOX Assay Method

Principle. Oxygen uptake was used by Ravdin and Crandal112 and Suda and Takeda 13 and is the most generally useful method of following the enzyme activity. It is essential to detect any spontaneous oxidation of the substrate. This can be assumed if the reaction mixture darkens or if the oxygen uptake exceeds the theoretical value. Reagents 0.02 M homogentisic acid. The unneutralized solution may be kept in the icebox for several weeks and is routinely used without neutralization. 0.1 M 2,4,6-collidine, pH 7.2. The collidine is dissolved and brought to pH 7.2 with dilute HC1. It can be used for several months, and is a more effective buffer at this pH than Tris. 14 0.05 M ascorbate. Sixteen milligrams of ascorbic acid is dissolved and neutralized in 2 ml. immediately before use.

Procedure. The reaction is run in Warburg vessels at 37 ° with NaOH in the center wells, and routinely with air as the gas phase. The more 11~Public H e a l t h Service Research Fellow of the N a t i o n a l I n s t i t u t e s of Health. 12 R. G. R a v d i n a n d D. I. Crandall, J. Biol. Chem. 189, 137 (1951). 13 M. Suda a n d Y. Takeda, J. Biochem. (Japan) 37, 381 (1950). 14 G. Gomori, Prec. Soc. Exptl. Biol. Med. 62, 33 (1946).

[38]

CONVERSION OF TYROSINE TO ACETOACETATE

293

rapid reaction with 100% oxygen is used only for special purposes, such as the preparation of maleylacetoacetate. The vessels contain 1 ml. of collidine buffer, 0.1 ml. of ascorbate, and enzyme, in a total volume of 3 ml. An amount of enzyme is used which causes an oxygen uptake of 5 to 50 t~l. per 10 minutes, and this activity is usually present in 0.5 ml. of the supernatant of 33% rat liver homogenate. After equilibration, 0.3 ml. of the unneutralized homogentisic acid is added from a side arm. When it is desirable to add neutralized substrate, its pH should be less than 6 to minimize its autoxidation in the side arm, the products of which inhibit the enzyme reaction. A blank without homogentisic acid is run simultaneously. A blank with homogentisic acid but without enzyme, or with inactivated enzyme, is not an effective guide to any spontaneous oxidation. The reaction occurs at a linear rate until about 90% of the substrate is oxidized and is routinely followed to completion. Only among reactions to which other additions have been made will spontaneous oxidation occur, and the results from these, distinguished by darkening and excessive oxygen uptake beyond 1 02 per homogentisate, must be discarded. Activity. The enzyme activity is expressed as the microliters of oxygen above that of the blank which would be taken up by 1 ml. of the enzyme in 10 minutes during the linear period of the reaction under the above conditions. The specific activity is the oxygen uptake per 10 minutes per milligram of protein. Application of Assay Method to Crude Tissue Preparations. The activity can be measured by this method in liver homogenates. The ascorbic acid is best omitted because of the high blank due to its oxidation by the mitochondrial elements. Very fresh crude preparations are often 20 to 30% less active than those a day old, owing to inhibition by the peroxide generated endogenously in the fresh material. Purification Procedure The enzyme is prepared from the undialyzed soluble fraction of rat liver which contains the over-all tyrosine-oxidizing system. The procedure is based on that of Ravdin and Crandall. 12 As described here, all three of the enzymes converting homogentisate to acetoacetate can be obtained separately. All steps are carried out at 4° or less. Step 1. Soluble rat liver fraction. 11 Step 2. Ethanol Precipitation. The undialyzed supernatant from step 1 is adjusted to pH 7, and ethanol is slowly added to a concentration of 32 % while the temperature is maintained below 0 °. The precipitate is centrifuged, carefully drained, and then washed by resuspension in a volume of cold 32% ethanol equal to the supernatant removed. The suspended

294

ENZYMES OF PROTEIN METABOLISM

[38]

precipitate is recentrifuged, drained, and dissolved in half the original volume of water. After adjusting to p H 7.5 it is dialyzed against water for 2 hours with efficient stirring. T h e p H is returned to 7.5, and the enzyme is frozen at - 10 °. On thawing, a h e a v y precipitate forms which can be separated and discarded. T h e enzyme keeps several months when frozen. The supernatant from the 32% ethanol precipitation can be stored at - 10 ° and used later for the preparation of isomerase and f u m a r y l a c e t o acetate hydrolase, the other two enzymes of the homogentisate system. Step 3. Ammonium Sulfate Precipitation. Ammoniacal saturated a m m o n i u m sulfate, p H 7.5 to 7.8, is added to the freshly thawed and centrifuged enzyme of step 2. T h e precipitates formed at 45 %, 60 %, and 75 % saturation are centrifuged down and dissolved in water. Only if the lower fractions are first removed stepwise will the highest fraction contain a reasonable a m o u n t of enzyme. T h e enzyme is much less stable at this stage. I t cannot be dialyzed in a routine manner because of loss of activity from the acidification, and it becomes inactive in several days at - 1 0 °. TABLE II HOMOGENTISATE OXIDASE PURIFICATION

Fraction Soluble, step 1 30% alcohol 45 % ammonium sulfate 60% ammonium sulfate 75% ammonium sulfate

Volume, Activity, Yield, Protein, Specific ml. ~l./ml./10 rain. Total % mg./ml, activity 68 50 8 10 10

263 163 118 213 254

17,900 8,1~0 945 2,130 2,540

-45 5 12 14

86 23 22.6 16.1 14.7

3.06 7.1 5.2 13.2 17.3

Properties Distribution. T h e enzyme is present in the livers and in lesser amounts in the kidneys 15 of all animals examined: rat, rabbit, guinea pig, ox, and horse. Alkaptonurics do not oxidize homogentisie acid, b u t the liver of such a patient has not been assayed for the presence of this enzyme. Specificity. Gentisie acid and benzoquinone acetic acid are not oxidized. The latter inhibits homogentisate oxidase, b u t the former does not. Effect of pH and Substrate Concentration. T h e p H o p t i m u m is about 7.5. T h e enzyme is saturated with 2 >( 10-4 M substrate. Activators and Inhibitors. T h e enzyme contains iron which can be .partially resolved b y t r e a t m e n t at p H 5. T h e activity is restored b y addi15D. I. CrandaU, Federation Proc. 12, 192 (1953).

[38]

CONVERSION OF TYROSINE TO ACETOACETATE

295

tion of Fe ++ but not Fe +++ or other metals. 15-17 Both ferric and ferrous reagents such as cyanide, azide, pyrophosphate, and a,a-dipyridyl inhibit the enzyme in low concentrations, but 80% CO does not. Orthophosphate and cysteine also inhibit. The enzyme contains an essential thiol group. After brief aging of the enzyme at step 3, it is activated by a variety of thiol compounds, although not regularly by cysteine, and also by other reducing agents such as ascorbic acid. This is the reason for including ascorbate in the assay. All preparations of the enzyme are inactivated by low concentrations oI thiol reagents such as p-chloromercuribenzoate, peroxide, quinones, cystine, and Hg ++. The effect of the latter two can be reversed by addition of mercaptoacetic acid. Spectrophotometric Assay. Enzyme preparations lacking an active isomerase (or its coenzyme glutathione) can be assayed by the maleylacetoacetate which accumulates during homogentisate oxidation. Preparations at step ,2 are suitable for this assay. The conditions are similar to those described for the assay involving oxygen uptake. The increase in optical density at 330 m~ at 1-minute intervals is proportional to the enzyme concentration.

E. Maleylacetoacetate Isomerase

/

O

O

H

II

C--CH2--C--CH~--COOH

HC

GSH

tl

HC

\ COOH

/

O

O

II

II

C--CH~--C--CH~--COOH

HC

KI

/

CH

HOOC By W. E. KNOX and S. W. EDWARDSua

Assay Method Principle. Maleylacetoacetate is converted by the enzyme to its trans isomer, fumarylacetoacetate, and only this isomer can be hydrolyzed 10B. Schepartz, J. Biol. Chem. 205, 185 (1953). 17M. Suda, Y. Takeda, K. Sujishi, and T. Tanaka, J. Biochem. (Japan) 88, 297 (1951).

296

ENZYMES OF PROTEIN METABOLISM

[38]

by the fumarylacetoacetate hydrolase added in excess. The reaction is followed by the disappearance of the near-ultraviolet absorption characteristic of the diketo acids.

Reagents Approximately 0.001 M maleylacetoacetate. This is prepared by the oxidation in 02 of homogentisic acid with homogentisate oxidase free of isomerase activity. 18 At the end of the reaction the solution is promptly chilled, deproteinized with 0.1 vol. of 20% MPA, filtered, and neutralized. The solution is kept frozen and can be used for several days. It slowly isomerizes to fumarylacetoacetate. The molar extinction coefficients at 330 mt~ of maleyl- and fumarylacetoacetates, respectively, are approximately: pH 1, 1.3, and 13.5 (X103); pH 13, 16, and 13.5 (X103). From these values the amounts of each in a mixture can be calculated. 0.005 M glutathione. This is diluted daily from an unneutralized 0.1 M stock solution kept frozen. Approximately 10 units/ml, of hydrolase, free of isomerase. 19 0.2 M phosphate, pH 7.5. Procedure. Maleylacetoacetate (0.3 ml. of 0.001 M) is added to a 1-cm. cell with 0.5 ml. of phosphate buffer, pH 7.5, and 1 unit of isomerase-free hydrolase, in a total volume of 3 ml. Any fumarylacetoacetate present in the substrate is rapidly hydrolyzed, and the constant optical density remaining at 330 mg represents maleylacetoacetate when read against its blank. The blank contains the blank filtrate from the maleylacetoacetate preparation. Enough maleylacetoacetate should be present to give an optical density of 0.8 to 1.6. Next 0.1 ml. of glutathione is added, and the blank rate of maleylacetoacetate disappearance is followed for 1 or 2 minutes. This depends on the amount of isomerase contaminating the hydrolase preparation and should be negligible. An amount of isomerase is then added which will cause about 0.3 density change the first minute, and readings are taken at 30-second intervals (ODt). Activity. The reaction follows first-order kinetics. The value k = log (ODt~/ODa) t2 -- tl is proportional to the isomerase concentration. Time is measured in minutes. The activity of an enzyme preparation is the k that 1 ml. of the enzyme in a 3-ml. volume would have when acting under the above conditions. The specific activity is the k per milligram of protein. is Preparation of homogcntisate oxidase, step 2, p. 293. 19 Purification of isomerase and fumarylacetoacetate hydrolase, step 2, p. 297.

[38]

CONVERSION OF TYROSINE TO ACETOACETATE

297

Application of Assay Method to Crude Tissue Preparations. Any optically clear preparations can be determined by this method. Purification

Procedure

The supernatant from the 32 % ethanol precipitation of homogentisate oxidase from rat liver 18 is used to prepare both the isomerase and hydrolase by further ethanol fractionation. The starting material can be stored for several weeks at - 10% All procedures must be carried out at 0 ° or below, and the enzymes kept frozen when not in use. Each fraction should be assayed for b o t h enzymes to ensure separation. Step 1. First Ethanol Fractionation. Ethanol is added to the 32% ethanol supernatant previously described, keeping the temperature between 0 and - 5 °. Precipitates are quickly centrifuged off at 60, 70, and 85% (v/v) ethanol and immediately dissolved in water. The waterinsoluble residues left after solution of these and also the second fractionation precipitates can be discarded. The 60% fraction can usually be discarded. The isomerase activity precipitates only with the higher ethanol concentrations, and t h a t in the 85% fraction is usually free of hydrolase activity (see Table III). Step 2. Hydrolase is obtained free from isomerase, and the yield of isomerase increased, by refractionation of the 60 to 75 % ethanol fraction of step 1. In the presence of higher ionic strength than used here, both enzymes will precipitate together in this fraction in step 1, but they can still be separated by a second or third fractionation. 1 M N a acetate is added to a final concentration of 0.03 M to the water-soluble protein of the 60 to 75% fraction of step 1. Ethanol is then added, keeping the temperature below 00, and precipitates collected at 40, 50, 60, 75, and TABLE III PURIFICATION AND SEPARATION OF ISOMERASE AND HYDROLASE

Hydrolase

Isomerase

Vol- Protein, Activity, Yield, Specific Activity, Yield, Specific Fraction ume mg./ml. ~OD/ml./min. % activity k % activity Soluble 320 First EtOH 60-75 % 40 75-85 % 35 Second EtOH 40-50% 13 50-60% 13 60-75 % 8

86

3.7

--

0. 043

58.2

--

0.68

19.4 5.6

28.6 0.04

97 --

1.47 --

75.0 5.6

16 1

3.87 1.0

9.4 ---

23.6 27.6 1.4

26 31 1

2.51 ---

0.1 1.3 3.1

298

ENZYMES OF PROTEIN METABOLISM

[38]

85% (v/v) ethanol and immediately dissolved in water. The 40%'fraction can usually be discarded. The other two lower fractions contain most of the hydrolase. One is usually sufficiently free of isomerase to use in the assay system. If not, it can be refractionated in the same way. The 85% fraction again contains the isomerase, but the recovery is poor. Only the isomerase fractions can be dialyzed against water, which inactivates the last traces of hydrolase. Both enzymes can be stored at - 1 0 ° for several months without loss of activity.

Properties Both glutathione and the enzyme must be present for isomerization to occur.

Specificity of Glutathione as Coenzyme. Cysteine, mercaptoacetic acid, and coenzyme A will not replace or inhibit the action of glutathione in the system. Oxidized glutathione is also inactive. The enzyme will isomerize maleylacetoacetate to fumarylacetoacetate in the absence of hydrolase. There is no indication that the reaction is reversible. The pH optimum of the reaction is between 8.5 and 9.0.

F. Fumarylacetoacetate Hydrolase

H

0

II

0

H HOOC--C-----C--COOH

II

H O O C - - C ~ C - - C - - C H 2 - - C - - C H ~ - - C O O H --* H

+

H

0

II

H3C--C--CH~--COOH B y S. W. EDWARDSlla and W. E. KNOX

Assay Method Principle. The absorption of fumarylacetoacetate at 330 m~ in neutral solution disappears as it is hydrolyzed to fumarate and acetoacetate. The reaction can be followed directly. Enzymes probably identical with the hydrolase have been described in liver by Connors and Stotz 2° (triacetic acid-hydrolyzing enzyme) and by Meister and Greenstein 21 (acylpyruvase). These were respectively measured manometrically by the hydrolysis of the f3,8-diketo acid and triacetic acid, and spectropho-

s0 W. M. Connors and E. Stotz, J. Biol. Chem. 178, 881 (1949). 2xA. Meister and J. P. Greenstein, J. Biol. Chem. 175, 573 (1948).

[38]

CONVERSION" OF TYROSINE TO ACETOACETATE

299

tometrically by the hydrolysis of a,~-diketo acids such as 3-acetylpyruvate. Hydrolase also hydrolyzes these compounds but acts much more rapidly on the physiologically occurring fumarylacetoacetate. Reagents

Approximately 0.001 M fumarylacetoacetate. Maleylacetoacetate prepared by the reaction of homogentisate oxidase ~2 is left in the acid filtrate after deproteinization without neutralization for at least 24 hours in the cold. The product is then largely isomerized to fumarylacetoacetate, which is kept frozen and can be used for several weeks. Fumarylacetoacetate can also be isolated as the silver salt. 12 Other reagents are those used in the assay of isomerase. ~ Procedure. An amount of fumarylacetoacetate (0.3 ml. of 0.001 M) which will have an optical density of approximately 1.2 is placed in a 1-cm. cell with 0.5 ml. of 0.2 M phosphate, pH 7.5, and water to a total volume of 3.0 ml. An amount of hydrolase is added which will produce about 0.3 optical density change per minute, and readings are taken at 330 m~ at 30-second intervals. The density decreases linearly until nearly all the substrate is hydrolyzed, and at a rate proportional to the enzyme concentration. Activity. The activity is expressed as the change in optical density per minute which would be produced by 1 ml. of the enzyme in a total volume of 3 ml. under these conditions. A unit of activity causes a change of 1.0 per minute. The specific activity is the density change per minute per milligram of protein. The amount of substrate hydrolyzed can be calculated from the molar extinction coefficient of fumarylacetoacetate under these conditions (e[330 m~, pH 7.5] = 13.5 X 103).

Purification Procedure The preparation of the enzyme and its separation from isomerase has been described.19 Connors and Stotz purified the enzyme from beef liver to a considerable extent, but the method involves a heat treatment step which can rarely be satisfactorily reproduced. 2° Meister and Greenstein have also purified the enzyme from rat liver. ~1

Properties Specificity. Maleylacetoacetate is hydrolyzed very slowly if at all by hydrolase. Fumaryl- and succinylacetoacetates are split very rapidly. a,~-Diketovalerate ~3 reacts more rapidly than any of the other a,~,- and

~2Preparation of maleylace¢oacetate,p. 294. 23A. Meister, J. Natl. Cancer Inst. 10, 75 (1949).

300

[39]

ENZYMES O F PROTEIN METABOLISM

f~,~-diketo acids tested, but only at about one-twelfth the rate of fumarylacetoacetate. Acetylacetone is not hydrolyzed. Activators and Inhibitors. The enzyme loses a certain portion of its activity on dialysis, and this cannot be restored by any additions yet tried. 2° Coenzyme A is ineffective, although the reaction mechanism might appear to be similar to the thiolysis of ~-keto f a t t y acids. Concentrations of iodoacetate, p-chloromercuribenzoate, Cu ++, and Hg ++ less t h a n those precipitating the protein do not inhibit the reaction, and no activation is observed with added thiol compounds or ascorbic acid. No essential thiol group appears to be involved in the reaction. It is assumed to be a purely hydrolytic reaction.

[39] E n z y m e s of A r o m a t i c B i o s y n t h e s i s B y BERNARD D. DAVIS, CHARLES GILVARG,1 and SUSUMU MITSUHASHI2

Studies with bacterial m u t a n t s 3 have revealed t h a t a number of aromatic metabolites (Fig. 1) are derived from a sequence of common

H0. C00H H0"~0H OH Tyl'osine

Quinic acid HO. .COOH 'O~OH

OR DHQ

COOH ) O ~ O H ' OH

COOH

~henylalanine • /STryptopha n

HO"~0H OH

DHS Shikimic acid FIG. 1. Scheme of aromatic biosynthesis.

~_ p-Amin:benzoic p-ttydroxybenzoic acid

precursors, proceeding from 5-dehydroquinic acid 4 to 5-dehydroshikimic acid ~ to shikimic acid. It has further been observed t h a t quinic acid can 1Fellow of the National Foundation for Infantile Paralysis. Fellow from the Institute for Infectious Diseases, University of Tokyo. 3 B. D. Davis, J. Bacteriol. 64, 729 (1952). 4 5-Dehydroquinic acid will be referred to as DHQ, and 5-dehydroshikimic acid as DHS.

[39]

ENZYMES OF AROMATIC BIOSYNTHESIS

301

replace D H Q as a growth factor for mutants of Aerobacter aerogenes 5 and t h a t Aerobacter extracts contain an enzyme for interconverting these two compounds. However, although it is clear that DHQ, DHS, and shikimic acid are directly on the p a t h of aromatic biosynthesis, the available evidence suggests t h a t quinic acid probably is not. 5 The bacterial enzymes promoting the reactions of Fig. 1, from quinic acid up to shikimic acid, will be described. These reactions are all reversible. A. 5-Dehydroshikimic Reductase from Escherichia coli 6 5-Dehydroshikimic acid + T P N H ~- H + ~-Shikimic acid + T P N +

Assay Method Principle. Although the reaction in the direction of shikimic acid is the one t h a t takes place biosynthetically and the one favored b y the equilibrium at p H 7 or 8, D H S and T P N H are less readily available reagents than shikimic acid and T P N . I t was therefore more convenient to follow the reverse reaction, measuring T P N H at 340 m~. T h e unfavorable equilibrium, however, limits the range of linearity with respect to enzyme concentration in this assay. A more satisfactory assay was therefore developed b y coupling reaction 1 with the essentially irreversible action of GSSG reductase 7 (2), giving the over-all reaction (3).

Shikimic acid -~- T P N + ~ D H S -~- T P N H ~- H + GSSG + T P N H ~- H + ~ 2GSH ~- T P N +

(1) (2)

Shikimic acid -~ GSSG ~ D H S ~- 2GSH

(3)

In the presence of an excess of GSSG reductase the D H S reductase activity becomes rate-limiting and hence can be assayed through the determination of sulfhydryl groups released. Reagents

1 M Tris-HC1 buffer, p H 8.0. 0.1 M ethylenediaminetetraacetic a c i d - - K O H , p H 8.0. s 6B. D. Davis and U. Weiss, Arch. exptl. Pathol. Pharmakol. 220, 1 (1953). 6 H. Yaniv and C. Gilvarg, J. Biol. Chem., in press (1955). 7T. W. Rall and A. L. Lehninger, J. Biol. Chem. 194, 119 (1952); see also Vol. II [127]. s The chelating agent is added to prevent SH removal by metal-catalyzed oxidations and SH formation by metal-requiring enzyme reactions.

302

ENZYMES OF PROTEIN METABOLISM

[89]

0.002 M T P N . 9 0.025 M G S S G 2 0.01 M shikimic acid 9,1° (1.74 mg./ml.). G S S G reductase. An acetone powder p r e p a r a t i o n of guinea pig liver is extracted with distilled w a t e r (1 ml. per 50 mg.) for 20 minutes at 0 °. T h e particulate m a t t e r is removed b y centrifugation at 5000 × g for 20 minutes. 7 T h e extract has to be prepared freshly, but the acetone powder remains active for m a n y m o n t h s when stored over a desiccant at 0 °. Enzyme. Dilute the solution with M / 3 0 Tris buffer, p H 8.0, so t h a t the sample being assayed contains ca. 0.005 unit.

Procedure. I n a test t u b e are placed 0.04 ml. of th~ buffer, 0.04 ml. of the ethylenediaminetetraacetate, 0.02 ml. of the T P N , 0.04 ml. of the GSSG, 0.10 ml. of the shikimic acid, and 0.04 ml. of the G S S G reductase. W a t e r is added to adjust the final volume to 0.60 ml., and the D H S reductase is added last. After incubation at 30 ° for 20 minutes 0.60 ml. of 6 % metaphosphoric acid is added. After centrifugation a 0.80-ml. portion of the s u p e r n a t a n t is analyzed for S H content. H As controls, SH should be determined at zero time and also after incubation without substrate. Occasionally a parallel t u b e is set up containing twice as m u c h guinea pig liver enzyme; the S H released should be the same in b o t h tubes if the G S S G reductase is not rate-limiting.~2 T h e manipulations can be simplified b y employing a composite stock solution containing all the reagents except the enzymes. Definition of Unit and Specific Activity. A unit of e n z y m e is defined as the a m o u n t t h a t catalyzes the f o r m a t i o n of 1 micromole of D H S per minute under the a b o v e conditions. Specific activity is expressed as units per milligram of protein. Protein is determined b y the m e t h o d of W a r b u r g and Christian. 13 Application of Assay Method to Crude Preparations. E x t r a c t s of E. coli, prepared as described below, usually contain ca. 0.2 unit of e n z y m e per 9 The reagents are best stored at - 15°. i0 Shikimic acid can be isolated from the dried fruits of the Chinese star anise, Illicium religiosum [S. Y. Chen, Am. J. Pharm. 101, 687 (1929)]. This compound can also be isolated from filtrates of a mutant (e.g., E. coli 83-24) blocked after it, and is commercially available from the California Foundation for Biochemical Research. 11R. R. Grunert and P. H. Phillips, Arch. Biochem. and Biophys. 30, 217 (1951). 12GSSG reductase is present in initial extracts of E. coli but is lost in the early stages of purification. 18 0. Warburg and W. Christian, Biochem. Z. $10~ 384 (1941).

[39]

ENZYMES OF AROMATIC BIOSYNTHESIS

303

milliliter and can be assayed directly. Such extracts neither contain significant amounts of SH nor yield it in the absence of shikimic acid. Extracts of some other organisms (e.g., yeast), which can also be assayed directly, require correction for their SH content. Purification Procedure

E. coli (strain W) is grown with aeration on minimal Medium A 14 supplemented with 0.2% Difco yeast extract, harvested after 24 hours, and lyophilized. 15 All further operations are carried out at 0 °. Extracts are prepared by grinding the bacteria with three times their dry weight of glass powder for 5 minutes and then for an additional 10 minutes with gradual addition of M/30 Tris buffer, pH 8.0 (20 ml./g. dry weight of bacteria). Centrifugation for 20 minutes at 5000 X g yields a clear yellow supernatant; this is adjusted to a protein concentration of 20 mg./ml. 16 In order to reduce the nucleic acid content of the extract, one-twentieth its volume of 1 M MnC12 is added over a period of 10 minutes with occasional stirring. 17 The resulting voluminous precipitate is removed by centrifuging at 5000 × g for 20 minutes. The supernatant is adjusted to pH 5.5 with 0.1 M acetic acid, 0.2 vol. of calcium phosphate geP 8 is added, and the whole is stirred for 20 minutes and then centrifuged. The precipitate is discarded. To the supernatant 1.2 vol. of gel is added, and the procedure is repeated except that the gel is retained. It is washed by being suspended in several volumes of water and centrifuged. The washed gel is eluted by being stirred for 20 minutes in a volume of M/IO phosphate buffer, pH 6.0, equal to the volume of the MnC12 supernatant. The eluted gel is removed by centrifugation. The supernatant is subjected to ammonium sulfate fractionation at pH 7.0. The salt is added as the solid over a period of 20 minutes, stirring is continued for an additional 20 minutes, and the precipitate is removed by centrifugation at 5000 × g for 20 minutes. The material precipitating between 32 and 45 % saturation is dissolved in M/30 Tris buffer, pH 8.0, and dialyzed overnight against the same buffer. A typical fractionation is summarized in the accompanying table. 14 B. D. Davis a n d E. S. Mingioli, J. Bacteriol. 60, 17 (1950). 15 Freshly harvested wet cells also yield the enzyme on extraction, b u t the response of such extracts to M n C l , (see later sections on dehydroquinase a n d quinic dehydrogenase) differed from t h a t of extracts from a b a t c h of lyophilized cells. 16 The cells m a y also be disrupted in a magnetostriction oscillator, b u t such extracts are more viscous a n d do not yield clear s u p e r n a t a n t s on centrifugation. 17 S. Korkes, A. del Campillo, I. C. Gunsalus, a n d S. Ochoa, J. Biol. Chem. 19.q, 721 (1951). is D. Keilin a n d E. F. Hartree, Proc. Roy. Soc. (London) B124, 397 (1938).

304

[39]

ENZYMES OF PROTEIN METABOLISM

The purified enzyme retained 80% of its activity after storage at 15° for 4 months. The product obtained by the above procedure has been freed of dehydroquinase, 19 the enzyme that forms DHS from DHQ. It was not possible to test for the enzyme that carries out the step subsequent to shikimic acid on the aromatic pathway, as this step is at present unknown. TABLE I SUMMARY OF PURIFICATION PROCEDURE (DHS REDUCTASE)

Fraction Initial extract MnCI~supernatant Calcium phosphate gel eluate (NH~)2SO~ fraction 0.32-0.45

Specific Protein, activity, mg./ml, units/mg, 20 8.4 2.2 2.9

0.0082 0. 0183 0. 025 0.078

Total activity, units

Recovery, %

7.9 6.0 2.21 1.18

100 76 28 15

Properties Specificity. The purified enzyme requires T P N as the electron acceptor, DPN being inactive. Dihydroshikimic acid, 5-epishikimic acid, 3-phosphoshikimic acid, 5-phosphoshikimic acid, and quinic acid are not oxidized by the enzyme plus TPN. Activators. There appears to be no multivalent metal ion requirement, since the enzyme is not inhibited by 0.006 M ethylenediaminetetraacetate. Effect of pH. The pH optimum is 8.5. About 50% of maximal activity is obtained at pH 10.5 or 7.5. Michaelis Constants. The Michaelis constants for TPN and shikimic acid, determined at pH 8.0, were 3.1 X 10-6 M and 5.5 X 10-5 M, respectively. Equilibrium Constant. The values of K' were 27.7 at pH 7.0 and 5.7 at pH 7.8. [Shikimic acid][TPN +] K ' = K,q[H +] = [DHS][TPNH] Hence the O/R potential of DHS/shikimic acid would equal that of T P N + / T P N H at about pH 8.5.

Distribution DHS reductase is present in E. coli, Aerobacter aerogenes, yeast, peas, and spinach leaves. It could not be detected in guinea pig liver. 19 See p. 305.

[39]

ENZYMES OF AROMATIC BIOSYNTHESIS

305

B. 5-Dehydroquinase from Escherichia coli 2° 5-Dehydroquinic acid ~ 5-Dehydroshikimic acid % H~O

Assay Method Principle. The method is based on the fact that DHS has an ultraviolet absorption peak at 234 m~, with a molar extinction coefficient21 of 11,900, whereas DHQ does not absorb significantly at this wavelength. 22

Reagents 3 mM. of DHQ (0.57 mg./ml.). Although this substance is sensitive to heat and very sensitive to alkali, solutions of the free acid can be stored at - 1 5 ° for months without change. DHQ is obtained from filtrates of mutants (e.g., E. coli 83-1) blocked immediately after it. 21 Such filtrates, which usually contain 200 to 300 rag./1, of DHQ (1 to 1.5 raM.), may be substituted for pure DHQ in the assay. The concentration of DHQ in a filtrate can be determined by means of enzymatic conversion to DHS, which proceeds almost to completion (see below); and as the converting enzyme one may use an extract of E. coli, prepared as described below. 1 M potassium phosphate buffer, pH 7.4. Enzyme. Dilute the solution so that the sample to be assayed contains about 0.005 to 0.02 unit.

Procedure. Place 0.1 ml. of the buffer, the enzyme sample, and enough water to make a final volume of 3.0 ml. in a silica cell having a 1-cm. light path. No change in 0D234 should take place over the next 2 to 3 minutes. Add 0.1 ml. of the DHQ, and read the optical density at 234 m~ at 30second intervals (room temperature, ca. 25°). Definition of Unit and Specific Activity. A unit of enzyme is defined as the amount that catalyzes the formation of 1 mieromole of DHS per minute under the above conditions. Thus 0.01 unit per 3.0 ml. would cause an 0D234 increment of 0.040 per minute. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Warburg and Christian. 18 Application of Assay Method to Crude Preparations. Extracts of E. coli or Aerobacter, prepared as described below, usually contain 0.2 unit of enzyme per milliliter and can be assayed directly. No side reactions 20 S. Mitsuhashi and B. D. Davis, Biochim. Biophys. Acta 15, 54 (1954). ~ I. I. Salamon and B. D. Davis, J. Am. Chem. Soc. 75, 5567 (1953). ~ U. Weiss, B. D. Davis, and E. S. Mingioli, J. Am. Chem. Soc. 75, 5572 (1953).

306

[39]

ENZYMES OF PROTEIN METABOLISM

appear to contribute to the 0D234 increment obtained with such extracts, or to destroy the D H S t h a t is formed. Although these extracts contain the enzyme 23 for converting D H S to shikimic acid, this reaction does not proceed significantly in the absence of an added source of T P N H .

Purification Procedure T h e extraction of enzyme from E. coli, t r e a t m e n t with MnC12, and t r e a t m e n t with 0.2 vol. of calcium phosphate gel were identical with those described for D H S reductase, 2~ except t h a t freshly harvested wet cells were used, and MnC12 was allowed to act for 30 minutes before centrifugation, is The subsequent adsorption on calcium phosphate gel was performed with 0.8 to 1.0 vol. The gel was washed with distilled water and stirred for 20 minutes with M/IO phosphate buffer, p H 6.1, in a volume one-twentieth t h a t of the original extract. T h e supernatant, which contained most of the D H S reductase, was discarded, and the dehydroquinase was eluted from the gel b y a similar t r e a t m e n t with buffer at p H 7.0. After removal of the gel b y centrifugation, the s u p e r n a t a n t was subjected to ammonium sulfate fractionation at p H 7.0, as described for D H S reductase. 23 T h e dehydroquinase was collected in the fraction between 50 and 60% saturation. T h e precipitate was dissolved in M/30 Tris buffer (pH 7.4) and dialyzed against the same buffer. TABLE II SUMMARY OF PURIFICATION PROCEDURE (DEHYDROQUINASE)

Fraction Initial extract MnCl~ supernatant Supernatant from first gel Eluate from second gel (NH4)2S04 fraction 0.50-0.60

Protein, mg./ml,

Specific activity, units/mg,

Total activity, units

Recovery, %

21.4 16.4 6.2 2.4 2.2

0.011 0. 014 0. 020 0. 055 0.089

23.6 19.6 13.0 6.2 3.0

100 80 55 26 12

A typical purification is summarized in the accompanying table. T h e product obtained b y this procedure had been freed of D H S reductase. With Aerobacter extracts there was no significant separation from quinie dehydrogenase, 24 which catalyzes a D P N - l i n k e d interconversion of D H Q and quinic acid. E. coli, however, lacks quinic dehydrogenase. 23See p. 301. ~ See p. 307.

[39]

ENZYMES OF AROMATIC BIOSYNTHESIS

307

The purified enzyme showed no significant loss of activity on storage at - 1 5 ° for 6 months and also was stable over a period of several days at 0%

Properties Specificity. Dehydroquinase, like fumarase and aconitase, converts an a-hydroxy acid to the corresponding a,~-unsaturated acid. The preparation described above, however, could not be shown to react with high concentrations of DL-malate, DL-isocitrate, or citrate. Furthermore, a purified preparation of fumarase 25 in high concentration did not react with DHQ. Purified dehydroquinase did not convert quinic acid to shikimic acid. Activators and Inhibitors. The enzyme does not appear to have any metal ion requirement, since it is equally active in Tris hydrochloride or in potassium phosphate buffer and is not inhibited by 0.05 M ethylenediaminetetraacetate in Tris buffer. The enzyme was 50 to 80 % inhibited by 0.005 M FeC13, ZnC12, CuSO4, or iodoacetamide. At the same concentration, Mg ++, Mn ++, arsenate, and azide were without effect, and Co ++, Fe ++, CN-, and hydroxylamine were slightly inhibitory. Effect of pH. The pH optimum lies at about 8.0, with 90% of optimal activity at pH 9.0, and 80% at 7.0. MichaeIis Constant. The dissociation constant for DHQ is 4.4 X 10-5 M. EQuilibrium Constant. The equilibrium constant at 29 ° and pH 7.4, neglecting the water term in the reaction, is D H S / D H Q = 15. Distribution The distribution observed for dehydroquinase was the same as that described for DHS reductase? 3 Certain mutants yield about ten times as high a concentration of the enzyme as their wild-type parent strains. These mutants include Aerobacter A170-143S1, selected for good growth response to quinic acid or DHQ, and E. coli 83-2, which accumulates 3 DHS.

C. Quinic Dehydrogenase from

Aerobacter

a e r o g e n e s 25~

Quinie acid & DPN + ~ 5-Dehydroquinic acid & D P N H & H +

Assay Method Principle. The method is based on the increase in absorption at 340 mu that results from the conversion of DPN to DPNH. Neither quinic acid ~5 Kindly furnished by Dr. S. Ratner. ~5~ S. Mitsuhashi and B. D. Davis, Biochim. Biophys. Acta I§, 268 (1954).

308

ENZYMES OF PROTEIN METABOLISM

[39]

nor DHQ absorbs at this wavelength. Cyanide is added to prevent reoxidation of D P N H by D P N H oxidase. An alternative method based on bioassay of DHS (formed enzymatically from DHQ) is described below in the section on "Crude Preparations."

Reagents 0.1 M quinic acid 26 (19.2 mg./ml.). 1 M potassium carbonate-bicarbonate buffer, pH 9.4. 0.01 M DPN. 1 M KCN. Enzyme. Dilute the sample with phosphate or Tris buffer (pH 7.4) to a concentration of 0.1 to 1 unit/ml.

Procedure. Add to the enzyme one-hundredth its volume of the KCN, and allow to stand at 0 ° for at least 5 minutes. (The cyanide-treated enzyme is stable for at least 12 hours.) Place 0.1 ml. of the buffer, 0.1 ml. of the DPN, 0.1 ml. of enzyme, and enough water to make a final volume of 3.0 ml. in a silica or Corex cell having a 1-cm. light path. No change in 0D34o should take place over the next 2 to 3 minutes. Add 0.1 ml. of the quinic acid, and read the optical density at 340 m~ at 30-second intervals (room temperature, ca. 25°). Definition of Unit and Specific Activity. A unit of enzyme is defined as the amount that catalyzes the formation of 1 micromole of DHQ (and of DPNH) per minute under the above conditions. If the molar extinction of D P N H at 340 m~ is taken as 6.22 X 103, 0.1 unit of enzyme per 3.0 ml. would cause an 0D34o increment of 0.207 per minute. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Warburg and Christian. 13 Application of Assay Method to Crude Preparations. The method described may be used with extracts of Aerobacter mutant A170-143S1, prepared as described below, which contain 2 to 3 units/ml. These extracts, as well as purified preparations (see below), also contain high concentrations of dehydroquinase, which converts DHQ to DHS; but this secondary reaction only improves the range of linearity of the assay, since a product of the assay reaction is removed without affecting the D P N H which is being measured. These extracts also contain nuc!eotide transhydrogenase, 27 traces of TPN, and TPN-linked DHS reductase. ~8 They g6 Quinic acid is commercially available from Light's (England) or Hoffman-La Roche. 27 S. P. Colowick, N. O. Kaplan, E. F. Neufeld, and M. M. Ciotti, J. Biol. Chem. 195, 95 (1952), N. O. Kaplan, S. P. Colowick, E. F. Neufeld, and M. M. Ciotti, ibid. 2059 17 (1953).

[39]

ENZYMES OF AROMATIC BIOSYNTHESIS

309

can therefore reoxidize D P N H by coupling it via T P N with the reduction of D H S to shikimic acid. However, in the extracts of the Aerobacter m u t a n t the concentration of quinic dehydrogenase was so high, compared with t h a t of the other factors, that this coupled reaction did not interfere with the assay. Because of this further conversion of D H S to shikimic acid, a bioassay method, which responds to both these substances, is generally more sensitive and reliable for crude preparations than the spectrophotometric method described above. In the bioassay, enzyme (0.002-0..02 unit), D P N (0.5 micromole), 2s phosphate buffer (pH 7.4, final concentration 0.1 M), and quinic acid (1 micromole) are incubated in a volume of 1.0 ml. for l0 to 30 minutes. One milliliter of 0.2 HC1 is added, the precipitated protein is centrifuged off, and 0.1- to 0.4-ml. portions of the supernatant are pasteurized (65 °, 10 minutes) and assayed 5 for D H S plus shikimic acid with E. coli m u t a n t 83-1. 29 The enzyme assays thus obtained o n purified preparations were in satisfactory agreement with those obtained spectrophotometrically; and the bioassay showed extracts of wild-type Aerobacter to contain about one-fiftieth as much quinic dehydrogenase as extracts of m u t a n t A170-143S1, although the spectrophotometric assay failed to reveal any of this enzyme in the wild-type extracts. Purification P r o c e d u r e

The enzyme was obtained from Aerobacter m u t a n t A170-143S1, which is blocked before D H Q and in addition has a secondary mutation 5 t h a t improves its growth response to quinic acid or DHQ. Since the mucoid growth of this organism interferes with centrifuging the cells, a derivative strain was used which had been selected for nonmucoid growth. The organisms were grown with aeration on Medium A 14 supplemented with 0.2 % yeast extract or 0.2 % casein hydrolyzate and harvested and washed with H20 after 24 hours. All further operations were carried out at 0 °. Extracts were prepared by grinding the wet bacteria with three times their weight of glass powder for 5 minutes, and then for an additional 5 minutes with the gradual addition of ten times their weight of M / 3 0 Tris buffer, p H 7.4. Centrifugation at 5000 × g for 10 minutes yielded a clear supernatant containing about 10 mg./ml, of protein. 2s With crude preparations that contain sufficient DPNH oxidase, one-fifth as much DPN may be used. ~9The inability of this organism to respond to DHQ does not vitiate this enzyme assay, since DHQ is almost completely converted to DHS by dehydroquinase, which has been present in excess in all crude or purified preparations of quinic dehydrogenase that have been tested.

310

ENZYMES OF PROTEIN METABOLISM

[39]

This supernatant was treated with 8% of its volume of 1 M MnC12,1~ twice with calcium phosphate gel, and with (NH4)2S04, as described for dehydroquinase. ~9A typical purification is summarized in the accompanying table. The purified enzyme had been freed of DHS reductase and T P N and so could not link quinic acid oxidation with DHS reduction. It had also been freed of D P N H oxidase and therefore did not require the presence of C N - in the spectrophotometric assay. We have not found it possible, however, by varying the concentrations of the reagents employed here, to effect separation from dehydroquinase. The purified enzyme showed negligible loss of activity in storage at - 1 5 ° for several weeks, and 30% loss in 6 months. TABLE III SUMMARY OF PURIFICATION PROCEDURE (QuINIC DEHYDROGENASE)

Fraction Initial extract MnCl~ supernatant Supernatant from first gel Eluate from second gel (NH~)2S04 fraction 0.50-0.60

Specific Protein, activity, reg./m1, units/mg, 10.8 7.1 2.2 1.1 2.0

0.25 0.37 0.64 1.57 2.15

Total activity, units

Recovery, %

270 254 160 83 22

100 94 59 30 8

Properties

Specificity. With the purified enzyme T P N could not replace D P N as the electron acceptor. With initial extracts T P N showed considerable activity, which could be accounted for by the combined action of the nucleotide transhydrogenase :7 and the traces of D P N that were present in these extracts. The purified enzyme did not catalyze the oxidation of shikimic acid to DHS by either D P N or T P N . Activators. There appears to be no multivalent metal ion requirement, since the enzyme is not inhibited by 0.05 M ethylenediaminetetraacetate. Effect of pH. The pH optimum is 9.8. About 50% of maximal activity is obtained at pH 8.5. Michaelis Constant. The dissociation constants at pH 9.4 for quinic acid and D P N are 4.9 X 10-4 M and 1.4 X 10-5 M, respectively. Equilibrium Constant. Since the purified enzyme contains dehydroquinase and so carries the reaction beyond DHQ to DHS, the equilibrium of quinic acid /DH Q was obtained by observing quinic aci d/ D H S values and correcting for the known ~9 D H S / D H Q equilibrium. T h e value obtained at 32 ° and pH 7.2 was

[40]

SULFUR TRANSFER ENZYMES (MAMMALIAN)

K' -

311

Ke~ _ [DPNH][DHQ] = 4.61 × 10-3 [H +] [DPN÷][quinic]

One would therefore expect the O / R potential of D H Q / q u i n i c to equal t h a t of D P N + / D P N H at about p H 9.7. Comparing the above equilibrium constant with those obtained for the D H Q ~ D H S and the D H S ~ shikimic acid reactions, and introducing the known value 3° of the nucleotide transhydrogenase equilibrium [ ( D P N + ) ( T P N H ) / ( D P N H ) ( T P N +) = 0.7], it can be calculated 25~ t h a t the equilibrium constant for shikimic/quinic is 1.0. This value has been verified experimentally b y allowing either quinic acid or shikimic acid to attain equilibrium in the presence of a mixture of the four enzymes plus traces of D P N and T P N . 30N. O. Kaplan, S. P. Colowick, and E. F. Neufeld, J. Biol. Chem. 205, 1 (1953).

[40] Cystathionine Cleavage Enzymes B y FRANCIS BINKLEY

A. Liver Enzyme Cystathionine--~ Cysteine ~- ~ - K e t o b u t y r a t e ~ Ammonia

Assay Method Principle. The cleavage of cystathionine m a y be followed b y the method of Sullivan and Hess I for the determination of cysteine, by methods of determination of ammonia, and by methods for the determination of keto acids. The method of Brand et al. 2 (modification of the Folin method for cystine) may be used for the determination as m a y any of the modifications of the nitroprusside method. With L-cystathionine as the substrate and with the mammalian enzyme, all methods are equally satisfactory. Reagents

L-Cystathionine, 3 0.02 M. Dissolve 520 mg. of the amino acid in 100 ml. of Tris buffer, p H 7.5. This solution is stable. 0.1 M Tris buffer, p H 7.5. 1 M. X. Sullivan and W. C. Hess, J. Biol. Chem. 116, 221 (1936). 2 E. Brand, G. F. Cahill, and B. Kassell, J. Biol. Chem. 133, 431 (1940). Prepared by the method of M. D. Armstrong, J. Org. Chem. 16, 433 (1951), or, better, by a method involving the addition of L-homocysteine to aeetylaminoacrylic acid followed by the separation of the isomers by fractional crystallization (J. Reid and F. Binkley, manuscript in preparation); see also A. Sch6berl and A. Wagner, Naturwissenschaflen 37, 113 (1950).

312

ENZYMES OF PROTEIN METABOLISM

[40]

0.1 M citric acid-NaOH buffer, pH 7.5. Enzyme. Dilute the enzyme as necessary with 0.85% saline to obtain about 1 unit/ml. (see below). Pyridoxal phosphate, 4 0.020 mg./ml, in 0.85% saline (freshly prepared).

Procedure. Four milliliters of Tris buffer, 2 ml. of citrate buffer, 1 ml. of pyridoxal phosphate, 2 ml. of L-cystathionine, and 1 ml. of enzyme are mixed and incubated at 38 ° for 30 minutes. One-half milliliter of 50% trichloroacetic acid is added, and the precipitate is removed by filtration. Cystine is determined in a 3-ml. aliquot of the filtrate by the procedure of Sullivan and Hess. (See Vol. I I I [84].) Definition of Activity. One unit of enzyme is defined as that amount necessary for the production of 1 mg. of eysteine in 30 minutes under the above conditions. Alternative Substrates. L-Lanthionine, meso-lanthionine, L-djenkolic acid, and D-allocystathionine may be used as alternative substrates under the conditions described above2 The mixture of isomers obtained by the methods of SchSberl for the preparation of lanthionine (L and meso) or of cystathionine (L- and D-allo) from L-homocysteine may be used. With preparations of the enzyme from liver tissue of rats the activities with L-lanthionine and L-cystathionine are nearly identical; with preparations of the enzyme from liver tissue of pigs the activity with L-lanthionine is about twice as great as that with L-cystathionine. Commercial preparations of the substrates (as of this date) are exorbitantly priced and are generally unsatisfactory. Purification Procedure The procedure outlined below is an unpublished method of our laboratory; it is suitable under ideal conditions for the preparation of crystalline, apparently homogeneous material from fresh pig liver. Step 1. Preparation of Crude Extract. Fresh or frozen pig liver is homogenized with 5 vol. of cold 0.85% saline in a Waring blendor. The homogenate is divided into 200-ml. portions in centrifuge bottles, heated to 55 to 58 ° in a water bath maintained at 60 °, cooled in an ice bath to near 10°, and centrifuged. The supernatant solution is decanted, and the combined supernatants are shaken with 0.5 vol. of cold chloroform for about 15 minutes, centrifuged, and the clear supernatant removed by suction, Step 2. Fractionation with Alcohol. One-half volume of ethanol is added to the clear solution (no special precautions as to temperature or rate of addition of alcohol are to be taken; somewhat better purification is ob4 See Vol. I I I [142].

[40]

SULFUR TRXNSFER ~.NZVMES (~AMMALIAN)

313

rained at room temperature), and the precipitate is removed b y centrifugation or filtration. Another 0.5 vol. of ethanol is added, and the precipitate is removed by centrifugation. The precipitate is mixed with 1 vol. (on the basis of the original weight of liver) of cold 0.85 % saline and 0.5 vol. of chloroform. The mixture is shaken for 15 minutes and then centrifuged; the clear supernatant is removed by suction. The t r e a t m e n t with heat and fractionation with alcohol are repeated, and, finally, the solution is dialyzed against frequent changes of distilled water. Step 3. Dialysis Precipitation. 5 In the course of prolonged dialysis, a considerable a m o u n t of inactive material separates from the solution. This material is removed by centrifugation and discarded; the dialyzed solution is refractionated with ethanol as described above. See the accompanying table for a summary of the purification methods. SUMMARY OF PURIFICATION PROCEDURE

Fraction 1. Heated extract 2. CHC1, treatment 3. 0.5-1.0 vol. alcohol fraction 4. (1) and (3) repeated 5. Dialysis precipitation

Total volume Total Protein, Specific Recovery, Units/ml. units mg./ml, activity % ml. 2000 2000

0.15 0.17

300 340

2.8 I. 1

0.05 0.16

---

200 200 50

0.61 0.54 1.26

122 108 63

0.31 0.17 0.11

1.97 3.17 11.4

41 36 21

Properties Specificity. There is considerable uncertainty as to the specificity of the activity. Present indications are t h a t the cleavage of thio ethers (lanthionine, cystathionine, djenkolie acid, methionine, S-methylcysteine, and other similar compounds) is catalyzed b y one enzyme. Desulfhydrase activity m a y be demonstrated in all preparations, but it is clear t h a t the bulk of desulfhydrase activity of liver tissue is due to rather specific desulfhydrases. Activators. With all preparations tested to date, citrate appears to have a special activating effect. P a r t of this activation appears to be due to inhibition of phosphatases (destruction of pyridoxal phosphate). (Versene will activate in a similar manner, and the cruder preparations are actiIn the course of the fractionation with alcohol in this step, crystalline material is sometimes obtained when fresh rather than frozen liver tissue is used in the preparation. This material appears to be homogeneous at pH 8.3 in the electrophoresis apparatus.

314

ENZYMES OF PROTEIN METABOLISM

[40]

vated to a greater extent than the purified preparations. There is, nevertheless, an effect of citrate in addition to that of Versene.) With substrates yielding volatile sulfides, glutathione (or other sulfhydryl compounds) is required; the extent of cleavage is found to be dependent upon the amount of glutathione present. Effect of pH. The enzyme exhibits an optimum for activity under alkaline conditions, and the optimum is dependent upon the time and temperature of digestion. For brief digestions (10 to 15 minutes) the optimum appears to be near pH 10, whereas for longer digestion periods an optimum near pH 9 is found. Similar behavior as to temperature of digestion has been observed. At lower temperatures the optimum pH is higher than at higher temperatures for any constant period of time of digestion. This behavior of the enzyme may be interpreted as evidence for the participitation of O H - ions in the cleavage; the optimum pH would be determined by the stability of the enzyme. B. Bacterial Enzymes Cystathionine --~ Homocysteine -t- Pyruvate -~- NH3 A cleavage enzyme responsible for the cleavage of L-cystathionine with the formation of L,homocysteine, pyruvate, and ammonia may be prepared from bacterial cells. Proteus morganii is a suitable organism; the harvested cells (from liquid media) are dehydrated by repeated treatment with acetone, suspended in citrate buffer, and subjected to a short digestion with trypsin (30 minutes to 1 hour). The extract is then treated in a manner identical to that described for the mammalian enzyme. In the assay of activity, the method of Brand et al. is used. In the determination of homocysteine a more alkaline buffer (pH 6.5) and twice the concentration of metabisulfite is necessary. The filtrate of the digest from the addition of trichloroacetic acid must be rendered alkaline and carefully readjusted to neutrality to ensure that the thiolactone is not present. Crystalline, homogeneous material has not been obtained from bacterial cells, although extracts with an activity of an order of magnitude near that of the preparations from liver tissue of mammals have been obtained.

[41]

DESULFHYDRASES AND DEHYDRASES

315

[41] Desulfhydrases and Dehydrases

By C. V. SMYTHE A. Desulfhydrases RCHSH

CHNH2COOH

-~ (RCH~CNH2COOH)

$

RCH~CO

COOH

~ H2S -k NH~

These enzymes take their name from the fact that they catalyze the removal of hydrogen sulfide from the substrate. The other product is thought to be an amino-unsaturated acid which is unstable and breaks down to the corresponding ~-keto acid and ammonia. Only the first step is believed to be enzymatic.

1. Cysteine Desulfhydrase The products produced by this enzyme are pyruvic acid, ammonia, and hydrogen sulfide. All three products can be determined readily, and for complete control all three should be determined. In special cases the determination of any one product may be sufficient. In most cases the hydrogen sulfide is the most characteristic product, so its determination is described in detail.

Assay Method The method 1 selected for description is adapted for use in standard Warburg vessels and for that reason may be more generally useful. Other methods such as the colorimetric one 2 based on measuring the methylene blue formed by reacting the sulfide with p-aminodimethylaniline or the one based on measuring the light scattered by colloidal lead sulfide may, of course, be used2 ,4 Principle. The hydrogen sulfide produced is absorbed in cadmium acetate with the production of insoluble cadmium sulfide. The sulfide is then dissolved in acid and oxidized to free sulfur by means of an iodine solution. The amount of iodine consumed is measured.

Reagents 1.0 M cadmium acetate. 0.2 N iodine in K I solution. 1.0 N HCI. 1 C. L. 3 C. 4 E.

V. Smythe, J. Biol. Chem. 142, 387 (1942). H. Almy, J. Am. Chem. Soe. 47, 1381 (1925). Fromageot and P. Desnuelle, Enzymologia 6, 80 (1939). A. Delwiche, J. Bacteriol 62, 717 (1951).

316

ENZYMES OF PROTEIN METABOLISM

[41]

0.01 N Na~S203. 0.10 M L-cysteine hydrochloride. M/15 phosphate buffer, pH 7.4 to 7.6. Enzyme solution, adjusted in concentration with phosphate buffer to give 25 to 200 units of activity per milliliter. (See definition below.)

Procedure. Place 2.0 ml. of enzyme solution in the main compartment of a regular conical Warburg vessel. Place 0.2 ml. of 0.1 M L-cysteine hydrochloride dissolved in water in the side arm of the vessel. Fill the gas space with nitrogen. After equilibration at 37 ° mix the solutions in the side arm and main compartment and allow the reaction to proceed for 2 hours. The inset will then contain yellow cadmium sulfide precipitate. Add to the inset 0.2 ml. of 0.2 N iodine solution and 0.1 ml. of 1.0 N HC1. Mix and allow the reaction to proceed until the cadmium sulfide is all dissolved. Transfer the contents of the inset with a pipet to a small titrating flask, wash the inset with water, and add the washings to the titration flask. Titrate the excess iodine with 0.01 N Na2S203. The difference in titration between a blank and the experimental flask represents the iodine used in oxidizing sulfide to free sulfur. Each milliliter of 0.01 N Na~S203 difference in titration equals 170 ~/of H2S. The solution in the main compartment of the vessel contains the other two products. The reaction may be stopped by lowering the pH to 5.0 or by adding trichloroacetic acid to a concentration of 5 % and deproteinizing the solution. The ammonia may be determined on the deproteinized solution by aerating it from an alkalinized solution into an HCI solution, nesslerizing 5 it, and comparing the color produced with standards. (See Vol. III [145].) The pyruvic acid may be determined by means of the hydrazone as described by Friedmann and Haugen 6 or enzymatically by means of carboxylase. 4 (See Vol. I I I [66].) Definition of Unit and Specific Activity. One unit of enzyme may be defined as that amount which produces 1 microgram of H~S under the above conditions. Specific activity is expressed 7 as units per milligram of Kj eldahl nitrogen. Purification Procedure

Step 1. Preparation of Crude Extract. 4 Fresh rat liver and twice its weight of 0.9% NaC1 (or Ringer phosphate) are ground with sand in a s p. B. Hawk and O. Bergeim, "Practical Physiological Chemistry," l l t h ed., p. 704, The Blakiston Co., Philadelphia, 1944. 6 T. E. Friedemann and G. E. Haugen, J. Biol. Chem. 147, 415 (1943). 7 j. M. Lawrence and C. V. Smythe, Arch. Biochem. 2, 225 (1943).

[41]

DESULFHYDRASES AND DEHYDRASES

317

mortar. Ordinary centrifugation yields an extract about equal in weight to the saline solution used. This extract contains the active enzyme and is relatively stable if kept at 0°. Step 2. Chloroform Treatment 8 of Extract. 4 The crude saline extract is treated with an equal volume of chloroform, and the mixture is vigorously shaken for 20 minutes at room temperature. After centrifugation the aqueous layer can be decanted. Step 3. Precipitation with Acetone. ~ The chloroform-treated extract is precipitated with 2 vol. of acetone at - 5 °. The precipitate is collected by filtration, washed with cold ether, and dried in vacuo. The dry powder is relatively stable, and active enzyme solutions can be prepared from it by extraction with water, saline, or phosphate buffer. Step 4. Adsorption on Ca~(P04)2 Gel. 7 The enzyme is adsorbed from solutions prepared as in step 3. If the volume of solution is about half that of the crude extract from which the precipitate was prepared, 12 rag. of Caa(PO4)~ gel per milliliter is sufficient. The enzyme is eluted by 80% glycerol at pH 8.0. TABLE I SUI~MARY OF PURIFICATION PROCEDURE

Product Saline extract of rat liver CHCl~--treated extract Solution from acetone precipitate Glycerol chlate from Ca.~(PO4)~

Activity, units/rag. N

Yield, % of original activity

25 70 97 376

100 81 57 40

Properties

Products prepared as above produce H2S from L-cystine as well as from L-cysteine. Probably the cystine is first reduced. Such preparations also produce H2S from homocysteine. This result may be due to contamination with a second enzyme (see below). They produce no H2S from D-cysteine, D-cystine, methionine, glutathione, thioglycollic acid, or a-amino-f~-thiolbutyric acid. The production of H2S from cysteine is reversible2 Optimal activity is at pH 7.4 to 7.6. Activity decreases gradually on both sides of the optimum. The action on cysteine is inhibited by other thiol compounds, by KCN, by As203 and by ketone reagents such as phenylhydrazine. s M. G. Sevag, Biochem. Z. 273, 419 (1934). 9 C. V. Smythe and D. Halliday, J. Biol. Chem. 144, 237 (1942).

318

ENZYMES OF PROTEIN METABOLISM

[41]

The enzyme occurs in all higher animals that have been tested. It is largely confined to the liver. Kidney and pancreas contain much smaller amounts. Other tissues contain very little or none. Tumors contain none. 10 Various bacteria contain highly active hydrogen sulfide-producing enzymes. Fromageot" now considers that the bacterial enzymes producing hydrogen sulfide from cysteine are essentially the same as the enzyme from animal tissues. They may differ in specificity, etc. 10 Pyridoxal phosphate has been established as the coenzyme 12,1~ of cysteine desulfhydrase. The stimulating effect of other ions 14 and compounds 3 on certain preparations has been described.

2. Homocysteine Desulfhydrase The reaction catalyzed by this enzyme is entirely analogous to that catalyzed by cysteine desulfhydrase. The acid produced is, of course, a-ketobutyric la instead of pyruvic. These two keto acids may be determined by the same method, so the products from this reaction may be determined in the same way as described above. As mentioned above, enzymes prepared as described there have some activity on homocysteine. It would be possible to ascribe this activity to lack of strict specificity by cysteine desulfhydrase, but Fromageot and Desnuelle 1~ found that different preparations from various animal sources had quite different ratios of activity on cysteine and homocysteine. They reported some preparations active on cysteine but inactive on homocysteine. They therefore concluded that two enzymes were involved. Later Kallio 13 prepared cell-free extracts from Proteus morganii and demonstrated that these extracts were considerably more active on homocysteine than on cysteine, although the cells from which the extracts were prepared showed the opposite ratio. He also showed that the curves for saturation with substrate were different and concluded that two enzymes were probably involved. The coenzyme appears to be the same. The preparation, assay, etc., for this enzyme from animal tissues is the same as described under cysteine desulfhydrase except, of course, that homocysteine is used in the assay instead of cysteine. For the preparation from a bacterial source Kallio's paper 13 is recommended. lo j. p. Greenstein, J. Natl. Cancer Inst. $, 491 (1943).

" C. Fromageot, in "The Enzymes" (J. B. Sumner and K. Myrbtick, eds.), Vol. 1, p. 1237, Academic Press, New York, 1951. 12 A. E. Braunshtein and R. M. Azarkh, Doklady Ak.ad. Nauk ~g.S.S.R. 71, 93 (1950); Chem. Abst. 44, 7900 (1950). la R. E. Kallio, J. Biol. Chem. 192, 371 (1951). 14F. Binkley, J. Biol. Chem. 150, 261 (1943). 1~C, Fromageot and P. Desnuelle, Compt. rend. 214, 647 (1942).

[41]

DESULFHYDRASES AND DEHYDRASES

319

3. Exocystine Desulfhydrase The reaction catalyzed by this enzyme is reported to be the formation of hydrogen sulfide from peptides which contain a terminal cystine.l~,~7The other product formed is thought to be a dehydropeptide. The dehydropeptide has not been isolated. Instead, pyruvic acid, ammonia, and whatever other product was attached to the cystine in the peptide have been isolated. It is assumed that a dehydropeptidase present in these tissues hydrolyzed the dehydropeptide resulting from the exocystine desulfhydrase action. That tissues possess dehydropeptidase activity has been amply demonstrated, but the possibility that the peptide hydrolysis may occur first and be followed by the action of cysteine desulfhydrase may not be completely ruled out. The action of the enzyme can be followed by measuring the products formed as described under cysteine desulfhydrase. Purification of the enzyme beyond the crude extract stage has not been reported. Crude extracts can be prepared as indicated under cysteine desulfhydrase.

B. Dehydrases RCHOHCHNH2COOH-~ (RCHzCNH2COOH) + H~O RCH2--CO--COOH + NH~ These enzymes take their name from the fact that the first step in the reaction is the removal of the elements of water from the substrate. It should be noted that the reaction is a strictly anaerobic one, and it should not be confused with aerobic deaminases of the same substrates. It is also to be noted that highly active L- and D-dehydrases occur in the same cell.

1. L-Serine and L-Threonine Dehydrase Although it is possible that these two substrates are attacked by different enzymes, present evidence 18,19 justifies considering that the same enzyme attacks both.

Assay Method Disregarding the postulated intermediate which appears to be unstable, the products from serine are pyruvic acid and ammonia, and from threonine, ~-ketobutyric acid and ammonia. These same products were le j . p. Greenstein a n d F. M. Leuthardt, J. Natl. Cancer Inst. §~ 209 (1944). 17 j . p. Greenstein a n d F. M. Leuthardt, J . Natl. Cancer Inst. 5, 223 (1944). 18 C. Yanofsky a n d J. L. Reissig, J. Biol. Chem. 202, 567 (1953). 1~ W. A. Wood a n d I. C. Gunsalus, J. Biol. Chem. 181, 171 (1949).

320

ENZYMES OF PROTEIN METABOLISM

[41]

considered under the desulfhydrases, and the methods described there are applicable here. Procedure. The assay procedure described by Yanofsky and Reissig TM is as follows. " E n z y m e assays were performed in a 1-ml. volume containing 3 X 10-3 M L-threonine (or 6 × 10-3 M L-serine), 10 ~, of calcium pyridoxal phosphate (assaying 28% pyridoxal phosphate), 0.5 ml. of 0.1 M pyrophosphate buffer at p H 9.3 and enzyme. Assay tubes were generally incubated at 37 ° for 20 minutes. The reaction was stopped b y the addition of 1 ml. of 10% trichloroacetic acid to each tube and the precipitate removed b y centrifugation. A 1.0-ml. portion of the supern a t a n t was then taken for either p y r u v a t e or k e t o b u t y r a t e determination. In some cases the complete sample was analyzed since the alkali added in the colorimetric determination was sufficient to dissolve all the precipitated protein. A control tube containing pyridoxal phosphate and substrate and a control tube with enzyme alone were run with each assay." Definition of Unit and Specific Activity.IS A unit of enzyme is defined as the a m o u n t required to form 0.1 micromole of a - k e t o b u t y r a t e or p y r u v a t e in 20 minutes under the conditions described above. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al. 2° Purification Procedure

The procedure described below is taken from the report of Yanofsky and Reissig. 18 Earlier workers prepared relatively crude preparations from animal tissues 21-23 and from several bacteria. 2~-27 Wood and Gunsalus 19 partially purified a similar enzyme from E. coli. Step 1. Preparation of Crude Extract. A wild-type strain (Em-5297a) of Neurospora crassa is grown in aerated bottles on minimal Neurospora medium 25 at 30 ° for 72 hours. The mycelium is collected by filtering through cheesecloth, washed twice with distilled water, and lyophilized. The powdered lyophilized solid is extracted with sixteen times its weight of 0.1 M phosphate buffer of p H 7.8 at 2 to 4 ° with constant shaking for 2 to 3 hours. The mixture is filtered through cheesecloth, and the filtrate 20O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 21E. Chargaff and D. B. Sprinson, J. Biol. Chem. 148, 249 (1943). ~2F. Binkley, J. Biol. Chem. 150, 261 (1943). 23E. Chargaff and D. B. Sprinson, J. Biol. Chem. 151, 273 (1943). 24E. F. Gale and M. Stephenson, Biochem. J. 32, 392 (1938). 2~H. C. Lichstein and W. W. Umbreit, J. Biol. Chem. 170, 423 (1947). ~s I-I. C. Lichstein and J. F. Christman, J. Biol. Chem. 175, 649 (1948). ~7D. E. Metzler and E. E. Snell, J. Biol. Chem. 198, 363 (1952). 28G. W. Beadle and E. L. Tatum, Am. J. Botany 32, 678 (1945).

[41]

DESULFHYDRASES AND DEHYDRASES

321

is then centrifuged at 12,000 r.p.m, for 30 minutes. The turbid supernatant is stored at - 1 5 °. Step 2. Treatment with Protamine Sulfate. Crude extract from step 1 (12 to 15 mg. of protein per milliliter) is adjusted to pH 7.2. One milliliter of a protamine sulfate solution (15 mg./ml, in phosphate buffer of pH 7.2) is added per 100 mg. of extract protein. The mixture is stirred for 15 minutes, centrifuged, and the supern~tant solution retained. Step 3. First Ammonium Sulfate Treatment. The supernatant solution from step 2 is made 0.34 saturated with (NH4)2S04, stirred, and centrifuged. The precipitate is retained and dissolved in half its original volume of 0.02 M phosphate buffer of pH 7.2. Step 4. First Calcium Phosphate Gel Treatment. To the solution from the preceding step 40 mg. of calcium phosphate gel (40 mg. dry weight per milliliter) is added for each 80 mg. of protein. The mixture is stirred and then centrifuged. The supernatant solution is then treated with twice the previous amount of calcium phosphate gel and again centrifugedl Step 5. Second Ammonium Sulfate Treatment. The supernatant solution from step 4 is made 0.24 saturated with (NH4)2S04. After 25 minutes the precipitate is removed by centrifugation and discarded. The supernatant solution is made 0.33 saturated with (NH4)2S04. After 25 minutes the precipitate is collected by centrifugation and dissolved in 0.02 M phosphate buffer of pH 7.2 in one-fifth the volume of the original crude extract. TABLE

II

SUM~\~ARY OF PURIFICATION PROCEDURE 15

Threonine dehydrase

Fraction Crude extract Supernatant after protamine treatment First (Ntt~)2S04 fraction Phosphate gel supernatant Second (Ntt4)2SO4 supernatant Phosphate gel supernatant

Ratio: Threonine Prounits / tein, Units/rag. Recovery, Serine mg. protein % units

Threonine dehydrase, units

Serine dehydrase, units

3500

940

1250

2.8

--

3.7

3280

850

815

4.0

94

3.9

2600

650

194

13.4

74

4.0

1940

500

97

20

55

3.9

1420

384

28

51

41

3.7

1320

350

20

66

38

3.8

322

ENZYMES OF PROTEIN METABOLISM

[41]

Step 6. Second Calcium Phosphate Gel Treatment. The solution from step 5 is treated with 4 mg. of calcium phosphate gel per 10 mg. of protein. After being stirred for 10 minutes the gel is removed and the treatment repeated. The supernatant solution is then dialyzed against 0.05 M phosphate buffer at pH 7.8 for 3 hours.

Properties The enzyme shows optimal activity in pyrophosphate buffer at pH 9.3 and in borate buffer at a slightly higher value. The activity drops rather sharply on the acid side of the optimum. Preparations prepared as described do not attack D-serine or D-threonine. They do not produce keto acids from a large group of amino acids including cysteine. They do have some action on tryptophan. The requirement for pyridoxal phosphate as coenzyme is thoroughly demonstrated in the paper quoted as well as in the previous publication by Reissig ~9 which also used Neurospora as enzyme source. The fact that other workers using enzymes from different sources lg,22-28,3° found other activators necessary for activity requires some further illumination. The amount of pyridoxal phosphate required for half maximal activity was found to be 4 X 10-~ M. The K~ values were found to be 5.5 X 10-3 M for L-serine and 3.3 × 10-8 M for L-threonine. 2. D-Serine and D-Threonine Dehydrase

The enzyme acting on these amino acids appears to be strictly analogous to that described above as acting on the corresponding L-acids. It has been studied chiefly in E. colt 3~ and in Neurospora crassa. 32 In both cases it occurs together with the enzyme specific for the corresponding L-amino acids.

Assay Method The products formed are the same as described above. The procedure for testing is the same except that D-serine or D-threonine is used as substrate.

Purification Procedure The most detailed report available is again due to Yanofsky, 82 and his procedure is given here in detail. ~9 j . L. Reissig, Arch. Biochem. and Biophys. 86, 234 (1952). ,0 V. R. Williams a n d J. F. Christman, J. Bacteriol. 65p 238 (1953). 31 D. E. Metzler a n d E. E. Snell, J. Biol. Chem. 198p 363 (1952). a2 C. Yanofsky, J. Biol. Chem. 198, 343 (1952).

[41]

DESULFHYDRASES AND DEHYDRASES

323

Step 1. Preparation of Crude Extract. This step is the same as that described above under L-serine and L-threonine dehydrase. Step 2. First Ammonium Sulfate Fraction. For each 100 ml. of crude extract 24.5 g. of solid (NH4)2SO4 is added with stirring. After 30 minutes the precipitate is removed by centrifugation, and 10.3 g. of (NH4)~S04 is added to the supernatant solution. After 30 minutes the precipitate is collected by centrifugation and taken up in three-tenths of its original volume of 0.05 M phosphate buffer of pH 7.8. Step 3. Acetone Fractionation. For each 30 ml. of solution from the preceding step 19 ml. of acetone ( - 1 0 °) is added. After 30 minutes the precipitate is removed and an additional 21 ml. of acetone is added to the supernatant solution. After 20 minutes the precipitate is collected and taken up in one-third its previous volume of phosphate buffer (one-tenth the volume of crude extract). Step ~. Second Ammonium Sulfate Fractionation. For each 10 ml. of solution from the preceding step 2.2 g. of (NH4)~S04 is added. The mixture is allowed to stand for 30 minutes and then centrifuged. An additional 1.2 g. of (NH4)~SO4 is added to the supernatant solution, and after 30 minutes the precipitate is collected by centrifugation. It is dissolved in one-half its previous volume (0.05 the volume of crude extract) of 0.1 M borate buffer of pH 8.2. The solution is dialyzed for 12 to 24 hours against 0.01 M borate buffer of the same pH. TABLE

III

SUMMARY OF PURIFICATION PROCEDURE 32

Serine dehydrase Fraction Crude extract First (NH4)~SO4 fractionation Acetone fractionation Second (NH4) 2SO4 fractionation

Serine dehydrase, Protein, units rag. 2800 2200 1850 1200

1500 350 115 19

Units/mg. protein

Recovery, %

1.9 6.3 16 63

79 66 43

Properties Pyridoxal phosphate is the coenzyme for this enzyme as well as for the one that acts on the corresponding L-amino acids and for the desulfhydrases that act on cysteine and homocysteine. The concentration required to give half maximal activity was found to be 3 X 10-6 M for the Neurospora enzyme and 1 X 10-e M for that from E. coll. In order to get

324

ENZYMES OF PROTEIN METABOLISM

[42]

enzyme preparations free of pyridoxal phosphate the mycelium was stored for several weeks at -15°. 83 No other activators were found. The enzyme prepared as described has no action on L-serine or L-threonine. The action on D-threonine is considerably slower than on D-serine. Keto acids are produced from DL-glutamic acid and DL-aspartic acid but not from the L-forms of these acids. Pyridoxal phosphate has no effect on the reaction with the dibasic acids. D-Amino acid oxidase does not appear to be present, for a number of other D-amino acids are not attacked. The Km value for D-serine was found to be 2.6 X 10-4 M for the Neurospora enzyme and 3 × 10-4 M for that from E. coli. Action of the enzyme is strongly inhibited by hydroxylamine, cyanide, L-cysteine, 8-hydroxyquinoline, and metal salts such as those of zinc, copper, and cobalt. 33 W. W. Umbreit, W. A. Wood, and I. C. Gunsalus, J. Biol. Chem. 165, 731 (1946).

[42] S u l f a t a s e s and D e s u l f i n a s e s By CLAUDE FROMAGEOT

I. Sulfatases The various enzymes which are collectively termed sulfatases are able to hydrolyze certain organic sulfate esters. According to the organic part of these esters, sulfatases are recognized as belonging to the following groups: arylsulfatases, alkylsulfatases, steroid sulfatases, glucosulfatases, chondrosulfatases, and myrosulfatases.

Assay Methods Sulfatases are studied by estimating one of the end products of hydrolysis: the liberated sulfuric acid or the organic part. Estimation of Liberated Sulfuric Acid. The more general methods are obviously the ones which concern the estimation of the liberated sulfuric acid. Dodgson and Spencer 1 have discussed several procedures. They describe a method which has proved satisfactory, within certain limits, for the study of sulfatases. Materials. BENZIDINE (" Pure," A.R.), twice recrystallized from 95 % ethanol (charcoal). For determining sulfate in tissue suspensions a 5% (w/v) solution of benzidine in ethanol is used. The solution is stored in a K. S. Dodgson and B. Spencer, Biochem. J. 55, 436 (1953).

[42]

SULFATASES AND DESULFINASES

325

warm room (otherwise the benzidine tends to crystallize) and is stable for 5 to 6 weeks. When determining sulfate in aqueous solution a 1% (w/v) ethanolic solution of benzidine is employed. All ethanol is filtered or redistilled immediately before use. Powdered glass is washed with distilled water, ethanol, and ether and then dried. CENTRIFUGE TUBES. For precipitating benzidine sulfate, 15-ml. centrifuge tubes of even taper with a tip of 4.5 mm. (i.d.) are used. All glassware is cleaned with nitric acid. TISSUE SUSPENSIONS. These are prepared according to Dodgson et al. ~ Determination of Sulfate in Aqueous Solution. In a 15-ml. centrifuge tube are placed 1 ml. of a solution of K~SO4 (containing from 10 to 70 ~,/ml. SO4--), 1 ml. of 20% (w/v) trichloroacetic acid, and 5 ml. of a 1% (w/v) cthanolic solution of benzidine. Mixing is by means of a let of air passing through a fine capillary which is subsequently washed with a few drops of ethanol. The tube is stoppered and allowed to stand for at least 2 hours at 0 °. A small pinch (3 to 5 mg.) of powdered glass is then added, and benzidine sulfate is separated by centrifuging at 2250 × g for 15 minutes. The supernatant is discarded. In the absence of powdered glass small particles constantly break away from the precipitate at this stage, but when powdered glass is present the precipitate packs down tightly on top of the glass and no such losses are experienced. The tube is inverted on filter paper and allowed to drain for 5 to 10 minutes. The mouth of the tube is wiped with filter paper, and the outside is washed with acetone. After standing for 1 minute on filter paper the mouth of the tube is washed with ethanol while the tube is held in an inverted position. After draining for a further 5 minutes on filter paper the mouth of the tube is again wiped with filter paper and about 10 ml. of ethanol is added, care being taken not to disturb the precipitate; otherwise large fragments break away which cannot effectively be washed. After the precipitate is broken up with a glass rod, the whole suspension is mixed and the glass rod is washed down with a little ethanol. The benzidine sulfate is separated by centrifuging, and after the supernatant solution is poured off the tube and contents are treated exactly as before except that, instead of adding 10 ml. of ethanol, the benzidine sulfate is dissolved in 3 ml. of N HC1. After a few minutes 1 ml. of a 0.1% (w/v) solution of NaNO2 is added. After mixing, 5 ml. of a 0.5% (w/v) solution of thymol in 7.5% (w/v) NaOH is added, and the tube is centrifuged to remove powdered glass. The intensity of the red color (~..... 500 m~) is measured against a reagent blank with the Hilger Spekker absorptiometer using blue glass filters (Chance 0.B.2) and 2.5-mm. cells. K. S. Dodgson, B. Spencer, and J. Thomas, Biochem. J. 53, 452 (1953).

326

ENZYMES OF PROTEIN METABOLISM

[49.]

The amount of sulfate is read from a calibration curve prepared by diazotizing and coupling known amounts of benzidine in similar volumes. In the range of 10 to 70 -y of SO~-- the calibration curve prepared in this way does not differ from that prepared by actual precipitation and determination of known amounts of K2S04. When present in amounts less than 10 ~, sulfate is not precipitated quantitatively.

Estimation of Sulfate in the Presence of Buffered Tissue Suspensions. At concentrations suitable for enzyme experiment, the amount of S04-present in tissue suspensions is usually less than 10 % It is therefore necessary to add sulfate to both tests and controls before precipitating with benzidine. Since sulfate inhibits certain sulfatases,3 the addition of sulfate is made after the incubation is complete. It is convenient to add the sulfate in the trichloroacetic acid solutions. In practice a control value of between 15 and 25 ~, SO4-- is used. A mixture of 0.6 ml. of acetate-buffered K~SO4 solution and 0.6 ml. of similarly buffered tissue suspension, suitably diluted, is placed in a 15-ml. stoppered centrifuge tube and mixed thoroughly. A buffered suspension without K2S04 is the control. After incubation 4.8 ml. of ethanol is added to the tube and the contents are mixed. Proteins and, in certain cases, polysaccharide material are precipitated, and, after standing for a few minutes to allow flocculation, the tube is capped and the precipitate separated by centrifugation. A 5-ml. portion of the supcrnatant is transferred to a 15-ml. centrifuge tube, and 1 ml. of a 25% (w/v) solution of trichloroacetic acid (containing about 15 ~, of SO4-- as K~SO4) followed by 1 ml. of a 5 % (w/v) solution of benzidine in ethanol is added to the tube. The contents are mixed by a stream of air as previously described. The tube is stoppered, allowed to stand overnight at 0 °, and the benzidine sulfate is separated and determined as before. The amount of sulfate in the test determination is read from the calibration curve, and the corresponding control value is then subtracted. Allowance is made for the contraction in volume which occurs when 1.2 ml. of an aqueous solution is mixed with 4.8 ml. of ethanol. 4 Sulfate can be recovered from a wide variety of tissue suspensions after incubation at 37 ° for 1 hour. Longer incubation periods do not affect the results, but unsatisfactory recoveries are obtained when the concentration of the tissue suspension in the incubation mixture exceeds

3% (w/v). EFFECT OF VARYING BUFFER CONCENTRATION AND P H . R e c o v e r i e s of

sulfate from incubated tissue suspensions in acetate buffers of varying molarity (0.1 to 0.75 M) and pH (4.2 to 7.2) are satisfactory. s A. B. Roy, Biochem. J. 65~ 653 (1953). 4 K. S. Dodgson and B. Spencer, Biochem. J. 65~ 444 (1953).

[42]

SULFATASES AND DESULFINASES EFFECT OF INORGANIC IONS ON SULFATE RECOVERIES.

327

K, Na, Mg, CN,

C1, and F at concentrations of 0.01 M, and Fe, Ca, and P04 at concentrations of 0.001 M, do not interfere with the recovery of sulfate from acetate-buffered suspensions. However, Fe, Ca, and P04 in higher concentrations cause considerable interference, and Ba, even in traces, renders the method invalid. EFFECT OF CERTAIN SULFATASE SUBSTRATES. Potassium p-acetylphenylsulfate (0.05 M), sodium chondroitinsulfate (1%, w/v), potassium myronate (0.01 M), and potassium glucose-6-sulfate (0.02 M) do not affect recoveries. Higher concentrations of glucose-6-sulfate result in high recoveries which are due to low blank values. Estimation of Liberated Organic Part

Arylsulfatases. The methods of estimation of the phenols liberated from arylsulfates are of two types: (1) using phenols which, in the anionic form, absorb light maximally at wavelengths in the visible or ultraviolet regions of the spectrum where the corresponding ethereal sulfates have negligible absorption; (2) using phenols which can be estimated colorimetrically after a chemical reaction which does not affect the respective arylsulfate. Methods of Type 1. The most convenient method is the one described by Dodgson et al., 2 as it has been applied to the study of arylsulfatase activity of rat tissues. The ice-cold enzyme solution (0.6 ml.), adjusted to the required pH, is pipetted into 15-ml. tapered centrifuge tubes and preincubated for 4 minutes before addition of 0.6 ml. of substrate solution (potassium p-acetylphenylsulfate dissolved in buffer at the same pH as the enzyme solution). The mixtures (1.2 ml.) are incubated at 37.5 ° for the desired period, and the enzyme action is stopped and protein precipitated by addition of 4.8 ml. of ethanol. After centrifuging, 1 ml. of N NaOH is added to 5 ml. of the clear supernatant, and the absorption of the liberated p-hydroxyacetophenone is measured in the spectrophotometer at 327.5 m~ against an ethanol blank. The estimations are duplicated, and controls, containing the enzyme solution with the substrate added after incubation followed immediately by ethanol, are run simultaneously. It is unnecessary to include controls in which substrate is incubated with buffer, since there is no hydrolysis of potassium p-acetylphenylsulfate at 37.5 ° under the experimental conditions established. Although the specific absorption by protein at 327.5 m~ is very slight, the ethanolic precipitation has been performed in order to clarify the incubation mixtures for the final measurement. The amount of p-hydroxyacetophenone liberated in the incubation

328

ENZYMES OF PROTEIN METABOLISM

[42]

mixture is given by the equation p-Hydroxyacetophenone -- ( E t -

Eo) × 43.86

where Et and Ec are the observed extinctions of the test and control solutions, respectively. ~ The percentage error involved in neglecting the decrease in absorption due to hydrolysis of the substrate can be ignored. Definition of Unit. One p-hydroxyacetophenone unit of arylsulfatase activity is that which liberates 1 ~, of p-hydroxyacetophenone in 1 hour from 0.007 M potassium p-acetyl phenylsulfate in the presence of 0.5 M acetate, pH 7.2, at 37.5 °. Remarks. (1) Arylsulfatases can be estimated with almost any phenolic ethereal sulfate hydrolyzed by sulfatase at a reasonable velocity, provided that the anion of the liberated phenol absorbs light at a different wavelength from the ethereal sulfate. For a discussion concerning the use of several phenolic ethereal sulfates, see Dodgson and Spencer 4 and Robinson et al. 5 Dipotassium 2-hydroxy-5-nitrophenylsulfate is also, in this respect, a particularly suitable substrate. 6 (2) The wide range of pH optima reported for arylsulfatases can be attributed largely to the use of different sources of the enzymes by the various authors. The fact that certain arylsulfatases are more or less inhibited by phosphates or by other substances points to the importance of the buffer. One of the more often used buffers is 0.5 M acetate-acetic acid. Methods of Type 2. The best procedure seems to be the one described by Abbott 7 as applied to arylsulfatase from takadiastase. Folin's reagent for the determination of phenol is used essentially as in the Gutman and Gutman modification s of the King and Armstrong procedure 9 for determining phenol in the phosphatase method. Reagents

Buffer-substrate. 0.02 M potassium phenylsulfate in 0.2 M sodium acetate-acetic acid at pH 6.2. Equal parts of solutions A and B are mixed as needed, and the pH is checked. Solution A. 2.12 g. of potassium phenylsulfate is dissolved in water and diluted to 250 ml. (kept cold). Solution B. 32.8 g. of sodium acetate (c.p., anhydrous) is dissolved in water and diluted to 1 1. Thirty milliliters of 0.4 M acetic acid 5D. Robinson, J. N. Smith, B. Spencer, and R. T. Williams, Biochem. J. 51, 202 (1952). 6A. B. Roy, Biochem. J. 57~ 465 (1954). 7L. D. Abbott, Arch. Biochem. 15~ 205 (1947). 8 E. B. Gutman and A. B. Gutman, J. Biol. Chem. 136, 201 (1940). 9 E. J. King and A. R. Armstrong, Can. Med. Assoc. J. 81~ 376 (1934).

[42]

SULFATASES AND DESULFINASES

329

is added, and the pH is checked. If necessary, the pH is adjusted to 6.2 with NaOH or acetic acid. Phenol reagent of Folin and Ciocalt~u. 1° This is diluted for use with 2 vol. of water. Phenol standard. A dilute phenol standard containing exactly 10 mg. of phenol per 100 ml. is made from the standardized stock solution (kept cold). Standard phenol solution plus reagent. One milliliter of the dilute phenol standard plus 3 ml. of diluted phenol reagent is made to a volume of 10 ml. in a graduated Klett tube. This solution is prepared for use just before color development. Procedure. Four test tubes, two for control (C) and two for test (T), each containing 10 ml. of buffer-substrate solution, and two test tubes (B), each containing buffer only (solution B and water, l : l ) , are kept in a water bath controlled at 50° for 5 minutes. After this time four tubes (T and B) are removed, and exactly 0.5 ml. of solution (or serum) to be tested is added to each tube. All tubes are replaced in the water bath at 50 ° after the contents have been carefully mixed and the tubes stoppered. One hour from the time the enzyme solution has been added to set T, or after the time interval used if longer incubations are found necessary, the four tubes containing the enzyme (T and B) are removed from the water bath; 4.5 ml. of diluted phenol reagent is added at once to T and to B and the contents of each tube mixed. One-half milliliter of enzyme solution, immediately followed by 4.5 ml. of diluted phenol reagent, is added to each control tube (C) and mixed. All tubes are then centrifuged or filtered. Exactly 6 ml. of each filtrate is transferred to test tubes for color development. These tubes with the standard phenol solution plus reagent are placed in a 37.5 ° water bath for 5 minutes, removed, and 20% sodium carbonate is added to each tube, 2.5 ml. to the standard tube, 1.5 ml. to each of the others. The contents of each are mixed immediately, and all tubes are replaced in the water bath (37.5 °) for 5 minutes to develop color. The tubes are then removed and, after standing 30 minutes at room temperature, are read in a Klett-Summerson photoelectric colorimeter (filter 42). Calculation. The difference (D) between the reading of the test and control filtrates represents the phenol liberated from the substrate by the enzyme in the time interval used. The reading of the standard (S) represents 0.1 mg. of phenol; the milligrams of phenol liberated by the sample taken is given by the following equation. D 7_5 15 D Mg. phenol = ~ × 0.1 × ~ × -6- = -S × 0.15 10 O. Folin and V. Ciocalt6u, J. Biol. Chem. 75, 627 (1927).

330

ENZYMES OF PROTEIN METABOLISM

[42]

If the sample is 0.5 ml. and calculation to 100 ml. of enzyme solution (or D serum) is desired, the above equation becomes ~ X 30 and represents the milligrams of phenol liberated per 100 ml. of solution tested during the time interval used. Definition of Unit. One phenol sulfatase activity unit is defined as that degree of phenolsulfatase activity which, at 50 °, will liberate 1 mg. of phenol in 1 hour from 0.02 M potassium phenylsulfate in 0.2 M sodium acetate-acetic acid at pH 6.2. Other Sulfatases. Alkylsulfatases. Substrate, sodium dichlorophenoxyethylsulfate. The measurement of activity is based on the solubility of the methylene blue-sulfate ester complex in chloroform. This blue complex is estimated colorimetrically. 11 Steroid Sulfatases. Substrate, sodium dehydroepiandrosterone sulfate. The liberated free ketosteroid is estimated by means of the Zimmermann reaction. 12,13 Glucosulfatases. Substrate, glucose-6-sulfate. The glucose liberated is estimated by quantitative fermentation by yeast; 14 the method seems complicated and time consuming. Preparation Preparations of Arylsulfatases A and B from Ox Liver (according to Roye,15.16). Fresh ox liver is cut into 2-cm. cubes, and 100-g. portions are homogenized with 500 ml. of acetone at 0 ° in a chilled blendor. The mixture is homogenized for 1 minute, filtered, and washed successively with 500-ml. portions of acetone and ether at 5 °. After being sucked dry the filter cake is broken: up and dried in vacuo over P205. The material is sufficiently d r y to powder and sieve within a few hours. When kept in vacuo at room temperature, the powder retains its enzymic activity for many weeks. Unfractionated Extract. Sixty grams of acetone powder is incubated for 1 hour at 37 ° with 400 ml. of water, and the insoluble material is removed by centrifuging. The debris is washed with a further 150 ml. of water, and the combined supernatants are clarified by centrifuging for 30 minutes at 8000 X g. Preparation of Sulfatase A.15,16 STAGE A. To 400 ml. of unfractionated extract is added 40 ml. of 0.2 M phosphate buffer, pH 7.0 (final pH 6.8), 11 A. J. Vlitos, Contribs. Boyce Thompson Inst. 17, 127 (1953). 12 S. R. Stitch a n d I. D. K. Halkerston, Nature 172~ 398 (1953). ~3 A. B. Roy, Biochim. et Biophys. Acta lfi, 300 (1954). 14 T. Soda, J. Fac. Sci. Tokyo Univ. 3, 150 (1936). 15 A. B. Roy, Biochem. J. 58, 12 (1953). is A. B. Roy, Biachem. J. §5~ 653 (1953).

[42]

SULFATASES AND DESULFINASES

331

and the volume is made up to 450 ml. After being brought to 0 °, 340 ml. of cold acetone is slowly added, the temperature being lowered to - 9 ° during the process. After equilibration at - 9 ° for 30 minutes, the precipitate containing sulfatase B is centrifuged off at the same temperature and removed. To the supernatant (650 ml.) at - 9 ° 15 ml. of the phosphate buffer is added, followed by a further 280 ml. of acetone. The mixture is equilibrated as before, and the precipitate of crude sulfatase A is centrifuged off at - 9 °, dissolved in 75 ml. of water and dialyzed overnight to give approximately 140 ml. of a clear red solution of sulfatase A. STAGE A-1. To 140 ml. of A is added 10 ml. of 0.5 M sodium acetate, pH 6.5 (final pH 7.0), and 1.5 ml. of 0.3 M CaC12. The solution is then precipitated with 115 ml. of acetone at - 9 ° as described above, and the inactive precipitate is discarded; the supernatant is kept at - 9 ° and treated with a further 115 ml. of acetone; the precipitate is centrifuged off as before, dissolved in 25 ml. of water and dialyzed overnight to give 40 ml. of a solution of sulfatase A-1. SUMMARY OF TYPICAL PREPARATION OF SULFATASE A a

Stage

Total activity, units

Activity, unit/rag. N

126,000 150,000 120,000 90,000 40,000 29,000

38 330 3000 -6700 40,000

Unfractionated extract A A-1 A-2 A-3 A-4 A. B. Roy, Biochem. J. 55p 653 (1953).

STAGE A-2. Sufficient solid (NH4)2SO4 is added to A-1 to make the solution 30% saturated (NH4)2SO4. After standing for 5 hours, the active precipitate is centrifuged off, dissolved in 10 ml. of water, and dialyzed overnight. STAGE A-3. A-2 is 20% saturated with (NH4)2SO4, and the inactive precipitate is centrifuged off. The supernatant is then 40% saturated with (NH4)2SO4, and the precipitate is separated, dissolved in 5 ml. of water and dialyzed overnight, giving 8 ml. of a clear, faintly strawcolored liquid. STAGE A-4. The clear solution of A-3 is dialyzed at 0 ° against twelve or more changes of distilled water for 48 hours, and the white, flocculent precipitate of the enzyme is centrifuged off. This is extracted for 24 hours

332

ENZYMES OF PROTEIN METABOLISM

[49.]

at 0 ° with 4 ml. of 0.1 M NaC1, during which time the bulk of the enzyme passes into solution. The insoluble residue is centrifuged off, washed with 0.1 M NaC1, and the combined supernatants stored at - 1 0 °. The concentrated enzyme solutions so obtained are perfectly stable, even at 0 °, but when diluted to a concentration suitable for assay the enzyme rapidly loses its activity. When so diluted, about 50% inactivation occurs on standing for 24 hours at 0 ° in the case of sulfatase A-4. Less highly purified preparations are considerably more stable. The number of sulfatase A units is defined as -y nitrocatechol (raised to the 2/~ power) liberated under the standard conditions described. 1~ Preparation of Sulfatase B. 6 Sulfatase B is obtained by the precipitation of 800 ml. of the unfractionated aqueous extract with 43% (v/v) acetoue in phosphate buffer, pH 7, at - 9 ° as described above. The precipitate so obtained is dissolved in water and dialyzed overnight against running tap water at room temperature, giving 400 ml. of sulfatase B (700,000 total units; 25 units/mg, protein). STAGE B-1. To 400 ml. of sulfatase B are added 25 ml. of 0.5 M sodium acetate, pH 6.5, and 4.5 ml. of 0.3 M CaC12. The enzyme is then precipitated by the addition of 220 ml. of acetone (34%, v/v, final concentration), the temperature being lowered to - 9 ° during the process. After equilibration at that temperature the precipitate is centrifuged off, dissolved in 100 ml. of water, and dialyzed as above. A copious inactive precipitate is separated; 250 ml. supernatant was obtained, containing sulfatase B (stage B-l; 500,000 units; 36 units/mg, protein). STAGE B-2. 250 ml. of B-1 is made 0.3 saturated with respect to (NH4)2S04 by the slow addition of 55 g. of solid (NH4)2SO4. After standing a few hours the precipitate is centrifuged off and discarded. The supernatant is made 0.5 saturated with respect to (NH,)2S04 by the addition of the calculated amount of solid, and after equilibration the active precipitate is centrifuged off, dissolved in water, and dialyzed overnight. This (NH4)2SO4 fractionation is then repeated on the dialyzate, giving a clear solution of sulfatase B-2 (200,000 total units; 190 units/mg. protein). Preparation of Sulfatases from Other Sources. Arylsulfatases from marine mollusks (Patella vulgata, Littorina littorea, etc.); see Dodgson et al. 17,1s Steroid sulfatase from Patella vulgata; see Roy. 1~ 16~ The enzymic activity is directly proportional to the enzyme concentration raised to the power intermediate between the first and second (approximately the power ~7 K. S. Dodgson, J. I. M. Lewis, and B. Spencer, Biochem. J. 55, 253 (1953). ~s K. S. Dodgson and B. Spencer, Biochem. J. §§, 315 (1953).

[42]

SULFATASES AND DESULFINASES

333

II. Desulfinases In animal tissues 19 and in bacteria 2° there are enzymic systems the activity of which results in the liberation of sulfite from cysteinesulfin~c acid. As part of these systems are transaminases which transform cysteinesulfinic acid into 2-sulfinylpyruvic acid; it is the latter which is decomposed according to the reaction HOOC.CO.CH2.SO2H--~ HOOC.CO.CH3 ~- SO2(-* SO,H2) At present it is not possible to decide whether desulfinases really exist, for we do not know whether the desulfination reaction is catalyzed enzymically or occurs spontaneously. Nevertheless it is only this desulfination reaction which will be considered in this discussion.

Assay Method The enzymic reaction on cysteinesulfinic acid is carried out anaerobically in flasks as represented by Fig. 1. As an example, each flask contains 25 ml. of M / 3 0 phosphate buffer, pH 7.3, in which are dissolved MgCl: (10-3 M), sodium cysteinesulfinate (10-2 M), ammonium ~-ketoglutarate (10 -2 M), and the enzymic preparation. The whole is maintained under pure nitrogen at 35 ° for 2 hours. The reaction is stopped by introduction of pure phosphoric acid (2 ml. per 25 ml. of reaction mixture) without any air being allowed to enter the flask. SOs is displaced by a nitrogen current, the flask being progressively heated up to the boiling point. It is collected in 0.1 N NaOH and titrated as usual by iodine and thiosulfate. If necessary, the sulfate formed by oxidation of SO3Na2 by iodine can be quantitatively estimated either as barium sulfate or as benzidine sulfate Fro. 1. Flask for assay (see pp. 325-326). of desulfinases.-~I 19F. Chatagner, B. Bergeret, and C. Fromageot, Biochim. et Biophys. Acta 9, 340 (1952). 20E. B. Kearney and T. P. Singer, Biochim. et Biophys. Acta 11, 276 (1953). ~1C. Fromageot, F. Chatagner, and B. Bergeret, Biochim. et Biophys. Acta 2p 294 (1948).

334

ENZYMES OF PROTEIN METABOLISM

[43]

[43] Rhodanese C N - + $203-- -o CNS- + SO3--

By B. H. SSR~O

Assay Method Principle. All published methods are based on the colorimetric determination of the thiocyanate formed in the reaction. The red color obtained in the presence of ferric ions is conveniently used for this purpose. The appearance of an interfering blue color (due to an iron-thiosulfate complex) is prevented in the present method 1 by the presence of formaldehyde. The method given here is applicable to rhodanese preparations of any degree of purity. Reagents 0.125 M Na2S203. 0.5% bovine serum albumin (stock solution). 0.20 M KH2P04. O.25 M KCN. 38% formaldehyde. Ferric nitrate reagent. 100 g. of Fe(NO3)~-9H20 + 200 ml. of 65% HN03 per 1000 ml. Enzyme. Dilute the enzyme in the presence of 0.0125 M thiosulfate and 0.025 % albumin (which protect the enzyme against inactivation by dilution) to obtain 0.3 to 1.6 R.U./ml. (See definition below.)

Procedure. One milliliter of Na~S~03, 0.5 ml. of KH2PO, and 0.5 ml. of KCN are mixed in a 50-ml. Erlenmeyer flask. One-half milliliter of the enzyme is added, and the reaction is stopped after 5 minutes at 20 ° by the addition of 0.5 ml. of 38% formaldehyde. Then 2.5 ml. of ferric nitrate reagent and 25 ml. of distilled water are added, and the optical density at 460 m~ is determined. One microequivalent of thiocyanate in the test gives the optical density, 0.104. The color is stable for at least 1 hour. A blank determination is always carried out by adding the formaldehyde to the test before the enzyme. With crude extracts or tissue homogenates it is necessary to remove the interfering turbidity by centrifugation or filtration before the optical density is determined. Definition of Unit and Specific Activity. One rhodanese unit (R.U.) is defined as that amount of enzyme which forms 10 microequivalents of 1B. H. SSrbo, Acta Chem. Scand. 7, 1129 (1953).

[43]

RHODANESE

335

thiocyanate under the above conditions. Specific activity is expressed as rhodanese units per milligram dry weight. With purified enzyme preparations protein is determined according to Bticher 2 instead of the dry weight. Purification Procedure

Crystalline rhodanese has repeatedly been obtained in this laboratory by the following procedure.1 Step 1. Extraction. Beef liver (fresh or frozen material can be used) is homogenized in a blendor with 2.5 1. of tap water per kilogram of liver, and 100 ml. of basic lead acetate solution (20%) is then added. The obtained precipitate is centrifuged off and discarded. Step 2. Fractionation with Ammonium Sulfate at pH 8.8. The following step is carried out in the cold room (+4°). Ammonium sulfate is added to 0.40 M concentration, and the pH is adjusted to 3.8 with 1 M HC1. The inactive precipitate is removed (by centrifugation or by suction filtration with the aid of Hyflo Super-Cel) and discarded. The ammonium sulfate concentration in the filtrate is raised to 1.33 M, and the obtained precipitate is removed after 3 hours and discarded. The enzyme is precipitated by raising the ammonium sulfate concentration to 1.91 M and is removed as before after 3 hours. The precipitate is suspended in a solution 0.05 M with respect to thiosulfate and Na2HPO4, with 60 ml./1. of fraction 1. Step 8. Fractionation with Ammoniacal Ammonium Sulfate. To fraction 2 is added 1.3 vol. of 3.25 M ammonium sulfate, adjusted to pH 7.9 with ammonia. After 3 hours at room temperature the precipitate is centrifuged off and discarded, and the enzyme is then precipitated from the supernatant by the addition of 100 g. of ammonium sulfate per liter. The precipitate is left in the cold room overnight and is then removed by centrifugation and dissolved in 0.01 M NaAc (1/~ vol. of fraction 2). Step 3. Dialysis. The remaining ammonium sulfate in fraction 3 is removed by dialysis against 0.01 M NaAc in the cold room. Step 5. Acetone Fractionation. The following step is carried out at - 5 °. Fraction 4 is adjusted with 0.1 M HAc to pH 4.9, and acetone is then gradually added to 35% by volume. The precipitate is removed by centrifugation, and the enzyme is precipitated from the supernatant by raising the acetone concentration to 50 %. The active precipitate is centrifuged off and dissolved in 0.01 M NaAc (1/~ vol. of fraction 4). If the first acetone precipitate contains more than 20% of the activity in fraction 4, it should be refractionated with acetone. 2 T. Biicher, Biochim. et Biophys. Acta 1, 292 (1947).

336

ENZYMES OF PROTEIN METABOLISM

[43]

Step 6. Dialysis. The remaining acetone in fraction 5 is removed by dialysis in the cold room against 0.01 M NaAc. Step 7. Ammonium Sulfate Fractionation at pH 4.5. The pH of fraction 6 is adjusted to 4.5 with 0.1 M HAc, and the enzyme is precipitated by the addition of an equal volume of 3.78 M ammonium sulfate. After 3 hours at room temperature the precipitate is centrifuged off and dissolved in 1 M ammonium sulfate of pH 7.8 (0.1 vol. of fraction 6). Step 8. Crystallization. The enzyme in fraction 7 is ready for crystallization if it has a specific activity of 200 R.U./mg. or better. Fraction 7 is adjusted to pH 7.8, and the enzyme is brought to crystallization by gradually increasing the concentration of ammonium sulfate. The best way to crystallize the enzyme is, however, to precipitate it with ammonium sulfate at pH 7.8 and then dissolve the amorphous precipitate in a small volume of 1 M ammonium sulfate, pH 7.8, at which point the enzyme immediately crystallizes. It can be recrystallized in the same way. The enzyme crystallizes in the form of rectangular plates or elongated prisms.

Properties Physicochemical Properties. When crystalline rhodanese was investigated in the Spinco ultracentrifuge, only one homogenous peak was observed. The molecular weight of 37,000 was obtained from the following data: sedimentation constant S, 3.0; diffusion constant, 7.5 × 10-7; and partial specific volume, 0.74. The enzyme showed a single peak and migrated anodically on electrophoresis in phosphate buffer at pH 7.4. At pH 6.5 and 5.5 partial denaturation occurred and the enzyme was no longer homogenous. The absorption spectrum of the crystalline enzyme showed only the usual protein band at 280 m~, with an extinction coefficient of 1.75/mg./ml./cm. at this wavelength. Specificity. The purified enzyme can use thiosulfate and different thiosulfonates 3 as sulfur donors but not colloidal sulfur 4 or other sulfur-containing compounds. Effect of Substrate Concentration, pH and Temperature. The optimal concentration of both thiosulfate and cyanide in the enzymatic reaction is 0.05 M in the case of the crystalline beef liver enzyme. 8 The optimal pH is 8.6, and the optimal temperature 50°. The apparent heat of activation is 7900 cal. These results differ somewhat from those reported by Saunders and Himwich 5 for rhodanese from other sources. 3 B. H. SSrbo, Acta Chem. Scand. 7, 1137 (1953). 4 B. H. SSrbo, Acta Chem. Scand. 7~ 32 (1953). 5 j . p. Saunders a n d W. A. Himwich, Am. J. Physiol. 163, 404 (1950).

[44]

GLUTAMINE

SYNTHESIS

337

Inhibitors. As rhodanese is strongly inhibited by sulfite and by incubation with cyanide in the absence of thiosulfate, it was suggested that the active group in the enzyme is a disulfide bond. 6 The enzyme is not inhibited by thiosemicarbazide, which makes the assumption of an active carbonyl group unlikely. Heavy metal enzyme inhibitors (except cyanide) do not inhibit the enzyme. 7 The enzyme is inhibited by some sulfhydryl reagents 5,7 but only incompletely and at rather high concerttrations of the inhibitors (0.001 M). SUMMARY OF PURIFICATION t)ROCEDURE a

Fraction 1. 2. 3. 4. 5. 6. 7. 8. 9.

Extract (NH4)2S04, p H 3.8 (NH4)2SO4, alc. Dialyzed Acetone Dialyzed (NH4)~SO4, pH 4.5 Crystals Recrystallized

Total volume, ml.

Total units, thousands

Specific activity, R.U./mg.

Yield, %

16,000 875 218 245 110 122 17.3 6.2 6.5

396 177 130 130 86.7 82.7 58.1 45.8 33.5

1.07 16.8 34.2 34.2 82.7 -201 257 267

100 45 33 33 22 21 14.7 11.5 8.5

B. H. SOrbo, Acta Chem. Scand. 7, 1129 (1953). e B. H. SSrbo, Acta Chem. Scan& 5, 1218 (1951). 7 B. H. SSrbo, Acta Chem. Scand. 5, 724 (1951).

[44] G l u t a m i n e S y n t h e s i s Glutamic acid W ATP + Ammonia--* Glutamine -t- ADP ~- Phosphate

By W. H. ELLIOTT The enzyme catalyzing the above reaction is called glutamine synthetase.

Assay Method Principle. The method devised by Speck I and Elliott 2 is based on the fact that the enzyme is capable of forming ~,-glutamyl hydroxamic acid ' J. F. Speck, J. Biol. Chem. 179, 1405 (1949). 2 W. H. Elliott, Biochem. J. 49, 106 (1951).

338

ENZYMES OF PROTEIN METABOLISM

[44]

(GHA) when ammonia in the above reaction is replaced by hydroxylamine. RCOOH Jr A T P -k NH~OH -~ R C O N H O H q- A D P q- H3P04 RCOOH = glutamic acid The hydroxamic acid is estimated by a slight modification of the method described by Lipmann and Turtle. 3 Two other methods of assay may be used. One of these depends on the measurement of phosphate released from A T P 1.2 but can be applied only to extracts free from ATP-ase. The other method is the direct estimation of glutamine formed.4 Reagents

0.05 M sodium ATP, pH 7.2. 0.5 M sodium glutamate, pH 7.2. M magnesium sulfate. M cysteine. The hydrochloride is neutralized with NaOH. 0.5 M glyoxaline-HC1 buffer, pH 7.2. M hydroxylamine. 5 ml. of a stock solution of 2 M NH~OH.HC15 is neutralized to pH 7.2 with 2 N NaOH and the volume adjusted to 10 ml. with water. Neutral solutions should be prepared freshly before use. Ferric chloride reagent. Equal volumes of 10% FeC13.6H20 in 0.2 N HC1, 24% TCA, and 50% (v/v) HC1 are mixed together. Procedure. Incubations are carried out at 30 ° in 12-ml. centrifuge tubes containing the following mixture: buffer, 0.5 ml.; ATP, 0.5 ml. ; glutamate, 0.5 ml.; MgS04, 0.1 ml. ; NH2OH, 0.1 ml.; cysteine, 60.1 ml.; enzyme q- H20 to final volume of 2.25 ml. The amount of enzyme is adjusted so that not more than 3 micromoles of GHA is formed, under which condition the amount of hydroxamic acid produced is proportional to enzyme added. After incubation for 20 minutes, 0.75 ml. of ferric chloride reagent is added to each tube, and the protein is removed by centrifuging. The hydroxamic acid in the supernatant is estimated by measuring the absorption in a photometer (Klett filter 54 or Evelyn filter 540). The main absorption is between 480 and 540 mg. Synthetic GHA 7 is used as standard.

3F. Lipmann and L. C. Tuttle, J. Biol. Chem. 159, 21 (1945); see Vol. III [39]. 4j. F. Speck, J. Biol. Chem. 179, 1387 (1949). Commercial NH2OH.HC1 is recrystallized from water. 6Cysteine activates only after certain steps in the enzyme purification. 7 N. Grossowicz, E. Wainfan, E. Borek, and H. Waelsch, J. Biol. Chem. 187, 111 (1950); see also Vol. II [36].

[44]

GLUTAMINE SYNTHESIS

339

Definition of Unit and Specific Activity. In the table one unit of enzyme is defined as that amount which produces, under the standard test conditions, 1.15 micromoles of GHA (equivalent to a Klett-Summerson photometer reading of 100). Specific activity is defined as units per milligram of protein, the latter being determined turbidimetrically. 8 Application of Assay Method to Crude Tissue Preparations. The method gives low values with crude tissue extracts, particularly those rich in ATP-ase. This is due to inhibition of glutamine synthetase by the ADP produced (see section on "Properties"). In addition, Schou et al. 9 have shown that extracts of Proteus vulgaris hydrolyze GHA. Purification Procedure

Purification from Sheep Brain. 2 This method, which gives a preparation almost free from ATP-ase, is easily reproducible and is recommended where an enzyme of high purity is not essential. A similar method using pigeon liver has been described by Speck.1 Acetone-dried cerebral cortex of sheep brain is extracted for 10 minutes with 10 vol. of distilled water with gentle stirring. The time of extraction is critical. After the suspension is centrifuged, the supernatant is filtered through cotton wool and cooled to 0°. Then 0.2 vol. of 0.1 M acetate buffer, pH 4.2, is added. The precipitate is centrifuged down, washed twice by resuspending in cold distilled water, and finally redissolved in water (half the original volume of extract) by the careful addition of 0.1 N NaOH to pH 6.8. A fourfold purification with 70 % recovery is achieved. Purification from Pea Seeds. 8 This method gives a highly purified preparation and can be used for large-scale isolation of the enzyme. The method has been repeated successfully a number of times by the author, and in another laboratory. Step I. Extraction. Dry green pea seeds (dwarf Blue Bantam variety) are pulverized to a fine powder in a hammer mill. Eighteen kilograms of the powder is added with stirring to 1441. of cold 0.1 M NaHCO3, and the extraction is continued for 30 minutes. Next 3.7 1. of 2 M MgSO4 is stirred in, and the precipitate is allowed to settle at 0 ° overnight.l° The supernatant is then decanted off, and the remaining suspension is centrifuged. (To avoid centrifuging large volumes the latter may be rejected with a loss of 25% of the enzyme.) The supernatants are combined. Step 2. Ammonium Sulfate Fractionation. The extract is adjusted to pH 6.5 with 2 M KH~PO4, and ammonium sulphate (300 g./1.) is added 8 w. H. Elliott, J. Biol. Chem. 201~ 661 (1953). g M. Schou, N. Grossowicz,and H. Waelseh, J. Biol. Chem. 192, 187 (1951). 10With smaller volumes the precipitate may be centrifuged off after 30 minutes.

340

ENZYMES OF PROTEIN METABOLISM

[44]

with stirring. After settling at 0 ° overnight ~° the precipitate is coliected b y decanting the s u p e r n a t a n t and centrifuging the remaining suspension. T h e precipitate is suspended in 6 1. of water, the p H adjusted to 7.2 with 2 M K2HPO4, and dialyzed for 36 hours against three changes of 20 1. of distilled water at 0 °. Step 3. Treatment with Protamine. T h e dialyzed extract is t r e a t e d with 2 % p r o t a m i n e sulfate solution until no further precipitate is formed. A b o u t 2 1. is required. T h e inactive precipitate is centrifuged off and discarded. Step 4. Nucleic Acid Precipitation. One-liter aliquots of the supern a t a n t f r o m step 3 are t r e a t e d at 0 ° with 10 ml. of M acetic acid (to p H 5.1) followed b y 60 ml. of a 2 % solution of nucleic acid adjusted to p H 5.5 with K O H . (The a m o u n t of nucleic acid required m a y v a r y with different batches of enzyme. Trial fractionation is therefore carried out on a 100-ml. aliquot to determine the m i n i m u m a m o u n t of nucleic acid needed to precipitate the e n z y m e completely.) T h e precipitate is centrifuged off at 0 ° and finely suspended in a b o u t 20 ml. of water. T h e p H is adjusted to 7.3 with M K2HP04, and the suspension stirred for 15 minutes. After centrifuging, the s u p e r n a t a n t is collected, and the precipitate is re-extracted with 20 ml. of 0.01 M K2HPO4. T h e milky s u p e r n a t a n t s are combined. Step 5. Second Ammonium Sulfate Fractionation. F o u r hundred milliliters of e n z y m e solution is mixed with 16 ml. of M potassium p h o s p h a t e SUM:M:ARY OF PURIFICATION PROCEDURE FOR PEA ENZYME

Fraction

Total Total units (thou- Protein, Specific Recovery, volume, Units/ml. ~ sands) mg./ml, activity % ml.

1. Extraction 109,000 2. (NH4)2SO4pptn. 8,700 3. Protamine fractionation 10,000 4. Nucleic acid pptn. 400 5. Second (NH4)~SO~ pptn. 75 6. Second nucleic acid pptn. 20

2.3 11.4

270 99

26 47

0.1 0.23

-30

12.1 227

121 91

23 40

0.53 5.7

37b 27

590

44

6.1

1760

35

8

97

13

220

11

These values were obtained from assays at pH 7.8. The activity will be slightly higher at pH 7.2, which is used in the assay method described above. b An increase in activity is usually obtained with protamine.

[44]

GLUTAMINE SYNTHESIS

341

buffer, pH 7.4, followed by 288 ml. of saturated ammonium sulfate solution. All solutions are previously cooled to 0 °. After standing for 15 minutes the precipitate is removed by centrifuging and a further 240 ml. of ammonium sulfate solution is added to the supernatant. After 20 minutes the precipitate is centrifuged down and redissolved in cold water with the addition of a few drops of M K~HPO4 to pH 7.3. The solution is dialyzed at 0 ° against three changes of 4 1. of distilled water for 24 hours. The precipitate which forms is centrifuged off and the supernatant collected. Step 6. Second Nucleic Acid Precipitation. Seventy milliliters of dialyzed solution is treated at 0° with 7.0 ml. of 1% potassium nucleate solution followed by 1.3 ml. of 0.2 M acetic acid. (Trial fractionation on a small scale is again required to determine the minimum amount of acetic acid required for enzyme precipitation.) The precipitate is collected and dissolved in 0.01 M phosphate buffer, pH 7.3.

Properties During the preparation described above, glutamine synthetase activity is almost exactly paralleled by glutamotransferase activity. The possibility exists that both activities are catalyzed by the same enzyme. Glutamotransferase is discussed elsewhere. 7 Studies on glutamine synthetase from sheep brain, pigeon liver, and peas have not revealed major differences in properties between the enzymes from these different tissues other than the effect of crystal violet mentioned below. Specificity. ATP cannot be replaced by ADP or AMP. The enzyme appears to be relatively unspecific with respect to glutamic acid. Although aspartic and other commonly occurring amino acids are inactive, recent work using preparations from both peas and sheep brain has shown that D-glutamic acid and a-aminoadipic acid will give hydroxamic acids. 11 The latter nevertheless is incapable of reacting with ammonia, a-Methylglutamic acid can also replace glutamic acid in the enzyme system from sheep brain. 1~ Ammonia, hydroxylamine, and hydrazine are all equally active in the pigeon liver and sheep brain enzyme systems when L-glutamic acid is the other substrate present. Other bases tested were found to be inactive. Activators and Inhibitors. ~V[g or Mn ions are necessary for activity. Mg ions activate maximally at 0.02 M with the brain and pea enzymes. ii L. Levintow and A. Meister, J. Am. Chem. Soc. 75, 3039 (1953). ~2N. Lichtenstein, H. E. Ross, and P. P. Cohen, J. Biol. Che~n. 201, 117 (1953).

342

ENZYMES OF PROTEIN METABOLISM

[45]

Mn ions at a concentration of 0.003 M give 30 % with the pea enzyme and 40% with the brain enzyme of the activity obtained in the presence of 0.02 M Mg ions. Higher concentrations of Mn ions cause decreased activity. The brain enzyme is inhibited by Ca ions, the latter apparently being competitive with Mg. The enzyme is completely inhibited by 10.3 M fluoride and by the same concentration of p-chloromercuribenzoate. The sulfoxide derived from methionine is a competitive inhibitor with respect to glutamic acid, the enzyme from Staph. aureus being ten times as sensitive to this compound as is the brain enzyme. Crystal violet inhibits the bacterial and pea enzymes but not those from brain and pigeon liver. ADP is a powerful inhibitor of glutamine synthesis, apparently being competitive with ATP; the sheep brain enzyme is 50% inhibited when the ratio A D P / A T P is 0.3. Effect of pH. The enzyme from sheep brain has an optimum at pH 7.2, the activity falling to zero at pH 4.5 and to 50% at pH 8.5. The pea enzyme behaves similarly, having an optimum at pH 7.5.

[45] Enzymatic Synthesis of Glutathione By JOHN E. SNOKE a n d KONRAD BLOCH Enzyme systems which catalyze the synthesis of GSH 1 from glutamic acid, cysteine, and glycine have been found in the livers of pigeons 2,3 and of various mammals. 4 The presence of the GSH-synthesizing system has also been demonstrated in extracts of E. coli. 5 The formation of GSH in pigeon liver proceeds in at least two consecutive steps and involves the formation of GlutCyst as an intermediate. 6 The formation of GlutCyst as well as the subsequent condensation of the dipeptide with glycine requires ATP. Of the two steps which are concerned with the total synthesis of GSH, only that which results in the formation of the tripeptide from GlutCyst has been extensively studied. The name glutathione synthetase is proposed for the enzyme which catalyzes the condensation of GlutCyst and glycine to form GSH. 1 The following abbreviations have been used: GSH, glutathione; GlutCyst, L-~.glutamyl-L-cysteine; ATP, adenosine triphosphate; ADP, adenosine diphosphat(,; 3-PGA, 3-phosphoglyceric acid; Tris, tris(hydroxymethyl)aminomethane. K. Bloch, J. Biol. Chem. 179, 1245 (1949). a R. B. Johnston, and K. Bloch, J. Biol. Chem. 188, 221 (1951). 4 S. Yanari, J. E. Snoke, and K. Bloch, J. Biol. Chem. 201~ 561 (1953). 5 p. j. Samuels, Biochem. J. 56~ 441 (1953). s j. E. Snoke and K. Bloch, J. Biol. Chem. 199, 407 (1952).

[45]

ENZYMIC SYNTHESIS OF GLUTATHIONE

343

I. Total Synthesis of GSH Assay Method The synthesis of GSH can be determined by means of the glyoxalase assay, 7 or more conveniently by measuring the incorporation of radioactive glycine or glutamic acid into the tripeptide. 3 GSH formation is strongly inhibited by ADP, 4 which is not only a product of the synthetic reaction but is liberated by the action of ATP-ase present in crude liver extracts. Therefore, for optimal rates of GSH synthesis ATP is continuously regenerated by adding 3-PGA. 4 The necessary glycolytic enzymes are present in crude pigeon liver preparations. The incubation mixture contains, in a total volume of 1.0 ml., 0.1 M Tris buffer, 0.1 M KC1, 0.01 M MgS04, 0.015 M KCN, 0.01 M 3-PGA, 0.01 M glutamie acid, 0.01 M cysteine, 0.015 M glycine, 0.003 M ATP, and 0.5 ml. of the enzyme preparation. The incubation is carried out at 37 ° and pH 7.8 for 1 hour.

Enzyme Preparation Fifty grams of acetone-dried pigeon liver 8 is ground to a fine powder in a mortar and stirred for 1 hour in a mixture of 500 ml. of 0.9% sodium chloride and 50 ml. of 0.4 M sodium bicarbonate. The supernatant obtained upon centrifugation is dialyzed for 20 hours against 20 1. of distilled water at pH 8.0. The dialyzed extract is brought to pH 5.80 by the addition of 1.0 M acetic acid, stirred for 10 minutes, and centrifuged2 All operations are carried out in the cold. The final supernatant, which contains the GSH-synthesizing system, can be stored in the frozen state for at least three months without loss of activity. When tested under optimal conditions, such preparations will synthesize from 1 to 2 micromoles of GSH per milliliter in 1 hour.

II. Glutathione Synthetase Assay Method The formation of GSH from GlutCyst and glycine can be followed by either the isotopic 3 or the glyoxalase assayJ In purified enzyme preparations when ATP-ase is absent, the determination of Pi released from ATP serves as a convenient means of assay. The incubation mixture con7 G. E. Woodward, J. Biol. Chem. 109, 1 (1935). 8 Preparations of similar activity m a y be obtained from acetone-dried livers of beef, pork, rat, and guinea pig. 4 9 On several occasions it has been observed t h a t the activity of the initial extract is low and t h a t a m a r k e d increase of total activity is obtained on acid precipitation of inactive protein. This finding is a t t r i b u t e d to the removal of interfering enzymes, among t h e m those which hydrolyze GSH. 2

344

ENZYMES OF PROTEIN" METABOLISM

[45]

tains, in a final volume of 1.0 ml., 0.1 M Tris buffer, 0.1 M KC1, 0.01 M MgS04, 0.015 M K C N , 0.015 M glycine, 0.002 M GlutCyst, 0.003 M A T P , and enzyme sufficient to synthesize approximately 0.5 micromole of G S H in 30 minutes. The incubation is carried out at 37 ° and p H 8.3. The rate of G S H formation is not linear with respect to time but falls off rapidly, owing to the liberation of ADP. If 3-PGA and a rabbit muscle preparation containing the necessary glycolytic enzymes 1° are added, the rate remains linear until the substrate concentration becomes limiting. Preparation from Pigeon Liver 11 The preparation described for the total synthesis of G S H is used as a starting material for G S H synthetase. T o the above supernatant, obtained after precipitation with acetic acid, is added 0.67 vol. of ammonium sulfate solution, saturated at 0 °, and the p H is adjusted to 5.2 with 1.0 M acetic acid. After 30 minutes of stirring, the precipitate is collected b y centrifugation, dissolved in 0.1 M bicarbonate, and dialyzed until free of salt. The dialyzed solution is adjusted to p H 4.0 with acetic acid, and a solution of protamine sulfate is added (0.1 rag. of protamine sulfate per milligram of protein). The p H is slowly brought to 5.7 b y the addition of 1.0 N sodium hydroxide. After 30 minutes of stirring, the precipitate is collected b y centrifugation and extracted with 0.1 M acetate buffer, p i t 4.5. T h e insoluble material is removed by centrifugation, and to the supernatant is added an equal volume of saturated ammonium sulfate. T h e mixture is stirred for 30 minutes. The precipitate is collected b y centrifugation, dissolved in 0.1 M bicarbonate, and the solution dialyzed against distilled water until salt-free. All the above operations are carried out in the cold. The enzyme preparation is stable if stored in the frozen state. The course of a typical fractionation is sumTABLE I PREPARATION OF PIGEON LIVER GLUTATHIONE SYNTHETASE 11

(The starting material was 50 g. of acetone-dried pigeon liver.) Step Dialyzed extract Acid ppt., pH 5.8 (NH4)2SO4 ppt. Protamine ppt.

Volume

Total activity ~

Specificactivity b

440 450 51 13

886 915 812 450

0. 065 0.11 0.61 3.47

a Micromoles of GSH synthesized per 30 minutes. b Micromoles of GSH synthesized per 30 minutes per milligram of protein. 10S. Ratner and A. Pappas, J. Biol. Chem. 179, 1183 (1949). 11j. E. Snoke, S. Yanari, and K. Bloch, J. Biol. Chem. 201, 573 (1953).

[45]

ENZYMIC SYNTHESIS OF GLUTATHIONE

345

marized in Table I. There is a fifty-fold increase of specific activity with a yield of approximately 50 %. Preparation from Brewer's Yeast Brewer's yeast is a more convenient source for the preparation of GSH synthetase. Dried brewer's yeast is pulverized for 24 hours in a ball mill. The yeast is suspended in 3 vol. of water and allowed to autolyze for 4 hours at 37 °. An additional 3 vol. of water is added, and, after 30 minutes of stirring, the autolyzate is centrifuged. To each liter of the supernatant is added 333 g. of solid ammonium sulfate, and the mixture is allowed to stand overnight. The precipitate is collected by centrifugation and dissolved in approximately one-fourth of the original volume of water. The concentration of ammonium sulfate is estimated by nesslerization, and sufficient solid ammonium sulfate is added so that the solution contains 260 g. of ammonium sulfate per liter. The pH is adjusted to 8.0 by the addition of 3.0 M ammonium hydroxide, and the mixture is allowed to stand overnight. The precipitate is removed by centrifugation, and the concentration of ammonium sulfate in the supernatant is brought to 350 g./1. by the addition of solid ammonium sulfate. After 1 hour of stirring, the precipitate is collected by centrifugation, dissolved in H20 to approximately one-fifteenth the original volume, and dialyzed overnight against running tap water. The dialyzed solution is adjusted to pH 4.5 by the addition of 0.2 N sulfuric acid and placed in a 42 ° water bath for 30 minutes. After cooling, the precipitate is removed by centrifugation, and 260 g. of solid ammonium sulfate is added per liter of supernatant. The mixture is stirred for 1 hour. The precipitate is collected by centrifugation, dissolved in a minimum of water, and the solution dialyzed briefly to remove the salt. To the dialyzed solution is added 0.54 vol. of saturated ammonium sulfate, and the pH is adjusted to 4.5 by the addition of 0.2 N H~SO4. The mixture is stirred for 1 hour and centrifuged. To the supernatant is added an additional 0.28 vol. of saturated ammonium sulfate, and the mixture is stirred for 1 hour. The precipitate obtained after centrifugation is dissolved in H20 and dialyzed. The protein concentration is adjusted to 10 mg./ml., and for every 10 ml. of solution there is added 0.2 ml. of a solution of nucleic acid (2.5 g. of nucleic acid and 3.75 ml. of 1 N sodium hydroxide in 50 ml.). The pH is adjusted to 5.3 by the addition of 0.05 N sulfuric acid, and the mixture is stirred for 30 minutes and centrifuged. The supernatant is adiusted to pH 4.9, and, after 30 minutes of stirring, the precipitate is collected by centrifugation, suspended in water, and dissolved by adjusting the pH to 7.0. A solution of protamine sulfate (2 mg./ml.) is added until a precipitate no longer forms. After centrifu-

346

[46]

ENZYMES OF P R O T E I N METABOLISM

gation, 2 vol. of saturated ammonium sulfate is added to the supernatant, and the precipitate is collected by centrifugation and dissolved in water. Unless otherwise noted, all operations are carried out in the cold. The enzyme at a n y stage of purification is stable when stored in the frozen state. During the assay, however, the enzyme is slowly inactiv a t e d and is therefore stabilized during incubation experiments by the addition of 0.1% bovine serum albumin. This purification procedure of the yeast enzyme yields a fraction with a specific activity from 1000 to 1500 times t h a t of the initial autolyzate with an approximate yield of 15%. A typical fractionation procedure is summarized in Table II. TABLE II PREPARATION OF YEAST GLUTATHIONESYNTHETASE (The starting material was 5.0 kg. of dried brewer's yeast.) Step Autolyzate First (NH4) :S04 fractionation Second (NH4) ~S04 fractionation Partial denaturation Third (NH4)2SO4fractionation Fourth (NH4) 2SO4fractionation Nucleic acid fractionation Protamine treatment

Volume

Total activity ~

Specificactivityb

22,300 5,800 1,380 1,320 264 120 22 8

202,000 166,000 122,000 82,000 80,000 58,000 38,000 29,000

0. 107 0.299 0.765 2.89 14.8 34.3 92.6 161

Micromoles of GSH synthesized per 30 minutes. b Micromoles of GSH synthesized per 30 minutes per milligram of protein.

[46] Hippuric Acid Synthesis Benzoic acid ~ Glycine

ATP ) Hippuric acid CoA

By H. CHANTRENNE T h e enzymes involved are associated with large cytoplasmic particles. T h e y have been obtained in solution and partially purified. Hippuric acid synthesis from glycine and benzoate involves at least two steps: (a) benzoyl activation with the formation of S-benzoyl-CoA at the expense of some source of energy; (b) condensation of benzoyl-CoA with glycine.

[46]

HIPPURIC ACID SYNTHESIS

347

Assay Method (Range: 10 to 100?/ml.)

Principle. Hippuric acid is extracted from its acid aqueous solution in an organic solvent which does not extract free glycine. The extracted hippuric acid is hydrolyzed and glycine determined in the hydrolyzate. Borsook and Dubnoff 1 extracted with ether and determined glycine by formol titration. 2 Leuthardt and Nielsen a determined glycine by the specific method of Alexander et al., 4 as modified by Krueger. ~The method described below has been used by the author 6 to his satisfaction. Accuracy: 5 %. Reagents Extraction fluid. Mixture of 1 vol. of n-butanol with 5 vol. of chloroform. 5 M sulfuric acid. 2 M KOH. 1 M KOH. Indicator. Bromocresol purple, 0.1% solution in water.

Procedure. Pipet a 1-ml. sample of the hippuric acid solution into a conical centrifuge tube (diameter 20 ram.), and add 0.2 ml. of 5 M H2SO4 and 2.5 ml. of extraction fluid. Shake for 2 minutes, and then centrifuge. Collect the organic (heavy) phase using a rubber bulb pipet, and transfer it into a dry test tube. Repeat the extraction twice, using 2.5 ml. of solvent each time. Filter the pooled extracts through a small filter paper to remove droplets of aqueous phase (important !). Wash the tube and the filter with 2 ml. of extraction fluid. Evaporate the filtered extract to dryness by blowing air into the tube. To the dry residue add 2 ml. of 2 M KOH solution and heat for 3 hours in a boiling water bath. After cooling, add 1 drop of bromocresol purple solution and, drop by drop, 5 M H2SO4 until the liquid turns yellow; adjust with M KOH to a bluish tinge (pH 5 to 6). Transfer into the distillation flask for determination of glycine according to Alexander et al.; ~-5 a total of 3 ml. of water is used for rinsing the tube. Application of Assay Method to Crude Enzyme Preparations. The method as described can be applied directly to mixtures containing soluble enzymes. The addition of sulfuric acid blocks the enzyme reaction, 1 H. Borsook and J. W. Dubnoff, J. Biol. Chem. 132, 307 (1940). See Vol. I I I [74]. F. Leuthardt and H. Nielsen, Helv. Chim. Acta 34, 1618 (1951). 4 B. Alexander, G. Landwehr, and A. M. Seligman, J. Biol. Chem. 160, 51 (1945). 5 R. Krueger, Helv. Chim. Acla 32, 238 (1949). H. Chantrenne, J. Biol. Chem. 189, 227 (1951).

348

ENZYMES OF PROTEIN METABOLISM

[46]

and after shaking with the extraction fluid and centrifuging, the flocculated proteins form a thin, compact layer between the two liquid phases, which does not interfere with the assay. For crude homogenates, it is necessary to remove the proteins first. Thus to the incubation mixture add 0.2 vol. of 10% metaphosphoric acid solution, ~ then centrifuge and determine hippuric acid in the supernatant. I t is recommended that the recovery of hippuric acid be checked in each case by adding known amounts of hippuric acid to the tissue preparation and determining hippuric acid immediately.

Preparation of the Enzymes Crude Homogenates. R a t or guinea pig livers are homogenized 7 in isotonic potassium phosphate at p H 7.5. According to Borsook and Dubnoff, 1 higher activities are obtained when 0.01 M benzoate and 0.02 M glycine are included in the buffer used for homogenization. Washed Particles. L e u t h a r d t and Nielsen 3 have isolated white particulate material (mitochondria) containing the enzymes, b y fractional centrifugation of a guinea pig liver homogenate in isotonic mannitol. This preparation was supplemented with 0.001 M benzoic acid, 0.0025 M fumaric acid, 0.0026 M MgCl2, 0.04 M KCI, 0.0005 M ATP, 0.0000145 M cytochrome c, made isotonic with potassium phosphate buffer at p H 7.5 and incubated at 38 ° in the air. Soluble Enzyme Preparation. 8 Homogenize ten rat livers in cold isotonic KC1 solution, and centrifuge for 15 minutes at 2000 X g in the cold. Wash the sediment twice with cold isotonic XC1. Suspend the washed sediment in 10 vol. of ice-cold acetone, centrifuge, and wash the precipitate with cold acetone twice. D r y the residue in the air as rapidly as possible b y spreading it on a large sheet of filter paper, and store at 0 ° in a v a c u u m desiccator. Suspend 1 g. of this acetone powder in 5 ml. of 0.02 M K2HPO4 solution, and keep it at room temperature for 10 minutes, with slow stirring. Centrifuge for 10 minutes at 10,000 X g. P u t the clear extract in the refrigerator, and re-extract the residue once as above. The pooled clear extracts can be kept frozen for at least two weeks without much loss of activity. As a rule they contain enough CoA to saturate the system.S Sehaehter and Taggart 9 have obtained an active preparation from pig

7 See Vol. I [2]. 8 It should be noted that the amount of CoA is considerably reduced and may become limiting in rats on a pantothenic acid deficient diet; A. E. Braunshtein and E. F. Yefimochkina, Doklady Akad. Nauk. S.S.S.R. 71, 347 (1950). 9D. Schachter and J. V. Taggart, J. Biol. Chem. 203, 925 (1953); Schachter and Taggart (J. Biol. Chem. in press) obtained a still purer preparation from an acetone powder of beef liver mitochondria prepared by the sucrose method. The

[46]

HIPPURIC ACID SYNTHESIS

349

kidney cortex, using the same method; they have purified it further by ammonium sulfate precipitation. The precipitate obtained between 30 and 40 % saturation is redissolved and dialyzed overnight against a large volume of 0.07 M KC1 previously adjusted to pH 8.0 with solid K2CO3. This preparation is completely CoA-dependent. The acetone powder extracts require A T P as a source of energy, and Mg++; 0.005 M cysteine is favorable. Typical incubation mixture: 0.01 M glycine, 0.0025 M benzoic acid, 0.006 M ATP, 0.0008 M MgSO4, 0.005 M cysteine, all in 0.02 M potassium phosphate pH 7.5. Soluble enzyme preparation: 10 mg. dry weight per milliliter of final volume of incubation mixture. CoA if required: half saturation of the rat liver enzyme system is obtained with about 5 Lipmann units I° of CoA per milliliter. Incubation in small test tubes at 37 ° for 1 to 3 hours. Hippuric acid formed: 0.2 micromole/ml. Removal of CoA from Soluble Enzyme 6 Treat a few grams of the anion exchange resin Dowex-1, 100 to 200 mesh grade, with N HC1 solution, wash with water until no acid is released, filter the resin, and blot it with filter paper. The resin can be kept in that semidry state in a stoppered vessel and is ready for use. Prepare a small column with this resin (height of resin 60 mm., diameter 5 to 7 mm.). Introduce 5 ml. of clear enzyme solution into the column, and let it pass slowly (20 drops per minute) through the resin. CoA is almost completely removed in one such operation. Spectrophotometric Method for Studying the Condensing Enzyme 9 S-Benzoyl-CoA ~ glycine--+ Hippurie acid ~- CoA-SH Principle. The absorption spectrum in the region 240 to 300 mt~ changes in the course of the reaction. The larger change in optical density occurs at about 280 m~. Procedure. 9 Into a 1-cm. Beckman silica cell introduce 0.09 micromole of S-benzoyl-CoA, 11 60 micromoles of glycine, 100 micromoles of potassium phosphate buffer of pH 7.5, and 0.6 rag. of purified pig kidney protein; make the final volume to 3 ml. Take readings every 10 minutes at 280 m~ in a Beckman DU spectrophotometer against a blank contain-

purification of the acetone powder extracts involves ammonium sulfate fractionation (30 to 50% saturation), selective adsorption of contaminating proteins with Ca phosphate gel, removal of additional impurities by precipitation with yeast nucleic acid at pH 5.4, and refractionation with ammonium sulfate (45 to 50 % saturation). The final preparation is 70 % homogenous as tested by electrophoresis and ultracentrifuge analysis and is essentially free of both the benzoate-activating enzyme and the benzoyl-CoAdeacylase. lo See Vol. III'[132]. 11See Vol. III [137].

350

ENZYMES OF PROTEIN METABOLISM

[47]

ing all the c o m p o n e n t s except benzoyl-CoA. A decrease in optical density of 0.73 corresponds to the cleavage of 0.1 micromole of benzoyl-CoA per milliliter. I n estimating hippuric acid synthesis b y this method, comparison m u s t be m a d e with a glycine free control, for some splitting of benzoyl-CoA m a y occur even in the absence of glycine. Cysteine, G S H , and other thiols interfere with hippuric acid f o r m a t i o n in this system.

Systems Closely Related to Hippuric Acid Synthesis p-Aminohippuric Acid Synthesis. T h e enzymes are obtained under essentially the same conditions, and their requirements are the same as for hippuric acid synthesis (compare refs. 9, 12, 13). Cohen and M c G i l v e r y 12 have described a m e t h o d for estimating differentially p-aminohippuric and p-aminobenzoic acids. p-Amino-ornithuric Acid Synthesis. T h e s y s t e m of enzymes has been studied b y M c G i l v e r y and Cohen, TM who h a v e described a colorimetric assay m e t h o d for p-aminobenzoyl derivatives of ornithine. 12 p. p. Cohen and R. W. McGilvery, J. Biol. Chem. 166, 261 (1946); 169, 119 (1947); 171, 121 (1947). 13R. K. Kielley and W. C. Schneider, J. Biol. Chem. 185, 869 (1950). 14R. W. McGilvery and P. P. Cohen, J. Biol. Chem. 183, 179 (1950).

[47] E n z y m a t i c Citrulline S y n t h e s i s Ornithine + CO2 -t- NH3--~ Citrulline B y SANTIAGO GRISOLI~_ A s s a y Method Principle. I n a properly fortified e n z y m e s y s t e m ornithine is quantit a t i v e l y converted into citrulline. 1 T h e over-all reaction ornithine--* citrulline, f r o m here on called reaction A, is composed of two main reactions, 2 A1 and A2, catalyzed b y enzymes E1 and E2, respectively: 3 C G 4 + COs ~- NH3

~ Intermediate El, Mg ++, ATP

(A1)

I n t e r m e d i a t e + Ornithine--~ Citrulline + C G E2

(A2)

i S. Grisolia and P. P. Cohen, J. Biol. Chem. 191, 189 (1951). 2 S. Grisolia and P. P. Cohen, J. Biol. Chem. 198, 561 (1952). 3 The two enzymatic systems required for reaction A will be called enzymes E, and E2 for convenience, although it is obvious that the partially purified preparations so far used may be composed of several enzymes. ~ * CG, carbamyl-L-glutamate.

[47]

ENZYMATIC CITRULLINE SYNTHESIS

351

Owing to the instability of the intermediate formed in reaction A1, it is best to s t u d y reaction A1 coupled with reaction A2. Reaction A2 is studied b y the addition of ornithine to an excess of the intermediate formed in reaction A~, f r o m here on called " I n t e r m e d i a t e , " followed b y citrulline estimation. Citrulline is measured b y a slight modification of the colorimetric m e t h o d of Archibald. 5

Reagents 0.04 M potassium A T P , p H 7.3. 0.1 M potassium 3-D-phosphoglycerate, p H 7.3. 0.2 M potassium phosphate buffer, p H 7.3. 0.1 M potassium carbamyl-L-glutamate, p H 7.3. 0.1 M L-ornithine. 0.3 M sodium bicarbonate. 1.0 M sodium bicarbonate. 0.3 M NH4C1. 0.2 M MgS04. 0.5 M HC104. 3 % diacetyl monoxime. H 2 S Q - - H 3 P 0 4 reagent (mix 1 p a r t of concentrated H2S04 and 3 parts of H3PO4). 6

Enzyme Preparations Muscle preparation, as described b y R a t n e r and Pappas. 7,8 R a t liver fraction B, ~-see below. R a t liver fraction E, 2 see below.

Procedure for the Study of Reaction A1 and Over-all Reaction A. R o u tinely it is convenient to mix an excess of the required a m o u n t of the components common to all samples, dilute to a convenient volume, and pipet aliquots into 12-ml. conical centrifuge' tubes. T h e final optimal concentrations of the components of the incubation mixture per milliliter and for a 20- to 30-minute incubation period (using up to 30 units of E~ with an excess of E~, see definitions below), expressed in micromoles, are as follows: A T P , 2; P G A , 25; NH~C1, 15; L-ornithine, 10; carbamyl-L5 R. M. Archibald, J. Biol. Chem. 156, 121 (1944).

6 We routinely use E. I. DuPont C. P. H2SO4 and Mallinckrodt A. R. 85 to 88 % H3PO4. Our results were erratic with other brands of phosphoric acid, apparently owing to differences in concentration of heavy metals in the reagents and the influence of these metals on the color reaction. Similar observations have been made by Dr. Sarah Ratner (personal communication). 7 S. Ratner and A. Pappas, J. Biol. Chem. 179, 1183 (1949). 8 For the preparation of the muscle system, see Vol. II [48].

352

ENZYMES OF P R O T E I N METABOLISM

[47]

glutamate, 5; phosphate buffer, 10; MgSO4, 5; and NaHCO3, 15. Bring the volume of each tube to 1.5 ml., incubate at 38 ° for 5 minutes, and complete with 0.5 ml. of the enzyme solution at definite intervals. (Immediately before testing take up in ice-cold water the desired amount of enzyme, rat liver fraction B, containing E1 and E~, and add 2 mg. of the muscle preparation per milliliter.) Incubate at 38 ° . Stop the reaction mixtures at definite intervals by the addition of 5 ml. of 0.5 M HCI04, and centrifuge in a clinical centrifuge at about 1000 X g for 5 minutes. Take appropriate aliquots of the protein-free supernatant (containing no more than 1 micromole of citrulline) in colorimeter tubes, bring the volume to 5 ml. with water, add 2 ml. of the sulfuric-phosphoric mixture, followed by 0.25 ml. of diacetyl monoxime, mix, and cap tubes with rubber stoppers fitted with capillary tubes. Heat in the dark in a boiling water bath for 15 minutes. Cool in the dark for 10 minutes, and read at 490 m~ within the next 20 minutes. The color reaction deviates from Beer's law, so a standard curve is necessary. Procedure for the Study of Reaction As. Bring a sample (containing no more than 5 to 6 micromoles of Intermediate prepared as described below) to a volume of 3.3 ml. with water. Add 0.1 ml. of 0.2 M phosphate buffer and 0.1 ml. of 0.1 M L-ornithine, bring samples to 38 ° at definite intervals and complete, also at definite intervals, with 0.5 ml. of E2, warmed to 38 ° just prior to addition, containing from 2 to 60 units of enzyme per milliliter. Incubate at 38 ° for 5 minutes, and stop the reaction by the addition of 5 ml. of 0.5 M HCI04. Analyze for citrulline as previously described. Method for Preparation of the Intermediate. Use 1 mg. of muscle preparation and about 30 units of E1 per milliliter of incubation mixture. Use the same concentrations of components of the reaction mixture as described for the study of reaction A1, except that ornithine is excluded, the concentration of carbamyl-glutamate is increased to 10 micromoles per milliliter of incubation mixture, the concentration of MgSO~ is increased to 20 micromoles and of NaHC03 to 40 micromoles per milliliter. Incubate for 30 to 40 minutes (since the intermediate decomposes spontaneously in solution, half-life is 46 minutes at 38 °, and incubation beyond this period is of little advantageS). Chill the samples to 0 to 1° in a ice-water bath, and add with vigorous mixing 0.2 ml. of ice-cold 0.5 M HCI04 per milliliter of incubation mixture. Wait for 5 minutes, then centrifuge at 2000 X g for 4 to 5 minutes at 0 °. Add 0.2 ml. of ice-cold 1 M NaHCO3 to each milliliter of supernatant if one wishes to test for the intermediate without further purification. If one wishes to isolate the intermediate it is best to adjust the pH of the deproteinized mixture to 8.0 with 2 M cold LiOH, add an excess of Ca ++, centrifuge off the precipitate~ and fractionate the supernatant with acetone. The fraction which is

[47]

ENZYMATIC

CITRULLINE

SYNTHESIS

353

obtained between 20 and 50 % acetone will give the intermediate of abort 20% purity. Further fractionation can be accomplished by refractionation with acetone, fractionation of the cyclohexylamine salt, and refractionation with butanol-ether. Either the calcium or cyclohexylamine salts are stable for many months if kept over a drying agent in a desiccator at 0 to 2°. Enzyme Units. For El, an enzyme unit is defined as the amount of enzyme E1 which in the presence of at least a 25-fold excess of E2 will catalyze the formation of 1 micromole of citrulline per hour, under the standardized conditions described, for a 20-minute incubation period at 38 ° during which time no more than 5 micromoles of citrulline should be formed. For E:, an enzyme unit is defined as the amount of enzyme which will catalyze the formation of 1 micromole of citrulline per hour under the standardized conditions described and for a 5-minute incubation period at 38 ° during which time no more than 3 micromoles of citrulline should be formed from 5 to 6 micromoles of Intermediate at pH 7.3 in a volume of 4 ml. Application of Assay Method to Crude Tissue Preparations. It is possible to study any of these reactions in extracts of acetone powders of tissues. The acetone treatment results in the solubilization of the enzymes of the citrulline synthesis system in animal tissues. Since extracts of mammalian liver acetone powder contain low concentrations of substrates, blank values are low, and there is little further metabolism of citrulline. Reaction A has been studied successfully with dialyzed or undialyzed extracts of whole liver acetone powder in this laboratory not only by research workers but also for the past several years by students in physiological chemistry. In testing for citrulline synthesis in tissues other than mammalian liver one must be sure first that citrulline is not metabolized, and second that both of the main enzymatic steps for citrulline synthesis are present in the tissue under examination. However, it is quite simple to test independently for both steps (1) by addition of preformed intermediate, and (2) by adding an excess of enzyme E~.

Purification Procedure All preparations described were performed in a cold room or a low temperature box. All centrifugations were carried out in a refrigerated centrifuge and always at a speed of 2000 X g. Step 1. Preparation of "Washed Residue" of Rat Liver. Disperse fresh rat livers in 5 parts of ice-cold isotonic KC1, using a Waring blendor at low

354

ENZYMES OF PROTEIN METABOLISM

[47]

speed for 1 minute. Centrifuge for 20 minutes at 0°. Discard supernatant, and bring residue to original volume with isotonic KC1. Repeat centrifugation and washing twice.

Step 2. Solubilization of Citrulline Synthesizing Enzyme System by Acetone Treatment. Take up the well-washed residue in a minimum volume of cold isotonic KCI, and mix with 10 to 15 vol. of cold ( - 2 0 °) acetone. Siphon off the clear upper layer without disturbing the protein precipitate, and then filter the latter on a Biichner funnel. When most of the water-acetone has been filtered off, fill the funnel with fresh cold acetone. When most of the acetone has been filtered off, cover the funnel with a rubber dam fastened with rubber bands and apply full suction with a water pump. In about 20 minutes the preparation will appear dry. Crumble the cake on filter paper. Transfer to a vacuum desiccator containing alumina and evacuate. Keep at 0 ° for at least 24 hours before step 3. Step 3. Alcohol Fractionation of Acetone Powder Extracts. The acetone powder is extracted for 30 minutes at 0° with 20 vol. of a mixture of 3 parts of isotonic KC1, 1 part of 0.1 M phosphate buffer, pH 7.4, and 1 part 0.1 M NaHCO3. Centrifuge for 20 minutes2 Fractionate the supernatant with ethanol under standard conditions. Bring the alcohol concentration to 18% and the temperature to - 4 ° , centrifuge after standing 30 minutes, and discard the precipitate. Bring the ethanol concentration to 27% and the temperature to - 9 ° , and centrifuge after standing for 30 minutes. The precipitate, called fraction B 2, is lyophilized. This fraction contains 90% of the E1 present in the acetone powder. It has approximately 4 units of E1 and 120 units of E2 per milligram of protein. After removal of fraction B, bring the alcohol concentration of the supernatant to 42% and the temperature to - 2 0 °. Centrifuge off, and lyophilize the precipitate, fraction C ~. This fraction has about 0.1 unit of E1 and about 258 units of E2. Step 4. Heat Treatment. Make a 4% solution of fraction C ~, and heat in a water bath kept at 56 °. Once the temperature inside the flask has reached 54°, keep at this temperature for 3 minutes. Cool in an ice-water bath, and dilute with 1 vol. of water. Centrifuge for 10 minutes. Discard the precipitate, and lyophilize the supernatant, called fraction E 2. When needed, extract fraction E for 10 minutes with water, centrifuge for 10 minutes, and use the supernatant. This fraction contains no enzyme E~ and about 385 units of E~ per milligram of protein. 9 These extracts h a v e approximately 2.4 units of E, a n d 90 units of E,, which represents a b o u t a threefold purification over the washed residue preparations, calculated on the basis of o p t i m u m citrulline synthesis reported b y P. P. Cohen a n d M. Hayano, J. Biol. Chem. 172, 45 (1948).

[47]

ENZYMATIC CITRULLINE SYNTHESIS

355

Properties of Enzymes El and E2 Specificity. The catalytic action of carbamyl-L-glutamate can be replaced by some other N derivatives. 10 From a consideration of the large number of compounds tested, it appears that the glutamic acid portion is required specifically and that the amino group substituent must possess an adjacent carbonyl group which apparently must be capable of enolization. The affinity of the enzyme system catalyzing reaction A for the active glutamic acid derivatives in decreasing order is as follows: acetyl, carbamyl, chloroacetyl, propionyl, and formyl. Whether or not the same enzyme system activates all these compounds is not known; however, some positive evidence is found in the competitive inhibition of citrulline synthesis by formylglutamate in the presence of carbamylglutamate. Activators and Inhibitors. Magnesium ions are required for reaction A1. It appears at present that reaction A2 requires no cofactors. ATP at concentrations over 5 X 10-3 M inhibits reaction A1; 1 X 10-2 M inhibits 60%. 2 Borate-KC1, phosphate, or glycylglycine buffers do not affect the over-all reaction A. Sodium fluoride up to 0.02 M, KCN up to 0.04 M, and DNP up to 0.0002 M (tested with optimum concentration of ATP and in the absence of the PGA-regenerating system) do not affect reaction A. Effect of pH. The optimum pH for both enzymes E1 and E~ is 7.2 to 7.4. Stability of Enzymes. Both enzymes are inactivated by heating in solution at neutral pH. Two minutes at 65° inactivates both enzymes; however enzyme E1 is more thermolabile. Heating at 38 ° for 30 minutes destroys 70% of the activity of El, and if prolonged for 60 minutes longer the activity is entirely lost. Similar treatment does not affect E2. 10S. Grisolia and P. P. Cohen, J. Biol. Chem. 9.04, 753 (1953).

356

ENZYMES OF PROTEIN METABOLISM

[48]

Enzymatic Synthesis of Arginine (Condensing and Splitting Enzymes) B y S. RATNER

A. Over-all Reaction 1,2 L-Citrulline + L-Aspartic acid + A T P ~ L-Arginine + Fumaric acid

IT Ornithine + Urea

Malic acid + ADP + P04

Assay Method Principle. An excess of arginase is provided during incubation, and the arginine formed is then measured as urea. A TP is inhibitory in substrate concentrations. It is therefore used in catalytic amounts and regenerated enzymatica!ly from PGA by a muscle extract which is provided to supply an excess of the necessary glycolyzing enzymes. Reagents

0.1 M L-citrulline adjusted to pH 7.4. DL-Citrulline will substitute for the over-all reaction but cannot be used to follow the activity of the condensing enzyme system. 0.1 M L-aspartic acid brought to pH 7.4 with 1 ml. of 1 N NaOH per 133 mg. in a final volume of 10 ml. 1.0 M potassium phosphate buffer, pH 7.4. 0.066 M MgSO4. 0.025 M ATP, pH 7.4, prepared by neutralizing 156 mg. of the acid sodium tetrahydrate salt in a final volume of 10 ml. 0.1 M PGA, 3 pH 7.4, prepared by dissolving 894 mg. of the acid barium dihydrate salt in 4 ml. of water with 4 ml. of 1 N HC1 in the cold. Add 5.6 ml. of 0.5 M K2SO4, and neutralize the supernatant with 6 to 7 ml. of 1 N K O H to pH 7.4. Adjust to a final volume of 25 ml. Arginase and lyophilized extract of rabbit muscle (see below, Preparation of Supplementary Enzymes). 1S. Ratner and A. Pappas, J. Biol. Chem. 179, 1183 (1949). 2 S. Ratner and B. Petrack, J. Biol. Chem. 200, 175 (1953). 3D-3-Phosphoglycerie acid can now be obtained commercially or can be prepared, employing glucose, according to C. Neuberg and It. Lustlg, Arch. Biochem. 1, 311 (1942-43); cf. Vol. III [34].

[48]

ENZYMATIC SYNTHESIS OF ARGININE

357

Procedure. The incubation mixture, made up in an ice bath, contains 0.1 ml. each of buffer, MgS04, ATP, arginase, and muscle solutions: 0.15 ml. each of the citrulline and aspartate solutions: 0.25 ml. of PGA; 0.5 to 1.0 ml. of enzyme; and water to a final volume of 2.0 ml. Incubate at 38 ° for 20 minutes. The reaction is stopped by adding 3.0 ml. of 8.5% TCA. Urea is estimated in the filtrate colorimetrically.4 Citrulline gives a small amount of color by this method and also depresses the urea color about 10%. These effects are compensated for by adding decreasing amounts of citrulline to the set of increasing urea standards so that the sum of the two equals 30 micromoles per 10 ml. of each standard solution. We have found it convenient to modify the concentration of the acid reagent employed so as to suit the range of the assay. It is made up to contain 90 ml. of concentrated H2SO~ and 270 ml. of concentrated H3PO4 in a final volume of 1 1. To 10 ml. of the mixture are added 0.5 ml. of 3% alcoholic a-isonitrosopropiophenone and 0.5 ml. of filtrate or standard. Application of Method to Crude Tissue Preparations. The method is primarily planned for crude and unfractionated extracts from sources such as liver, kidney, or yeast, whether or not high arginase activity is present in the source material. The direct estimation of arginine, by one of the Sakaguchi methods, 5 with preparations which lack arginase is less convenient. Most crude preparations contain glycolizing enzymes, and the stimulation by muscle extract may at this stage be small, particularly when arginine synthesizing activity is low. Definition of Unit and Specific Activity. One unit catalyzes the formation of 1 micromole of arginine per hour. Specific activity is defined as units per milligram of protein. Protein is determined by biuret color according to Gornall et al., e scaled down one-half. The condensing enzyme system and the splitting enzyme together catalyze the over-all reaction. In crude preparations, the assay reflects the activity of the rate-limiting component. In fresh extracts of liver and kidney, this is usually the splitting enzyme. Preparation of Supplementary Enzymes. A preparation of arginase, free of arginine-synthesizing enzymes, may he prepared by extracting minced beef liver with 1.5 vol. of water containing 0.5% Mn ++ with intermittent stirring for 98 hours in the cold. Acetone is added to the clarified extract at 0 °. The fraction precipitating between 40 and 60% acetone is converted to a powder with a large excess of acetone and stored dry at 0°. A solution of suitable activity is prepared by extracting 120 mg. 4 R. M. Archibald, J. Biol. Chem. 157, 507 (1945). 5 j. W. Dubnoff, J. Biol. Chem. 141~ 711 (1941). 6 A. G. Gornall, C. J. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949).

358

ENZYMES OF PROTEIN METABOLISM

[48]

of the powder with 10 ml. of 0.1 M phosphate buffer for 15 minutes at room temperature followed by heating for 20 minutes in a 60° bath. The clarified extract keeps well at - 2 0 °. It should contain about 8 to 10 mg. of protein per milliliter and about 10 to 15 Van Slyke-Archibald7 units per milligram (cf. Vol. II [49]). A stable preparation of rabbit muscle, free of inhibitory ammonium sulfate and moderately low in ATP-ase, can be made from an extract of the minced tissue by fractionating with ammonium sulfate, according to Racker. 8 The 50 to 72 fraction is dialyzed for 3 hours against running tap water, then overnight at 0° against several changes of distilled water, and finally against 0.01 M potassium phosphate buffer, pH 7.4, for another day. It is then clarified, lyophilized, and stored under anhydrous conditions at 0 °. For incubation, 0.1 ml. of a solution containing 12 mg./ ml. usually provides the necessary excess. The activity may vary considerably, however, and for condensing enzyme assay particularly it is best to determine for each lot the amount which gives maximum assay. The solution keeps for about 2 days at 0°.

Enzyme Procedure Preparation of Crude Extracts. The enzymes are readily extracted from homogenized tissue. However the ATP-ase content of whole homogenates, and even of the supernatant fluid, is often high enough to lower the rate of arginine synthesis. Extracts of mammalian tissue are therefore best prepared after drying with acetone. Fresh beef liver or pig kidney, chilled at the slaughterhouse, is cut into small pieces, minced for a minute in a Waring blendor with 2 vol. of acetone, transferred to 8 vol. of acetone with brief stirring, and filtered rapidly on a large Biichner. The cake is resuspended in 10 vol. of acetone and filtered as before. The procedure is carried out at 0 °. The well-packed cake is rapidly spread out at room temperature and broken up finely, until dry. The activity slowly decreases on storing at 2 ° . The powder is extracted with 10 vol. of 0.02 M potassium phosphate buffer, pH 7.4, with mechanical stirring for 45 minutes at 0 °, and centrifuged cold. Such extracts have 25 to 30 mg. of protein per milliliter and a specific activity of 1 to 1.5. Fractionation. In general, fractionation procedures lead to the separation of the several enzymes which catalyze the over-all reaction. With concentrated extracts, a cut can be obtained by alcohol fractionation, which is somewhat higher than the extract in over-all activity.1

D. D. Van Slyke and R. M. Archibald, J. Biol. Chem. 165, 293 (1946). 8E. Racker, J. Biol. Chem. 167, 843 (1947).

[48]

ENZYMATIC SYNTHESIS OF ARGININE

359

Properties These will be given under the individual enzymes. It need only be mentioned here that activity is strongly inhibited by fluoride and falls off about 50% on overnight dialysis. Except in low concentrations, ammonium sulfate is inhibitory.

B. Condensing Enzyme System 9,1° L-Citrulline + L-Aspartic acid + ATP --~ L-Argininosuccinic acid + ADP + PO4

Assay Method

Principle. Formation of argininosuccinic acid is followed from the rate of citrulline disappearance. Citrulline is estimated colorimetrically,tl before and after incubation. A T P is regenerated as in the over-all assay. Pyruvate formation (from P G A ) or P04 liberation (Tris buffer m a y be used) exceeds the synthesis 30 to 80%, depending on conditions, and is therefore less reliable for assay. Also the ATP-ase blanks are likely to be high early in the purification procedure. Reagents. The reagents are the same as for the over-all system except that arginase is not needed. Procedure. The composition of the incubation mixture is the same as for the over-all system except that arginase is omitted and MgSO~ is increased to 0.2 ml. About 7 to 15 units of enzyme should be used. Since citrulline differences are being measured, the L-aspartic acid and L-citrulline may be reduced to 0.1 ml. each when low activity is expected or when the yeast component is assayed. Incubate for 20 minutes at 38 °, and deproteinize with TCA just as before. Citrulline is estimated in 0.1 ml. of the filtrate (Levy-Lang constriction pipette) by the method of Archibald. It We have found that color production depends on trace metals, probably Cu ++, and technical H2SO4 should be used in making the acid reagent. It is convenient for the range of assay to make the acid reagent from a mixture containing 1 part of concentrated H2SO4 and 3 parts of concentrated H3PO4; 100 ml. of the mixture is diluted to 250 ml. with water. To 5.0 ml. of the dilute acid are added 0.1 ml. of sample and 1.0 ml. of 0.75% aqueous diacetylmonoxime. The boiling period is 15 minutes. Definition of Unit and Specific Activity. One enzyme unit catalyzes the disappearance of 1 micromole of citrulline (or formation of 1 micromole of 9 S. R a t n e r a n d B. Petrack, J. Biol. Chem. 191, 693 (1951). ~0 S. R a t n e r a n d B. Petrack, J. Biol. Chem. 200, 161 (1953). 11 R. M. Archibald, J. Biol. Chem. 156, 121 (1944).

360

ENZYMES OF PROTEIN METABOLISM

[48]

argininosuccinic acid) per hour. Specific activity is expressed as units per milligram of protein. 6 After separation of the two condensing enzyme system components, specific activity is calculated from the protein of the limiting component. Activity is proportional to enzyme concentration provided that no more than half the citrulline has been utilized. Application of Method to Crude Tissue Preparations. Urea gives high color intensity with the citrulline method. Although the presence of splitting enzyme does not interfere with the assay when the preparation lacks arginase, it does interfere when splitting enzyme is present along with some arginase. In such cases, as for example in crude liver extract, condensing enzyme can be assayed as described for the over-all system with the addition of an excess of splitting enzyme. Splitting enzyme is removed during the first fractionation with ammonium sulfate. Purification Procedure

Two Components of Condensing Enzyme System. During purification, two enzymes become separated with loss of the less stable component. The latter may be replaced by a corresponding enzyme from yeast. Separation is evident after the gel step, and the incubation mixture employed for assay of the surviving liver component must then be supplemented with an excess of the yeast enzyme (50 units). The fraction obtained after step 2 of the yeast enzyme purification is suitable. The yeast enzyme can be assayed only when extensively purified liver component is available, substantially free of the less stable component. Complete separation has not been achieved. In the presence of an excess of purified liver enzyme, the activity of the yeast enzyme is proportional to concentration. However, owing to contamination of the liver enzyme by the component being assayed, the range of proportionality is restricted to rather narrow limits. It is desirable that the amount of liver enzyme be chosen so as to give a base line of 1 micromole; 0.25 mg. of liver enzyme (specific activity 12 without and 56 with yeast enzyme) will provide this condition. Proportionality then holds in the range of about 3 micromoles above the base line. Purification of Liver Enzyme 1° The enzyme does not withstand dialysis or acidification below pH 6, and it is sensitive to alcohol. At all purity levels, activity can be retained only by storing concentrated solutions, such as are obtained after ammonium sulfate precipitation, at - 2 0 ° . Dilutions for assay are made with 0.006 M MgSO4 immediately prior to testing. To avoid dialysis, the inhibitory effects of salts on adsorption were partially overcome by dilu-

[~:8]

ENZYMATIC

SYNTHESIS

OF

ARGININE

361

tion and the use of Tris buffer. All manipulations are carried out at 0 °, and each of the three steps is completed in a single day. Step 1. Extraction and Ammonium Sulfate Fractionation. Two hundred grams of beef liver acetone powder is extracted within a week of peparation with 10 vol. of buffer as described for the over-all system. The clear extract is brought to a volume of 1800 ml. with buffer and fractionated by the successive additions of 380, 126, and 126 g. of ammonium sulfate, corresponding to 30, 40, and 50% saturation. For each fraction, the salt is added over a half-hour interval with mechanical stirring which is continued for a total of 2 hours before centrifuging at 13,000 r.p.m, for 15 minutes in the Servall. The 0 to 30 fraction is taken up in 140 ml. of 0.05 M potassium phosphate buffer, pH 7.4, and kept for purification of the splitting enzyme. The 30 to 40 fraction is discarded, and the 40 to 50 fraction is taken up in 120 ml. of the same buffer, 0.02 M. Step 2. Adsorption and Elution with Alumina. The last fraction is diluted with 90 ml. of 0.066 M MgSQ, 450 ml. of 0.1 M NaHC03, and water to a final volume of 1260 ml. A suspension of alumina C~ (243 ml.) containing 23 mg. of dry weight per milliliter 12 is added, and the mixture is stirred intermittently for 20 minutes. The residue containing about 35% of the protein and 15% of the units is discarded, and the supernatant again treated with 720 ml. of alumina suspension as above. The residue adsorbs about 90% of the remaining units and protein. It is eluted first with 1350 ml. of 0.02 M potassium phosphate buffer, pH 7.5, with intermittent stirring for 15 minutes at 5° and the eluate discarded. Three additional elutions, each with 1350 ml. of 0.05 M buffer, remove 65% of the adsorbed units with a 2.5-fold purification. The combined eluates, 4050 ml., are brought to 0.6% saturation with 1715 g. of ammonium sulfate. After 2 hours the precipitate is removed by suction, for which a 9-cm. paper and 0.75 g. of Celite filter aid for each 2 liters are employed. The combined cakes are extracted four times each with 7.5 ml. of 0.1 M Tris buffer, pH 8.4, from which a combined supernatant of 37.5 ml. is obtained. Step 3. Adsorption and Elution with Calcium Phosphate Gel. The solution from the above step is diluted with 15.5 ml. of 0.066 M MgSO4 and water to a volume of 380 ml. Adsorption with 244 ml. of prechilled, aged, calcium phosphate gel, 13 containing 24 mg.'°of dry weight per milliliter, removes 90% of the units and 60% of the protein. Successive fractional elutions are carried out with dipotassium phosphate by use of 312 ml. of 0.01 M, 250 ml. of 0.02 M, and 187 ml. of 0.02 M for the first three; these are discarded. The next three elutions are carried out each with 250 ml. 15 For the preparation of alumina C~ gel, see Vol. I [11]. ~3 For the preparation of calcium phosphate gel, see Vol. I [11].

362

ENZYMES OF P R O T E I N METABOLISM

[48]

of a 1:1 mixture of 0.05 M dipotassium phosphate and 2 % ammonium sulfate, and the seventh elution with 250 ml. of a 1:3 mixture of 0.1 M phosphate and 2 % a m m o n i u m sulfate. The last four eluates, which contain about 65 % of the adsorbed units, have a combined volume of 1 1. and, after addition of 20 ml. of 0.066 M MgS04, are brought to half saturation with 360 g. of a m m o n i u m sulfate. T h e precipitate is centrifuged off at 13,000 r.p.m, and taken up in 6 ml. of 0.1 M Tris buffer, p H 8.4. I t contains two-thirds of the protein of the combined eluates and most of the units. If a phosphate-free enzyme is not specifically desired, it is preferable, for stability, to take up the last precipitate in 0.02 M phosphate buffer. TABLE

I

SUMIVrARY OF PURIFICATION OF LIVER ENZYME

Yeast enzyme Yeast enzyme omitted added Fractionation step

Total Volume, protein, Specific Total Specific Total Recovery, ml. mg. activity units activity units %

1. Extract of acetonedried liver 1800 57,600 1. (NH4)~S04 precipitation, 40-50 % fraction 137 11,400 2. Alumina C~ gel; elution followed by (NH~).~SO4 fractionation 37.5 1,900 3. Ca3(PO4)2gel; elution followed by (NH4)2SO~ fractionation 15.4 376

1.6 92,160

1.6

92,160

8.2

8.2

93,500

93,500

101

13.3 25,270 27.4 52,200

56.6

4,440 56.6 21,280

23.1

11.8

The preparation contains some myokinase and inorganic pyrophosphatase activity but only traces of ATP-ase.

Purification of Yeast Enzyme T h e enzyme is relatively stable in the earlier steps of the procedure and withstands dialysis and acidification to p H 5. I t becomes less stable with purification, and the final fraction loses about half the activity in two weeks on storage at - 2 0 °. Storage conditions are the same as for the liver enzyme. T h e initial extract has only traces of the complement a r y component.

[48]

ENZYMATIC SYNTHESIS OF ARGININE

363

Step 1. Extraction and First Ammonium Sulfate Fractionation. Two hundred grams of slowly dried Fleischmann's commercial baker's yeast is extracted with 600 ml. of 0.066 M dipotassium phosphate for 2 hours at 38 ° and 3 hours at 21 °, centrifuged for 1 hour at 2000 r.p.m., and the cloudy supernatant (300 ml.) diluted to 600 ml. with water. All subsequent steps are carried out at 0 °. Ammonium sulfate (212 g.)'is added to half saturation, the precipitate obtained by centrifuging at 13,000 r.p.m. discarded, and the supernatant brought to 90% saturation with 169 g. of ammonium sulfate. The precipitate is taken up in 80 ml. of 0.02 M potassium phosphate, pH 7.5, and dialyzed overnight against the same buffer. Step 2. Removal of Inactive Proteins with Calcium Phosphate Gel. The dialysate, 130 ml., is diluted with an equal volume of water to bring the protein concentration to 14.2 mg./ml., and 2.6 ml. of 1 N acetic acid is slowly added to bring the pH to 5.2. After centrifugation, the supernarant is treated with 50 ml. of calcium phosphate gel as above, centrifuged, and the supernatant adjusted to pH 7.2 with 4 ml. of 1 M K2HPO4. Step 3. Second Fractionation with Ammonium Sulfate. All the above supernatant, 290 ml., is brought to 0.55% saturation with 112 g. of ammonium sulfate, the precipitate discarded, and the supernatant brought to 70 % saturation by the further addition of 30 g. This precipitate is dissolved in 15 ml. of 0.02 M potassium phosphate, pH 7.5. Step ~. Adsorption and Elution with Alumina C.r Gel. The above solution is diluted with 203 ml. of 0.02 M acetate buffer, pH 5.0, and 79 ml. of alumina C~ gel (18.9 mg./ml.) is added as above. After centrifugation, eight successive elutions are carried out with potassium phosphate buffer, pH 7.5, as follows: (a) 106 ml. of 0.005 M, (b) 106 ml. of 0.01 M, (c) 99 ml. of 0.02 M and 7 ml. of 0.066 M MgSO~, (d) same as (c), (e) same as (c) TABLE II SUMMARY OF PURIFICATION OF YEAST ENZYME

Fractionation step 1. Extract of dried yeast 1. (NH4)~S04 precipitation, 50-90% fraction 2. Ca3(PO4)2 gel; negative adsorption 3. (NH~):SO4 precipitation, 55-70% fraction 4. Alumina C~ gel; elution followedby salt fractionation

Total Volume, protein, Specific Total Recovery, ml. rag. activity units % 300

8880

80 710,000

102 290

3320 2030

136 451,000 195 396,000

64 56

22.5

1310

307 402,000

57

13.8

274

1050 288,000

40

364

ENZYMES OF PROTEIN METABOLISM

[48]

except that the mixture contained 2 g. of ammonium sulfate, (f) same as (e), (g) 99 ml. of 0.05 M buffer, 7 ml. of MgS04 solution, and 2 g. of ammonium sulfate. The last four eluates are combined (424 ml. in all) and brought to 0.85 saturation with 255 g. of ammonium sulfate. The precipitate obtained on centrifugation at 13,000 r.p.m, is taken up in 8 ml. of the same buffer, 0.02 M, to which 0.1 vol. of 0.066 M MgSO4 has been added. The preparation contains appreciable inorganic pyrophosphatase activity.

Properties Specificity. Only L-citrulline and L-aspartic acid are active as the amino acid substrates. Other high-energy phosphate donors cannot replace ATP as the specific donor. Dependence on Substrate Concentration and pH. The Km for both citrulline and aspartic acid is 1.2 × 10-3 M, and for Mg++, which also exerts a protective effect, the Km is 1.0 × 10-3 M. In the presence of excess PGA, 1.25 )< 10-3 M ATP gives maximum rates. Inhibition is observed at higher concentrations. In the absence of PGA, the highest rate (about one-half the maximum) is attained at 3.5 X 10-3 M ATP. Under the latter conditions, the pH optimum is 8.6. Inhibitors. Fluoride, calcium, and manganous ions are strongly inhibitory at 0.001 M. At this concentration cyanide has no effect, and only slight inhibitions are produced by ferric, ferrous, and zinc ions. C. Splitting Enzyme ~4 L-Argininosuccinic acid ~ L-Arginine + Fumaric acid

Assay Method Principle. Cleavage of argininosuccinic acid is followed by the rate of arginine formation. An excess of arginase is provided during incubation, and arginine is estimated as urea (cf. Over-all Reaction, p. 357). Fumarase is removed during the heat step. Arginase, when originally present as in liver extracts, becomes limiting at an early stage of purification. Direct estimation of arginine is less convenient and inadvisable, since the initial rate is only maintained for about 10 minutes in the absence of arginase or fumarase.

Reagents 0.01 M argininosuccinic acid, pH 7.4, prepared by dissolving 65 to 90 mg. of the assayed barium salt 15 in 9.0 ml. of water and add14 S. Ratner, W. P. Anslow, Jr., a n d B. Petraek, J. Biol. Chem. 204, 115 (1953). 15 F o r p r e p a r a t i o n a n d assay of argininosuccinic acid, see Vol. I I I [93].

[48]

ENZYMATIC SYNTHESIS OF ARGININE

365

ing 1.0 ml. of 0.5 M K~SO4. The clarified solution must contain at least 10 micromoles per milliliter. 1.0 M potassium phosphate buffer, pH 7.4. Arginase containing at least 6 Van Slyke-Archibald units 7 per 0.1 ml. (cf. Preparation of Supplementary Enzymes p. 357).

Procedure. Incubation is carried out in a final volume of 1.0 ml. The mixture contains 0.5 ml. of argininosuccinic acid, 0.05 ml. of buffer, 0.1 ml. of arginase and 1 to 6 units of suitably diluted enzyme. After 20 minutes of incubation at 38 °, the reaction is stopped with 2.0 ml. of 7.5% TCA. Urea is estimated as described for the over-all system except that 1.0 aliquot of the sample or 1.0 ml. of suitable standards is used. Citrulline should, of course, be omitted from the standards. Activity is proportional to enzyme concentration, provided that no more than 40 % of the substrate has been utilized. Definition of Unit and Specific Activity. One unit of enzyme catalyzes the formation of 1 micromole of arginine per hour. Specific activity is expressed as units per milligram of protein. 6 Application of Assay Method to Crude Tissue Preparations. Since arginase is provided, the method is applicable to a variety of crude tissue preparations, and fumarase, which is almost always present in crude preparations, cannot interfere. Purification Procedure The procedure is applicable in detail to pig kidney, ~ as well as to beef liver. Fumarase is completely removed in the heat step, and with liver, arginase is largely removed during the first ammonium sulfate fractionation; the final preparation is arginase-free. The enzyme withstands short periods of exposure to pH 5.0 and withstands dialysis against phosphate buffer but not against Tris buffer. With purification, the enzyme becomes unstable to Tris buffer. The enzyme keeps well when stored at - 2 0 ° as a concentrated solution such as is obtained after each ammonium sulfate precipitation. Dilution with 0.02 M buffer is made just prior to assay. Purification may be interrupted after each ammonium sulfate fractionation, but all the subsequent steps should be completed in a single day. With the exceptions noted, all manipulations are carried out at 0 °, and potassium phosphate, pH 7.4, is employed as buffer throughout. Steps 1-3. Second Ammonium Sulfate Fractionation. The 0 to 30 fraction in 190 ml., obtained during preparation of the condensing enzyme system, is diluted with 475 ml. of 0.02 M buffer and brought to 20% saturation with 93.9 g. of salt over a 30-minute period with mechanical

366

[48]

ENZYMES OF PROTEIN METABOLISM

stirring. After 2 hours, the mixture is centrifuged at 13,000 r.p.m, in the Servall centrifuge for 10 minutes. T h e s u p e r n a t a n t fluid is brought to 3 0 % saturation in a similar m a n n e r with 46.9 g. of salt. The precipitate obtained on centrifugation is dissolved in 32 ml. of 0.02 M buffer. Step ~. Removal of Inactive Protein by Heat. T h e 20 to 30 % saturation fraction is diluted with 294 ml. of 0.04 M sodium acetate buffer, p H 5.0, at room t e m p e r a t u r e and heated for 4 minutes, with swirling, in a large bath kept at 60 ° . In this time the inside t e m p e r a t u r e has reached 53.5 ° and the outside b a t h has dropped to 59 ° . The mixture is then rapidly chilled in a - 5 ° cooling bath and centrifuged at 2000 r.p.m, in the International centrifuge for 30 minutes. This step usually removes 65 to 75 % of the protein with a loss of 15 to 25% of the total activity. Steps 5-6. Treatment with Calcium Phosphate Gel. I t is desirable t h a t the s u p e r n a t a n t fluid, after heat treatment, contain close to 3.0 mg. of protein per milliliter. One-tenth volume (29 ml.) of aged calcium phosphate gel I3 containing 24.4 mg./ml., d r y weight, is immediately added to the s u p e r n a t a n t fluid, and the mixture is stirred i n t e r m i t t e n t l y for 20 minutes. The residue is discarded after centrifugation at 2000 r.p.m, for 15 minutes. This step usually removes from 40 to 50% of the protein and 5 to 10 % or less of the activity. T h e supernatant fluid (310 ml.) is again treated with 156 ml. of gel as above, and the solution obtained after centrifugation is discarded. I t contains 167 mg. of protein and 4 % of the activity present prior to the second addition of gel. T h e positive adsorption step varies in removing from 65 to 80% of the protein and 95% or more of the activity. T h e residue is washed with 85 ml. of 0.01 M buffer, and the washing is discarded. F o u r successive elutions are carried out, each with 75 ml. of TABLE III SUMMARY OF PURIFICATION OF SPLITTING ENZYME FROM LIVER

Fractionation step 1. Acetone powder extract 2. First (NH,)2S04 precipitation, 0-30 % fraction 3. Second (NH4) 2SO4precipitation, 20-30 % fraction 4. Heat treatment at pH 5.1 5. Ca3(P04)2 gel, supernatant 6. Fractional elution from Cas(P04)~ gel followed by (NH4)~SO, precipitation

Total Volume, protein, Specific Total Recovery, ml. mg. activity units % 1800

52,800

1.5 79,200

190

9,080

8.8 79,900

101

43 290 310

3,500 931 474

14.8 51,800 47 43,700 91 43,200

65 55 54

6.8

105

193

20,300

25

[48]

ENZYMATIC SYNTHESIS OF ARGININE

367

0.02 M buffer, with intermittent stirring at 5 to 10°, followed by centrifugation at 2000 r.p.m, for 15 minutes. The combined eluates (300 ml.) are brought to 50 % saturation by the addition of 105.9 g. of ammonium sulfate; the mixture is centrifuged at 13,000 r.p.m, for 15 minutes, and the residue is taken up in 5.0 ml. of 0.02 M buffer. The fractional elution varies in removing from 50 to 80% of the activity and 40 to 60% of the protein present.

Properties Specificity. The only dicarboxylic acid which acts as substrate is fumaric acid. As the second substrate, only L-arginine, TM and at a lower rate canavanine, 18 is active. The latter forms, with fumaric acid, the compound canavaninosuccinic acid. Dependence on Substrate Concentration and pH. The Km for argininosuccinic acid is 1.5 X 10-3 M, and 15 × 10-3 M for both arginine and fumaric acid. The pH optimum is 7.4 in the direction of cleavage and approximately the same for reversal. Equilibrium and Velocity. The equilibrium constant calculated for the (arginine) (fumaric acid) expression K = (arginiuosuccinic acid) is 11.4 X 10-a; (moles per liter at 38 °) and pH 7.5. When the forward and reverse reactions are compared at substrate saturation for each, formation of argininosuccinic acid is 1.4 times as fast as cleavage. At low substrate concentrations, the rate is very much higher in the forward direction. rnhibitors. At concentrations equimolar to argininosuccinic acid (5 micromoles per milliliter) arginine and fumaric acid each inhibit 30%, owing most probably to reversal. At 0.005 M, some inhibition is caused by ferric, ferrous, cobaltous, and zinc ions. 16j. B. Walker, J. Biol. Chem. 204, 139 (1953).

368

ENZYMES OF PROTEIN METABOLISM

[49]

[49] Arginase NH

tl

HOOC--CHNH2--(CH2)2--CH--NHCNtt2 + H20 Arginine H O O C - - C H N H 2 - - ( C H 2 ) , - - C H N H , ~- O~C(NH2)2 Ornithine Urea

By D. M. GREENBERG Assay Method

Arginase Unit. A unit of arginase activity is that amount which in 1 minute at 25 ° and pH 9.5 (glass electrode) and with a substrate concentration of 0.285 M arginine will liberate 1 ~M. (0.06 mg.) of urea containing 0.028 mg of N. This unit, proposed by Van Slyke and Archibald, 1 is the most sound for the measurement of arginase activity because arginine itself is an excellent buffer at the above pH (pK of s-amino group is 9.04); with the high substrate concentration the reaction will be zero order and the urea formed is proportional to the amount of enzyme present; furthermore, ornithine inhibition is minimized. Reagents 0.85 M arginine solution, pH 9.5. To 9.00 g. of arginine monohydrochloride add 1.6 ml. of 18 M NaOH (CO2-free) and make up to 50 ml. with H~O. Check the pH with a glass electrode. Acetic acid, 87 % by volume. Sulfuric acid, 50 % by volume. Maleic acid--MnSO4 solution. Dissolve 5.8 g. of maleic acid in 400 to 500 ml. of H20, and adjust the pH to 9.7 to 9.8 with 0.1 M NaOH. Slowly add this solution, with mixing, to 25 ml. of 2 M MnSO4 in a 1-1. volumetric flask and make the volume up to the liter mark. The pH will be at about 7.0. The maleate keeps high concentrations of Mn ++ in solution, preventing deposition of MnO2. Xanthydrol reagent. Xanthydrol is prepared from xanthone accordD. D. Van Slyke and R. M. Archibald, J. Biol. Chem. 165, 293 (1946).

[49]

ARGINASE

369

ing to the procedure of Werner. 2 Mix 1.0 g. of xanthone 3 and 1.5 g. of zinc dust in 15 ml. of 95% ethanol. To this add 1.4 ml. of saturated NaOH and 0.6 ml. of H20. Heat the mixture in a water bath at 70 ° for 15 minutes, then filter into about 100 ml. of cold water and allow to stand in the refrigerator overnight in order to precipitate the xanthydrol. Filter onto a weighed filter paper and dry on the paper in vacuo over calcium chloride or P205. After weighing, scrape the xanthydrol from the paper into a small volume of methanol, filter the solution into a glass-stoppered 50-ml. volumetric cylinder to remove the paper fibers, and add additional methanol to adjust the concentration to 5 %. Store in the refrigerator. (A slight yellow color may appear, or a few, small, yellowish cubical crystals may form on standing in the refrigerator. These do not interfere with the determination.) The xanthydrol solution should be made up fresh about every two weeks. Dixanthylurea wash solution. In each of four 50-ml. centrifuge tubes place 5 ml. of a solution containing about 0.6 mg. of urea, 5 ml. of glacial acetic acid, and 1 ml. of the 5% xanthydrol reagent. Stir, and allow to stand for 30 minutes. Then add 20 ml. of methanol, stir the solution, and centrifuge. Wash the precipitate with 20 ml. of methanol, centrifuge, and decant. This precipitate should be sufficient to saturate l-liter each of methanol and 3:1 methanol-water at room temperature. Procedure. The enzyme preparation, in not less than about 0.1% protein solution; this enzyme dilution is activated before assay by incubation at about 37 ° for 3 to 4 hours in the presence of Mn ++ (0.05 M MnSO4, 0.05 M maleate at pH 7). For assay, the activated enzyme is introduced into a 1:50 dilution of the stock maleate-manganous sulfate solution. This enzyme dilution should contain in the range of 0.03 to 0.3 A./ml. (For example, extract A, p. 371, should be diluted about 1 : 500.) As soon as this dilution is made, 1 ml. of it is added to 0.5 ml. of 0.85 M arginine at pH 9.5 in a 15 X 125-mm. Pyrex test tube previously brought to 25 °, mixed, and incubated for 10 minutes at 25 ° in a constant temperature bath. The reaction is stopped by the addition of 2.0 ml. of 87 % acetic acid, and the urea determined as described below. A control for the reagent blank is prepared by adding the acid to the arginine before, instead of after, the addition of arginase. In our hands linearity was best in the enzyme concentration range given, where the maximal hydrolysis was only 0.7%. Van Slyke and Archibald show a nearly linear proz A. E. Werner,~"The Chemistry of Urea," p. 183, Longmans, Green and Co., London, 1923. 3 Commercial xanthydrol can be made suitable for use in the analysis by being completely reduced in the same manner.

370

EI~ZYMES OF PROTEIN METABOLISM

[49]

portionality between enzyme and urea formed up to 3.0 A. per 1.5 ml. of reaction mixture. Determination of Urea. It is desirable to determine urea directly rather than first hydrolyze it to ammonia with urease, as is commonly done. The reasons for this are that urease preparations are apt to contain arginolytic activity, and heat inactivation of the arginase in the samples being assayed causes increased enzyme activity with resultant further hydrolysis, whereas acid inactivation followed by readjustment to pH 6.8 and addition of KCN for good urease action has a number of undesirable features. The colorimetric determination of urea by the procedure of Engel and Engel 4 suffers from two sources of error, namely, the general unsuitability of commercial xanthydrol and the mechanical loss of dixanthylurea during washing. These sources of error are reduced by employing purified xanthydrol as described above and by carrying out the entire procedure in 15 X 125-mm. Pyrex test tubes rather than centrifuge tubes. After stopping the arginase action with acetic acid, add 0.2 ml. of the 5% xanthydrol reagent in methanol directly to the acetic acid mixture. Mix the contents of each tube well, taking care to keep it from getting upon the walls of the tube. Then place the tube in a refrigerator for 12 to 18 hours. After removal from the refrigerator, allow the tubes to warm to room temperature, add 2 vol. of filtered methanol, saturated with dixanthylurea (by standing over solid dixanthylurea), and stir frequently for 15 minutes. The contents of the tubes should present a silky appearance. Care must be taken that the wash solution does not deposit dixanthylurea through evaporation following filtration. Centrifuge the tubes in a large horizontal head of an International No. 2 centrifuge for 10 minutes at 2500 r.p.m., and siphon off the supernatant fluid carefully down to about 0.5 ml. with a capillary tube, employing a slight vacuum. Wash the precipitate twice more in a similar fashion using, however, 10 ml. of a 3:1 methyl alcohol-water solution saturated with dixanthylurea as above. The small volume of wash solution remaining in each tube after the last wash can be removed in a few hours by placing all tubes at an angle in a desiccator attached to a vacuum and suitably trapped. To each tube then add 10 ml. of 50% sulfuric acid (a dispensing buret is convenient). Stir the contents well, and allow to stand for 20 to 30 minutes. Read the color intensities in standard colorimeter tubes in a Klett-Summerson photoelectric colorimeter with a blue filter or in a Coleman Junior spectrophotometer. For a blank, 50 % sulfuric acid is employed. Colorimeter 4 M. G. Engel a n d F. L. Engel, J. Biol. Chem. 167~ 535 (1946).

[49]

ARGINASE

371

readings should not be below 100 or above 500 to 600 for accurate results. A calibration curve m a d e from known a m o u n t s of urea gave a straight line plot having the equation Urea (~,) = 0.21 X colorimeter reading (Klett-Summerson) T h e nitrogen content of the arginase p r e p a r a t i o n is determined b y the micro-K]eldahl method, the a m m o n i a being t i t r a t e d in cruder preparations and being distilled and determined b y nesslerization in preparations of high activity.

Purification Procedure The method of preparation of arginase described below yielded a 400-fold increase in activity over t h a t of the original extract. 5,6 Fresh livers from male horses ~ should be secured, d a r k in color and free from a yellowish coloration. The highest arginase content was found in livers of animals sacrificed in the late spring or early s u m m e r months. T h e livers were packed in d r y ice and k e p t frozen until used. When r e a d y to work up t h e y were thawed, cleaned, and ground in a m e a t grinder. T h e effect of each step of the isolation procedure on the p u r i t y is shown in the table. Step 1. Starting with 3 or 4 kg., 2 1. of water is added per kilogram of fresh ground liver. With vigorous stirring, the p H is adjusted to a b o u t 7.6 with 0.1 M N a O H . N e x t 75 ml. of 2.0 M MnSO4 is added in small portions (15 to 20 ml.), alternating with sufficient 0.1 M N a O H to keep the p H between 6.5 and 7.7 (at the end of the M n S Q addition the p H was 7.6). W a t e r is now added to bring the total volume of liquids added up to 3 1./kg. of liver. T h e n 50 ml. of toluene is stirred in. T h e mixture is allowed to extract overnight in the cold room and then is strained through a layer of cheesecloth (extract A). For assay a small aliquot is filtered through paper. Step 2. Equal portions of extract A and cold redistilled acetone are mixed with stirring and centrifuged i m m e d i a t e l y for 20 minutes at 850 × g. at 3 °. The s u p e r n a t a n t is discarded. T h e precipitate is d r a i n e d for 5 minutes and is extracted with 25 ml. of 0.05 M maleateS-0.05 M 5 E. A. Bagot, Ph.D. Thesis, University of CMifornia, 1952. 8 O. A. Roholt, Jr., Ph.D. Thesis, University of California, 1953. 7 Horse liver is now used in our laboratory because of the excessively high cost of beef liver. It appears not to be inferior to beef liver. s Owing to the oxidation of manganous hydroxide, the addition of even very dilute (0.001 M) NaOH to a more or less neutral solution containing Mn ++ results in the formation of brown manganic hydroxide. To prepare the more dilute solutions used above, it is convenient to prepare a solution of 11.6 g. of maleic acid in 400 to 500 ml. of H~O, adjusting the pH to about 9.7 to 9.8 with 1 M NaOH. This solution is quantitatively added to 100 ml. of 2 M MnSO4 in a liter volumetric flask and

372

ENZYMES OF PROTEIN METABOLISM

[49]

1V[nS04 solution at pH 7.0 for each 100 ml. of extract A originally used. The resuspended precipitates are then centrifuged as above for 1.5 hours, and the precipitates are discarded. The supernatant (extract B) is clear and red, becoming turbid at room temperature. Step 3. Extract B in 250-ml. lots is heated in 1-1. Erlenmeyer flasks for 20 minutes in a 60 ° water bath with constant agitation. The flasks are cooled under tap water, and the extract is filtered on a Biichner funnel covered with a rubber dam (extract C). Step 4. Extract C is dialyzed thoroughly against 0.001 M maleate0.001 M MnSO4 solution, pH 7.0, and lyophilized, giving powder D (3.5 to 4.0 g./kg, of liver). Step 5. 0.05 M MnS04 is saturated with (NH4)2S04 in the cold and adjusted to p H 7.2 with concentrated NH4OH. An 8% solution of powder D is prepared in 0.2 M maleate-0.05 M MnS04 solution, pH 7.45. The resulting solution should be at pH 7.0. A drop of caprylic alcohol is added, and 81.8 ml. of the above (NH4)2SO4 solution per 100 ml. of protein solution is added slowly with stirring. The solution is allowed to stand for 1/~ hour and then is centrifuged for 1~ hour at 15,000 X g. The precipitate is discarded, and 37.5 ml. of (NH~)SO, is added per 100 ml. of supernatant fluid and centrifuged as above. The supernatant is discarded. The precipitate is taken up in 0.05 M MnSO4-0.05 M maleate solution of pH 7.0 and dialyzed against 0.001 M maleate-0.05 M MnSO4 solution of pH 7.0. The protein solution is lyophilized, giving powder E. Step 6. A 5% solution of powder E is prepared in water. The pH should be 7.0. Ninety-four milliliters of the MnSO4-(NH~)~SO, solution of step 5 is added to 100 ml. of protein solution and treated as in the first (NH,):SO4 fractionation. Thirty-six milliliters of the (NH,)2SO4 is added per 100 ml. of supernatant and centrifuged as before. The precipitate is taken up in and dialyzed thoroughly against 0.005 M maleate-0.01 M MnSO4, pH 7.0. After thorough dialysis the protein solution is diluted to 0.4 mg. of protein N per milliliter with the same solution (solution F). brought to volume. The pH will then be about 7. Solutions of the desired concentration can be prepared by adding to this maleate or MnSO4 solutions of the proper concentration and diluting with water. The maleate was adopted for use with arginase because it keeps high concentrations of manganese in solution. In previous work, in which other buffers were employed, deposition of MnO2 precipitates was a frequent annoyance. The value for the second dissociation constant of maleic acid is in the neighborhood of pH 5.8, thus being somewhat out of the buffer range used in our work. In our work this complication can be neglected, since control experiments indicate that the enzyme preparations used maintained their pH values throughout the experiments.

[49]

ARGINASE

373

Step 7. At 0 °, 13.7 ml. of methanol per 100 ml. of protein solution is slowly added with stirring to solution F, and then the solution is cooled to - 5 ° and centrifuged for i hour at - 5 ° at 1600 X g. The small precipitate is discarded, and the supernatant is placed in a - 9 ° ice bath. To prevent its freezing, the slow addition of 29.4 ml. of methanol per 100 ml. of supernatant is commenced immediately with stirring. The solution is allowed to reach - 9 ° and is centrifuged as before at - 9 °. A small amount of cold 0.005 M maleate-0.01 M MnS04 solution, pH 7.0, is added to the precipitate, and it is lyophilized directly, giving powder G. Step 8. Powder G is dissolved in and thoroughly dialyzed against 0.01 M maleate-0.03 M Mn ++ solution, pH 6.9. After dialysis the protein solution is diluted to 0.5 mg. of protein N per milliliter with the same solution. At 0 ° 27 ml. of cold redistilled carbitol acetate (diethylene glycol monoethyl ether acetate) per 100 ml. of protein solution is slowly added with stirring. The solution is cooled to 0 ° and centrifuged at 650 X g. for 45 minutes at 0 °. The precipitate is dissolved, and the supernatant is placed in a 0 ° ice bath until thermal equilibrium is reached. Next 26 ml. of carbitol acetate per 100 ml. of protein solution is slowly added with stirring. The solution is centrifuged at 0 ° for 45 minutes at 650 × g. The supernatant is discarded, and the precipitate is dissolved in a small amount of 0.03 M MnSQ-0.01 M maleate solution at pH 6.9. This supernatant mixture is thoroughly dialyzed against the same solution so as to remove any residual carbitol acetate and then lyophilized to give powder H.

Properties Arginase purified by the above method is a colorless and compgratively stable protein. Aqueous solutions of the enzyme at pH 7.0 can stand indefinitely at average refrigerator temperatures (4°) without loss of activity, although this does not apply for room temperatures (~20°). At pH values below 6.0 and above 9.0 the enzyme becomes very unstable and quickly loses activity even at refrigerator temperatures. The purified enzyme is unstable at high dilutions but is stabilized by low concentrations of ornithine (0.0025 M) or glycine (0.02 M) and by arginine at higher concentrations (0.285 M). Arginase can be lyophilized to a white powder from neutral aqueous solutions and redissolved without any apparent loss of activity. The lyophilized preparations can be maintained unchanged indefinitely at room temperatures. The molecular weight is about 140,000. The turnover number, in molecules of substrate decomposed per molecule of enzyme per minute at 25 ° and pH 10.2, is 1.3 X 105. Physical-chemical tests of purity yielded the following results. In the analytical centrifuge at pH 7 there was one large and a very small second,

374

ENZYMES OF PROTEIN METABOLISM

[50]

faster-moving b o u n d a r y ; electrophoresis at p H 6.90 showed a slow-moving main b o u n d a r y comprising something over 90% of the total with a very small faster-moving b o u n d a r y as well as a small slower-moving boundary. INCREASE IN ARGINASE ACTIVITY WITH PURIFICATION

(Average Values) Preparation

Protein N per ml. or mg.

A./ml. or mg.

Extract A Extract B Extract C Powder D Powder E Solution F Powder G Powder H

50 . 90 25 80 725 350 425

.

4.0 . 0.70 0.09 0.09 0.4 0.1 0.1

A.P.

Yield, a %

12

100

125 225 1000 1800 3500 4250

78 75 50 40 30 25

.

a Based on extract A units.

[50] Arginine Dihydrolase B y EVELYN L. OGINSKY Arginine dihydrolase, the enzyme system converting arginine to ornithine, has recently been found to consist of two distinct enzymatic reactions: (1) arginine desimidase, which hydrolyzes arginine to citrulline and NH3, and (2) citruUinase, which decomposes citrulline to ornithine, NH~, and COs.

•Reaction 1: Arginine desimidase H O O C - - C H - - ( C H ~ ) ~ - - N H - - C - - N H ~ --~

~'H2

~TH HOOC--CH--(CH2)3--NH--C--NH~

I

Reaction 2: Citrultinase

+ NH3

II

NH~

0

(or citrulline ureidase) H O O C - - C H - - ( C H ~ ) 3--NH~

f

NH2

-t- NH3-b CO~

[50]

ARGININE DIHYDROLASE

375

A. Arginine Desimidase Assay Method Principle. Measurement of both end products, citrulline and NH3, is recommended when crude cell preparations are employed as enzyme source. The activity of cell extracts free of other enzymes attacking arginine or citrulline can be measured by estimation of either end product. Reagents 0.1 M L-argin~ne. Adjust the hydrochloride to pH 6.5. 0.2 M phosphate buffer, pH 6.5. Enzyme. Bacterial cells, intact or acetone-dried, 25 mg. of dry weight per milliliter; or cell-free extract, 2 mg. of protein or 300 ~, of N per milliliter.

Procedure. Mix in test tubes 0.4 ml. of arginine solution, 1.0 ml. of buffer, 0.2 ml. of enzyme, and water to 3.0 ml., and incubate at 37 °. Stop the reaction in replicate tubes at ]5-minute intervals with 0.2 ml. of 70% perchloric acid, and after centrifugation assay aliquots of the supernarants for citrulline 1 and NH3. 2 Purification Procedure Step 1 is based on the methods employed by Roche and Lacombe using baker's yeast, 3 Schmidt et al. using C. perfringens, 4 and Oginsky and Gehrig using S. faecalis. 5 Step 2 has been reported for the arginine desimidase from baker's yeast. 3 Step 1. Preparation of Crude Extract. Acetone-dried bacterial or yeast cells are made up in a suspension in either water, 1% NaC1, or 0.033 M phosphate buffer, pH 6.5, and incubated at room temperature for 1 hour, preferably with shaking. The suspension is stored in the refrigerator overnight, and the cellular debris is removed by centrifugation at 0% Lyophilized preparations of the supernatant enzyme extract are stable for several months in the refrigerator. Step 2. Ethanol Precipitation. The crude extract from baker's yeast obtained by step 1 can be fractionated with ethanol at pH 5.7 to 5.8 and 1°; arginine desimidase precipitates almost completely between 20 and 30% ethanol. 1 R. M. Archibald, J. Biol. Chem. 156, 121 (1944); see Vol. I I I [92]. 2 j. A. Russell, J. Biol. Chem. 156, 457 (1944); see also Vol. I I I [145]. a j. Roche and G. Lacombe, Biochim. et Biophys. Acta 9, 687 (1952). 4 G. C. Schmidt, M. A. Logan, and A. A. Tytell, J. Biol. Chem. 198, 771 (1952). 5 E. L. Oginsky and R. F. Gehrig, J. Biol. Chem. 198, 799 (1952).

376

ENZYMES OF PROTEIN METABOLISM

[50]

Properties Specificity. T h e e n z y m e is specific for L-arginine and is inactive on citrulline, canavanine, glycocyamine, creatinine, creatine, urea, and agmatine. Activators and Inhibitors. T h e a c t i v i t y of the e n z y m e e x t r a c t p r e p a r e d as a b o v e is stable to prolonged dialysis and has not been shown to require a n y cofactors. Only the e n z y m e in y e a s t juice obtained b y NaC1 plasmolysis 3 requires Co ++ (at 1 × 10 -3 M) or Ni ++ (at 2 X 10 -4 M) after dialysis. T h e e n z y m e is strongly inhibited b y several cations (Cu ++, M g ~+, H g ++, and Zn ++) at 1 X 10 -~ M. A p p r o x i m a t e l y 50 % inhibition of a c t i v i t y is produced b y 1 X 10 -3 M arsenite, 1 X 10 -3 M hydroxylamine, or 1 X 10 -2 M semicarbazide. N o effect is observed with the following at 1 X 10 -3 M : A1+++, Co ++, 5/In ++, Fe +++, cyanide, arsenate, fluoride, azide, or d i e t h y l d i t h i o c a r b a m a t e . Effect of p H . T h e p H o p t i m u m depends on the source of the enzyme; t h a t f r o m y e a s t is 6.2 to 6.5, t h a t f r o m S. faecalis 6.8. Distribution. T h e e n z y m e has been reported to be present in b a k e r ' s yeast, 3 Clostridium perfringens, 4 Streptococcus faecalis, 8 Lactobacillus fermenti, 7 Lactobacillus mesenteroides, 7 Chlorella pyrenoidosa, 7 Pseudomonas aeruginosa, 8 Staphylococcus aureus, 9 and Streptococcus lactis. 1°

B. Citrullinase Assay Method Principle. T h e simplest m e t h o d of measuring e n z y m a t i c b r e a k d o w n of citrulline 1~-14 is the m a n o m e t r i c determination of CO2 formation. E n z y m e a c t i v i t y can also be determined b y colorimetric m e t h o d s for either of the two other reaction products, ornithine ~5 and NH3. 2 Inorganic o r t h o p h o s p h a t e is t a k e n up during the course of the reaction, and equivalent a m o u n t s of A7 P are formed. A T P generation can

6 T. Sekine, J. Japan. Biochem. Soc. 19, 79 (1947) [Chem. Abstr. 44, 10789 (1950)]. J. B. Walker, J. Biol. Chem. 204, 139 (1953). s F. Horn, Z. physiol. Chem. 216, 244 (1933). 9 I. Lominski, R. B. Morrison, and I. A. Porter, Biochem. J. 51, xvii (1952). 10M. Korzenovsky and C. H. Werkman, Arch. Biochem. and Biophys. 41, 233 (1952). 11V. A. Knivett, J. Gen. Microbiol. 8, v (1953). 1~H. D. Slade, Arch. Biochem. and Biophys. 42, 204 (1953). 13 M. Korzenovsky and C. H. Werkman, Arch. Biochem. and Biophys. 46, 174 (1953). 14E. L. Oginsky and R. F. Gehrig, J. Biol. Chem. 204, 721 (1953). 15F. P. Chinard, J. Biol. Chem. 199, 91 (1952).

[50]

ARGININE DIHYDROLASE

377

be demonstrated either by coupling of the reaction with hexokinase 16 or by labeling of ATP with p32 from radioactive orthophosphate. 1~

Reagents 0.5 M acetate buffer, pH 5.8. 0.01 M 5-AMP or ADP, adjusted to pH 5.5 to 6.0. 0.1 M MgC12. 0.1 M K phosphate, pH 5.8. 0.1 M arsenate, adjusted to pH 5.8. 0.2 M DL-citrulline, or 0.1 M L-citrulline, adjusted to pH 5.5 to 6.0. 2.0 M H~S04. Enzyme. Acetone-dried bacterial cell preparations suspended in H~O to give 20 mg. per milliliter, or cell-free enzyme preparations, described below, containing 3 to 5 rag. of N per milliliter.

Procedure. In the center cup of double-side arm Warburg flask, place 1.0 ml. of acetate buffer, 0.5 ml. of 5-AMP or ADP, 0.1 ml. of MgC12, 0-5 ml. of K phosphate, and 0.5 ml. of enzyme preparation. In one side arm, place 0.2 ml. of citrulline; in other side arm, 0.2 ml. of H2SO4. Flush the flasks with N2 for 5 minutes, and incubate at 37 °. Tip in citrulline at zero time, and measure CO: release. Manometer readings should be made at intervals of 5, or at the most 10, minutes to obtain valid estimation of maximum reaction rates. H2S04 tipped in at the end of the reaction interval releases bound CO2. Control flasks, iacking only citrulline, provide endogenous COs correction. The 5-AMP or ADP, MgC12, and phosphate requirements are replaceable by 0.5 ml. of arsenate alone. Purification Procedure Two methods can be employed to obtain cell-free extracts from bacterial cells. Intact cells in H:O or phosphate buffer, pH 5.6 to 5.8 (20 to 30 mg. dry weight per milliliter), are subjected to disintegration by (1) sonic oscillation in the Raytheon oscillator at a frequency of 9 kilocycles for 90 to 180 minutes, 12,13 or (2) shaking with Ballotini glass balls in the Mickle sonic disintegrator at 50 cycles per second for 10 minutes. 1~ The clear supernatant obtained after either procedure contains both arginine desimidase and citrullinase.

Properties

Specificity. The enzyme is specific for L-citrulline and is inactive on D-citrulline or urea. 16 H. D. Slade, C. C. Doughty, and W. C. Slamp, Arch. Biochem. and Biophys. 48, 338 (1954). 17 V. A. Knivett, Biochem. J. 66, x (1953).

378

ENZYMES OF PROTEIN METABOLISM

[51]

Activators and Inhibitors. Dialysis of cell-free extracts or of acetonedried cells results in preparations which are completely inactive unless supplemented as above with 5-AMP or ADP, Mg ++, and orthophosphate. The Mg ++ requirement can be replaced by Mn ++, Co ++, or Zn ++. When the phosphate requirement is replaced by equivalent amounts of arsenate, neither Mg ++ nor 5-AMP (or ADP) is then required. The enzyme is inhibited by fluoride, which produces 50% inhibition of 10 mg. of S. faecalis acetone-dried cells at 1 × 10-3 M. This inhibitor is effective in the presence or absence of added Mg ++, i.e., on phosphorolysis or arsenolysis. The citrullinase reaction is inhibited by excess ornithine and runs to only 75 to 85% completion unless this product is removed (as by ornithine decarboxylase16). Hg ++ and organic mercurials are potent inhibitors at 1 X 10-4 M or higher; this inhibition is relieved by BAL or GSH. Arsenite, IAA, and DNP at 1.7 X 10-8 M are without effect. Phosphate inhibits the reaction run in the presence of arsenate. Effect of pH. The pH optimum of the cell-free enzyme extract, with either the phosphate or arsenate system, is 6.2 to 6.3. Distribution. The enzyme has been reported to be present in S. faecalis, 11.12.14S. lactis, 13 Pseudomonas,16 and C. perfringens.4

[51] U r e a s e

By JAMES B. SUMNER Jack B e a n M e a l

It is usually possible to purchase satisfactory jack bean meal from American supply houses. The author has purchased jack beans high in urease from Charles Martin, Waldron, Arkansas, or from Ernest Nelson, Route 1, Waldron, Arkansas. The beans can be ground in a Mikro Sample Mill, sold by the Pulverizing Machinery Company of Summit, New Jersey. The beans must be "bone d r y " when ground. Preparation of Crystalline U r e a s e 1

Place 100 g. of jack bean meal, finely powdered and rich in urease, in a 1000-ml. beaker. Add 500 ml. of 32 % c.p. acetone at 28 °. At once stir for 3 or 4 minutes, using preferably a wooden stick. Pour on 32-cm. Whatman No. 1 pleated filter paper. When 100 to 150 ml. of extract has filtered through, place the filtering material in the ice chest at 0 to 5 °, and allow to stay overnight. The next day centrifuge the urease crystals 1j. B. Sumner, J. Biol. Chem. 69, 435 (1926).

[51]

UREASE

379

in the filtrate, using a refrigerated centrifuge. Decant the supernatant liquid, and dissolve the urease crystals in water. The water employed for this preparation should be distilled from pyrex glass.

RecrystaUization of Urease The method of Dounce ~ is as follows: Dissolve the crystals of crude urease by adding 3 ml. of water for every 100 g. of jack bean meal. Centrifuge the urease solution practically clear in a refrigerated centrifuge, or else filter and refilter it until it is practically clear in the ice chest. Then for every 20 ml. of urease solution add 1 ml. of 0.5 M citrate buffer, pH 6.0. Next add with stirring 0.2 vol. of pure acetone. Place the preparation in the ice chest. Crystallization of the urease will be complete in about 30 minutes. The crop of urease crystals can be increased by adding acetone, a few drops at a time, until the acetone concentration is about 25 %. Urease Paper Centrifuge down crystals of urease and dissolve in water, using about 10 ml. for the crystals from 100 g. of jack bean meal. Add about 100 mg. of neutral cysteine and 10 mi. of 0.5 M citrate buffer, pH 6.2, and mix. Moisten strips of filter paper with this solution, and dry. Place the dry strips in a brown glass bottle, and stopper. Acting on urea, one square centimeter of urease paper should turn 1 ml. of 0.5 M citrate buffer, pH 6.0, alkaline to phenol red in 6 minutes at 25 °. This urease paper will remain serviceable for several years. About 0.3 sq. cm. will be sufficient for each analysis of blood urea. Estimation of Urease Activity 3 Place 1 ml. of properly diluted crystalline urease in a test tube that is free from heavy metals, immerse this tube in a thermostat bath at 20 °, and allow it to come to this temperature. Then from a 1-ml. pipet blow into the urease solution 1 ml. of 3 % urea, 9.6 % phosphate, pH 7.0, which is at 20 °, starting a stop watch at this time. Mix, and allow to digest for 5 minutes. At this time add 1 ml. of N hydrochloric acid, and mix. Transfer the solution to a 200-ml. volumetric flask, dilute to about 150 ml., and add 10 ml. of Nessler solution. Dilute to mark, mix, and read in a photoelectric colorimeter, using a green glass filter. The milligrams of ammonia nitrogen present represent urease units. The amount present should be 0.2 to 0.5 mg. If crude urease is being analyzed, it will not be possible to nesslerize directly, but the ammonia will first have to be aerated into acid. 2A. L. Dounce, J. Biol. Chem. 140, 307 (1941). J. B. Sumner and D. B. Hand, J. Biol. Chem. 76, 1t9 (1928).

380

ENZYMES OF PROTEIN METkBOLISM

[52]

[52] G l u t a m i n a s e , A s p a r a g i n a s e , and ~ - K e t o A c i d - c o - a m i d a s e

By

ALTON M E I S T E R

A. L-Glutaminase from Escherichia coli CONH2

I

(CH2)2

I

CHNH2

I

COOH

COOH

I

+ H~O--. (CH~)~

-}- NH3

I

CHNH2

I

COOH

Assay Method

Principle. Enzyme activity may be measured by determinations of ammonia or of L-glutamate. 1 The assay procedure described below is based on determination of the rate of ammonia liberation. Reagent L-Glutamine (0.08 M), freshly prepared in 0.1 M sodium acetate buffer, pH 4.9.

Procedure. Enzyme solution (0.25 ml.) and L-glutamine (0.25 ml.) are incubated for 5 to 60 minutes at 37 °. The reaction is stopped by addition of 0.5 ml. of 15% trichloroacetic acid. Ammonia is determined by aeration in the presence of potassium carbonate into sulfuric acid traps. ~ Blanks containing glutamine alone and enzyme preparation alone are employed. Definition of Unit. One unit is defined as the quantity of enzyme which catalyzes the formation of 1 aM. of ammonia per hour under the assay conditions described above. Purification Procedure ~

Step 1. Starting Material. Escherichia coli (Strain W) is grown on a medium consisting of 0.3 % KH2P04, 0.7% K~HP04, 0.05% sodium citrate.2 H20, 0.01% MgS04-7 H20, 0.1% (NH4)~S04, 0.2% enzymatic digest of casein, and 0.5% glucose. 4 After incubation at 37 ° for 18 hours, 1 Assay based on determination of L-glutamate is not reliable in crude extracts of E. coli owing to the presence of L-glutamic acid decarboxylase. 2 j. p. Greenstein and F. M. Leuthardt, J. Natl. Cancer Inst. 5, 209 (1944) ; see Vol. I I I [145] for details of NH3 procedure. Based in part on D. R u d m a n and A. Meister, J. Biol. Chem. 200, 591 (1953). 4 B. D. Davis and E. S. Mingioli, J. Bacteriol. 60, 17 (1950).

[52]

OMEGA-AMIDASES

381

the cells are harvested in the Sharples centrifuge, washed twice with water by centrifugation, and lyophilized. The cells may be stored at - 2 0 ° for several months without loss of glutaminase activity. Step 2. Preparation of Cell-Free Extract. The dried cells are thoroughly ground in a mortar with 3 parts of Alumina (No. A-301, Aluminum Company of America) for about 30 minutes. The ground material is then vigorously shaken with 20 parts of cold distilled water on a shaking machine at 5 ° for 20 minutes, after which the mixture is centrifuged at 18,000 × g for 20 minutes. The slightly turbid cell-free supernatant contains 60 to 80 % of the original activity of the cells and is about twice as active as the cells on the basis of total nitrogen (Kjeldahl). 5 Step 3. Adsorption on and Elution from Calcium Phosphate Gel. A solution of calcium phosphate gel (100 ml. ; dry weight, 15 mg./ml.) is centrifuged, and the sedimented gel is mixed with the extract obtained from 5 g. of cells. After shaking for 30 minutes at 24 to 28 °, the mixture is centrifuged at 600 × g for 20 minutes. The supernatant solution is discarded, and the gel is thoroughly mixed with 40 ml. of 0.1 M KH2PO4 (pit 4.5). The mixture is shaken for 20 minutes at 5 °, followed by centrifugation at 5 °. Two additional elutions are carried out, and the eluates are combined and lyophilized. 6 The lyophilized material may be stored at 5 ° for several months without loss of activity. Solutions of the enzyme lose considerable activity when kept at 5 ° for three weeks. The preparation at this stage contains 20 to 40 % of the total activity of the extract and exhibits an activity of 6000 to 8000 units/mg, of protein nitrogen, representing a 15- to 20-fold increase in specific activity over the cellfree extract. Step 4. Fractionation with Sodium Sulfate. The specific activity may be doubled with the loss of approximately half of the total activity by suspension of the lyophilized powder obtained in step 3 in saturated Na2SO4 solution: 50 rag. of powder is suspended in 5 ml. of Na2SO~ solution, shaken for 20 minutes at 24 to 28 °, and centrifuged. The pellet, which represents the more active fraction, is dissolved iu 1 ml. of cold water. This solution loses activity after storage at 5 ° for several days. Properties

Specificity. The following amides are hydrolyzed at rates less than 1% of that observed with L-glutamine: D-glutamine, L-isoglutamine, L-asparagine, n-asparagine, L-isoasparagine, L-homoglutamine (a-aminoadipamic 5 A representative fractionation is described in Table I. L-Glutamic decarboxylase may be eluted from the calcium phosphate gel by buffer of p H 5.5. 3

382

ENZYMES OF ]PROTEIN METABOLISM

[52]

acid), a-ketoglutaramate, and a-ketosuccinamate. I n contrast to the crude extract, the purified preparation does not decarboxylate L-glutamic acid. 6 Effect of p H . The optional p H range (in 0.1 M acetate buffer) is 4.7 to 5.1.

Other M e t h o d s of Preparation 7 The preparation and properties of glutaminase isolated from C. welchii 8 have been described. Aqueous extracts of certain animal tissues

(e.g., liver, kidney, brain, and spleen of the rat, rabbit, and mouse) exhibit glutaminase activity in the presence of phosphate or arsenate2 -11 TABLE I SUMMARY OF PURIFICATION PROCEDURE FOR GLUTAMINASE

Fraction Extract Eluate from gel Na2S04 fraction

Total Total Specific volume, Units/ml., units, Nitrogen, activity, Recovery, ml. thousands thousands mg./ml, units/mg. N % 82 120 10

1.44 0.314 1.77

118 37.6 17.7

3.66 0. 0485 0. 149

394 6,470 11,900

-32 15

This enzyme (designated glutaminase I) exhibits a p H o p t i m u m at 8.0 and has been purified three-fold from rat liver. 12 I t is associated with particulate material. A soluble preparation capable of deamidating glutamine in the presence of a-keto acids has also been obtained from rat liver. 12 This reaction appears to involve transamination of glutamine 13 yielding a-ketoglutaramate, which is hydrolyzed b y an amidase to a - k e t o g l u t a r a t e and ammonia. 14 A similar mechanism is involved in the a-keto acid-mediated deamidation of asparagine: x4,15 (See a-keto acid~-amidase, below.) A n u m b e r of animal tissues possess asparaginase activity, which m a y be distinguished from asparagine transaminase b y differential heat inactivation. For a recent review, see C. A. Zittle, in "The Enzymes" (J. B. Sumner and K. Myrb/ick, eds.), Vol. I, Part 2, p. 922, Academic Press, New York, 1951. 8 D. E. Hughes and D. H. Williamson, Biochem. J. 51, 45 (1952). 9 C. E. Carter and J. P. Greenstein, J. Natl. Cancer Inst. 7, 433 (1947). 10j. p. Greenstein and F. M. Leuthardt, Arch. Biochem. 17, 105 (1948). 11M. Errera and J. P. Greenstein, J. Biol. Chem. 178, 495 (1949). i~ M. Errera, J. Biol. Chem. 178, 483 (1949). See also the most recent work of M. C. Otey, S. M. Birnbaum, and J. P. Greenstein, Arch. Biochem. and Biophys. 49, 245 (1954). is A. Meister and S. V. Tice, J. Biol. Chem. 187, 173 (1950). 14A. Meister, J. Biol. Chem. 200, 571 (1953). l.~ A. Meister, H. A. Sober, S. V. Tice, and P. E. Fraser J. Biol. Chem. 197, 319 (1952).

[52]

OMEGA-AMIDASES

383

B. L-Asparaginase from Guinea Pig Serum

CONH2 CH~

I CHNH2

I

COOH

COOH CH2

+ tt~O--~ I

+ NH3

CHNH~

I

COOH

Assay Method

Principle. Enzyme activity may be conveniently measured by determinations of ammonia.

Reagents L-Asparagine monohydrate (0.04 M). Sodium borate buffer (0.010 M), pH 8.5.

Procedure. Enzyme solution (0.5 ml.), substrate (0.5 ml.), and buffer (1.0 ml.) are mixed and incubated for 5 to 60 minutes at 37 °. The reaction is stopped by addition of 0.5 ml. of 15% trichloroacetic acid, and ammonia is determined as described above. 2 Definition of Unit. One unit is defined as the quantity of enzyme which catalyzes the formation of 1 ~M. of ammonia per hour under the conditions of the assay. Purification Procedure 16

Step 1. Fractionation with Na2S04. Fresh guinea pig serum (100 ml.) is mixed with an equal volume of 30 % Na~SQ. The mixture is allowed to stand at 24 to 28 ° for 30 minutes and then centrifuged at 1000 X g for 60 minutes. The supernatant solution is discarded, and the precipitate is dissolved in 50 ml. of cold water. This preparation is four to five times as active as the original serum. Step 2. Adsorption of Impurities by Calcium Phosphate Gel. The final solution prepared in step 1 is mixed with the residue obtained by centrifuging an equal volume of calcium phosphate gel (dry weight, 15 mg./ ml.). After shaking at 24 to 28 ° for 30 minutes, the gel is removed by centrifugation. The solution may be stored in the frozen state without loss of activity over a period of several weeks. Properties Specificity. The purified preparation does not hydrolyze L-isoasparagine, L-glutamine, D-glutamine, DL-C~-methylasparagine, L-homogluta,6 A representative fractionation is described in Table II.

384

ENZYMES OF PROTEIN METABOLISM

[52]

mine (a-aminodipamic acid), a-ketosuccinamate, and a-ketoglutaramate. I t hydrolyzes D-asparagine at about 3 % of the rate observed for the L-isomer. Effect of pH. T h e optional p H range (in 0.1 M sodium borate buffer) is 7.5 to 8.5. TABLE II SUMMARY OF PURIFICATION PROCEDURE FOR ASPARAGINASE

Fraction

Specific Total volume, Total Nitrogen, activity, Recovery, ml. Units/ml. units mg./ml, units/mg. N %

Serum Step 1 Step 2

100 50 47

168

272 247

16,800 13,600 11,600

8.45 3.20 1.72

19.9 85.1 144

-81 69

Other Methods of Preparation 7 T h e preparation of yeast asparaginase has been described. 17

C. a-Keto Acid-~-Amidase from Rat Liver CONH2 COOH

] I

J I

+ H20-~

C--O

+ NH3

C:O

I

I

COOH

C00H n=l,

2

Assay Method

Principle. T h e reaction is followed b y determination of the ammonia or a-keto acid formed. Reagent Sodium a - k e t o g l u t a r a m a t e or a-ketosuccinamate (0.03 M), freshly prepared in 0.1 M sodium borate buffer, p H 9.0.

Procedure. E n z y m e solution (1 ml.) and substrate (1 ml.) are incubated for 15 to 60 minutes at 37 ° . Ammonia is determined as described above. * Definition of Unit. One unit is defined as the q u a n t i t y of enzyme which catalyzes the formation of 1 uM. of ammonia per hour under the assay conditions. 17 W. G r a s s m a n n a n d O. Mayr, Z. physiol. Chem. 214, 185 (1933).

[52]

385

OMEGA-AMIDASES

Purification P r o c e d u r e 14,1s

Step 1. Forty-five grams of minced fresh rat liver is blended in a Waring blendor with 90 ml. of ice-cold water for 2 minutes. After centrifugation at 600 X g at 5 ° for 2 hours, the supernatant solution is dialyzed against running water at 10 ° for 18 hours and centrifuged again. Step 2. Differential Heat Denaturation. The solution (100 ml.) obtained above is heated at 60 ° 19 in a water bath for 40 minutes. T h e solution is cooled in ice, and the coagulated protein is removed by centrifugation. Step 3. Adsorption on and Elution from Calcium Phosphate Gel. The solution obtained in step 2 is mixed with 67 ml. of calcium phosphate gel (15 mg. dry weight per milliliter), and the mixture is shaken for 30 minutes at 24 to 28 °. After centrifugation, the gel is drained free of the supern a t a n t solution and is washed twice b y centrifugation with 20 ml. of water. The gel is then shaken successively with two 20-ml. portions of 0.2 M potassium phosphate buffer (pH 5.5). The gel is treated in the same manner successively with buffer of p H 6.0 and p H 6.5. T h e most active fraction is obtained b y elution at p H 6.5. This solution is adiusted to p H 7.5 with 0.5 N N a 0 H , and stored at 0 ° or frozen. The preparation maintains its original activity for only one or two weeks. Lyophilization or dialysis results in inactivation. TABLE III SUMMARY OF PURIFICATION PROCEDURE FOR a-KETO ACID-co-AMIDASEa

Fraction Liver homogenate Step 1 Step 2 Step 3 (gel eluate, pH6.5)

Total Specific volume, Total Nitrogen, activity, Recovery, ml. Units/ml. units mg./ml, units/mg. N % 136 100 91

104 87.4 85.3

14,100 8,740 7,750

10.2 4.51 2.63

40

42.3

1,690

0.12

10.2 19.4 32.4 353

-62 55 12

° Substrate, a-ketoglutaramate. P r o p e r t i e s 14

Specificity. The enzyme does not deamidate the optical isomers of asparagine and glutamine, L-homoglutamine, or a-ketoadipamate. Effect of pH. T h e optional p H range (in 0.1 M sodium borate buffer) is 8.5 to 9.5. is A representative fraetionation is described in Table III. ~9A thermometer is placed in the enzyme solution and gentle stirring is carried out throughout the heating procedure.

386

ENZYMES OF PROTEIN METABOLISM

[53]

[53] Aspartase L-Aspartic Acid ~ Fumaric Acid -~ NH3

(1)

By ARTTURI I. VIRTANEN and NILS ELLFOLK Assay Method

Principle. The method is based on the measuring of the amount of ammonia liberated. Reagents Stock solution of L-aspartic acid (0.1 M), pH 7.2. Phosphate buffer (M/15), pH 7.2. Enzyme preparation with initial velocity of 2 to 5 % NH3-N per minute per 10 ml.

Procedure. Two milliliters of aspartic acid solution, 2 ml. of phosphate buffer, and 6 ml. of distilled (0,3 ml. of toluene as antiseptic) water are incubated at 37 ° . Two-milliliter samples are taken for ammonia determination in an apparatus of Pucher et al. The ammonia is trapped in the receiver by 0.01 N H2S04 and is titrated iodometrically. The solution to be analyzed is made alkaline in the distillation flask with sodium carbonate buffer (5 g. of Na2C03 + 5 g. of NaC1 in 100 ml. of distilled water). Initial Velocity. Initial velocity is graphically obtained from the completed experimental curve and expressed in micrograms of NH3-N formed at 37 ° per minute at the pH and in the buffer designated. Purification Procedure 1

A. Propionic Acid Bacteria The nutrient solution for the propionic acid bacteria is prepared as follows. Fifty liters of skim milk is coagulated at 35 ° with rennet, heated to 96 ° to destroy the enzyme, filtered, and sterilized at 120 ° . After chilling to 45 ° the whey is inoculated with Lactobacillus helveticus. The lactic acid produced is neutralized with sterilized chalk. The whey culture is incubated at 42 ° until all the lactose is fermented to lactic acid, which is Checked with the Fehling test. The incubation has to be continued for 5 to 6 days. To 50 1. of fermented whey 1000 g. of pressed yeast and 100 g. of peptone are added. The mixture is heated to 120° for 30 minutes in an autoclave and centrifuged. The pH is adjusted to 6.8, and the solution is portioned out into 8-1. Erlenmeyer flasks and sterilized at 1 N. Ellfolk, Acta Chem. Scand. 7, 824 (1953).

[~3]

ASPARTASE

387

120 ° for 30 minutes. Before the inoculation of the propionic acid bacteria, the p H of the solution is controlled and adjusted to 6.8. The culture of the bacterial mass is performed in these Erlenmeyer flasks for the first 20 hours at 37 ° and then for 25 hours at 25 ° . The bacteria are separated from the solution with an air-driven Sharples supercentrifuge. The washed mass is dried on porous plates and ground to a dustlike powder. Preparation of Crude Extract. The dry bacteria are stirred thoroughly with n-butanol at a low temperature (0 to - 2 °) for 30 minutes and then separated by centrifuging at a low temperature ( - 2 °) and suspended in water at 0 °. The butanol is removed by dialyzing against tap water of low temperature ( + 2 ° ) . After 2~ hours of dialysis the bacteria are centrifuged off, the aspartase is obtained in a cell-free solution. Nucleic Acid Precipitation. By lowering the p H of the crude enzyme solution with 5 % acetic acid to 4.5-4.3, a strong opalescence was formed. After centrifugation the precipitate was easily dissolved when neutralized with solid CaCO3 (pH 6.5). Excess carbonate was removed by centrifugation. A quantitative recovery of aspartase was obtained in a colorless solution which has a high concentration of nucleic acids. 1~

B. Pscudomonas In culturing Ps. fluorescens a nutrient solution of the following composition is employed, at pH 7:100 1. of tap water, 800 g. of meat extractpeptone powder (Baeto Nutrient Broth Dehydrated, Difco Laboratories), 300 g. of K2HPOt, 100 g. of MgS04-7 H20. The culture of the bacterial mass is performed in large vats or in Roux flasks at 25 °. After 48 hours the bacteria are harvested b y means of a milk separator and washed with tap water. The bacterial mass is dried on porous plates, and the dry mass is ground to a dustlike powder. Preparation of Crude Extract. The extract is prepared with n-butanol in exactly the same way as described above. Aspartase is precipitated from the extract b y lowering the p H to 4.5 with 0.1 M acetic acid at 0 °. The precipitate should immediately be dissolved in neutral buffer (m/15 phosphate, pH 7.2).

Properties Specificity. The enzyme is strictly specific to L-aspartie acid at~d fumarie acid, having no action on D-aspartic acid, 2 L-cysteic acid, 3 a,$-diaminosuccinic acid, 3 or other amino acids tested (glycine, 4 alanine, ~ leux, N. Ellfolk, Acta Chem. Scand. 9, in press (1955). 2 A. I. V i r t a n e n a n d T. Laine, Suomen Kemistilehti B9, 28 (1936). 3 N. Ellfolk, Acta Chem. Scand. 8, 151 (1954). 4 j . H. Quastel a n d B. Woolf, Biochem. J. 20, 545 (1926). 5 R. P. Cook a n d B. Woolf, Biochem. J. 22, 474 (1926).

388

ENZYMES OF PROTEIN METABOLISM

[53]

cine, ~ phenylalanine, 7 tyrosine, 7 dioxyphenylalanine, 7 histidine, 7 glutamic acid4). Ammonia was not added to maleic, 4 glutaconic, 4 crotonic, 8 mesaconic, ° aconitic, ~ sorbic acid, ~ or the diamide 6 and mono -9 and diethyP ester of fumaric acid. Inhibitors. 1° T h e enzyme of propionic acid bacteria is inhibited by citrate, oxalate, and ethylenediaminetetraacetic acid and pyrophosphate. A weak inhibition is observed with cyanide, azide, and acetonitrile. Practically no inhibition is produced b y fluoride and sodium sulfide. A metal seems to be responsible for the activity of this enzyme. As to the thiol group reagents, strong inhibition power is observed with p-chloromercuribenzoate, whereas weaker effects are observed with oxidizing agents (ferricyanide, o-iodosobenzoate, iodine) and different arsenicals (phenylarsine oxide and 3-amino-4-hydroxydichloroarsine hydrochloride). Iodoacetamide produced a rather weak inhibition. BAL (2,3-dimercaptopropanol) has an antidotal effect on the action of p-cloromercuribenzoate. Certain h e a v y metal ions inhibit the enzyme. Strong inhibitory effect is observed with Ag, Hg, Zn, Cd, and Co. A weaker inhibition is observed with Pb and Ni. These observations point to the existence of an essential thiol group in the enzyme. Effect of pH. The enzyme exhibits a sharp optimum for activity at p H 7.5 in phosphate buffer. 8

Distribution Aspartase is a typical bacterial enzyme, being present, e.g., in Escherichia coli,4 Pseudomonas fluorescens,5 Ps. pyocyaneus,5 Ps. aeruginosa,1 Proteus vulgaris, 5 Serratia marcescens, 5 Propionibacterium sp., 6 Lactobacillus helveticus, ~ Diplococcus pneumoniae, ix Micrococcus aureus, ll Sarcina spp., 11 Salmonella enteriditis. ~1 B o t t o m yeast, 12 brewer's yeast, 1~ and molds (PeniciUium notatum) ~ also have aspartase activity. In higher plants 8 and their seedlings 1~ the activities are weak or cannot be demonstrated. No definitive evidence of the presence of this enzyme in animal tissues has been reported. 5 s A. I. V i r t a n e n a n d J. T a r n a n e n , Bioehem. Z. 250, 193 (1932). K. P. Jacobsohn a n d M. Soares, Compt. rend. soc. biol. 125, 554 (1937). s T. Ichihara, Hukuoka Acta Med. 24, 1231 (1931) [Chem. Abstr. 9.6, 3539 (1932)].

9 K. P. Jacobsohn and M. Soares, Enzymologia 1, 183 (1936). 10N. Ellfolk, Acta Chem. Scand. 7, 1155 (1953). 11H. Saito, J. Biochem. (Japan) 34, 49, 103 (1941) [Chem. Abstr. 4[i, 1199 (1951)]. II H. Haehn and H. Leopold, Biochem. Z. 292, 380 (1937). is y. Sumiki, Bull. Japan. Soc. Ferment. 23, 33 (1928) [Chem. Abstr. 23, 2531 (1929)]. 14N. Tsuda, Japan. J. Nutrition 8, 108 (1950) [Chem. Abstr. 45, 8591a (1951)]. 15 M. Damodaran and S. S. Subramanian, Proc. Indian Acad. Sci. 27B, 47 (1948).

[53]

ASPARTASE

389

Quantitative Determination of L-Aspartic Acid Aspartase can be used for the quantitative determination of L-aspartic acid, although the enzyme preparations contain fumarase and the ammonia formation depends not only on reaction 1 b u t also on reaction 2 Fumaric acid W H20 ~ Malic acid

(2)

Three parallel experiments are needed: (1) the actual experiment; (2) one to which has been added approximately the same a m o u n t of aspartic acid as is found in the solution to be investigated; and (3) an experiment with enzyme preparation alone. The following example illustrates the method (Virtanen and LouhivuorP6). Take six test tubes, containing 5 ml. of 0.067 M phosphate buffer (pH 7.0) and 2 ml. of toluene per tube. Suspend 500 rag. of dried powder of Ps. fluorescens carefully in each solution. Add to three of the tubes 10 ml. of solution with 20.0 mg. of neutralized L-aspartic acid, and to the other three 10 ml. of distilled water. I n c u b a t e at 37 ° with occasional shaking. After equilibrium is reached, place the tubes in ice water for interruption of the reaction.

Aspartic Expt. acid, mg. I II III

--

IV V VI

20 20 20

Aspartic acid-N, mg. ----

Average 2.10 2.10 2.10 Average

0.01 N NHs-N H2SO4 formed, ml. mg.

NH3-N NH3-N formed liberated Found from from aspartic aspartic aspartie acid-N, % acid, mg. acid-N, % of added ~

17.20 17.46 17.38

2.408 2.444 2.433

----

----

----

26.53 26.96 26.79

2.428 3.714 3.774 3.751

1.286 1.346 1.323

62.19 64.09 63.00

98.6 101.6 99.9

3.746

63.09

Calculated from the mean value of ammonia formed from aspartic acid. The accuracy of the ammonia determination decides how small an a m o u n t of L-aspartic acid can be determined b y the method. B y using dry preparations or strong enzyme solutions (amorphous powder after lyophilization is quite stable) the enzyme concentration can be raised so high t h a t the equilibrium is reached in some hours. 16A. I. Virtanen and A. Louhivuori, Acta Chem. Scand. 1, 799 (1947).

390

ENZYMES OF PROTEIN METABOLISM

[54]

Aspartase can well be applied to determination of L-aspartic acid in protein hydrolyzates. When determination is performed in solutions containing dicarboxylic acids (oxalic acid, citric acid, fumaric and malonic acids), ~° however, these acids must be removed from the solution before the determination, since, being inhibitors to aspartase, an equilibrium is established on the basis of which no calculations on the content of aspartic acid can be made.

[E4] A m i n e

Oxidases

A. Amine Oxidase from Steer Plasma ~ RCH2NH2 + 05--~ RCHO + H202 + NH3 B y CELIA WHITE TABOR, HERBERT TABOR, and

SANFORD M. ROSENTHAL Assay M e t h o d The oxidative deamination of amines is usually followed by the measurement of oxygen consumption or ammonia production. 2 The spectrophotometric assay used here involves the measurement of the benzaldehyde produced during the oxidative deamination of benzylamine and depends on the difference in the absorption spectrum of benzaldehyde and benzylamine at 250 m~. The molar extinction coefficient of benzaldehyde is 13,000, whereas that of benzylamine is <200. Reagents

0.2 M potassium phosphate buffer, pH 7.2. 0.1 M benzylamine sulfate. 1.07 g. of redistilled benzylamine and 5 ml. of 2 N I-I2S04 are made up to 100 ml. with distilled water. Enzyme. About 10 to 50 spectrophotometric units (see below) are used for each determination. Procedure. One milliliter of 0.2 M phosphate buffer, 0.1 ml. of 0.1 M benzylamine sulfate, enzyme, and water (final volume of 3.0 ml.) are placed in a silica cell having a 1-cm. light path. A blank cell is made up

1The method reported here has been described in J. Biol. Chem. 208, 645 (1954). 2Literature has been reviewed by E. A. Zeller in "The Enzymes" (J. B. Sumner and K. Myrb~ck, eds.), Vol. II, p. 536, Academic Press, New York; and by H. Blaschko, Pharmacol. Revs. 4, 415 (1952).

[54]

AMINE OXIDASES

391

similarly except for the omission of benzylamine. Readings at 250 m~ are taken initially and every minute for 5 minutes. Definition of the Spectrophotometric Unit and Specific Activity. One spectrophotometric unit of enzyme is defined as the a m o u n t of enzyme which produces an initial rate of change in optical density at 250 m~ of 0.001 per minute at 30 °. Protein is measured by the absorption at 280 m~ (see Vol. I I I [73]). Specific activity is expressed as spectrophotometric units per milligram of protein.

Purification Procedure

Step 1. Steer blood, obtained at the slaughterhouse, is treated immediately with 1/6 vol. of citrate solution (containing 8 g. of citric acid and 26.7 g. of Na citrate.5.5 H20, per liter of solution). The plasma is separated by centrifugation in an International Centrifuge2 Step 2. Fractionation with Saturated Ammonium Sulfate. 4 To 3930 ml. of plasma, 2118 ml. of saturated ammonium sulfate solution is added with stirring (final concentration 1.4 M). After cooling to 0 to 3 ° the precipitate is removed by filtration through a fluted filter paper. To the filtrate 3760 ml. of saturated ammonium sulfate is added (final concentration approximately 2.4 M). The precipitate is collected by filtration and dissolved in water (final volume 800 ml.). This fraction contains most of the activity; as determined by conductivity measurements the concentration of ammonium sulfate in this solution is approximately 1 M. A second ammonium sulfate fractionation is carried out on this material, with the addition of the following volumes of saturated ammonium sulfate, and collection of each precipitate by filtration: (I) 185 ml. (final concentration approximately 1.66 M ammonium sulfate); (II) 85 ml. (final concentration 1.78 M); (III) 75 ml. (final concentration 2.04 M); (IV) 100 ml. (final concentration 2.2 M); and (V) 100 ml. (final concentration 2.36 M). Each precipitate is taken up in water and tested for specific activity. Precipitates I I I and IV (from 1.78 to 2.2 M ammonium sulfate concentrations) are found to have the highest specific activities. These are pooled (total volume 214 ml.) and dialyzed for 48 hours against For large volumes, we have found that separation in the Sharples centrifuge (cream separator attachment) is satisfactory. 4 The ammonium sulfate solution used is saturated with ammonium sulfate at room temperature and is approximately 4 M (NH4)~SO4.The ammonium sulfate concentrations of the various fractions are checked by conductivity measurements in a Barnstead purity meter (PM-2). The solutions are diluted 1:50,000, and the conductivity readings compared with suitable standards.

392

ENZYMES OF P R O T E I N METABOLISM

[54]

three changes of 0.01 M sodium acetate (2 1. each). The final volume of the dialyzed solution is 285 ml. 5 Step 3. Alcohol Precipitation. Portions (50 ml.) of this dialyzed material are heated rapidly in a water b a t h to 65 ° with stirring and maintained at this t e m p e r a t u r e for 10 minutes. T h e y are rapidly cooled to 0 ° and combined. All alcohol precipitations are carried out at 0 ° with alcohol cooled to - 1 0 ° or below. After addition of 28.3 ml. of 0.1 M MnCl~, 283 ml. is fractionated with ethanol. The following alcohol fractions are obtained b y slow addition of the stated volumes of alcohol, with mechanical stirring: (I) 142 ml. of 25% alcohol (10.2% alcohol); (II) 71 ml. of 25% alcohol (14.6% alcohol); (III) 71 ml. of 25% alcohol and 14.2 ml. of absolute alcohol (21.5% alcohol); (IV) 71 ml. of absolute alcohol (33.4% alcohol); and (V) 57 ml. of absolute alcohol (40.7% alcohol). These precipitates are collected b y centrifugation in the refrigerated centrifuge, taken up in 30 to 40 ml. of cold water, and the specific activity of each determined. The fraction showing the highest specific activity (usually fraction IV) is then subiected to a second alcohol fractionation. T o this fraction (volume 37.5 ml.) is added 3.75 ml. of 0.1 M MnCl~. T h e following successive additions of cooled absolute alcohol are made: (I) 1.91 ml. (4.43% alcohol); (II) 1.3 ml. (7.2% alcohol); (III) 1.13 ml. (9.5%); (IV) 1.63 ml. (12.7%); and (V) 1.63 ml. (15.6%). T h e precipitates are collected b y centrifugation in the refrigerated centrifuge and dissolved in a b o u t 10 ml. of cold water. The highest specific activities are found in fractions III, IV, and V. These are combined for the next step (final volume 30.3 ml.). TABLE I SUMMARY OF PURIFICATION PROCEDURE OF AMINE OXIDASE

Step Citrated plasma Ammonium sulfate Alcohol~ Calcium phosphate b

Total volume, ml. Units/ml. 3930 214 30.3 212

103 440 1873 189

Specific Total Protein, activity, units mg./ml, units/rag. 405,000 69.2 94,100 24.8 56,800 16.0 40,200 0.86

1.49 17.8 117 219

Recovery, % 23.3 14.1 9.9

a This step follows dialysis and heating. b As indicated in the text, this step was carried out on aliquots; the data given here represent the total units obtained for all the aliquots.

Step 4. Calcium Phosphate Absorption and Elution. Six-milliliter portions of the active alcohol fractions (containing approximately 90 rag. of 5 The adequacy of the dialyses is checked by conductivity measurements. Inadequate dialysis interfered with the alcohol fractionation.

[54]

AMINE OXIDASES

393

protein) are mixed with 21 ml. of a calcium phosphate gel (over 4 months old, and containing 14 mg. of solid per milliliter). (See Vol. I [11] for preparation of this gel.) After high-speed centrifugation, the enzyme is eluted with 42 ml. of cold 0.01 M K2HP04.

Properties The enzyme is stable (for at least four months) in the frozen state at - 2 0 °. It is also conveniently stored as a lyophilized powder after the alcohol fractionation step. The enzyme (particularly the crude plasma and ammonium sulfate fractions) has been stored at 0 to 3 ° for two to three weeks without any loss in activity. Substrate Activity. The enzyme oxidatively deaminates the following amines in order of decreasing activity: 8 spermine, spermidine, heptylamine, hexylamine, homosulfanilamide, butylamine, benzylamine, dodecylamine, decamethylenediamine. Others such as tyramine, mescaline, amylamine, furfurylamine, and propylamine are attacked to a lesser degree. No activity is observed with epinephrine, norepinephrine, histamine, and putrescine. Inhibitors. The enzyme is inhibited by cyanide, semicarbazide, and hydroxylamine at about 10-8 concentration. With lower concentrations of cyanide a latent period is observed before inhibition is complete. 1-Isonicotinylhydrazine (isoniazid) and 1-isonicotinyl-2-isopropylhydrazine (iproniazid) inhibit the enzyme completely at 10-4 and 10-3 M, respectively. Complete inhibition with these compounds requires a period of preincubation of the enzyme and inhibitor. Other inhibitors are pyridoxamine, quinacrine, dibenamine, pyribenzamine, and benadryl. Effect of pH. The pH optimum of the enzyme (measured at a substrate concentration of 2.4 micromoles per milliliter) varies with the substrate. The optimum for spermine is 6.2; for benzylamine, 7.5.

Other Preparations Amine oxidases have been described from various sources. The one most commonly used is the particulate monoamine oxidase from liver. The preparation usually consists of high-speed centrifugation of the active particles, which are then washed a number of times by centrifugation. Although this preparation resembles the amine oxidase described here, it exhibits considerable differences in inhibitor and substrate specificity. The literature on amine oxidase has recently been reviewed by Zeller 2 and Blaschko. 2 6 The oxidation of these substrates was followed in the Warburg apparatus at p H 7.2 and a substrate concentration of 2.4 micromoles per milliliter. The relative activities vary with the p H and with the substrate concentrations.

394

ENZYMES OF PROTEIN METABOLISM

[54]

B. Diamine Oxidase (Histaminase) 7 from Hog Kidney RCH2NH2 ~- H20 + O2--~ RCHO + H202 ~- NH3 By H E R B E R T TABOR Assay Method

Principle. Diamine oxidase 8 catalyzes the oxidation of histamine and various diamines with the consumption of 1 mole of oxygen and the production of 1 mole of aldehyde, I mole of NH3, and 1 mole of H202. In the presence of catalase the H20~ is converted to H20 and 0.5 mole of 02. Assay of diamine oxidase, therefore, has depended on disappearance of histamine, consumption of oxygen, production of ammonia, or the coupled oxidation of indigodisulfonate.9 The assay described below is based on the oxygen consumption obtained with histamine as the substrate in the presence of added catalase. Reagents Histamine dihydrochloride (0.02 M). Thirty-five milligrams of commercial histamine dihydrochloride is dissolved in water, neutralized with approximately 0.4 ml. of 1 N NaOH, and made up to 10 ml. in volume. 0.2 M potassium phosphate buffer, pH 7.2. Catalase. Dilute crystalline catalase (see Vol. II [137]) to contain 1 unit/ml.

Procedure. 1.5 ml. of phosphate buffer, 0.25 ml. of catalase, and aliamine oxidase solution are placed in a Warburg vessel; 0.2 m]. of the histamine solution is placed in the side arm. After temperature equilibration, the histamine is added, and the initial oxygen consumption is measured. A blank flask is also run without any substrate. The measurements are carried out at 37.5 ° with air as the gas phase. Definition of Unit and Specific Activity. One unit of activity is defined as the amount of enzyme giving an oxygen consumption of 1 cmm. per hour. Specific activity represents units per milligram of protein. Protein concentration is determined by the method of Lowry et al. (see Vol. III [73]). Application of Assay Method to Crude Tissue Preparations. The assay method is applicable to crude tissue preparations, although the rate of oxygen consumption is relatively low. 7 This presentation is based on material published in J. Biol. Chem. 188, 125 (1951). s I n this presentation diamine oxidase a n d histaminase will be considered as one enzyme, 7,9 even t h o u g h there is some evidence t h a t two enzymes m a y be involved; R. Kapeller-Adler, Arch. exptl. Pathol. Pharmakol. 219, 419 (1953). 0 E. A. Zeller, in " T h e E n z y m e s " (J. B. S u m n e r a n d K. M y r b a c k , eds.), Vol. II, p. 544, Academic Press, New York.

[54]

AMINE OXIDASES

395

Purification Procedure Hog kidney acetone powder is a convenient source of diamine oxidase 9 and is used as the starting material in the purification method described here. Step 1. Acetone Powder. Fresh hog kidneys are packed in ice at the slaughterhouse. Two to four hours later, 100-g. portions are homogenized with 500 ml. of acetone (previously chilled to - 10°) in a Waring blendor for 1 minute, and quickly filtered with suction. The filter cake is homogenized in another 500 ml. of cold acetone, filtered, quickly air-dried at room temperature, and sifted through wire mesh. This powder is stored at 2 °. Under these conditions it is stable for 6 months but gradually loses activity thereafter. Steps 2 to 5 are carried out at room temperature, the remainder at 2°. Step 2. Acetone Powder Extract. 150 g. of this powder is extracted with 3000 ml. of 0.2 M potassium phosphate buffer (pH 7.2) for 20 minutes at room temperature with occasional stirring. After centrifugation the residue is discarded. Step 3. To this extract 14.5 g. of anhydrous sodium sulfate is added for each 100 ml. of the above extract. After all the salt has dissolved, the solution is filtered through a fluted paper. To the filtrate additional sodium sulfate (7 g. per each original 100 ml.) is added. The precipitate is collected by filtration, dissolved in 275 ml. of 0.1 M phosphate buffer (pH 6.8), and dialyzed for 1 hour against running tap water. Step 4. The dialyzed solution (in 100-ml. test tubes) is heated in a 60 ° water bath (mechanically stirred) for 20 minutes. The precipitate is centrifuged and washed with 100 ml. of water. Step 5. C~ Adsorption and Elution. 109 ml. of aluminum hydroxide gel C~ (11.1 mg./ml.) is added to the combined supernatants. After 10 minutes the mixture is centrifuged and the supernatant discarded. The gel is washed with 60- and 30-ml. portions of 0.2 M phosphate buffer (pH 7.2). The enzyme is eluted with 1 M K2HP04 (portions of 200, 100, 100 ml.). Forty milliliters of 0.5 M KH2PO4 is added to the combined eluate, and the solution is dialyzed overnight against 4 1. of cold distilled water. Step 6. The enzyme is precipitated by the addition of 40 g. of ammonium sulfate per 100 ml. of the dialyzed solution. The precipitate is collected by centrifugation in an angle centrifuge at 12,000 r.p.m, for 10 minutes and extracted with 20 ml. of 0.2 M potassium phosphate buffer (pH 7.2). Step 7. First pH Precipitation. After dialysis against cold, running 0.04 M sodium acetate for 4 hours, the solution is mixed with 6 ml. of

396

ENZYMES OF PROTEIN METABOLISM

[54]

0.2 M sodium acetate buffer (pH 5.0). After 30 minutes the precipitate is collected by centrifugation and dissolved in 6.5 ml. of 0.1 M phosphate buffer (pH 7.2). Step 8. Second pH Precipitation. Three milliliters of 0.2 M sodium acetate buffer (pH 5.0) is added to the above solution; after 40 minutes the precipitate is collected by centrifugation and dissolved in 6.5 ml. of 0.1 M phosphate buffer (pH 7.2). TABLE II SUMMARY OF PURIFICATION PROCEDURE OF DIAMINE OXIDASE

Step Acetone powder extract Na2SO~ fractionation Heating Gel adsorption and elution First pH ppt. Second pH ppt.

Specific Total protein, activity, Recovery, Total units mg. units/mg. % 21,300 17,800 14,300 6,160 3,360 1,230

27,900 5,050 2,430 76 25 6.8

0.76 3.5 5.9 81 134 183

84 67 29 16 6

Properties The purified preparations can be Stored at - 1 0 °. Under these conditions there is no loss in enzyme activity over at least several weeks. The purified preparation contains no catalase activity. The purified preparation has been tested with histamine, putrescine (1,4-diaminobutane), and cadaverine (1,5-diaminopentane) ; all three substrates are actively degraded. Other substrates, including ethylenediamine, hexamethylenediamine, propylenediamine, and agmatine, have been tested with other preparations and found to be degraded2 Oxidation is prevented by substitutions in the a-position, i.e., the carbon adiacent to the amino group, as in a-methylhistamine (imidazoleisopropylamine), Diamine oxidase is inhibited by cyanide and various carbonyl reagents, by various amines and guanidines, and by 1-isonicotinylhydrazinc. 1° Reference to these and other studies on substrate and inhibitor specificities are found in the review by Zeller2 ~0E. A. Zeller, J. Barsky, J. R. Fours, W. F. Kirchheimer, and L. S. van Orden, Experientia 8, 349 (1952).

[55]

AMINO ACID AMIDASE FROM HOG KIDNEY

397

[55] Amino Acid Amidase from Hog Kidney NH~

NH2

I

!

R - - C H - - C O N H 2 --* R - - C H - - C O O H ÷ N H .

By

SANFORD M . BIRNBAUM

Assay Method Either the amino acid or the ammonia liberated can be measured as an assay of enzymic activity. The latter has proved to be more convenient, particularly for a large series of determinations.

Reagents 0.2 M tris(hydroxymethyl)aminomethane--HC1 buffer, pH 8. 0.1 M MnC12. 0.05 M L-amino acid amide solution, pH 8. 1.0 M acetic acid. Saturated K2C03. 2 % boric acid solution with indicator to color composed of 10 parts of 0.1% bromocresol green in alcohol and 2 parts of 0.1% methyl red in alcohol. Standard HC1, usually 0.05 N.

Procedure. For each assay incubate two test tubes containing 0.7 mh of buffer, 0.3 mh of MnC12, and 1.0 mh of properly diluted enzyme at 37 ° for 5 to 10 minutes to bring to temperature and allow any protein metal interaction to proceed. At zero time add 1.0 mh of substrate to one of the tubes and continue the incubation for a definite period (t), usually 1 hour. At t add 1 mh of substrate to the blanks and as rapidly as possible 0.4 mh of N acetic acid to both. Heat tubes in boiling water bath for 10 minutes, and set aside for the determination of enzymatically formed ammonia at the operator's convenience. Measurement of Ammonia. The digest tubes and the blanks are set up in a train with alternating receivers composed of 20 ) 150-mm. test tubes containing 2 mh of the boric acid-indicator solution. 1 Each tube is fitted with an aeration device consisting of a No. 2 two-hole rubber stopper with a 5-mm. glass tubing inlet reaching to the bottom of the tube and an outlet protruding 4 to 5 ram. below the stopper. The tubes are connected in series by short lengths of rubber tubing. 1 As an alternative procedure the ammonia can be t r a p p e d in 5 ml. of 2 N H2SO4 diluted to 20 mh and nesslerized. The caprylic alcohol t h a t distills over m u s t first be removed by boiling.

398

ENZYMES OF PROTEIN METABOLISM

[55]

In the presence of manganese ions in strongly alkaline solution and air: the amino acid amides are unstable and ammonia is liberated, resulting in high, variable blank values. Consequently the ammonia distillation is accomplished by passing nitrogen rather than air through the train. Starting with the tube adjacent to the nitrogen source, 1 or 2 drops of caprylic alcohol and 2 ml. of saturated potassium carbonate are added to each digest and blank, and the stopper is inserted immediately. The distillation is continued for 60 minutes after which the tubes are disconnected starting with the tube furthest from the nitrogen source. The inlet tubes of the traps are rinsed down with distilled water, and all are diluted to a roughly similar volume, 8 to 10 ml. The titration is best accomplished with a Gilmont ultramicroburet, the solution being mixed by a stream of clean air. The end point is compared with 2 ml. of boric acid-indicator mixture diluted to 8 to 10 ml. Activity Unit. The hydrolysis is directly proportional to time throughout most of the course of the reaction; consequently the activity can be quoted directly in micromoles hydrolyzed per milligram of protein nitrogen per hour. More precise zero-order kinetics are obtained if the hydrolysis is not allowed to exceed 40 % of the available substrate. Concentration Fresh frozen hog kidney is thawed and defatted, and 1000 g. of the diced material is homogenized in the Waring blendor with 2 1. of ice water. The homogenate is strained through cheesecloth and centrifuged at 2500 r.p.m, for 20 minutes to remove cellular debris. The specific activity against L-leucinamide at this stage is 200 micromoles per milligram of nitrogen per hour, and the total activity is 3.1 moles per hour. For the next step, a portion of the supernatant that can be handled in one centrifuging is measured out. This amount is chilled at 0 °, and the pH is dropped to 5.0 by the careful addition of 2 N tiC1. The thick suspension is immediately centrifuged at 0 ° at 4400 r.p.m, for 30 minutes, and the clear, red supernatant is quickly decanted and brought to pH 6.5 by the addition of 2N NaOH. When the entire homogenate has been taken through this step, solid ammonium sulfate is added to the combined supernatant in the amount of 350 g./1. After about 30 to 60 minutes of standing at 5 ° the precipitate is spun off3 and discarded. An additional 220 g. of solid ammonium sulThis is probably an oxidation of the a-amino group b y the oxides of manganese which are readily formed on bubbling air through alkaline solutions of M n ++ ions. 3 The total yield and specific activity are dependent to a degree on the thoroughness with which the a m m o n i u m sulfate fractions are separated from their supernatants. The figure quoted is obtained through the use of the Spinco b a t c h bowl at 20,000 r.p.m.

[55]

AMINO ACID AMIDASE FROM HOG KIDNEY

399

fate is added to each liter of supernatant, and the mixture is allowed to stand at 2 to 5 ° for at least 60 minutes. 4 The precipitate is centrifuged down, 3 taken up in a small a m o u n t of ice water, and the clear, cherryred solution is dialyzed free of ammonia and lyophylized. The specific activity against L-leucinamide at this stage is 3200 micromoles per hour per milligram of nitrogen and the total activity is 2.7 moles per hour. 5

Properties Specificity. This preparation has a wide range of activity against L-amino acid amides and dipeptide amides. The table is illustrative. The presence of a free amino group is essential, and separation of the amino group from the terminal amide, as in glycyl L-phenylalanine amide, is inhibitory. INITIAL HYDROLYTIC RATES WITH HOG KIDNEY AMIDASEa

Amide L-Leucinamide D-Leucinamide Acetyl L-leucinamide L-Alaninamide L-Phenylalaninamide Glycyl L-phenylalaninamide L-Prolinamide L-Tertiary leucinamide ~

Rate, t~M./hr./mg. N 3200 0 0 128 1950 493 7 12

In terms of micromoles of substrate hydrolyzed at 37° per hour per mi[ligram of protein N. N. Izumiya, S.-C. J. Fu, S. M. Birnbaum, and J. P. Greenstein, J. Biol. Chem. 205, 221 (1953). M e t a l Activation. Although the homogenate is only slightly activated b y M n ++ ions, the purified product is activated about 5-fold. Unlike the intestinal leucine amino peptidase reported b y Smith and Bergmann, 6 this effect is practically immediate. M g ++ ions are slightly less effective than Fin ++, giving a b o u t 4- to 4.5-fold activation. Co ++ and Ca ++ are without effect, and Zn ++ is inhibitory. All ions were added as the chlorides in 0.01 M final concentration.

4The enzyme is extremely stable at this stage and can be left overnight at 5°. 5 Further purification and considerable decolorization can be accomplished by adsorption at neutral pH values onto the experimentally determined amount of Caa(PO4)~ gel and elution with phosphate buffer, pH 7. The product is unstable, however, and cannot be lyophylized without extensive loss of activity. The ratio of the rates on the various amides listed in the table is not changed by this procedure. 6 E. L. Smith and M. Bergmann, J. Biol. Chem. 138, 789 (1941).

400

ENZYMES OF PROTEIN METABOLISM

[56]

Effect of pH. The pH optimum determined with a 60-minute incubation period is about 8. This optimum is not sharp and is probably related to enzyme stability, as much higher values are obtained with homogenares in which the enzyme is presumably more stable. 7 7 M. E. Mayer and A. E. Greco, J. Natl. Cancer Inst. 12, 37 (1951).

[56] N i t r o e t h a n e O x i d a s e

CH3CH2NO2

+ O~ + H20--~ CH3CHO

By

+ HNO2 + H202

H E N R Y N . LITTLE

Assay Method

Principle. Measurement of oxygen uptake, aldehyde liberation, and nitrite liberation has been used to determine enzymatic activity. I In the method described below, activity is estimated by determining the optical density of the color formed when the enzymatically liberated nitrite reacts with sulfanilic acid and a-naphthylamine. Reagents 0.5 M nitroethane. Dissolve 375 mg. of nitroethane in 0.2 M phosphate buffer, adjust the pH to 7.0, and dilute to 10 ml. Reagent should be prepared fresh daily. 0.2 M phosphoric acid-NaOH buffer, pH 7.0. 0.01 M phosphoric acid-NaOH buffer, pH 7.0. 5 X 10-3 M copper sulfate. Sulfanilic acid. Dissolve 3.3 g. of sulfanilic acid in 750 ml. of hos water, and add 250 ml. of glacial acetic acid. a-Naphthylamine. Dissolve 0.5 g. of recrystallized a-naphthylamine in 250 ml. of glacial acetic acid and dilute to 1 1. with water. Enzyme. Dilute the stock enzyme with 0.01 M phosphate buffer to obtain 24 to 48 units of enzyme per milliliter. (See definition below.)

Procedure. To 0.5 ml. of the properly diluted enzyme contained in an 18 X 150-mm. test tube add 0.4 ml. of 0.2 M phosphate buffer. Add 0.1 ml. of 0.5 M nitroethane, and agitate the tube for exactly 5 minutes in a mechanical shaker. Stop the enzymatic reaction by addition of 0.2 ml. of 5 × 10-~ M copper sulfate. Remove a 0.1-ml. aliquot of the reaction mixture, place it in 4.9 ml. of water, and add 0.5 ml. each of the sulfanilic acid and a-naphthylamine reagents. After 15 minutes deter1 H. N. Little, J. Biol. Chem. 193, 347 (1951).

[56]

NITROETHANE OXIDASE

401

mine the intensity of the color by means of a Klett-Summerson pho~oelectric colorimeter equipped with a 540-m~ filter. Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which will liberate enough nitrite to give a Klett reading of 100 when the assay is performed as described. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al. 2 Appraisal of Assay. This assay may be successfully applied to crude tissue preparations, provided that appropriate control tubes are included to correct for the turbidity of the enzyme. Since nitroethane interferes with the production of the red color in this assay the Klett readings may not be directly correlated with the quantitative amount of nitrite released. The density of the color developed, however, is found to be directly proportional to the amount of the enzyme present. Duplicate assays agree within + 5 %. Purification Procedure

Nitroethane oxidase has not been highly purified. The preliminary purification steps indicated below yielded consistent results in the author's laboratory. 1 The fractionation is carried out at 5°, although the enzyme is relatively stable at room temperature for short periods of time. Step 1. Preparation of Crude Extract. ~ a t e r i a l suitable for preparation of the enzyme is the mycelium of Neurospora crassa (5297a) which has been cultured for 4 to 6 days on a nitrate medium2 The mycelium is pressed dry, washed with water, and thoroughly macerated in five times its weight of 0.01 M phosphate buffer, pH 7, by means of a TenBroeck homogenizer. The insoluble debris is removed by centrifugation. Step 2. First Fractionation with Ammonium Sulfate. To the above supernatant solution, sufficient solid ammonium sulfate is added to raise the concentration to 40% saturation. The precipitate is removed by centrifugation, and additional ammonium sulfate is added until a concentration of 60% saturation is reached. The precipitate is recovered by centrifugation, resuspended in the minimum volume of 0.01 M phosphate buffer, pH 7.0, and dialyzed for 24 hours against the same buffer. Step 3. Second Fractionation with Ammonium Sulfate. The dialyzed sample is adjusted to pH 11.0 with sodium hydroxide and allowed to O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951); see Vol. I I I [73]. 3 Medium contains, in units per liter, NaNOs, 3 g.; Na tartrate, 5 g.; KH2PO4, 3 g.; MgSO4.TH20, 0.5 g. ; NaC1, 0.1 g. ; CaC12, 0.1 g. ; biotin, 5 v; sucrose, 20 g. ; Na tetraborate, 8.8 × 10 -5 g.; (NH4)sMo~024, 6.4 X 10 -8 g.; FeC13.6H20, 9.6 X 10 -4 g.; ZnSO4.TH20, 8.8 × 10 -8 g.; CuCl~, 2.7 × 10 -4 g.; MnC14.4H~O, 7.2 X 10 -s g.

402

ENZYMES OF P R O T E I N METABOLISM

[56]

stand for 1 hour. The ammonium sulfate concentration is then brought to 40% saturation with solid ammonium sulfate. The precipitate is removed by centrifugation, and, by the addition of more ammonium sulfate, the supernatant solution is adjusted to 60% saturation. The resultant precipitate is recovered by centrifugation, dissolved in the minimum amount of 0.01 M phosphate buffer, pH 7.0, and then dialyzed exhaustively against the same buffer. When stored at - 2 0 ° this preparation loses its activity only slowly over a period of weeks. SUMMARY OF PURIFICATION PROCEDURE a

Fraction

Total Specific volume, Total Protein, activity, Recovery, ml. Units/ml. units mg./ml, units/mg. %

1. Phosphate extract 140 2. First (NH4)~SO4 fraction 0.4-0.6 43 3. Second (NH4).~S04 fraction 0.4-0.6 9.2

94.8 "216 435

13,300 12

7.9

--

9,290 10

21.6

70

4,000

2.2

198

30

a H. N. Little, J. Biol. Chem. 193, 347 (1951).

Properties Substrate Concentration and Specificity. The optimum substrate concentration for nitroethanase is 0.05 M. Less than 10% of maximum activity is obtained at 0.005 M nitroethane. The enzyme is inactive toward cyclic nitro compounds and attacks only a limited number of aliphatic nitro compounds. The enzyme liberates nitrite at approximately the same rate from nitroethane, f~-nitropropionic acid, 1-nitropropane, and 2-nitropropane. Nitromethane, nitroacetic acid, and 1-chloro-l-nitropropane are only slightly attacked. Activators and Inhibitors. There are no known activators of this enzyme. Complete activity is maintained after exhaustive dialysis against phosphate buffer. At 0.01 M final concentration of inhibitor, the enzyme is less than 50% inhibited by sodium cyanide, sodium azide, sodium fluoride, and hydroxylamine. At 1 X 10-4 M, copper sulfate causes about 90% inhibition, and high concentrations (0.05 M) of sodium, potassium, and ammonium chloride and sodium and potassium nitrate cause 40 to 85 % inhibition. Effect of pH. The optimum pH for the enzyme is 7.0. Activity is rapidly and irreversibly lost by incubation at pH's below 5.5, whereas the enzyme is relatively stable under alkaline conditions.

[57]

ORGANIC NITRATE REDUCTASE

403

[57] Organic Nitrate Reductase By LEON A. HEPPEL Assay Method

Principle. Organic nitrate reductase catalyzes the reduction of various nitrate esters by glutathione. Inorganic nitrite is liberated and the assay procedure is based on its rate of formation. Reagents Nitroglycerine. An ethanolic solution is available from various pharmaceutical concerns. It can be stored at 3 ° for at least three months. 0.067 M KH2PO4-Na2HPO4 buffer, pH 7.4. GSH (0.2 M). Potassium cyanide (0.2 M). Sulfanilamide, 0.2 % in water. Keeps one month at 3 °. HC1, 1:1 dilution (volume) of concentrated solution. NaNO2, 0.1% solution, standardized with permanganate. Ammonium sulfamate, 0.5% in water. N-(l-Naphthyl)ethylenediamine dihydrochloride, 0.1% solution in water, kept in a dark bottle.

Procedure. An amount of ethanolic solution containing 1.6 rag. of nitroglycerine is evaporated in 30-ml. test tubes by means of a stream of air. Then the tubes are packed in ice, and the following additions made: 1.44 ml. of phosphate buffer, 0.08 ml. of GSH, 0.08 ml. of KCN, enzyme, and water to make 2 ml. The tubes are placed in a water bath at 37 °, and samples are removed for nitrite analysis at 5 and 10 minutes. Blank tubes are incubated without enzyme, and the rate of the spontaneous reaction is subtracted from the total rate. For nitrite analysis a mercuric chloride filtrate is prepared. 1 An aliquot of the filtrate containing up to 0.01 mg. of nitrite is treated with 0.2 ml. of 50% HC1 and 1 ml. of 0.2% sulfanilamide and then allowed to stand for 3 minutes. Next 0.2 ml. of ammonium sulfamate is added, followed in 2 minutes by 0.2 ml. of N-(1-naphthyl)ethylenediamine dihydrochloride. The volume is made up to 10 ml., and the density is measured at 500 mt~ in the Coleman spectrophotometer or similar instrument. A calibration curve is constructed with the standard nitrite solution. 2 1 E. W. Scott, J. Ind. Hyg. Toxicol. 25, 20 (1943). M. B. Shinn, Ind. Eng. Chem. Anal. Ed. 13, 33 (1941).

404

ENZYMES OF PROTEIN METABOLISM

[57]

Definition of Unit and Specific Activity. One unit of enzyme activity is defined as that amount which causes the formation of 1 micromole of inorganic nitrite per hour under the test conditions. Specific activity is expressed as units per milligram of protein. Protein is determined nephelometrically with the Beckman model DU spectrophotometer at 340 m~. 3 Application of Assay Method to Crude Tissue Preparations. The method is applicable to crude tissue preparations. Such preparations frequently form inorganic nitrite from nitroglycerine without addition of GSH, but after dialysis GSH becomes a necessary component of the assay system. Purification Procedure

This is taken from the work of Heppel and Hilmoe. 4

Step 1. Preparation of Crude Extract. Fresh hog liver, kept on ice and used within several hours, is homogenized in a Waring blendor with 5 vol. of cold (0 °) acetone and filtered on a suction funnel. This procedure is repeated on the filtered cake, after which the material is pressed through a wire screen and dried in a current of air. These operations are performed at room temperature. Subsequent manipulations are at 2 °, except as indicated. Fifty-six grams of acetone powder is suspended in 360 ml. of 0.067 M phosphate buffer, pH 7.4. After a brief interval the mixture is centrifuged for 7 minutes at 13,000 × g. Step 2. First Ammonium Sulfate Fractionation. The supernatant solution is adjusted to pH 5.1 with 1 N acetic acid, an inactive precipitate is removed by centrifuging, and solid ammonium sulfate is added to give 0.5 saturation. ~ This precipitate is rejected, and a second precipitate, obtained by increasing the ammonium sulfate concentration to 0.9 saturation, is collected, dissolved in 0.067 M phosphate buffer, pH 7.4, and dialyzed for 3 hours against running demineralized water. Step 3. Second Ammonium Sulfate Fractionation. The pH is again adjusted to 5.1, and the precipitate obtained between 0.6 and 0.8 saturation with ammonium sulfate is collected, dissolved, and dialyzed. Step 4. Ethanol Fractionation. The dialyzed ammonium sulfate fraction is diluted with distilled water until the protein concentration is 10 mg./ml. Then the pH is adiusted to 5, and fractions are collected between the following limits of ethanol concentration (v/v) : 0 to 0.39; 0.39 3 T. Bficher, Biochim. et Biophys. Acta 1, 292 (1947); see Vol. I I I [73]. 4 L. A. Heppel a n d R. J. Hflmoe, J. Biol. Chem. 183, 129 (1950). 6 To o b t a i n s a t u r a t i o n s of 0.5 a n d 0.6, a d d 29.1 a n d 36.1 g., respectively, of a m m o n i u m sulfate to 100 ml. of solution. To increase t h e s a t u r a t i o n of 100 ml. from 0.5 to 0.9, a d d 26.8 g.; to increase it from 0.6 to 0.8, a d d 12.9 g.

[57]

ORGANIC N I T R A T E REDUCTASE

405

to 0.54; 0.54 to 0.65. The solution is kept at - 8 ° as soon as danger of freezing is passed. The last precipitate contains the bulk of the enzyme activity, and it is dissolved in 0.067 M phosphate buffer, pH 7.4. Step 5. Third Ammonium Sulfate Fractionation. The solution in phosphate buffer is treated directly with solid ammonium sulfate to give 0.6 saturation. The precipitate is rejected, and the fraction obtained by increasing the concentration to 0.8 saturation is dissolved in phosphate buffer and dialyzed 4 hours against demineralized water. Step 6. Adsorption on Calcium Phosphate Gel. The dialyzed solution is adjusted to p H 6.3 with dilute acetic acid and then diluted to give a protein concentration of 0.5 mg./ml. T o every 10 ml. is added 5 ml. of aged calcium phosphate gel 6 (8.2 mg. dry weight per milliliter), after which the gel is eluted successively with 10-ml. portions of M/150, M/90, M/45, and M/15 phosphate buffer, pH 7.4. The last two eluates (III and IV, see below) show the best purification. SUMMARY OF PURIFICATION PROCEDURE ~

Total vohlme,

1. 2. 3. 4. 5. 6.

Specific activity, Recovery, Total units units/mg, protein %

Fraction

ml.

Crude extract First ammonium sulfate Second ammonium sulfate Ethanol Third ammonium sulfate Calcium phosphate gel Eluate III Eluate IV

315 133 85 40 35

13,010 12,900 9,350 4,$50 4,040

85 84

1,565 545

I. 1 4.3 9.4 37 55 80 98

100 99 72 37 31 12 4

L. A. tIeppel and R. J. Itilmoe, J. Biol. Chem. 183~ 129 (1950).

Properties Specificity. The purified enzyme has been found to be active with both nitroglycerin and erythritol tetranitrate. For the enzyme-catalyzed reaction GSH cannot be replaced b y cysteine. However, there is a slow spontaneous reaction between both GSH and cysteine, on the one hand, and the nitrate esters. This occurs more rapidly in alkali. Balance studies show t h a t 2 micromoles of GSH are oxidized for every micromole of nitrite formed. No physiological substrate has been found as yet. The purified enzyme has no glyoxalase activity, does not catalyze the reduction of cyto6 D. Keilin and E. F. Hartree, Proc. Roy. Soc. (London) B124, 397 (1938).

406

ENZYMES OF PROTEIN METABOLISM

[58]

chrome c by glutathione, and does not stimulate the oxidation of glutathione by air. Inhibitors. The enzyme is inhibited to the extent of 50% by 10 -4 M cupric sulfate. A concentration of 0.1 M sodium fluoride is without effect. Incubation for 5 minutes at 70 ° destroys activity completely. Effect of pH. The enzymatic reaction between nitroglycerin and GSH shows a pH optimum between pH 7 and pH 8. The nonenzymatic reaction between these compounds is slow at neutral pH, but its rate increases rapidly as the pH becomes more alkaline. Substrate Optima. Optimal substrate concentrations for the enzymecatalyzed reaction are 5 X 10-3 M GSH and 3 X 10-3 M nitroglycerin. With excess nitroglycerin the rate is proportional to the concentration of GSH between the limits of 2.5 X 10-4 M and 1.6 X 10-5 M.

[GS]

N i t r o a r y l R e d u c t a s e f r o m Neurospora crassa

DPNH NO2--CsH4--N02 -~ [NHOH--C6H4--N02] --* NH~--CeH4--N02

By MILTON ZUCKER and ALVIN NASON Assay M e t h o d

The reduction of trinitrotoluene to hydroxylaminodinitrotoluene and aminodinitrotoluene by xanthine oxidase and pig heart and liver extracts has been studied by Bueding and Jolliffe. 1 They employed an alcoholic potassium hydroxide-ether reagent to assay for trinitrotoluene and its monohydroxylamino derivative and used the standard diazo-coupling assay of Bratton and Marshall 2 for amine determination. With m-dinitrobenzene as substrate, a system in Neurospora very similar to t h a t found in pig heart and liver extracts has been characterized. Both nitrophenylhydroxylamine and nitroaniline have been identified as products of the in vivo reduction. Substances giving reactions similar to these compounds are also products of the in vitro reduction of dinitrobenzene. Therefore, m-nitrophenylhydroxylamine and m-nitroaniline have been used as standards for the assay of the in vitro system. The reagents of Bueding and Jolliffe cannot be used for determination of dinitrobenzene and its hydroxylamino derivative. The assay for m-nitrophenylhydroxylamine described below is based on its reaction with pentacyanoammine ferroate to give a purple color, a No suitable assay 1 E. Bueding and 1q. Jolliffe, J. Pharmacol. and Exptl. Therap. 88, 300 (1946). z A. C. Bratton and E. K. Marshall, Jr., J. Biol. Chem. 125, 537 (1939). a F. Feigl, " S p o t Tests," 1st ed., Nordman Co., New York, 1937.

[58]

NITROARYL REDUCTASE FROM NNUROSPORA CRASSA

407

for dinitrobenzene was found. Nitroaniline is assayed for by the diazocoupling method of Bratton and Marshall? Since reduced pyridine nucleotides serve as electron donors, the reaction can also be followed spectrophotometrically by measuring the disappearance of the 340-my absorption band of the reduced nucleotides upon their oxidation.

Reagents 0.1 M m-dinitrobenzene (DNB). Dissolve 168 mg. of the compound, recrystallized from EtOH, in 10 ml. of 95% EtOH. 3 X 10-3 M dinitrobenzene. Dissolve 50.4 mg. in 100 ml. of distilled H20 with heating. 0.1 M sodium pyrophosphate-HC1 buffer, pH 9.0. DPN, 4 mg./ml. (Sigma Chemical Company, 90% pure). Crystalline yeast alcohol dehydrogenase. 4 Dilute 1:40 with 0.1 M K2HP04. Boiled pig heart extract. Grind 10 g. of acetone-dried pig heart in 70 ml. of cold 0.1 M phosphate buffer, pH 7.5. Centrifuge, and boil the supernatant in a water bath for 5 minutes in the dark. Recentrifuge, and store the supernatant in the cold, protected from light. Not a reliable source of flavin after two to three weeks of storage. FAD, 2 × 10-5 M (Sigma Chemical Company, 40% pure). Dissolve enough FAD in distilled water to give an extinction of approximately 0.200 at 455 my. The extinction coefficient at this wavelength is 1.13 × 107 sq. cm/mole. 5 Store frozen and protected from light.

Colorimetric Assay 1% aqueous NaNO2 (prepared weekly). 1:4 dilution of concentrated HC1. 1% aqueous ammonium sulfamate (prepared weekly). 0.02 % aqueous a-naphthylethylenediamine dihydrochloride. 0.05% aqueous pentacyanoammine ferroate reagent (at leas~ 1 day old). The ferroate reagent is prepared according to Feigl. 8 Ten Na nitroferricyanide is dissolved in 30 ml. of concentrated (28%), loosely stoppered, and stored overnight at 4 °. EtOH until no more yellow precipitate is formed. The precipitate is 4 E. Racker, J. Biol. Chem. 184, 313 (1950). BO. W a r b u r g and W. Christian, Biochem. Z. 298~ 150 (1938).

grams of NH4OH is added collected

408

ENZYMES OF PROTEIN METABOLISM

[58]

by filtration, washed with EtOH, and air-dried. The resulting yellow ferroate compound is stable for months at room temperature.

Spectrophotomelric Assay 6 X 10-3 M D P N H . Prepared enzymatically by reduction of DPN. 6 2 X 10-3 M T P N H . Prepared enzymatically by reduction of T P N /

Procedure for the Colorimetric Assay. The reaction is started by addition of 0.05 ml. of 0.1 M D N B in 95% E t O H to 0.6 ml. of pyrophosphate buffer containing 0.05 ml. of DPN, 0.05 ml. of ADH, and 0.05 ml. of boiled pig heart or FAD and 0.2 ml. of enzyme. After 15 to 30 minutes of incubation at room temperature in a desiccator evacuated and flushed with N2, the 1-ml. reaction mixture is divided into a 0.65-ml. aliquot for the hydroxylamino assay and a 0.35-ml. aliquot for the amine assay. To the 0.65-ml. sample, 0.2 ml. of ferroate reagent is added, and the volume is brought to 2 ml. with distilled H~O. To the 0.35-ml. sample, 1 ml. of HC1 and 0.1 ml. of NaNOs are added, rapidly mixed, followed by addition of 0.5 ml. of ammonium sulfamate. The mixture is shaken vigorously for 30 to 60 seconds, and 0.5 ml. of naphthylethylenediamine dihydrochloride is added. The volume is brought to 3 ml. with distilled H20. After 30 to 60 minutes the intensities of both the purple ferroate and pink diazo-coupling colors are measured in a Klett-Summerson colorimeter with a No. 54 filter. In a test volume of 2 ml., 0.09 sM. of m-nitrophenylhydroxylamine gives a reading of 100 Klett units. A similar reading is given by 0.014 ~M. of m-nitroaniline in a 3-ml. volume. Turbidity corrections are made with a blank containing only enzyme and buffer in the above procedures. Nitrophenylhydroxylamine on diazotization gives a yellow color which slowly fades to pink. It is 2 % as intense as an equivalent amount of amine coloration, and suitable corrections are made when necessary. Procedure for the Spectrophotometric Assay. To a mixture containing 2.1 ml. of 0.1 M pyrophosphate buffer, 0.05 ml. of D P N H , and 0.05 ml. of boiled pig heart or FAD in a 3-ml. cuvette with a light path of 1 cm., 0.2 ml. of enzyme is added. Readings of the optical density at 340 mp are made at 30-second intervals over a period of 2 to 3 minutes to determine the endogenous rate of D P N H oxidation. The reaction is then started by the addition of 0.5 ml. of 3 X 10-3 M D N B in H20, and readings are made for 2 minutes at 30-second intervals beginning 45 seconds after addition of substrate. Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which results in the production of 0.1 uM. of NO~PhNHOH e See Vol. I I I [127] for enzymatic reduction of D P N . 7 A. Nason a n d H. J. Evans, J. Biol. Chem. 202, 655 (1953).

[58]

NITROARYL REDUCTASE FROM NEUROSPOR& CRASSA

409

or 0.1 ~M. of NO2PhNH2 per hour under the above conditions. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al. 8 Application of Assay Methods to Crude Tissue Preparations. Amine formation from dinitrobenzene is catalyzed by extracts from a number of plant species, microorganisms, and animal tissue extracts when fortified with D P N H and boiled pig heart. Care must be exercised in interpretation of the data, since a number of enzymes, particularly flavoproreins such as xanthine oxidase and aldehyde oxidase, can catalyze the reduction of dinitrobenzene. In plant extracts very little hydroxylamino compound is formed. Reduction of D N B in crude extracts of Neurospora can be demonstrated in all mycelia regardless of the nitrogen source used for growth. Purification Procedure

Step 1. Preparation of Crude Extract. Culture conditions and preparation of cell-free extracts of Neurospora crassa have been described2 All steps described below were carried out at 0 to 4 °, and precipitates were centrifuged at 3000 X g. Step 2. Fractionation with Ammoniacal Ammonium Sulfate. Sixtyseven milliliters of saturated ammonium sulfate adjusted to pH 8.5 with concentrated NH4OH is added to 100 ml. of crude extract, making the mixture 40 % saturated. The resultant precipitate is centrifuged off, and the supernatant is brought to 60 % saturation by addition of 83 ml. more of ammoniacal ammonium sulfate. After 10 minutes the precipitate is collected by centrifugation and dissolved in 40 ml. of cold distilled H~O (fraction 2). This fraction contains both the partially purified nitrophenyl-hydroxylamine- and nitroaniline-forming systems. Further fractionation, as outlined below, leads to an almost complete loss of the amine-forming system. A further purification of this system has not been attained. Step 3. Adsorption and Elution of the Enzyme from Calcium Phosphate Gel. Twenty milliliters of calcium phosphate gel ~° (20 mg./ml, dry weight) is added to fraction 2 and mixed for 15 minutes. The gel precipitate is collected by centrifugation and eluted for 15 minutes by suspending it in 20 ml. of cold 0.5 M K:HP04. The eluate (fraction 3) is obtained by centrifugation and represents a 10 to 15 fold purified enzyme. 8 0 . H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193,

265 (1951). 9 See Vol. I I [60] for culture conditions. i ° See Vol. I [11] for preparation of calcium phosphate gel.

410

[58]

ENZYMES OF PROTEIN METABOLISM SUMMARY OF PURIFICATION PROCEDURE

Units/ml.

Fraction 1. Crude extract 2. (NH4)2S04 fraction, 0.40-0.60 3. Eluate from calcium phosphate gel

Total volume, ml. A° 100

]9

Bb

Specific activity, units/mg.

Total units A

B

Protein, mg./ml.

A

B

Recovery, % A

B

7.0

1900 700

6.0

3.2

1.2

45

23 5.3

1030 240

2.0

11.5

2.7

54

34

20

34 3.0

.8

42.5

3.7

36

9

680

60

a A, units in terms of nitrophenylhydroxylamine formation. b B, u~its in terms of nitroaniline formation. Properties

Specificity. T h e partially purified enzymes f r o m step 2 or 3 will reduce other polynitroaryl c o m p o u n d s in addition to m-dinitrobenzene. These include p-dinitrobenzene, 3,5-dinitrobenzoic acid, 1,3,5-trinitrobenzene, and 2,4,6-trinitrotoluene. 2,4-Dinitrophenol and p-nitrobenzoic acid are reduced in vivo b u t not in vitro. N o other m o n o n i t r o a r y l compounds are reduced b y whole tissue or extracts. Nitroglycerin, ethylnitrate, aliphatic nitro compounds, and oximes do not stimulate D P N H oxidation. Aryl hydrazides and azo compounds are also inactive. I n o r ganic nitrogen compounds cannot serve as substrates for this system, although nitrite and hydroxylamine-reducing enzymes are present in the purified fractions (see below). Both D P N H and T P N H serve as electron donors. T h e Km's are 3.3 X 10 -4 M and 4.1 X 10 -4 M, respectively, as determined spectrophotometrically. Similar results are obtained when nitroaniline f o r m a tion is assayed. Activators and Inhibitors. If 0.5 M p h o s p h a t e buffer at p H 7.0 is used to elute the e n z y m e in step 3, boiled pig heart is required for b o t h D P N H oxidation and f o r m a t i o n of n i t r o p h e n y l h y d r o x y l a m i n e . F A D (5 × 10 -7 M final concentration) completely replaces the boiled pig h e a r t requirement. F M N will not substitute for F A D . B o t h cysteine and K C N (5 X 10 -~ M final concentration) stimulate the f o r m a t i o n of n i t r o p h e n y l h y d r o x y l a m i n e two- to fourfold. Cysteine has no effect on nitroaniline formation. H o w ever, K C N completely inhibits the f o r m a t i o n of amine at this concentration. NaN3 is also inhibitory to the amine-forming system. These results suggest a m e t a l requirement for this system, although other chelating agents h a v e little effect.

[59]

NITRATE REDUCTASE FROM NEUROSPORA

411

Stability. The enzyme system from fraction 2 loses 75 % of its activity over a period of a week even at - 2 0 °. Fraction 3 is somewhat more stable and shows measurable activity after several weeks at - 2 0 °. Both fractions are stored best between p H 7.5 and 9.0. Neither cysteine nor glutathione stabilizes the enzymes. Both fractions lose 90% of their activity when stored at 0 to 4 ° overnight. Thus dialysis over periods longer than several hours is impractical owing to the lability of the enzymes. Over 70 % of the activity of both fractions 2 and 3 is destroyed at 60 ° for 5 minutes. Effect of pH. The optimum p H for both nitrophenylhydroxylamine and nitroaniline formation lies between 8.0 to 9.0 with pyrophosphate, Tris, or phosphate buffers. At p H ' s lower than 8.0, D P N H itself rather than the yeast alcohol dehydrogenase and D P N must be used. Contamination with Other Enzymes. Fraction 3 also contains a nitrite and a hydroxylamine reductase. Since both enzymes are adaptive and are not present in extracts of mycelia grown on NH4C1 as a sole source of nitrogen, the dinitrobenzene system can be prepared free of these contaminants from such extracts. Neither xanthine nor succinate serves as electron donor for the dinitrobenzene system.

[59] N i t r a t e R e d u c t a s e

f r o m Neurospora

T P N H q- H + q- NO3---+ T P N + q- NO~- q- H~0

By ALVIN NASON and HAROLD J. EVANS Assay Method

Principle. E n z y m a t i c activity is best determined colorimetrically by testing for nitrite.' The measurement for nitrite is based on the formation of a red-colored azo compound; this involves first the reaction of sulfanilamide and nitrous acid to form a diazonium salt, followed b y the coupling of the salt to an aromatic amine to yield the red azo dye. The activity of purified enzyme preparations m a y also be followed b y measuring the rate of T P N H oxidation spectrophotometrically as observed b y the decrease in optical density at 340 m~. Reagents T P N H solution (2 micromoles per milliliter), reduced enzymatically 2 with pig heart isocitric dehydrogenase, or chemically with hydrosulfite2 1F. D. Snell and C. Snell, "Colorimetric Methods of Analysis," 3rd ed., Vol. 2, D. Van Nostrand Co., New York, 1949. A. Nason and H. J. Evans, J. Biol. Chem. 202, 655 (1953). 3 N. O. Kaplan, S. P. Colowiek, and E. F. Neufeld, J. Biol. Chem. 199, 107 (1952).

412

ENZYMES OF PROTEIN METABOLISM

[59]

0.1 M KNO3. Dissolve 1.01 g. of the salt in 100 ml. of distilled H20. F M N (500 ~//ml.). Dissolve 50 mg. of the solid in 100 ml. of distilled H20, and store in an amber reagent bottle at 4 °. 0.1 M pyrophosphate buffer, pH 7.0. Dissolve 4.46 g. of sodium pyrophosphate (Na4P20~.10H~O) in 100 ml. of distilled H~O, and adjust to pH 7.0 with HC1. 1% sulfanilamide. Dissolve 1 g. of the solid in 100 ml. of an acid solution made up with 75 ml. of distilled H20 and 25 ml. of concentrated HC1. Store in an amber reagent bottle. 0.02 % N-(1-naphthyl)ethylenediamine hydrochloride. Dissolve 20 mg. of the solid in 100 ml. of distilled water. Store in an amber reagent bottle. Enzyme. Dilute the stock enzyme with pyrophosphate buffer to obtain 200 to 700 units of enzyme per milliliter. (See definition below.)

Procedure. Add 0.05 ml. of enzyme at zero time to give a final reaction mixture of 0.5 ml. containing 0.1 ml. of nitrate, 0.04 ml. of FMN, 0.04 ml. of TPNH, and 0.27 ml. of pyrophosphate. After 5 minutes of incubation at room temperature, add 0.9 ml. of H20 and 0.5 ml. of sulfanilamide reagent to stop the reaction, followed by 0.5 ml. of the naphthylethylenediamine reagent to develop the color. After 10 minutes, read the density of the color on a Klett colorimeter with a green (540-m~) filter. Control tubes lacking T P N H are used to correct for the turbidity caused by the enzyme. Definition of Unit and Specific Activity. One unit of nitrate reductase is defined as that amount of enzyme which results in the formation of 10-3 t~M. of nitrite (approximately a reading of 10 on the Klett-Summerson colorimeter) under the above conditions of assay. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al. 4 Application of Assay Method to Crude Tissue Preparations. With extracts or homogenates nitrite reductase activity is frequently present. Under such conditions the reaction mixture should include :KCN at 5 × 10-5 M final concentration. This inhibits 85 to 95% of the nitrite reductase and only 10 to 20% of the nitrate reductase when F1V[N is the flavin. Nitrite reductase is removed in the first step of the enzyme purification. 40. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 198, 265 (1951).

[59]

NITRATE REDUCTASE FROM NEUROSPORA

413

Purification Procedure

Step 1. Preparation of Crude Extract. The mycelial mats grown in the presence of nitrate or nitrite nitrogen are collected on a Bfichner funnel, washed with distilled water, and frozen for 1 to 3 hours at - 1 5 °. They are then homogenized in a TenBroeck homogenizer in three times their weight of cold 0.1 M K~I-IPO4, and centrifuged at 20,000 × g for 10 minutes at 4 °. The supernatant solution, which is the crude extract, usually contains 85% or more of the nitrate reductase activity of the homogenates. All subsequent steps of the purification procedure are carried out at 0 to 4°; centrifugations are performed at approximately 3000 X g; and ammonium sulfate is used only as the saturated solution, at 20 to 25 °, adjusted with sodium hydroxide to pH 7.0 to 7.5. Step 2. Fractionation with Ammonium Sulfate. To 100 ml. of crude extract (fraction I) is added 75 ml. of ammonium sulfate solution to give a final concentration of 43% saturation. After standing for 15 minutes, followed by centrifugation, the supernatant solution is discarded, and the precipitate is dissolved in 40 ml. of cold 0.1 M phosphate buffer, pH 7.0 (fraction II). To fraction II (40 ml.) is added 12.5 ml. of ammonium sulfate solution, and after 15 minutes the precipitate (24% saturation) is collected by centrifugation and discarded. The supernatant solution is treated with 21.5 ml. of ammonium sulfate solution, and after 15 minutes the precipitate (46% saturation) is collected by centrifugation and dissolved in 16 ml. of cold 0.1 M phosphate buffer, pH 7.0 (fraction III). Step 3. Adsorption of Nitrate Reductase on Calcium Phosphate Gel. To fraction III (16 ml.) is added 7.2 ml. of calcium phosphate gel, 5 aged nine months or longer (11 rag. of dry weight per milliliter). After 15 minutes the precipitate is collected by centrifugation, washed twice with 5-ml. portions of cold 0.1 M phosphate buffer, pH 7.5, and eluted once with 4.0 ml. of cold 0.1 M pyrophosphate buffer, pH 7.0 (fraction IV). Step 4. Further Fractionation with Ammonium Sulfate. To fraction IV is added 6.0 ml. of ammonium sulfate to give 60% saturation. After standing for 15 minutes at 0 °, the precipitate is collected by centrifugation and dissolved in 2.0 ml. of cold 0.1 M phosphate buffer, pH 7.0. The protein in this final fraction (V) represents 10% of the units in the crude starting material and an over-all purification of 60- to 70-fold, as shown in the table, which gives a summary of the purification procedure. In terms of turnover number fraction V catalyzes the reduction of 80 moles of nitrate per mole of protein per minute, assuming a molecular weight for the enzyme of 100,000. 6 For the p r e p a r a t i o n of calcium p h o s p h a t e gel, see Vol. I [11].

414

ENZYMES OF P R O T E I N METABOLISM

[69]

T h e pyridine-nucleotide nitrate reductase from soybean leaves has also been purified approximately 70-fold b y calcium phosphate and ammonium sulfate fractionation techniques, e SUMMARY OF PURIFICATION OF PROCEDURE

Fraction I. II. III. IV. V.

Total Total protein, Specific activity, Recovery, units mg. units/mg, protein %

Crude extract 24,800 414 0-43% ppt., ammonium sulfate 12,700 105 24-46% ppt., ammonium sulfate 5,000 12 Calcium phosphate gel eluate 2,420 0.9 0-60% ppt., ammonium sulfate 2,400 0.6

60 121 417 2690 4000

51.1 20.0 9.8 9.7

Properties

Stability. Of the various fractions, fraction I I is the most stable, losing only 10 to 20% of its activity after a m o n t h at - 1 5 °, the optimal p H for storage being 7.0. T h e addition of glutathione (10 -3 M final concentration) to the enzyme further enhances enzyme stability during storage. The enzyme is almost completely inactivated on 2 minutes' contact with dialyzing m e m b r a n e unless the latter has been previously soaked overnight in a 0.1 M phosphate buffer containing glutathione at 10 -3 M final concentration. The enzyme can be dialyzed, provided t h a t the abovetreated m e m b r a n e is used with a 0.1 M phosphate dialyzing solution containing glutathione or cysteine at 10-a M or 10-~ M final concentrations, respectively. ~ p H Optimum. The enzyme exhibits a sharp optimum for activity at p H 7.0 in pyrophosphate buffer. The activity is lower in phosphate buffer unless chelating agents such as Versene, pyrophosphate, or triphosphate are present, suggesting the presence of a metal inhibitor in the reaction mixture. Specificity. T h e r e is a marked specificity for T P N H as the electron donor. With high concentrations of enzyme the specificity for T P N H is shown to be relative, since D P N H under these conditions also acts as an electron source. The maximal rate of activity achieved with T P N H is a b o u t t w e n t y times t h a t obtained with D P N H . The dissociation constants (Kin) for the T P N H - and D P N H - e n z y m e complexes are 7 >( 10 -5 and 1.4 >( 10-4 in moles per liter, respectively. With the soybean enzyme, however, T P N H and D P N H are almost equally effective as elec6 H. J. Evans and A. Nason, Plant Physiol. 28, 233 (1953). 7 D. J. D. Nicholas and A. Nason, J. Biol. Chem. 207, 353 (1954).

[59]

NITRATE REDUCTASE FROM NEUROSPORA

415

tron donors for nitrate reduction. The Km for the enzyme-nitrate complex is 1.4 × 10-3 mole per liter. Activators and Inhibitors. Nitrate reductase has a definite flavin requirement which can be fulfilled by boiled pig heart extract, 2 FAD, or in part by FMN, resulting in a 2- to 5-fold increase in enzyme activity. Although FAD has been established as the prosthetic group of the enzyme, FMN, which gives only half as much reactivation, serves as a cheap and convenient flavin for the assay. The dissociation constants (Kin) for the FAD- and FMN-enzyme complex are 3.2 × 10-7 and 30 × 10-7 in moles per liter, respectively. The sulfhydryl nature of the enzyme is shown by its complete inhibition by 5 × 10-8 M p-chloromercuribenzoate; this is almost completely restored by glutathione or cysteine. Iodoacetate at 10-2 M final concentration has no effect. Various metal-binding agents such as cyanide, azide, thiourea, 8-hydroxyquinoline, potassium ethyl xanthate, and o-phenanthroline are inhibitory, indicating a heavy metal constituent. The latter has been identified as molybdenum. 7 The addition of molybdate to the reaction mixture does not stimulate activity unless the enzyme has been freed of its metal by previous dialysis against cyanide. Nor does the addition of Fe ++, Fe +++, Mn ++, Zn++, Mg ++, BOa--, or Cu ++ to the reaction mixture stimulate activity. Cu ++ is markedly inhibitory (65%) at 10-4 M final concentration. Adaptive Nature of the Enzyme. Nitrate reductase activity is present in mycelia grown in the presence of nitrate or nitrite. There is no activity in mycelia grown in ammonia or alanine as a sole nitrogen source. Equilibrium Constant. From the E0' at pH 7.0 for T P N H : T P N +, which is probably about -0.28 volt on the assumption that it is close to that of D P N H : D P N +8 and E0' at pH 7.0 for NO3-:N02- of +O. 54, 9 the free energy change and equilibrium constant of the nitrate reductase reaction are calculated to be -30,000 calories and 1027, respectively. Mechanism of Action. With purified nitrate reductase from Neurospora it has been shown 1° that during the enzymatic transfer of electrons from T P N H to nitrate both FAD (or FMN) and molybdenum function as electron carriers. The reduction sequence mediated by the enzyme in the absence or presence of added indophenol dye is as follows: T P N H --* FAD (or FMN) --* Mo --* NO~2,3',6-trichloroindophenol s H. Borsook, J. Biol. Chem. 133, 629 (1940). gL. Anderson and G. W. E. Plaut, in "Respiratory Enzymes" (H. A. Lardy, ed.), rev. ed., p. 84, Burgess Publishing Co., Minneapolis, 1949. 1o D. J. D. Nicholas and A. Nason, J. Biol. Chem. 211, 183 (1954).

416

ENZYMES OF PROTEIN ~ETABOLIS~

[60] H y d r o x y l a m i n e R e d u c t a s e f r o m Neurospora

[60]

crassa

NH20H + D P N H + H +--~ NH~ + DPN + -~ H~O

By MILTON ZUCKER and ALVIN NASON Assay Method Principle. The method is based on the fact that the absorption of D P N H at 340 mt~ disappears on oxidation. The reaction may be followed by measuring the rate of D P N H oxidation spectrophotometrically.

Reagents NH~OH.HC1 (0.4 M). Dissolve 69.5 mg. in 2.5 ml. of H20. The solution is prepared daily. No nitrite is present in the Eastman Kodak product. D P N H (approximately 6 X 10-3 M), enzymatically reduced. 1 Boiled pig heart extract. Grind 10 g. of acetone-dried pig heart in 70 ml. of cold 0.1 M phosphate buffer, pH 7.5. Centrifuge, and boil the supernatant in a water bath for 5 minutes in the dark. Recentrifuge, and store the supernatant in the cold, protected from light. Not a reliable source of flavin after two to three weeks of storage. FAD, 2 X 10-5 M (Sigma Chemical Company, 40% pure). Dissolve enough FAD in distilled water to give an extinction of approximately 0.200 at 455 m~. The extinction coefficient at this wavelength is 1.13 X 10-7 sq. cm./mole. 2 Store frozen and protected from light. 0.1 M sodium pyrophosphate-HC1 buffer, pH 8.0. Enzyme. When necessary, dilute stock enzyme with 0.5 M K~HPO4 to obtain 1000 units or less per milliliter. (See definition below.)

Procedure. Mix 2.60 ml. of p i t 8.0 buffer, 0.05 ml. of DPNH, and 0.05 ml. of boiled pig heart extract or FAD in a cuvette having a 1-cm. light path. Take readings at 340 m~ at 30-second intervals after addition of 0.2 ml. of enzyme for 2 to 3 minutes to obtain an endogenous rate of D P N H oxidation. Start the reaction by addition of 0.1 ml. of NH~OH.HC1, and continue readings at 15- to 30-second intervals for 2 minutes, beginning 45 seconds after addition of substrate. Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which causes an initial rate of change (AE~40) of 0.001 per 1See Vol. III [127] for enzymatic reduction of DPNH. O. Warburg and W. Christian, Biochem. Z. 298, 150 (1938).

[60]

ttYDROXYLAMINE REDUCTASE FROM NEUROSPORA CRASSA

417

minute after correction for the endogenous rate of oxidation. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al2 Application of Assay Method to Crude Tissue Preparations. In other wild-type Neurospora strains, i.e., strain 146, the enzyme is detected only in the purified fraction, presumably as a result of inhibitors in the crude preparation. T P N H must be used as the electron donor. Hydroxylamine reductase has also been detected in crude extracts of bacillus organisms by measuring the disappearance of NH2OH 4 or by measuring D P N H oxidation. 5 NH2OH is assayed for according to Czaky. 6 In Neurospora the enzyme is found only in extracts of mycelia grown in the presence of nitrate or, to a lesser extent, nitrite.

Purification Procedure

Culture Conditions. Mycelial mats of Neurospora crassa (wild type, macroconidial strain Em5297a) are grown from spore innoculums on 125 ml. of a modified Fries minimal medium 7 in 500-ml. Erlenmeyer flasks at 30 ° . Four- and five-day-old mats are used as the source of enzyme. The procedures described below have been repeated successfully a number of times in this laboratory. All steps were carried out at 0 to 4 °, and precipitates were centrifuged at 3000 × g. Step 1. Preparation of Crude Extract. Mycelial mats are collected on a Biichner funnel, washed with distilled water, and frozen at - 1 5 ° for 1 to 3 hours. The frozen mats are coarsely powdered in a cold mortar and pestle, homogenized in four times their weight of cold 0.1 M K2HP04 with a Tenbrock glass homogenizer, and centrifuged at 3000 × g at 0 ° for 20 minutes. The resulting supernatant solution, designated as the crude cell-free extract, contained 85 % of the total protein. Step 2. Ammonium Sulfate Fractionation. Eighty-three milliliters of saturated ammonium sulfate solution adjusted to pH 7.0 to 7.3 with concentrated ammonium hydroxide is added to 100 ml. of crude cell-free 30. H. Lowry, N. J. Rosebrough, ~. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 4 S. Taniguchi, H. Mitsui, J. Toyoda, T. Yamada, and F. Egami, Japan. J. Biochem. 40, 175 (1953). R. Klausmeyer and R. Bard, J. Bacteriol. 68, 129 (1954). T. Z. Czaky, Acta Chem. Scand. 2, 450 (1948). 7 In units per liter: sodium tartrate, 5 g. ; NH4NO3, 3 g. ; I4H2PO~, 3 g. ; MgSO4-7H~O, 0.5 g. ; NaC1, 0.1 g. ; CaCl2, 0.1 g. ; sucrose, 20 g. ; biotin, 5 tLg.; 1 ml. of a complete race element solution containing sodium tetraborate 8.8 × 10-5 g. ; (NH4)4 MoTO24.6H20, 6.4 × 10 -5 g.; FeCI~-6H~O, 9.6 × 10 -4 g.; ZnSO4.7HsO, 8.8 × 10 -3 g.; CuC12, 2.7 X 10 -5 g.; M n C l r 4 H : O , 7.2 X 10 -8 g.

418

ENZYMES OF PROTEIN METABOLISM

[60]

extract making the mixture 45% saturated. After standing for 5 minutes the precipitate is removed by centrifugation, and the supernatant solution is brought to 65% saturation by addition of 104 ml. more of ammonium sulfate solution. After 10 minutes the resulting precipitate is collected by centrifugation and dissolved in 40 ml. of cold distilled water (fraction 2). Step 3. Absorption of Reductase by Ca3(P04)2 Gel. Twenty milliliters of Caa(PO4)2 gel 8 containing 20 mg./ml, dry weight is added to the enzyme solution (fraction 2). After intermittent stirring for 15 minutes, the gel precipitate is collected by centrifugation and eluted by suspending in 20 ml. of cold 0.5 M K~HPO4 buffer for 15 minutes. The supernatant solution obtained from subsequent centrifugation is used as the purified enzyme fraction (fraction 3). Fraction 3 also contains a pyridine-nucleotide nitrite reductase which parallels hydroxylamine reductase activity during fractionation as well as under various conditions and treatments. As yet no separation of the two activities has been obtained. The ratio of the two activities varies in different extracts, and addition of hydroxylamine to a system saturated with nitrite usually produces a small stimulation in the rate of D P N oxidation. However, when extracts of strain 146 are carried through the above procedure, hydroxylamine reductase preparations free of nitrite reductase activity are obtained. SUMMARY OF PURIFICATION PROCEDURE

Fraction

Total Total Specific volume, Units/ml., units, Protein, activity, Recovery, ml. thousands thousands mg./ml, units/mg. %

1. Crude extract 100 2. (NH4)~SO4 fraction 45 3. Ca~.(PO4)~geleluate 20

0.20 0.40 0.50

20 18 10

3.4 2.1 1.4

56 190 360

-90 50

Properties Specificity. The purified enzyme is specific for NH2OH and will not catalyze the reduction of O-methyl hydroxylamine, hydrazine, or nitrophenylhydroxylamine. Both D P N H and T P N H serve as electron donors in the reaction. The Km's calculated from Lineweaver-Burke plots are 0.7 X 10-4 M for D P N H and 1.0 × 10-4 M for T P N H . The maximum velocity obtained with D P N H is one-half that obtained with T P N H . The turnover number of hydroxylamine reductase is approximately 250 moles of T P N H oxidized per minute per mole of enzyme, assuming a molecular 8 See Vol. I [11] for preparation of Caa(PO,), gel.

[60]

HYDROXYLAMINE REDUCTASE FROM NEUROSPORA CRASS&

419

weight of 100,000 for the enzyme. The K~ for NH20H is 3.8 × 10-3 M. Activators and Inhibitors. FAD (3 X 10-7 M, final concentration) but not F M N stimulates the rate of DPNH oxidation two- to threefold and completely replaces the stimulation obtained with boiled pig heart extracts. A number of chelating agents inhibit the oxidation of DPNH, KCN being the most effective (50% at 5 X 10-6 M KCN). Other inhibitors at a final concentration of 5 X 10-3 M are salicylaldoxime, potassium ethyl xanthate, diethyldithiocarbamate, o-phenanthroline, a,a-dipyridyl, and sodium azide. Cysteine and glutathione have no effect alone, nor are they effective in reversing the inhibition obtained with p-chloromercuribenzoate. Iodoacetate at 10-2 M has no effect on the enzyme. Bisulfite (1.6 X 10-3 M, final concentration) completely inhibits the enzyme. Stability of Enzyme. The enzyme is most stable between pH 7.5 and 9.0, losing all its activity after a week at - 1 5 °. There is a complete loss of activity on standing overnight at 4 °. The enzyme loses all activity after 5 minutes at 50 °. Hydroxylamine reductase can be dialyzed against 3 X 10-3 M sodium pyrophosphate and 10-3 M cysteine for at least 2 hours, losing less than half its activity during this time. However, there is a complete loss of activity after 1 hour if 10-2 M phosphate, pH 8.0, is the dialyzing solution. This loss of activity on dialysis cannot be restored by addition of boiled enzyme, boiled pig heart extract, FAD, or FMN (10-6 M final concentration). At a final concentration of 10-~ M, ferrous, ferric, zinc, calcium, molybdate, cupric, manganous, borate, and magnesium ions did not restore activity either. Effect of pH. The effect of pH on enzymatic activity was determined by use of various buffers of suitable pH values. There is maximum enzyme activity between pH 8.0 and 9.0 when pyrophosphate or tris(hydroxymethyl)aminomethane is the buffer. Little or no activity is obtained with phosphate buffer unless 10-3 M Versene is included in the reaction mixture, suggesting the removal of a metal inhibitor.

420

ENZYMES OF PROTEIN METABOLISM

[61]

[61] Nitrite M e t a b o l i s m Enzymatic Formation of Nitrogen Gas (N~) from Nitrite and Nitric Oxide Gas (NO) 2 N O ~ - ~ N~ + 4 0 2 NO--~ N2 + 2 0

Formation of Nitric Oxide Gas from Nitrite N02 --~ NO + 0 By VICTOR A. NAJJAR

Assay Method

Principle. The method of measuring nitrogen and nitric oxide formation from nitrite is based on the fact that gas (N~ and NO) is formed which can be measured manometrically by the increase in pressure and that NO can be absorbed from the gas phase by alkaline sulfite. However, the formation of nitrogen from nitric oxide results in a decrease in pressure, as NO is also in the gas phase and two molecules of NO form one molecule of N2. Reagents NaNO2, 0.1 M. Hydrogen donor system. 0.3 % Difco yeast extract, glucose-6-phosphate 0.1 M, malate 0.1 M, and cofactors (see below). KOH, 20%. Nitric oxide gas, oxygen-free nitrogen gas. Phosphate buffer, pH 6.8, 0.2 M; tris(hydroxymethyl)-aminomethane buffer, pH 8.0, 0.2 M.

Procedure. Buffer (0.7 ml.), nitrite (0.1 ml.), and hydrogen donor (0.1 ml.) are placed in the main compartment, and the enzyme (0.5 ml.) in the side bulb. Alkali (0.2 ml. of 20 % KOH) is pipetted into the center well to absorb COs that might be formed. The manometers, while shaking, are flushed adequately for 15 to 20 minutes with oxygen-free nitrogen gas. After a period of equilibration the reaction is started by tipping in the enzyme. The increase in pressure as a function of time is recorded, and the amount of gas evolved is calculated. The bath is kept at a convenient temperature, 25 to 37% For the measurement of nitrogen (N2) formation from nitric oxide (NO) gas, NO is introduced into the gas phase after the system is thoroughly flushed with oxygen-free N2. It is essential that oxygen be rigor-

[61]

NITRITE METABOLISM

421

ously excluded from the whole system, as nitric oxide reacts readily ~Tith oxygen to form nitrogen dioxide gas (2 NO + 02 --* 2 NO2 ~ N204) which can be recognized by the red color of NO~ gas only if the latter is present in considerable amounts. The nitrogen dioxide formed reacts with water to produce nitrous and nitric acids (2 NO2 + H20--~ H N Q + HN02). This can occur to such an extent as to tax the capacity of the buffering system. The pH may drop to such a level as to precipitate and inactivate the enzyme. The decrease in pressure resulting from the conversion of NO to N2 is recorded as a function of time, and the amount of NO uptake can thus be calculated. Since 1 micromole of N2 gas is formed from 2 micromoles of NO gas, the resulting decrease in volume is a direct measure of the nitrogen formed. Nitric oxide gas is commercially available and can be generated readily in the laboratory in one of two reactions. I~NO~ + H I -~ ~/~ I~ + NO + H~O 3 C u + 8 H + + 2 N O 3 - - ~ 3 C u ++ + 4 H 2 0 + 2 N O

(1) (2)

In both reactions oxygen should be excluded rigorously to avoid the formation of nitrogen dioxide. The latter reaction is conveniently carried out by dropping 6 N HNO~ over copper wire in a closed system filled with N2 gas. The NO generated is allowed to bubble through water over which it is collected in order to remove traces of NO2 gas that is also formed. The reaction is preferably carried out in an ice bath.

Preparation of the Enzymes The enzymes are obtained from denitrifying bacteria, ~ Pseudomonas stutzeri, and a thermophilic strain of Bacillus subtilis (strain 115). These organisms are grown anaerobically in 0.3% (Difco) yeast extract and 0.5% KNO3. The latter is incubated at 50 to 55 ° for 8 hours, and the former at 25 ° for 24 to 38 hours or at 37 ° for 16 to 20 hours. A standard inoculum consisting of a 16-hour culture is used for inoculating fresh cultures (10 to 15 ml./1.). Cells are harvested by centrifugation and washed once with water. The enzymes are prepared by grinding P. stutzeri cell paste with alumina powder in the cold and extracting with 1 vol. of water at 4 to 6 °. This is then centrifuged for 30 minutes at 2100 × g to remove alumina, intact cells, and large cell fragments. The turbid supernatant is the source of enzyme. B. subtilis is lysed by adding a few crystals of lysozyme (Nutritional Biochemicals) to the cell paste and incubating at room temperature until complete lysis takes place. A volume of water is then added, and the nonlysed cells are centrifuged down. This supernatant is i V. A. Najjar and M. B. Allen, J. Biol. Chem. 206, 209 (1954).

422

ENZYMES OF PROTEIN METABOLISM

[61]

also a source of enzyme. Crude extracts so prepared produce 1 to 2 micromoles of N2 per milliliter of extract per hour at 25 ° in a complete system containing yeast extract as hydrogen donor and nitrite in phosphate buffer, pH 6.8. When the extracts are centrifuged at 15,000 X g for 30 to 60 minutes at 6 ° to separate the small particles, the resulting supernatant shows little or no activity. The activity is restored, however, by the addition of the particles. 1In this system DPN and TPN have no stimulating effect. Nitrogen formation from nitric oxide is similarly dependent on the particles which presumably contain the reducing system. Crude extract of P. stutzeri produces NO gas from nitrite in the complete system described above. The NO formed is trapped in alkaline sulfite to form Na~N~O2SO3 (5% Na2SOa in 0.1 N NaOH). The extent of NO formation is appreciated by the difference in the amount of gas formed in duplicate vessels, one of which contains 0.2 ml. of the sulfite in one side arm. The amount of NO trapped in the sulfite can be obtained by liberating N20 upon the addition of 0.1 ml. of 5% H2S04. Purification Procedure

The enzymes from P. stutzeri have been purified to some extent by ammonium sulfate fractionation. ~,3 Nitrogen formation from nitrite can be obtained with the highest activity in an ammonium sulfate fraction obtained at 0.4 to 0.55 saturation. After 48 hours of dialysis against water this fraction shows little activity with the complete system. However, with the addition of excess T P N H or glucose-6-phosphate and catalytic amounts of TPN, the activity is regenerated. That the enzyme has glucose-6-phosphate dehydrogenase can readily be shown in the Beckman spectrophotometer. The enzyme activity is also stimulated by the addition of D P N H or malate and catalytic amounts of DPN, but to a lesser extent than that exhibited by the TPN system. In these extracts the reduction of DPN by malate can also be demonstrated. The reduction of NO to N2 by an ammonium sulfate fraction obtained at 0.4 to 0.7 saturation is likewise poorly active after a 48-hour dialysis against distilled water. 2,3 The activity is regenerated by TPN and glucose-6-phosphate and to a lesser extent by DPN and malate. Properties 3

The reduction of nitrite to nitrogen has a pH optimum around 6.8 in phosphate buffer. However, the pH optimum for the formation of nitrogen from nitric oxide is at 8.0 in tris(hydroxymethyl)-aminomethane 2 C. W. C h u n g a n d V. A. Najjar, Federation Proc. 15 t 192 (1954). a C. W. C h u n g a n d V. A. Najjar, u n p u b l i s h e d observations.

[61]

NITRITE METABOLISM

423

buffer. At a concentration of 1 × 10-3 M, Ca ++, Mg ++, Mo, and Co ++ have no effect on nitrite reduction. Cyanide at a concentration of 1 X 10-3 M is slightly inhibitory. Further supplementation with FAD or FMN produces many-fold stimulation of NO to N2 conversion. The host of enzymes described above belong to the class of adaptive enzymes. It is necessary to have nitrate or nitrite in the medium for the development of these enzymes. Nitrate has proved consistently superior, since nitrite is inhibitory to cell growth when present in necessarily large amounts.

[62]

RIBONUCLEASES

427

[62] Ribonucleases By

MARGARET R. McDoNALD

Enzymes capable of hydrolyzing R N A are present in a great variety of cells. Only the ribonuclease (RNase) of bovine pancreas 1 has been purified and crystallized. 2 Its properties and mode of action have been extensively studied. There is no basis for assuming t h a t the RNases of other tissues have the same properties or mode of action, although their gross effect on R N A m a y be similar. In fact, studies on crude tissue extracts show m a n y dissimilarities between crystalline pancreatic RNase and RNases from other sources. The hydrolysis of R N A by pancreatic RNase is accompanied b y the gradual formation of free acid groups without any significant liberation of free phosphoric acid. 3,~ The split products, unlike the undigested RNA, are not precipitable b y acetic acid, hydrochloric acid, 4 or a solution of uranium salt in trichloroacetic acid. 2 T h e y readily diffuse through collodion or cellophane membranes t h a t are impermeable to the undigested RNA. 2 Digestion of R N A b y RNase is accompanied b y a shift in the ultraviolet absorption spectrum of the substrate toward the shorter wavelengths. This shift is most distinct in the region of 290 to 305 m~. 5 Most of these phenomena have been utilized in developing methods of RNase assay. 6 The one described here has been found b y the author to be the most convenient for the routine assay of pancreatic RNase. I t is essentially Kunitz's modification 2 of the "acid-soluble phosphorus" method of Dubos and T h o m p s o n J

Assay Method P r i n c i p l e . The method is based on the fact that, during digestion of I~NA b y RNase, 40% of the total nucleic acid phosphorus is converted into a form soluble in acid uranium acetate.

1Partially purified RNase has been prepared from sprouted soybeans [M. Schlamowitz and R. L. Garner, J. Biol. Chem. 163, 487 (1946)], but its properties have not been studied. M. Kunitz, J. Gen. Physiol. 24, 15 (1940). 8 W. Jones, Am. J. Physiol. 52, 203 (1920). 4 R. J. Dubos, Science 85, 549 (1937). 5 M. Kunitz, J. Biol. Chem. 164, 563 (1946). a See J. S. Roth and S. W. Milstein [J. Biol. Chem. 196, 489 (1952)] for detailed literature references. 7 R. J. Dubos and R. H. S. Thompson, J. Biol. Chem. 124, 501 (1938).

428

ENZYMES OF NUCLEIC ACID METABOLISM

[62]

Reagents Substrate. RNA, 8 dissolved in 0.1 M acetate buffer, p H 5.0, to a concentration of 0.5 rag. of total P per milliliter. The solution should be fresh and the p i t carefully adjusted in order to obtain reproducible results. M a c F a d y e n ' s reagent2 0.25% uranium acetate in 2.5% trichloroacetic acid. Procedure. One milliliter of RNase is mixed with 1 ml. of substrate, the mixture is left for 10 minutes at 25 °, then 2 ml. of M a c F a d y e n ' s reagent is added with thorough mixing. T h e suspension is left at 25 ° for 30 minutes, then filtered through 7-cm. Whatm.an No. 42 filter paper, and 2 ml. of the filtrate is analyzed for total phosphorus, l° This is designated as soluble phosphorus. With partially purified RNase preparations, ultraviolet absorption measurements at 260 m~ of a fivefold aqueous dilution of the filtrate, with a tenfold dilution of M a c F a d y e n ' s reagent as the blank solution, can be satisfactorily substituted for the more time-consuming phosphorus determinations. Definition of Unit and Specific Activity. The R N a s e activity unit is defined as the activity which gives rise under the standard conditions described above to t h e formation of 1 X 10-4 mg. of soluble phosphorus per milliliter of digestion mixture per minute in a range of concentrations of enzyme where the a m o u n t of soluble phosphorus formed is proportional to the concentration of enzyme used. For convenience, a standard curve is plotted--soluble phosphorus vs. activity u n i t s - - f r o m data obtained b y measuring the activity of a series of dilutions of ribonuclease of a known enzyme content. T h e activity of any unknown solution of RNase is then determined from a single measurement b y means of the standard curve. Specific activity is expressed as RNase units per milligram of protein. Protein is determined b y the m e t h o d of L o w r y et al. 11 Application of Assay Method to Crude Preparations. Of the various procedures 6 for the assay of RNase in crude tissue homogenates, the one described above is probably most applicable, provided t h a t control determinations are simultaneously made without added substrate. The press If commercial RNA is used, it should be purified by the procedure of G. E. Woodward [J. Biol. Chem. 156, 143 (1944)] to remove inhibitory mononucleotides. 9 D. A. MacFadyen, J. Biol. Chem. 107~ 297 (1934). 10By the method of E. J. King [Biochem. J. 26, 292 (1932)] or a similar procedure; also see Vol. III [114]. 11O. H. Lowry, N. J. Rosebrough, A. L. Farr. and R. J. Randall, J. Biol. Chem. 193, 265 (1951); also see Vol. III [73].

[62]

RIBONUCLEASES

429

ence of phosphatases which, by removing the formed products, would tend to increase the rate of the reaction, can be detected by assaying the filtrate for inorganic phosphorus. I°

Purification of Pancreatic RNase The procedure described below is essentially t h a t of Kunitz 2 as modified b y McDonald. 12 The method has proved to be very reproducible, both in the hands of the author and in m a n y other laboratories. I t has been used for the preparation of radioactive RNase. 1~ The saturated (NH4)2S04 is prepared at 20 to 25 ° (760 g. of salt per liter of H20). Determinations of p H are made on a test plate by mixing 1 drop of the appropriate 0.01% indicator with 1 drop of the solution to be tested and comparing the color with t h a t found by mixing 1 drop of the same indicator with 1 drop of 0.1 M standard buffer of the desired pH. This gives only apparent p H values but is adequate for reproducing the necessary conditions. All filtrations, unless otherwise specified, are done with suction on Bfichner funnels. Step 1. Preliminary Purification. About 20 pounds of fresh TM beef pancreas is collected in ice-cold 0.25 N H2S04. The glands are drained, freed of fat and connective tissue, and minced in a meat grinder. The ground pancreas is suspended in 2 vol. of ice-cold 0.25 N H2S04 and left at 0 to 5 ° for 18 to 24 hours, with occasional stirring. The suspension is then strained through cheesecloth, and the strained fluid is saved. The residue is resuspended in an equal volume of cold 0.25 N H2SO4 and restrained after 1 hour. The residue is discarded. The combined extracts are brought to 0.65 saturation of (NH,)2S04 by the addition of 430 g. of salt per liter of strained fluid. The suspension is filtered by gravity through 50-cm. fluted filter papers (Eaton-Dikeman No. 612 or W h a t m a n No. 12), at 0 to 5 °, and the clear filtrate is saved. The residue is suspended in a volume of cold H20 equal to that of the original minced pancreas, and 430 g. of (NH4)2SQ is added per liter of H20 used. The mixture is refiltered through fluted paper. This filtrate 15 is combined with the first one, and 105 g. of (NH4)~SO, is added per liter of filtrate (final concentration of (NH4)2S04, 0.8 saturation). The resulting precipitate is all3 M. R. McDonald, J. Gen. Physiol. 32, 39 (1948). 13C. B. Anfinsen, J. Biol. Chem. 185, 827 (1950). 14Frozen pancreases, obtainable from any of the large slaughterhouses, can also be used if only ribonuclease (or deoxyribonuclease) is to be prepared. They should be thawed by leaving them immersed in 0.25 N H2SO, at 5 °. 15The residue on the paper can be used for the isolation of deoxyribonuclease (M. Kunitz, J. Gen. Physiol. 33, 349 (1950); see also Vol. II [63]), chymotrypsinogen, trypsinogen, trypsin, and trypsin-inhibitor compound (M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19, 1002 (1936) ; see also Vol. II [2, 3, 4]).

430

ENZYMES OF NUCLEIC ACID METABOLISM

[62]

lowed to settle for 2 days at 0 to 5°; the settling is greatly facilitated by occasional stirring of the mixture and removal of foam during the first day of standing. The clear supernatant fluid is siphoned off and discarded; the remaining suspension is filtered through hardened paper (Schleicher and Schuell No. 576). The yield is about 4 g. per liter of ground pancreas used. Step 2. Removal of Proteolytic and Potential Proteolytic Activity. Each gram 18 of filter cake is dissolved in 5 ml. of H~O, and the resulting solution is poured into 20 ml. of boiling 0.2 saturated (NH4)2SQ previously adjusted with H2SO4 to pH 3.0 (methyl orange). The mixture is stirred for 5 minutes at 95 to 100 °, cooled quickly to 25 °, and left at 20 to 25 ° for approximately 1 hour. The suspension is filtered through soft paper (Eaton-Dikeman No. 617) with the aid of 10 g. of Standard Super-Ce117 per liter, and the filter cake is washed three times with small quantities of 0.2 saturated (NH4)2SO4. The residue is discarded. The filtrate is brought to 0.5 saturation of (NH4)2SO4 by the addition of 188 g. of salt per liter of filtrate; 10 g. of Standard Super-Cel is then added per liter, and the suspension is filtered with suction through soft paper. The residue is again discarded. The filtrate is brought to 0.8 saturation of (NH4)2SQ by the addition of 210 g. of salt per liter, and the resulting suspension is filtered on hardened paper; the yield is approximately 3 g./1. of ground pancreas used. The filtrate is discarded. Each gram 16 of filter cake is dissolved in 5 ml. of H~O, the pH of the solution is adjusted to 4.8 (methyl red or bromocresol green) with a few drops of 5 N NaOH, and 5 ml. of saturated (NH4)2S04 is added. The mixture is filtered through soft paper with the aid of 1 g. of Standard Super-Cel per 100 ml. of suspension. The residue is discarded. The filtrate is adjusted to pH 4.2 (bromocresol green) with 1 N H2SO4, after which 67 ml. of saturated (NH4)2SO4 is added slowly with constant stirring for each 100 ml. of solution (final concentration of (NH4)2SO4, 0.7 saturation). The suspension is filtered with suction on hardened paper. The yield is approximately 2 g./1. of minced pancreas originally used. The filtrate is discarded. TM Step 3. Crystallization. Each gram of filter cake is dissolved in 1 ml. of H20. The solution is filtered through soft paper with the aid of 5 g. of Standard Super-Cel per 100 ml. of solution; the residue on the paper 16 This expression denotes the relative a m o u n t s of material used. I t does not m e a n t h a t each gram of m a t e r i a l is processed separately. 17 Supplied b y Johns-Manville, 22 E a s t 40th Street, New York. 18 The yield of ribonuclease can be increased b y adding (NH4)2SO, to this filtrate to 0.8 saturation, filtering on h a r d e n e d paper, a n d reworking t h e filter cake with the next b a t c h of material being processed for the removal of proteolytic c o n t a m i n a n t s according to t h e second p a r a g r a p h of step 2.

[62]

RIBONUCLEASES

431

is washed several times with small quantities of H20 and then discarded. The combined filtrate and washings are brought with H20 to a final volume of 2 ml. Saturated (NH4)~S04 is then added slowly, with stirring, until a v e r y faint t u r b i d i t y appears (approximately 40 ml. per 100 ml. of solution is required), and the p H of the mixture is adjusted immediately to 4.6 (methyl red or bromocresol green) with a few drops of 1 N N a O H . T h e solution clears rapidly and is left at 20 to 25 °. Crystals of ribonuclease gradually form. 19 T h e y are filtered on hardened paper after 3 days; the yield is approximately 1.2 g./1. of minced pancreas originally used. The filtrate is adjusted to p H 4.2 (bromocresol green) with 1 N H2SO4 and brought to 0.8 saturation of (NH4)2SO4 by the slow addition, with constant stirring, of saturated (NH4)2SO4. The suspension is filtered on hardened paper, and the filtrate is discarded. The yield of filter cake is about 0.4 g./1. of minced pancreas. Additional ribonuclease crystals can be obtained b y reprocessing this filter cake according to the second paragraph of step 2. Step 4. Recrystallization. Each gram of filter cake of crystals is dissolved in 2 ml. of H20. The solution is filtered through soft paper with the aid of 0.1 g. of Standard Super-Cel. The residue is washed several times with small amounts of H~O. T h e combined filtrate and washings are brought to 3 ml. with H20. Saturated (NH4)2SO4 is added slowly, with stirring, until the solution becomes very faintly turbid (about 4 ml. per 10 ml. of solution is required). The mixture is left at 20 to 25°; crystals form rapidly. T h e y are filtered on hardened paper after 2 days; the yield is about 0.6 g./g. of filter cake. The filtrate is adjusted to p H 4.2, and saturated (NH4)2SO~ is added slowly to 0.8 saturation. The suspension is filtered on hardened paper, and the filtrate is discarded. The yield is about 0.3 g./g. of filter cake. Additional ribonuclease crystals can be obtained from this filter cake by processing it as described in step 3. Step 5. Recrystallization in Ethanol. Ribonuclease is recrystallized twice b y means of (NH4)~SO4, as described in the preceding section. Each gram of filter cake from the third crystallization is dissolved in 1.5 ml. of H20, and the solution is dialyzed 2° in collodion or viscose din19If too much (NH4),S04 has been added, an amorphous precipitate will form rapidly. This may change within 1 or 2 days into a mass of fine crystals; if not, water should be added dropwise until the amorphous material dissolves and typical "silkiness" is seen when the solution is stirred. Almost complete crystallization should then occur within the next 3 days. If no "silkiness" is seen the solution shouId be adjusted to pH 4.2, brought to 0.8 saturation with saturated (NH4)2SO,, and filtered with suction through hardened paper; the filter cake should again be processed as described in step 3. 90 M. Kunitz and It. Simms, J. Gen. Physiol. 11, 641 (1927-28). If the dialyzer of Kunitz and Simms is not available, it is advantageous to dialyze with slow mechani-

432

ENZYMES OF NUCLEIC ACID METABOLISM

[62]

lyzing tubing at 0 to 5 ° against cold H 2 0 for 24 hours. The dialyzed solution is diluted with H : O to 5 ml. and cooled to 5 °, and 60 ml. of 95% ethanol of the same t e m p e r a t u r e is added with stirring. A h e a v y a m o r phous precipitate i~ formed, which, on standing at 10 to 20 °, changes within several hours into a mass of fine fan-shaped rosettes of rectangular or needle-shaped crystals. T h e crystals are filtered on hardened p a p e r after 2 days and washed several times with cold 9 5 % ethanol. T h e y are then dried for 24 to 72 hours Over CaC12 in a desiccator. T h e d r y powder can be stored in a cool place indefinitely; the yield is a b o u t 0.3 g. of d r y crystals per g r a m of filter cake. SUMMARY OF PURIFICATION OF BOVINE PANCREATIC R I B O N U C L E A S E a

Fraction 1. Preliminary purification Acid extract 0.65-0.8 sat. (NH4)2S04 2. Removal of proteolytic contaminants 0.5-0.7 sat. (NH4)2S04 3. Crystallization Crystals Mother liquor 4. Recrystallization 2 X crystallized 3 X crystallized 5. Crystallization from ethanol Dialyzed ribonuclease Crystals

Total units, b )< l0 s

Specific activity, units/mg. protein

Yield, %

2000 1200

5 82

100 60

886

119

44

585 289

135 92

29 14

472 315

151 166

23 16

267 255

167 166

13 12

M. R. McDonald, J. Gen. Physiol. 32, 39 (1948). Based on 12.5 1. of ground pancreas. P r o p e r t i e s of P a n c r e a t i c R N a s e Specificity. Crystalline pancreatic R N a s e hydrolyzes R N A b u t not polymerized or depolymerized D N A . 4,21,22 D e a m i n a t e d R N A is degraded b y RNase, showing t h a t NH2 groups are not essential for its action. 2~ T h e action of R N a s e appears to involve specifically pyrimidine nucleo-

cal stirring for 48 hours against 2 1. of cold distilled H20 which is changed twice daily. 21L. M. Gilbert, W. G. Overend, and M. Webb, Exptl. Cell Research 2, 138 (1951). 22 M. C. Durand and R. Thomas, Biochim. et Biophys. Acta 12, 416 (1953). ,8 L. Vandendriessche, Compt. rend. tray. lab. Carlsberg. Sdr. chim. 27, 342 (1951).

[6 9.]

RIBONUCLEASES

433

tides. ~4 Cyclic pyrimidine ribose nucleotides (2',3'-monohydrogen phosphate esters of nucleosides) are digested by RNase to give 3'-riboside phosphate, whereas the analogous purine derivatives are not. 2~ RNase appears to be a specific phosphodiesterase which hydrolyzes only secondary phosphate esters of pyrimidine riboside 3'-phosphates. 26 Recent publications state that it degrades polyribophosphate 27 and thymic acid. 22 Kinetics. A mathematical analysis of the kinetics of the hydrolysis of RNA by RNase is complicated by the complexity of the reaction and by the fact that the enzymatic reaction is always accompanied by a significant amount of spontaneous hydrolysis of the substrate. The time required for any amount of digestion is inversely proportional to the concentration of enzyme in solution, whereas the ultimate amount of digestion is independent of the amount of enzyme used. 2 A marked rate reduction of enzyme activity is observed when the substrate concentration is increased, with a definite lag in attainment of the maximal rate of digestion. 28 The rate of formation of titratable acid groups is much slower than the rate of formation of acid-soluble split products. 2 Activators and Inhibitors. No specific activators are required for the enzymatic activity of RNase; no specific inhibitors are known. Its activity, as determined spectrophotometrically5 or turbidimetrically, 29 is inhibited by Mg ++, Ca ++, and Mn ++, the minimal inhibitory concentration being less than 0.0005 M. This inhibition is not reduced by F1- or citrate ions; it is not suppressed by Na + or NH4 +, which, in the concentration range of 0.0005 to 0.1 M, stimulate ~'ibonuclease activity. Higher concentrations of Na + and NH4 + have an inhibitory action, 0.33 M NaC1 decreasing the initial rate of reaction about 40 %.2s The liberation of acid groups and the formation of acid-soluble split products by RNase is enhanced by 0.1 M NaC1 or 0.1 M MgC12, the latter being the more effec~4 R. A. Bolomey and F. W. Allen, J. Biol. Chem. 144, 113 (1942); H. S. Loring, F. H. Carpenter, and P. M. Roll, J. Biol. Chem. 169, 601 (1947); G. Schmidt, R. Cubiles, and S. J. Thannhauser, Cold Spring Harbor Symposia Quant. Biol. 12, 161 (1947); C. E. Carter and W. E. Cohn, J. Am. Chem. Soc. 72, 2604 (1950); G. Schmidt, R. Cubiles, N. Z611ner, L. Hecht, N. Strickler, K. Seraidarian, M. Seraidarian, and S. J. Thannhauser, J. Biol. Chem. 192, 715 (1951); B. Magasanik and E. Chargaff, Biochim. et Biophys. Acta 7, 396 (1951). 25 R. Markham and J. D. Smith, Biochem. J. 52, 552 (1952); D. M. Brown, C. A. Dekker, and A. R. Todd, J. Chem. Soc. 1952, 2715. ~6R. Markham and J. D. Smith, Biochem. J. 52, 558 (1952); E. Volkin and W. E. Cohn, J. Biol. Chem. 205, 767 (1953). 2TS. Zamenhof, G. Leidy, P. L. FitzGerald, H. E. Alexander, and E. Chargaff, J. Biol. Chem. 203, 695 (1953). 2s C. Lamanna and M. F. Mallette, Arch. Biochem. 24, 451 (1949). ~* M. McCarty, J. Exptl. Med. 88, 181 (1948).

434

ENZYMES OF NUCLEIC ACID METABOLISM

[62]

tive. 8°,31 NaC1 or MgC12 does not liberate t i t r a t a b l e acid groups in the absence of the enzyme. 31 R N a s e - c a t a l y z e d liberation of acid groups is m a r k e d l y inhibited b y 2 X 10 -8 M Cu ++ or Zn ++ and slightly inhibited b y Ni ++ and Ag + of the same concentration; Co ++, Cd ++, Fe +++, and H g ++ h a v e a negligible effect. 3~ Higher concentrations (1 X 10 -3 M) of Co ++ and H g ++ inhibit RNase. 33 S t r e p t o m y c i n has been reported as an a c t i v a t o r s° and an inhibitor a4 of R N a s e ; penicillin 35 and basic dyes such as acridine 34 are also inhibitors. T h e enzyme is inhibited b y t r e b u r o n a6 (a synthetic sulfated polygalacturonic acid) and b y heparin 36,37 b u t not b y chondroitin-sulfuric acid, hyaluronic acid, or alginic acid. as This inhibition is reversible, the a c t i v i t y of the inhibited R N a s e being restored b y acid hydrolysis at 80°. a7 Mononucleotides 39 and D N A 4° inhibit RNase. Benzimidazole, 2-aminobenzimidazole, and 5,6-dimethylbenzimidazole accelerate R N a s e action, s~ I n c u b a t i o n of R N a s e with N a p-chloromercuribenzoate first increases then decreases its enzymatic activity; 33 incubation with iodoacetate, iodoacetamide, 32 periodic acid, 41 formaldehyde, ninhydrin, and phenylisocyahate 42 inactivates RNase. I t is also i n a c t i v a t e d b y X - r a y s 43 and b y O H radicals. 44 R N a s e is readily i n a c t i v a t e d b y digestion with pepsin. 2,4 Physicochemical Properties. Crystalline pancreatic R N a s e is a protein of the albumin t y p e with the following e l e m e n t a r y composition in per cent d r y weight: C, 48.2; H, 6.2; N, 16.1; S, 3.6 (partly inorganic); P, trace; residue, 0.1. 2 I t s amino acid composition, expressed as grams per 100 g. of protein (ash-, sulfate-, and moisture-free), is as follows: arginine, 5.2; aspartic acid, 14.2; cysteine, 0.6; cystine (half), 6.5; glutamic acid, 30 G. Ceriotti, Nature 163, 874 (1949). 3z C. E. Carter and J. P. Greenstein, J. Natl. Cancer Inst. 7, 29 (1946-47). Electrolytes, in the absence of RNase, do degrade RNA to dialyzable components. 32 C. A. Zittle, J. Biol. Chem. 163, 111 (1946). 33y. Miura and Y. Nakamura, Compt. rend. 232, 1874 (1951). a4L. Massart, G. Peeters, and A. Lagrain, Arch. intern, pharmacodynamie 76, 72 (1948) [Chem. Abstr. 42, 6383 (1948)]. a3L. Massart, G. Peeters, and A. Vanhoucke, Experientia 3, 494 (1947). 33j. S. Roth, Arch. Biochem. and Biophys. 44, 265 (1953). 37 N. ZSllner and J. Fellig, Am. J. Physiol. 173, 223 (1953). 33L. Ledoux, Biochim. et Biophys. Acta 10, 190 (1953). a9 C. A. Zittle, J. Biol. Chem. 160, 527 (1945). 40 M. R. McDonald in Carnegie Inst. Wash. Year Book No. 47, 148 (1948). 4i W. F. Goebel, P. K. Olitsky, and A. C. Saenz, J. Exptl. Med. 87, 445 (1948). 4~C. A. Zittle, J. Franklin Inst. 246, 266 (1948). 43D. Lea, K. M. Smith, B. Holmes, and R. Markham, Parasitology 36, 110 (1944); E. S. G. Barron, S. Dickman, J. A. Muntz, and T. P. Singer, J. Gen. Physiol. 32, 537 (1949); B. Holmes, Nature 165, 266 (1950). 44E. Collinson, F. S. Dainton, and B. Holmes, Nature 166, 267 (1950).

[69.]

RIBONUCLEASES

435

13.0; glycine, 1.3; histidine, 4.2; hydroxyproline, 0; isoleucine, 3.1; leucine, 0; lysine, 10.4; methionine, 4.4; phenylalanine, 3.6; proline, 3.6; serine, 12.0; threonine, 9.0; tryptophan, 0; tyrosine, 7.9; valine, 7.3; amide NH~, 2.5. 45 No indication of a special prosthetic group is evident from its ultraviolet absorption spectrum which shows a maximum molecular extinction coefficient of 11,540 near 280 m~ and a minimum of 6160 (5000) at 252 m~. 46 The following physical constants have been obtained for the enzyme: isoelectric point (by electrophoresis), ca. pH 7.8; 47 diffusion coefficient 2 at 20 ° in 0.5 M (NH4)RSQ, 0.092 sq. cm./day; diffusion coefficient47 at 25 ° in 0.5 M (NH4)2S04, 0.117 sq. cm./day; sedimentation constant 47 at 25 ° in 0.5 M (NH4)2SO4, 1.85 X 10-13; specific volume 47 at 25 °, 0.709; molecular volume 2 (calculated from diffusion coefficient), 14,850; molecular weight 47 (calculated from sedimentation and diffusion data), 13,000; molecular weight 2 (by osmotic pressure measurements), 15,000 + 1000; molecular weight (by X-ray analysis), 15,700 ± 300, 4s 13,400; 49 optical rotation per milligram of N at 25 ° (5 % aqueous solution), -0.470. 2 Ribonuclease is a good antigen despite its low molecular weight. 5° Aqueous solutions of crystalline pancreatic ribonuclease are quite stable 2 over a wide range of pH when kept at temperatures below 25 °. Heating to higher temperatures causes gradual loss in enzymatic activity. The rate of inactivation varies, however, with the pH of the solution, the concentration of the enzyme, and the concentration of electrolytes present. 51,52 The region of maximum stability is between pH 2 and 4.5. Effect of pH and Temperature. 2 The optimum pH for the action of RNase is ca. 7.7 (7.2 to 8.2); the optimum temperature is ca. 60 °. Crystalline RNase appears to be homogeneous from electrophoretic and ultracentrifugal studies. 47 Solubility studies 2,1~ indicate the possible presence of small amounts of impurities. Chromatographic fractionation has revealed the presence of two enzymically active components. 63,54 4~ E. Brand, as cited in J. H. Northrop, M. Kunitz, and R. M. Herriott, "Crystalline Enzymes," 2rid ed., p. 26, Columbia University Press, New York, 1948. 4~ F. M. Uber and V. R. Ells, J. Biol. Chem. 141, 229 (1941); D. Shugar, Biochem. J. 52, 142 (1952). 47 A. Rothen, J. Gen. Physiol. 24, 203 (1940). 4s I. Fankuchen, J. Gen. Physiol. 24, 315 (1940); not corrected for solvent of crystallization. 49 C. H. Carlisle and H. Scouloudi, Proc. Roy. Soc. (London) A207, 496 (1951) [Chem. Abstr. 46, 316 (1952)]. 50 j. Smolens and M. G. Sevag, J. Gen. Physiol. 26, 11 (1942). 5~ M. R. McDonald, J. Gen. Physiol. 32, 33 (1948). 52 A. Kleczkowski, Biochem. J. 42, 523 (1948). 53 A. J. P. Martin and R. R. Porter, Biochem. J. 49, 215 (1951). ~4 C. H. W. Hirs, S. Moore, and W. H. Stein, J. Biol. Chem. 200, 493 (1953).

436

ENZYMES OF NUCLEIC ACID METABOLISM

[62]

Whether the two components possess the same enzymatic specificity and properties has not been determined. The degree of inhomogeneity of a given preparation probably depends on the exact experimental conditions employed in the isolation and crystallization of the enzyme, sinc~ some preparations are more nearly homogeneous than others. Chromatographically homogeneous samples of the predominant fraction can be isolated either from crystalline preparations or directly from acid extracts of pancreas utilizing preparative scale chromatography. 54

Properties of Other RNases The following observations, made with tissue homogenates or crude extracts, indicate that RNases differing markedly in their properties from the digestive one of pancreas and from each other occur in various cells. Polynucleotide fractions, obtained by exhaustive digestion of RNA with crystalline RNase and resistant to further incubation with it, are hydrolyzed with phosphatase-free RNase preparations from beef spleen. 55 The pH optima for the action of diverse tissue RNases vary, values of 4.5, 6.0, 7.8, and 7.0 having been obtained for calf thymus, s6,s7 calf spleen, 57 rat liver, rat kidney, 58 and chick erythrocytes. 59 The heat lability of the various preparations also vary, as do their reaction to electrolytes. 57,58 More than one RNase may be present in the same tissue. 5s Hirs et al. 54 were unable to prepare, by the chromatographic procedure successfully used for pancreas, RNase from acid extracts of beef liver, spleen, and thymus. These findings, together with the always possible and almost probable fact of zymogens and inhibitors being present in tissues, make the comparison of the RNase content of various tissues, when assayed under the same conditions, extremely unreliable. It would appear that each tissue RNase (as with all enzymes) should be studied as an individual entity and that generalizations based on one isolated example are extremely hazardous. ~ G. Schmidt, R. Cubfles, and S. J. Thannhauser, J. Cellular Comp. Physiol. 38, Suppl. 1, 61 (1951). 56K. D. Brown, G. Jacobs, and M. Laskowski, J. Biol. Chem. 194, 445 (1952). 57M. E. Mayer and A. E. Greco, J. Biol. Chem. 181, 861 (1949). 58j. S. Roth, Biol. Bull. 103, 288 (1952). 69Z. B. Miller and L. M. Kozloff,J. Biol. Chem. 170, 105 (1947).

[63]

DEOXYRIBONUCLEASES

437

[63] D e o x y r i b o n u c l e a s e s B y MARGARET R. MCDONALD

Several enzymes (deoxyribonucleases) capable of hydrolyzing highly polymerized DNA occur in various cells. Although some of their properties are similar, others are markedly dissimilar. Only pancreatic deoxyribonuclease (DNase) has been highly purified and crystallized. Its preparation and properties will be discussed first. Methods for the partial purification of thymus, spleen, yeast, and streptococcal DNase will then be given, followed by a comparison of their properties.

Assay Method When solutions of DNA are hydrolyzed by DNase, their viscosity decreases and their specific absorption of ultraviolet light increases. Titratable acid groups are liberated without the formation of free phosphoric acid. The split products are not precipitable by mineral acids, proteins, or alcohol; they diffuse through collodion or cellophane membranes. Methods of assaying DNase based on all these phenomena have been extensively used; their relative merits have been discussed. L,2 Kunitz's spectrophotometric procedure 3 is probably the most convenient for routine measurements of purified DNase. The procedure described here has been found by the author to be the most generally useful in studies on DNase, applicable to both tissue homogenates and purified preparations. It is essentially Allfrey and Mirsky's modification2 of Laskowski's acidsoluble method. 4 Principle. The method is based on the colorimetric determination of the acid-soluble deoxypentose compounds released in the course of enzyme action. Reagents

Substrate. 200 mg. of Na-DNA 5 in 100 ml. of H20 or 0.05 M MgSO4, depending on the Mg ++ requirement of the DNase being assayed. 1N. B. Kurnick, Arch. Biochem. 29, 41 (1950). 2V. Allfrey and A. E. Mirsky, J. Gen. Physiol. 36, 227 (1952). 3 M. Kunitz, J. Gen. Physiol. 33, 349 (1950). 4 M. Laskowski, Arch. Biochem. 11, 41 (1946). 5Highly polymerized Na-DNA is obtainable from the Worthington Biochemical Sales Co., Freehold, New Jersey. For methods of isolation and purification of this compound, see Vol. III [103].

438

ENZYMES OF NUCLEIC ACID METABOLISM

[63]

0.2 M buffer. The composition and pH of the buffer is determined by the DNase being assayed. 3.0 M trichloroacetic acid.

Procedure. One milliliter of substrate plus 1 ml. of buffer is incubated with 1 ml. of enzyme solution at 35 ° for various times, after which 1 ml. of 3.0 M trichloroacetic acid is added. The mixtures are left in an icewater bath for 15 minutes, then filtered through 7-cm. Whatman No. 42 paper. Aliquots of the filtrate are analyzed by Dische's diphenylamine procedure, 6 and the optical densities obtained are converted to deoxypentose-P equivalents by comparison with those obtained from a standard solution of DNA. Definition of Unit and Specific Activity. The DNase activity unit is defined as that quantity of enzyme which catalyzes the formation of 1 "~ of acid-soluble deoxypentose-P per hour under the conditions described above. Specific activity is expressed as DNase units per milligram of protein. Protein is determined by the method of Lowry et alJ I. Crystalline Pancreatic Deoxyribonuclease Purification Prccedure The procedure described here is essentially that of Kunitz2 The yield of crystalline DNase is low, owing partly to the fact that at pH 2.8, which is most favorable for crystallization, the enzyme is gradually denatured. From 3 to 5 mg. of dry first crystals are usually obtained for each kilogram of ground pancreas extracted. The method has been found to be reproducible in several laboratories. The saturated (NH4)2S04 solution is prepared at 20 to 25 ° (760 g. of salt per liter of H20). All filtrations, unless otherwise specified, are done with suction. Step 1. Preliminary Purification. 8 Fresh *~beef pancreases are collected in ice-cold 0.25 N H2SO4. The glands are drained, cleaned of fat and connective tissue, then minced in a meat grinder. The minced pancreas is suspended in an equal volume of ice-cold H20, and ice-cold 0.25 N H2SO4 is added with stirring until the pH of the suspension is approximately 3.0 (tested with 0.01% methyl orange on a test plate); a volume of acid equal to half that of the H20 added is generally required. The suspension e Z. Dische, Mikrochemie 8, 4 (1930); see also Vol. I I I [99]. 7 O. H. Lowry, N. J. Rosebrough, A. L. Farr, a n d R. J. Randall, J. Biol. Chem. 193, 265 (1951); see Vol. I I I [73]. 8 Based on the procedure of M. M c C a r t y , J. Gen. Physiol. 29, 123 (1946). 9 Frozen pancreas, obtainable from a n y of the large slaughter-houses, can also be used if only deoxyribonuclease (or ribonuclease) is to be prepared. T h e y should be t h a w e d b y leaving t h e m immersed in 0.25 N H~S04 a t 5 °.

[63]

DEOXYRIBONUCLEASES

439

is left at 2 to 5 ° for 18 to 20 hours. I t is then strained through cheesecloth. The residue is resuspended in 1 vol. of ice-cold H20 and again strained. The residue is then discarded, and the combined filtrates are brought to 0.2 saturation of (NH4)2SO4 b y the addition of 114 g. of salt per liter of filtrate. The precipitate formed is filtered through a rapid filtering paper (such as E a t o n - D i k e m a n No. 617) with the aid of 10 g. of Celite No. 503 10 and 10 g. of Standard Super-Cel ~° per liter of solution. The filter cake is discarded. The clear filtrate is brought to 0.4 saturation of (NH4)2SO4 b y the addition of 121 g. of salt per liter and refiltered with the aid of 3 g. of Celite No. 503 per liter through double paper, E-D No. 612 on top of No. 617. The residue H is suspended in five times its weight of water, the suspension is brought to 0.3 saturation of (NH4)2S04 by the addition of 176 g. of salt per liter of H~O used and refiltered on E-D No. 617 paper; the filtrate is discarded. Step 2. Incubation at 37 ° Followed by Fraetionation with A m m o n i u m Sulfate. The residue is suspended in ten times its weight of H20, and the suspension is brought to 0.15 saturation of (NH4)2SO4 b y the addition of 83.7 g. of salt per liter of H20. The solution is titrated to p H 3.2 (glass electrode) with about 2 ml. of 5 N H2SO4 per liter. It is heated to 37 ° and left for 1 hour at t h a t temperature. I t is then cooled to 20 ° and filtered through E-D No. 617 paper with the aid of an additional 5 g. of Celite No. 503 per liter of suspension. The residue is discarded. The filtrate is titrated to p H 5.3 (glass electrode) with 5 N N a O H (about 2 ml./1.) and brought to 0.5 saturation of (NH4)2S04 by the addition of 220 g. of salt per liter. The precipitate formed, designated 0.5 precipitate, is filtered on E-D No. 617 paper with the aid of 5 g. of Celite No. 503 per liter of solution. The clear filtrate is titrated with a few drops of 5 N H2SO4 to p H 4.0 (tested with bromocresol green on a spot plate) and brought to 0.7 saturation of (NH4)2S04 by the addition of 135 g. of salt per liter. The scant precipitate formed, designated 0.7 precipitate, is filtered on E-D No. 612 paper with the aid of 2 g. of Standard Super-Cel per liter and stored. The filtrate is discarded. The 0.5 precipitate is resuspended in ten times its weight of water and step 2, including the incubation at 37 °, is repeated several times until no appreciable 0.7 precipitate is formed. lo Supplied by Johns-Manville, 22 East 40th Street, New York. 11The filtrate, when adjusted to 0.25 N H2SO4 by the addition of 7 ml. of concentrated H2SO~ per liter of H20 used in the extraction and washing of the ground pancreas, can be utilized for the preparation of chymotrypsinogen, trypsinogen, trypsin, trypsin-inhibitor compound (M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19, 1002 (1936) ; see also Vol. II [2, 3, 4]) and for ribonuclease (M. Kunitz, J. Gen. Physiol. 24, 15 (1940); see also Vol. II [62]).

440

ENZYMES OF NUCLEIC ACID METABOLISM

[63]

The 0.7 precipitates are combined and suspended in ten times their weight of H~O and filtered through E-D No. 612 paper. The residue is washed with H20 until the washing is water clear. Step 3. Fractionation with Ethanol. The combined filtrate and washings are diluted with H~O to a concentration of approximately 1% protein (the approximate concentration of protein can be determined spectrophotometrically at 280 m~, the optical density being 1.2 per milligram of protein per milliliter). The pH of the solution is adjusted with 5 N H2SO4 to pH 3.8 (tested with methyl orange on a spot plate), and 2 ml. of saturated (NH4)2SQ is added per 100 ml. of solution. The mixture is cooled in an ice-salt bath to 2 °, and one-quarter of its volume of ice-cold 95% ethanol is added slowly, with stirring, keeping the temperature of the solution between 2 and 5 °. The mixture is stored for 24 hours at 2 to 5 ° and is then centrifuged at the same temperature. The residue is discarded, and the clear supernatant is left at - 10° for 24 hours, after which it is centrifuged at the same temperature. The supernatant is discarded. Step 4. Crystallization. The precipitate is dissolved in approximately ten times its volume of ice-cold H20, after which it is brought to 0.38 saturation by the addition of 60 ml. of saturated (NH4)2S04 per 100 ml. of solution. The precipitate formed is filtered with suction on hardened paper (such as Schleicher and Schuell No. 576) at 5 to 10 °. It is then suspended in three times its weight of ice-cold H20 and dissolved by the slow addition of several drops of 0.25 N NaOH, keeping the pH of the solution below 4.8. If the solution is turbid it is centrifuged clear at about 5 °, then adjusted to pH 2.8 (glass electrode) with several drops of 0.2 N H2SOt. The heavy precipitate, which usually forms at approximately pH 3.5, dissolves readily as the pH of the solution reaches 3.0 or lower. The clear solution is left at 5 ° overnight and then at approximately 20 ° for 6 to 8 hours. Crystals appear during the latter step. Step 5. Recrystallization. The suspension of crystals is centrifuged. The residue is snspended in approximately 3 vol. of 0.02 saturated (NH4)2SO4 and dissolved with the aid of a few drops of 0.2 N NaOH at a pH of about 4.6. The solution is centrifuged if turbid, titrated to pH 2.8 (glass electrode), and left at 20 °. Crystals of DNase form within an hour. They are filtered on hardened paper at 5 °, then washed, first with ice-cold acidified 30% ethanol (1 drop of 5 N H2SO4 per 100 ml.), then with ice-cold acetone, and dried at room temperature for several hours. The mother liquors in steps 4 and 5 yield additional crystals when treated as follows: The solution is diluted threefold with ice-cold H20 and titrated with 0.2 N NaOH to pH 4.6 (tested with bromocresol green on a spot plate). Any insoluble material formed is removed by centrifugation. The clear supernatant is titrated with 0.2 N H2SO~ to ptI 4.0

[63]

DEOXYRIBONUCLEASES

441

and t h e n b r o u g h t to 0.38 s a t u r a t i o n of (NH4)~S04, as described in step 4, which is t h e n followed t h r o u g h in e v e r y detail. TABLE I SUMMARY OF PURIFICATIONPROCEDUREa OF PANCREATICDEOXYRIBONUCLEASE Fraction 1. 2. 3. 4.

Precipitate from 0.3 saturated (NH4)~S04 Filtrate, after 1 hour of incubation at 37 ° Precipitate from 20% alcohol at - 1 0 ° First crystals First mother liquor 5. Second crystals Second mother liquor

Specific activity b 0.2 3-5 5-6 8-10 5-6 8-10 8-10

Yield' 100 30 15 5 10

M. Kunitz~ J. Gen. Physiol. 33, 349 (1950). b The specific activity, i.e., activity per milligram of protein, is expressed in terms of that of the best preparations, which is taken as equal to 10. c The yield is given in per cent of the activity of the first fraction, precipitated in 0.3 saturated (NH4)~SO4.

Properties Specificity. Crystalline pancreatic D N a s e h y d r o l y z e s h i g h l y polymerized D N A and D N A which has been d e n a t u r e d so t h a t its physical properties, b u t n o t its chemical composition, are altered. 12 D e t a c h m e n t of a p o r t i o n of the purines 12,13 inhibits the h y d r o l y s i s ; apurinic acid 12 is n o t h y d r o l y z e d b y D N a s e . it ( M g ++ alone causes disintegration of apurinic acid. 12) D N a s e does n o t h y d r o l y z e R N A . 8,15 Analysis of the p y r i m i d i n e a n d purine c o n t e n t of the p r o d u c t s of D N a s e hydrolysis of D N A shows the p y r i m i d i n e - p u r i n e ratio of the dialyzable p r o d u c t s to be higher t h a n the p a r e n t s u b s t r a t e ; t h a t of the nondialyzable " c o r e , " lower. ~6,17 This does n o t necessarily m e a n t h a t D N a s e preferentially h y d r o l y z e s p y r i m i d i n e nucleotide groupings, ~6 and until m o r e is k n o w n a b o u t the composition a n d s t r u c t u r e of the nucleotides formed, little can be said a b o u t the b o n d specificity of D N a s e . Kinetics. T h e various changes in the physical and chemical properties

1~C. Tamm, H. S. Shapiro, and E. Chargaff, J. Biol. Chem. 199, 313 (1952). 18 C. A. Zittle, J. Franklin Inst. 243, 334 (1947). 14This has been confirmed cytochemically by A. Howard and S. R. Pelc, working in London, and by H. Gay in the author's laboratory. 1~L. M. Gilbert, W. G. Overend, and M. Webb, Exptl. Cell Research 2, 138 (1951). 16S. Zamenhof and E. Chargaff, J. Biol. Chem. 178, 531 (1949); 187, 1 (1950). 17 M. G. Overend and M. Webb, J. Chem. Soc. 1950, 2746.

442

ENZYMES OF NUCLEIC ACID METABOLISM

[63]

of D N A catalyzed b y D N a s e occur at unequal rates. 17-19 T h e change in viscosity and ultraviolet absorption generally precedes a n y noticeable change in the precipitability of the nucleate with strong acids or the liberation of acid groups. W h e n the concentration of s u b s t r a t e is low, the hydrolysis approxim a t e s closely a reaction of the first order, 18,2° the unimolecular constant being independent of the concentration of enzyme. At relatively higher concentrations of substrate, the initial rate of reaction decreases rapidly with increase in s u b s t r a t e concentration. 18,2°,2~ T h e products of the reaction are also inhibitory. 21 Activators and Inhibitors. M g ++ 4.8.22 (or other divalent ions ~3) are obligatory for the enzymic action of pancreatic DNase. T h e concentration of M g ++ required increases with increasing concentration of s u b s t r a t e and is practically independent of the concentration of enzyme. ~s,2° T h e relative concentrations of M g ++ and of D N A for the optimal rate of activation are such t h a t there is always a considerable excess of M g ++ over the a m o u n t necessary to change N a - D N A into M g - D N A stoichiometrically. T h e concentration-activation function for M g ++ on D N a s e passes through a m a x i m u m ; at concentrations of M g ++ a b o v e 0.02 M there is a decrease in the rate of digestion. ~s This inhibitory effect is also shown b y NaC1. 4,~8 Arginine, lysine, and histidine 24,25 h a v e been reported as activators ~-6 of D N a s e when used in concentrations ranging from 0.001 to 0.01 M ; at concentrations greater t h a n 0.01 M the effect decreases and lysine becomes inhibitory. 24 Fluoride, citrate, s arsenate, 2~ borate, and selenite ions 27 inhibit the action of D N a s e , owing p r o b a b l y to their ability to remove the a c t i v a t ing M g ++. Thioglycolic acid, Na2S, Cu ++, Zn ++, Fe ++, Fe +++, Cr ++, and Ni ++ are inhibitory 27,28 in the presence of M g ++. N a - u s n a t e 28 inhibits 18 M. Kunitz, J. Gen. Physiol. 33, 363 (1950). 19G. Jungner, I. Jungner, and L. G. Allg4n, Nature 164, 1009 (1949); R. Vercauteren, Nature 165, 603 (1950). 20 j. Gr4goire, Compt. rend. 231, 384 (1950). 2, L. F. Cavalieri and B. Hatch, J. Am. Chem. Soc. 75, 1110 (1953). 22F. G. Fischer, I. BSttger, and H. Lehmann-Echternacht, Z. physiol. Chem. 271, 246 (1941). 23 C. E. Carter and J. P. Greenstein, J. Natl. Cancer Inst. 7, 29 (1946); T. Miyaji and J. P. Greenstein, Arch. Biochem. and Biophys. 32, 414 (1951). 24 W. Frisch-Niggemeyer and O. Hoffmann-Ostenhof, Monatsh. 81, 607 (1950) [Chem. Abstr. 44, 9497 (1950)]. 2~V. L. Nemchinskaya and V. S. Shapot, Biokhimiya 18, 210 (1953) [Chem. Abstr. 47, 8132 (1953)]. 26This activation may be due to impurities, since a commercial sample of arginine which activated DNase did not do so after purification. ~ 27L. M. Gilbert, W. G. Overend, and M. Webb, Exptl. Cell Research 2, 349 (1951). 2s A. Marshak and J. Fager, J. Cellular Comp. Physiol. 85, 317 (1950).

[63]

DEOXYRIBONUCLEASES

443

DNase in the presence, but not the absence, of Co ++. Some tissues contain a protein(s) which markedly inhibits pancreatic DNase. 29 Physicochemical Properties. Crystalline DNase is a protein of the albumin type with the following elementary composition in per cent dry weight: C, 50.16; H, 6.91; N, 14.88; S, 1.09; P, 0; ash, 0.47. 3 No indication of a special prosthetic group is evident from its ultraviolet absorption spectrum which shows a maximum molecular extinction of 70,000 at 280 m~ and a minimum of 26,000 at 250 m~. 3 I t contains about 8 % tyrosine and 2 % tryptophan. 3 Its molecular weight calculated from diffusion measurer~.ents (assumed specific gravity, 1.33) is 63,000;3 from inactivation by deuteron and electron bombardment, 62,000. 30 The isoelectric point of DNase is in the region of p H 4.7 to 5.0. 3 The stability of solutions of DNase depends markedly on their concentration. Solutions containing > 0.1 mg. of protein per milliliter in dilute buffer in the p H range of 4.0 to 9.0 are stable for at least a week at 50. 3 Solutions containing <0.01 mg. per milliliter require the presence of gelatin or peptone as stabilizers, s The p H of optimum stability is from 5 to 6. Dilute solutions of the enzyme at p H 2.8 are reversibly denatured when heated for 5 minutes at 900. 3 When inactivated in the dry state by heat, the entropy of activation is - 3 7 cal./mole °K, the heat of activation 16,000 cal./mole. 8° Effect of pH. The optimal p H range for the hydrolysis of D N A by DNase is 6.0 to 7.0. 8 Purity. The specific activity of DNase becomes constant after one recrystallization. Crystalline preparations are free of measurable traces of trypsin, chymotrypsin, and ribonuclease. All attempts to determine the purity of the preparations by solubility tests failed becaus'e of the continuous formation of denatured protein at the salt concentrations required for the test.3

II. Partially Puxified Deoxyribonucleases Purification of Thymus Deoxyribonuclease 31 Step 1. Extraction and Activation. Four hundred grams of calf t h y m u s is freed from fat and connective tissue, cooled in ice, and homogenized in a high-speed mixer for 2 minutes with 1.5 1. of 0.14 M NaC1. The suspension is strained through cloth and centrifuged. The turbid supernat a n t solution (total viscosimetric 8 units, 800) is covered with 5 ml. of 29W. Dabrowska, E. J. Cooper, and M. Laskowski, J. Biol. Chem. 177, 991 (1949); E. J. Cooper, M. L. Trautmann, and M. Laskowski, Proc. Soc. Exptl. Biol. Med. 73, 219 (1950); H. H. Henstell and R. I. Freedman, Cancer Research 12, 341 (1952). 30C. L. Smith, Arch. Biochem. and Biophys. 45, 83 (1953). 31M. Webb, Exptl. Cell Research 5, 27 (1953).

444

ENZYMES OF NUCLEIC ACID METABOLISM

[64]

toluene and left at 4 ° for 24 to 48 hours. It is then adjusted to pH 5.0 to 5.2 with 0.1 N HC1. A heavy white precipitate forms. This is removed by centrifugation, and the supernatant is filtered through cotton wool to remove lipid material. Step 2. Concentration and Purification. The filtrate (total units, 25,000) 32 is adjusted to pH 6.4 with 0.1 N NaOH and dialyzed for 24 hours against 10 1. of H20 at 4 °. The formed precipitate is removed by centrifugation, and the clear supernatant (total units, 24,800) is concentrated tenfold by pervaporation in 3/~-in. diameter cellophane dialysis tubes. The solution (total units, 27,000) is centrifuged, and the supernatant (pH 5.2) is left at 35 ° for 10 minutes. A heavy white precipitate forms. The suspension is centrifuged, and the supernatant (total units, 13,200; units/rag. N, 10) is dialyzed for 48 hours at 4 ° against three changes of H20 (total volume of H20, 20 1.). The formed precipitate is removed by centrifugation. Step 3. Ethanol Fractionation. The supernatant (total units, 9000; units/mg. N, 50) is cooled in a freezing mixture, and cold ( - 10 °) ethanol is added to a final concentration of 29% (44 ml. per 100 ml. of solution), keeping the temperature of the mixture at - 8 °. The resulting suspension is centrifuged, and the precipitate is discarded. The supernatant is again cooled in a freezing mixture, and cold ( - 1 0 °) ethanol is added as above to a final concentration of 37.5% (15 ml. per 100 ml. of solution). The resulting suspension is centrifuged at 0 °, and the supernatant is discarded. The precipitate is dissolved in 40 ml. of H~O (total units, 3400; units/mg. N, 500) and dialyzed for 6 hours against 1 1. of H20 at 4 °. The solution is diluted to 200 ml. with H20 and refractionated with alcohol as before. The precipitate is suspended in H20 and dialyzed to remove the ethanol. The solution is then dried in vacuo from the frozen state, yielding 20 rag. of a pale cream-colored powder containing 15.8% N (total units, 2300; units/mg. N, 750; yield, 9%). III. Purification of Spleen Deoxyribonuclease ~3

Unless otherwise specified, all operations are performed at 1 to 5 ° and all filtrations are done with suction. Step 1. Extraction. Twenty pounds of frozen calf spleen is thawed for 24 to 48 hours, then minced in a meat grinder. The mince is suspended in 20 1. of 0.075 N H2SO4; the suspension is stirred for 1 hour and then left for 18 to 24 hours. It is then filtered by gravity through fluted paper 32 This marked increase in activity by the acid treatment (or by prolonged incubation at 37 °, M. Webb, Exptl. Cell Research 5, 16 (1953)) has not been observed by the author using frozen thymus or spleen. 83 M. R. McDonald, unpublished data.

[63]

DEOXYRIBONUCLEASES

445

(Whatman No. 12) with the aid of 10 g. of Standard Super-Cel and 10 g. of Filter-Cel per liter of suspension. The filtrate is saved. The residue is Suspended in 10 1. of 0.03 N H2S04 and refiltered. The residue is discarded. Step 2. Fractional Precipitation with (NH4)~S04. The filtrates are combined (total acid-soluble P units, 2 9 million; 34 units/mg, protein, 55), brought to pH 7.5 with 5 N NH40H (about 5 ml./1.), and then to 0.4 saturation of (NH4)2SO4 by the addition of 243 g. of salt per liter of filtrate. The resulting suspension is filtered with the aid of 5 g. of Standard Super-Cel per liter through soft paper (Eaton-Dikeman No. 616). The residue is discarded. The filtrate is brought to 0.75 saturation of (NH4)~SQ by the addition of 241 g. of salt per liter. The resulting suspension is filtered through hardened filter paper (Schleicher and Schuell No. 576), and the filtrate is discarded. The filter cake (about 140 g.) is dissolved in three times its weight of cold H20 (total units, 8.3 million; units/mg, protein, 140), and 66.7 ml. of saturated (NH4)2S04 is added per 100 ml. of H~O (final concentration of (NH4)2SO4, 0.4 saturation). The suspension is filtered through soft paper with the aid of 2 g. of Standard Super-Cel per 100 ml., and the residue is discarded. The filtrate is brought to 0.75 saturation by the addition of 140 ml. of saturated (NH4)2S04 per 100 ml. of filtrate, and the resulting suspension is filtered through hardened paper. The filtrate is discarded. Step 3. Heat Fractionation. The filter cake (about 55 g.) is dissolved in twenty-five times its weight of H20 (total units, 7.7 million; units/rag. protein, 215), and the solution is brought to pH 4.3 with 1 N HC1. It is then heated rapidly to 62 ° and left at 62 ° for 10 minutes, after which it is cooled to 25 ° and left at room temperature for 1 hour. The suspension is filtered through soft paper with the aid of 10 g. of Standard Super-Cel per liter; the residue is discarded. The filtrate is brought to pH 7 with 1 N NaOH and then to 0.75 saturation of (NH4)~S04 by the addition of 516 g. of salt per liter. The suspension is filtered, and the filtrate is discarded. The filter cake (about 20 g.) has 75% of the activity originally extracted (total units, 6.7 million; units/mg, protein, 700). IV. Purification of Yeast Deoxyribonuclease 35

Step 1. Extraction and Activation. 350 g. of fresh baker's yeast is washed with H20, centrifuged, suspended in 60 ml. of H20 and crushed in a bacterial mill. The mixture is centrifuged for 2 hours at 1900 >< g. 3~Over 90% of the activity present in the minced (or homogenized) spleen is found in the extract. s5S. Zamenhofand E. Chargaff,J. Biol. Chem. 180, 727 (1949).

446

ENZYMES OF NUCLEIC ACID METABOLISM

[63]

The residue 86 is suspended in 300 ml. of 1 M NaC1, and the viscous mixture is left at 4 ° for four months, during which period its viscosity disappears completely and its DNase activity increases markedly (0.6, 0.6, 17, 25, 30, and 30 viscosimetric unitsS/ml, after 0, 14, 19, 52, 90, and 120 days, respectively). Step 2. Concentration and Purification. The mixture (total units, 15,200) is then centrifuged at 1900 X g for 1 hour, dialyzed with rocking against ice-cold H20 for 4 hours, and dried in vacuo from the frozen state. The residue (total units, 15,000; units/rag, protein, 14) is suspended in 30 ml. of H20 and centrifuged at 31,000 × g for 2 hours. The residue is discarded, and (NH4)2SO4 is added to the yellow supernatant to 0.6 saturation. The resulting precipitate is collected by centrifugation at 31,000 X g, drained with suction on Whatman paper No. 50, suspended in 45 ml. of H20, and dialyzed with rocking against ice-cold H20 for 7 hours. The dialyzed mixture (total units, 10,200) is centrifuged at 31,000 × g for 1 hour, and the supernatant is discarded. The sediment is washed with H20, then extracted, first with 30 ml. and then again with 12 ml. of 1 M NaC1. The combined extracts (total units, 6200) are centrifuged at 31,000 X g for 1 hour, and the supernatant is dialyzed for 6 hours against ice-cold H20. The dialyzate is then dried in vacuo from the frozen state, yielding 27 rag. of pale yellow fluff, insoluble in H20 and soluble in salt solutions (total units, 4300; units/rag, protein, 1250; yield, 35%). V. Purification of Streptococcal Deoxyribonuclease 37

Group A hemolytic streptococci produce, during growth, appreciable amounts of extracellular DNase. It is readily precipitated from the culture medium by (NH~)2SO4. Step 1. Elaboration. Fifteen liters of neopeptone broth 38 is inoculated with a strain of group A hemolytic streptococcus (H105) and incubated at 37 ° for 20 hours. The cells are then removed by centrifugation in a Sharples centrifuge. Step 2. Concentration and Purification. The slightly turbid superuatant is brought to 0.4 saturation of (NH4)2SO4 by the addition of 243 g. of salt per liter of supernatant. The suspension is filtered with suction with the aid of 1 g. of Filter-Cel and 1 g. of Hyflo Super-Cel per liter. The residue is discarded, and the filtrate is brought to 0.8 saturation by the addition of 281 g. of (NH4)2SO4 per liter. The resulting precipitate is recovered by filtration and dissolved in 100 ml. of H20. (This solution 3s The s u p e r n a t a n t can be used for the preparation of yeast D N a s e inhibitor. 35 ~7 M. McCarty, J. Exptl. Med. 90, 543 (1949). as V. P. Dole, Proc. Soc. Exptl. Biol. Med. 63, 122 (1946).

[63]

DI~OXYRIBONUCLEASES

447

contains a l m o s t all the original D N a s e activity.) T h e solution is b r o u g h t to 0.4 s a t u r a t i o n of (NH4)~S04, filtered, a n d the residue discarded. T h e filtrate is b r o u g h t to 0.5 s a t u r a t i o n of (NH4)~S04, a n d the p r e c i p i t a t e is recovered b y filtration. I t is dissolved in a small v o l u m e of H~O, dialyzed against H20, a n d dried i n vacuo f r o m the frozen state. T h e dried m a t e rial (170 mg.) contains the bulk of the original D N a s e a c t i v i t y a n d has 25,000 viscosity s u n i t s / m g . Properties

& c o m p a r i s o n of some of the properties verse sources is p r e s e n t e d in :Fable I I . T h e fied D N a s e f r o m spleen a n d m o u s e leukemic e x t r a c t s of m a n y o t h e r m a m m a l i a n tissues, t h y m u s D N a s e . 2,~'I.s9,4°

of several D N a s e s f r o m diproperties of partially puritissues, a n d of crude D N a s c a p p e a r to resemble those of

TABLE II SUMMARY OF PROPERTIES OF VARIOUS DEOXYRIBONUCLEASES

Deoxyribonuclease from StreptoPancreas ~ Thymus b Serum c Yeast ~ coccus* 1. pit optimum 2. Mg ++ requirement 3. Inhibition by: F1 or citrate ions Bacterial RNAI Yeast protein s Pancreatic DNase antisera" Streptococcal DNase antisera'

7.0 -F

5.2 --

7.5 +

-{-

--

+

-+

--

6.0 +

7.5 + + +

4+

M. McCarty, J. Gen. Physiol. 29, 123 (1946). b M. Webb, Exptl. Cell Research 5, 27 (1953). c F. Wroblewski and 0. Bodansky, Proc. Soc. Exptl. Biol. Med. 74, 443 (1950). S. Zamenhof and E. Chargaff, J. Biol. Chem. 180, 727 (1949). M. McCarty, J. Expll. Med. 90, 543 (1949). s A. W. Bernheimer, Trans. N. Y. Acad. Sci. [II] 14, 137 (1952). 89 M. E. Mayer and A. E. Greco, J. Biol. Chem. 181, 861 (1949). 40 K. D. Brown, G. Jacobs, and M. Laskowski, J. Biol. Chem. 194, 445 (1952).

448

ENZYMES OF NUCLEIC ACID METABOLISM

[64]

[64] Preparation of Nucleoside Phosphorylase from Calf Spleen I By

VINCENT E . PRICE, M . CLYDE OTEY, a n d PAUL PLESNER

Purine nucleoside phosphorylase was first described by Kalckar 2,3 in 1945 and shown to catalyze the equilibrium Inosine -~ phosphate ~- Hypoxanthine ~- ribose-l-phosphate The equilibrium was shown to greatly favor synthesis of the nucleoside. 4 Both ribosides and deoxyribosides of hypoxanthine and guanine are rapidly attacked by the phosphorylase. Xanthosine and deoxyxanthosine are phosphorolyzed at a much slower rate. 5 Pyrimidine nucleosides are attacked by a different enzyme, pyrimidine nucleoside phosphorylase. 6,7 Nucleoside phosphorylase has been very useful in the synthesis of new intermediates. Friedkin 8 has used nucleoside phosphorylase in the synthesis of the ribosides and deoxyribosides of 8-azaguanine. Korn et al. 9 have recently used a purified preparation of beef liver nucleoside phosphorylase in the synthesis of 4-amino-5-imidazolecarboxamide riboside. Rowen and Kornberg have presented evidence 1° indicating that purine nucleoside phosphorylase is active in the phosphorolysis and synthesis of nicotinamide riboside (pyridinium N + riboside). With this substrate the equilibrium is far toward free nicotinamide and ribose-l-phosphate formation. If hypoxanthine is removed from the above phosphorylase reaction by its oxidation to uric acid with xanthine oxidase, ribose-l-phosphate can be isolated. This is the basis for an excellent preparative method for 1 This is a hitherto unpublished method based on work initiated by Dr. Vincent E. Price while working with Dr. Herman Kalckar at the University of Copenhagen in 1951 and completed with Mr. M. Clyde Otey at the National Cancer Institute. The method has been checked by Dr. Paul Plesner and Dr. Hans Klenow of the University of Copenhagen. H. M. Kalckar, J. Biol. Chem. 158, 723 (1945). It. M. :Kalckar, Federation Proc. 4, 248 (1945). 4 H. M. Kalckar, J. Biol. Chem. 167, 477 (1947). M. Friedkin, J. Am. Chem. Soc. 74, 112 (1952). 6L. A. Manson and J. O. Lampen, J. Biol. Chem. 193, 539 (1951). M. Friedkin and D. Roberts, J. Biol. Chem. 207, 245 (1954). 8 M. Friedkin, J. Biol. Chem. 209, 295 (1954). 9 E. D. Korn, F. C. Charalampous, and J. M. Buchanan, J. Am. Chem. Soc. 75, 3611 (1953). 10 j. W. Rowen and A. Kornberg, J. Biol. Chem. 198, 497 (1951).

[64]

NUCLEOSIDE PHOSPHORYLASE FROM CALF SPLEEN

449

this acid-labile ester. 4 Friedkin H has more recently used the phosphorolysis of guanine deoxyriboside in the p r e p a r a t i o n of the extremely labile deoxyribose-l-phosphate. I n this case guanine was removed b y its conversion to xanthine b y the guanase in the phosphorylase preparation. Purine nucleoside phosphorylase is also an i m p o r t a n t enzyme in the methods for the differential s p e c t r o p h o t o m e t r y of purine compounds developed b y Kalckar. 12 For these analytical techniques a highly purified phosphorylase preparation is invaluable. Assay Method

P r i n c i p l e . The assay method is based on the determination of h y p o xanthine formed during the phosphorolysis of inosine b y nucleoside phosphorylase. The hypoxanthine is oxidized to uric acid b y xanthine oxidase and m a y be followed b y differential s p e c t r o p h o t o m e t r y as described b y Kalckar. 12 E n z y m e . X a n t h i n e oxidase is prepared according to Ball ~ with the modification of K a l c k a r et al., TM or according to Horecker and Heppel. 1~ One enzyme unit is defined as the a m o u n t of enzyme which causes an increase in optical density of 0.001 per minute at 293 mt~ in a cuvette with a light p a t h of 1 cm. under the following standard conditions:

Phosphate buffer, 0.05 M, pH 7.4 Hypoxanthine, 0.0075 M Enzyme T water to a volume of

2.7 ml. 0.2 ml. 3.0 ml.

Procedure. The assay is done under the following conditions:

Phosphate buffer, 0.05 M, pH 7.4 Inosine, 0.0075 M Xanthine oxidase, seven- to tenfold excess in units as determined using hypoxanthine as substrate Enzyme sample -{- water to

2.7 ml. 0.2 ml. 3.0 ml.

Unit. One unit of nucleoside phosphorylase is defined as the a m o u n t of enzyme which under the above conditions causes an increase in optical density of 0.001 per minute at 293 m~ in the initial rate when read in a cuvette with a light p a t h of 1 cm.

11 M. Friedkin, J. Biol. Chem. 184, 449 (1950). 1~H. M. Kalckar, J. Biol. Chem. 167, 429, 445, 461 (1947). la E. G. Ball, J. Biol. Chem. 128, 51 (1939). 14H. IV[. Kalckar, N. O. Kjeldgaard, and H. Klenow, Biochim. et Biophys. Aeta 5, 575 (1950). 1~B. L. Horecker and L. A. Heppel, J. Biol. Chem. 178, 683 (1949); see Vol. II [73].

450

ENZYMES OF NUCLEIC ACID METABOLISM

[64]

Preparation

Reagents

Acetate buffer, pH 4.0:0.02 mole of Na acetate and 0.1 mole of acetic acid made up to 1000 ml. Acetate buffer, pH 5.2:0.02 mole of Na acetate adjusted to pH 5.2 with acetic acid and made up to 1000 ml. Ethanol-acetate buffer solution, 60 %: 600 ml. of absolute ethanol made up to 1000 ml. with the acetate buffer, pH 5.2. Ethanol-acetate buffer solutions, 12%, 9%, and 6%: 120 ml., 90 ml., and 60 ml. of absolute ethanol, respectively, made up to 1000 ml. with the acetate buffer, pH 5.2. Glycine-acetate buffer: 0.5 mole of glycine and 0.02 mole of Na acetate in 800 ml. of H20, adjusted to pH 5.3 with acetic acid and taken to 1000 ml. Procedure. 2600 g. of calf spleen is homogenized in a Waring blendor with 2.5 vol. of cold distilled water and filtered through gauze; the residue is washed with 0.5 vol. of water. The pH is adjusted to 5.2 with 1300 ml. of the acetate buffer, pH 4.0, and the homogenate is allowed to stand for 1 hour in an ice bath. After centrifugation in a refrigerated centrifuge at 0 °, the active supernatant, $1, is taken to 20% ethanol by addition of 0.5 vol. of the 60% ethanol-acetate buffer solution, which is cooled to - 1 0 ° before addition, and then allowed to stand overnight at - 5 °. In the morning, most of the supernatant, $2, above the precipitate formed is siphoned off and discarded after assaying for phosphorylase. The precipitate, P~, is centrifuged from the remainder of the supernatant and collected in eight 100-ml. plastic tubes. The precipitate of each tube is submitted to fractional extraction 16 as described below. A plastic Potter-Elvehjem homogenizer plunger made to fit the 100-ml. tubes is recommended for suspension of the precipitate at each step. Each tube of P2 is homogenized with 65 ml. of the 12% ethanol-acetate buffer solution, allowed to extract for 30 minutes at - 5 °, and centrifuged, giving $3 and P3. Each tube of Pa is homogenized with 65 ml. of the 9% ethanol-acetate buffer solution and after 30 minutes at 0 ° centrifuged, giving $4 and P4. Each tube of P4 is homogenized with 33 ml. of the 6% ethanol-acerate buffer solution and after 30 minutes at 0 ° centrifuged, giving $5 and Ps. 10E. J. Cohn, F. R. N. Gurd, D. M. Surgenor, B. A. Barnes, R. K. Brown, G. Derouaux, J. M. Gillespie, F. W. Kahnt, W. F. Lever, C. H. Liu, D. Mittelman, R. F. Mouton, K. Sehmid, and E. Uroma, J. Am. Chem. Soc. 72, 465 (1950).

[64]

NUCLEOSIDE

PHOSPHORYLASE

$:x, ¢~ ¢q

~9 O

Z O

° °

~9

Q ¢g

c~ o O

FROM

CALF

SPLEEN

451

452

ENZYMES OF NUCLEIC ACID METABOLISM

[64]

To extract the phosphorylase the precipitate, Ps, in each tube is homogenized with 33 ml. of glycine-acetate buffer, and after 30 minutes at 0 ° centrifuged, giving $6 and Pe. To each 100 ml. of $6 is added 20 g. of ammonium sulfate (182 g./1. final concentration) and after 30 minutes at 0 ° centrifuged, giving $7 and PT. The supernatant, $7, is the active enzyme preparation, and gives 133-fold purification based on the extinction at 280 m~, and about 65-fold purification based on nitrogen. A summary of the fractionation is presented in the table. Notes and Comments on the Preparation

1. The solutions used should always be made up at room temperature and then cooled down to the desired temperature. The hydrogen ion concentration and the temperatures of the buffers given must be strictly followed; a difference of a few degrees of temperature or of 0.1 unit of pH from those stated will considerably alter the fractions obtained. 2. The fractional extractions with ethanol-acetate buffers remove the more soluble contaminating proteins. The phosphorylase is then extracted with glycine-acetate buffer, leaving behind a dense fatty pellet which is nearly immiscible even in 0.02 M phosphate buffer, at pH 5.2, and only slowly goes into solution. From its solubility properties 18 it is thought to be largely fl-lipoprotein. Fractional extraction 1~ permits more rapid equilibration than fractional precipitation, and by keeping the enzyme in a precipitated state while extracting away other proteins denaturation of the phosphorylase is minimized. In fractional extraction the volume of the eluting solution used in a given extraction is very important. If the volume is too small the concentration of protein in the eluate will be high and will cause the phosphorylase to be extracted at the ethanol-acetate buffer steps, resulting in a low yield of enzyme. 3. Phosphate, 0.02 M, at pH 5.2, may be used in place of the glycineacetate buffer to extract the phosphorylase from the lipoprotein precipitate. T h e use of phosphate gives a somewhat higher yield but extracts some lipoprotein as well which requires further ammonium sulfate fractionation for its removal. 4. Only the early steps of the fractionation procedure are described. These provide a simple reproducible method of obtaining a very active preparation. Further fractionation steps (unchecked) are: (1) Precipitate the activity of $7 by adding 20 g. more of ammonium sulfate to each 100 ml. of volume. (2) Centrifuge and wash the pellet twice with the same volume of 30% ethanol in 0.02 M succinate at pH 5.8 and - 5 ° to remove ammonium sulfate. (3) Extract the 30% ethanol pellet twice with

[64]

NUCLEOSIDE PHOSPHORYLASE FROM CALF SPLEEN

453

the same volume of 10 % ethanol in 0.02 M succinate at pH 5.8. (4) Centrifuge and treat the combined active supernatants with 0.1 M Zn acetate in 10 % ethanol-succinate buffer. A large portion of the phosphorylase precipitates at 0.002 M Zn ++ concentration. A smaller fraction precipitating between 0.002 M and 0.010 M Zn ++ is very highly active with an activity about 800 times the original homogenate based on absorption and about 400 times based on nitrogen. Alternative Methods of Preparation. An alternative preparation of purine nucleoside phosphorylase from beef liver has been carried out by Korn in Buchanan's laboratory. 17 An acetone powder extract of the liver is subjected to three successive ethanol precipitations followed by fractionation with ammonium sulfate and silica gel. The purity of the preparation is about 200-fold based on the absorption of the various fractions at 280 mu. Recently this preparation has been used in the synthesis of 4-amino-5-imidazolecarboxamide riboside2 Suggestive evidence was obtained that adenine could also be utilized by their preparation. The preparation of nicotinamide (N +) riboside phosphorylase from beef liver acetone powder has been described by Rowen and Kornberg. 1° The powder was extracted with 0.1 M Na:HP04 and carried through successive steps using ammonium sulfate, calcium phosphate gel, and alumina gel/C~, with 60-fold purification. This preparation was also active in the phosphorolysis of inosine, aud ino.sine markedly inhibited the phosphorolysis of nicotinamide riboside. It was suggested that the same enzyme may be active in the phosphorolysis of both inosine and nicotinamide riboside. Purine nucleoside phosphorylase from yeast has been purified and separated from hydrolase activity by tteppel and Hilmoe, TM using ammonium sulfate and calcium phosphate gel with some 55-fold purification. This enzyme resembles Kalckar's phosphorylase from animal sources with a pH optimum near 7.0 and having activity against inosine, guanosine, and nicotinamide riboside only in the presence of free phosphate or arsenate.

Eaitor's note: For the preparation of pyrimidine nucleoside phosphorylase (thymidine phosphorylase from horse liver), see Vol. III [26]. ~7j. M. Buchanan, in "Phosphorus Metabolism" (McElroy and Glass, eds.), Vol. 2, p. 419, Johns Hopkins Press, Baltimore, 1952. ~8L. A. Heppel and R. J. Hilmoe, J . Biol. Chem. 198, 683 (1952); see Vol. II [66].

454

ENZYMES OF NUCLEIC ACID METABOLISM

[65]

[65] Nicotinamide Riboside Phosphorylase 1 Nicotinamide Riboside + + Phosphate ~ Nicotinamide Ribose-l-phosphate W H +

By ARTHUR KORNBERG Assay Method Principle. Nicotinamide riboside but not nicotinamide yields a fluorescent condensation product with acetone;2 the cleavage of nicotinamide riboside is measured b y this fluorometric method. Reagents

Nicotinamide riboside (NR), 0.003 M. See Vol. I I I [129]. KH2PO4-K2HPO4 buffer, 0.1 M, p H 7.4. Procedure. The incubation mixture (0.50 ml.) contained 0.05 ml. of NR, 0.10 ml. of phosphate buffer, enzyme (0.5 to 0.7 unit), and water. After 10 minutes at 38 °, the reaction was stopped b y a twentyfold dilution with ice-cold water and an aliquot of 0.20 ml. was immediately assayed fluorometrically. 2 Definition of Unit and Specific Activity. One unit of enzyme is defined as t h a t a m o u n t which causes the cleavage of 1 micromole of N R in 1 hour. Specific activity is expressed as units per milligram of protein. Protein was determined b y the m e t h o d of Lowry et al.3

Purification Procedure Ten grams of beef liver acetone powder 4 was extracted with 100 ml. of 0.1 M Na2HPO4 for 10 minutes with gentle shaking. This and subsequent operations were carried out at 2 °. T o 80 ml. of extract (see the table) were added an equal volume of 0.1 M acetate buffer, p H 5.0, and 48 g. of ammonium sulfate. After 10 minutes the precipitate was removed b y centrifugation and 12 g. of ammonium sulfate was added to the supernarant. The resulting precipitate was collected in the centrifuge and dissolved in 13 ml. of water (fraction 2). 1j. W. Rowen and A. Kornberg, J. Biol. Chem. 193, 497 (1951). 2j. W. Huff and W. A. Perlzweig, J. Biol. Chem. 167, 157 (1947); for a description of fluorometric assay, see Vol. III [128]. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193~ 265 (1951). 4 A. Kornberg, J. Biol. Chem. 182, 779 (1950).

[65]

NICOTINAMIDE RIBOSIDE PHOSPHORYLASE

455

This fraction was diluted with water to 45 ml. and treated with 52 ml. of aged calcium phosphate gel 6 (10 mg./ml., dry weight). The gel was collected b y centrifugation and washed once with 65 ml. of water, and the enzyme was eluted twice with 32.5-ml. portions of phosphate buffer (0.005 M, p H 7.5) (fraction 3). The addition to 65 ml. of fraction 3 of 6 ml. of 0.1 M acetate buffer, p H 5.0, reduced the p H to 6.2. Then 14.2 ml. of alumina gel C~ 6 (1.6 m g . / ml., dry weight) was added. The gel was collected in the centrifuge and washed with 71 ml. of water; the enzyme was eluted with two 35.5-ml. portions of 0.02 M phosphate buffer, p H 7.5. This final fraction represents a sixtyfold purification over the crude extract, with a yield of 17 %. Fractions 2 and 3 lose no activity after storage for several weeks at - - 1 0 % SUMMARY OF PURIFICATION PROCEDURE

Fraction 1. 2. 3. 4.

Crude extract Ammonium sulfate Calcium phosphate gel eluate Alumina gel eluate

NR splitting activity, units

Protein, mg./ml,

Specificactivity, units/rag, protein

6912 3159 1890 1140

20.2 21.0 0.17 0. 067

4.3 11.5 162.0 ~ 255.0

a In repeated preparations the specific activity of this fraction varied between 144 and 180 units.

Properties pH Optimum. The p H optimum of the enzyme is approximately 8; the reaction rates at p H 7 and 9 are about 80% of the value at p H 8. The Michaelis constant for N R i s 1.1 × 10-3 M. The constant for phosphate is 2.8 × 10-4 M. Specificity. The purified preparation did not a t t a c k N M N , D P N , T P N , or methylnicotinamide, nor were there any inhibitory effects of these compounds at equimolar concentration on the rate of N R splitting. The purified enzyme fractions are active in the phosphorolysis of inosine, and, in addition, inosine markedly inhibited N R phosphorolysis. Although the question as to whether one enzyme is responsible for both activities has not been settled, available evidence does suggest identity. The ratio of specific activities of N R and inosine splitting for fractions 1, 2, and 3 were 1.3, 1.4, and 1.8, respectively. 5 D. Keilin and E. F. Hartree, Proc. Roy. Soc. (London) B124, 397 (1938). R. Willst~ttter and H. Kraut, Ber. 56, 1117 (1923).

456

ENZYMES OF NUCLEIC ACID METABOLISM

[66]

The Michaelis constant (Ks) for inosine was shown to be 1.3 X 10-~ M, as compared with a value of 1.7 × 10-5 M obtained by Friedkin and KalckarJ The affÉnity of inosine as an inhibitor of N R phosphorolysis was determined at several concentrations of inosine and also at various levels of N R and was found to be approximately 3.5 X 10-5 M. Since the experimental conditions under which the K8 and KI values were determined, were for technical reasons, different, it is difficult to evaluate properly the disparity between them. 7 y[. Friedkin and H. M. Kalckar, J. Biol. Chem. 184, 437 (1950).

[66] H y d r o l y t i c N u c l e o s i d a s e s PuR 1 + H~O ~ Pu 1 + R 1 P y R I + H20--~ pyl + R 1

By T. P. WANG I. LactobaciUus pentosus Assay Method Principle. The products of action of the hydrolytic nucleosidase from Lactobacillus pentosus are the nitrogenous base, purine or pyrimidine, and the free ribose. Demonstration of the appearance of one of these products constitutes the basis of assay of this enzyme. However, in view of the fact that the nitrogenous base is also one of the products of phosphorolytic cleavage of nucleosides, the appearance of free ribose, as determined by the reducing sugar method, would be the more reliable method for assay of hydrolytic nucleosidase.

Reagents a. PuR. Any of the PuR such as AR, GR, HxR, UAR, or X R 1 can be used for this purpose. Tris buffer (0.05 M), pH 7.5. Extract, 1 ml., equivalent to 200 mg. of wet cells. b. PyR. Either CR or UR. 1 Arsenate or phosphate buffer (0.05 M), pH 7.5. Extract. ~The following abbreviations are used in this article: PuR, purine riboside; PyR, pyrimidine riboside; Pu, purine base; Py, pyrimidine base; AR, adenosine; GR, guanosine; H×R, inoglne; UAR, uric acid riboside; XR, xanthosine; CR, cytidine; UR, uridine; A, adenine; G, guanine; Hx, hypoxanthine; UA, uric acid; X, xanthine; C, cytosine; U, uracil; R, ribose; R-l-P, ribose-l-phosphate; and R-5-P, ribose-5phosphate.

[66]

HYDROLYTIC NUCLEOSIDASES

457

Procedure. Six micromoles of P u R (or P y R ) is incubated at 37 ° wit5 3 ml. of a cell-free extract of L. pentosus and 2 ml. of Tris buffer (or arsenate or phosphate buffer in the case of P y R ) . At the end of 2 hours, two 1-ml. samples are taken from the mixture, one deproteinized by an equal volume of 4 % HC104 (ice-cold), and the other b y 1 ml. of Ba(OH)~ (0.3 N) and 1 ml. of 5 % ZnSO4'6H20 2 in the order mentioned. The precipitate is removed b y filtration or centrifugation, and the protein-free filtrates are analyzed for reducing sugar a n d / o r the nitrogenous base. The perchloric acid filtrate should be kept in an ice bath and neutralized immediately with solid K H C Q before any analysis is made. Any K C 1 Q fo~med can be removed by centrifugation. 1. Reducing Sugar. This determination is made with 0.2 to 0.5 ml. of the perchlorie acid filtrate or 0.3 to 0.75 ml. of the Ba-Zn filtrate according to Nelson's colorimetric method. 3 The perchloric acid filtrate would contain free ribose and ribose phosphate, if any is formed, whereas the Ba-Zn filtrate would contain only the free ribose. Since the nucleoside is split hydrolytically by the L. pentosus extract, no phosphate ester of ribose should be formed. The reducing value obtained from the perchloric acid filtrate should thus be the same as that obtained in the Ba-Zn filtrate. The activity of the extract is as follows: for Pyr, 6 micromoles of substrate will be completely cleaved by 0.3 ml. of extract (equivalent to 60 mg. of wet cells) in 2 hours at 37 °. For PuR, 70 to 80 % of the 6 micromoles of nucleoside (in the case of XR, about 40 %) will be split. 2. Nitrogenous Base. (.~) CHROMATOGRAPHIC OR IONOPHORETIC METHOD.4 All the Pu or P y bases can be distinguished from their nucleosides and from each other b y either chromatographic or ionophoretic methods. For instance, when a reaction mixture containing G R is applied to a strip of W h a t m a n No. 1 filter paper which is then developed by the 5 % Na~HPO4-isoamyl alcohol system of Carter, the newly formed G will stay around the origin with an Rs value of 0.02, differing quite distinctively from the remaining G R which has an Rs value of 0.62 in the same system. (B) SPECTROPHOTOMETRICMETHODS. In the case of PuR, the formation of free Pu base can be demonstrated with suitable enzyme or a mixture of enzymes in a Beckman spectrophotometer. See following table:

2 Method of M. Somogyi, described in footnote 3. N. Nelson, J. Biol. Chem. 153, 375 (1944). 4Paper chromatography: R. J. Block, E. L. Durrum, and G. Zweig, "A Manual of Paper Chromatography and Paper Eleetrophoresis," Chapter 9, Academic Press, New York, 1955; Ionophoresis: W. C. Werkheiser and R. J. Winzler, J. Biol. Chem. 204, 971 (1953).

458

ENZYMES OF NUCLEIC ACID METABOLISM

Base Adenine Guanine Hypoxanthine Xanthine Uric acid

[66]

Change in millimolar extinction for complete reaction

Enzyme X a n t h i n e oxidase Guanase a n d x a n t h i n e oxidase X a n t h i n e oxidase X a n t h i n e oxidase Uricase

+15.53 + 7.25 +10.88 +10.03 - 12.17

at at at at at

305 290 290 290 290

mt~ 5 mt~ ~ m~ 6 m~ ~ m~ 6

Since the substrate specificity of xanthine oxidase is rather broad, it is essential to use other independent methods to determine the exact nature of the base formed if such a determination is necessary. In addition to the chromatographic methods mentioned in the previous section, such procedure as taking a general spectrum in the ultraviolet region of the reaction mixture is frequently used, since all the Pu bases have their characteristic absorption spectra and can be distinguished from each other by such. For instance, in a reaction mixture containing AR, if the solution gives a maximum absorption at 260 m~ in addition to a positive reaction with xanthine oxidase, it can be assumed that one of the products is A instead of Hx. The Py bases differ from their nucleosides by having higher absorption at 300 m~ in alkaline solution. The following values are calculated from the absorption data given by Hotchkiss. 7 MILLIMOLAR EXTINCTION CHANGES AT 300 m ~ WHEN THE REACTION SOLUTION I s CHANGED FROM NEUTRAL TO ALKALINE

C +0.81

CR -0.28

U + i . 93

UR Almost no change 8

In addition, the appearance of free Py bases can also be demonstrated by enzymatic methods. Base Cytosine Uracil

Enzyme

Assay M e t h o d

Cytosine deaminase Uracil oxidase

After a decrease in absorption a t 280 m ~ 9 Measuring oxygen u p t a k e manometrically 1°

H. Klenow, Biochem. J. 50, 404 (1952). H. M. Kalekar, J. Biol. Chem. 167, 429 (1947). R. D. Hotchkiss, J. Biol. Chem. 175, 315 (1948). 8 j . M. Ploeser and H. S. Loring, J. Biol. Chem. 178, 431 (1949). 9 A decrease in absorption of 58% at 280 m~ will be observed when cytosine is deamiHated to uracil, according to the d a t a of Hotchkiss. ~ lo T. P. Wang a n d J. O. Lampen, J. Biol. Chem. 194, 785 (19527.

[66]

HYDROLYTIC NUCLEOSIDASES

459

Preparation of Enzyme Preparation of L. pentosus Cells. L. pentosus 124-2 is the source of the hydrolytic nucleosidase. Stock of this organism is kept in agar stabs containing 5 g. of Difco yeast extract, 5 g. of Difco peptone, 10 g. of glucose, 10 g. of NaAc, 0.5 g. of KH~P04, 0.2 g. of MgSO4.7H20, 0.01 g. each of FeSO4"7H20, MnSO4.4H~O, and NaC1, and 20 g. of Difco agar per liter of distilled water. Stabs are stored at 5 ° and transferred monthly. Liquid medium of the same composition as listed above, with the omission of agar, is used for preparing resting cells. The inoculum is made by transferring cells from the stab to 10 ml. of the liquid medium. After incubation at 37 ° for 20 hours, the inoculum is added to a liter of medium and the culture is again incubated at 37 ° for 20 hours. The cells are then harvested by centrifugation at 4500 r.p.m, for 15 minutes and washed once with 1% KC1. The yield of wet packed cells is around 5 g./1. of medium. Preparation of Cell-Free Extracts. Cell-free extracts are prepared by grinding the wet cells with either powdered glass 11 or alumina A-303 12 or by breaking the cells with sonic oscillations. With the alumina grinding method, the resultant paste is extracted with 5 ml. of water per gram of initial wet cells. In the case of sonic disintegration, the cells are suspended to a concentration of 1 g. per 5 ml. of water and disrupted for 30 minutes in a magnetostriction sonic oscillator (type R-22-3, Raytheon Manufacturing Company). The insoluble residue is removed by centrifugation at 20,000 r.p.m, for 20 minutes. All operations involved in the preparation of cell-free extracts are made at a temperature between 0 and 5 °. The clear extracts are stored around - 2 0 ° in a deep-freeze. Properties

Specificity. Regarding substrate specificity, the hydrolytic nucleosidase(s) from L. pentosus differs from the phosphorolytic nucleosidase in (1) its ability to act on AR, CR, UAR, and XR in addition to GR, HxR, and UR, and (2) its inability to act on deoxyribosides of C, Hx, and U. Thymidine also is not attacked. UAR, a riboside found in beef erythrocytes, is split by the L. pentosus nucleosidase, whereas thymine riboside, a synthetic compound, is not cleaved. Stability. The rate of splitting PyR by the extract is faster in phosphate, arsenate, sulfate, and succinate than in Tris, KC1, or KNO3. This apparent stimulation of phosphate, arsenate, etc., is due to the fact that these anions have the ability to stabilize the pyrimidine nucleosidase. On the other hand, the monovalent anions do not possess this ability. Thus, ~1G. Kalnitsky, M. F. Utter, and C. H. Werkman, J. Bacteriol. 4.9, 595 (1945). ~2H. McIlwain, J. Gen. Microbiol. 2, 288 (1948).

460

ENZYMES

OF NUCLEIC

ACID METABOLISM

[66]

the activity of the enzyme is retained if a 10-minute incubation in arsenate precedes addition of the P y R substrate. Almost complete loss of activity results if the enzyme is preincubated with Tris under similar conditions. The possibility of inhibition by the Tris is unlikely in view of the fact that the enzyme is fully active if it is incubated in a mixture of arsenate and Tris buffers. Contrary to the behavior of the pyrimidine nucleosidase, the purine nucleosidase is stabilized by the monovalent anions and rapidly loses its activity in arsenate or phosphate buffers. The inactivation of purine nucleosidase in the presence of phosphate ion cannot be prevented by the addition of bovine serum, cysteine, Versene at pH 7.0, pyrophosphate at pH 8.0, or a boiled extract of an active nucleosidase preparation. The striking difference between the purine and the pyrimidine nucleosidases toward inorganic anions suggests that two types of enzyme are present in the L. pentosus extract, one specific for PuR and the other for PyR. Attempts toward purification of these enzymes have been made but with little success. The crude extract is rather stable. No significant loss of activity occurs when the extract is stored at - 20 ° for a year. Repeated freezing and thawing also do not appreciably affect the activity. Mechanism of Action. Because of the wide distribution of the phosphorolytic nucleosidase in animal tissues and in microorganisms, the existance of a hydrolytic enzyme is, in a way, not expected. Efforts therefore have been made to study the actual mechanism of action of L. pentosus nucleosidase. Evidence for a hydrolytic cleavage is briefly presented as follows. 1. As mentioned in the assay section, the reducing sugar value obtained from either the perchloric acid filtrate or the Ba-Zn filtrate is the same. This indicates that no phosphate ester is formed or at least that it does not accumulate. 2. Neither R-1-P nor R-5-P is dephosphorylated by the extract. Addition of a phosphatase inhibitor, such as NaF (final concentration, 0.1 M) to the reaction mixture does not affect the reducing sugar value obtained in the perchloric acid filtrate. Therefore, the lack of accumulation of phosphate ester in the reaction mixture cannot be a result of the presence of phosphatase in the extract. 3. The extract does not catalyze the arsenolysis of R-1-P. If R-1-P is the product, as in the case of phosphorolytic nucleosidase, R-1-P should remain as such. However, all efforts indicate that free ribose instead of R-1-P is the product found in the reaction mixture. 4. R-5-P is degraded only slowly in extracts of cells grown on glucose but is metabolized rapidly in extracts prepared from cells grown on xy-

[66]

HYDROLYTIC NUCLEOSIDASES

461

lose. 13 Free ribose is not degraded b y either type of extract. When U R or H x R is incubated with the extracts from cells grown on xylose, quantitative recovery of free ribose is obtained at the end of incubation. Thus, free ribose rather than the phosphate ester of ribose must be the p. ~uct of action of L. pentosus nucleosidase. 5. When a reaction mixture containing R-1-P is deproteinized by the cold perchloric acid method, no significant a m o u n t of reducing sugar can be demonstrated in the protein-free filtrate. The recovery of free ribose from nucleoside cleavage in perchloric acid filtrate, therefore, cannot be due to a secondary splitting of the sugar phosphate b y the acidic deproteinizing agent.

II. Yeast A. Uridine Nucleosidase 14

URWH~O--*U~-R

Assay Method Principle. Two methods, both based on the differential spectrophot o m e t r y of U R and U, are employed. The first method consists in measuring the absorption increase at 290 m~ in alkaline solution (0.01 N N a O H ) when U R (Era = 30) 1~ is split to U (Era = 5.4 X 103). The second m e t h o d measures the decrease of absorption at 280 m~ and p H 7.0, since U R has an Em of 3.5 X 103, and U has an Em of 1.4 X 103 at these conditions. Reagents

UR, 20 mg./ml. Buffer (phosphate, borate, glycine, or Veronal), 0.1 M, p H 7.0. Enzyme. Procedure. M e t h o d 1. In the first method, 0.025 ml. of U R is incubated with 0.1 ml. of enzyme and 0.2 ml. of buffer at 38 ° for 30 minutes. F o u r milliliters of 0.01 N N a O H is then added, and the solution is read at 290 m~ in the Beckman spectrophotometer. The increment in absorption at this wavelength read against a zero time blank is a measure of the U formed. This method, rapid and sensitive, is employed during purification of the enzyme. M e t h o d 2. Incubation is conducted in a 3-ml. cuvette at 26 °. The incubation mixture consists of 0.05 to 0.2 ml. of enzyme, 0.01 to 0.03 ml. of UR, and 3 ml. of buffer. Readings at 280 m~ are t a k e n at various time intervals to follow the cleavage of U R to U. This method is used for kinetic study of enzyme activity. i~ j. O. Lampen and H. R. Peterjohn, J. Bacteriol. 62, 281 (1951). 14C. E. Carter, J. Am. Chem. Soc. 73, 1508 (1951). 15E~ = molar extinction.

462

ENZYMES OF NUCLEIC ACID METABOLISM

[66]

Purification Procedure

Steps 2 to 4 are conducted at 4 to 10 °. Step 1. Three pounds of Fleischmann baker's yeast is plasmolyzed in toluene according to Kunitz. 16 Step 2. To the clear plasmolyzate, solid (NH4)2SO4 is added to the concentration of 445 g./1. After standing for 1 hour, the precipitate is collected by centrifugation, dissolved in distilled water, and dialyzed against distilled water. Step 3. To 350 ml. of this solution, 385 ml. of saturated (NH4)2SO4 is added. After standing for 1 hour, the precipitate is centrifuged, dissolved, and dialyzed as above. The dialysis runs for 48 hours with frequent change of water. Step 4. The fraction is adjusted to pH 4.7, and the precipitate is discarded. The supernatant solution, adjusted to pH 7.0 with dilute NaOH, contains about 60 % of the original activity and represents a purification of ten- to fifteenfold. Properties

Specificity. The purified enzyme is specific for UR and has no activity on AR, GR, HxR, CR, or TDR. 17 Uridylic acid also is not attacked. Kinetics. When 227 ~, of U R is incubated with 0.2 ml. of enzyme (containing 400 ~/of protein) and 3 ml. of 0.1 M buffer, pH 7, at 26 °, the reaction follows first-order kinetics up to 83 % of hydrolysis of the substrafe. Addition of 200 ~ of U to the reaction mixture produces 27 % inhibition, whereas addition of 3000 ~ of R produces only 30 % inhibition. pH Optimum. There is a well-defined optimum at pH 7.0 in either phosphate, glycine, or Veronal buffers. B. Purine Nucleosidase P u R + H 2 0 - ~ Pu + R Assay Method

Principle. Spectrophotometric methods are generally used. Reducing sugar determinations are also made on occasion. Reagents and Procedures. See hydrolytic nucleosidase from L. pentosus section. Purification Procedure 18

Step 1. Fresh baker's yeast (Fleischmann) is rapidly dried in a thin layer (1/~ inch) at 22 to 25 ° overnight with the aid of a fan. Slow drying 16 M. S. Kunitz, J. Gen. Physiol. 29, 393 (1947). 17 T O R - T h y m i d i n e deoxyriboside or thymidine. 18 L. A. Heppel and R. J. Hilmoe, J. Biol. Chem. 198, 683 (1952).

[66]

HYDROLYTIC NUCLEOSIDASES

463

destroys the hydrolytic nucleosidase. Autolyzates are prepared by mixing 500-g. portions of yeast with 1500 ml. of 0.2 M acetate buffer, pH 5.1, incubating for 6 hours at 37 °, and centrifuging. Step 2. 140 ml. of autolyzate is mixed with 27.2 g. of (NH4)~SO4, and the pH is adjusted to 4.6 with 17.2 ml. of 2 M acetic acid. Another 13.3 g. of (NH4)2SO4 is then added (0.45 saturation). After 15 minutes, the mixture is centrifuged for 8 minutes at 13,000 X g. The supernatant (164 ml.) is brought to 0.55 saturation by the addition of 9.7 g. of (NH4)2SO4. The precipitate is collected by centrifugation dissolved in 0.1 M acetate buffer, pH 6.0, and dialyzed for 6 hours against flowing 0.01 M acetate buffer, pH 6.0. This fraction is then centrifuged to remove any precipitate formed during dialysis. Step 3. To 25 ml. of the clear solution, adjusted to pH 7.3 with 1 ml. of 0.2 M NH4OH, is added 10.1 g. of (NH4)2SO4 (0.6 saturation). The precipitate is removed by centrifugation, and the supernatant (27.5 ml.) is brought to 0.7 saturation with 1.7 g. of (NH4)~SO4. The second precipitate is collected by centrifugation and dissolved in 0.02 M NaAc to give a volume of 15.5 ml. Step 3- A 1-ml. portion of the last fraction is treated with 2.5 ml. of distilled water and 0.2 ml. of aged calcium phosphate gel 19 (10.2 mg. of solid per milliliter). After the gel is removed by centrifugation, the supernatant shows a 55-fold purification compared with the original autolyzate. The over-all yield is 21 to 22%. The purity of the enzyme can be doubled by adsorbing the enzyme on calcium phosphate gel and eluting successfully with water M/600 and M/300 phosphate buffers, pH 7.4. But the yield for this step is low (14%) and considered "not useful" by the investigators of this method. Is Steps 2 to 4 are carried out at 2 °. The activity of the enzyme is expressed in a unit which represents the amount of enzyme causing the cleavage of 1 micromole of substrate per hour.

Properties Specificity. The enzyme splits AR, GR, HxR, XR, and a number of synthetic PuR. In addition, nicotinamide riboside is cleaved to nicotinamide ribose. CR, UR, and uridylic acid are not attacked. The hydrolysis of HxR is competitively inhibited by AR and GR. The same enzyme for all these three ribosides is suggested. Other Properties. Phosphate and arsenate are not required. Phosphate buffers (0.02 M) with a pH value below 6.7 have no effect on the rate; ~9For preparation of calcium phosphate gel, see Vol. I [11].

464

ENZYMES OF NUCLEIC ACID METABOLISM

[67]

more alkaline phosphate buffers are inhibitory when compared with glycine and glycylglycine buffers. The purified enzyme is relatively unstable, b u t the (NH4)~S04 fractions can be stored for several m o n t h s at - 1 0 °. T h e p H of the optimal activity of enzyme is around 7 to 8. With C14-adenine, it has been demonstrated t h a t the hydrolytic nucleosidase from yeast does not catalyze either the synthesis of nucleoside from free ribose and a purine base or the exchange reaction between a nucleoside and a purine base. PURIFICATION OF YEAST PURINE, NUCLEOSIDASEHYDROLITE

Step 1. 2. 3. 4.

Autolyzate (NH4)2SO4fraction (NH4)2SO4fraction Calcium phosphate gel supernatant

[67] N u c l e o s i d e

Volume, ml.

Total units

140 27.5 15.5

2800 1730 700

54.5

595

Over-all yield, % 62 25

1.7 16.2 31

21.2

94

Transdeoxyribosidase

+R t

,.

Specific activity against HxR, units/mg, protein

from Bacteria

~

+R

OH OH R and R r represent certain of the naturally occurring purines and pyrimidines.

By WALTER S. MCNUTT Assay Method

Principle. The m e t h o d employed consists in the quantitative estimation of deoxyribosides b y their specific growth-promoting effect upon the microorganism, Thermobacterium acidophilus R26. This bacterium responds equally in growth to equimolecular amounts of the deoxyribosides of adenine, guanine, hypoxanthine, thymine, uracil, and cytosine. T h e microbiological process, described b y Hoff-JCrgensen, 1 is very sensitive, estimating 1 ~, of deoxyriboside with an error of about 10%. T h e micro1 E. Hoff-JCrgensen, Biochem. J. 50, 400 (1951); see Vol. III [110].

[67]

NUCLEOSIDE TRANSDEOXYRIBOSIDASE FROM BACTERIA

465

biological analysis is more reproducible than are most microbiological procedures. To separate the individual deoxyribosides in a mixture, one-dimensional, ascending chromatography on paper may be carried out in glass jars 11 X 48 cm. The compounds are placed along Whatman No. 4 filter paper in quantities of 10 to 20 ~/of purine or pyrimidine per centimeter. The solvent (25 to 30 ml.) is added to the dry vessel; the paper is suspended above the solvent in the closed system, and the system is equilibrated at room temperature. The distance transversed by the solvent is 45 cm. No special precautions were taken to ensure a constant temperature (usually about 23°), and Rf values varied somewhat on independent runs. The following systems were employed: (1) n-Butanol-water-ammonia: 1 ml. of 15 N NH40H was added to 100 ml. of n-butanol saturated with water. After thorough shaking the clear supernatant was used. Equilibrated for 11 hours. Time to traverse 45 cm., 1.5 to 2 days. (2) n-Butanolwater-acetic acid: 25 ml. of n-butanol saturated with water -4- 5 ml. of glacial acetic acid. Equilibrated for 8 hours. Time to traverse 45 cm., 1.5 to 2 days. (3) n-Butanol-water-ethyl acetate-morpholine: 5 ml. of n-butanol saturated with water A- 5 ml. of ethyl acetate saturated with water -4- 10 ml. of morpholine. Equilibrated for 24 hours. Time to traverse 45 cm., 2 to 2.5 days. (4) n-Butanol-water-morpholine-methylglycol: 10 ml. of n-butanol saturated with water -4- 10 ml. of morpholine + 5 ml. of methylglycol -4- 2 ml. of water. Equilibrated for 24 hours. Time to traverse 45 cm., 3 to 3.5 days. The Rf values of the deoxyribosides and certain related compounds in the several systems are shown in Table I. The purines, pyrimidines, and their deoxyribosides, if present in sufficient quantity (about 5 ~, of purine or pyrimidine per 1-cm. spot), show up as dark areas on the dry chromatogram when examined beneath ultraviolet light (h = 247 mg). The appropriate areas are cut from the sheet and eluted with warm water, the filtered solution being assayed microbiologically. The difference in microbiological activity between the corresponding areas in the control and in the experimental samples may be used to calculate the amount of a given deoxyriboside which has formed or disappeared. Procedure. (Example: Uracil deoxyribosido -t- adenine ~ adenine deoxyriboside -4- uracil.) Uracil deoxyriboside (0.44 gM. in 10 gl.), adenine (500 3'), 0.2 M phosphate buffer (500 gl., pH 8.04), and Lactobacillus helvetieus enzyme (2.0 mg. of dry bacterial substance, dialyzed for 3 days, 500 pl.) were incubated under toluene for 9 hours at 37 °. Ethanol (7ml., 97 %) was added to stop the action of the enzyme. The control tube differed from the experimental in having the uracil deoxyriboside added

466

[67]

ENZYMES OF NUCLEIC ACID METABOLISM

a f t e r t h e a d d i t i o n of t h e e t h a n o l . T h e v o l u m e s w e r e r e d u c e d t o a b o u t 500 ~l. a t 55 °, a n d t h e walls of t h e t u b e w e r e w a s h e d w i t h t h e liquid. T h e p r o t e i n was r e m o v e d b y p r e c i p i t a t i o n t h r o u g h t h e a d d i t i o n of 7 ml. of e t h a n o l . T h e s u p e r n a t a n t w a s c o l l e c t e d , a n d t h e p r e c i p i t a t e was w a s h e d t w i c e m o r e w i t h 2 ml. e a c h t i m e of w a r m 9 5 % e t h a n o l . T h e c o m b i n e d s u p e r n a t a n t w a s r e d u c e d t o d r y n e s s a t 55 ° a n d t a k e n up in 500 ~l. of w a t e r , 100 ~l. of t h i s s o l u t i o n b e i n g p l a c e d a l o n g a 4-cm. d i s t a n c e on t h e p a p e r c h r o m a t o g r a m . T h e c h r o m a t o g r a m w a s d e v e l o p e d in t h e n - b u t a n o l w a t e r - a m m o n i a s y s t e m . S p o t s of u r a c i l d e o x y r i b o s i d e , a d e n i n e d e o x y r i boside, a n d h y p o x a n t h i n e d e o x y r i b o s i d e w e r e p l a c e d a l o n g s i d e t h e s a m ples ( c o n t r o l a n d e x p e r i m e n t a l ) . A s s a y of t h e e l u t e d a r e a s s h o w e d t h a t 0.37 ~IV[. of a d e n i n e d e o x y r i b o s i d e ( R / = 0.40) w as f o r m e d ; 0.45 ~ M . of u r a c i l d e o x y r i b o s i d e ( R / = 0.34) d i s a p p e a r e d ; a n d less t h a n 0.01 ~ M . of h y p o x a n t h i n e d e o x y r i b o s i d e ( R / = 0.15) w a s f o r m e d . TABLE I R$ VALUES OF DEOXYRIBOSIDES AND RELATED COMPOUNDS

Rf values in system

Compound Adenine Guanine Hypoxanthine Cytosine Uracil Thymine 4 (5) -Amino-5 (4) -imidazole carboxamide Adenine deoxyriboside Guanine deoxyriboside Hypoxanthine deoxyriboside Cytosine deoxyriboside Uracil deoxyriboside Thymine deoxyriboside Cytidine 5-Methylcytosine Inosine Uric acid

n-Butanolwaterammonia 0.40 0.15 . 0.19 0.28 0.33 0.50 0.32 0.41 0.18 0.17 0.26 0.34 0.48 0.15 0.36 O. 08 0.02

n-Butanolwateracetic acid --

n-Butanol- n-Butanolwater-ethyl wateracetatemorpholinemorpholine methyl glycol 0.45

-----

0.48 . 0.36 ----

----0.35 0.44 0.56 -----

0.44 0.70 0.57 0.54 0.70 0.88 0.94 -----

-0.66 0.58 0.55 0.68 0.77 0.85 -----

.

.

-----

It has been pointed out by R. Markham and J. D. Smith [Biochem. J. 45, 294 (1949)] that the capacity of the system for guanine is very limited.

[67]

NUCLEOSIDE TRANSDEOXYRIBOSIDASE

FROM BACTERIA

467

Preparation of the Enzyme Enzymes capable of catalyzing the above reaction have been prepared from Lactobacillus helveticus S, Lactobacillus delbriickii ATCC 9649, and Thermobacterium acidophilus R26. All these organisms respond to one or more of the deoxyribosides as growth factors. The cells from which the TABLE II COMPOSITION OF THE MEDIA USED IN THE CULTIVATION OF Lactobacillus helveticus S AND Thermobacterium acidophilus R26

Component Glucose H C l - h y d r o l y z e d casein E n z y m a t i c a l l y digested casein Sodium acetate S o d i u m citrate, 5 H 2 0 KH:PO4 K2HPO4 NaC1 MgSO4-7H~O MnSO4.4H20 FeSO4.7H20 L-Asparagine L-Cystine DL-Tryptophan Dl~-Cysteine T w e e n 80 Adenine Guanine Uracil Thymine Cytidylic acid Oleic acid Riboflavin Calcium p a n t o t h e n a t e Niacin Nicotinic acid p - A m i n o b e n z o i c acid T h i a m i n e chloride.HC1 Pyridoxal.HC1 Pyridoxamine.2HC1 P t e r o y l g l u t a m i c acid Biotin

Lactobacillus helveticus S,

Thermobacterium acidophilus R26,

g./1.

g./1.

10 5 0.6 10 10 3 3 5 2.8 0.56 0.14 0.1 0.2 0.1 -1 mg./1, 10 10 10 --10 0.4 0.4 0.4 -0.2 0.2 0.2 0.2 0.01 0. 002

15 12.5 2.5 15 -1 1 -0.1 0.02 0.01 --0.1 0.25 0.5 mg./1, 10 10 -10 25 -0.5 0.5 -0.5 0.5 ---0. 025 --

468

ENZYMES OF NUCLEIC ACID METABOLISM

[67]

enzymes were prepared were obtained from growth in so-called" synthetic media" supplemented with thymidine. 2 The enzyme from Lactobacillus helveticus S is prepared by growing the organism in 250-ml. lots of complete basal medium, 3 supplemented with thymidine at a level of 1 mg. per 250 ml. (see Table II for composition of the medium). After incubation for 24 hours at 37 ° the cells from 500-ml. portions of medium are collected by centrifugation and washed twice with 75 ml. of 0.05 M citrate buffer at pH 6.0. The washed cells are suspended in 15 ml. of the same buffer and.are broken up by agitation with glass beads for 30 minutes in the Mickle electric shaker. During this operation the cell suspension is kept cool by intermittent cooling in ice water. The turbid solution is transferred to a dialyzing membrane and dialyzed against distilled water at 3 ° for several days. The insoluble material which settles out is removed by centrifugation at 3000 r.p.m., and the slightly turbid supernatant solution is used. No further purification of the enzyme is required in order to obtain essentially uncomplicated results. The enzyme preparation does, however, split the deoxyribosides to a slight extent (17 to 20%).

Properties These bacterial extracts retain their transdeoxyribosidase activity over a period of a month or more when preserved in the frozen state. The enzyme functions over a wide range of pH values (5 to 9). Specificity. Extracts from Lactobacillus helveticus S, prepared as described above, catalyze the exchange with the following purine acceptors: adenine, guanine, hypoxanthine, and xanthine. The pyrimidines, thymine, uracil, cytosine, and 5-methylcytosine act as acceptors in this system as does, also, 4-amino-5-imidazole carboxamide, although only a low yield of microbiologically active compound is obtained. Uric acid, 2,6diaminopurine, and dihydrothymine are inactive. The transfer of the deoxyribosyl group from one purine or pyrimidine to another does not proceed through deoxyribose-l-phosphate or through hydrolysis followed by resynthesis, since the enzyme preparation is incapable of catalyzing the synthesis of deoxyribosides when incubated with deoxyribose-1phosphate or deoxyribose in the presence of an appropriate purine or pyrimidine. 2 W. S. M c N u t t , Biochem. J. 50, 384 (1951). 3 W. S. M c N u t t a n d E. E. Snell, J. Biol. Chem. 182, 557 (1950).

[68]

5-ADENYLIC ACID DEAMINASE FROM MUSCLE

469

[68] 5'-Adenylic Acid Deaminase from Muscle 5-AMP -t- H20--, 5-IMP W N H ,

By GORDON NIKIFORUK and SIDNEY P. COLOWICK

Assay Method Principle. The method originally used by Schmidt 1 was based on the measurement of ammonia liberation. The method described below was developed by Kalckar 2 and is based on the fact that deamination of adenine compounds is accompanied by a shift in the ultraviolet absorption spectrum. At 265 m~ the molecular extinction of 5-IMP is only 40 % of that of 5-AMP. Reagents 5'-AMP stock solution (0.04 M). Dissolve 139 mg. of the free acid 8 in 10 ml. of 0.08 M NaHC03. This solution may be stored at - 1 5 ° for many months without change. 0.01 M citric acid--NaOH buffer, pH 6.5. 0.01 M succinic acid--NaOH buffer, pH 5.9. Enzyme. Dilute the stock enzyme with succinate or citrate buffer to obtain 200 to 1000 units of enzyme per milliliter. (See definition below.)

Procedure. Dilute the stock solution of 5'-AMP 1 : 1000 in the succinate buffer, and place 3 ml. of the resulting solution in a quartz cell having a 1-cm. light path. Take readings at 265 m~ before and at 30-second intervals after mixing with 0.1 ml. of enzyme. Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which causes an initial rate of change in optical density (AE265) of 0.001 per minute under the above conditions. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al. 4 The values reported in the table are for activities determined in succinate buffer, pH 5.9. The activity in citrate buffer, pH 6.5, is about three times as high. (See section on "Properties. ") 1 G. Schmidt, Z. physiol. Chem. 179, 2~3 (1928); 208, 185 (1932); 219, 191 (1933). 2 H. M. Kalckar, J. Biol. Chem. 167, 429, 461 (1947). 3 Crystalline 5-adenylic acid is obtainable commercially from various sources. For methods of isolation and purification of this compound, see Vol. I I I [119]. 4 0. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951); see Vol. I I I [73].

470

ENZYMES OF NUCLEIC ACID METABOLISM

[68]

Application of Assay Method to Crude Tissue Preparations. With extracts or homogenates of most tissues, the a m o u n t of material required is such t h a t the light absorption b y the enzyme preparation itself is too high to p e r m i t direct spectrophotometric measurements. F o r such preparations, it is recommended t h a t a high concentration of substrate (1 : 10 dilution of stock A M P ) be used and t h a t the reaction mixtures be fixed at appropriate intervals b y addition of an equal volume of 10 % perchloric acid. The filtrates, after appropriate dilution, m a y be analyzed either spectrophotometrically at 265 mu, or b y ammonia determination. Certain precautions are necessary in the interpretation of results with crude tissue preparations. The presence of phosphatase plus adenosine deaminase m a y readily simulate 5-adenylie deaminase activity. Purification Procedure Steps 1 and 2 of the following procedure are based largely on the m e t h o d originally described b y Schmidt. ~ Step 3 is adapted from the m e t h o d of Kalckar, ~ and step 4 is based in principle on the reports of Tiselius 5 and Mitchell et al. ~ The procedure through step 3 has been carried out successfully m a n y times in several laboratories. Step 4 has not been explored as thoroughly and is not recommended as a routine procedure. Step 1. Preparalion of Crude Extract. Rabbit muscle (from hind limbs and back) is chilled, passed through a meat grinder, and washed four times b y occasional stirring with 4 vol. of cold 0.85% NaC1 for 20 minutes and squeezing through a cheesecloth3 The deaminase is extracted from the colorless residue b y occasional stirring with 1 vol. of cold 2 % NaHCO3 for 1 hour and filtering through a folded W h a t m a n No. 12 filter paper. Longer periods of extraction with N a H C 0 3 lead to a loss of activity. Step 2. Adsorption of Deaminase by Alumina. a Alumina C~ (dry weight 0.025 g./ml.) is added slowly to fraction 1 with continuous stirring. F i f t y milliliters of alumina per liter of fraction 1 is usually sufficient for complete adsorption, b u t occasionally as much as five times this q u a n t i t y is required. T h e suspension is k e p t at 8 ° for 15 minutes, with occasional stirring. I t is then centrifuged in the cold, the supernatant fluid dis5 A. Tiselius, Arkiv Kemi, Mineral. Geol. 26B, No. 1 (1948-49). e H. K. Mitchell, M. Gordon, and F. A. Haskins, J. Biol. Chem. 180, 1071 (1949). 7The ground muscle may be extracted once with 1 vol. of 0.03 N KOH prior to the four washings with 0.85 % NaCl. This step does not decrease the yield of deaminase and has the advantage that the KOH extract may be used as starting material for the preparation of other muscle enzymes. See Vol. I [39, 60]. 8 For the preparation of alumina C~, see Vol. I [11].

[68]

5 - A D E N Y L I C ACID DEAMINASE FROM MUSCLE

471

carded, and the deaminase eluted at 25 to 30 ° with 1.0 M Na2HP04, 9 using 50 ml./1, of fraction 1. The eluate is stored in the cold to permit crystallization of the bulk of the Na~HP04, which is discarded. Step 3. Fractionation with Ammoniacal Ammonium Sulfate. A solution of ammonium sulfate, saturated at 0 ° and adjusted to p H 7.6 with 0.01 vol. of 18% ammonia, is added slowly to the eluate at 0 ° to give 0.27 saturation. After 10 minutes the precipitate is removed b y centrifugation in the cold at 15,000 r.p.m. The supernatant fluid is brought to 0.45 saturation, and, after centrifugation, the 0.27 to 0.45 fraction is dissolved in the least possible volume of 0.1 M Na2HPO4 (approximately 0.5 ml./1, of fraction 1). This preparation can be kept for at least two months at 4 ° without loss of activity. Step ~. Separation on Filter Paper. W h a t m a n No. 1 filter paper (50 X 40 cm.) is rolled into a cylinder, and the edges are stapled together. The enzyme solution (fraction 3) is deposited on the paper around the cylinder at a distance of 2 inches from the bottom. The paper is placed in a glass vessel containing 0.4 saturated ammonium sulfate at p H 7.6 (depth of solution about 1 inch). I t is i m p o r t a n t to perform this procedure rapidly, as even the slightest drying imparts lyophobic properties to the area containing the deposited enzyme. About 7 to 9 hours is required for ascension of the solvent. The cylinder is then unfolded and cut horizontally into four equal strips. These are extracted with 2 % N a H C Q , using ten times the volume of fraction 3 originally deposited. The extracts, although varying in total activity, are combined, since each shows higher specific activity than t h a t in fraction 3. The purification achieved here appears to be due to selective elution of deaminase from the paper by NaHCO3. T h e resulting material is unstable and cannot be further purified b y repetition of steps 2, 3, or 4. SUMMARY OF PURIFICATION PROCEDURE a

Fraction

Total Total Specific volume, Units/ml., units, Protein, activity, Recovery, ml. thousands thousands mg./ml, units/mg. %

1. NattCO3 extract 5300 2. Eluate from alumina 200 3. (NH4)~SO4fraction, 0.27-0.45 2.3 4. Eluate from paper 14

0.38

2000

1.46

260

--

7.6

1520

9.2

820

76

60.0 3.0

200 42

22.0 0.30

2,700 10,000

10 2.1

G. Nikiforuk and S. P. Colowiek, J. Biol. Chem. in press. g As reported earlier by Schmidt, 1lower phosphate concentrations (0.1 M) are in effective in eluting deaminase and, when followed by elution at higher phosphate concentrations, result in a low recovery of enzyme.

472

ENZYMES OF NUCLEIC ACID METABOLISM

[68]

The table summarizes the results of the purification procedure. The yields were in most cases better than those reported here.

Properties Specificity. The purified enzyme is absolutely specific for 5'-AMP, having no action on adenine, adenosine, 2'-AMP, 3'-AMP, ADP, ATP, DPN, or TPN. Activators and Inhibitors. The activity of the enzyme, when tested in succinate or malonate buffer at pH 5.9, can be increased by addition of certain anions, including citrate, chloride, acetate, and lactate. The concentrations of citrate and chloride required to double the rate are 8 )< 10-4 M and 3 X 10-2 M, respectively, but the maximum degree of stimulation by either citrate or chloride is about 2.5-fold. A system maximally stimulated by either anion is not further activated by addition of the other. The nature of the cation is immaterial in these experiments; the chlorides of magnesium, sodium, and potassium show practically identical stimulatory effects, whereas the sulfates of these three cations are uniformly without effect, even at concentrations as high as 0.1 M. Certain anions, including fluoride, phosphate, and pyrophosphate, are strongly inhibitory. The inhibition by fluoride is increased by raising the substrate concentration, whereas the inhibition by phosphate is decreased at higher substrate concentration. Thus, at low substrate concentration (4 X 10-~ M AMP) the concentrations of fluoride and phosphate required to produce 50% inhibition are 5 × 10-3 M and 3 )< 10-3 M, respectively; at high substrate concentration (4)< 10-3 M AMP), the concentrations required for 50% inhibition are 0.2 X 10-3 M and 8 X 10-3 M, respectively. The per cent inhibition by fluoride or phosphate is approximately the same in the absence or presence of stimulatory anions. The competitive nature of inhibition by phosphate has also been noted by Conway and Cooke. 1° Although inhibition by fluoride suggests participation of a metal in the reaction, no evidence for a metal requirement could be obtained. 4 Various metal-binding agents, including ethylenediaminetetraacetate, 8-hydroxyquinoline, cyanide, and thiocyanate (as well as citrate), failed to inhibit the enzyme. Addition of Mg or Mn ions failed to increase the activity. Certain heavy metal ions inhibit the enzyme. Mercuric ions at 1 >( 10-~ M cause 50 % inhibition. This inhibition is reversible by cysteine. Iodoacetate, even at 0.01 M, causes no detectable inactivation after incubation with the enzyme for 30 minutes at 25 ° in succinate buffer o.f pH 5.9. 10 E. J. Conway a n d R. Cooke. B~:oehem, J. 38, 479 (1939),

[691

SFECIFIC ADENOSINE' DE AMINASE FROM INTESTINE

473

Effect of pH. The enzyme exhibits a sharp optimum for activity at pH 5.9 in succinat~ and other 1 buffers, the activity falling to one-half of optimal at pH 5.5 or 6.4. In citrate buffer (0.1 M), no.t only is the activity increased markedly (see above), but the optimum pH is shifted to pH 6.5, the activity falling to one-half of optimal at pH 5.6 or 7.3. The degree of inhibition of the enzyme by fluoride is also dependent on pH. Thus, 0.01 M fluoride, which produces 96% inhibition of the enzyme in succinate buffer at pH 5.6, causes no inhibition of the enzyme at pH 6.7. Similarly, in citrate buffer, 0.01 M fluoride causes 83% inhibition at pH 5.6, but no inhibition at pH 7.3. Effect of Substrate Concentration. The Michaelis-Menten constant, Kin, determined from a Lineweaver-Burk plot which gave a straight line over the concentration range 4 X 10-~ to 4 X 10-3 M AMP, was 6.0 X 10-6. Degree of Purity. The absolute percentage purity of the most active fraction is not known, since none of the criteria for homogeneity have been applied. The turnover number under favorable conditions (fraction 4 in citrate buffer, pH 6.5, 4 X 10-3 M AMP, 25 °) may be calculated to be about 2500 moles of substrate per 10~ g. of protein per minute. The purity of fraction 3 or 4 in terms of separation from interfering enzymes is satisfactory. None of the following activities is detectable: adenosine deaminase, hexokinase, myokinase, ATPase, or nucleotide pyrophosphatase. These fractions are therefore suitable for use in spectrophotometric analysis H of compounds which can give rise to 5'-AMP, such as ATP, ADP, and DPN. 11 See Vol. I I I [111].

[69] Specific Adenosine Deaminase from Intestine By

NATHAN O. KAPLAN

Adenosine + H20 --~ Inosine + NH3 The deaminase from intestine, unlike the enzyme from takadiastase (see Vol. II [70]), is specific for adenosine and will not deaminate other adenine derivatives. Kalckar ~ has separated the deaminase from the potent phosphatase present in the intestine. Kornberg and Pricer, ~ however, have described a method in which separation of the phosphatase is not essential for the assay of adenosine, the phosphatase being inhibited by addition of phosphate. Both of these procedures will be outlined. 1 H. M. Kalckar, J. Biol. Chem. 167, 445 (1947). A. Kornberg a n d W. E. Pricer, Jr., J. Biol. Chem. 193, 481 (1951).

474

ENZYMES OF NUCLEIC ACID METABOLISM

[69]

Principle of Assay. Deamination of adenosine is followed by measurement of the decrease at 265 m~ and is based on the same principle as that for 5'-AMP deaminase (see Vol. II [68]). Assay Procedure. Thirty-six micrograms of adenosine is introduced into 0.05 M glycylglycine or phosphate, pH 7.6 (see below), in a total volume of 3 ml. The deaminase is then added, and the change at 265 m~ followed. No specific units have been defined for the enzyme. Separation of Deaminase from Phosphatase by the Method of Kalckar.1 The first steps are identical with those outlined by Schmidt and Thannhauser for the preparation of intestinal phosphatase. 3 The mucosa from calf intestine is digested with trypsin in the presence of toluene for 36 hours and then filtered through a cake of Hyflo. The filtrate is then treated with 600 g. of ammonium sulfate per liter; the precipitate is collected on a film of Hyflo and dissolved in 0.1 M ammonium acetate, pH 8.5. Solid ammonium sulfate is then added to the solution to bring the concentration to 500 g./1. The precipitate is redissolved in the ammonium acetate and dialyzed overnight at 0 ° against 0.025 M ammonium acetate, pH 8. The adenosine deaminase is separated from phosphatase activity by addition of 0.1 vol. of alumina (containing 25 mg. of aluminum hydroxide per milliliter) to the above dialyzed fraction. After 10 minutes of stirring at room temperature the mixture is centrifuged, and to the supernatant 0.05 vol. of alumina is added. Both precipitates are combined and eluted with 0.2 M phosphate (pH 8) and dialyzed against 0.02 M ammonium acetate, pH 8. The supernatant from the alumina mixture contains all the phosphatase and about 35 to 40 % of the adenosine deaminase. The eluates contain almost no phosphatase and about one-half of the deaminase activity. 5'-Adenylic acid is deaminated at about 2 % of the rate of adenosine by the eluates; hence the eluates can be used for adenosine determination. About 0.4 to 1.0 ~' of the purified protein per milliliter can be used for a spectrophotometric determination. Adenosine Assay in the Presence of Phosphate. Kornberg and Pricer 2 describe the following procedure for obtaining the intestinal deaminase without the necessity for separating the phosphatase from the deaminase. One hundred milligrams of Armour intestinal phosphatase (stated to contain 15 Schmidt-Thannhauser units/mg.) is dissolved in 10 ml. of ammonium acetate buffer (0.02 M, pH 8.0). The solution is dialyzed against 0.04 M sodium acetate at 2 ° for 3 hours. This solution can be stored in the deep-freeze without loss in activity. Adenosine is deaminated very rapidly when the reaction is carried out in 0.05 M phosphate a G. Schmidt and S. J. Thannhauser, J. Biol. Chem. 149, 369 (1943).

[70]

NONSPECIFIC ADENOSINE DEAMINASE FROM TAKADIASTASE

475

(pH 7.4). Under identical conditions, no detectable deamination of 5'-AMP, 3'-AMP, or 2'-AMP takes place. pH Optimum. The intestinal deaminase is active over a very wide pH range. Although the pH optimum is near the neutral point, the activities at pH 9 and pH 6 are about two-thirds of that at the optimal pH. Use of Deaminase to Assay for Adenosine Derivatives. When the assay with the preparation of Kornberg and Pricer is carried out in phosphate, an unknown can be assayed specifically for adenosine. However, when the reaction is carried out in a nonphosphate medium, the total bound adenosine can be determined (i.e., coenzyme A, TPN, DPN, etc.). This is due to the fact that the crude fraction contains a pyrophosphatase as well as a monoesterase. Therefore, it is possible to determine total bound adenosine by means of the nonphosphate medium. 4,~ 4 T. P. Wang, L. Shuster, and N. O. Kaplan, J. Biol. Chem. 206, 299 (1954). 5 L. Shuster, N. O. Kaplan, and F. E. Stolzenbach, J. Biol. Chem. 215, 195 (1951).

[70] Nonspecific Adenosine Deaminase from Takadiastase Adenosine compound -~ H20 --* Inosine compound ~ NH3

By NATHAN O. KAPLAN Assay Method 1

Principle. The method is based on the change in absorption at 265 mp which manifests the conversion of adenosine to inosine. The principle of the procedure is identical to that used for following the specific 5'-AMP deaminase (see Vol. II [68]). Reagents 0.1 M phosphate, pH 6.8. 0.004 M adenosine. Enzyme. Diluted in 0.1 M phosphate, pH 6.8, if necessary.

Procedure. To 3 ml. of phosphate, add 0.05 ml. of 0.004 M adenosine. After observing the absorption at 265 m~, start the reaction by the addition of approximately 20 units of enzyme. Take readings at 15 and 120 seconds after addition of enzyme. Definition of Unit and Specific Activity. A unit represents the change in optical density of 0.01 in the 15- to 120-second interval. Specific act N. O. Kaplan, S. P. Colowick,and M. M. Ciotti, J. Biol. Chem.194, 579 (1952).

476

ENZYMES OF NUCLEIC ACID METABOLISM

[70]

tivity is expressed as units per milligram of protein, determined by the procedure of Lowry et al. 2 Application of Assay Method to Crude Extracts. Since the crude takadiastase extracts are quite pigmented, it is difficult to determine the activity by the procedure outlined above. However, some estimation of the enzyme content can be determined by ammonia release. This method has been used in assaying the crude extract in the purification procedure outlined below. Purification Procedure The first step in the purification procedure is taken principally from the method of Mitchell and McElroy2 The further steps are essentially from the procedure of Kaplan et al., 1 with some modifications introduced by Astrachan (personal communication). Step 1. One hundred grams of takadiastase 4 is added to 2000 ml. of H20 and 200 g. of Permutit. The precipitate is centrifuged and washed with 300 ml. of water. The supernatant and wash are combined. Alcohol is then added at 0 ° to 33%. After centrifugation, the resulting precipitate is discarded. Alcohol is then added to 65%, and, after standing for 20 minutes, the precipitate is collected by centrifugation at 0 ° and dissolved in 500 ml. of H20. The solution is passed through a charcoal Permutit pad (60 g. of charcoal plus 60 g. of Permutit); this removes a considerable amount of pigmented material. The pad is washed with about 250 ml. of H20. The washings are combined to give a total volume of approximately 600 ml. This solution is the product of step 1 as outlined in Table I and has an activity of 33 units/rag, of protein. Step 2. Precipitation with Acetone. Acetone is added at 0 ° to the above solution (dissolved 65% alcohol precipitate) to 23 %. The resulting precipitate is removed by centrifugation at 0°; this precipitate contains only a negligible amount of activity. The solution is then brought to 40 % acetone; the resulting precipitate contains most of the activity, although some activity is present in a further fraction with 50 % acetone. The 40 % acetone precipitate is dissolved in a 100 ml. of water, and the specific activity is 219 units/mg, of protein. Step 3. Fractionation with Ethanol. Ethanol is added to the solution from step 2 to 10 % at 0 °. The resulting precipitate contains only a slight amount of activity. The supernatant is then chilled to - 1 2 °, and as a result a second precipitate ensues. The precipitate is dissolved in 25 ml. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951); see also Vol. I I I [73]. 3 H. K. Mitchell and W. D. McElroy, Arch. Biochem. 10, 351 (1946). 4 Takadiastase powder can be obtained from Parke, Davis and Company.

[70]

NONSPECIFIC A D E N O S I N E DEAMINASE FROM TAKADIASTASE

477

of phosphate (0.1 M, p H 6.8) and contains the deaminase. This step gives approximately a threefold purification. Step 4. Ammonium Sulfate Fractionation. Some further purification is achieved b y bringing the active fraction to 70 % ammonium sulfate. The precipitate is colored and discarded. A fraction obtained between 70 and 100% ammonium sulfate contains the enzyme with a considerable increase in purity. However, the yield is quite low, and it has been our general practice to stop the purification at step 3. The advantage of the ammonium sulfate fractionation is t h a t this procedure removes phosphatase which is still present in small amounts in the ethanol precipitate. TABLE I SUMMARY OF PURIFICATION

Step Crude water extractsa of takadiastase (I00 g.) I. 66 % alcohol precipitate II. 40 % acetone precipitate III. - 12° ethanol precipitate IV. 100% ammonium sulfate fraction

Total units Units/mg. Recovery, % 522,000 227,000 70,800 32,420 8,100

4 33 219 614 1110

-43.0 13.5 6.2 1.5

The activity of crude preparations varies considerably. The, activity of the crude extract was determined by ammonia release from adenosine. Properties

Stability. The enzyme is quite stable and can be kept in the deepfreeze for over six months without loss in activity. The dissolved precipitate of step 3 has been lyophilized and kept as a dry powder for over a year with no decrease in activity. pH Optimum. Mitchel and M c E l r o y a report a broad p H optimum (5 to 8) in phosphate. This occurs with high and low levels of adenosine. However, with low substrate levels in succinate, a sharp p H optimum of 6.3 is found. Specificity. The enzyme, unlike the adenosine deaminase from intestine, is not specific. I t will deaminatc 5'-AMP, 3'-AMP, ATP, ADP, D P N and adenosine diphosphate ribose (ADPR), as well as adenosine. I I t does not deaminate 2'-AMP, T P N , or adenine. 3',5'-Diphosphoadenosine is deaminated, but 2',5'-diphosphoadcnosine is not. 5 I t is of interest to note t h a t the " s y n t h e t i c " 3'-isomer of T P N is deaminated at a slow rate. 6 The takadiastase enzyme deaminates adenosine twice as fast as 5'-AMP and approximately four times as fast as 3'-AMP. D P N , ADP, and A T P are deaminated at considerably slower rates. 5 T. P. Wang and N. O. Kaplan, J. Biol. Chem. 206, 311 (1954). e L. Shuster and N. O. Kaplan, J. Biol. Chem. 215, 181 (1955).

478

[71]

ENZYMES OF NUCLEIC ACID METABOLISM

K,~. A s u m m a r y of the affinities of various substrates for the deaminase is given in T a b l e I I . Distribution. As yet, the nonspecific deaminase described a b o v e has been found only in takadiastase. TABLE II AFFINITIES OF VARIOUS SUBSTRATES FOR DEAMINASE

Substance

Approximate Km

ATP DPN 5'-Adenylic acid ADP 3'-Adenylic acid ADPR Adenosine

M X 10-~ 1.2 1.8 0.8 0.7 1.7 1.5 0.6

[71] Cytosine Nucleoside Deaminase from Escherichia

coli

C R 1 ~ H20--~ U R 1 -~ NH3 C D R i ~ H 2 0 --* U D R 1 -t- NH3

(1) (2)

By T. P. WANG

Assay Method Principle. S p e c t r o p h o t o m e t r i c m e t h o d s are used in following the act i v i t y of this enzyme, since the deamination of the cytosine c o m p o u n d s is accompanied b y a decrease in absorption of 55 % at 282 m~. T h e molecular extinctions of C R and U R at 282 m~ are 6000 and 2700, respectively. 2 T h e absorption spectra of the corresponding deoxyribosides are not m a t e rially different from those of the ribosides. Reagents C R or C D R , a n y suitable concentration. E. coli extract, 1 ml., equivalent to 50 to 100 mg. of wet cells. Tris buffer (0.1 M), p H 7.5.

Procedure. Place in a 3-ml. silica B e c k m a n cuvette 1.5 ml. of Tris buffer and a solution of C R or C D R containing a b o u t 0.3 micromole of the 1 CR, UR, CDR, and UDR stand for cytidine, uridine, cytosine deoxyriboside, and uracil deoxyriboside, respectively. 2 T. P. Wang, H. Z. Sable, and J. O. Lampen, J. Biol. Chem. 184, 17 (1950).

[71]

CYTOSINE NUCLEOSIDE DEAMINASE FROM ESCHERICHI~_ COLI

479

nucleoside. Make up to 2.9 ml. with water, and take an initial reading at 282 m~. Then add 0.1 ml. of the enzyme. After a quick stirring, take readings at 30-second intervals. The reaction will be finished in about 30 minutes.

Preparation of Enzyme 2

Preparation of E. coli Cells. E. coli strain 15 (9723 of the American Type Culture Collection) is the source of this enzyme. Stock culture of E. coli is kept on agar slants containing 0.3% Difeo beef extract, 0.2% Difco yeast extract, 0.7 % Difco peptone, 0.4% glucose, and 1.5 % Difco agar. An inoculum is made by transferring a loopful of bacteria from the slant to 10 ml. of medium of the same composition as listed above except the agar. The inoculum is then incubated for 24 hours at 37 °. The cells are collected by centrifugation at 4500 r.p.m, for 15 minutes. Preparation of Cell-Free Extract. The packed wet cells of E. coli, washed once with 0.9 % NaC1, are ground in an ice-chilled mortar with two and one-half times their weight of alumina powder (A-303 or A-301 of the Aluminum Company of America) according to McIlwain. 3 The paste is then mixed with 10 to 20 vol. (with respect to the original cells) of cold 0.05 M Tris buffer, pH 7.5, allowed to stand at 2 ° for 30 minutes, and centrifuged at 20,000 X g for 15 minutes in a Servall centrifuge. The supernatant is slightly opaque and light yellow in color. Attempts have been made to purify the enzyme by alcohol and (NH4):SO4 fractionations. The efforts were unsuccessful. Properties

Specificity. The cell-free extract is specific for the cytosine nucleosides. No action is observed when adenine, adenosine, cytosine, isocytosine, cytidylic acid, guanine, and guanosine are tested. The deamination is faster with CDR than with CR. The Km for CR is 1.74 X 10-4 M, and that for CDR, 8.9 X 10-5 M. General Properties. The enzyme is not inactivated by prolonged dialysis or by freezing and thawing. Preparations kept for several months at - 2 0 ° retain their original activity. The enzyme has a broad pH oPtimum between 6.5 and 8.5. Products of the Reaction. The products of the reaction are uracil nucleosides and ammonia. The former can be identified by their absorption spectra in ultraviolet region and by paper chromatographic or ionophoretic methods. Ammonia can be easily demonstrated by any of the standard methods such as by use of Nessler's reagent. H. McIlwain, J. Gen. Microbiol. 2, 288 (1948).

480

ENZYMES OF NUCLEIC ACID METABOLISM

[72]

When a demonstration of the f o r m a t i ~ of uracil nucleosides is desired, it is preferable to use a thoroughly dialyzed extract. Because of the presence of a pyrimidine nucleoside phosphorylase which requires inorganic phosphate for its activity in the extract, 2 it is essential to remove any inorganic phosphate present in the extract to prevent any splitting of the uracil nucleosides formed from the deamination of cytosine nucleosides.

[72] Guanase Guanine ~ H~O --* Xanthine ~ NH3

By Louis SHUSTER Assay Method Principle. When guanine is deaminated to xanthine, there is a shift in the ultraviolet absorption spectrum, the greatest change being a decrease of about 50% in the extinction at 245 m~. This change is the basis for the method of Roush and Norris. 1 The method of Kalckar, 2 which is more commonly used, involves measurement of the xanthine produced in the reaction by oxidation with xanthine oxidase. This oxidation is followed spectrophotometrically by measuring the increase in optical density at 290 m~ due to the formation of uric acid. The increase obtained is roughly sixfold, which makes this method more sensitive than that of Roush and Norris.

Reagents Guanine. A stock solution of 0.001 M can be made up by dissolving 15 rag. of free guanine in a few milliliters of 1 N N a O H and diluting up to 100 ml. 0.1 M glycylglycine or tris (hydroxymethyl)aminomethane buffer, pH 8.0. Xanthine oxidase, prepared from milk (see Vol. II [73]). An aliquot of 0.1 ml. should contain enough enzyme to oxidize 50 ~/of hypoxanthine per milliliter per hour. Guanase. The enzyme is diluted with glycylglycine or Tris buffer to contain 200 to 1000 units of enzyme per milliliter (see definitions below). i A. R o u s h a n d E. R. Norris, Arch. Biochem. 29, 124 (1950). z H. M. Kalckar, J. Biol. Chem. 167, 461 (1947).

[72]

GUANASE

481

Procedure. Dilute the stock solution of guanine 1 : 15 with buffer, and place 2.9 ml. of the resulting solution in a quartz cell having a 1-cm. light path. By the method of Roush and Norris, add 0.1 ml. of guanase solution and follow the decrease in optical density at 245 mr*. By Kalckar's method add 0.05 to 0.10 ml. of xanthine oxidase, and read the optical density at 290 mu. Then add 0.1 ml. of guanase solution, and read at 30-second intervals after mixing. Application to Crude Tissue Preparations. Changes in turbidity during the course of the reaction may be checked at 320 mtL where no change ascribable to uric acid occurs. If uricase is present (this can be checked by adding 10 y of uric acid and noting any decrease at 290 m#), the reaction should be followed at 270 m,, the xanthine peak, where the optical density decreases about 80% during oxidation to uric acid, and this is unaffected by the action of uricase. Purification Procedure The following procedure is given by Kalckar, 2 who presents no data on specific activities or yields. Step 1. Preparation of Crude Extract. Rat liver freed of blood is blended with 2 vol. of cold distilled water and extracted for 15 minutes in a mechanical shaker. The homogenate is centrifuged at low speed (2000 r.p.m, for 5 minutes), and the precipitate is discarded. The supernatant is then centrifuged at high speed (15,000 r.p.m, for 20 minutes at 0°), and the precipitate is again discarded. The clear supernatant is used for the succeeding steps. Step 2. Fractionation with Ammonium Sulfate. Saturated ammonium sulfate is added to fraction 1 to give 0.4 saturation. The resulting precipitate is removed by centrifugation and discarded. The supernatant is brought to 0.6 saturation with more saturated (NH4)~SO4: The precipitate is centrifuged down and dissolved in a small volume of water. Step 3. Fractionation with Ethanol. Fraction 2 contains both guanase and nucleoside phosphorylase. Almost complete separation of these activities can be achieved by fractionation with alcohol at - 5 °. Fraction 2 is adjusted to pH 5.5 with 0.3 vol. of 0.1 M succinate-acetate buffer. Ethanol is added to a concentration of 15%. The resulting precipitate, which contains both guanase and nucleoside phosphorylase, is discarded. More ethanol is added to the supernatant to give a concentration of 40%. The precipitate is centrifuged down and extracted with 0.1 M glycine buffer, pH 9.1. Any insoluble residue is discarded. The clear glycine extract still shows slight nucleoside phosphorylase activity, but in the ,absence of large amounts of inorganic phosphate this preparation can be

482

ENZYMES OF NUCLEIC ACID METABOLISM

[73]

used to measure guanine in the presence of guanosine. N o unit is available for the enzyme.

Properties Specificity. B o t h guanine and 8-azaguanine are d e a m i n a t e d b y this enzyme. 1 Guanosine and guanylic acid are not attacked. When acting on guanine the e n z y m e exhibits a broad p H o p t i m u m in the range of p H 6 to 10. When acting on 8-azaguanine the p H o p t i m u m is 6.3, and points of 5 0 % a c t i v i t y are a t p H values 5.5 and 7.5. T h e Km values for guanine and 8-azaguanine at p H 6.5 in 0.05 M p h o s p h a t e buffer are 5 X 10 -e and 7 X 10 -~, respectively.

[73] Xanthine Oxidase from Milk H y p o x a n t h i n e + 2 02 ~ - Uric Acid + 2 H202 B y B. L. HORECKER and L. A. HEPPEL

Assay Method P r i n c i p l e . X a n t h i n e oxidase m a y be assayed b y m e t h y l e n e blue reduction, 1 oxygen uptake, ~ uric acid formation, 3 and c y t o c h r o m e c reduction. 4 T h e m e a s u r e m e n t of uric acid f o r m a t i o n b y the absorption at 290 mg, as originally introduced b y Kalckar, is p e r h a p s the m o s t convenient of these methods. However, since the purification procedure described here was developed with the aid of the c y t o c h r o m e reduction test, the results are given in t e r m s of this assay. T h e rate of a p p e a r a n c e of the reduced band of c y t o c h r o m e c at 550 mg is expressed as the firstorder velocity constant for the reaction. Reagents

C y t o c h r o m e c (2.5 X 10 -4 M). P r e p a r e b y the m e t h o d of Keilin and Hartree, 5,6 and analyze b y the m e t h o d of Theorell. 7 1 M. Dixon, Biochem. J. 20, 703 (1926). 2 E. G. Ball, J. Biol. Chem. 128, 51 (1939). 8 H. M. Kalckar, J. Biol. Chem. 167, 429 (1947). 4 B. L. Horecker and L. A. Heppel, J. Biol. Chem. 178, 683 (1949). 5 D. Keilin and E. F. Hartree, Biochem. J. 39, 289 (1945); see Vol. II [133]. 6 Commercial preparations are available which are satisfactory for the assay. 7 H. TheoreI1, Biochem. Z. 285, 207 (1936).

[73]

XANTHINE OXIDASE FROM MILK

483

Catalase, 5.0 units/ml. Prepare by the method of Sumner and Dounce. 8.9 Hypoxanthine (0.05 M). Dissolve 68 mg. in 10 ml. of 0.05 M NaOH. Albumin. Dissolve 60 rag. of crystalline bovine serum albumin (Armour Laboratories) in 10 ml. of water. 0.1 M phosphate buffer, pH 7.4. Enzyme. Dilute in water to obtain 0.25 to 2.5 units of enzyme per milliliter. (See definition below.)

Procedure. To 1.0 ml. of buffer in a 1.0-cm. absorption cell add 0.1 ml. of cytochrome c, 0.1 ml. of catalase, 0.1 ml. of albumin, 0.2 ml. of water, and 0.04 ml. of diluted enzyme. Take readings at 550 mu at 1-minute intervals after mixing with 0.01 ml. of hypoxanthine. After 7 minutes add about 1 mg. of solid Na2S204 and take a final reading. Definition of Unit and Specific Activity. Calculate the concentration of oxidized cytochrome c (ferricytochrome) from the equation dR -Ferricytochrome - 1.96 × dt 104 moles per liter where d~ and dt are the density readings after addition of Na2S204 and at any time t during the rate determination, respectively. A unit of enzyme is that quantity which will give a value of 1.0 for (A log ferricytochrome)/At, the first-order velocity, where t is expressed in minutes. The specific activity is the number of units per milligram of protein in the test. Protein is determined by the turbidimetric method of Bficher. 1° Purification Procedure Steps 1 and 2 are based on the procedure of Ball. 2

Step 1. Preparation of Buttermilk. Churn 2 quarts of fresh raw cream (40 to 42 % butter fat) in a mechanical mixer at 2 ° until the butter separates as fine hard particles. Strain through several layers of cheesecloth. Step 2. Trypsin Digestion and Heating. To the strained buttermilk (780 ml.) add 0.2 M Na2HP04 (470 ml.) to bring the pH to 7.5 and 2.9 g. of trypsin (Wilson 1:300), dissolved in 50 ml. of water. Incubate at 37 ° for 31/~ hours. Cool the incubation mixture to 20 °, and test aliquots t o determine the minimum amount of 0.5 M CaC12 required to produce a nearly clear supernatant solution. Generally 0.12 to 0.15 vol. is sufficient; further addition will greatly reduce the yield of enzyme. To the bulk of the digestion mixture (1280 ml.) add 190 ml. of 0.5 M CaCI~, s j . B. Sumner and A. L. Dounce, J. Biol. Chem. 19.7, 439 (1939). 9 Commercial catalase preparations may be used. i0 T. Bficher, Biochim. et Biophys. Acta 1, 292 (1947); see also Vol. I I I [73].

484

ENZYMES OF NUCLEIC ACID METABOLISM

[73]

incubate for 15 minutes at 20 °, and centrifuge. Warm the slightly opalescent supernatant solution to 60 ° in 3 minutes, keep at this temperature for 5 minutes, and cool as rapidly as possible. Step 3. Ammonium Sulfate Fractionation. To the heated fraction (1200 ml.) add 271 g. of ammonium sulfate, and filter overnight. Add 72 g. of ammonium sulfate to the filtrate, collect the precipitate by centrifugation, and dissolve in water. Step 4. Aluminum .Hydroxide Gel Adsorption and Elution. Add the ammonium sulfate fraction (273 ml.) to 153 mg. of aluminum hydroxide gel C~/11 which has been previously centrifuged. Stir to suspend the gel, centrifuge, and discard the supernatant solution. Elute the gel with three 12-ml. portions of 0.5 M phosphate buffer, pH 7.5. Step 5. Calcium Phosphate Gel Adsorption and Elution. Combine the eluates, and add 54 ml. of ammonium sulfate solution, saturated at 0 °. Collect the precipitate, and dissolve in 60 ml. of water. Add 99 mg. of calcium phosphate gel (aged about 3 months and centrifuged before use). Suspend the gel thoroughly, centrifuge, and discard the supernatant solution. Wash the gel twice with 12-ml. portions of 0.1 M phosphate buffer, p i t 6.2, and elute the enzyme with two 12-ml. portions of 0.5 M phosphate buffer, pH 6.2. To the combined eluates add 36 ml. of saturated ammonium sulfate solution, collect the precipitate by centrifugation, and dissolve in 30 ml. of water. SUMMARY OF PURIFICATION PROCEDURE

Fraction 1. 2. 3. 4. 5.

Buttermilk Heated fraction (NH4)~SO4fraction AI(OH)3eluate Ca3(PO4)2eluate

Total Specific volume, Total Protein, activity, Recovery, ml. Units/ml. units mg./ml, units/mg. % 780 1200 267 36 41

1.67 0.67 2.33 11.1 5.40

1300 49.0 800 1.73 624 2.58 400 2.85 221 0.98

0.034 0.39 0.90 3.89 5.51

-62 48 31 17

Properties Specificity. The purified enzyme will oxidize hypoxanthine, xanthine, and aldehydes. 1 The ratio of rate of oxygen uptake to cytochrome c reduction remains constant during the purification procedure. ~ Activators and Inhibitors. Xanthine oxidase is slowly and irreversibly inactivated by cyanide, 12 and this effect is now attributed to its metal 11R. Willstatter and H. Kraut, Ber. 66, 1117 (1923). 1~A. Szent-GySrgyi,Biochem. Z. 173, 275 (1926)~

[74]

URICASE

485

content. 13 The enzyme contains FAD. The inactivation on dialysis reported by BalF can be reversed by sulfhydryl compounds. TM The enzyme is inhibited by buffers and salts such as phosphate, imidazole, and sodium and potassium chloride. 15 Stability. The heated fraction m a y be stored at 2 ° for several weeks with little loss in activity. I t can be kept in the frozen state indefinitely. The final solution slowly loses activity when frozen and stored at - 1 6 °. i8 D. E. Green and H. Beinert, Biochim. et Biophys. Acta 11, 599 (1953). 14H. M. Kalckar, N. O. Kjeldgaard, and H. Klenow, Biochim. et Biophys. Acta 5, 575 (1950). is D. B. Morell, Biochem. J. 51, 666 (1952).

[74] U r i c a s e Uric Acid + 02 + 2 H20 --~ Allantoin + H202 + CO2 B y ENZO LEONE

Assay Method Principle. The rate of 02 uptake b y the enzyme is measured manometrically, in the presence of uric acid. The method was originally developed by Keilin and Hartree, ~ who used Barcroft differential manometers; a dangling cup, containing uric acid, is tipped in after equilibration. The procedure described below applies to Warburg manometers. Reagents

Uric acid, lithium salt. Dissolve 500 mg. of uric acid in 31.25 ml. of boiling 0,1 N LiOH; cool; bring to 100 ml. with water. This solution, of which 0.4 ml. contains 2 mg. of uric acid, must be prepared daily. 0.1 M boric acid-KC1/NaOH buffer, p H 9.0. Enzyme. Amounts of enzyme are chosen so as not to exceed an uptake of 60 to 65 pl. of 02 during the first 15 minutes. Procedure. In Warburg manometer flasks, place 1 ml. of borate buffer, enzyme, and water up to a final volume of 2.6 ml. In the center well place 0.3 ml. of 20% K O H adsorbed on a filter paper roll; in the side arm, 0.4 ml. of uric acid solution. T e m p e r a t u r e 39°; gas phase, air. After equilibration, tip in uric acid, and take readings every 5 minutes for the first 15 minutes. F r o m the O~ uptake during the first 15 minutes calculate Che corresponding u p t a k e per hour. i D. Keilin and E. F. Hartree, Proc. Roy. Soc. (London) Bllg~ 114 (1936).

486

ENZYMES OF NUCLEIC ACID METABOLISM

[74]

Definition of Unit and Specific Activity. One unit of enzyme is defined as the amount which takes up 1 ~1. of O2 per hour, under the above conditions; the same value, divided by the volume, in milliliters, of the preparation used, represents the concentration of enzyme. The specific activity is expressed as Qo, i.e., units, calculated as above, per milligram dry weight of nondialyzable material. Application of Assay Method to Crude Tissue Preparations. The manometric assay is the method of choice, especially when examining unknown biological material for uricase activity. Allowance must be made for the 02 uptake of the control, in the absence of uric acid. It is also advisable, when dealing with tissues in which the presence of uricase is uncertain, to perform a colorimetric determination of residual uric acid, after an incubation period of at least 1 hour. Brown's 2 phosphotungstic acid or Fearon's 3 dichloroquinone chlorimide color reactions are suitable for this purpose. In crude tissue preparations or in fractions prepared by differential centrifugation, uricase can also conveniently be studied by the spectrophotometric method developed by Kalckar 4 (as described by Schneider and Hogeboom~) ; this method is based on the decrease in the ultraviolet absorption spectrum which takes place during uric acid oxidation; although in this procedure the 02 tension and the temperature are less rigorously controlled, it is of value owing to its simplicity and the relatively small amounts of enzyme required. Purification Procedure

Highly purified preparations of uricase (Qo2 -- 2000 to 6000) can be obtained by the procedure originally described by Davidson 6 and later modified and improved by Holmberg, 7 but the final yield is small and not always reproducible. Thus, although the above procedure may be followed to obtain a highly purified enzyme, the following method (Leone s) is recommended when a Qo, value not exceeding 700 to 800 is aimed at and a good yield of enzyme is required. Step 1. Borate-Butanol Extraction of Ox Kidneys. Ox kidneys, which can be stored for several weeks in the frozen state without much loss of activity, are freed from adhering fat and tissue and passed through a H. Brown, J. Biol. Chem. 158, 601 (1945). 3W. R. Fearon, Biochem. J. 38, 399 (1944). 4H. M. Kalckar, J. Biol. Chem. 167, 461 (1947). 5 W. C. Schneider and G. H. Hogeboom, J. Biol. Chem. 195, 161 (1952). J. N. Davidson, Bioehem. J. $2, 1386 (1938); 36, 252 (1942). C. G. Holmberg, Biochem. J. 83, 1901 (1939). 8 E. Leone, Biochem. J. 54, 393 (1953).

[74]

URICASE

487

mechanical meat mincer. The mince is then homogenized for 5 minutes in a Waring blendor with 0.1 M borate buffer, pH 10, and n-butanol (300 ml. of borate buffer and 10 ml. of n-butanol to every 100 g. of mince)2 The borate is preheated to about 40 ° before homogenization, and the suspension is brought to pH 10 by the addition of 0.1 N NaOH. The whole suspension is incubated at 37 ° for 16 to 18 hours, cooled down, and centrifuged for 30 minutes at 3000 r.p.m.; a yellow, cloudy supernarant is obtained, which is decanted and preserved; more 0.1 M borate buffer, pH 10, is added to the residue to bring the mixture to the original volume; the suspension is well stirred and centrifuged for 30 minutes at 3000 r.p.m. This is repeated twice, and the four supernatants are combined. See the table for a summary of yield and increase in purity. Step 2. Calcium Phosphate Gel Treatment (Alkaline). To fraction 1, about one-fifth of its volume of calcium phosphate gel is added; the gel (prepared according to Keilin and Hartree 1°) should have a dry weight content of about 25 mg./ml. The mixture is centrifuged after 10 to 15 minutes. The supernatant is preserved, and the residual calcium phosphate cake is thrice extracted with small amounts of a one-fifth-saturated solution of (NH4)2SO4. The three (NH4)2SO4 extracts are added to the supernatant from the first centrifugation.

Step 3. (NH4)2S04 Precipitation and Further Purification by Dialysis. The concentration of (NH4)~SQ in fraction 2 is increased to 50% saturation by the addition of solid salt. The mixture, after some time in the cold, is centrifuged. The bulk of the supernatant fluid is siphoned off; a further period at 0 ° and repeated centrifugation may be needed to separate the (NH~)~SO4 precipitate. This is next suspended in distilled water, made up to a volume corresponding to about one-fifth of the original extract from step 2, and dialyzed for 12 to 20 hours at room temperature against running tap water, until a brown precipitate settles out in the cellophane bag, while the fluid portion becomes clear. The dialyzed preparation is centrifuged at about 5000 r.p.m. The precipitate is ground in a precooled mortar with ice-cold 0.1 M phosphate buffer, pH 7.3, and centrifuged in the cold at about 10,000 r.p.m. The clear, almost colorless 9 This proportion is the optimal one, since, a l t h o u g h it ensures a good Qoi value, it is still possible to perform the (NH4)2S04 precipitation directly on the centrifuged extracts, w i t h o u t a n y dialysis to remove n-butanol. However, if small a m o u n t s of a n enzyme preparation with a Qol value of a b o u t 70 to 100 are required, especially for analytical use, one can increase the b u t a n o l concentration up to 22 to 25 ml. per 100 g. of mince a n d 260 ml. of borate buffer. In this way after incubation a n d centrifugation a n extract is obtained which can either be used as such or can be dialyzed against r u n n i n g t a p water for 12 to 16 hours, if f u r t h e r t r e a t m e n t such as freeze-drying is intended. 10 D. Keilin a n d E. F. Hartree, Biochem. J. 49, 88 (1951).

488

[74]

ENZYMES OF NUCLEIC ACID METABOLISM

phosphate extract is decanted, and the protein residue is ground with 0.1 M borate buffer, p H 10, and centrifuged at high speed, at room temperature, for a b o u t 10 minutes. The resulting extract, slightly opalescent but almost colorless, is preserved, and the borate extraction is repeated three times. The borate extracts are combined. Step ~. Calcium Phosphate Gel Treatment (Acid). Fraction 3 is cooled in an ice bath, and enough 0.5 N acetic acid is added, with continuous stirring, to give a faintly acid reaction (methyl red just pink). Next, calcium phosphate gel, 0.1 vol., is added, and the p H is adjusted again. After 10 to 15 minutes, the mixture is centrifuged. T h e s u p e r n a t a n t is decanted, and the calcium phosphate precipitate with the adsorbed enzyme is washed with distilled water, care being taken to maintain a weakly acid reaction. After centrifugation and washing, the precipitate is ground in a m o r t a r with a one-fifth-saturated solution of (NH4)2S04, at a neutral to slightly alkaline pH, and centrifuged again. The (NH4)2SO4 extraction of the calcium phosphate gel is repeated two or three times; the water-clear and almost colorless supernatants are combined. Step 5. (NH4)2S04 Precipitation and Dialysis. Fraction 4 is brought to half-saturation with (NH4)~S04, and the resulting precipitate is centrifuged down and washed in the centrifuge tubes with a half-saturated solution of (NH4):S04. The final precipitate is dissolved in 0.1 M borate buffer, p H 10, corresponding to no more than one-tenth of the volume of the extract from step 4, and dialyzed in the cold against three changes of 0.01 M borate buffer, p H 10. After 36 hours, a small a m o u n t of brownish precipitate is removed by centrifugation. SUMMARY OF PURIFICATION PROCEDURE

Fraction 1. Borate-butanol extract 2. Extract after alkaline treatment with calcium phosphate gel 3. Borate extracts after (NH4)~SO4 precipitation and dialysis 4. Extract after acid treatment with calcium phosphate gel 5. Borate extract after second (NH4)2SO4 precipitation and dialysis

Total units, Dry Specific RecovTotal thou- weight, activity, ery,• volume, Units/ml. sands mg./ml. Qo~ % ml. 5000

252

1260

6.25

40

90

5000

227

1135

4.54

50

81

1600

420

672

1.40

300

48

1600

262

419

0.37

708

30

160

2530

405

3.10

816

29

With reference to the uricase content of the original suspension (1400 thousands of units).

[74]

URICASE

489

At all stages of purification, uricase activity remains unchanged for many weeks in alkaline extracts. At the first step of the purification procedure, satisfactory stability can also be achieved by freeze-drying the preparation.

Properties Specificity. Keilin and Hartree 1 have shown the specificity of uricase for uric acid; the enzyme is not active toward substituted uric acids, such as mono-, di-, and tri-methyl or -ethyl derivatives, or the corresponding riboside (Schulern). Activators and Inhibitors. Several substances have activating properties, like dithizone, phenanthroline, a,a'-dipyridyl, thiourea, and sodium diethyldithiocarbamate (Davidson6). It appears most probable that the activation is due to the metal-binding capacity of these substances; a similar mechanism is presumably involved in the activation by ethylenediaminetetraacetate (Leonel~). Cyanide is an uricase inhibitor, in 10-4 M concentration. The cyanide inhibition is reversible, 1 and full activity can be restored if cyanide is removed by dialysis. B Most metals, such as Cu, Mn, Zn, Co, Ni, and Fe, have an inhibitory effect. Effect of pH and Temperature. Optimum pH is 9.25; 1 45 ° is the optimum temperature (Ro 13). Nature of the Enzyme. The most highly purified preparation by Davidson 6 contained 0.10 to 0.20% Fe and 0.06 to 0.09% Zn, together with traces of other metals. Holmberg's 7 preparation had a content of 0.025 % of Fe and 0.13 % of Zn. Praetorius TM has reported a Zn content of less than 0.04% in a uricase preparation, purified according to Holmberg and dialyzed against BAL. The likelihood of uricase's being a metal-containing enzyme is strongly indicated by its reversible inhibition by cyanide, but the identity of the metal remains uncertain. Mahler, Baum, and Htibscher 15 have recently reported a new procedure for the purification of uricase. Starting with pig liver mitochondria and using alkaline extraction, isoelectric precipitation and ammonium sulfate fractionation, they have obtained a preparation homogeneous on ultracentrifugation and electrophoresis. There is a gradual increase of the copper content during the purification and assuming 1 Cu a t o m / M enzyme the observed Cu content of 0.055 per cent or the purest preparation leads to a molecular weight of 122000. H W. Schuler, Z. physiol. Chem. 208, 237 (1932). 12 E. Leone, unpublished observation. 13 K. Ro, J. Biochem. (Japan) 14, 361 (1931). 14 E. Praetorius, Biochim. et Biophys. Acta 2, 590 (1948). 16 H. R. Mahler, H. Baum, and G. Hiibscher, Federation Proc. 14, 249 (1955).

490

ENZYMES OF NUCLEIC ACID METABOLISM

[75]

[75] Pyrimidine Oxidase and Related Enzymes By OSAMU HAYAISHI I. Uracil-Thymine Oxidase 1,2 Uracil + 1/~ O5 -* Barbituric Acid Thymine + ~ 05-~ 5-Methylbarbituric Acid

Assay Method Principle. Since the extinction of the oxidation products is approximately three times as great as that of the substrates, the rate of increase in optical density serves as the basis for activity measurement. Reagents 0.001 M uracil or thymine solution. 2.67 × 10-3 M methylene blue solution. 0.02 M Tris buffer, pH 8.5.

Procedure. The test system (at 22 to 25°), in a quartz cell having a 1-cm. light path, contains 0.3 ml. of substrate, 0.3 ml. of the methylene blue solution, 2.3 ml. of the Tris buffer, and 0.1 ml. of enzyme. Readings are taken at 2-minute intervals (at 225 mt~ in the case of uracil and at 270 mt~ in the case of thymine) in a Model DU Beckman spectrophotometer. Definition of Unit and Specific Activity. A unit of enzyme is defined as the amount producing a density increase of 0.100 during the first 10 minutes, and the specific activity is defined as units per milligram of protein. Protein is determined by the method of Lowry et al2 With one unit or less of 'enzyme, the reaction rate is linear for about 30 minutes. Proportionality is observed between rate and the amount of enzyme in the range of 0.5 to 5 units.

Purification Procedure A strain of Mycobacterium 4 is cultured in a medium containing thymine (0.1%), K2HPO4 (0.15%), KH~PO4 (0.05%), and MgSO4'7H20 1 O. Hayaishi and A. Kornberg, J. Am. Chem. Soc. 73, 2975 (1951). 2 O. Hayaishi and A. Kornberg, J. Biol. Chem. 197, 717 (1952). 8 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 4 This strain, originally isolated from rabbit feces by Dr. Schatz, Dr. Savard, and Dr. Pintner, was classified and kindly furnished to the author by Dr. T. Stadtman. It is available at the Department of Microbiology, Washington University School of Medicine, St. Louis, Missouri.

[75]

P Y R I M I D I N E OXIDASE AND R E L A T E D ENZYMES

491

(0.02%). Large-scale cultures can be conveniently made in 20-1. glass carboys, each containing 10 1. of the medium at 26 ° for 40 hours with constant shaking. The inoculum is usually made by growing the cells in 100 ml. of the same medium for 24 hours at 26 °. The cells are harvested by centrifugation in a Sharples supercentrifuge and washed once with a 0.5% NaC1-0.5% KC1 solution, The yield is approximately 0.5 to 1.0 g. (wet weight) per liter of medium. The cells can be stored at - 1 0 ° without loss of activity for a period of at least six months. Cell-free extracts prepared by grinding the cells with three times their weight of alumina (Alcoa A-301) and extracting with Tris buffer (0.02 M, pH 9.0, ten times the weight of the wet cells) are treated with ammonium sulfate (24.5 g. per 100 ml. of extract). The precipitate is removed by centrifugation, and more ammonium sulfate is added to the supernatant (10.5 g. per 100 ml. of extract). The resulting precipitate, collected by centrifugation, is dissolved in Tris buffer (0.02 M, pH 9.0) to a volume corresponding to one-twentieth of that of the original extract. This fraction contains 35.6 units/ml, and 1.2 mg. of protein per milliliter. The specific activity of crude cell-free extracts is usually about 10.

Properties Specificity. Under the conditions described above, the enzyme does not act on the following pyrimidines: barbituric acid, isobarbituric acid, 5-methylbarbituric acid, 6-methyluracil, dihydrothymine, dihydrouracil, 2-thiouracil, 2-thio-5-methyluracil, or cytosine. Effect of pH. The optimum pH of the reaction for uracil oxidation is between 8.5 and 9.0; that for thymine oxidation is between 9.0 and 9.5. Below pH 8.0 and above pH 10.0, both activities are greatly decreased. The enzyme is most stable at about pH 9.0. There is no appreciable loss of activity for at least six months on storage at pH 9.0 at - 1 0 °. Substrate A~nity. The Michaelis constants are 0.35 X 10-4 and 1.31 X 10-4 M for thymine and uracil, respectively. Identity of Uracil and Thymine Oxidase. The fact that the ratio of the rate of thymine to uracil oxidation is almost identical in cell-free extracts from either uracil- or thymine-grown cells, or in the partially purified enzyme preparations, suggests the identity of the two oxidase activities. Further indication is provided by kinetic analysis of the competitive inhibitory action of uracil and thymine. The affinity of uracil for the enzyme is the same whether it is determined with uracil as a substrate (1.31 X 10-4 M) or as a competitive inhibitor of thymine oxidation (1.10 X 10-4 M); similar results are obtained with thymine.

492

ENZYMES OF NUCLEIC ACID METABOLISM

[76]

II. Barbiturase 2 Barbituric Acid -t- 2H~O--~ Urea + Malonic Acid Assay Method

Principle. The method is based on the fact that the cleavage of the pyrimidine ring is accompanied by the disappearance of the ultraviolet absorption spectrum. Reagents 0.02 M sodium barbiturate solution. 0.2 M glycylglycine buffer, pH 8.3. 2.5% solution of crystalline bovine serum albumin. 0.02 M phosphate buffer, pH 7.0.

Procedure. The test system (22 to 25 °) contains, in 1.0 ml., 0.1 ml. of barbiturate solution, 0.3 ml. of glycylglycine buffer, 0.1 ml. of albumin solution, and 0.2 mi. of enzyme. At 10-minute intervals 0.1-ml. aliquots are removed and the reaction is stopped by dilution to 3.0 ml. with phosphate buffer. Readings are made at 255 m~ in a Model DU Beckman spectrophotometer. Definition of Unit and Specific Activity. A unit of enzyme is defined as the amount producing a density decrease of 0.100 in a 10 minute interval. Specific activity is expressed as units per milligram of protein. Protein is measured by the method of Lowry et al. 3 Purification Procedure

Step 1. Preparation of Crude Extracts. Mycobacteria are cultured under essentially the same conditions as for the preparation of pyrimidine oxidase with the exception that uracil (0.1%) and glucose (0.2%) provide the sole nitrogen and carbon sources. The inclusion of glucose increases the yield of cells to about 1.5 g. (wet weight) per liter of culture medium and also increases the yield and specific activity of the enzyme three- to fourfold. Cell-free extracts prepared by grinding cells with three times the weight of dry alumina and extracting with phosphate buffer (0.02 M, pH 6.65) are lyophilized and stored at --10 °. Five hundred milligrams of lyophilized powder (obtained from 5.3 1. of culture medium) is dissolved in 20 ml. of distilled water, and insoluble material is centrifuged off and discarded. Step 2. Protamine Treatment. To the supernatant are added 20 ml. of phosphate buffer (0.02 M, pH 7.0) and 4 ml. of protamine sulfate (10 mg./ ml.). After 3 minutes, the precipitate is collected by centrifugation and extracted with 20 ml. of 0.5 M K2HPO~. The opalescent extract is diluted

[75]

PYB,IMIDINE OXIDASE AND RELATED ENZYMES

493

with 60 ml. of water. Removal of the resulting precipitate by centrifugation yields a clear, colorless solution (protamine fraction). Although this step yields little or no purification on a protein basis, it succeeds in removing essentially all the nucleic acid which is present in the cell-free extract. Step 3. Dowex Column Chromatography. Ten milliliters of the protamine fraction is adsorbed on a Dowex-1 formate column (8 cm. × 1 sq. cm.) and eluted with 0.1 M K2HPO4 at a rate of 0.3 ml./min. The eluate is tested for both urease and barbiturase activity, and the fraction between 18 and 22 ml. is observed to possess the highest specific activity of barbiturase and practically no urease activity; the urease activity appears in a later eluate. TABLE I SUMMARY OF PURIFICATION PROCEDURE

Fraction Crude extract Protamine treatment Dowex chromatography Eluate between 13.5 and 36.0 ml. Eluate between 18 and 22.5 ml.

Total activity units

Specificactivity, units/mg, protein

234 173

12.9 13.9

168 56

77.0 94.0

Properties Specificity. There is no action, as judged spectrophotometrically, on the following compounds: 5-methylbarbituric acid, orotic acid, barbital, pentobarbital, 2qthiobarbituric acid, or isobarbituric acid. Effect of pH. The enzyme exhibits a fairly sharp optimum for activity between p H 8 and 9. The Michaelis Constant. The K~ is approximately 3.37 X 10 -3 M. III. DihydroSrotic Dehydrogenase 5 0rotic Acid + D P N H + H + ~ DihydroSrotic Acid + D P N

Assay Method Principle. D P N H is generated b y the addition of glucose dehydrogenase, glucose, and D P N . The reaction is followed b y the decrease in optical density at 280 m~ in the Beckman D U spectrophotometer. Orotic acid absorbs strongly at this wavelength, whereas the product, dihydro5rotic acid, has no absorption. 5I. Lieberman and A. Kornberg, Biochim. et Biophys. Acta 12, 223 (1953).

494

ENZYMES OF NUCLEIC ACID METABOLISM

[75]

Reagents Glucose dehydrogenase, approximately 2500 units/ml., prepared according to the method of Strecker and Korkes.6 0.15 M MgCI:. 1 M potassium phosphate buffer, pH 6.1. 0.01 M sodium orotate. 0.1 M cysteine, pH 7.0. 0.001 M DPN. 1 M glucose.

Procedure. The test system contains 0.1 ml. of MgC12, 0.1 ml. of phosphate buffer, 0.2 ml. of glucose, 0.04 ml. of sodium orotate, 0.2 ml. of cysteine, 0.03 ml. of DPN, 250 units of glucose dehydrogenase, and the enzyme preparation in a volume of 3.0 ml. All the components except the glucose dehydrogenase are mixed and incubated at room temperature for 5 minutes. The glucose dehydrogenase is then added, and the rate of orotate removal is followed in the Beckman DU spectrophotometer by the decrease in optical density at 280 mp. Definition of Unit and Specific Activity. A unit of enzyme is defined as the amount producing an optical density decrease of 0.100 in a 6-minute interval. In general, not more than 4 units of activity is used for the assay. Specific unit is defined as units of activity per milligram of protein, as measured by the method of Lowry et al2 Under the conditions of the assay, the rate of orotate reduction is proportional to the amount of enzyme, and, in the absence of glucose, glucose dehydrogenase, or D P N , no removal of orotate is observed. Purification Procedure

Step 1. Preparation of the Cell-Free Extract. An obligate anaerobic bacterium, Zymobacterium oroticum, 7 is grown in a medium containing 2% tryptone, 0.05% Difco yeast extract, 0.2% orotic acid, and 0.05% sodium thioglycolate. For the preparation of stock test-tube cultures, the medium is adjusted to pH 7.0 with 1 M KOH prior to autoclaving (15 minutes at 15 pounds pressure). Anaerobic conditions are maintained with a pyrogallol-Na2CO3 seal. Large cultures are grown in Erlenmeyer flasks (1 to 6 1.) without an anaerobic seal. After autoclaving for 20 to 30 minutes at 15 pounds pressure, the medium is cooled, neutralized with a sterile 50% K2CO3 solution, and sterile water is added to the neck 6H. J. Strecker and S. Korkes, J. Biol. Chem. 196, 769 (1952) ; see Vol. I [44]. Available at the Department of Microbiology, Washington University School of Medicine, St. Louis~ Missouri.

[75]

PYRIMIDINE OXIDASE AND RELATED ENZYMES

495

of the flask. The inoculum (one or two fresh stock cultures) is added promptly. Growth appears to be complete in 16 to 20 hours at 30 ° . The cells are harvested in a Sharples supercentrifuge and resuspended in 0.01 M sodium orotate (7 ml./1, of culture), potassium phosphate buffer (1 M, pH 7.0, 0.4 re.l/1, of culture), and cysteine (0.1 M, pH 7.0, 0.4 ml./1, of culture). This cell suspension is incubated in vacuo at 26 ° for 20 minutes. More active extracts appear to be obtained when this step is included in the procedure. After centrifugation, the cells are suspended in ice-cold water (about 5 ml./1, of culture), and an aliquot of the suspension (about 6 ml.) is shaken with 6 g. of glass beads s (0.10 to 0.15 ram. in diameter) in a Mickle vibrator for 15 minutes at 2 °. The mixture is centrifuged in a Servall centrifuge (at ca. 10,000 X g), and the precipitate is washed once with cold water. The volume of the combined cell-free extract is adjusted to 10 ml./1, of culture. If the purification is not init ated at once, the extract is acidified to pH 6.0 with 2 M sodium acetate buffer (pH 6.0) and stored at - 1 0 °. Step 2. Protamine Fraction. Purification of the enzyme is carried out at 0 to 2 °. One hundred milliliters of freshly prepared cell-free extract is diluted with an equal volume of water, and 15 ml. of a 1% solution of protamine sulfate (Eli Lilly) is added with stirring. After 5 minutes, the precipitate is collected by centrifugation and the supernatant solution discarded. One hundred milliliters of sodium citrate buffer (0.5 M, pH 6.0) is added to the hard and difficultly soluble precipitate. After 12 to 24 hours, the softened precipitate is dissolved to a considerable extent by homogenization with a glass pestle, 200 ml. of water is added with stirring, and the resultant stringy precipitate is discarded after centrifugation. The supernatant solution (protamine fraction) is essentially free of nucleic acid as indicated by the ratio of optical densities at 280 and 260 m~ (0.98). Step 3. Ammonium Sulfate Fraction. To the protamine fraction is now added, with stirring, 100 g. of ammonium sulfate. After 5 minutes the precipitate is removed by centrifugation, and on further addition of 25.2 g. of ammonium sulfate to the supernatant solution another precipitate is formed. This precipitate is collected by centrifugation and dissolved in 120 ml. of water (ammonium sulfate fraction). Step 4. Acid Ammonium Sulfate Fraction. Thirty milliliters of sodium formate buffer (0.5 M, pit 4.2) and then 36 g. of ammonium sulfate are added to the ammonium sulfate fraction with stirring. After 5 minutes the precipitate is removed by centrifugation, 18 g. of ammonium sulfate is added to the supernatant solution, and the precipitate that forms is 8 Obtained from Minnesota Mining and Manufacturing Company, St. Paul, Minnesota.

496

ENZYMES OF NUCLEIC ACID METABOLISM

[75]

collected and dissolved in 62 ml. of sodium acetate buffer (0.01 M, pH 6.0)(acid ammonium sulfate fraction). Purification at this stage is about eightfold with an over-all yield of about 40 %. Further purification of the enzyme (three- to fivefold) could be obtained by subjecting the acid ammonium sulfate fraction to column chromatography with Dowex-1 (formate, 2% cross-linked), and eluting with phosphate buffer. The yield was too variable to warrant inclusion of this step in the routine purification procedure. TABLE II SUI~I~IARY OF PURIFICATION PROCEDURE

Fraction Cell-free extract Protamine Ammonium sulfate Acid ammonium sulfate

Total activity, units

Specificactivity, units/rag, protein

4590 3180 2420 1850

22.8 26.5 84.1 175

Properties

Substrate Specificity. No activity and no inhibition of orotate reduction is observed with uracil, cytosine, 5-methylcytosine, or thymine. Coenzyme Specificity. With TPN, the reaction rate is less than 2 % of that observed with DPN, and no inhibitory effect on the reaction with DPN is observed. Activators and Inhibitors. Although Mg ion has little effect on the initial rate of the reaction, an effect is apparent when the reaction proceeds for longer periods. Thus with 0, 2 × 10-3 M, and 5 × 10-3 M Mg ++, the decreases in optical density at 280 mu in 6 minutes are found to be 0.225, 0.240, and 0.261, respectively; at 20 minutes the density decreases are 0.355, 0.426, and 0.503, respectively. Cysteine. Freshly prepared cell-free extracts show little or no stimulation on the addition of cysteine. The reaction rate with purified enzyme preparation, however, is increased up to twofold in the presence of 0.002 to 0.007 M cysteine. Larger amounts of cysteine seem to have an inhibitory effect. Effect of pH. The optimum pH for the reaction is around 6.5. At pH 5.5 and 7.8, the initial rates of reaction were 13 and 65%, respectively, of that at pH 6.5. Further Metabolism of Dihydroiirotic Acid 9 DihydroSrotie acid is further hydrolyzed by the action of an enzyme, dihydroSrotase, to yield ureidosuccinic acid. Ureidosuccinic acid is reey9I. Lieberman and A. Kornberg~ J. Biol. Chem. in press.

[76]

ADENOSINE PHOSPHOKINASE

497

clized to 5-(acetic acid)-hydantoin; the enzyme responsible for the latter reaction is referred to as 5-(acetic acid)-hydantoinase. Ureidosuccinic acid is also converted to aspartic acid, NH3, and CO2 by an enzyme system referred to as ureidosuccinase. All these reactions are shown to be reversible, except the last-mentioned one, but only ureidosuccinase has been purified.

[76] A d e n o s i n e P h o s p h o k i n a s e Adenosine + ATP--~ 5'-AMP + ADP 2-Aminoadenosine + ATP --~ 2-Amino-5'-AMP + ADP

By ARTHUR KORNBERG Assay Method Principle. The phosphorylation of the nucleoside is initiated with small quantities of ATP which are immediately regenerated by the action of pyruvate phosphokinase (added in large excess) on phosphopyruvate. In the presence of myokinase, this leads to the accumulation of the nucleoside mono-, di-, and triphosphates. adenosine kinase Adenosine + A T P - - - ~ 5'-AMP ~- ADP pyruvate phosphokinase Phosphopyruvate + ADP ) Pyruvate ~- ATP myokinase 5'-AMP + ATP ~ * 2 ADP The amount of ADP and ATP formed is estimated spectrophotometrically as follows: hexokinase ATP ~- glucose ) Glucose-6-P + ADP myokinase 2 ADP ~ ~ ATP + 5-AMP glueose-6-phosphate dehydrogenase Glueose-6-P q- TPN ) 6-Phosphogluconate + TPNH Thus, for every mole of ATP present 2 moles of TPN are reduced, and for every mole of ADP present 1 mole of TPN is reduced. The extent of TPN reduction can be measured at 340 mg; the molecular extinction coefficient for T P N H of 6.22 X 106 sq. cm./mole is employed. 1 1B. L. Itoreeker and A. Kornberg, J. Biol. Chem. 175, 385 (1948).

498

ENZYMES OF NUCLEIC ACID METABOLISM

[76]

Reagents Succinate buffer (0.33 M, pH 6.0). MgC12 (0.1 M). MnC12 (0.02 M). Glutathione (0.16 M). ATP (0.002 M). Phosphopyruvate (0.06 M). Myokinase. 2 Pyruvate phosphokinase-(acetone I). 3

Procedure. The incubation mixture contained 0.1 ml. of succinate buffer, 0.05 ml. of adenosine, 0.01 ml. of MgC12, 0.01 ml. of MnC12, 0.02 ml. of glutathione, 0.01 ml. of ATP, 0.025 ml. of phosphopyruvate, 0.02 ml. of myokinase, 0.05 ml. of pyruvate phosphokinase, adenosine phosphokinase, and water to a final volume of 0.50 ml. After 20 minutes at 21 to 23 °, the incubation mixture was placed in a boiling water bath for 3 minutes, centrifuged, and an aliquot of the supernatant fluid analyzed for ADP and ATP. 3,4 Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which leads to the accumulation of 1 pM. of kinaselabile phosphate during the test period. (ADP contains 1 mole of kinaselabile phosphate, and ATP contains 2.) Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al. ~ Purification Procedure 3

Autolyzates of dried baker's yeast and of several dried beer and ale yeasts were all found to be active. Twenty-five grams of dried lager beer yeast, which yielded the most active autolyzate, was suspended in 75 ml. of 0.1 M sodium bicarbonate and incubated for 6 hours at 34 °. The mixture was centrifuged and yielded approximately 40 ml. of clear yellow autolyzate (51.2 units/ml., 1.1 units/mg, of protein). All subsequent operations were performed at 2 °, unless otherwise specified. To 40 ml. of autolyzate were added 40 ml. of water, 20 ml. of 1 N acetic acid, and then 8 ml. of nucleic acid solution (Merck, 50 mg./ml., pH 5.0). After 5 minutes, 40 ml. of 2 N acetic acid was added, and the precipitate was removed by centrifugation. The pH of the clear supernatant, which was S. P. Colowick and H. M. Kalckar, J. Biol. Chem. 148, 117 (1943); see Vol. I I [99]. 3 A. Kornberg and W. E. Pricer, Jr., J. Biol. Chem. 193, 481 (1951); see Vol. I [66]. 4 A. Kornberg, J. Biol. Chem. 182, 779 (1950). 50. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 198, 265 (1951); see also Vol. I I I [73].

[76]

ADENOSINE PHOSPHOKINASE

499

approximately 4.4, was raised to 6.3 by the addition of about 12 ml. of 2 N NaOH. Salmine sulfate (Lilly, 100 mg. in 4 ml.) was added; 5 minutes later the precipitate was removed by centrifugation, yielding 120 ml. of supernatant (12.6 units/ml., 3.8 units/mg, of protein). The pH was adjusted to 5.1 with 7.0 ml. of 2 N acetic acid, the solution cooled to - 0 . 5 °, and ethanol added with mechanical stirring. The temperature was maintained just above the freezing point during the early ethanol addition and at - 5 ° thereafter. The precipitates were centrifuged at - 5 °. Forty-five milliliters of 50% ethanol and then 7.0 ml. of zinc chloride (0.5 M) were added. After 5 minutes the precipitate was removed by centrifugation. To the supernatant was added 10 ml. of 50 % ethanol, and after 5 minutes the precipitate was again removed by centrifugation. The addition of 60 ml. of ethanol to the supernatant produced a precipitate which was collected by centrifugation and dissolved in citrate buffer (0.05 M, pH 6.2) to a volume of 50 ml. (14.5 units/ml., 13.1 units/mg, of protein). Lyophilization yielded 930 mg. of a white powder which, when stored in a vacuum desiccator over CaCl~ at 2 °, was stable for eight months or more. (In the liquid state at 2 ° about one-third of the activity was lost overnight.) The dry preparation was used throughout these studies. The optical density, at 280 mtL in a light path of 1 cm., of a purified enzyme solution containing 1 mg. of protein per milliliter was 6.4. The ratio of the density at 280 m~ to that at 260 m~ was 0.53, a value corresponding to that of nucleic acid. Efforts to remove this nucleic acid, as by the use of metal salts, protamine, and ion exchange resins, were unsuccessful. Properties Adenosine phosphokinase has also been demonstrated by Caputto 6 to be present in liver and kidney. Specificity. A large number of related nucleosides were found to be inactive under conditions which resulted in the phosphorylation of adenosine and 2-aminoadenosine (Table I). (In this experiment the amount of pyruvate released from phosphopyruvate was a measure of the ability of a nucleoside to serve as a phosphate acceptor.) These compounds, when tested at equimolar levels in the presence of adenosine, showed only slight inhibitory effects (13 to 30%) on the rate of adenosine phosphorylation. Other Properties. Rates of adenosine phosphorylation by ATP, estimated by adenosine removal, were maximal at the lowest substrate concentrations which could be conveniently tested. The levels were 5 X 10-4 M for ATP and 2 × 10-4 M for adenosine. I t is noteworthy that, 6 R. Caputto, J. Biol. Chem. 189, 801 (1951).

500

[76]

ENZYMES OF NUCLEIC ACID METABOLISM

TABLE I 0.8 ~M. of test compound was used. The values are expressed as micromoles of pyruvate formed, corrected for the blank. Results with the test compounds are compared with those obtained with adenosine in the same experiment. (We are indebted to Dr. J. Davoll and Dr. G. B. Brown for many of the nucleosides used in this study.) Test compound 2-Oxy-9-~-D-ribofuranosyladenine (crotonoside) 9-~-D-2-Deoxyribofuranosyladenine 9-f~-D-Ribopyranosyladenine 9-f~-D-Glucopyranosyladenine 9-a-D-Arabofuranosyladenine 9-a-L-Arabofuranosyladenine 2,6-Diamino-9-~-D-ribofuranosylpurine 2,6-Diamino-9-~-D-xylofuranosylpurine 2-Chloro-9-f~-D-ribofuranosyladenine 2,8-Dichloro-9-~-D-ribofuranosyladenine 2-Methylthio-9-/~-D-ribofuranosyladenine 2-Acetamido-9-f~-D-ribofuranosyladenine 6-Oxy-9-fl-D-ribofuranosylpurine (inosine) 2-Amino-6-oxy-9-f~-D-ribofuranosylpurine (guanosine) Uridine Cytidine D-Ribose Yeast-adenylic acid

0.02, 0.00 0.00 0.00 0.00 0.00 0.22, 0.00 0.05, 0.03, 0.00 0.00 0.00 0.00 0.00 0.02 0.00 0.00

0.00

0.26 0.05 ~ 0.01

Adenosine 1.52, 1.41 0.91 1.18 1.18 1.34 1.34 1.41 1.34 0.68, 1.28 0.68, 1.28 0.68 1.18

0.91 1.35 1.35 1.35 0.62 1.49

This value may be attributed to an impurity of adenosine; prolonged incubation resulted in no increase of this value. when adenosine phosphokinase a n d m y o k i n a s e , t h e r e m o v a l of c o m p l e t e d , d e p e n d i n g on w h i c h M g ++ w a s r e q u i r e d a n d w a s

is c o u p l e d w i t h p y r u v a t e p h o s p h o k i n a s e a d e n o s i n e or p h o s p h o p y r u v a t e is r e a d i l y s u b s t a n c e is l i m i t i n g . p a r t i a l l y r e p l a c e d b y M n ++ ( T a b l e I I ) .

TABLE I I Mg ++ REQUIREMENT OF ADENOSINE PHOSPHOKINASE (The results are in micromoles.) Mg ++,M X 10a Mn ++, M × 108 A-Adenosine

0.33 0.29

0.54

1.7 0.62

3.3 0.70

0.33

1.7

3.3

1.7 0.33

0.46

0.55

0.50

0.62

T h e p H o p t i m u m of t h e r e a c t i o n d e t e r m i n e d e i t h e r w i t h y e a s t a u t o l y z a t e s ( p h o s p h o p y r u v a t e as d o n o r ) or w i t h p u r i f i e d a d e n o s i n e p h o s p h o k i n a s e ( A T P as d o n o r ) w a s a p p r o x i m a t e l y 6.0; a b o u t 50 % of t h e a c t i v i t y w a s o b s e r v e d a t p H 5.0 a n d 7.0. C i t r a t e b u f f e r w a s i n h i b i t o r y .

[77]

NUCLEOTIDE SYNTHESIS BY TISSUE EXTRACTS

501

[77] Nucleotide Synthesis by Tissue Extracts Adenine -4- R-5-P -]- ATP--~

AMP -I- ADP -~- P or 2AMP -[- PP

By MURRAY SAFFRAN

The formation of 5-adenylic acid from adenine, ribose-5-phosphate (R-5-P), and ATP proceeds in two steps. In the first step, R-5-P is phosphorylated by ATP. In the second step, the phosphorylated R-5-P reacts with adenine to form 5-adenylic acid. Saffran and Scarano 1 have suggested that R-5-P is phosphorylated to ribose-l,5-diphosphate; Kornberg et al. 2 have evidence for the formation of 5-phosphoribosyl pyrophosphate.

Assay Method Principle. The incorporation of C14-adenine into the acid-soluble nucleotide fraction is determined by incubating the tissue preparation with 8-Cl~-adenine, R-5-P, and ATP, precipitating the proteins with perchloric acid, isolating the acid-soluble nucleotides by barium-alcohol precipitation, washing with neutral ethanol, and determining the radioactivity in the nucleotides. Reagents

8-C~4-adenine. Dissolve about 1 mg. (about l0 Gcounts per minute) of 8-C14-adenine'HC1 (prepared according to Clark and Kalckar 3) in 1 ml. of water. Store at - 2 0 °. 0.05 M Ribose-5-phosphate. Suspend about 250 rag. of Ba ribose5-phosphate t in 4 ml. of water, and dissolve with about 25 #1. of concentrated H2SO4. Spin down the undissolved solid, and carefully remove the supernatant. Neutralize the supernatant by careful addition of 4 M KOH. Analyze the solution for ribose by the orcinol reaction, s and dilute to 0.05 M. Store the solution at - 2 0 °. 0.05 M Na ATP. 6 10% perchloric acid. 1 M. Saffran a n d E. Scarano, Nature 172, 949 (1953). 2 A. Kornberg, I. Lieberman, and E S. Simms, J. Am. Chem. Soc. 76, 2027 (1954). 3 V. M. Clark a n d H. M. Kalckar, J. Chem. Soc. 1950, 1029. 4 Available commercially or m a y be easily prepared; see Vol. I I I [28]. 5 See Vol. I I I [13]. 6 Available commercially.

502

ENZYMES OF NUCLEIC ACID METABOLISM

[77]

Concentrated ammonia. 25% solution of Ba(OAc)~. Ethanol brought to neutrality with concentrated ammonia. 1 M HC1. 0.25 saturated (NH4)2S04. Tissue extract. Active extracts of pigeon liver and breast muscle are consistently obtained by the following procedure. Homogenize the tissue in the cold in 10 vol. of a medium consisting of 0.13 M KC1, 0.015 M KH2PO4, and 0.01 M MgC12, brought to pH 7.2 with 1 M K O H (about 12.5 ml./1, of medium). Spin the homogenate at 80,000 X g 7 in the cold, and dialyze the particle-free supernatant against four changes of the homogenizing medium in the cold room. It is convenient to allow the extract to dialyze overnight against the last change of medium. Divide the dialyzed extract into small volumes, and freeze. Store at - 2 0 °. The frozen extracts retain activity for a few weeks. Procedure. Thaw the tissue extract. Pipet 1 ml. of the extract into a centrifuge tube containing 10 ul. of 8-C14-adenine, 50 ul. of 0.05 M R-5-P, and 50/~l. of 0.05 M ATP. Incubate at 37 ° for 1 or 2 hours. Chill the tubes in ice for a few minutes, and add 300 ul. of 10 % HC104. After 5 minutes, spin down the proteins. Remove a 1-ml. aliquot of the supernatant liquid to another centrifuge tube, and neutralize with concentrated NH~. Add an excess (100 ~l.) of 25% Ba(OAc)2 and 4 ml. of neutralized ethanol. Stir with a thin glass rod. Let stand in the cold for 10 minutes, then spin. Decant, and discard the supernatant liquid. Wash the precipitate twice with 2 ml. of neutralized ethanol, spinning the precipitate down well after each wash. Suspend the precipitate in 400 ul. of H20. Add 100 ul. of 1 M HC1 to dissolve the precipitate. Precipitate the Ba ++ with 50 ul. of 0.25 saturated (NH4)2SO4, testing the supernatant liquid with a trace of (NH4)2SO4 after spinning to ensure that all the Ba ++ is removed. Plate 100 ul. of the supernatant liquid, and determine the radioactivity. Purification Procedure

Scarano s has described the partial purification, by alkaline (NH4)2SO4 fractionation, of the enzyme that phosphorylates R-5-P. Kornberg et al." have reported the preparation of the enzymes involved in the synthesis of nucleotides. However, these methods of concentrating the activities of either the R-5-P-phosphorylating enzyme or of the nucleotide-forming enzyme have not been described in detail. Williams and Buchanan 9 have outlined procedures for concentrating the enzymes needed for the 7 A Spinco p r e p a r a t i v e ultracentrifuge was used. s E. Scarano, Nature 172, 951 (1953). 9 W. J. Williams a n d J. M. B u c h a n a n , J. Biol. Chem. 203, 583 (1953).

[77]

NUCLEOTIDE SYNTHESIS BY TISSUE EXTRACTS

503

incorporation of hypoxanthine into IMP, in the presence of R-5-P and ATP. These enzymes may or may not be identical with those that form other nucleotides. Their preparation is described as an example of the concentration of a nucleotide-forming system. The following scheme is taken from Williams and Buchanan2 Step 1. Preparation of Crude Extract. Decapitate and bleed several pigeons. Excise the livers quickly, chill in cracked ice, and free them from gross connective tissue. Mince the liver with scissors and measure the mince by displacement of homogenizing medium in a graduated cylinder. Homogenize 1 part of the mince in 1.5 parts of a medium consisting of 0.035 M sodium phosphate buffer (pH 7.4), 0.13 M KC1, 0.04 M KHCO~, and 0.01 M MgC12. Spin the homogenate for 30 minutes at approximately 100,000 X g. Remove the supernatant carefully into other vessels. The above steps are carried out in the cold, with ice-cold medium and chilled instruments and containers. Step P. Fractionation with Ethanol. Fraction 1. Chill the particle-free extract to 0 ° in a dry ice-acetone bath. Add slowly 90% ethanol to 15% concentration by volume. During the addition of the ethanol, maintain the temperature of the extract-ethanol mixture just above the freezing point by cooling in the dry ice-acetone bath. Spin off the precipitate (fraction 1) in the cold, and keep at - 1 5 ° until lyophilized. Step 3. Fractionation with Ethanol. Fraction 2. To a second aliquot of the pigeon liver extract prepared by step 1, add ethanol slowly, as in step 2, to a concentration of 20 %. Spin down the precipitate. Decant off the supernatant carefully into another container. Discard the precipitate. Increase the ethanol concentration of the supernatant to 45% by the slow addition of 90% ethanol. Spin down the precipitate (fraction 2), and keep at - 1 5 ° until lyophilized. During the addition of ethanol in this step, maintain the temperature of the mixture just above the freezing point, as in step 2. Step 4. Lyophilization, Storage, and Use. Dissolve each fraction in small quantities of distilled ~ater. Lyophilize the solutions, maintaining the apparatus at - 1 5 °. Keep the lyophilized powders at - 1 5 ° ; they are stable for at least several days. Just before use, dissolve the dried powder in the incubating medium in a volume equal to one-half the volume of the original pigeon liver extract.

Properties Specificity. The crude extract will form nucleotide from adenine, ATP, and R-5-P or R-l-P, hut the purified enzyme will not utilize the 1-ester. 2,s Ribose-l,5-diphosphate, made by the system phosphoglucomutase

Glucose-l,6-P2 + R-1-P ¢__

) R-1,5-P2 ~- glucose-6-P

504

ENZYMES OF NUCLEIC ACID METABOLISM

Glucose-6-P + TPN

Zwischenferment

[78]

) 6-Phosphogluconic acid + T P N H + H +

will replace R-5-P and ATP in the dialyzed extract.1 The base specificity of the system has not been explored. Hypoxanthine,9 adenine, 1,2 and orotic acid 2 have been reported to be incorporated into nucleotides by similar systems. Activators and Inhibitors. The unfractionated system requires Mg ++ and P O t - - - for activity. The optimal concentration of Mg ++ is about 0.01 M. In the crude extract, the optimal concentration of adenine is about 0.002 M. Fluoride has no appreciable effect on the system. In the presence of low concentrations of R-5~P, adenosine phosphates decrease the rate of incorporation of adenine. Stability to Heat. The R-5-P phosphorylating enzyme is heat sensitive and is inactivated by heating at 60 ° for 5 minutes. The adenine-incorporating activity is much more resistant to this treatment. The activity of fraction 2 in the IMP-forming system also survives heating at 60 ° for 5 minutes. 1° Other properties of the IMP-forming enzymes have not been described. ~0E. D. Korn and J. M. Buchanan, Federation Proc. 12, 233 (1953).

[78] Some Methods for the Study of de Novo Synthesis of P u r i n e Nucleotides B y DAVID A. GOLDTHWAIT a n d G. ROBERT GREENBERG

A soluble enzyme system capable of synthesizing a purine nucleotide de novo can be found in pigeon liver extracts. A tentative scheme for the biosynthesis of inosine-5'-phosphate may be outlined as follows: P--O--P--O

H

\/

Ribose-5-P

C

i -[- glutamine

A+ TP

H CIO H

I ATP, glycine o I ]

I

HCOH

I

HC I

CH20--P 5-Phosphoribosylpyrophosphate (I)

H,C--NH2 CI

O

NH

\

Ribose-5-P

Glycinamide ribotide (II)

[78]

de Novo SYNTHESIS OF PURINE NUCLEOTIDES

H--C--N

H2C--NH--CHO

t

ATP

glutamine

O

~

--~

C

" 1-C unit"

/

NH

/

\

Ribose-5'-P Formylglycinamide ribotide (III)

NH2

0

/\

+C02

--* aspartic acid

\

Ribose-5'-P 5-Amino imidazole ribotide (IV) OH C

C--N

/

C--N

CH

I

II

C

H2N

505

C~N

N

%CH / \

ATP ) "1-C unit"

H2N Ribose-5'-P 5-Amino-4-imidazoleearboxamide ribotide (V)

C--N

% CH / HC C--N %/ \ N Ribose-5'-P Inosine-5'-phosphate (VI)

Discussion of the methods of studying purine biosynthesis will be divided into six sections: (1) over-all de novo synthesis of inosinic acid (VI) by extracts of an acetone powder of pigeon liver; (2) synthesis of formylglycinamide ribotide and glycinamide ribotide (II, III) by the same preparation; (3) isolation of 5-amino-4-imidazolecarboxamide riboside from E . coli cultures; (4) conversion of 5-amino-4-imidazolecarboxamide riboside to the ribotide by a yeast enzyme; (5) activation of the l-carbon unit as a folic acid derivative; and (6) transformylation of this active l-carbon unit to glycinamide ribotide (II) and to 5-amino-4imidazole-carboxamide ribotide (V). de Novo Synthesis of Inosinic Acid P r i n c i p l e . Because of the large endogenous production of purine nucleotides in this system, it is necessary to use the incorporation of a radioactive precursor as the index of de novo synthesis. One of the simplest methods of determining purine synthesis is the incorporation of C 14 formate into a non-acid-hydrolyzable fraction. This is measured by counting a dried aliquot of the reaction filtrate as an infinitely thin layer in a glass planchet. The synthesis of methionine and serine in this preparation under these conditions is negligible. If there is incorporation of C14-formate, the hypoxanthine moiety may be isolated either by crystallization of the silver nitrate salt or by ion exchange chromatography, and the C ~4 activity of the purified material determined. The molecule may

506

ENZYMES OF NUCLEIC ACID METABOLISM

[78]

be degraded by sulfuric acid hydrolysis to obtain C-2 and C-8 together, and C-6, separately.

Reagents C14-sodium formate, 0.1 M. Specific activity approximately 10,000 counts/min./micromole as an infinitely thin layer using an endwindow (2 mg./cm. ~) counter. Glutamine, 0.2 M (Nutritional Biochemical Corp., Cleveland, Ohio). Ribose-5-phosphate, 0.05 M potassium salt. Dissolve 7 millimoles of barium ribose-5-phosphate (Schwarz Laboratories, Inc., N. Y.) in 15 ml. of water and 7 ml. of 1 N HC1 solution. Pass this through a Dowex 50 column (H + form, 1.2-cm. diameter X 10 cm.), and wash the column with 50 ml. of water. Take an aliquot for pentose analysis I with 5-AMP as a standard. The solution is neutralized with KOH and diluted with water to make a 0.05 M solution. 3-Phosphoglyceric acid, potassium salt, 0.14 M. Dissolve the commercial barium salt (Schwarz Laboratories, Inc., N. Y.) in hydrochloric acid solution. Remove the barium ion either by precipitation as BaSO4 or by passage through a Dowex 50 column (H + form), and neutralize the solution with KOH. ATP, 0.04 M (Pabst Laboratories, Milwaukee, Wis.), disodium salt neutralized with NaOH. MgCl~, 0.1 M. KHCO3, 1.0 M. Glycine, 0.1 M. DL-Homocysteine, 0.1 M (General Biochemical Inc., Chagrin Falls, Ohio). Dissolve in water immediately prior to use. Boiled extract of pigeon liver. Mince 25 g. of fresh pigeon liver, place immediately in 50 ml. of boiling distilled water in a small metal Waring blendor, and homogenize for 2 minutes while the blendor is heated from the side with a small burner. Centrifuge the homogenate for 30 minutes at 5000 X g, decant the supernatant fluid, and store at - 1 3 °. Procedure. Incubation mixture: C14-Na formate 0.05 ml., glutamine 0.05 ml., ribose-5-phosphate 0.10 ml., 3-phosphoglycerate 0.20 ml., ATP 0.05 ml., MgCl~ 0.05 ml., KHCO8 0.05 ml., glycine 0.05 ml., homoeysteine 0.05 ml., boiled extract 0.2 ml., enzyme 0.5 ml. Incubation time, 30 minutes at 38 °. The reaction is stopped by the addition of 0.5 ml. of 20% trichloroacetic acid. Approximately 0.2 micromole of C14-formate is incorporated into hypoxanthine. i W. M e j b a u m , Z. physiol. Chem. 258, 117 (1939).

[78]

de Yoyo

SYNTHESIS OF PURINE NUCLEOTIDES

Analytical Methods.

DETERMINATION

OF

MEASUREMENT OF FIXATION OF C14-FORMATE.

PURINE

507

SYNTHESIS

Hydrolyze0 . 2

BY

m l . of t h e

trichloroacetic acid filtrate by adding 0.1 ml. of 3 N HC1 and heating at 100 ° for 15 minutes. Dilute to 2.0 ml. with water, and pipet a 0.5-ml. aliquot into a glass planchet (Tracerlab, Inc.). Add 5 drops of 95% ethanol, and take carefully to dryness under an infrared lamp. DETERMINATION OF PURINE SYNTHESIS BY ISOLATION OF THE SILVER

aliquot of t r i c h l o r o a c e t i c acid filtrate, add 1.0 ml. of water and 0.8 ml. of 3 N HNO3, heat at 100 ° for 45 minutes, and then add 3 ml. of carrier hypoxanthine solution (5 mg./ml, in dilute nitric acid). Neutralize with KOH to a bromothymol blue end point, and add 1 drop of 0.1 N HNO3. After addition of 1.0 ml. of 0.2 M AgNO3, allow to stand at 0 ° with occasional stirring for 20 minutes. Centrifuge. Dissolve the precipitate in 7 ml. or less of 17% HNO3 [1 vol. of COliC. HN03 (69 to 71%) and 3 vol. of water] by heating at 100 °, filter off silver chloride at 100 °, and allow the silver nitrate salt of hypoxanthine to crystallize at 0 ° for 20 minutes. Centrifuge, and wash the precipitate once with 1.0 ml. of cold 17 % HNO3. Dissolve the precipitate in 5 ml. of 17% nitric acid at 100 °, and recrystallize at 0 °. Wash the precipitate again, as before. Repeat the recrystallization and washing. After the final recrystallization wash the precipitate twice with 1.0 ml. of cold water and suspend it in 1.5 ml. of water. Decompose the silver nitrate salt by vigorous bubbling of H2S through the suspension for 15 minutes. Aerate for 15 minutes, filter off the precipitate, and dilute the filtrate to a final volume of 5 ml. Pipet 1.0 ml. of this into a glass planchet, and dry for counting. Dilute a 0.1-ml. aliquot to 10 ml. with potassium phosphate buffer (0.02 M, pH 7.0), and read in the Beckman spectrophotometer at 248 m~. Calculate the concentration of hypoxanthine using a molecular extinction coefficient of 10,500. Then calculate the specific activity of the hypoxanthine which was counted as well as the total amount of hypoxanthine synthesized per reaction vessel. NITRATE SALT OF HYFOXANTHINE. TO a 0 . 5 - m l .

DETERMINATION OF PURINE SYNTHESIS BY ISOLATION OF HYPOXANTHINE ON D O W E X 5 0 COLUMNS AND DEGRADATION BY SULFURIC ACID

HYDROLYSIS. One milliliter of trichloroacetic acid filtrate plus 1 ml. of 2 N HC1 solution is heated at 100 ° for 30 minutes to hydrolyze the purine nucleotides. Five milligrams of carrier hypoxanthine (5 mg./ml, of 1.2 N HC1) is added, and the solution is put through a Dowex 50 column (H + form, 12% cross linkage, 50 to 100 mesh, 1.5-era. diameter × 7 cm.). The hypoxanthine is eluted with 1.2 N HC1 and appears in the fractions between 75 and 225 ml. These fractions are combined and diluted to 200 ml., a 1.0-ml. aliquot is neutralized, diluted to 10 ml., and the concentration of hypoxanthine is determined from the optical density at

508

ENZYMES OF NUCLEIC ACID METABOLISM

[78]

248 mu. T o the hypoxanthine isolated by the above method, hypoxanthine carrier is added to make a total of 0.2 millimole. The solution is taken to dryness in the degradation vessel (Fig. 1, C) on a steam bath. A degradation train is set up as in Fig. 1. T o the dry hypoxanthine sample in vessel C, 100 ml. of sulfuric acid (7 Vol. of conc. H2S04 plus 3 Vol. of water) is added. The vessel is heated to 150 ° and aerated by the application of v a c u u m to the outlet on F for 15 minutes. The v a c u u m is then applied to the bubbler, I, the t e m p e r a t u r e is raised to 192 to 197 °, and

i

O

E

F

G

H

I

FIG. 1. Degradation apparatus. A, U tube containing Ascarite; B, mercury valve; C, reaction vessel; D, condenser; E, Cellosolve-dry ice trap or a bead tower containing

1 N H~SO4 solution; F, U tube containing anhydrous magnesium perchlorate; G, bubbler containing 3 ml. of 2 N COs-free NaOH, and drying tube with anhydrous magnesium perchlorate; H, U tube with iodic sulfate (2); I, bubbler similar to G. For other details of purine and pyrimidine determination, see E. D. Korn, Vol. IV [26]. aeration is continued at this t e m p e r a t u r e for 30 minutes. T h e bubblers are detached, weighed, and aliquots of the alkali solutions are taken for plating as BaC03 and counted. The C02 collected in bubbler G arises from the 6-carbon (yield, 0.2 millimole); the C02 in bubbler I is derived from the iodic sulfate ~ oxidation of the CO arising from carbons 2 and 8 (yield, 0.4 millimole). Experiments with isotopically labeled adenine 3 indicate t h a t this acid hydrolysis does not result in randomization of C-4 and C-6 similar to t h a t reported to occur with uric acid. 4 Preparation of Extract. Adult pigeons (Palmetto Pigeon Plant, Sumpter, S.C.) are decapitated, their livers removed, washed, chilled in ice water, and weighed. Fifty grams of liver is homogenized in 500 ml. of M. Schutze, Ber. 77B, 484 (1944).

a W. H. Marsh, Doctoral Thesis, Western Reserve University, 1951. 4 C. E. Dalgliesh and A. Neuberger, J. Chem. Soc. 1954, 3407.

[78]

de Novo SYNTHESIS OF PURINE NUCLEOTIDES

509

acetone (Merck, reagent, - 1 3 °) in a Waring blendor at full speed for 30 seconds at - 13°. The acetone solution is filtered off through a Biichner funnel, and the cake is rehomogenized with 250 ml. of acetone for 15 seconds and again collected. The homogenization with 250 ml. of acetone is repeated once. The powder is partially dried on the Bfichner funnel and then placed i n vacuo at 4 ° over concentrated }I2SO4 for 1 day. The connective tissue is screened out, and the remaining acetone powder (12.5 g.) is stored i n vacuo at - 1 3 °. One gram of acetone powder is extracted with 10 ml. of 0.05 M K2HPO4 by slow stirring at 0 ° for 30 minutes. After centrifugation at 5000 × g for 20 minutes at 0 °, the supernatant solution is used immediately in the reaction mixture, or it may be lyophilized. The protein concentration varies between 30 and 40 mg./ml. Further details of the de novo synthesis of inosinic acid may be found in the references listed, s-7

Biosynthesis of Formylglycinamide Ribotide and Glycinamide Ribotide s,9 Principle. The formyl group of the formylglycinamide ribotide is readily hydrolyzed by heating with dilute acid. Therefore the synthesis of this compound can be estimated by the incorporation of C14-formate into an easily acid-hydrolyzable form. The biosynthesis of the formylglycinamide ribotide requires a folie acid derivative. Glycinamide ribotide accumulates if the extract is treated with Dowex 1 to remove the folic acid derivative. The formation of the glycinamide ribotide is estimated by employing glycine-l-C 14 as a precursor. The amide linkage in glycinamide ribotide is not affected by heating to 100° at pH 5.4. Therefore, on treatment of an aliquot of the reaction filtrate with ninhydrin, the residual glycine-l-C TM radioactivity is converted to C1402, and the remaining radioactivity represents synthesis of the glycinamide ribotide (or formylglycinamide ribotide). Procedures are also described for the separation of the ribotides on an anion exchange column and the preparation of crude barium salts of these compounds. Reagents

Glycine-l-C TM, 0.1 M, 15,000 to 20,000 eounts/min./micromole. 5 G. R. Greenberg, J. Biol. Chem. 190, 611 (1951). 6 j. M. Buchanan, J. Cell. Comp. Physiol. 38, Suppl. 1, 143 (1951). 7 j. M. Buchanan, B. Levenberg, J. G. Flaks and J. A. Gladner, "Amino Acid Metabolism," p. 743, Johns Hopkins Press, Baltimore, 1955. s D. A. Goldthwait~ R. A. Peabody, and G. R. Greenberg, J. Am. Chem. Soc. 76, 5258 (1954). 9 S. C. Hartman, B. Levenberg, and J. M. Buchanan, J. Am. Chem. Soc. 77, 501

(1955).

510

ENZYMES OF NUCLEIC ACID METABOLISM

[78]

Leucovorin, 4 mg. of the calcium salt (American Cyanamide Co.) per milliliter of water. This solution is stable in the frozen state. Tetrahydrofolic acid. Prepare according to O'Dell et al. lo as modified by Jaenicke and Greenberg. ~1 Dissolve 3 mg. of the free acid of tetrahydrofolic acid in 1 ml. of 0.05 M KHCOa and 0.2 % E D T A under petroleum ether, and keep at 0 °. It is best to make the solution fresh for each assay. Other reagents are described in the section "De Novo Synthesis of Inosinic Acid." FORMYLGLYCINAMIDE RIBOTIDE. Procedure. Incubation mixture: C14-Na formate 0.05 ml., glutamine 0.05 ml., glycine 0.05 ml., ribose-5phosphate 0.10 ml., ATP 0.02 ml., 3-phosphoglycerate 0.10 ml., MgC12 0.05 ml., leucovorin or tetrahydrofolic acid 0.05 ml., enzyme 0.2 ml. Total volume, 0.7 ml. Incubation time, 30 minutes at 38 °. One-half milliliter of 20 % trichloroacetic acid is added to stop the reaction. When tetrahydrofolic acid is employed, it may be advantageous to carry out the reaction under anaerobic conditions. Analytical Methods. To determine the total formate incorporation, pipet 0.05 ml. of the trichloroacetic acid filtrate into a glass planchet, add 5 drops of water and 5 drops of ethanol, dry, and count. The nonacid-hydrolyzable formate (counts after hydrolysis) is determined as indicated in the section "De Novo Synthesis of Inosinic Acid." Micromoles of C ~4 activity in formylglycinamide ribotide Total counts/vessel--counts after hydrolysis/vessel Counts/micromole of formate It should be emphasized that there are other compounds derived from C~4-formate which comprise approximately 5 to 10% of the total C 14 activity incorporated. Under these conditions, 0.5 to 1 micromole of the ribo tide is synthesized. GLYCINAMIDE RIBOTIDE. Procedure. Incubation mixture: C14-glycine 0.05 ml., glutamine 0.05 ml., ribose-5-phosphate 0.10 ml., ATP 0.02 ml., 3-phosphoglycerate 0.10 ml., MgC12 0.05 ml., enzyme 0.2 ml. Final volume, 0.7 ml. Incubation time, 30 minutes at 38 °. The reaction is stopped by the addition of 0.5 ml. of 20% trichloroacetic acid. Analytical Method. A 0.1-ml. aliquot of the trichloroacetic acid filtrate is pipetted carefully into the bottom of a small test tube and neutralized with 1 N N a O H using bromothymol blue. One milliliter of 1.0 M potas10 B. L. O'DeU, J. M. Vandenbelt, E. S. Bloom, and J. J. Pfiffner, J. A m . Chem. Soc. 69, 250 (1947). 11Lo Jaenicke and G. R. Greenberg, unpublished studies.

[78]

de Novo SYNTHESIS OF PURINE NUCLEOTIDES

511

sium phosphate buffer, pH 5.4, 0.1 ml. of carrier glycine (0.1 M), and 1.0 ml. of ninhydrin solution (30 mg./ml.) are added. A marble is placed on the tube, which is then heated at 100 ° for 30 minutes. After cooling, 1 drop of caprylic alcohol is added, and the mixture is aerated with C02 for 15 minutes and diluted to 10 ml. A 2-ml. aliquot is pipetted into a glass planchet, dried under an infrared light, and counted. The factor to correct to an infinitely thin layer in a glass planchet has been found to be approximately 2.4, but it should be determined in each laboratory by adding in a blank run a C144abeled compound which does not react with ninhydrin. All the glycine-l-C 14 incorporated into forms not decarboxylated by ninhydrin can be accounted for by ion exchange chromatography in the fractions corresponding to glycinamide ribotide and formylglycinamide ribotide as described in the next section. ISOLATION OF GLYCINAMIDE RIBOTIDE AND FORMYLGLYCINAMIDE RIBOTIDE. The ribotides are isolated in partially purified form by chromatography on a Dowex 1 column. The elution of the compounds is followed either by radioactivity or by analysis for pentose. For large-scale preparation of the ribotides the components of the reaction mixture are increased 200-fold. The trichloroacetic acid filtrate and combined washings (5% trichloroacetic acid) of the precipitate are extracted several times with ether to remove the trichloroacetic acid and to bring the pH to at least 4. The solution is then adjusted to pH 8.2 with KOH, and 0.5 M barium acetate is added until no further precipitation of barium-insoluble material occurs (approximately 8.5 ml.). The precipitate is redissolved in a minimum amount of dilute HC1 and adjusted to pH 8 to 9 by addition of NH4OH. The precipitate is separated and washed three times with a total of 45 ml. of H~O. The combined supernatant solution and washings are diluted threefold with water and passed through a Dowex 1 column (4% cross linkage, 250 to 400 mesh, formate form, 2-cm. diameter X 15 cm.). Glycine and an unidentified compound containing C 14 from glycine are eluted at pH 8.5 with 0.02 Mammonium formate. Glycinamide ribotide is eluted with 0.05 M ammonium formate at pH 6.5 between 1800 and 2400 ml. The formylglycinamide ribotide, synthesized on a comparable scale, is also isolated by column chromatography by a similar procedure. The column is washed successively with 700 ml. of 0.05 M ammonium formate at pH 6.5 and with 600 ml. of 0.05 M ammonium formate at pH 5.2, and then the compound is eluted with 0.05 M ammonium formate at pH 5.0 in two components between the volumes 650 ml. and 2300 ml. These components are considered to be isomers, but the exact nature of the isomerization is not known. After the solutions are concentrated by lyophilization, the barium salts of both ribotides may be prepared and

512

ENZYMES OF NUCLEIC ACID METABOLISM

[78]

precipitated at pH 8.2 by the addition of 4 vol. of alcohol. It must be emphasized that the barium salts of both ribotides obtained by this procedure are only partially pure. Preparation of Extract. The enzyme system employed for formylglycinamide and glycinamide ribotide synthesis is prepared from the pigeon liver acetone powder extract (see Synthesis of IMP) as follows. The extract from 10 g. of acetone powder is passed through a Dowex 1 column (bicarbonate form, 4% cross linkage, 2.9-cm. diameter × 15 cm.) over a period of 2 hours, dialyzed overnight against 0.05 M K2HP04, and lyophilized. One hundred milligrams of lyophilized powder is dissolved in 1 ml. of water. Preparation of 5-Amino-4-imidazolecarboxamide Riboside 12,13

Principle. In E. coli under sulfonamide bacteriostasis the following sequence of reactions is considered to take place: sulfa

Glycine and other inhibition --~ 5-IRMP ) Purine ribonucleotides precursors I R + P~ Amino imidazolecarboxamide riboside (IR) accumulates in the culture medium and is isolated by adsorption on charcoal, purification on ion exchange columns, and crystallization. I R has been prepared by another procedure. TM Procedure. Escherichia coli, strain B, is carried on Difco nutrient agar slants and transferred approximately monthly. The glucose-salts 15 solution is prepared by dissolving the following compounds in 500 ml. of distilled water: NH4CI 0.5 g., (NH4)2S04 0.05 g., NaC1 0.1 g., MgCI~6H~O 0.1 g., Na2HPO4.7H~O 11.4 g. and KH2P04 3.0 g. The solution is autoclaved and an equal volume of an autoclaved solution of 0.8% glucose added. For small-scale studies 10 ml. of the glucose-salts solution is pipetted into 21 × 175-ram. tubes, the additions made, the tubes plugged with cotton and autoclaved for not more than a total of 10 minutes at 15 lb. The inoculum is grown in stationary culture for 16 hours at 37 °. A reading of 150 to 170 in the Klett colorimeter with a 540-mt~ filter represents normal growth. To each liter of the culture medium containing 12Abbreviations: IR, 5-amino-4-imidazolecarboxamide riboside; 5-IRMP, 5-amino4-imidazolecarboxamide-5'-phosphoriboside; EDTA, ethylenediaminetetraacetate; FAH,, tetrahydrofolie acid. xa G. R. Greenberg, Federation Proc. IS, 745 (1954). x4E. D. Korn, F. C. Charalampous, and J. M. Buchanan, J. Am. Chem. Soe. 75, 3610 (1953). x5j. Spizizen, J. C. Kenney, and B. Hampil, J. Bacteriol. 62, 323 (1951).

[78]

de Nolo SYNTHESIS OF PURINE NUCLEOTIDES

513

30 mg. of glycine and 112 mg. of sulfadiazine is added 40 ml. of inoculum. The incubation is carried out in stationary culture at 37 ° for 11 hours with 3 1. of medium per 12-1. Florence flask. At the end of the incubation the Klett colorimeter reading should be about 50 to 80. The procedure has been carried out in 200-1. lots in a large kettle with a liquid depth of about 18 inches. The following operations are performed at room temperature. T o each liter of medium is added 5 g. of Filter-Cel (Johns Mansville). After stirring, the suspension is filtered on W h a t m a n No. 1 paper. Analysis of the clear filtrate shows 80 to 100 micromoles of nonacetylatable, diazotizable amine per liter. An aliquot of the filtrate m a y be concentrated by lyophilization and chromatographed on paper to determine that the riboside is formed (see Table I). The carboxamide compounds are detected by diazotization on the paper. TABLE I PAPER CHROMATOGRAPHY OF I R

(RI value or centimeters from starting line)

IR I Inosine D-Ribose L-Arabinose

(1)

(2)

(3)

0.42 0.56 0.34 0.53 0.45

0.24 0.56 0.17 0.28 0.20

6.7 13.4 2.4 8.2 6.8

Solvents: (1) n-Butanol:/~,¢'-dihydroxyethyl ether:water (4:1:1) and in the presence of 1 M NH~OH. (2) Solvent 1 saturated by boric acid. (3) n-Butanol saturated by water and in the presence of 1 M NH4OH. [Solvent allowed to go beyond edge of paper (40 cm.) and therefore data are recorded in centimeters.] T o the filtrate Norit A is added (1 g./1.), and the mixture is stirred for 45 minutes. All the amine is adsorbed. The Norit A is collected and dried on a Bfichner funnel. The cake is extracted by shaking for 3 hours with ten times its weight of a mixture of ethanol:concentrated ammonium h y d r o x i d e : w a t e r in the volume proportions of 5:3:2. The charcoal is again collected on a Bfichner funnel and the extraction repeated two additional times. The combined filtrates are concentrated to an oil i n vacuo with a water pump. T o dissolve the oil, 10 ml. of water is added per liter of original medium, and the solution is brought to p H 10 to 11 by addition of ammonium hydroxide. The deep amber solution is passed through a Dowex 1 (formate form, 10% cross linkage) column. For 0.5 millimole of riboside a 1.2-cm. diameter × 7-cm. column is employed.

514

ENZYMES OF NUCLEIC ACID METABOLISM

[78]

The riboside is washed through the column with water, while most of the dark-colored material is retained on the column. The eluted riboside solution is a light amber color. In some experiments small quantities of the ribotide can be recovered on the column by elution with 0.2 M ammonium formate at pH 4.18. The solution is concentrated .to an oil by drying from the frozen state. The residue is dissolved in 0.01 N HC1 (100 ml. per 0.5 millimole of riboside). The acidified solution is placed on a Dowex 50 (NH4 + form) column. Since the capacity of the resin for the riboside is low, a large column is necessary. For 0.5 millimole a 2-cm. diameter X 40-cm. column is used. The column is washed with a volume of 0.01 N HC1 equal to the volume of the acid solution of riboside and then with an equal volume of distilled water. The riboside is readily eluted from the column with 0.1 N NH4OH, and the compound is measured by the optical density at 267 m~. The solution of the riboside is reduced to a small volume in vacuo and then to dryness by lyophilization. This preparation is adequate for conversion to the ribotide. The residue is dissolved in a small amount of water (less than 10 ml.) by gentle heating in a water bath and transferred to a small beaker. The riboside crystallizes at 4 ° over a period of several days. Recrystallization is brought about by dissolving the compound in a minimal quantity of water, decolorizing if necessary by heating with a very small quantity of Darco G-60, and allowing the solution to stand at 4 ° for 48 hours or more. Crystallization may be hastened by freezing the solution and then bringing it to 4 °. The crystals show slight amber color. A yield of about 30% is obtained. Analytical Methods. Non-acetylatable, diazotizable amine is determined by the method of Ravel et al. 16 The riboside may be detected on paper chromatograms by the ultraviolet method, or the diazotizable amine may be detected by spraying with a fresh solution of 8 vol. of 0.2 N HNO3 and 1 vol. of 0.1% NAN02, after 5 minutes spraying with 0.5% ammonium sulfamate and then after 3 minutes with 0.1% N-(1naphthyl)ethylenediamine dihydrochloride. Care should be taken not to get the paper too wet in the process. Data on the chromatographic behavior of I R are shown in Table I. Conversion of 5-Amlno-4-imidazolecarboxamide Riboside to the 5'Phosphoriboside ATP + I R --* ADP + 5-IRMP Principle. The reaction is catalyzed by brewer's yeast. 5-IRMP is isolated from the reaction mixture by ion exchange chromatography. (5-IRMP has been prepared enzymatically from IMP. 17) 16 j . M. Ravel, R. E. E a k i n a n d W. Shive, J. Biol. Chem. 172, 67, 1948. 17 j . G. Flaks a n d J. M. B u c h a n a n , J. Am. Chem. Soc. 76, 2275 (1954).

[78]

de Novo SYNTHESIS OF PURINE NUCLEOTIDES

515

Reagents Na-phosphoglycerate and Na4 ATP. See section "De novo synthesis of Inosinic Acid." Muscle enzyme fraction, is Procedure. The following additions are made to an Erlenmeyer flask: 15 ml. of yeast autolyzate (55 mg. of lyophilized powder per milliliter of water), 105 micromoles of 5-amino-4-imidazolecarboxamide riboside, 96 micromoles of MgC12, 168 micromoles of sodium-3-phosphoglycerate, 60 micromoles of Na4ATP, 6 mg. of muscle enzyme fraction, 600 micromoles of potassium phosphate buffer, pH 7.4, and water to a volume of 21 ml. The reaction mixture is incubated for 60 minutes at 38 °, cooled and transferred to a centrifuge tube, and the flask rinsed three times with 2 to 3 ml. of water. To the combined mixture 2 ml. of 20% trichloroacetic acid is added. After centrifugation the protein precipitate is washed four times with 5 ml. each of 1% trichloroacetic acid. The combined supernatant fractions (50 to 60 ml.) are extracted in a separatory funnel three times with 150- to 200-ml. portions of ether. The ether dissolved in the aqueous phase is aerated out with N2 gas. A saturated solution of Ba(OH)2 is added to pH of 8.5 or until no further precipitation occurs. The precipitate which contains the major part of the added ATP, and also PGA and inorganic phosphate, is collected by centrifugation, redissolved in 10 ml. of 0.1 M acetic acid, adjusted to pH 8.5, and the precipitate collected by centrifugation. This procedure is repeated once more, and the precipitate is discarded. The combined supernatant fractions (about 88 ml.) containing about 81 micromoles of diazotizable amine are passed through an ion exchange column. The column is prepared from Dowex 1 (acetate form, 4% cross linkage, 200 to 400 mesh) and has the dimensions of 1.4 cm. 2 by 23 cm. The solution is added slowly to the column which is then washed with water. Unreacted riboside is found in the water eluate. Elution of the 5'-phosphoriboside is effected by 0.20 M ammonium acetate, pH 4.18, at a flow rate of 1 to 2 ml./min. The first 150 ml. of eluate contains compounds having maxima at 275 mu and 280 m~. The phosphoriboside is eluted after about 1000 ml. It is characterized in the eluates by a maximum at 267 m~ and a ratio of optical densities at 267 m~/260 m~ of 1.08-1.11 and a ratio of 1.04-1.06 for 267 m~/272 mt~. Fractions having values outside these ranges are rejected. 5-AMP is eluted directly after the carboxamide ribotide. Ammonium acetate is removed by lyophilization. The process is repeated two times by redissolving the ribotide and the remaining ammonium acetate in a small volume of water. The ammonium salt of the ribotide kept as the frozen aqueous solution has been used for most of our studies of the enzymatic is S. Rather and A. Pappast J. Biol. Chem. 179, 1183 (1949).

516

ENZYMES OF NUCLEIC ACID METABOLISM

[78]

f o r m a t i o n of inosinic acid. A yield of a b o u t 50 to 5 5 % of 5 - I R M P is obtained, based on the original I R . A b o u t 2 0 % of the u n r e a c t e d I R is recovered in the w a t e r wash. 5 - I R M P has been isolated as the calcium a n d b a r i u m salts. At p H 7, 5 - I R M P shows a m a x i m u m absorption at 268 m~ and a molecular extinction coefficient of 12,800. Assay of the reaction is m a d e as described under the p r e p a r a t i v e conditions with 0.5 ml. of the y e a s t e n z y m e and with the other reagents in proportion. T h e reaction time is 30 minutes. A known volume of the T C A filtrate is c h r o m a t o g r a p h e d directly with 77 % ethanol as a solvent. T h e 5 - I R M P is determined b y diazotization after elution f r o m the paper. Some c h r o m a t o g r a p h i c d a t a of 5 - I R M P are shown in T a b l e I I . TABLE II R/

VALUES AND RELATIVE MIGRATION RATES Of COMPOUNDSa

5-IRMP 5-AMP 5-IMP 3-AMP IR

5-IRMP

AND SOME RELATED

(1)

(2)

(3)

(4)

0.23 0.24 0.21 -0.45

0.76 0.65 0.81 0.56 0.60

12.5 11.9 7.5 14.9 30.2

0.51 0.34 0.58 ---

Numbers of lcss than 1 refer to R/values, and those greater than 1 to centimeters from starting line. In the latter case the solvent was allowed to flow off the end of the paper. The chromatography is descending unless otherwise stated. The solvents are in volume proportions: (1) 77% ethanol. (2) 5% K2HPO4 layered with isoamyl alcohol (ascending). (3) n-Butanoh 50% acetic acid, 1 : 1. (4) Saturated (NH4)~S04:0.2 M Na acetate (pH 5.9):isopropanol, 79:19:2 (ascending).

Preparation of Extract. T h e e n z y m e for converting the riboside to the ribotide is obtained f r o m washed, l o w - t e m p e r a t u r e dried y e a s t (AnheuserBusch). Brewer's y e a s t is autolyzed for 31//~ hours. 19 T h e a u t o l y z a t e is dialyzed a t 4 ° against running distilled w a t e r for 24 hours and lyophilized. This almost white powder is stable indefinitely a t 4 ° in a v a c u u m desiccator. Activation of a 1 - C a r b o n Unit T h e formylation of the purine precursors m a y be divided into two steps: (1) the activation of the 1-carbon unit f r o m f o r m a t e or from serine and (2) the t r a n s f o r m y l a t i o n to glycinamide ribotide or to 5 - I R M P . T h e e n z y m e source is the same in each case. I n these experiments t e t r a h y d r o folic acid is used as the cofactor. Boiled extracts contain a cofactor(s) 19A. Kornberg and W. E. Pricer, Jr., J. Biol. Chem. 193, 481 (1951).

[78]

de Noyo SYNTHESIS OF PURINE NUCLEOTIDES

517

which is more stable than tetrahydrofolie acid. General references to studies on this problem are listed. 7,13,~7,20 REACTION OF FORMATE AND TETRAHYDROFOLATE (TETRAHYDROFOLATE

FORMYLASE). Principle. Pigeon liver extract catalyzes the over-all reaction :1 HCOOH ~- folate derivative ~- ATP Mg++

Formylfolate derivative ~ ADP ~ P~ The folate compound is the easily oxidizable tetrahydrofolic acid employed in substrate quantity, and the product has the properties of N ~°-formyltetrahydrofolic acid. The reaction is measured by the fixation of C14-formate into a nonacid-volatile form when tetrahydrofolic acid and ATP are present.

Reagents Tetrahydrofolic acid. See "Biosynthesis of Formylglycinamide Ribotide and Glycinamide Ribotide." Sodium formate-C TM. 20,000 counts/micromole as counted with a 2 m g . / ~ . 2 end-window counter in a glass planchet at infinite thinness. Enzyme. The enzyme is used as obtained after dialysis or by dissolving the lyophilized powder in water (p. 519).

Procedure. To a test tube in an ice bath add the following: 0.05 ml. of enzyme, 0.05 micromole of ATP, 0.7 micromole of PGA, 1.8 micromoles of Na formate-C TM, 10 micromoles of KHCO3, 3 micromoles of MgCl2, 1.25 micromoles of FAH4 (0.2 ml.), and water to a volume of 0.5 ml. About 1 cm. of petroleum ether is layered over the reaction mixture before the addition of FAH4. Incubate for 15 minutes at 37 °. Stop the reaction by addition of 1 ml. of 10% TCA. Pipet 0.05 to 0.1 ml. into a planchet, and measure C 14 as total formate fixation as described under "Biosynthesis of Formylglycinamide Ribotide and Glyeinamide Ribotide" (Analytical 5~ethod). Appropriate controls are carried out without FAH4 and at zero time. Calculation: Micromoles FAH4.C14HO Counts fixed X filtrate volume (ml.) Counts/micromole formate-C 14 X volume of sample (ml.) Approximately 0.2 micromole of FAH4.CHO is synthesized in 15 minutes per 0.05 ml. of enzyme. ~0W. Sakami, in "Amino Acid Metabolism," p. 658, Johns Hopkins Press, Baltimore, 1955; D. A. Goldthwait, R. A. Peabody, and G. R. Greenberg, ibid., p. 765.

518

ENZYMES OF NUCLEIC ACID METABOLISM

[78]

The reaction may be assayed by measurement of FAH4 CHO spectrophotometrically (see below), but this must be carried out with another protein precipitant such as 4% HC104. Liberation of orthophosphate may also be measured with appropriate controls because of apyrase activity. In this case the reduced folic compounds are removed batchwise from the HC104 filtrate on Dowex 50 (H + form) exchanger, since these compounds interfere with the phosphate analysis. Paper chromatography of the reaction filtrate (prepared by acidification with TCA anaerobically) with 1 M formic acid as a solvent (Whatman No. 1 paper) yields a compound at Rs = 0.37 which corresponds both in R / a n d , after elution with acid, in its absorption spectrum to the NS-Nl°-imidazolinium derivative of formyltetrahydrofolic acid (ACF). This compound contains more than 90% of the C 14 activity; a small quantity is found in N~°-formylfolic acid, RI 0.69, which is derived by oxidation of the product. CON'VERSION OF H-CARBON OF SERINE TO CHOFAH4.11 Principle. Serine -[- FAH4 -b T P N --* T P N H ~- NI°-CHOFAH4 ~ glycine One glycine is formed per mole of N~°-CHOFAH4. It is not known whether adenine nucleotide and phosphate are involved. NI°-CHOFAH4 is measured by conversion to ACF. Procedure. To a test tube in an ice bath add the following under a layer of petroleum ether: 0.05 ml. of enzyme, 5 micromoles of L-serine, 0.1 micromole of TPN, 1.25 micromoles of FAH4, 0.3 micromole of ATP, 10 micromoles of inorganic phosphate, 3 micromoles of MgC12, and 2.5 micromoles of MnS04.15 The reaction volume is 0.5 ml. Time 15 minutes, temperature 38 °. The reaction is stopped by addition of 0.5 ml. of 4% HC104. The protein precipitate is centrifuged down and allowed to stand under petroleum ether for 30 minutes in order to convert the reaction product to ACF. An aliquot of the filtrate (0.1 to 0.2 ml.) is transferred to a 1-ml. Beckman cuvette of 1-cm. light path, water is added to a final volume of 1 ml., and the solution is layered with petroleum ether. A reading is made at 360 m~. An aliquot is measured in the same way at zero time along with other controls. The difference in the optical density represents ACF. A change in optical density of 22 per milliliter for a 1-cm. light path is equivalent to 1 micromole of ACF. About 0.1 to 0.2 micromole of FAH4"CHO is formed in this reaction in the time given. Conversion of 5-IRMP to IMP

Principle. Transformylation from N~°-formyltetrahydrofolic acid to 5 - I R M P is measured by the disappearance of the diazotizable amine. The NS-N~°-imidazolinium salt of formyltetrahydrofolic acid is used, as it is more easily obtained than Nl°-formyltetrahydrofolic acid and under

[78]

de ~OYO SYNTHESIS OF PURINE NUCLEOTIDES

519

anaerobic conditions at neutral p H is rapidly converted to the latter. The Nl°-derivative can be synthesized directly b y reduction of N l°formylfolic acid. l°,11 The conversion of 5 - I R M P to I M P can be measured in the over-all system in the presence of C~4-formate, an ATP-regenerating system, and catalytic quantities of FAH4 by measuring C14-formate fixation and the disappearance of diazotizable amine. When serine is the 1-carbon source in the presence of catalytic quantities of cofactor the disappearance of diazotizable amine is measured. These experiments can be carried out with glycinamide ribotide as the 1-carbon acceptor. Reagents

Anhydroleucovorin (anhydrocitrovorum, ACF) (Calco Chemical Division, American Cyanamide Co.). The compound can be prepared from leucovorin by acidification and crystallization. 21 Three milligrams of ACF is dissolved in 1 ml. of 0.05 M K H C 0 3 and 0.2% E D T A under petroleum ether. I R M P - 5 , ammonium, K or Na salt, 4 ~moles/ml. Procedure. Incubation mixture: 0.1 ml. of extract, 0.08 ml. of ACF solution, 0.03 ml. of 5-IRMP. The reaction is carried out under petroleum ether at 38 ° for 20 minutes. Stop with I ml. of 10% TCA. Analyze 0.5 ml. of filtrate for non-acetylatable, diazotizable amine. Appropriate controls are made with I R M P and without ACF and vice versa and with zero time values. Some diazotizable amine color is due to the folic acid compounds. Preparation of Extract. Pigeon liver is homogenized at 0 ° with 5 vol. of 0.25 M sucrose solution containing 0.1% E D T A and centrifuged at 80,000 X g for 30 minutes. The supernatant extract is frozen overnight, allowed to stand for 2 hours at room temperature, and any precipitate is removed by centrifugation. The fraction precipitating between 0.25 and 0.60 saturation with ammonium sulfate is dialyzed against 0.01 M KHCO3 overnight at 4 °. Ammonium sulfate solution is prepared as follows: 71.5 g. of ammonium sulfate plus 98 ml. of water at room temperature, brought to p H 7.0 with NaOH, and water added to bring to a total volume of 100 ml. of solvent. T h e extract is stable to freezing and thawing.22, 2a

21D. B. Cosulich, B. Roth, J. M.: Smith, Jr., M. E. Hultquist, and R. P. Parker, J. Am. Chem. Soc. 74, 3252 (1952) ; M. May, T. J. Barrios, F. L. Barger, M. Lansford, J. M. Ravel, G. L. Sutherland, and W. Shire, ibid. 78~ 3067 (1951). 22The structure of amino imidazole ribotide (IV in Figure 1) is based on studies by S. H. Love and J. S. Gots, J. Biol. Chem. 210, 395 (1955); B. Levenberg, S. C. Hartman, and J. M. Buchanan, Federation Proc. 14~ 243 (1955). ~3Dr. Lothar Jaenicke and Mr. Richard A. Peabody kindly provided the authors with some of the procedures described here.

[79]

ACID PROSTATIC PHOSPHATASE

523

[79] Acid Prostatic Phosphatase

By G~RHARDSCHMIDT Prostatic phosphomonoesterase was discovered in 1935 by Kutscher and his associates, 1-3 who traced the frequent appearance in human urine of considerable amounts of a phosphatase with a pH optimum in the range between 5 and 6 to the admixture of semen. A systematic examination of the tissues of the male urogenital tract resulted in the detection of very high concentrations of this phosphatase in the prostate gland. In comparison with the phosphatase content of this gland and its secretion, those found in the other tissues of the male urogenital system were negligible.1 Distribution. Normal prostate glands of adults, hypertrophic glands, primary carcinoma of the prostate and their metastases contain the enzyme in concentrations of similar orders of magnitude. 4-~ The enzyme is secreted in the seminal fluid. Its concentration in normal semen is always high and often of a similar range as that in the prostate gland. 7 Before puberty, prostate glands of man and of Rhesus monkeys contain only negligible amounts of acid phosphatase, s Injections of testosterone (but not of ovarian hormones) resulted in several hundredfold increases of the concentrations of the enzyme in the prostate glands of immature Rhesus monkeys. 8 Relatively much smaller concentrations of this enzyme were found in many other human and animal tissues. Skeletal muscle and heart, however, are practically devoid of acid phosphatase. So far, the abundance of this enzyme in prostatic tissue appears to be a characteristic property of man and of monkeys. The prostate glands of dog, bull, ram, and rat were found to contain only small amounts of the enzyme2 ,s Very considerable amounts of acid phosphatase were found, however, in the preputial glands of rats 9 and in the seminal vesicles of guinea pigs. 1° The concentration of acid phosphatase in rat preputial glands did not increase on injections of testosterone, but the total yield of phosphatase 1 W. Kutscher and H. Wolberg, Z. physiol. Chem. 256, 237 (1935). 2 W. Kutscher and A. WSrner, Z. physiol. Chem. 239, 109 (1936). 3 W. Kutscher and J. Pany, Z. physiol. Chem. 255~ 169 (1938). 4 j. Fischmann, H. A. Chamberlin, R. Cubiles, and G. Schmidt, J. Urol. 59, 1194 (1948). 5 j. D. Fergusson, Lancet 251, 551 (1946). 6 A. B. Gutman and E. B. Gutman, J. Clin. Invest. 17, 473 (1938). 7 A B. Gutman and E. B. Gutman, Endocrinology 28, 115 (1941). 8 A. B. Gutman and E. B. Gutman, Proc. Soc. Exptl. Biol. Med. 41, 277 (1939). 9 A. B. Gutman and E. B. Gutman, Proc. Soc. Exptl. Biol. Med. 39, 528 (1938). ~0 H. A. Bern and R. S. Levy, Am. J. Anat. 90, 131 (1952).

524

ENZYMES I N PHOSPHATE METABOLISM

[79]

from the p r e p u t i u m was much higher after such injections, owing to the enhanced growth of glandular tissue. N o r m a l serum contains only v e r y small amounts of prostatic acid phosphatase; in serum of patients suffering from carcinoma of the prostate, elevated values of the enzyme are frequently encountered, whereas no such elevations occur in cases of benign h y p e r t r o p h y of the prostate gland. Determinations of this enzyme in serum are i m p o r t a n t for the diagnosis of prostate carcinoma, as well as for the evaluation of therapeutic measures. 11 I t is not yet clear whether an appreciable elevation can be caused b y a beginning prostate carcinoma. Specificity22

According to our present knowledge, acid prostatic phosphatase is a phosphomonoesterase in respect to the known phosphoric acid esters of living organisms, a-L-Glycerylphosphorylcholine, a-L-glycerylphosphorylethanolamine, and a-L-glycerylphosphorylserine as well as the phospholipids and the nonterminal phosphoryl groups of polynucleotides are not hydrolyzed b y the enzyme. TM Among synthetic diesters of phosphoric acid, diphenylphosphate is resistant to prostatic phosphatase, but the corresponding paranitro derivative, bis-p-nitrophenylphosphate, is hydrolyzed at a considerable rate b y the enzyme. Cohn and Volkin 14 reported t h a t another phosphatase which acts as a specific phosphomonoesterase toward the phosphoric acid esters previously examined, namely barley 3'-nucleotide phosphatase, hydrolyzes di(dinitrophenyl)phosphate. I t is conceivable t h a t nitrophenyl diesters of phosphoric acid behave exceptionally toward phosphomonoesterases because of the enhancing influence of the nitro groups on the dissociation of the hydroxyl group. Furthermore, not all phosphoric acid monoesters are substrates of prostatic phosphatase, and those which are hydrolyzed are cleaved at very different rates. 2'- and 3'-Nucleotides are hydrolyzed at faster rates than any other biological phosphoric acid monoesters. T h e hydrolysis rate of monophenylphosphate is similar to t h a t of the 2'- and 3'-nucleotides. 2'- and 3'-Adenylic acid are hydrolyzed at very similar rates; this is also the case for the 2'- and 3'-uridylic acids. Adenosine-5'-phosphate, 11A detailed discussion of the clinical application of phosphatase determinations in serum is beyond the scope of this chapter. References pertinent to this field will be found in the papers quoted in footnotes 4 and 21. 1~Some of the data on specificity of acid prostatic phosphatase are based on unpublished observations of G. Schmidt, K. Seraidarian, M. J. Bessman, and L. M. Greenbaum. is G. Schmidt, R. Cubiles, N. ZSllner, L. Hecht~ N. Strickler, K. Seraidarian, M. Seraidarian, and S. J. Thannhauser, J. Biol. Chem. 192, 715 (1951). 14W. E. Cohn and E. Volkin, J. Biol. Chem. 203, 319 (1953).

[79]

ACID PROSTATIC PHOSPHATASE

525

ribose-5-phosphate, and both glycerophosphates are hydrolyzed approximately at one-third of the rate of the 2 ~- and 3'-nucleotides. Robison ester is hydrolyzed at least five times more slowly than these nucleotides, and fructose diphosphate is completely resistant toward prostatic phosphatase. Phosphorylcholine, phosphorylethanolamine, and phosphorylserine are rapidly hydrolyzed. The rate of hydrolysis of ATP is at least thirty times slower than that of adenosine-3'-monophosphate. Tile hydrolyses of monophenylphosphate and of the 2'- and 3'-nucleotides are unimolecular reactions at least to a degree of hydrolysis of 50 %. For the other esters, the rates fall more rapidly even at early phases of hydrolysis.

Behavior of Prostatic Phosphatase toward High-Molecular Phosphorus Compounds. Owing to the growing importance of methods for end-group determinations and to the lack of a convenient chemical method for the differentiation of primary and secondary phosphoryl groups, a pure monophosphoesterase would be a highly valuable tool for the analysis of the structure of nucleic acids and phosphoproteins. The preceding paragraphs demonstrate that prostatic phosphatase has the requirements for such a tool to a limited extent--limited because an ideal end-group reagent should hydrolyze all phosphomonoester groups and none of the phosphodiester groups. Prostatic phosphatase, as stated before, is practically inactive toward the phosphomonoester groups of fructose diphosphate and hydrolyzes glucose-6-phosphate so slowly that its complete cleavage would require unreasonably long digestion times. On the other hand, it hydrolyzes nitrophenyl diesters of phosphoric acid. These observations add weight to the argument that in a substrate such as ribonucleic acid the behavior of some phosphoryl groups against phosphatase might be determined by structural conditions other than their terminal or nonterminal positions. Conclusions pertinent to the structure of the substrate must be based on the analysis of the organic fragments as well as on the amount of hydrolyzable or resistant phosphoryl groups. Whenever feasible, the interpretation of phosphatase-labile phosphoryl groups as phosphomonoester groups should be verified by titration. If the inorganic phosphate formed by prostatic phosphatase originates exclusively from phosphomonoester groups, no additional acidic groups appear in the range between pH 5 and pH 8.5. The only change of the titration curve during the titration is a slight displacement toward the alkaline side, owing to the higher pK~ of inorganic phosphoric acid in comparison to that of phosphoric acid esters. Any increase of the amount of acidic groups within the range mentioned indicates the transformation of phosphodiester groups to phosphomonoester groups or to inorganic phosphate.

526

ENZYMES IN PHOSPHATE METABOLISM

[79]

1. NUCLEIC ACIDS. Deoxyribonucleic acid prepared by any modification of E. Hammarsten's procedure is practically resistant to prostatic phosphatase. This is in agreement with the negligible amounts of terminal secondary phosphoryl groups present in highly polymerized DNA molecules. Ribonucleic acid, prepared by mild procedures in the laboratory, is slowly hydrolyzed by prostatic phosphatase until approximately 10% 13,1s,16 of its total phosphorus is liberated as inorganic phosphate. Continuation of the incubation for several days, however, results in an additional but much slower formation of inorganic phosphate which finally may amount to 40 % of the total phosphorus. This is largely due to the presence of small amounts of ribonuclease in all preparations of prostatic phosphatase available at present. Although these preparations are very useful as end-group reagents for the polynucleotide mixtures of exhaustive ribonuclease digests or for ribonuclease resistant oligonucleotides, they cannot be applied as end-group reagents for ribonucleic acids. 2. PHOSPHOPROTEINS. The action of phosphatases on the phosphoryl groups of phosphoproteins is of interest for the elucidation of the structural significance of these groups. It is obvious that phosphatase preparations used for the study of the enzymatic dephosphorylation of phosphoproteins must be carefully checked for the absence even of small amounts of proteolytic enzymes. This is particularly important in view of the fact that prolonged incubation periods are usually necessary in such experiments. Ovalbumin. Perlmann iv found that 46% of the phosphoryl groups of the ovalbumin component A1 (which has a higher electrophoretic mobility than the second component A2) are hydrolyzed by prostatic phosphatase. The ovalbumin A1 is transformed by the action of this enzyme to a protein of a lower electrophoretic mobility which resembles that of A~. Casein. Casein was fraetionated by Warner is into two components, a-casein and/~-casein. The solubility properties ~9 and the electrophoretic behavior 2° of casein suggest an even more complex composition of the mixture represented by ordinary casein preparations. According to Perlmann, 2~,~ 42% of the phosphoryl groups of a-casein are hydrolyzable by 15G. Schmidt, R. Cubiles, and S. J. Thannhauser, J. Cellular Comp. Physiol. 88, Suppl. 1, 61 (1951). 16R. Markham and J. D. Smith, Biochem. J. 52, 565 (1952). 1TG. E. Perlmann, J. Gen. Physiol. 35, 711 (1952). 18R. C. Warner, J. Am. Chem. Soc. 66, 1725 (1944). 19 K. LinderstrCm-Lang,Compt. rend. tray lab. Carlsberg 17, No. 9 (1929). 200. Mellander, Biochem. Z. 800, 240 (1939). 21G. E. Perlmann, J. Am. Chem. Soc. 74, 3191 (1952). 22G. E. Perlmann, in " A Symposium on Phosphorus Metabolism" (W. D. McElroy and B. Glass~eds.), Vol. 2, p. 167, Johns Hopk~na Press~ Baltimore, 1952.

[79]

ACID PROSTATIC PHOSPHATASE

527

prostatic phosphatase, whereas E-casein is resistant to this enzyme. Additions of f~-casein to solutions of a-casein inhibit the dephosphorylation of the latter by prostatic phosphatase. The question as to whether the phosphatase-resistant groups of phosphoproteins are diesterified requires further investigation. p H Optimum. The pH optimum of acid prostatic phosphatase is in the region between 5.3 and 5.6 for most substrates; Lundquist found, however, that the hydrolysis of calcium phosphocholine by prostate phosphatase has a pH optimum of 6.5. 23 Isoelectric Point. The isoelectric point of acid prostatic phosphatase is as pH 4.4. 3 Michaelis-Menten Constants. The Michaelis-Menten constants of acid prostatic phosphatase were determined by M. Seraidarian 24 for the hydrolysis of the following phosphoric acid esters: a-glycerophosphate, 2.96 X 10-3; ~-glycerophosphate, 2.1 X 10-3 (Ohlmeyer2~ found a Km value of 4.7 X 10-3 for the hydrolysis of f~-glycerophosphate at pH 4.5) ; yeast adenylic acid (mixture of 2'- and 3'-adenosinephosphoric acid), 2.2 X 10-3; yeast uridylic acid (mixture of 2'- and 3'-uridine phosphates), 2.3 X 10-3. Inhibitors. In its behavior to some enzyme inhibitors, prostatic phosphatase differs sharply from some alkaline phosphatase, such as intestinal phosphatase. Fluorides, which have no effect on alkaline phosphatase at low concentration, inhibit prostatic phosphatase completely at 0.01 M concentration. 2 On the other hand, cyanides, cysteine, and hydrogen sulfide, which are powerful inhibitors of alkaline phosphatase, are without appreciable influence on prostatic phosphatase. 2 Kutscher and WSrner 2 found strong and irreversible inhibitory affects of many "narcotics" such as alcohols and urethans on acid prostatic phosphatase. The use of the comparatively strong inhibitory effect of ethyl alcohol on prostatic phosphatase has been suggested as a means for the differentiation of acid prostatic phosphatase of serum from other acid phospharases (e.g., erythrocyte phosphatase)36 At present, however, the practical application of this principle for clinical tests is still in the experimental stage. In a comparative study on the acid phosphatases of the human prostate gland and of human erythrocytes Abul-Fadl and I(ing 27 reported that the former was specifically and practically completely inhibited by 25I. Lundquist, Acta Scan& Physiol. 14, 263 (1947). 24M. Seraidarian, Thesis, Science Faculty, Tufts College, 1952. 25p. Ohlmeyer, Z. physiol. Chem. 282, 1 (1945). 2sF. G. Herbert, Quart. J. Med. 39, 221 (1946). ~7M. A. M. Abul-Fadl and E. J. King, Biochem. d. 45, 51 (1949).

528

ENZYMES IN PHOSPHATE METABOLISM

[79]

0.02 M sodium L-tartrate but not appreciably influenced by 0.5% formaldehyde. The characteristic behavior of prostatic phosphatase toward both these substances appears to be promising as a criterion regarding the prostatic origin of the acid phosphatases of serum in clinical cases. 28-30 Magnesium ions are without appreciable affect on prostatic phosphatase. 2 Prostatic phosphatase is completely inactivated by heating its solutions at neutral or weakly acid solutions at 60 ° during 5 minutes. It is unstable at room temperature, even at slightly alkaline pH ranges as well as below pH 5, but it can be kept for many months at neutral or weakly acid reaction (pH 6) in the refrigerator. The enzyme is usually inactivated by precipitation with alcohol or acetone. It has been lyophilized in active form. E n z y m e Units and Activity Determinations. Schmidt et al. 1~ defined as one unit of acid prostate phosphatase the amount of enzyme which forms 0.1 mg. of inorganic phosphorus within 15 minutes at 37 ° from a solution of " y e a s t " adenylic acid (Schwarz Laboratories; the substance is a mixture of the 2'- and 3'-adenylic acids). The incubation mixture contains 50 mg. of sodium adenylate in 10 ml. of 0.1 M sodium acetate buffer (pH 5.6) and 1 ml. of the enzyme solution. One gram of moist prostate tissue contains approximately 1500 enzyme units; for activity determination, 1 ml. of a hundredfold diluted stock solution (obtained by homogenizing the glands in 5 vol. of water) is usually a suitable amount. Phosphatase values in serum are usually expressed in King-Armstrong units per milliliter of serum. 4-9 One King-Armstrong unit is the amount of phosphatase which liberates 1 mg. of phenol from sodium phenyl phosphate at 37 ° within 1 hour; the other conditions of the hydrolysis (nature of buffer system, total volume) vary in different investigations. A procedure for phosphatase determinations in serum with sodium glycerophosphate as substrate has been described by Shinowara et al. ~1 Huggins and Talaley 3: described an assay method in which phenolphthalein phosphate was used as substrate. This substrate can be used only in comparatively low concentrations (0.3 X 10-4 M) because it inhibits the enzyme in higher concentrations. This is a serious disadvantage for kinetic studies. The use of nitrophenylphosphates as substrates for acid phosphatase in clinical determinations in serum might be adapted for clinical purposes in the future, but no reference as to their suitability can be given at present. ~8 W H. Fishman and F. Lerner, J. Biol. Chem. 200, 89 (1953). ~9 W. H Fishman, R. M. Dart, C. D. Bonner, W. F. Leadbetter, F. Lerner, and F. Homburger, J. Clin. Invest. 23, 1034 (1953). 90 E. P. Kintner, J. Lab. Clin. Med. 37, 637 (1951). 31 G. Y. Shinowara, L. N. Tones, and H. L. Reinhart, J. Biol. Chem. 142, 921 (1942). a, C. Huggins and P. Talaley, J. Biol. Chem. 1§9, 398 (1945).

[79]

ACID PROSTATIC PHOSPHATASE

529

Preparation of Purified Solutions of Prostatic Phosphatase. Prostate glands obtained by surgical enucleation (material obtained by transurethral cauterization is usually inactive) are used fresh or stored in a deepfreeze in which the full activity is preserved for years. The thawed glands are cut into small pieces with scissors and homogenized in a Waring blendor for 2 minutes. After addition of a few drops of toluene, the suspension is kept overnight in the refrigerator. The suspension is centrifuged, and the turbid supernatant is dialyzed overnight against distilled water in the cold room. To the strongly opalescent solution is added N acetic acid dropwise until a copious flocculent precipitate forms. Great caution during the acidification is essential for the success of this step during which the pH of the solution must not decrease below 5.5. This pH is considerably higher than that of the isoelectric point of prostatic phosphatase which was found to be at pH 4.4 by Kutscher and Pany2 Between pH 5.5 and 6.5, no loss of activity occurs despite the fact that a slight excess of acidity beyond the critical value of pH 5.5 results in the immediate and complete loss of the enzymatic activity. After centrifugation, a clear and usually almost colorless supernatant is obtained. This solution usually contains 300 to 400 units of the enzyme per millil i t e r - a n activity which is sufficiently high for the use of such enzyme solutions as a reagent of secondary phosphoryl groups. The solutions can be concentrated by precipitation in 0.9 saturated ammonium sulfate solution and by dissolving the precipitate in a small volume of water and subsequent removal of the salt by dialysis against distilled water in the cold. Solutions of prostatic phosphatase can be stored in the refrigerator in the presence of some toluene for many months without loss of activity. They are practically free of purine or pyrimidine deaminases, nucleotidases, phosphodiesterases, 5'-nucleotidase, or proteolytic enzymes, but they contain small amounts of heat-stable ribonuclease. So far, it has not been possible to remove the latter contamination. Further purification of the enzyme can be achieved by adsorbing contaminations on suspensions of aluminum hydroxide which is added dropwise under stirring. Prostatic phosphatase has very little affinity to this adsorbent, and it is easy to obtain in this way colorless solutions of the enzyme without appreciable loss of activity. A similar procedure for the purification of acid prostatic phosphatase was recently described by Derow and Davidson2 3 The behavior of acid prostatic phosphatase toward some reagents used for the purification of enzymes has recently been studied by London and Hudson2 4 In agreement with the observations of Schmidt, they 83 M. A. Derow a n d M. M. Davison, Science 118, 247 (1953). 34 M. London a n d P. B. Hudson, Arch. Biochem. and Biophys. 46, 141 (1953).

530

ENZYMES IN PHOSPHATE METABOLISM

[80]

found that the enzyme (similarly to the behavior of alkaline intestinal phosphatase) is not appreciably adsorbed on aluminum hydroxide gels. It is adsorbed on kaolin or Fuller's earth at pH 4.5 when these adsorbents are applied in relatively large quantities (25 rag. per 28 mg. of protein). A considerable part of the activity can be recovered from the adsorbate by elution with citrate buffers of pH 7.0.

[80]

Intestinal Phosphomonoesterase R-O-P

+ H20 -~ P 4- ROH

By LEON A. HEPPEL Assay Method

Principle. The assay depends on measuring the formation of inorganic phosphate from sodium/~-glycerophosphate at an early stage in the reaction when the rate is linear. Reagents Sodium ~-glycerophosphate solution (0.1 M). Ethanolamine-HC1 buffer, pH 9.5 (0.1 M). Magnesium acetate (0.005 M or 0.05 M).

Procedure. The lower concentration of magnesium acetate is used in testing the crude enzyme preparation, since higher concentrations are inhibitory. Mix 1 ml. each of buffer, magnesium acetate, and sodium ~-glycerophosphate and make up to 4.8 ml. Mix this with an amount of enzyme such that hydrolysis never exceeds 5%. Incubate for 15 minutes at 38 °, and analyze for inorganic phosphate. I The reaction is stopped by the addition of 2.5 ml. of 25% (w/v) trichloroacetic acid. Any precipitate is removed by filtration through Whatman No. 42 paper, and P~ is determined on the filtrate. Definition of Unit and Specific Activity. The unit is defined as the amount of enzyme which liberates 1 ~ of inorganic phosphate per rainute at 38 °. Specific activity is expressed as units per milligram of protein N. Purification Procedure

This procedure is the method of Morton. ~ Calf intestines are collected from the slaughterhouse as soon as possible after killing, and the 1C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925). 2R. K. Morton, Biochem. J. §7, 595 (1954).

[80]

INTESTINAL PHOSPHOMONOESTERASE

531

mesenteric membranes are removed. The mucosa is then obtained by rinsing the lumen with water and scraping the mucosa with the edge of a plastic spatula. The mucosa is cooled to 0 ° in an ice bath. One must proceed quickly. Step 1. The mucosa is dispersed in three times its volume of distilled water (at 0°), using a Waring blendor for 2 minutes, and adjusted to pH 7.5 by slow addition of N NaOH. After hard mechanical stirring for about 30 minutes at 0 °, the dispersion is centrifuged (1800 X g, 40 minutes) in aluminum cups (1300 cc. capacity) in an International serum centrifuge (13 1.). The supernatant is poured off and filtered through a layer of well-washed cotton wool on a Bflchner funnel to remove loose fat and other aggregates. The filtrate is immediately cooled to 0 ° in a stainless steel container held in a cold bath at - 10°. The precipitate is discarded. Step 2. The filtrate at 0 to 2 ° is adjusted to pH 5 by addition of 2 M acetate buffer, pH 4.0, held for 45 minutes at 0 °, and then centrifuged (1800 × g, 40 minutes, room temperature). The white precipitate is then thoroughly dispersed in 8 1. of 0.15 M NaC1. The suspension is adjusted to pH 7.5 with 0.5 M Na~CO3, cooled to 0 °, and stirred gently to 0 to 2 ° overnight. The enzyme is then reprecipitated at pit 5 as before and similarly collected by centrifuging. At this stage the supernatant is waterclear and almost free of soluble protein. The precipitate is then washed in the cups by rubbing up into 8 1. of distilled water (at 0 °) and immediately recentrifuging. By this means the ionic strength of the material is lowered, and the last traces of soluble protein removed. The precipitate is again dispersed in distilled water at 0 ° and made up to 5 1. Step 3. About 2 1. of n-butanol is slowly added over a period of 15 minutes with vigorous mechanical stirring. The material is then heated to 38 ° for 5 minutes and immediately centrifuged in 1-1. glass cups (1800 X g, 30 minutes). The water-clear, colorless aqueous layer is removed by suction and filtered through a thick layer of Hyflo Super-Cel over Whatman No. 1 paper on a 1-1. Bfichner funnel. The filtrate is then adjusted to pH 8.5, held at 0 ° overnight, and again filtered through Hyflo Super-Cel to remove a small precipitate. Step 4. The filtrate is adjusted to pH 6.4, cooled to - 5 °, and the enzyme directly precipitated at - 5 ° by addition of ether (10% v/v) and acetone (60% v/v). After settling overnight at - 7 °, the supernatant is removed by suction, and the precipitate collected by centrifuging at - 5 °. It is often convenient to dry the precipitate at this stage and to accumulate material from several batches of mucosa before proceeding further with purification. For this purpose the precipitate is washed twice with dry acetone at - 1 5 ° followed by dry anesthetic ether at - 1 5 °, and

532

ENZYMES IN P H O S P H A T E METABOLISM

[80]

then dried, initially in a s t r e a m of nitrogen and finally in vacuo over H2SO4 and CaCl~. T h e d r y powder m a y be stored over N a O H and CaCl~ in vacuo at 0 °. I t retains activity for at least 12 months. F o r further purification it is t r e a t e d exactly as the undried precipitate. Usually, drying is omitted and the 60 % ( v / v ) acetone precipitate is dissolved in a m i n i m u m of 0.05 M Veronal-HC1 buffer at p H 6.4 and dialyzed against 0.015 M m a g n e s i u m acetate, at p H 6.4. Insoluble m a t e rial is removed b y centrifuging (20,000 X g, 1 hour, 2°). Step 5. Following addition of ether (10 % v / v ) the enzyme was precipit a t e d at - 5 ° with acetone between 35 and 48 % ( v / v ) . This fraction was dissolved in 0.05 M veronal-HC1 buffer at p H 6.4 and dialyzed against glass-distilled w a t e r (at 0 °) until free of organic solvent. Step 6. T h e solution is adjusted to p H 4.9 with 0.05 N acetic acid and heated rapidly to 48 ° for 2 minutes. A slight opalescence appears. Magnesium acetate (0.5 M) is added to give a final concentration of 0.015 M , the solution adjusted to p H 6.4, and the e n z y m e precipitated at - 5 ° with acetone between 40 and 5 0 % ( v / v ) . The slight precipitate between 40 and 5 0 % ( v / v ) acetone is dissolved in buffer and dialyzed free of organic solvent as for step 5. Step 7. T h e solution is adjusted to p H 5.5 with 0.5 N acetic acid, and successive 20-mg. lots of washed charcoal 3 are slowly added after mixing to a thick slurry in distilled water. The precipitate is removed b y centrifuging (5000 X g, 15 minutes, 2°), and the procedure is repeated until SUMMARY OF ~°URIFICATION PROCEDURE a

Step

Volume, ml.

Activity, units/ml.

Total units (X 103)

1 2 3 4 5 6 7 8

12,000 5,000 4,450 55 20 20 32 10

205 216 166 12,360 26,250 18,900 7,340 14,200

2460 1080 739 680 525 378 235 142

Total N, mg./ml, 2.12 0.82 0.03 2.05 1.96 0.94 0.11 0.17

Specific activity, units/mg. N 97 263 5,533 6,029 13,393 20,106 66,727 83,529

Yield, % 100 44 30 28 21 15 10 6

R. K. Morton, Biochem. J. in press. a Preparation of activated charcoal: 180 g. of animal charcoal is treated successively with 5 1. of 0.1 N HC1, 2 1. of 5% potassium chloride, 200 ml. of 0.1 M barium hydroxide, and finally 21. of 5% potassium chloride. After each treatment the charcoal is sucked dry over Whatman No. 42 paper on a large Biichner funnel. After the last treatment it is dried at 100° in an oven and stored in an airtight jar.

[81]

PHOSPHOMONOESTERASE OF MILK

533

about 30% of the enzymic activity is adsorbed. The solution is then centrifuged (14,000 × g, 30 minutes), adjusted to piI 8 with 0.1 N N a 0 H , and filtered on a Biichner funnel through a thin layer of magnesium carbonate over a Whatman No. 1 filter paper in order to remove colloidal material (charcoal). Step 8. The enzyme solution is adjusted to pH 6.4 with 0.1 h r acetic acid and dialyzed at 2 ° against 0.015 M magnesium acetate for 4 hours with continuous stirring. The solution is cooled to 0 °, ether added to 10% (v/v), and the enzyme precipitated at - 5 ° with acetone between 40 and 48 % (v/v). The precipitate is dissolved in glass-distilled water at 0 ° and dialyzed against glass-distilled water at 0 ° for 4 hours with frequent changes of water.

Properties Specificity. Alkaline phosphatase catalyzes the hydrolysis of various orthophosphomonoesters, phosphoamides, etc. For example, creatine phosphate, phenyl phosphate, glucose-6-phosphate, and f~-glycerophosphate are split. Pyrophosphates, such as ATP and ADP, are not split. R N A is not attacked. The enzyme also catalyzes transferase reactions. Thus, it stimulates a reaction of creatine phosphate and glucose to give glucose-6-phosphate and creatine. Effect of Inhibitors. 4 Phosphate ion inhibits the enzyme. Cyanide inhibits 50% at a concentration of 3 mM. Fluoride has no effect up to 50 raM. There is a slight enhancing effect of magnesium ions. 4 G. Schmidt and S. J. Thannhauser, J. Biol. Chem. 149, 369 (1943).

[81] P h o s p h o m o n o e s t e r a s e Typical Reaction:

of M i l k

Glycerol P ~- H~O -~ P~ -~ Glycerol

By ROBERT K. MORTON

Assay Method Principle. The rate of hydrolysis of a suitable phosphate ester is determined by estimation of liberated inorganic phosphate (P~) or of liberated alcoholic moiety (such as phenol). Kay and Graham 1 originally used sodium f~-glycerol P and later 2 phenyl P as substrates, p-Nitrophenyl P ~ has also been used. 1 H. D. Kay and W. R. Graham, J. Dairy Research 5, 63 (1933). 2 H. D. Kay and W. R. Graham, J. Dairy Research 6, 191 (1935). 3 R. Aschaffenburg and J. E. Mullen, J. Dairy Research 16, 58 (1949).

534

ENZYMES IN PHOSPHATE METABOLISM

[81]

Reagents Sodium ~-glycerol P (0.1 M). Ethanolamine-HC1 buffer 4 (0.5 M), pH 9.95 at 20 ° (pH 9.65, 38°). 5'6 Magnesium acetate (0.2 M). TCA (30%). Enzyme. Dilute the preparation so that 0.1 ml. hydrolyzes no more than 5% of the substrate in 5 minutes at 38 °.

Procedure. Prepare a stock solution containing the following reactants: sodium ~-glycerophosphate, 0.02 M; ethanolamine-HC1 buffer, 0.05 M; magnesium acetate, 0.01 M (or 0.001 M for testing crude ext r a c t s - s e e below). The final pH should be 9.95 at 20 °. Pipet 5.0 ml. into suitable test tubes, and bring to 38 °. Add 0.1 ml. of the diluted enzyme preparation, and incubate for 5 minutes at 38 °. Stop the reaction with 2 ml. of 30% TCA. After 5 minutes, remove any precipitate by centrifuging and wash the precipitate twice with 5 % TCA. Estimate the liberated inorganic phosphate in the combined supernatant and washings by the method of Fiske and SubbaRow 7 or by any other suitable method (see Vol. I I I [147]). With partially purified enzyme preparations (after step 3 of purification procedure) no precipitate occurs on addition of TCA, and this may be omitted. The reaction is stopped by addition of the molybdate-sulfuric acid reagent of Fiske and SubbaRow, 7 and phosphate is then estimated directly. For the control tube, add TCA or molybdate-sulfuric acid to the buffered substrate prior to addition of the enzyme. Alternatively, phenyl P may be used as substrate and the liberated phenol estimated by a suitable method (see Vol. II [88]). Definition of Unit and Specific Activity. One unit of enzyme is defined as the amount which liberates 1 ~ of inorganic phosphate phosphorus per minute from ~-glycerol P under the above conditions. Because of the different activity of the enzyme with different substrates, the substrate must always be specified. Specific activity is expressed as units per milligram of protein nitrogen, determined by a micro-Kjeldahl procedure on dialyzed material (see Vol. III [145]) or as the Qp value (Engelhardt and LyubimovaS), i.e., the equivalent microliters of phosphorus liberated by 1 mg. of enzyme preparation in 1 hour at 38 °. The factor 6.25 is used to convert nitrogen values to dry weight. 4 C. A. Zittle a n d E. S. Della Monica, Arch. Biochem. 26, 112 (1950). Note t h a t all alkaline buffers change p H values markedly with temperature. 6 Other suitable amine compound~, as well as Veronal, are satisfactory as buffers. C. H. Fiske a n d Y. SubbaRow, J. Biol. Chem. 66, 375 (1925). 8 V. A. E n g e l h a r d t a n d M. N. Lyubimova, Nature 144, 668 (1939).

[81]

PHOSPHOMONOESTERASE OF MILK

535

Application of Assay Method to Crude Preparations. Whole milk or b u t t e r m i l k should be dialyzed against distilled w a t e r for 12 hours to remove inorganic p h o s p h a t e which inhibits the enzyme and causes a high b l a n k value. T h e final concentration of m a g n e s i u m should not exceed 0.001 M, higher concentrations being inhibitory to crude enzyme preparations2 Purification P r o c e d u r e

T h e purification procedure described here is t h a t of Morton. 10,11 Zittle and Della Monica 12 h a v e also published a purification based on the use of butanol. I° Step 1. Preparation of Buttermilk. C r e a m of high b u t t e r f a t content (65 to 70%) is obtained b y separation of fresh whole milk at 35 ° . I t is diluted with distilled w a t e r to 45 to 5 0 % b u t t e r f a t , cooled to i0 °, and held overnight, if necessary. I t is churned to b u t t e r in a barrel churn or a reciprocating shaking machine. W h e n a fine b u t t e r grain appears, the b u t t e r m i l k is removed and the b u t t e r washed twice with minimal quantities of distilled water (at 0°). T h e b u t t e r m i l k and washings arc combined and strained through cheesecloth. Commercial b u t t e r m i l k m a y be used provided t h a t it is obtained f r o m fresh unpasteurized cream of high b u t t e r f a t content. Step 2. Treatment with Butanol. Alkaline p h o s p h a t a s e in milk and b u t termilk is not in true solution b u t associated with microsomal particles (Mortong,l~.14). These m a y be sedimented b y high-speed centrifuging (60,000 X g, 1 hour), and, where suitable e q u i p m e n t is available, this m a y be introduced into the purification procedure with considerable advantage. ~5 T h e enzyme is released from the microsomes into true solution as follows. T h e b u t t e r m i l k is heated to 32 °, and n-butanol (30 % v / v ) is added slowly with stirring. T h e emulsion is raised to 35 ° for approxim a t e l y 5 minutes and then adjusted to p H 4.95 b y cautious addition of 0.25 N acetic acid. On centrifuging, the material separates into distinct layers comprising a precipitate, a clear yellow-green aqueous layer over9 R. K. Morton, Ph.D. Thesis, University of Cambridge, 1952; Biochem. J. in press. 10R. K. Morton, Nature 166~ 1092 (1950). LIR. K. Morton, Biochem. J. 55~ 795 (1953). L2C. A. Zittle and E. S. Della Monica, Arch. Biochem. and Biophys. 35, 321 (1952). la R. K. Morton, Nature 171, 734 (1953). 14R. K. Morton, Biochern. J. 55~ 786 (1953). ~5The buttermilk is adjusted to pH 6.5 and centrifuged (60,000 × g, 1 hour). The precipitate is washed once by suspending it in 0.85 % NaC1 (at pH 6.5) and reeentrifuging. The sediment is suspended in distilled water and treated as described for buttermilk. Steps 7, 8, and 9 may then be omitted.

536

ENZ~ES

IN PHOSPHATE METABOLISM

[81]

laid b y a butanol-saturated material, and excess butanol. 16 The aqueous layer is removed b y suction and filtered through a thick layer of Hyflo Super-Cel (Johns-Manville Corp.) over W h a t m a n No. 1 filter paper on a large Bfichner funnel. T h e filtrate is brought to p H 8.5 with 0.2 N N a O H and held overnight at 5 °, when a bulky white precipitate settles. T h e sup e r n a t a n t is largely decanted, and the remainder is recovered b y centrifuging (2000 X g, 15 minutes). Step 3. Precipitation with Ether-Acetone. The clear serum is adjusted to p H 6.3 with 0.1 N acetic acid and cooled to 0 °. I t is treated in batches with addition of diethyl ether (10% v / v , - 1 5 °) and then acetone (45% v / v , - 1 5 ° ) , the t e m p e r a t u r e being maintained at - 3 to - 7 ° in a suitable low-temperature bath. T h e material is held overnight at about - 5 °, the s u p e r n a t a n t cautiously decanted, and the pink-colored precipitate collected b y centrifugation (2000 >( g, 20 minutes, - 5 ° ) . Step 4. Fractionation with Ammonium Sulfate. The precipitate is dissolved in a minimum of distilled water at 0 ° and either centrifuged (20,000 X g, 20 minutes, 0 °) or filtered overnight at 0 ° through Whatman No. 542 filter paper. T h e red-colored, water-clear solution is dialyzed against distilled water at 0 ° to remove organic solvents. Solid ammonium sulfate is added to 0.63 saturation, the solution being maintained at p H 7.1 b y cautious addition of 0.1 N N a O H . T h e red-colored precipitate is collected b y centrifugation (4000 X g, 40 minutes, room temperature) and discarded. T h e clear s u p e r n a t a n t is brought to 0.85 saturation b y addition of solid ammonium sulfate. After standing for 1 hour, the precipitate is collected b y centrifugation as before. I t is dissolved in a minimal q u a n t i t y of distilled water and dialyzed against distilled water at 0 ° to remove ammonium sulfate. Step 5. Adsorption by Charcoal. T h e solution is brought to p H 8.6 with 0.1 N NaHCO3 and diluted to about 5 mg. of protein per milliliter with distilled water. Animal charcoaP 7 is added as a slurry in distilled water. T h e material is held for about 30 minutes and then centrifuged (2000 X g, 15 minutes). The charcoal should be added cautiously in small amounts (about 2.5 g. per 100 ml. of enzyme solution), and the procedure repeated until about 10% of the enzyme is adsorbed. The p H should be maintained at about 8.6 t h r o u g h o u t this step. Peptized charcoal is finally 16The excess butanol may be recovered by distillation and is quite suitable for further preparative work. 17The charcoal is prepared as follows: About 180 g. of animal charcoal (British Drug [Houses Ltd.) is treated successively with 51. of 0.1 N HC1, 21. of 5% KC1, 200 ml. of 0.1 M BaOH, and finally 21. of 5% KC1. After each treatment the charceal is sucked dry on a Whatman No. 42 filter paper on a large Btichner funnel. After the last treatment it is dried at 100°.

[81]

PHOSPHOMONOESTERASE OF MILK

537

removed by filtration of the enzyme solution through a thin layer of BaC03 overlying Hyflo Super-Cel on a Whatman No. 1 paper on a Btichner funnel. Step 6. Fractionation with Ether-Acetone. The solution is dialyzed against distilled water, adjusted to pH 6.4, and fractionated by addition of diethyl ether (10 % v/v) followed by acetone (40 to 49 % v/v) at - 5 ° (see step 3). The 40 to 49 % acetone precipitate is dissolved in the minimal amount of distilled water at 0 ° and dialyzed at 0 ° against distilled water to remove organic solvents. Any precipitate is removed by centrifuging (20,000 X g, 1 hour). Step 7. Adsorption with Charcoal. The solution is brought to pit 8.6, and step 5 is repeated, sufficient charcoal being added to adsorb about 20 % of the phosphatase activity. Step 8. Fractionation with Ammonium Sulfate. Step 4 is repeated, the fraction obtained at pH 7.2 between 0.69 and 0.82 saturation with ammoSUMMARY OF P U R I F I C A T I O N PROCEDURE a

Fraction

Specific Total Total Protein activity, Recovvolume, Units/ml. units nitrogen, units/rag, ery, ml. thousands thousands mg./ml. N %

1. Buttermilk and washings 32,500 2. Serum from butanol treatment 27,000 3. Ether-acetone precipitate 1,480 4. (NH4)~SO4 fraction, 0.63-0.85 127 5. Supernatant from charcoal adsorption 280 6. Ether-acetone fraction (40-49 % acetone) b 75 7. Supernatant from charcoal adsorption 89 8. (NH4)~SO4 fraction, 0.69-0.82 12 9. Ether-acetone fraction (42-48% acetone) b 10

80

2,600

2.80

28.6

--

65

1,755

0.20

325.0

67

1,092

1,616

1.20

910.0

62

9,230

1,172

2.25

4,102.2

45

3,340

935

0.46

7,261

36

9,800

735

1.04

9,423

28

5,730

510

0.49

11,694

20

29,000

348

2.04

14,216

13

26,800

268

1.75

15,314 ~

10

R. K. Morton, Biochem. J. 55, 795 (1953). b Limits of fractionation should be determined by small-scale trials. The figures given here should be taken as a guide only, since small changes of ionic strength, etc., may considerably alter fractionation. c Q~ 106,000. The purification compared to whole milk was 5660 times, or 535 times compared with buttermilk.

538

ENZYMES IN PHOSPHATE METABOLISM

[81]

nium sulfate being retained. The precipitate, dissolved in the minimal amount of distilled water, is dialyzed against distilled water until free of salt. Step 9. Fractionation with Ether-Acetone. The solution is adjusted to pH 6.5 and to 0.015 M magnesium acetate, and the enzyme is precipitated by addition of diethyl ether (10% v/v) and acetone (42 to 48% v/v) at - 5 ° (see step 3). The 42 to 48 % acetone fraction is dissolved in the minimal amount of distilled water, frozen and dried in vacuo.

Properties Stability. The enzyme may be stored at 0 ° in vacuo over NaOH for about four months without loss of activity beyond an initial decline which occurs on drying the preparation. A slow, irreversible decline occurs with longer periods of storage. Dilute solutions of the enzyme may lose activity owing to irreversible denaturation if stored frozen. They may be stored more satisfactorily at 0 ° and at pH 7.2 under toluene vapor to exclude bacterial contamination. Specificity. It has been shown 9 that the purified enzyme hydrolyzes orthophosphomonoesters and related compounds such as phosphoenolpyruvate and phosphoamides (e.g., creatine phosphate). It is probable that acyl phosphates and thiophosphates are also hydrolyzed. However, phosphodiesters and pyrophosphates are not hydrolyzed by the pure enzyme. The rates at which various phosphomonoesters are hydrolyzed differs considerably (see also Effect of pH, below). For example, under optimal conditions for both substrates, the rate of hydrolysis of phenyl P is about 1.6 times that of E-glycerol P. Activators and Inhibitors. The purified enzyme is activated by several divalent metals. As indicated in the assay procedure, magnesium (10-2 M), gives maximum activation. Optimal concentrations for other metals are: zinc 10-8 M, calcium 10-~ M, and manganese, 10-3 M. Zinc and beryllium are inhibitory at 10-4 M and higher concentrations. The enzyme is inhibited 9,18 by various anions, such as phosphate, pyrophosphate, arsenate, carbonate, and borate, and by iodine. Inhibition may be both competitive and noncompetitive, and the amount of inhibition depends on both the substrate concentration and the pH of the test system. Certain amino acids, such as glycine, alanine, and cysteine, are inhibitory at high concentrations, as are other metal chelating agents. Effect of pH. The optimal ptI for hydrolysis varies with the experimental conditions, being dependent on the substrate, the concentration of the substrate, and the nature of the buffer (should this be inhibitory). 18 C. A. Zittle and E. S. Della Monica, Arch. Biochem. 26~ 135 (1950).

[82]

PHOSPHOMONOESTERASE OF BONa

539

Since different alkaline buffer systems have different temperature coefficients, it is important to make activity and pH determinations at the one temperature or to make accurate temperature corrections. When the activity is determined with either Veronal-acetate-HCl (0.05 M) or ethanolamine-HC1 (0.04 M) buffer, with 0.01 M magnesium, the following are the optimal pH values with the various substrate concentrations as shown: phenyl P (2.5 X 10-3 M), pH 10.05; ~-glycerol P (2 X 10-2 M), pH 9.65; adenosine-5-P (4 X 10-4 M), pH 9.5; creatine P (9 X 10-3 M), pH 9.55. Transphosphorylation Activity. The enzyme catalyzes the transfer of the phosphate group from suitable substrates (such as phenyl P) to certain aeceptors (such as glucose or glycerol) to form new phosphate esters 19 (see Vol. II [88]). Chemical Constitution2 The purified enzyme appears to be an unconjugated protein. It contains 16.2 % nitrogen. Tests for phosphate, carbohydrate, and nucleotide were negative2 No evidence of a dialyzable or dissociable group (except a divalent metal) essential for enzymic activity has been obtained. Relationship to Other Phosphomonoesterases. The milk enzyme differs from the alkaline phosphatase of calf intestinal mucosa (which has also been purified by a procedure similar to that described above2°). The specific activity of the purified milk enzyme is about one-fifth of that of the pure intestinal phosphatase. The optimal pH for hydrolysis of different substrates also differs for the two enzymes. The milk phosphatase is identical with the phosphomonoesterase of mammary gland and of kidney TM and either identical with, or closely related to, the phosphomonoesterases of bone and of liver. 19 R. K. Morton, Nature 172, 65 (1953). 2o R. K. Morton, Biochem. J. 57, 595 (1954). 21 S. J. Folley and H. D. Kay, Biochem. J. 29, 1837 (1935).

[82] Phosphomonoesterase of Bone By

ELLIOT VOLKIN

Assay Method

Principle. The monoesterase specifically hydrolyzes singly esterified phosphoryl groups, as in ~-glycerophosphate, monophenylphosphate, and mononucleotides, and does not attack diesterified phosphoryl groups, as in diphenylphosphate or the internucleotide phosphoryl groups of poly-

540

ENZYMES IN PHOSPHATE METABOLISM

[82]

nucleotides. Generally speaking, a disadvantage in its use is its relatively low specific activity as compared with monoesterases from other sources (prostate, semen, potato, barley). The enzyme is activated by magnesium ion and shows optimal activity around pH 8.6 to 9.0. Protocol. One milliliter each of adenosine-2'-, -3'-, and -5'-phosphate and monosodium diphenylphosphate, each containing 100 ~, of phosphorus per milliliter; 1 ml. of 0.1 M NH4C1-NH3 buffer, pH 9.0, containing 0.005 M MgS04; 1 ml. of phosphomonoesterase preparation from beef metatarsal bone (0.10 mg. of nitrogen per milliliter); water to 5 ml. At the time intervals indicated in the table, 1 ml. was removed for inorganic phosphate assay. 1 Procedure. The protocol and table demonstrate the typical hydrolysis of the mononucleotides of adenylic acid by bone monoesterase. The absence of contaminating diesterase activity is evident by the failure of the preparation to liberate inorganic phosphate from diphenylphosphate.

Preparation Principle. No important changes have been forthcoming in the preparation of this enzyme since it was first described in 1938 by Gulland and Jackson. 2 The method consists in (a) autolysis of metatarsal bone for the extraction of total phosphatase activity 3 and (b) a preferential elution of monoesterase from diesterase activity from Norit charcoal, which adsorbs both enzymes. Increased activity results from removal of the eluting borate ion, and concentration of the enzyme by precipitation with alcohol: ether yields a more workable enzyme solution concentration. It should be noted that the method is not designed for the purification of monoesterase free of enzymes other than diesterase. Procedure. The beef foreleg bones are skinned, scraped free of tissue, sawed or crushed into small nuggets, then freed of marrow. About five times their weight of water containing a little chloroform is added, and the bones are allowed to autolyze for 7 days at room temperature. The phosphoesterases of the autolyzate are adsorbed on Norit A charcoal, by stirring 6 g. of the charcoal with 1 1. of extract at 0 to 5 ° for 1.5 hours. The charcoal is collected, washed with water, and eluted with 25 ml. of pH 8.6 Clark and Lubs borate buffer. The eluate, containing monoesterase but no diesterase, is dialyzed with cold distilled water, then precipitated at - 5 ° by the addition of 5 vol. of 2:3 methanol:ether. The precipitate is 1C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925); see also E. Volkin and W. E. Cohn, in "Methods of Biochemical Analysis," Vol. I, pp. 287-305, Interscience Publishers, New York, 1954. J. M. Gulland and E. M. Jackson, Biochem. J. 32, 590 (1938). s M. Martland and R. Robison, Biochem. J. 25, 237 (1929).

[83]

GLUCOSE-6-PHOSPHATASE FROM LIVER

541

collected, redialyzed against cold, distilled water, and a n y remaining insoluble material removed by centrifugation. The enzyme preparation m a y be stored frozen without loss of activity. DEPHOSPHORYLATIONOF THE ADENYLICACID MONONUCLEOTIDESBY BONE PHOSPHOMONOESTERASZ Percentage of hydrolysis Time, rain. Adenylic-2' 20 4O 60 240

50 8O 96 --

Adenylic-3' 42 72 95 --

Adenylic-5' Diphenylphosphate 50 78 95 --

0 0 0 0

[83] G l u c o s e - 6 - p h o s p h a t a s e f r o m Liver Glucose-6-P A- H20--* Glucose -]- P04

By MARJORIE A. SWANSON Assay Method

Principle. T h e method is based on the incubation of the specific substrate with the enzyme and determination of the liberated orthophosphate. Reagents G~6-P stock solution (0.1 M). Suspend 260 mg. of the barium salt I in 2 ml. of distilled water, and dissolve in the minimum a m o u n t of 1 N HC1. Add 72 mg. (0.5 mM) of anhydrous Na2SO4. R e m o v e the precipitated BaS04 b y centrifugation, and test the supernarant solution for complete precipitation with a v e r y small a m o u n t of Na~SO4. Bring the supernatant solution to p H 6.5 with NaOI-I, and make to 5 ml. Buffer. Dissolve 116 mg. of maleic acid in water, bring to p H 6.5 with NaOH, and make to 10 ml. Enzyme. 5 % to 10% homogenate of liver (ice-cold).

Procedure. I n t o a conical 12- or 15-ml. calibrated centrifuge tube measure 0.1 ml. of substrate solution and 0.3 ml. of buffer and bring to 37 ° in The crystalline salt G-6-P, Ba7H20 is now available commercially. For methods of preparation see Vol III [19].

542

ENZYMES IN PHOSPHATE METABOLISM

[83]

a water bath. Add 0.1 ml. of homogenate, mix by swirling, incubate for 15 minutes, stop with 1 ml. of 10% TCA, and chill in ice. After 5 minutes, dilute to 2.5 ml., and centrifuge. Take 2-ml. aliquots of supernatant solution for determination of inorganic phosphate. No unit of activity has been defined. Under the conditions described, about 7 rag. of inorganic phosphate is liberated per gram of rat liver.

Purification Attempts to purify the enzyme have been limited by its extreme instability and its insolubility. It appears to be bound to the microsomes. These can be sedimented from isotonic sucrose at 30,000 X g, or agglutinated at pH 5.4. The precipitated microsomes may be washed at this pH to remove phosphoglucomutase. Hexose isomerase, ATPase, AMPase, and a feeble glycerophosphatase are not removed by the washing. Solubilization by means of detergents or bile salts has not been attempted. Occurrence This specific phosphatase has been found only in liver, kidney, and small intestine. ~

Properties The enzyme is apparently specific for G-6-P. After removal of the mutase by washing, G-1-P is split not at all, and fructose disappears in the same proportion that inorganic phosphate is liberated from F-6-P. Galactose phosphate and mannose phosphate have not been tested. It has an optimum pH of 6.5 and appears to require no metallic activator. Molybdate and arsenate inhibit to some extent; fluoride gives very inconsistent results. 3 Under the conditions specified, the enzyme does not appear to be completely saturated with substrate. About 10 % greater activity is obtained if the substrate concentration is doubled. A much greater decrease in activity is noted if the substrate concentration is halved. Activity is not proportional to the dilution of the enzyme; the maximum activity (for rat liver) is found with 10 to 20 mg. of tissue per milliliter of incubation mixture. It decreases with both higher and lower concentrations of enzyme. The rate of the reaction decreases with time, but not sufficiently to be a first-order reaction. Change in temperature has only a small effect, a Q10 (26 to 36 °) of 1.2 being found in the assay system described. ~ G-6-Pase is apparently located solely in the microsomes. 2'4 It there2 H. G. Hers and C. de Duve, Bull. soc chim. biol. 32, 20 (1950). 8 M. A. Swanson, J . Biol. Chem. 184, 647 (1950). M. A. Swanson, unpublished data.

[84]

FRUCTOSE-1,6-DIPHOSPItATASE FROM LIVER

543

fore becomes an indicator for the presence of microsomes, as in mitochondria prepared from liver or kidney. Homogenates prepared from liver in isotonic sucrose, KC1, NaCl, or in distilled water have the same G-6-Pase activity, but mitochondria prepared from sucrose have no G-6-Pase activity, and those prepared from salt solutions may contain 10 % to 20 % of the total tissue activity.

[84] Fructose-l,6-diphosphatase from Liver FDP + H20 --~ Pi + F-6-P

By R. W. McGILVERY

Assay Method Principle. The method is that of Pogell and McGilvery' in which the P~ liberated from FDP is determined. Reagents 0.05 M FDP. Prepare a solution of the sodium salt at pH 7.4 preferably from the purified cyclohexylammonium salt? 0.05 M boric acid-NaOH buffer, pH 9.5. 0.05 M MgS04. 0.005 M MnC12. 0.05 M cysteine-NaOH, pH 9.5. Prepare this solution daily. 0.1 M trichloroacetic acid. Enzyme. Dilute the solution to be tested to a concentration of 5 to 20 units/ml, with water (see definition below). Crude preparations should be adjusted to pH 9.5 immediately before the assay. Procedure. Place 0.i ml. of FDP, 0.4 ml. of borate buffer, 0.I ml. of MgSO4, and 0.i ml. of MnCl~ in a centrifuge tube. Warm the tube and a container with the enzyme sample in a 38 ° bath for 5 minutes. Then add 0.2 ml. of enzyme to the tube, followed immediately by 0.i ml. of cysteine previously warmed to 38 °. After 20 minutes of incubation add 1 ml. of trichloroacetic acid and determine the Pi liberated, 8 which should not exceed 1.25 micromoles from 5 micromoles of FDP. Definition of Unit and Specific Activity. One unit of the enzyme is de1B. M. Pogell and R. W. McGilvery,J. Biol. Chem. in press. See Vol. III [22]. See Vol. III [147].

544

ENZYMES IN PHOSPHATE METABOLISM

[84]

fined as the amount which will liberate 1 micromole of P~ with linear kinetics in 1 hour under the above conditions. Specific activities are given in units per milligram of protein, estimated as 6.25 × Kjeldahl N for crude preparations and as (1.55E2s0 - 0.76E260) mg./ml, for transparent solutions. Application of Assay Method to Crude Preparations. No exact assay of crude extracts containing nonspecific phosphatases active at pH 9.5 can be made. A rough correction for contaminating activities can be made by substituting F-6-P for F D P in the assay and subtracting the value thus obtained from the FDPase assay. Preparation of the extracts at pH 4.5 (see Purification Procedure) may destroy most of the interfering enzymes, but tests should be made. In most cases, Mn ++ and cysteine should be omitted (see Properties). Purification Procedure Step 1 is essentially according to Gomori, 4 and the remaining steps are those of Pogell and McGilvery.1 Step 1. Preparation of Autolyzed Extract. Homogenize the livers from exsanguinated rabbits (6 to 8 make a convenient amount) for 2 minutes in a Waring blendor with 4 ml. of 0.005 M sodium lactate buffer, pH 3.5, per gram of tissue. Centrifuge the homogenate (specific activity of 1.3 units/rag.) for 30 minutes at 1000 X g. Transfer the supernatant, which should be near pH 4.5, to a flask in a 38 ° bath. Keep the flask in the bath for 8 hours after the temperature of the solution reaches 37 °. Chill the flask to 4 ° in ice, and adjust the solution to pH 7.0 with 1 N NaOH. Remove and discard insoluble materials by centrifugation for 60 minutes at 2500 X g. Step 2. First Ammonium Sulfate Fractionation. Bring the autolysate (specific activity of 20 to 25 units/mg.) to 2.28 M by the slow addition of solid ammonium sulfate and then to 2.75 M by the dropwise addition of 3.89 M ammonium sulfate solution. Remove and discard the precipitate by centrifugation for 20 minutes at 2500 X g. Raise the concentration of ammonium sulfate to 3.00 M by dropwise addition of 3.89 M solution. Collect the precipitated enzyme (45 to 65% from the autolysate, specific activity of 50 to 100 units/mg.) by centrifugation. Step 3. Second Ammonium Sulfate Fractionation. Dissolve the precipitate, and dilute the solution to a protein concentration of 4 to 4.5 mg./ml. with a measured volume of water. Measure the total volume, and calculate the salt concentration of the diluted solution. Remove a slight amount of insoluble material by centrifugation for 1 hour at 18,000 X g. 4 G. Gomori, J. Biol. Chem. 148, 139 (1943).

[84]

FRUCTOSE-1,6-DIPttOSPHATASE FROM LIVER

545

Adjust the supernatant to p H 7.0 with 1 N N a O H , and raise the salt concentration to 1.90 M with solid ammonium sulfate and then to 2.48 M by dropwise addition of 3.89 M solution. Centrifuge for 30 minutes at 2500 X g, and discard the precipitate. Raise the supernatant to 3.00 M ammonium sulfate b y addition of the 3.89 M solution. Collect the precipitate (25 to 35% from the autolyzate, specific activity of 100 to 120 units/mg.) b y centrifugation for 1 hour at 2500 X g. Step ~. Alumina Adsorption. Dissolve the precipitate, and dilute it to a concentration of 5 mg. of protein per milliliter with water. Bring the solution to p H 4.0 with 1 N lactic acid, and add an a m o u n t of C~ alumina 5 suspension containing an a m o u n t of alumina equal to the weight of protein. After 10 minutes of gentle stirring, collect the gel b y centrifugation. Successively wash the gel b y centrifugation with portions of 0.005 M sodium lactate, p H 4.1, and 0.1 M sodium borate, p H 8.5, equal in volume to the protein solution before addition of the alumina. Elute the enzyme b y three extractions of the gel with 0.1 M sodium borate, p H 9.25, at the centrifuge, using a volume one-third t h a t of the original solution before adsorption for each extraction. In occasional preparations, the use of more alumina with an eluting buffer of higher p H will be required. The eluate should contain ca. 2000 units per rabbit liver (10 to 15% of the activity from the autolysate) with a specific activity near 400 units/mg, of protein, representing an increase in specific activity of 300-fold over the original homogenate. SUMMARY OF PURIFICATION PROCEDUREa

Step

Fraction

1. Homogenate Autolysate 2. 2.75-3.00 M (NH4)2SO4 3. 2.48-3.00 M (NH4)~S04 4. Eluate from alumina

Totalb volume, ml.

Total units b

Specific activity, units/mg.

Recovery, %

1980 1320 --75

-122,000 56,000 40,000 16,800

1.3 20-25 50-100 100-120 400

-100 45-65 25-35 10-15

B. M. Pogell and R. W. McGilvery, J. Biol. Chem. in press. b The total values are for a preparation from 400 g. of rabbit liver. The other columns give the ranges encountered in various trials.

Properties Specificity. The enzyme has no effect on G-l-P, G-6-P, F-6-P, PGA, or L-sorbose-l-P. F-1-P and L-sorbose-l,6-di-P are hydrolyzed at rates of 6 See Vol. I [11] for preparation of alumina C~.

546

ENZYMES IN" PHOSPHATE METABOLISM

[85]

0.009 and 0.03, respectively, the rate of F D P hydrolysis, indicating a specificity toward the phosphate bond bearing a 1-relation to a 2,5-furanose ring of D-arabino configuration. Although only one of the P groups of F D P is hydrolyzed, occasional purified enzyme preparations give products having higher acid-labile P contents than does F-6-P. The cause of this anomaly is unknown. Activators and Inhibitors. Mg ++ or Mn ++ is required for activity. In crude extracts, Mg ++ is better than Mn ++, and addition of Mn ++ or cysteine in the presence of Mg ++ causes inhibition. At all stages beyond step 1, addition of Mn ++ or cysteine in the presence of Mg ++ results in stimulation and is necessary in order to demonstrate recovery of the enzyme on fractionation. The individual effects vary from one preparation to another and from step to step. The conditions given in the assay method represent a compromise. Preparations stimulated by 0.0005 M Mn ++ are slightly stimulated by Fe ++ and inhibited by Co ++, Ni ++, Cu ++, and Zn ++ at the same concentration? Fluoride at 0.01 M inhibits 60%. 4 Stability. Preparations carried through step 3 have been stored in the frozen state for 15 months with loss of less than half of the activity. After step 4, the stability is markedly lowered. The enzyme from step 2 can be incubated for 2 hours at 38 ° without substrate or activators at any pH between 4 and 9 with loss of less than half of the activity. Heating the crude extract at pH 4.6 in a 100 ° bath until its temperature rises to 70 ° causes 10 to 15% loss. At 75 ° the loss is about one-third, and at 80 ° it is greater than 90%. pH Optimum. The enzyme is most active under the assay conditions at p H 9.3 to 9.5. Serine, barbital, and borate buffers give equal values.

[85] "5" Nucleotidases 5-AMP + H~O --~ Adenosine + P By LEON A. HEPPEL and R. J. HILMOE " 5 " Nucleotidases 1 are enzymes which hydrolyze phosphate esterified at carbon 5' of the ribose or deoxyribose portions of the nucleotide molecule. Thus far, only mononucleotides have ~,een available as substrates; polynucleotides containing phosphomonoester groups at carbon 5' of the sugar have not been reported. 1j. Reis, Bull. soc. chim. biol. 16, 385 (1934).

[85]

" 5 " NUCLEOTIDASES

547

I. "5" Nucleotidase of Seminal PLasma Assay Method Reagents 1.0 M glycine-NaOH buffer, pH 8.5. 0.1 M MgC12. 5-AMP solution, kept frozen. Enzyme solution, kept frozen.

Procedure. A total volume of incubation mixture of 1.24 ml. is made to contain 0.1 ml. of buffer, 0.1 ml. of MgC12, 3 micromoles of 5-AMP, and enzyme. Incubation is for 15 minutes at 37 °, and the reaction is stopped by the addition of trichloroacetic acid in a final concentration of 5%. If necessary, the mixture is centrifuged. An aliquot is analyzed for inorganic p.2 The amount of enzyme can be varied widely because the reaction is linear until nearly all the 5-AMP is utilized. A substrate blank is usually necessary. Definition of Unit and Specific Activity. A unit of activity corresponds to the liberation of 1 micromole of P per hour, and specific activity is defined as units per milligram of protein. Protein is determined by the method of Lowry et al. 3

Purification Procedure This is from the work of Heppel and Hilmoe. ~ Bull seminal plasma is obtained, usually from an Artificial Breeder's Association. It may be stored at - 8 ° for at least six months. The following steps are at 2 °, except as noted. Step 1. Treatment with Protamine Sulfate. 110 ml. of bull seminal plasma is mixed with a solution of 550 mg. of protamine sulfate in 330 ml. of distilled water. A precipitate is removed by centrifugation and discarded. Step 2. First Ammonium Sulfate Fractionation. The supernatant is brought to pH 6.7, and the volume is adjusted to 490 ml. Ninety-five grams of ammonium sulfate is added, and the pH is adjusted to 4.2 by means of 66 ml. of 1 N acetic acid. Then 139 ml. of 0.2 M acetate buffer, pH 4.2, and 84.5 g. of ammonium sulfate are added. After 15 minutes the suspension is centrifuged for 7 minutes at 13,000 r.p.m, in a Servall type SS-1 angle centrifuge. The precipitate is discarded. To 745 ml. of super2 C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925). 30. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 4L. A. Heppel and R. J. Hilmoe, J. Biol. Chem. 188, 665 (1951).

548

ENZYMES IN P H O S P H A T E METABOLISM

[85]

n a t a n t is added 225 g. of ammonium sulfate, and the precipitate is collected by centrifugation, dissolved in 45 ml. of distilled water, and dialyzed against running 0.01 M acetate buffer, pH 6.0, for 4 hours. Step 3. Second Ammonium Sulfate Fractionation. The dialyzed material from step 2 measures 74 ml. and is turbid. I t is clarified by centrifugation and mixed with 8.2 ml. of 1 M acetate buffer, pH 6.0, and 26.4 g. of ammonium sulfate. The precipitate is removed by centrifugation and discarded. To 91.5 ml. of supernatant is added 5.6 g. of ammonium sulfate. The precipitate is collected by centrifugation, dissolved, and dialyzed as before. Step ~. Ethanol Fractionations. The dialyzed ammonium sulfate fraction (35 ml.) is brought to pH 4.6 with 4.5 ml. of 0.1 N acetic acid, and enough 1 M acetate buffer, pH 4.6, is added to bring the concentration of acetate to 0.1 M. The solution (41 ml.) is cooled to - 0 . 5 °, and successive additions of absolute ethanol are made in the following amounts (milliliters) : 3.5, 0.7, 0.5, 0.5, 0.3, 0.5, 1.0, and 2.0. The mixture is rapidly stirred during the ethanol additions and is maintained near its freezing point. After each addition there is a 5-minute wait before a brief centrifugation. The precipitates are dissolved in 0.02 M acetate buffer, pH 6.5. The best fractions are combined, and the ethanol step is repeated. No dialysis is carried out. Step 5. Heating, Followed by Alumina Gel. The ethanol fraction is adjusted to pH 6.8, warmed to 60 ° over a period of 90 seconds, and maintained at this temperature for 20 minutes. This t r e a t m e n t removes contaminating phosphatase activity. After rapid cooling small aliquots are mixed with different quantities of aluminum hydroxide gel C~ ~ (11 mg. dry weight per milliliter, aged three months at least), centrifuged, and SUMMARY OF PURIFICATION PROCEDURE a

Step Seminal plasma 1. Protamine sulfate 2. Ammonium sulfate 3. Ammonium sulfate 4. First ethanol Second ethanol 5. Heating and gel "

Volume, ml.

Total units

110 419 82 35.5 37 18.3 80

310,000 290,000 133,000 74,000 61,270 18,710 12,710

Specific activity, Yield, units/mg. % 50 111 274 476 986 1350 2500

100 93 43 24 19.8 6 4

L. A. Heppel and R. J. Hilmoe, J. Biol. Chem. 1 8 8 , 665 (1951).

5 R. Willsti~tter and H. Kraut, Ber. 56, 1117 (1923); see also Vol. I [11].

[85]

"5"

NUCLEOTIDASES

549

the supernatant solutions tested for activity and protein content. The maia lot is then treated with the amount of gel found to give the best purification and a high yield.

Properties

Specificity. Purification of bull seminal plasma with respect to the splitting of 5-AMP resulted in no change in the relative activities toward the 5' esters of inosine, uridine, nicotinamide riboside, and cytidine. Adenosine-2',5'-diphosphate is not split. A low activity against ATP and ADP remains after purification. Stability. The purified enzyme is stable for eight weeks as a frozen solution. Lyophilized samples can be stored at - 8 ° for at least six months with little loss of activity. Effect of pH. The pH optimum is 8.5. The reaction rate is proportional to the concentration of enzyme over a wide range. Substrate A~nity. The 5'-phosphates of adenosine, inosine, uridine, and cytidine are relatively tightly bound, with dissociation constants below 10-4. For nicotinamide mononucleotide the value is 1.6 X 10-3. Metal Requirements and Inhibitors. In the absence of MgC12 the rate of reaction is decreased by 70%. Neither CaC12 nor MnC12 can replace MgC12. In the presence of MgC12 the reaction is inhibited by CaC12, and this effect can be overcome by excess MgC12. Sodium fluoride inhibits the rate of splitting of 5-AMP by bull semen enzyme to the extent of 98% at 0.1 M and 73% at 0.01 M. Borate buffer (8 × 10-2 M) inhibits 85 % compared with glycine buffer of the same pH. II. "5" Nucleotidase of Snake Venom Assay Method The assay method is the same as for seminal plasma.

Purification Procedure This is taken from Hurst and Butler. 6 Ten milligrams of lyophilized venom is dissolved in 10 ml. of water and chromatographed on a column of 1 g. of shredded filter paper (Whatman No. 5) 10 cm. long and 0.8 cm. in diameter. The flow rate is i ml./min. Fifty-five milliliters of water is run through the column, followed by 80 ml. of 0.1% NaC1 and 30 ml. of 1.0% NaC1. Fractions of 10 ml. are collected, and their optical density at 260 m~ measured. The elution peak obtained with 1% NaC1 contains "5" nucleotidase with a considerable amount of phosphodiesterase removed. e R. O. Hurst and G. C. Butler, J. Biol. Chem. 193, 91 (1951).

550

ENZYMES IN PHOSPHATE METABOLISM

[85]

Properties The unfractionated venom from many species of snakes has been tested and found to be highly specific in hydrolyzing 5'-nucleotides, but not the other isomers. 7 As is true for the bull semen enzyme, substrate affinity is high. The following enzyme-substrate dissociation constants have been obtained for Crotalus atrox venom: 5-AMP, <10-4; nicotinamide mononucleotide, 8 × 10-4; ribose-5-phosphate, 1.7 × 10-8. Venom does not split adenosine 2',5'-diphosphate.

III. Potato Adenosine-5-phosphatase This enzyme fraction has the advantage of being able to remove P esterified at carbon 58 of adenosine 2',5'-diphosphate. The following description is taken from Kornberg and Pricer. 8 One kilogram of peeled Maine potatoes is homogenized in a Waring blendor with 2 vol. of distilled water (in five separate batches). To 2.8 1. of filtrate is added 950 g. of ammonium sulfate; the precipitate is removed by filtration, and to the filtrate (2.5 1.) is added 550 g. of ammonium sulfate. This precipitate is collected by filtration, dissolved in water, and dialyzed for 90 minutes against cold, distilled, running water. The dialyzed fraction (61 ml.) is acidified to pH 4.5 with 2.7 ml. of 0.1 N acetic acid and fractionated with 95 % ethanol at - 5 ° to - 10 °. The precipitate obtained with 30 ml. of ethanol is discarded; that obtained after a further addition of 100 ml. is dissolved in 15 ml. of water. This fraction as compared with crude potato extracts is twenty to fifty times as active in splitting 5-AMP on a protein basis, but only five times as active in splitting adenosine-3'-phosphate. At pH 5.0 (0.1 M acetate, 0.01 M MgC12) 5-AMP is split only 2.1 times as fast as adenosine-3'phosphate, but by carrying out the reaction at an alkaline pH it is possible to limit the reaction to 5-AMP. Thus, at pH 9.4 (0.1 M glycine, 0.01 M MgCl~) the hydrolysis of adenosine-3'-phosphate and of the 2'-isomer is completely inhibited, whereas about one-third of the adenosine-5-phosphatase activity remains. 7 E. A. Zeller, personal communication. A. Kornberg and W. E. Pricer, Jr., J. Biol. Chem. 186, 557 (1950).

[86]

3t-NUCLEOTIDASE FROM RYE GRASS

551

[86] 3 ' - N u c l e o t i d a s e f r o m R y e G r a s s Nucleoside-3'-P + H20 --* Nucleoside + P

By Lovis SHUSTER and NATHAN O. KAPLAN

Assay Method The release of inorganic phosphate is measured by the method of Fiske and SubbaRow.1 An alternate method, with 3'-adenylic acid as substrate, is to measure the release of adenosine with adenosine deaminase prepared from Armour's intestinal phosphatase, by the procedure of Kornberg and Pricer. 2 In this case an aliquot of 0.05 to 0.1 ml. is removed from the incubation mixture (see below) into 2.9 ml. of 0.1 M phosphate buffer, pH 6.8, and the decrease in optical density upon the addition of 0.03 ml. of deaminase is followed at 265 m~ (see Vol. II [70]). The phosphate buffer successfully inhibits any phosphatase present in the deaminase preparation.

Reagents 3'-Adenylic acid solution (0.04 M). This is made up by dissolving 139 mg. of the free acid (obtainable from the Schwarz Laboratories, Inc., as yeast adenylic acid, isomer B) in 10 ml. of 0.1 M NaHCO~. This solution may be stored in the deep-freeze for many months. 0.1 M Tris(hydroxymethyl)aminomethane-HC1 buffer, pH 7.5. Enzyme. The aliquot used should contain 2 to 4 units of enzyme. (See definition below.)

Procedure. The following mixture is incubated for 15 minutes at 37°: 0.1 ml. of 0.04 M 3'-adenylic acid, Tris buffer, and enzyme to a volume of 2.0 ml. The reaction is stopped by the addition of 1.0 ml. of 20 % trichloroacetic acid. The control consists of the same mixture with TCA added prior to the addition of enzyme. If a protein precipitate forms, it should be removed by centrifugation or filtration. Usually so little protein is required for a test that the phosphate determination may be applied directly to the incubation mixture. Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which causes the initial release of 1 micromole of inorganic phosphate per hour under the conditions of the assay. Specific acC. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925). 2 A. Kornberg and W. E. Pricer, Jr., J. Biol. Chem. 193, 481 (1951).

552

ENZYMES IN PHOSPHATE METABOLISM

[86]

tivity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al2 Application of Assay Method to Crude Preparations. Many biological materials contain nonspecific phosphatases capable of dephosphorylating 3'-adenylic acid at pH 7.5. It is, therefore, advisable to test for such enzymes until their absence is assured by using as substrate 2'-adenylic acid (obtainable from Schwarz Laboratories, Inc., as yeast adenylic acid, isomer A) and subtracting any activity with this substrate from apparent 3'-nucleotidase to give the actual 3'-nucleotidase activity. For data on the relative amounts of 3'-nucleotidase and nonspecific phosphatase in some crude preparations, see the paper by Shuster and Kaplan. ~ Purification Procedure

The first four steps of the following procedure have been successfully repeated by several workers. The last two steps, although not presenting any special difficulties, have not been carried out often enough to allow any conclusions as to their reproducibility. Domestic rye grass (obtainable from any seed store) has proved to be a consistently satisfactory starting material, and crude extracts contain a negligible amount of nonspecific phosphatase. Step 1. Preparation of Crude Extract. About 500 g. of domestic rye grass seed is soaked in a shallow vessel with enough water to keep it thoroughly wet for 2 to 3 days at room temperature. Then it is homogenized in a Waring blendor with 1 1. of cold water. Blending is carried out in batches and for 3 minutes at a time. The homogenate is squeezed through cheesecloth, and the resulting juice can be used to blend subsequent portions of seed. The crude extract is centrifuged to remove starch and cell debris (250-ml. polyethylene bottles are convenient containers). Usually this extract is deeply colored with anthocyanins and other pigments, but these do not interfere in the assay. Step 2. Ammonium Sulfate Precipitation. In order to concentrate the extract, solid ammonium sulfate is added to reach 90 % saturation. The resulting dark brown precipitate, which floats to the surface, is then removed by filtration or centrifugation and dissolved in 150 ml. of cold water. This solution is dialyzed against cold water overnight. Step 3. First Adsorption on Alumina Gel. Alumina C7 (dry weight 22 mg./ml.) is added to fraction 2 with continuous stirring. About 100 to 150 ml. of the gel is usually sufficient to adsorb 90 % of the enzyme. The suspension is kept at 8 ° for 10 minutes, then centrifuged in the cold. The 30. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 195, 265 (1951). 4 L. Shuster and N. O. Kaplan, J. Biol. Chem. 201, 535 (1953).

[86]

3'-NUCLEOTIDASE FROM RYE GRASS

553

gel is washed twice with 150 ml. of water, then twice with 150 ml. of 0.5 M ammonium sulfate. The enzyme is then eluted from the gel with four 150-ml. portions of 1.0 M NaHC03. The amber-colored eluate is dialyzed against cold tap water overnight. Step 4. Adsorption on Calcium Phosphate Gel. About 100 to 150 ml. of calcium phosphate gel (dry weight 13 mg./ml.) is added to fraction 3 with continuous stirring. The suspension is allowed to stand for 10 minutes at 8 °, then centrifuged in the cold. The gel is washed twice with 150 ml. of distilled water, and the enzyme is eluted with five 50-ml. portions of 1.0 M ammonium sulfate. This eluate is not dialyzed previous to the next step. Step 5. Second Adsorption on Alumina Gel. About 50 ml. of alumina C~ gel is added to fraction 4 with continuous stirring. The suspension is kept at 8 ° for 30 minutes, then centrifuged in the cold. The enzyme is adsorbed with some difficulty from 1.0 M (NH4)2SO4, but under these conditions about 80% should go on the gel. The gel is washed three times with 50 ml. of H20, then elutcd with five 50-ml. portions of 1.0 M NaHCO3. The eluate is dialyzed overnight against cold water. Step 6. Second Adsorption on Calcium Phosphate Gel. Ten milliliters of calcium phosphate gel is added to fraction 5 with stirring, and the suspension is kept for 10 minutes in the cold prior to centrifugation. The gel is washed twice with 40 ml. of H20; the enzyme is eluted with three 10-ml. portions of 1.0 M (NH4)2S04, and the eluate is dialyzed overnight in the cold against 4 1. of distilled water. This preparation can be stored for at least several months at 4 ° or - 1 8 ° with little loss in activity.

Properties Specificity. The purified enzyme acts only on 3'-nucleotides and 3'-nucleotide groupings. There is no appreciable split of the other phosphate monoesters which have been tested. 4 Relative Rates. Of the various 3'-nucleotides, 3r-adenylic acid is attacked most rapidly. Initial rates of cleavage for inosinic acid, uridylic acid, and coenzyme A are about one-half the rate for adenylic acid; the guanylic acid rate is about one-third, and the rate for cytidylic acid is about one-tenth. There is some similarity between relative rates and enzyme-substrate affinities, the Km values for adenylic, uridylic, and cytidylic acids being 0.3 X 10-3, 2.5 X 10-3, and 2.5 X 10-3, respectively. Ribonuclease Activity. Although the purification procedure removes considerable ribonuclease, fraction 6 still contains enough ribonuclease to degrade about 0.03 mg. of yeast RNA per hour per unit of 3'-nucleotidase under the conditions of the 3r-nucleotidase assay. Up to one-half of this residual ribonuclease can be removed by several passages through

554

ENZYMES IN PHOSPHATE METABOLISM

[86]

a column of Dowex-1 resin in the f o r m a t e f o r m with little loss of 3'-nucleotidase activity. Since this ribonuclease does not f o r m 3'-nucleotides, it need not interfere with the e n z y m a t i c assay of such nucleotides in the presence of R N A . Effect of pH. T h e p H o p t i m u m is 7.5. Points of 50 % a c t i v i t y are a t p H 6.0 and 9.0. T h e e n z y m e is relatively stable to acid a n d can withstand 0.1 N HC1 at r o o m t e m p e r a t u r e for 5 to 10 minutes with little loss in activity. I n some cases up to 80 % of the original a c t i v i t y has been recovered after precipitation with 5 % trichloroacetic acid in the cold. Activators and Inhibitors. Cyanide and thiols inhibit at p i t 7.5 and above. T h u s cysteine a t 10 -3 M and K C N and glutathione at 5 × 10 -3 M all inhibit a b o u t 50 %. At a p H below 6.0 these produce no inhibition in this concentration range, and at p H 4.5 t h e y m a y s t i m u l a t e up to a maxim u m of a b o u t 100 %. I t is, therefore, advisable to e m p l o y a p H of a b o u t 5.0 to 5.5 when 3'-nucleotidase is used for the dephosphorylation of reduced c o e n z y m e A. 5 I n h i b i t i o n b y thiols can be reversed b y dialyzing the inhibited enz y m e against distilled w a t e r or b y oxidation of the thiol through continuous exposure to air. This inhibition is not reversed b y the addition of MgCI~, MnC12, FeC13, or ZnS04 in the same concentration as the inhibitor. Sodium fluoride, sodium azide, p o t a s s i u m ethyl x a n t h a t e , sodium versenate, and iodoacetate are ineffective as inhibitors in concentrations of 10 -3 to 10 -2 M. T h e e n z y m e is c o m p e t i t i v e l y inhibited b y 2'- and 5'-nucleotides. Heat Stability. Crude p r e p a r a t i o n s are heat-labile, m o s t of the a c t i v i t y being destroyed b y heating for 1 m i n u t e a t 100 °. During purification there is a p p a r e n t l y some r e m o v a l of a factor responsible for this heat lability, since m a n y purified p r e p a r a t i o n s are heat-stable to v a r y i n g degrees; some p r e p a r a t i o n s h a v e been found to retain 25 % of their a c t i v i t y SUMMARY OF PURIFICATION pROCEDURE

Fraction 1. 2. 3. 4.

Crude extract (NH~)~S04 ppt. Eluate from alumina Eluate from calcium phosphate 5. Eluate from alumina 6. Eluate from calcium phosphate

Total Specific volume, Total Protein, activity, Recovery, ml. Units/ml. units mg./ml, units/mg. % 800 170 680

69.8 234.0 31.6

55,800 39,900 21,400

1.64 4.25 0.095

42.5 55 332

200 250

42.9 16.5

8,600 4,130

0.054 0.015

792 1100

15 7

50

47.0

2,350

0.028

1680

4

5 T. P. Wang, L. Shuster, and N. O. Kaplan, J. Biol. Chem. 206, 299 (1954).

-72 38

[87]

PREPARATION AND PROPERTIES OF ACETYL PHOSPHATASE

555

after heating for 30 minutes at 100 ° in 0.05 M Tris buffer, pH 7.5. The addition of crude extract to such preparations makes them heat labile. The factor responsible for heat lability is as yet unknown.

[87] P r e p a r a t i o n a n d P r o p e r t i e s of A c e t y l P h o s p h a t a s e

By D. E.

KOSHLAND, JR.

Preparation Principle. The purification follows the procedure of Lipmann and is based on the stability of acetyl phosphatase in the presence of such strong denaturing agents as hot acidic solutions and cold trichloroacetic acid. Procedure. Ground horse meat (2.5 kg.) is added to 7.5 1. of boiling 1% KC1-0.067 M HC1 (final pH ca. 3.0). After 10 minutes, the solution is cooled to room temperature as rapidly as possible, and the mixture is filtered through cheesecloth. The filtrate is neutralized to pH 6 with 1 M sodium bicarbonate, and the precipitate which forms is removed by centrifugation. After the solution has cooled to less than 5 °, 400 ml. of cold 50 % TCA solution is added with stirring, and the precipitation is allowed to proceed for 1 hour. The bulk of the solution is removed by decantation, the small volume (ca. 1 1.) containing the precipitate is neutralized to pH 6.5 with sodium carbonate, and the precipitate is centrifuged in the cold. The precipitate is then treated with approximately 120 ml. of 0.1 M acetic acid. The soluble portion contains approximately 50% of the enzyme with an over-all purification factor of ca. 37. Fraction Initial extract Acetic acid solution

Total units of enzyme

Units of enzyme per mg. of total protein

33,700 16,000

0.65 24.0

Properties Physical Properties. The enzyme is nondialyzable and is destroyed by pepsin. 1 It is resistant to hot acidic (pH 3) solutions and to cold trichloroacetic acid. 1 It is presumed to be a basic protein because of its solubility in acidic, and its insolubility in basic, solutions. 1 Occurrence. The enzyme is present in a wide variety of animal tissues and to a smaller extent in bacteria. ~,2 1 F. Lipmann, Advances in Enzymol. 6, 231 (1946). B. Shapiro and E. Wertheimer, Nature 156, 690 (1945).

556

ENZYMES IN PHOSPHATEMETABOLISM

Source Pigeon muscle Beef kidney E. coli L. delbri~ckii

[88]

Units of enzyme per g. of tissue or per ml. of bacterial juice 80 36 O. 7 O. 1

Substrate Action. The enzyme hydrolyzes all acyl phosphates so far tested: acetyl, propionyl, butyryl, succinyl, octanoyl, and palmityl. 1,3 That it is an acyl phosphatase and not a general phosphatase is indicated by its failure to act on glycerophosphate, its presence in tissue which is generally low in nonspecific phosphatase activity,, and its stability in the presence of acid. ~,3 The enzyme causes splitting of the anhydride at the phosphorus-oxygen bond and is activated somewhat by magnesium ion.3.4 It shows no transferase activity to either AMP or ADP. 3 Inhibition. The enzyme is inhibited strongly by phosphate, pyrophosphate, hexosephosphate, nucleic acid, and hyaluronic acid. 1 It is less strongly inhibited by sulfate, citrate, and oxalate, and slightly inhibited by fluoride. ~-3 Cyanide has no effect at a concentration of 0.1 M. 2 Assay. One unit of acetyl phosphatase is defined as that amount of enzyme which causes 50% decomposition of a 0.006 M acetyl phosphate solution in 20 minutes at 37 ° in 0.066 M acetate buffer at pH 5.4. Acetyl phosphate concentration is measured by the hydroxamic acid method of Lipmann and Turtle. 5 A. L. Lehninger, J. Biol. Chem. 162, 340 (1946). R. Bentley, J. Am. Chem. Soc. 71, 2765 (1949). 5 F. Lipmann and L. C. Tuttle, J. Biol. Chem. 159, 21 (1945) ; see Vol. I I I [39].

[88] Transphosphorylation by P h o s p h a t a s e s Typical Reactions:

Phenyl P ~ Glucose --~ G-6-P ~ Phenol Phenyl P -~ Glycerol -~ Glycerol P -~ Phenol

(1) (2)

By ROBERT K . MORTON Assay Method Principle. The group-specific acid and alkaline phosphatases (cf. Vol. II [79 to 82]) catalyze a direct transfer of the phosphate group from suitable " d o n o r s " (substrates) to certain "acceptors" to form new phosphate esters. The mechanism of this transphosphorylation has been elu-

[88]

TRANSPHOSPHORYLATION BY PHOSPHATASES

557

cidated by Morton, 1 who showed that, in general, the substrate-specific phosphatases (cf. Vol. II [83 to 86]) do not catalyze a similar reaction. Some hydrolysis (liberation of inorganic P) always accompanies transphosphorylation (synthesis of new ester P). The method of assay of transphosphorylation activity described below is that of Morton. 2 A donor (substrate) chosen so that the liberated molecule may be readily estimated is used at a high concentration and at the optimal pH for transfer (and hydrolysis). The percentage transfer (see Definition of Activity) is independent of the donor concentration, but the optimal pH for hydrolysis is a function of donor concentration. 2 Hence the pH used is markedly suboptimal for hydrolysis of the ester formed by phosphate transfer. A specific enzymic method is employed for estimation of the synthesized ester, which should always be less than l0 -4 M concentration at the completion of the reaction. In this way the true initial rate of transphosphorylation is measured. The acceptor is used at the optimal concentration for transphosphorylation. The concomitant hydrolysis is measured by liberated inorganic P.

Reagents Disodium phenyl P (0.1 M). Anhydrous glucose or glycerol. TCA (30%). NaOH (0.2 M).

Required for alkaline phosphatases: Ethanolamine-HC1 buffer 3 (0.5 M), pH 10.15 at 20 ° (pH 9.80 at 38 ° 4) for intestinal phosphatase, or pH 10.40 at 20 ° (pH 10.10 at 38 ° 4) for other phosphatases. 5 Magnesium acetate (0.2 M).

Required for acid phosphatases: Acetic acid-NaOH buffer (0.5 M), pH 5.50. 8 Acetic acid (0.1 M). Enzyme. Dilute the preparation with distilled water so that 0.1 ml. liberates about 0.5 micromole of phenol under the test conditions. Refer also to Application of Assay Method to Crude Tissue Preparations. 1R. K. Morton, Nature 172, 65 (1953). R. K. Morton, Ph.D. Thesis, University of Cambridge, 1952. C. A. Zittle and E. S. Della Monica, Arch. Biochem. 26~ 112 (1950). 4 Note that all highly alkaline buffers change pH markedly with temperature. Intestinal alkaline phosphatase has a lower pH optimum than the other tissue alkaline phosphatases (see footnote 2). e Citric aeid-NaOH buffer (0.25 M) could probably be used. This buffer activates human prostatic acid phosphatase.

558

ENZYMES IN PHOSPHATE METABOLISM

[88]

Procedure. Suitable reaction tubes (approximately 6 mm. in diameter by 60 mm.) are graduated at 0.40 ml. and at 2.00 ml. They are closed with close-fitting rubber stoppers, previously extracted with boiling water and then with 5 % TCA. The stoppers are boiled in glass-distilled water and dried just prior to use. ALKALINE PHOSPHATASES. The final concentrations of reactants (in 0.5 ml.) are: glucose, 2 M, or glycerol, 4 M; phenyl P, 0.01 M; Mg(CH3C00)2, 0.2 M; ethanolamine buffer, 0.05 M. The stock solution is prepared by dissolving 3.60 g. of anhydrous glucose or 3.68 g. of glycerol in 1 ml. of phenyl P (0.1 M), 1 ml. of Mg(CH~CO0)2 (0.2 M), and 1 ml. of ethanolamine buffer (0.5 M), adding sufficient 0.2 M N a O H to adjust to the required pH, 5 and making up to 8 ml. The stock is stored at - 1 5 °. The solution is added to the 0.4-ml. mark in the reaction tube, equilibrated for 5 minutes at 38 °, 0.1 ml. of the enzyme (at 38 °) is added, the tube stoppered, and the contents thoroughly mixed by inversion. The reaction is stopped after 5 minutes of incubation at 38 ° by addition of 0.1 ml. of 30% TCA. After mixing and holding for about 1 minute, a predetermined volume of 0.2 M N a 0 H is added to adjust to pH 7.4, and the volume is made up to 2 ml. The tube is held in an ice bath until the assays are completed. A control tube is treated identically with the enzyme tube, except that 0.1 ml. of distilled water is added in place of enzyme. The enzyme is then added immediately after addition of TCA. With the extracts obtained by the assay procedure of Morton 2 (see below), and with all purified phosphatase preparations, no precipitate occurs with the TCA. With crude homogenates, the protein precipitate should be removed by centrifugation and then washed with 5 % TCA. The combined supernatant and washings should then be adjusted to pH 7.4 and made up to volume (2 ml.). If aliquots greater than 1 ml. are used for assay of hexose P or glycerol P, the TCA may become inhibitory to the enzymes of the assay system. Perchloric acid should then be substituted for TCA, and insoluble KC104 removed by centrifugation after adjustment to p H 7.4 with 0.2 M KOH. ACID PHOSPHATASE. The procedure is essentially as described above, except that the final concentrations of reactants (in 0.5 ml.) are as follows: glucose, 3 M, or glycerol, 5 M; phenyl P, 0.001 M; acetate buffer, 0.1 M. Appropriate adjustments are therefore made in the preparation of the stock reaction solution. The reaction is carried out at 38 ° for 5 minutes at pH 5.5. ~ Measurement of Reaction Products. Suitable aliquots-(0.t.' to 0.5 ml.) are used in the following procedures.

[88]

TRANSPI-IOSPHORYLATION BY PHOSPHATASES

559

PHENOL. The method of Folin and Ciocalt~u ~ as adapted b y King s is modified to give a final volume of 10 ml., and protein precipitation is omitted. INORGANIC P. The Weil-Malherbe and Green 9 modification of the method of Martin and D o r y 1° is used. GLYCEROL P. The method is that of M o r t o n 2 and is based on the specific enzymic reduction of ferricytochrome c b y L-a-glycerol P, catalyzed by the glycerol P dehydrogenase of insoluble particles of rabbit skeletal muscle. 11 Cuvettes (1-cm.) are prepared, each containing ferricytochrome c, 0.1 micromole; MgC12, 30 micromoles; glycylglycine buffer, p H 8.0, 150 micromoles; and the enzyme preparation 11 (0.2 ml.) in a volume of 2.5 ml. Optical densities at 550 m~ are determined before and after the further addition of the aliquot (0.5 ml.) to the one cuvette and of water (0.5 ml.) to the control. The contents of the cuvettes should be mixed before readings, which are continued until constant for at least 10 minutes. The reaction m a y take up to 40 minutes for completion, owing to the poor affinity of the enzyme for its substrate.12 Since the product formed in the transphosphorylation reaction is DLs-glycerol p,1.2 the micromoles of ferricytochrome c reduced are converted to equivalent L-s-glycerol P and multiplied b y a factor of 2. If the final volume in the cuvette is 3 ml. and a slit width of 0.05 m~ is used, then Glycerol P = (Ferrieytochrome c reduced X 0.5) X 2 = Change in optical density (AE550) X 0.154 micromole 13 O. Folin and V. Cioealt~u, J. Biol. Chem. 73, 627 (1927). 8E. J. King, "Micro-Analysis in Medical Biochemistry," 2nd ed., Churchill, London, 1952. H. Weil-Malherbe and R. It. Green, Biochem. J. 49, 286 (1951). 10j. B. Martin and D. M. Doty, Anal. Chem. 21, 965 (1949). 11The residue after extraction of rabbit skeletal muscle in 0.003 M KOH 14is repeatedly dispersed in tap water and squeezed dry. The residue is ground with sand and Na~HPO4 (0.2 M) and then dispersed in 2 vol. of distilled water. After centrifugation (2500 X g, 30 minutes), the supernatant is filtered through cotton-wool and recentrifuged (12,000 X g, 1 hour). The supernatant is cooled to 0°, adjusted to pH 5.5 with acetic acid (0.2 M), and centrifuged (12,000 X g, 30 minutes). The white precipitate is dispersed in about 10 ml. of glycylglycine buffer (0.1 M, pH 7.5), adjusted to pH 7.5 with NaOH (0.1 M), and thoroughly dispersed in a PotterElvehjem homogenizer at 0°. It is centrifuged (12,000 X g, 10 minutes), and the cloudy supernatant stored at 0°. Activity rapidly declines after 2 to 3 dayJs. 12D. E. Green, Biochem. J. 30, 629 (1936). 13With a slit width greater than 0.05 m~, the appropriate factor for converting AE~50 to micromoles of ferrocytochrome e should be determined under the experimental conditions used. The ferricytochrome c added is fully reduced by addition of a few

560

ENZYMES IN PHOSPHATE METABOLISM

[88]

HEXOSE P. The specific enzymic method of Slater 14 (see also Vol. III [19]) is used. Definition of Activity. It is clear from consideration of the reaction that moles of ester P formed equals moles of phenol minus moles inorganic P. The complete assay system thus provides a check on the estimations. The specific enzymic methods should always be used for estimation of ester P synthesized. One unit of enzyme catalyzing phosphorylation may be defined as that amount which synthesizes 1 micromole of ester phosphate (hexose P or glycerol P) per minute under the above conditions, and specific activity as units per milligram of total N (or dry weight). The efficiency of transphosphorylation is expressed by the Percentage transfer =

Ester P formed X 100 Phenyl P utilized Hexose P (or glycerol P) X 100 Phenol liberated

Application of Assay Method to Crude Tissue Preparations. The studies on which these methods are based were carried out with essentially homogeneous preparations of alkaline phosphatases 2,15 and partially purified acid phosphatases. 2 However, the methods should be suitable for assay of transphosphorylase activity of any phosphatase preparation, provided that due precautions are exercised. Preparations containing certain other enzymes, for example, specific glucose-6-phosphatase (which has a high affinity for G-6-P), should be assayed with glycerol as acceptor. It is desirable, whenever possible, to assay with both glycerol and glucose. However, the relative stability of the enzymes used for estimation of hexose P makes this method generally more convenient. Because of the numerous objections to assaying alkaline phosphatase in crude tissue homogenates 16 it is recommended that the extraction procedure of Morton 2 be followed. The tissue homogenate (at pH 7.5) is thoroughly mixed with an equal volume of n-butanol and held at 35 ° for 15 minutes. After centrifugation, the aqueous material is removed. The extraction is repeated, with butanol-saturated NaHCO3 (0.05 M). The aqueous extracts are combined, centrifuged (20,000 X g, 30 raincrystals of sodium succinate or, alternatively, of Na2S204. This enzyme system is virtually free of cytochrome oxidase and is valuable for spectrophotometric assay of succinate.2 Succinate must therefore be rigorously excluded from the trausphosphorylation system. 14E. C. Slater, Biochem. J. 53, 157 (1953). 16R. K. Morton, Nature 166, 1092 (1950); see also Vol. II [80, 81]. le j. C. Mathies, Biochim, et Biophys. Acta 7, 387 (1951).

[89]

PHOSPHODIESTERASE FROM SNAKE VENOM

561

utes) to remove insoluble material, and dialyzed overnight against 0.05 M NaHCO3. The extract is then made to a known volume. Such preparations are virtually free of specific glucose-6-phosphatase and may be used for assay of transphosphorylation activity with glucose as acceptor. Since acid phosphatases are readily extracted into solution in dilute salt solutions, the butanol extraction method has not been applied to these enzymes.

[89] Phosphodiesterase from Snake Venom R - - O - - P ( O 2 H ) - - O - - R ' + H20--, R--O--PO3H2 + R'OH

By G. C. BUTLER Assay Method Gulland and Jackson 1used diphenyl phosphate as substrate and measured the liberation of phenol. Sinsheimer and Koerner 2 measured the liberation of p-nitrophenol from bis(p-nitrophenyl)phosphate. The latter method is the most convenient procedure available at present for measuring phosphodiesterase activity. Neither of these methods will, however, detect the presence of 5'-nucleotidase, since this enzyme does not hydrolyze phenyl phosphates. The 5'-nucleotidase found in the venoms of Russell's viper, diamond rattlesnake, and water moccasin may be assayed by the liberation of inorganic phosphate from 5-AMP. The activities of both enzymes may be measured in a single digest by using as substrate the mixed deoxyoligonucleotides resulting from the action of deoxyribonuclease on DNA. The conversion of "uranium-insoluble" to "uranium-soluble ''~ phosphate is a measure of phosphodiesterase activity, and the liberation of inorganic phosphate indicates 5'-nucleotidase activity. This method of measuring diesterase activity is unreliable in the presence of appreciable amounts of 5'-nucleotidase because the latter enzyme removes the product of action of the former and may thus effect an activation of the diesterase. If the diesterase is to be used for the hydrolysis of polynucleotides, it is advisable to test its activity on the oligonucleotides.

Reagents Magnesium oligonucleotide (MgON) solution (0.5%). Dissolve 500 mg. of the dry powder, prepared by the method of Little 1 j. M. Gulland and E. M. Jackson, Biochem. J. 82, 590 (1938). 2 R. L. Sinsheimer and J. F. Koerner, J. Biol. Chem. 198, 293 (1952). R. O. Hurst, J. A. Little, and G. C. Butler, J. Biol. Chem. 188, 705 (1951).

562

ENZYMES IN PHOSPHATE METABOLISM

[89]

and Butler, 4 in 100 ml. of 0.05 M Na barbiturate-Na2C03 (Veronal-carbonate) buffer, pH 9.25. ~ Uranyl acetate-trichloroacetic acid reagent. Dissolve 1.56 g. of uranyl acetate in 100 ml. of 10 % trichloroacetic acid. Enzyme. For assay, dilute the enzyme solutions so that the phosphodiesterase gives, under the test conditions prescribed below, less than 75 % hydrolysis in 1 hour.

Procedure. Since magnesium oligonucleotide contains some "uraniumsoluble" nucleotides, the extent of this solubility must be measured before the action of the enzyme can be tested. To 2 ml. of the uranyl acetate solution (above) add 2 ml. of the MgON solution, stir the mixture, allow it to stand for !0 minutes, and then centrifuge it. Filter the supernatant solution through Whatman No. 44 paper. Dilute 1 ml. of the filtrate to 10 ml. with water, and determine the total phosphorus in 1 ml. by King's method2 After the total phosphorus content of the MgON solution has been determined, the percentage solubility in the uranium reagent may be calculated and used as the blank value for enzymic digestion. To 5 ml. of the MgON solution add 1 ml. of enzyme solution, and after 1 or 2 hours at 37 ° measure the percentage of phosphate soluble in the uranium reagent as described above. This value is a measure of the phosphodiesterase action. In another sample of the diluted uranium filtrate determine the inorganic phosphate which gives a measure of the phosphomonoesterase action. For rough assessment of enzyme activity the uranium precipitate may be observed visually, a small amount of precipitate indicating extensive phosphodiesterase action. This method is often useful in selecting samples, and in judging the dilution they need, for more precise measurement of enzymic activity. Purification Procedure

Principle. The method to be described is based on the finding of Hurst and Butler 7 that both the phosphodiesterase and the 5'-nucleotidase of snake venoms may be adsorbed from aqueous solutions onto packed cellulose fibers. Dilute solutions of neutral salts elute both these enzymes, the diesterase somewhat more readily than the 5'-nucleotidase. The following directions describe how Mr. I. G. Walker has applied this 4j. A. Little and G. C. Butler, J. Biol. Chem. 188, 695 (1951); see Vol. I I I [107]. s E. J. King and G. E. Delory, Enzymologia 81 278 (1940). e E. J. King, Biochem. J. 26, 292 (1932). 7 R. O. Hurst and G. C. Butler, J. Biol. Chem. 195, 91 (1951).

[89]

PHOSPHODIESTERASE FROM SNAKE VENOM

563

principle to the separation of the phosphatases in the venom of the water moccasin (Agkistrodon piscivorus), s The previously published procedure ~ has been modified to make it more versatile. The modified method can be applied to any snake venom by making, when necessary, adjustments in the concentration of the NaC1 solution used for elution. In general it is advisable to elute the phosphodiesterase with the weakest possible NaC1 solution. Preparation of the Cellulose Column. Tear up fifteen sheets of Whatman No. 5 filter paper 12.5 cm. in diameter into 0.5-inch squares, and disperse them in 700 ml. of water by 1 minute's agitation in a blendor. Pour about one-third of the resulting slurry into a glass tube 35 mm. in diameter and 50 cm. long, closed at the bottom with a coarse sintered glass disk 20 ram. in diameter. Remove air bubbles by stirring the contents of the tube with a long wire, adding water from time to time as required to dilute the suspension. When all the air bubbles are removed, fill the column with water and apply air pressure at the top. Permit water to pass out the bottom of the column until more slurry can be added, then repeat the whole process. After all the filter paper has been added in this way, pack the column of cellulose by forcing water through under air pressure until the rate of packing becomes very slow. Then cover the cellulose column with a fiat disk of filter paper, and continue packing with a weighted piston (a rubber stopper on the end of a glass rod with 500 g. applied at the top) until the column of cellulose is 15 cm. long. During this final packing keep the vessel full of water. When the piston is removed, siphon off the excess water. Preparation of Phosphodiesterase. Pour a solution of 150 mg. of lyophilized moccasin venom in 5 ml. of water onto the cellulose column, and as soon as the solution has soaked into the filter paper begin washing with 0.03 % NaC1 solution. Pass this NaC1 solution through the column (with gravity) at a rate of 0.5 to 1.0 ml./min., and collect the filtrate as successive 15-ml. fractions. Measure the optical densities of the fractions at 280 mu. Test all fractions having an optical density greater than 0.1 for enzymic activity. Pool those samples displaying both a strong phosphodiesterase and a weak 5'-nucleotidase action. Although some workers have reported verbally that they are unable to obtain the results reported by Hurst and B u t l e r / a number of others, personally instructed in the separation procedure, have used it with success. The most probable causes of failure are inadequate packing of the cellulose, use of too little filter paper per milligram of venom, and too rapid passage of the salt solution through the column. 8 The dried venom is obtained from Ross Allen's Reptile Institute, Silver Springs, Florida.

564

ENZYMES IN PHOSPHATE METABOLISM

[89]

Properties

Stability. Solutions prepared in the manner described above retain their diesterase activity during several months' storage at 5 ° if mold and bacterial growths are prevented. Merthiolate at a concentration of 0.01% is a suitable preservative, since it does not inhibit the enzyme. Merthiolate will, however, interfere with measurements of ultraviolet absorption. Activity. The phosphodiesterase from 300 mg. of lyophilized moccasin venom hydrolyzed completely 5 g. of MgON to mononucleotides in 5 hours with the liberation of only 2.4 % of inorganic phosphate. Specificity. The purified enzyme acts on polynucleotides (both ribose and deoxyribose types) to produce 5'-mononucleotides and on phenol diesters of phosphoric acid to liberate phenol. No inorganic phosphate is liberated from either of these two types of substrate. Effect of pH. The phosphodiesterase from Russell's viper venom, acting on a 0.3% solution of MgON in Veronal-carbonate buffer, shows maximum activity at p H 9.3. The activity falls off sharply below p H 9.1 and above p H 9.6. Inhibitors. The phosphodiesterase from rattlesnake venom is not inhibited b y sodium arsenate (0.002 M) or b y beryllium sulfate (0.001 M). The diesterase from Russell's viper venom is not inhibited b y sodium citrate (0.001 M), but its activity is completely suppressed b y cysteine hydrochloride (0.15 M). TABLE I MONOESTERASE AND DIESTERASE ACTIVITIES FROM MOCCASIN VENOM

Conversion of MgON, % Optical density, Time, U-insol. Total P to Concentration 280 m~ hr. to U-sol. inorganic P

Sample Whole venom Filtrate fraction no. 7 (15-ml. fractions) 8 9 10 11 12 13 Combined fractions 9-12

0.7 mg./ml. \ I From 150 mg. ~ of venom 1 From 150 mg. of venom

-0.20 0.54 1.72 1.88 0.62 0.22 0.12

1 1 1 1 1 1 1 1 1 2 3 24

35 0 14 67 92 53 31 21 72 92 100 --

17 0 0.27 0.64 0.27 0.32 0.14 0 --0.52 2.3

[90]

SPLEEN AND INTESTINAL PHOSPHODIESTERASES

565

TABLE II PHOSPHODIESTERASE ACTIVITY OF DIFFERENT VENOMS

Four samples of lyophilized venom were assayed for phosphodiesterase activity by the method of Sinsheimer and Koerner. 2 Ten milligrams of each sample was dissolved in 10 ml. of water; the resulting solutions gave the following relative phosphodiesterase activities after both 1 hour and 2 hours of incubation.

Venom Russell's viper Rattlesnake Water moccasin 1952• Water moccasin 1953~

Supplier

Relative phosphodiesterase activity

Haitkine Inst. Ross Allen Ross Allen Ross Allen

1.0 0.84 0.55 0.56

It was not possible to separate the phosphodiesterase satisfactorily from the 1952 sample on a cellulose column, although exactly the same procedure gave excellent results with the 1953 sample.

Pyrophosphatase Activity. C. W. Helleiner has found t h a t phosphodiesterase fractions eluted from a cellulose column containing w a t e r moccasin v e n o m h a v e parallel activities in hydrolyzing sym-diphenylpyrop h o s p h a t e to monophenylphosphate, and m a g n e s i u m oligonucleotide to mononucleotides.

[90] Spleen and Intestinal Phosphodiesterases By

LEON A. HEPPEL and R. J. HILMOE

I. Spleen Spleen contains several diesterase fractions which differ in heat stability, substrate specificity, and other properties. Only one fraction has been extensively purified, and it is described here.

Assay Method

Principle. The assay is a modification of the procedure introduced b y M a c F a d y e n . I As polynucleotide is hydrolyzed b y the nuclease, an increasing fraction becomes soluble in uranium acetate-perchloric reagent. The test involves incubating the diesterase with R N A or other polynucleotide, adding the reagent, centrifuging the precipitate, and measuring the optical density of the s u p e r n a t a n t solution at 260 mtz. 1 D. A. MacFadyen, J. Biol. Chem. 107, 299 (1934).

566

ENZYMES IN PHOSPHATE METABOLISM

[90]

Reagents Ribonuclease-resistant "core." "Core" is made b y exhaustive digestion of yeast sodium nucleate with ribonuclease, followed by dialysis for 2 days at 2 ° against running distilled water. T h e dialyzed solution is concentrated b y lyophilization, neutralized to p H 7, and made up to a concentration of 17 mg./ml. I t can be kept at - 10 ° for a n u m b e r of months. Uranium acetate-perchloric acid solution. 250 mg. of uranium acetate and 4.17 ml. of 60% perchloric acid are dissolved in water and diluted to 100 ml. 0.25 M sodium suceinate-HC1 buffer, p H 6.5.

Procedure. T o 0.04 ml. of succinate buffer and 0.05 ml. of " c o r e " solution in a 10 X 75-mm. test tube are added 0.04 to 0.07 unit of enzyme and water to a final volume of 0.2 ml. The test tube is stoppered, and the mixture is incubated for 30 minutes at 37 ° . At the end of incubation, 0.2 ml. of the uranium acetate-perchloric acid solution is added with mixing. T h e tube is placed in an ice bath for 5 minutes and then centrifuged for 5 minutes at 2000 r.p.m, in an International No. 2 centrifuge. One-tenth milliliter of the supernatant fluid is diluted to 4 ml., and the optical density is determined in the Beckman Model D U spectrophotometer at 260 m~ using quartz cells with a 1-cm. light path. With each set of assays a control is run containing only buffer and " c o r e , " and this blank is subtracted from the experimental readings. Definition of Unit and Specific Activity. One unit of enzyme is defined as t h a t a m o u n t which causes a change in optical density (AE260) of 2.0 in the test as carried out above. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of L o w r y et al. ~ Purification P r o c e d u r e

This is the m e t h o d of Hilmoe and Heppel2

Step 1. Preparation of Acetone Powder Extract. Two thousand grams of calf spleen, obtained within 6 hours of slaughter and kept on ice, is cut into small pieces and mixed with 2000 ml. of cold water, 2000 g: of ice, and 4000 ml. of 17 % sucrose. Portions of 100 g. are worked up at a time. The mixture is homogenized for 2 minutes in a Waring blendor. All subsequent manipulations are carried out in a room at 3 °, except as noted. T h e homogenate is centrifuged for 10 minutes at 1300 × g to remove gross connective tissue fragments and cellular debris. T h e n 2 o. H. Lowry, N. J. Rosebrough, A. L. Farr~ and R. J. Randall, J. Biol. Chem.193, 265 (1951). See also Vol. III [73]. 3 R. J. Hilmoe and L. A. Heppel, unpublished.

[90]

SPLEEN AND INTESTINAL PHOSPHODIESTERASES

567

8500 ml. of turbid supernatant liquid is adjusted to pH 5.1 with 63 ml. of 2 M acetic acid and centrifuged for 30 minutes at 1700 × g. The supernatant solution is discarded and replaced by an equal volume of cold 8.5% sucrose, which is used to wash the precipitate. The precipitate is then mixed with 5 vol. of acetone ( - 1 0 °) and homogenized in a Waring blendor for 15 seconds. The suspension is filtered with suction on a large Bfichner funnel and washed several times with cold acetone. The pad of precipitate is then broken up and dried at room temperature. The acetone powder so obtained is stored at 3 °. Forty grams of acetone powder is extracted with 800 ml. of 0.2 M acetate buffer, pH 6.0, at 2 ° for 30 minutes. The suspension is centrifuged for 8 minutes at 13,000 X g. Step 2. First Ammonium Sulfate Fractionation. 670 ml. of the extract is mixed with 130 g. of ammonium sulfate. The mixture is adjusted to pH 4.9 with 36 ml. of 2 M acetic acid, followed by an additional 52 g. of ammonium sulfate. A precipitate is removed by filtering through a folded Schleicher and Schuell No. 588 filter paper, and the filtrate (765 ml.) is mixed with 119 g. of ammonium sulfate. This is filtered as above, and the precipitate is scraped off the paper and dissolved in 0.05 M acetate buffer, pH 6.0. Step 3. Heating and Dialysis. 99 ml. from step 2 is mixed with 49.5 ml. of distilled water (2°). The pH of this mixture is 5.2. It is heated in a boiling water bath to 55 ° (requires 1 minute) and held at this temperature for 5 minutes before cooling in an ice-water bath. The suspension is centrifuged for 8 minutes at 13,000 X g. The supernatant solution (138 ml.) is dialyzed against running distilled water (2 °) for 7 hours with gentle agitation. A precipitate is formed and removed by centrifuging for 7 minutes at 13,000 X g. Step 4. Second Ammonium Sulfate Fractionation. 159 ml. from step 3 is adjusted to pH 8 with 7.3 ml. of 0.2 M ammonium hydroxide and mixed with 265 ml. of saturated (at 2 °) ammonium sulfate solution containing ammonium hydroxide so that its pH is 8.0 (measured with the Model G Beckman meter on a sample diluted fivefold with cold water). After 15 minutes the precipitate is removed by centrifuging 8 minutes at 13,000 X g. The precipitate is dissolved in 0.01 M sodium succinate-HC1 buffer, pH 6.5, and dialyzed at 2 ° against running distilled water with gentle agitation for 6 hours. The precipitate which forms is removed by centrifugation. Step 5. Treatment with Aluminum Hydroxide Gel. The supernatant solution from step 4 (45 ml.) is adjusted to pH 4.8 with 0.1 M acetic acid and mixed with about 0.4 vol. of aluminum hydroxide gel C~ 4 (dry 4 R. Willst~tter a n d H. Kraut, Bet. §6~ 1117 (1923); see also Vol. I [11].

568

ENZYMES I N PHOSPHATE METABOLISM

[90]

weight 9.9 mg./ml.). The exact a m o u n t of gel to use is determined by trial on small aliquots; 90 to 95% of the activity should be adsorbed. The precipitate is washed successively with 45 ml. of water and 45 ml. of 0.001 M pyrophosphate buffer, p H 8.6; and the enzymatic activity is then eluted with 45 ml. of 0.01 M pyrophosphate buffer, p H 8.6. SU~IMARY OF PURIFICATION PROCEDURE a

Fraction 1. 2. 3. 4. 5.

Acetone powder extract First ammonium sulfate Heating and dialysis Second ammonium sulfate Gel eluate

Specific Volume, Total Protein, activity, Yield, ml. Units/ml. units mg./ml, units/mg. % 675 101 170 45 45

5.5 25.6 8.0 13.5 6.3

3720 2590 1365 600 282

4.6 9.7 2.0 1.4 0.25

1.2 2.6 4.0 9.6 25.2

100 70 37 16 7.6

R. J. Hilmoe and L. A. Heppel, unpublished. Properties Specificity. The purified enzyme has a wide specificity. Hydrolysis of the following substances is catalyzed: RNA, the ribonuclease-resistant " c o r e " (see above), various dinucleotides such as adenylyl uridylic acid and cytidylyl cytidylic 5 acid, alkyl esters of both purine and pyrimidine mononucleotides, ~ dinucleoside monophosphates, cyclic mononucleotides, and cyclic dinucleotides. 6~ In general, activity against purine nucleoside 3P-phosphodiester linkages is stronger than against pyrimidine linkages. Thus far, there has been no success in a t t e m p t s to dissociate these two types of activity. T h e enzyme preparation has the curious p r o p e r t y of hydrolyzing cyclic phosphodiester linkages in a different m a n n e r from other phosphodiester bonds. 7 F r o m cyclic structures 2'-phosphates are formed; from other diester bonds only the 3'phosphates result. Thus, cyclic cytidylic acid is split to give cytidine-2'-phosphate. On the other hand, cytidylyl cytidylic acid and cytidine-3'-benzylphosphate are hydrolyzed to give cytidine-3'-phosphate. The cyclic dinucleotide of cytidylic acid contains

5L. A. Heppel, R. Markham, and R. J. Hilmoe, Nature 171, 1152 (1953). 8D. M. Brown, L. A. Heppel, and R. J. Hilmoe, J. Chem. Soc. 1954, 40. ~ Recently, by further purification, fractions have been obtained which are nearly free of activity against cyclic nucleotide linkages. These preparations are fully active in splitting non-cyclic, internucleotide bonds and the synthetic esters listed above. M. E. Mayer and A. E. Greco, Federation Proceedings 13, 261 (1954) have briefly described the preparation of highly active nucleases from spleen. 7p. R. Whitfeld, L. A. Heppel, and R. Markham, unpublished.

[90]

SPLEEN AND INTESTINAL PHOSPHODIESTERASES

569

one 3',5'-phosphodiester bond and one cyclic bond. Accordingly it is hydrolyzed to give one molecule of cytidine-3'-phosphate and one of cytidine-2'-phosphate. Effect of pH. The pH optimum is near 7.0. Stability. Frozen solutions may be kept for a year. Heat Stability. Incubation at 60 ° for 20 minutes destroys most of the activity, whether at neutral or acid pH. Other nuclease fractions from spleen show a much greater stability. II. Intestinal Phosphodiesterase

This is a diesterase fraction from calf intestine which is found in the supernatant solution when the initial suspension is brought to pH 5.1 and centrifuged. The fractionation here described removes phosphomonoesterase activity. The preparation is inactive with "core," and RNA is used as a substrate in the assay, which is otherwise the same as for spleen diesterase. Purification Procedure s

Calf intestine, obtained shortly after slaughter and kept on ice, is rinsed with tap water and the mucosa is scraped off. To 210 ml. of mucosa are added an equal volume of cold water and 420 ml. of 17 % sucrose. The suspension is then homogenized at 2 ° for 1 minute in a Waring blendor. The pH is adjusted to 5.1 with 5.2 ml. of 2 M acetic acid, after which the mixture is centrifuged for 25 minutes at 1700 X g. The temperature is maintained at 0 to 5 ° for this and all subsequent operations. The precipitate is discarded, and the supernatant solution, measuring 530 ml., is mixed with 137 g. of ammonium sulfate. The pH is adjusted to 4.6 with 3.4 ml. of 2 M acetic acid, after which 14 ml. of 2 M acetate buffer, pH 4.6, is added, followed by 4.4 g. of ammonium sulfate. The precipitate is removed by centrifugation for 7 minutes at 13,000 × g, and 35.3 g. of ammonium sulfate is added to the supernatant solution (597 ml.). A second precipitate is removed in the same way. Then 56 g. of ammonium sulfate is added to form a third precipitate, also discarded. Finally, 60 g. of ammonium sulfate is added to the supernatant solution (now measuring 610 ml.). This brings the saturation with respect to ammonium sulfate from 0.7 to 0.85. The precipitate which forms is collected by centrifugation and dissolved in 0.05 M acetate buffer, pH 6.2, to a volume of 38 ml. (fraction A). Refractionation is carried out in much the same way. To 32 ml. of fraction A are added 32 ml. of water and 11 g. of ammonium sulfate. 8 R. J. Hilmoe and L. A. Heppel, unpublished.

570

ENZYMES IN PHOSPHATE METABOLISM

[91]

The p H is adjusted to 4.6 with 0.7 ml. of 2 M acetic acid, followed by 0.15 g. of ammonium sulfate. A precipitate is removed by centrifugation. Then 10.3 g. of ammonium sulfate is added to the supernatant solution (68 ml.), and a second precipitate is removed. Next 4.5 g. of ammonium sulfate is added to the supernatant solution (now 72 ml.), and a third precipitate is removed. Finally, 7.2 g. of ammonium sulfate is added to the supernatant solution (73 ml.), and this time the precipitate is collected by centrifugation and dissolved in 0.05 M acetate buffer, pH 6, to a volume of 5 ml. This preparation is best kept as a lyophilized powder at - 10 °. It is also stable for three to six months as a frozen solution kept at - - i 0 °.

Properties

Specificity. The final preparation shows only a small purification with respect to protein, but certain activities present in the original extract have been removed, including the hydrolysis of phosphomonoesters and "core." All four cyclic ribomononucleotides are rapidly split to the corresponding 3'-mononucleotides. Alkyl esters of both purine and pyrimidine nucleotides are also cleaved to give the 3'-mononucleotides. For example, adenosine-3'-benzylphosphate is hydrolyzed to adenosine-3'phosphate and benzyl alcohol. Effect of pH. The enzyme preparation shows a broad pH optimum around 7.

[91] I n o r g a n i c P y r o p h o s p h a t a s e f r o m Y e a s t HP~O~--- ~ H20 = 2 H P O 4 - - + H +

By LmON A. HEPPEL Assay Method Principle. The method is based on the formation of inorganic orthophosphate from inorganic pyrophosphate under suitable conditions of pH, temperature, concentration of substrate, and concentration of activating ion. Reagents 0.1 M Veronal--HC1 buffer, pH 7.2, stored at 36 °. 0.01 M Na4P207. 0.01 M MgCI~.

Procedure. The incubation mixture contains 4 ml. of buffer solution, 1 ml. of Na4P2OT, 1 ml. of MgCI,, and 1 ml. of enzyme in 0.02 M buffer,

[91]

INORGANIC PYROPHOSPHATASE FROM YEAST

571

pH 7.2. After 15 minutes at 30 °, samples of 1 ml. are pipetted into 15-ml. volumetric flasks containing 1 ml. of 1 N H~SO4 and 5 ml. of H20 for orthophosphate determination by the method of Fiske and SubbaRow.1 Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount liberating 1 mg. of orthopbosphate P per minute at 30 ° and pH 7.2. Specific activity is expressed as milligrams of P liberated per minute per milligram of protein.

Purification Procedure (Method of Kunitz 2) Step 1. PlasmoIysis of Yeast with Toluene at 38 to 40 °, Followed by Extraction with Water at 5 °. Twelve pounds of fresh Fleischmann's baker's yeast is crumbled by hand into small fragments in a 12-1. enameled vessel and covered with 3 1. of warm toluene of about 50 °, under a hood. The vessel is placed in a warm bath of 60 to 70 °. The yeast is macerated continuously by means of a wooden paddle, and the temperature is allowed to rise gradually to 38 to 40 ° when the yeast liquefies and begins to "work." A rapid liberation of C02 takes place, and the volume of the emulsified yeast-toluene mixture increases considerably. The vessel containing the yeast is removed from the hot water bath and left in the hood for 3 hours at 20 to 25 °. The emulsion is stirred every 15 minutes in order to facilitate the liberation of C02 and thus to prevent sudden discharge of the gas which often results in overflow of the emulsion. The vessel is afterwards placed in an ice-water bath, and the yeast emulsion is cooled to about 10°. The emulsion is distributed in two 10-1. jars, and 3 1. of distilled water of about 5 ° is added to each jar and mixed. The jars are covered tightly and left for 18 to 20 hours at 5 °. A layer of a thick emulsion of yeast stromata in toluene gradually forms above the suspension of yeast in water. The yeast suspension is siphoned off from underneath the toluene-stromata emulsion and filtered in the hood with suction on three 32-cm. Btichner funnels with the aid of 100 g. of Johns-Manville Standard Super-Cel or Filter-Cel per liter of fluid, using either a single sheet of Eaton-Dikeman No. 617 filter paper or a double sheet of Schleicher and Schuell No. 595 filter paper. The residue on each funnel is washed once with 1000 ml. of cold water. The residues are rejected. Step 2. Fractionation with Ammonium Sulfate. The filtrate and washings of step 1 are combined and brought to 0.5 saturation of ammonium sulfate (314 g./1. of filtrate)2 The light precipitate formed is filtered with 1 C. H. Fiske a n d Y. SubbaRow, J. Biol. Chem. 66, 375 (1925). M. Kunitz, J. Gen. Physiol. 35, 423 (1952). 8 The weight of solid a m m o n i u m sulfate required in changing the saturation is calculated as follows: X --- 53.3($2 - $1) 1 - 0.3S2

572

E~ZYMES IN PHOSPHATE METABOLISM

[91]

suction on 32-cm. E a t o n - D i k e m a n No. 617 p a p e r with the aid of 10 g. of S t a n d a r d Super-Cel plus 10 g. of Celite No. 503 (Johns-Manville) per liter of solution. T h e residue is rejected. T h e filtrate is brought with solid a m m o n i u m sulfate to 0.7 s a t u r a t i o n (135 g./1. of filtrate). T h e precipitate formed is allowed to stand for 16 to 18 hours at 5 to 10 ° and is then filtered with suction on 32-cm. hardened paper, S. and S. No. 575. T h e weight of the cake is a b o u t 200 g.

Step 3. Autolysis at 5 ° Followed by Refractionation with Ammonium Sulfate. T w o hundred grams of filter cake of step 2 is dissolved in 1000 ml. of cold w a t e r at a b o u t 5 °. T h e solution is t i t r a t e d with 0.5 N H2S04 to p H 5.35 (glass electrode) and is stored for 8 days in the refrigerator at a b o u t 5 ° . Considerable loss of protein occurs b y autolysis. T h e solution is b r o u g h t to 0.7 s a t u r a t i o n b y addition of 472.4 g. of solid a m m o n i u m sulfate. T h e precipitated protein is filtered with suction a t 10 ° on 18- to 24-cm. S. and S. No. 575 hardened paper. T h e filtrate is discarded. T h e weight of the filter cake is usually a b o u t 70 g. This is dissolved in 210 ml. of w a t e r of 3 to 5 °, and 210 ml. of s a t u r a t e d a m m o n i u m sulfate of 20 to 25 ° is added slowly with stirring. T h e mixture is allowed to stand for 1 hour a t 10 ° and is afterwards filtered at 10 ° with suction on 18-cm. E - D No. 617 soft paper, with the aid of 30 g. of Celite No. 503. T h e residue on the p a p e r is washed twice with 50 ml. of 0.5 s a t u r a t e d a m m o n i u m sulfate of 10 °. T h e semidry residue is rejected. T h e combined filtrate and washings are b r o u g h t to 0.7 s a t u r a t i o n of a m m o n i u m sulfate (135 g. of solid a m m o n i u m sulfate to 1000 ml. of liquid) and filtered with suction on 18-cm. S. and S. No. 575 paper. T h e yield is a b o u t 50 g. of s e m i d r y filter cake. T h e filtrate is rejected. Step ~. Adsorption of Impurities on Cas(P04)2 Gel. FIRST ADSORPTION. F i f t y g r a m s of filter cake f r o m step 3 is dissolved in 1000 ml. of cold water, and 250 ml. of Ca3(PO4)2 gel 4 is stirred in. T h e mixture is filtered with suction at 10 ° on 15- to 18-cm. E - D No. 612 paper. T h e residue on the p a p e r is washed twice with 50 ml. of cold water. T h e residue is rejected. T h e combined filtrate and washings are t i t r a t e d with 0.5 N H~SO4 to p H 5.2 (glass electrode) and b r o u g h t to 0.7 s a t u r a t i o n with where X = gram of solid ammonium sulfate to be added to 100 ml. of solution of saturation $1 to change it to saturation $2. $1 and $2 are fractions of saturation at 23°. 4 Preparation of calcium phosphate gel: 100 ml. of 1 M CaC12 is mixed with 3 to 4 1. of water in a tall jar; 133.4 ml. of 0.5 M Na~PO~ is added with stirring. The precipitate formed is washed several times with water by decantation until the supernatant liquid reaches a pH of 8. The washed precipitate is filtered in an 18-cm. Biichner funnel on soft paper without suction. The well-drained gel is scraped off the paper and brought to 400 g. with water. It is used at once or stored at 5°; see also Vol. I [11].

[91]

INORGANIC PYROPHOSPHATASE FROM YEAST

573

solidammonium sulfate (472.4 g. added to 1000 ml. of fluid). The precipitate formed is filtered with suction on 15-cm. hardened paper. The filtrate is rejected. SECOND ADSORPTION. The filter cake, 30 g., is redissolved in 600 ml. of cold water, and 150 ml. of Ca(PO4)2 is added. The mixture is filtered with suction, washed, titrated to pH 5.2, and brought to 0.7 saturation of ammonium sulfate, as described for the first adsorption. The final filter cake, 20 g., is dissolved in 60 ml. of cold water; 60 ml. of saturated ammonium sulfate is added slowly with stirring; the precipitate formed is removed by filtration at 10° with suction on 15-cm. hardened paper and washed twice with 10 to 15 ml. of cold 0.5 saturated ammonium sulfate. The residue is then rejected. The combined filtrate and washings are brought to 0.7 saturation with solid ammonium sulfate (13.5 g. added to 100 ml. of fluid) and filtered at 10° on 18-cm. hardened paper. The final yield is about 12 g. of semidry cake. Step 5. Dialysis. The filter cake (12 g.) is dissolved in 60 ml. of cold water. The solution is transferred to a cellophane tube provided with a large glass bead and is dialyzed in a rocking machine for 18 to 20 hours at 5 ° against 20 1. of slowly running distilled water. Step 6. Crystallization in 22% Ethyl Alcohol. The dialyzed solution of step 5 is brought with cold water to a final volume of 100 ml. The pH is adjusted to about 5.2 (tested on a drop plate with 0.01% methyl red solution) with the aid of several drops of 0.5 M NaOH. The solution is placed in an ice-salt bath and cooled to about 2 ° . Ten milliliters of cold 95 % alcohol is added slowly with stirring. The temperature of the solution is not allowed to rise above 5 °. The solution, if turbid, is centrifuged at 3 to 5 °. The clear supernatant solution is titrated with 1 M NaOH to a pH of about 6.5 (drops tested colorimetrically; yellow to 0.01% methyl red and to 0.01% phenol red). Any precipitate formed at pH 6.5 is centrifuged off at 3 to 5 °. The clear supernatant solution is placed again in an ice-salt bath, and 20 ml. of 95 % alcohol is added slowly, while the temperature of the solution is kept below 5 °. The solution is then titrated colorimetrically with 0.1 M HC1 to pH 5.2 and stored at - 8 °. Bundles of fine crystals appear within several hours. The solution is left at - 8 ° for 5 days. Step 7. First Recrystallization. The crystallization mixture of step 6 is heated with stirring to 15°. The solution is centrifuged immediately for 5 minutes at 3 °. The supernatant solution is stored at - 8 °. The sediment of crystals is resuspended in 50 ml. of 22% alcohol of about 15° and recentrifuged. The supernatant alcohol solution is rejected. The residue is suspended in 30 ml. of cold water and centrifuged at 3 °. The supernatant solution is stored at 3 °. The residue is resuspended in 20 ml. of cold water

574

ENZYMES

IN P H O S P H A T E

[91]

METABOLISM

and recentrifuged. T h e final residue is rejected. T h e combined supernar a n t solutions are made up with water to 60 ml., cooled to 2 to 3 °, adjusted to p H 5.3 with several drops of 0.1 M N a O H , after which 6 ml. of 9 5 % alcohol is added. The solution, if turbid, is centrifuged clear. The s u p e r n a t a n t solution is adjusted with 0.1 N N a O H to p H 6.5 at 3 °, and 12 ml. of 95% alcohol is then added. The solution is titrated with 0.05 M HC1 to p H 5.3 and stored at - 8 ° for 2 to 3 days. Fine needlelike crystals appear rapidly. Step 8. Second Recrystallization. T h e suspension of crystals of step 7 is centrifuged at - 8 °. T h e s u p e r n a t a n t solution is rejected. T h e residue of crystals is dissolved in 50 ml. of cold water. The solution is titrated with several drops of 0.1 M N a O H to p H 5.2. Five milliliters of 95% alcohol is added at 3 ° . T h e solution, if turbid, is centrifuged at 3 ° . The p H of the clear s u p e r n a t a n t solution is adjusted with 0.1 N N a O H to p H 6.5 and recentrifuged if necessary. Another 5 ml. of alcohol is added slowly at 3 to 5 °, and the p H is brought back with 0.05 M HC1 to 5.3. T h e solution is left for 18 hours at 2 to 3 °. Well-formed long prismatic crystals are gradually formed. The yield of crystals is increased on readj u s t m e n t of the p H to 5.3, further addition of 5 ml. of alcohol, and storing of the mixture at - 8 ° for 24 hours. The crystals are centrifuged and are recrystallized in the same m a n n e r as described for the second recrystallization. TABLE I SUMMARY OF PURIFICATION PROCEDURE (KUNITZ) a

Step

Preparation

1 Original extract 2 0.7 saturated ammonium sulfate fraction 3 After autolysis 4b Before dialysis 5 After dialysis 6 First crystals First mother liquor 7 Recrystallized once 8 Twice recrystallized Three times recrystallized Four times rccrystallized Five times recrystallized

Total protein, mg.

Specific activity

Total activity, Yield, enzyme unitsb %

69,000 42,500

0.28 0.40

19,000 17,000

100 89

16,200 5,200 3,800 360 1,500 225 125 90 75 60

0.80 1.75 2.35 16.0 1.70 24.0 34.0 38.0 40.0 39.0

13,000 9,200 9,000 5,800 2,600 5,400 4,300 3,400 3,000 2,350

68 48 47 30 (14) 28 23 18 16 12

M. Kunitz, J. Gen. Physiol. 85, 423 (1952). b Units are in milligrams of P liberated per minute.

[91]

INORGANIC PYROPHOSPHATASE FROM YEAST

575

Step 9. Drying and Storing. The suspension of crystals is centrifuged at - 8 ° and washed by centrifugation with a small amount of cold 22 % alcohol. The residue of crystals is resuspended in 2 to 3 ml. of 22 % alcohol and poured into 75 ml. of acetone at - 8 °. The mixture is filtered immediately with suction on 5-cm. hardened paper; the crystals are washed with 15 ml. of cold acetone and dried in a vacuum desiccator for 1 hour at room temperature. The dried crystals are stored in a stoppered vial at about 5 °. The yield of twice-recrystallized material is 10 to 15 mg./lb. of yeast.

Properties The purified enzyme is quite specific for inorganic pyrophosphate. It does not hydrolyze adenosine diphosphate, adenosine triphosphate, or thiamine pyrophosphate. Inorganic pyrophosphatase requires the presence of Mg, Co, or Mn ions as activators. These ions are antagonized by calcium ions. Mg is also antagonized by Co or Mn ions. The approximate conditions for optimum rate are 40 ° and pH 7.0 at 3 to 4 × 10-3 M Na4P207 and equivalent Mg. The reaction is complete and irreversible. Alternative Purification Procedure The following alternative procedure is presented because it involves fewer steps and is simpler when only small quantities of enzyme are required. It is based on the work of Heppel and Hilmoe. ~ Step 1. Ammonium Sulfate Fractionation. Baker's yeast is crumbled and dried in a 0.5-in. layer at 22 to 25 ° for several days. A mixture of 700 g. of dry yeast and 2100 ml. of 0.1 M NaHCO3 is incubated for 13 hours at 34 °. It is cooled to 2 ° and diluted to 6300 ml. with cold distilled water, followed by 1833 g. of ammonium sulfate. The mixture is filtered overnight at 2 ° through fluted paper (Schleicher and Schuell No. 588). To 5460 ml. of filtrate is added 868 g. of ammonium sulfate. This is filtered through a single 50-cm. fluted paper with an arrangement for automatic refilling. Moistened papers are laid over the filter funnel to prevent drying of the precipitate during the filtration. The precipitate is scraped from the paper and dissolved with water to a volume of 180 ml. The solution is dialyzed against running distilled water for 6 hours at 2 °. The precipitate which develops is removed by centrifugation. The volume of supernatant is 220 ml. Step 2. Ethanol Fractionation. To 72 ml. of supernatant is added 20.9 ml. of 2 M acetate buffer, pH 6.0. The solution is placed in a beaker 5 L. A. Heppel a n d R. J. Hilmoe, J. Biol. Chem. 192~ 87 (1951).

576

ENZYMES ZN PHOSPHATE M~TABOLISM

[91]

immersed in a b a t h at - 1 0 °, and, with mechanical stirring, 77 ml. of absolute alcohol, also a t - 1 0 °, is added f r o m a s e p a r a t o r y funnel. T h e first 10 ml. is introduced in 2 minutes; then the rate of flow is sharply reduced so t h a t a p p r o x i m a t e l y 30 minutes is needed for completion. T h e mixture is centrifuged for 3 minutes at 13,000 X g in a Servall t y p e SS-1 centrifuge k e p t in a room at - 1 0 °. T h e precipitate is discarded. I n a similar manner, 75 ml. of alcohol is added to the s u p e r n a t a n t , and a second precipitate is removed b y centrifugation. The s u p e r n a t a n t is cooled to - 2 1 °, and 87 ml. of alcohol is added over a 5-minute period. T h e mixture is k e p t at this low t e m p e r a t u r e for 10 minutes and then centrifuged as before. T h e s u p e r n a t a n t is discarded, and the plastic tubes containing a small a m o u n t of pink precipitate are allowed to drain for several minutes a t - 1 0 % T h e precipitate is dissolved b y adding 7 ml. of 0.1 M acetate, adjusted to p H 7, and w a t e r up to 35 ml. T h e step requires little attention, and several lots can be fractionated simultaneously. Step 3. Aluminum Hydroxide Adsorption. T o 107 ml. of the ethanol fraction (three lots) is added sufficient aged a l u m i n u m hydroxide gel C~ ~ (3 mg. of solids per milliliter) to adsorb 90 to 98 % of the inorganic p y r o p h o s p h a t a s e activity. M t e r centrifugation and removal of the supernatant, the precipitate is treated with 107 ml. of 0.001 M p y r o p h o s p h a t e , adjusted to p H 7.2. T h e colorless eluate is the final preparation. I t can be stored as a suspension in 0.6 s a t u r a t e d a m m o n i u m sulfate at - 8 ° for at least 6 m o n t h s w i t h o u t loss of activity. I n T a b l e I I specific a c t i v i t y is defined as micromoles of orthophosp h a t e liberated per hour per milligram of enzyme. TABLE II SUMMARY OF PURIFICATION PROCEDURE a

Step

Total units

Volume, ml.

Specific activity, units/mg)

Autolyzate 1. Ammonium sulfate 2. Ethanol 3. Gel eluate

17,200,000 15,800,000 4,000,000 3,200,000

220 107 107

450 1,200 43,500 ~ 86,000

L. A. Heppel and R. J. Hihnoe, J. Biol. Chem. 192, 87 (1951). b Units are in micromoles of inorganic phosphate liberated per hour. To convert to Kunitz units, divided by 1860. c Recently, preparations have been tested after storage for 4 years. Material carried through the ethanol step and stored at 3° as a suspension in saturated ammonium sulfate lost only 30 % of its original activity. Another sample of the ethanol fraction (step 2) had been taken to Dr. Kunitz who kindly crystallized it by his method. After 4 years at --8 ° the crystals had full activity. 6 R. Wfllstatter and H. Kraut, Ber. 56, 1117 (1923).

[92]

METAPHOSPHATASE

577

[92] Metaphosphatase B y IN~.S MANDL and CARL NEUBERG

Two distinct metaphosphatases have been described: (1) an enzyme which produces orthophosphate from relatively low molecular weight substrates, ( N a P Q ) 3 or (NaPQ)6, was announced from the laboratory of the senior author of this chapter by Kitasato 1,2 and described b y Neuberg and Jacobsohn 3 and Neuberg and Fischer. t I t occurs in yeasts, molds, and various animal organs; (2) an enzyme, analogous to nucleic acid depolymerases, which depolymerizes metaphosphates of mol. wt. > 1,000,000 to smaller fragments without splitting off orthophosphate was found by Ingelmann and Malmgren 5-7 and occurs in some strains of Aspergillus and P e n i c i l l i u m and also in Proteus vulgaris.

Assay Methods The production of orthophosphate from tri- or hexametaphosphate can be followed b y measuring the reduction of phosphomolybdate at acid p H according to the Fiske and SubbaRow determination s or any of its modifications, e.g., L o h m a n n and Jendrassik 9 as recommended b y Kitasato, 2 use of aminonaphtholsulfonate as recommended b y Neuberg and Fischer, 1° use of ascorbic acid according to Lowry and Lopez 11 and recommended b y Ingelmann and Malmgren, 5 or Sumner's procedure 1~ as used by Krishnan and Bajaj. 13 This test is not given b y pyrophosphate, metaphosphate, or triphosphate. P present in the enzyme preparation can be removed b y previous precipitation with Mg-acetate and N H 4 0 H or cerrected for b y blank determination. Procedure (Low Molecular Weight Substrates). The substrate can be made by melting of NaH2P042 and rapid cooling of the melt, ~ or commercial tri- or hexametaphosphate preparations m a y be used if they are crystalline. The enzyme-rich cell extract is incubated in approximately IT. Kitasato, Biocliem. Z. 197, 257 (1928). 2 T. Kitasato, Biochem. Z. 201, 206 (1928). C. Neuberg and K. P. Jaeobsohn, Biochem. Z. 199, 499 (1928). 4 C. Neuberg and H. A. Fischer, Compt. rend. tray. lab. Carlsberg 22~ 366 (1938). 5 B. Ingelmann and H. Malmgren, Acta Chem. Scand. 2, 365 (1948). 6 B. Ingelmann and H. Malmgren, Acta Chem. Scand. 3, 157 (1949). 7 H. Malmgren, Acta Chem. Scand. 3, 1331 (1949). 8 C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925). 9 K. Lohmann and L. Jendrassik, Biochem. Z. 178, 419 (1926). 19C. Neuberg and H. A. Fischer, Enzymologia 2, 241 (1937). i~ O. H. Lowry and J. A. Lopez, J. Biol. Chem. 162, 421 (1946). 12j. B. Sumner, Science 100, 413 (1944). 18p. S. Krishnan and V. Bajaj, Arch. Biochem. and Biophys. 42, 175 (1953).

578

ENZYMES

IN

PHOSPHATE

METABOLISM

[92]

0.5% solution of the substrate at the desired pH at 37 °. Samples are withdrawn at intervals, the reaction stopped with 5 to 10 % trichloroacetic or perchloric acid, filtered immediately, and the supernatant tested colorimetrically or spectrophotometrically for the amount of P present. First-order kinetics are followed if incubation does not exceed 1 hour. 14 This method is not applicable to the metaphosphate depolymerases which produce an insignificant amount of P only. The breakdown of the high molecular weight substrates is followed by measuring the decrease in viscosity. 5 Procedure (High Molecular Weight Substrates). The substrates are prepared by heating KH2PO4--~ (KPOs)n, where n = 15,000 to 20,000. The water-insoluble K salt is dissolved in buffers with excess Na + ions so that the actual substrate is (NaPO3)n. Samples of 5 ml. of 0.5% substrate solution in acetate or phosphate buffer +1.17 % NaCl (total ionic strength 0.3, of which 0.2 is due to NaC1) + 1 ml. of enzyme solution are set up in capillary Ostwald viscosimeters in a thermostat at 25 °. Viscosity measurements are taken relative to that of the buffer and spe[

\

cific viscosities (~o - 1 = vs,.) calculated. Since the viscosity of metaphosphate solutions is affected by concentration and species of salt present, an ionic factor was introduced. 5 In comparison experiments for the same substrate and substrate concentration, this factor is proportional to m~. at time = 0, and z, the relative measure of enzyme activity, is deThe value of z is found most easily as the fined as z = (~/,~.)t=0 • d(1/v,p.) dt slope of the straight line obtained by plotting the ratio of the specific vis(~.~.)~=0 cosities at time t = 0 and t = t, i.e. (~ls,.)t=t' VS. t, where t is the time from the addition of the enzyme to the beginning of the viscosity measurement plus half the outflow time. Purification Procedure Animal tissue extracts or yeast plasmolyzates containing metaphosphatase have been acetone-dried or precipitated with acetone, without further purification. A. niger mycelia were grown at 30 ° by Mann 1~ on culture media of 10 % glucose, 0.5 % NaNO3, 0.1% MgSO4-7H~O, and variable amounts of K2HP04. They were then extracted with water, dialyzed, precipitated with 3 vol. of cold acetone, redissolved in water, and purified by adsorption on Caa(PO4)2 gel A- alumina C,. The culture media separated from mycelia also show metaphosphatase activity. Dialyzed i~ O. Meyerhof, R. Shatas, and A. Kaplan, Biochem. et Biophys. Acta 12, 121 (1953). 15T. Mann, Biochem. J. 38, 339 (1944).

[92]

METAPHOSPHATASE

579

commercial takadiastase (Parke-Davis) arid clarase (Takamine) were used as the A. oryzae metaphosphatase source; A. niger and PeniciUium expansum molds grown on typical media 4,5 were simply well ground up in buffer, filtered, and the solution dialyzed. P. vulgaris 6 grown on agar plates was treated with acetone to remove lipoids, then dried and extracted by grinding with sand in water or buffer. No pure metaphosphatase has been prepared as yet. Partial purification of a metaphosphatase from rice bran, extracted with water and precipitated with ethanol, was reported by Yoshida. le On fractional precipitation with acetone this author obtained a purified enzyme in the 30 to 50 % acetone fraction.

Properties Stability. Yeast metaphosphatase loses up to 40 % of its activity after incubating for 3 hours at 33 °, but its activity is completely preserved in 50% glycerol solution, pH 7. 3,1°,u Activators and Inhibitors. Divalent metals, especially Co ++ and Mn ++ but also Fe ++ and Mg ++, activate metaphosphatase in acteone-dried liver extracts. 17 A. niger metaphosphatase activity is enhanced by Zn, Mn, Ca, Mg, and Pb and completely inhibited by Ag and Hg. 7 On the other hand, Zn ++ in A. niger growth medium prevents or reduces development of metaphosphatase activity.13 Yeast metaphosphatase is affected qualitatively the same way, but activation is less. Mg, then Mn, Co, and Zn, are potent activators. The pH optimum is displaced in the case of A. niger metaphosphatase, since activation is better at more alkaline pH if other factors remain the same. N a F inhibition of the enzyme is very slight; iodoacetie acid, taurocholic acid, and formaldehyde have no effect. NaCN almost completely inhibits the yeast enzyme, but has very little effect on A. niger activity; arsenite reduces metaphosphatase activity of yeast to 30 to 40 % without any effect on the A. niger enzyme. 7 NAN03, which strongly inhibits glucose oxidase at pH 5 in A. niger, does not affect metaphosphatase. 15 Heating at 50 ° for 10 minutes inactivates metaphosphatase without affecting pyrophosphatase. TM A purified yeast metaphosphatase is appreciably inhibited by N a F and KCN, to a lesser extent by glutathione, azide, and arsenate. Effect of pH. Optimum pH varies for metaphosphatases from different sources. Various Penicillium strains show pH optima between 4.5 and 4.8, as does P. vulgaris with a pH optimum of 4.7. 8 A. niger acts 16 A. Yoshida, J. Chem. Soc. Japan~ Pure Chem. Sect. 72, 677 (1951) [Chem. Abstr. 46, 6677 (1952)]. 17 E. Bamann and E. Heumiiller, Naturwissenschaften 9-8, 535 (1940). ~s A. Schi~ffner and F. Krumey, Z. physiol. Chem. 255, 145 (1938).

580

ENZYMES IN PHOSPHATE METABOLISM

[93]

optimally at p H 5.7 6 against high molecular weight substrates, at p H about 415 against (NaPOa)~ or (NaPO3)~, although some strains have an optimum p H around 6 against trimetaphosphate. 13 A. oryzae is active at p H 6.6 to 7; S. pombe has a weak effect at p H 5.6, but S. cerevisiae (yeast) shows highest metaphosphatase activity at p H 7 to 7.2. 4,1° The isoelectric point of A. niger metaphosphatase has been reported as 3.1. 5 Molecular Weight Determination. Only A. niger metaphosphatase has been investigated for its physicochemical constants. The molecular weight found is approximately 33,000; sedimentation constant $20 = 3.2S; diffusion constant D20 = 8.8 × 10-7 cm.2/sec.

[93] T r i p h o s p h a t a s e B y INES MANDL and CAI~L NEUBERG

2H20 Inorganic triphosphatases splitting NasP3010 ~ 2Na2HPO4 + NaH2P04 (3 moles of orthophosphate) have been found in yeast, I A . oryzae takadiastase, and normal animal tissues (kidney, muscle)2 as well as in plants 3 and cancerous tissues. 3 Yeast appears to contain at least two different triphosphatases of this type. A . oryzae furthermore contains anH~O other type of triphosphatase which splits NasP3010 ) NaH2P04-[Na4P2OT. 4 I t is possible t h a t the triphosphatase found in myosin 5,6 is of the same type, b u t this has not been proved; however, pyrophosphate was actually isolated after cleavage b y A . oryzae takadiastase. 4 Assay Method

The production of orthophosphate can be followed b y the m e t h o d described for metaphosphatase assay (Vol. II [92]), since triphosphate, like m e t a p h o s p h a t e and pyrophosphate, does not give this reaction. Procedure. Triphosphate substrate can be prepared according to Huber's method, 7 or some commercial triphosphates m a y be used; if i C. Neuberg and H. A. Fischer, Enzymologia 2, 241 (1937). 2 C. Neuberg and H. A. Fischer, Enzymologia 2, 360 (1937). a L. Frankenthal, I. S. Roberts, and C. Neuberg, Exptl. Med. and Surg. 1~386 (1943). 4 C. Neuberg, A. Grauer, and I. Mandl, Enzymologia 14, 157 (1950). 5j. Needham, A. Kleinzeller, M. Miall, M. Dainty, D. M. Needham, and A. S. C. Lawrence, Nature 150, 46 (1942). 6 M. Dainty, A. Kleinzeller, A. S. C. Lawrence, M. Miall, J. Needham, D. M. Needham, and S.-C. Shen, J. Gen. Physiol. 27, 355 (1944). 7 H. Huber, Z. anorg, u. allgem. Chem. 230, 123 (1937).

[93]

TRIPHOSPHATASE

581

they are crystalline they are mostly monomolecular. The product must be recrystallized several times and the pH adjusted to the optimum which differs with the enzyme source. Solutions having a final substrate concentration, of 0.4 to 0.5% after addition of the enzyme are incubated at 37 °. Samples are withdrawn, diluted with 5 ml. of 7% trichloroacetic acid per milliliter, shaken, filtered, and tested for p.1

Purification Procedure Animal organs such as hog kidney or beef muscle and also tumor tissues are freed from foreign matter, chopped, and ground with sand in ten times their weight of water or dried in vacuo, then chopped and suspended in one hundred times their weight of water. 3 (Drying causes some loss of activity.) One hundred grams of fresh organs is cut up, minced for 2 hours with 300 ml. of tap water, and centrifuged after remaining in the icebox for 48 hours.2 One hundred milliliters of the aqueous extract is then precipitated with 250 ml. of acetone, filtered, dried, and taken up in 25 ml. of H20, centrifuged, and washed with another 25 ml. of H20. Both the solid (1.39 g.) and the solution (50 ml.) show triphosphatase activity, 0.2 g. of solid being more active than 7 ml. of solution. 2 Triphosphatase may also be obtained by plasmolysis of top yeast by ether or ethyl acetate2 Fresh and intact bottom yeast does not yield any triphosphatase on plasmolysis, but a solution obtained by shaking dried bottom yeast with glycerol 1:5 for 48 hours at room temperature or maceration juices prepared from this material have triphosphatase activity. 1,3,8~9 Commercial A. oryzae takadiastase (Parke-Davis) and crude potato phosphatase are dissolved in water and after filtration dialyzed.l,8,8

Properties Activators and Inhibitors. Acetone-dried liver extract triphosphatase is activated by Mg but (unlike metaphosphatase) not by Mn. Fe ++ and Co ++ activate but to a lesser extent. 1° Triphosphatase activity of myosin is doubled by Ca ++ 5.6 Of the two triphosphatases present in yeast, that obtained from dried bottom yeast is active only if Mg ions are added, but that obtained from top yeast seems to be independent of the presence of Mg2 The latter enzyme is inactivated by alkali (like pyrophosphatase and a-glycerophosphatase). Heating for one-half hour at 50 ° destroys triphosphatase activity without affecting pyrophosphatase. 9 Top yeast plasmolyzate triphosphatase is completely inactivated by

s C. Neuberg and H. A. Fischer, Enzymologia 2, 191 (1937). 9 A. Schi~ffnerand F. Krumey, Z. physiol. Chem. 255, 145 (1938). 10E. Bamann and E. Heumiiller, Naturwissenschaften 28, 535 (1940).

582

ENZYMES IN PHOSPHATE METABOLISM

[94]

10 minutes of heating at 100°.3 Triphosphatase of frozen tumor" tissues retains its activity for months2 Effect of pH. The Mg-dependent triphosphatase from bottom yeast has an optimum pH between 7 and 8; top yeast enzyme has a more acid pH optimum2 Tumor tissue triphosphatase tested in veronal-acetate buffer acts best at pH 5.5 to 6. 3 Potato triphosphatase and A. oryzae triphosphatase are most active at neutral pH. A. oryzae enzyme is active also at pH 2.9, s but at pH 8.25 only slight cleavage is produced by takadiastase 8 or by tumor tissues (5 to 6% vs. 100% in 4 days)2 Cleavage of triphosphate to pyrophosphate and orthophosphate by A. oryzae enzyme is optimal at pH values of 6 to 6.3. 4

[94]

Myosin Adenosinetriphosphatase ATP ~- H . 0 -~ ADP ~- H,P04

By S. V. PERRY Assay Method Principle. The assay of myosin adenosinetriphosphatase (ATPase) is most simply carried out by estimating colorimetrically the inorganic phosphate liberated by the enzyme from ATP in the presence of Ca ++ under specified conditions. Reagents 0.05 M ATP (3.1 mg. of 7-minute P per milliliter) Na salt, pH 6.8. This solution may be stored at - 1 5 ° for many months without appreciable hydrolysis. 0.1 M CaCl~. 0.2 M glycine-NaOH buffer, pH 9.1, at 25 °. Enzyme. Myosin stock solution diluted with 0.5 M KC1.

Procedure. The incubation medium, containing 1 ml. of 0.2 M glycine buffer, pH 9.1, 0.2 ml. of 0.1 M CaC12, 0.3 ml. of 0.05 M ATP, and distilled water to bring the volume up to 1.8 ml., is warmed to 25 °, and 0.2 ml. of myosin solution in 0.5 M KCI is rapidly added. The contents of the tube are mixed immediately and incubated for 5 minutes at 25 °. The reaction is stopped by the rapid addition of 1 ml. of 15% trichloroacetic acid. After centrifuging or filtering, an aliquot (1 to 2 ml.) is taken for the estimation of inorganic phosphate by the method of Fiske and SubbaRow 1 or of Allen. 2 C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925). 2 R. J. L. Alien, Biochem. J. 84, 858 (1940).

[94]

MYOSIN ADENOSINETRIPttOSPHATASE

583

Definition of Unit of Activity. The extent of ATP hydrolysis is plotted against myosin concentration, and from the gradient of the linear portion of the graph may be derived a unit of activity in terms of the volume of a hypothetical gas (in microliters) equivalent to the amount of P (in micrograms) liberated by 1 mg. of myosin in 1 hour2 For example,

QP =

(

PX~X--

+ (rag. myosin)

The concentration of protein is 6 × total N content of the myosin 8 solution determined by the micro-Kjeldahl technique. Application of Assay Method to Crude Tissue Preparations. In homogenates and crude tissue extracts satisfactory assays cannot be carried out unless it is possible to make corrections for the activity of other ATPases, apyrases, and systems which simulate ATPase activity. Nuclei have been reported to contain a Ca++-activated ATPase, and in some muscles, e.g., pigeon breast, the ATPase associated with the sarcosomes shows Ca ++ as well as Mg ++ activation. Simulated ATPase activity is shown by enzymic systems which require ATP for the synthesis of various metabolites, and muscle extracts rich in creatine will form creatine phosphate on the addition of ATP. Creatine phosphate ~ and any other acid-labile phosphate esters formed in the homogenate will be estimated as inorganic phosphate under Fiske-SubbaRow or Allen conditions. In homogenates and crude tissue extracts myosin may be present partly as actomyosin and care must be exercised in comparing the enzymic activities of this complex with that of purified myosin preparations assayed under similar ionic conditions (see Properties). For a method of differentiating between the myofibrillar (myosin) and the granular ATPases of rabbit muscle, see Perry. 5 Purification P r o c e d u r e

Myosin is most frequently prepared from rabbit skeletal muscle, and although the procedure given below is designed for use with this tissue, it can be adapted with slight modification for the extraction of myosin from skeletal and cardiac muscle of other animals. The best published preparations are Weber's ~ L-myosin and Szent-GySrgyi's7 "crystalline" myosin, but even these preparations, which have identical propers K. Bailey, Biochem. J. 86, 121 (1942). 4 C. F. Cori, J. Biol. Chem. 165, 395 (1946). 5 S. V. Perry, Biochim. et Biophys. Acta 8, 499 (1952). 6H. H Weber and H. Portzehl, Advances in Protein Chem. 7, 161 (1952). TA. Szent-GySrgyi,"Muscular Contraction." Academic Press, New York~ 1947.

584

ENZYMES IN PHOSPHATE METABOLISM

[94]

ties, have been shown to contain small amounts (up to 10 to 15%) of impurities. 8,9 Based on the procedures of Edsall 1° and Bailey, 3 the method also incorporates features from Szent-GySrgyi's preparation and is in general similar to that of Mommaerts and Parrish. 8 Muscle is extracted with KCl-potassium phosphate buffer, pH 6.5, as recommended by Guba and Straub, 1~to give a myosin solution containing only small amounts of actomyosin, which complex can be removed by bringing the ionic strength to 0.3 as suggested by Portzehl et al. ~2 Sarcoplasmic proteins are then removed by repeated precipitation by dilution. Step 1. Preparation of Crude Extract. To obtain myosin preparations of high enzymic activity the preparation should be carried out at 0 °, and ice-cold distilled water freed from heavy metal contamination must be used throughout. An adult rabbit is killed by stunning, bled, and rapidly skinned. The dorsal and leg muscles are quickly dissected out and chilled in crushed ice. When completely cold the muscle is minced once in a meat grinder and immediately extracted with 3 vol. of cold KCl-potassium phosphate buffer, pH 6.5 (0.3 M KC1, 0.10 M KH2PO4 and 0.05 M K2HPO4). The suspension is stirred slowly for 15 minutes, and the extract is then centrifuged for 10 minutes at 600 X g. Clarification of the crude myosin extract is achieved by gentle filtration of the supernatant through a paper-pulp pad which has previously been washed with the KCl-potassium phosphate medium. At this stage 100 g. of minced muscle will give 200 to 250 ml. of extract. Step 2. Precipitation of Crude Myosin. The slow addition, with stirring, of the crude extract to 14 vol. of distilled water brings the myosin down as crystalloid particles which give a characteristic sheen to the suspension. Overnight the myosin settles well and can be removed by centrifugation. The resultant gel is transferred to a measuring cylinder, using a little of the supernatant if necessary, the volume of precipitate is noted, and the myosin is brought into solution by addition of sufficient solid KC1 to bring the ionic strength to 0.5. Step 3. Removal of Actomyosin. The pH of the solution is adjusted if necessary to approximately pH 6.6 with a small amount of solid NaHCO~ and with bromothymol blue as external indicator. Water is added to bring the ionic strength of the solution to 0.3. Any precipitate of actomyosin is then removed by centrifugation, and the clear or slightly turbid solution is reprecipitated twice as follows. s W. F. H. M. Mommaerts and R. G. Parrish, J. Biol. Chem. 188, 545 (1951). 9T. C. Tsao, Biochim. et Biophys. Acta 11, 368 (1953). 1oj. T. Edsall, J. Biol. Chem. 89, 289 (1930). ~l F. Guba and F. B. Straub, Studies Inst. Med. Chem. Univ. Szeged 3, 46 (1943). 1~H. Portzehl, G. Schramm, and H. H. Weber, Z. Naturforsch. 5b, 61 (1950).

[94]

MYOSIN ADENOSINETRIPttOSPItATASE

585

Step 4. Reprecipitation of Myosin. To induce the formation of crystalloid needles (Szent-GySrgyi's "crystalline" myosin), distilled water is added to the myosin solution slowly with stirring (glass stirrer) until the ionic strength is 0.04. Some denaturation is liable to occur at this stage, but this can be avoided b y carrying out the " c r y s t a l l i z a t i o n " within 15 minutes at 0 °. The p u r i t y of the preparation is not increased b y "crystallization," but in view of the fact t h a t it does not take place so readily with impure myosin it can be considered to some extent a control of the purity of the preparation. The precipitate is allowed to settle, centrifuged off, and the gel dissolved up b y the addition of solid KC1 to u -- 0.5. The p H is adjusted to 6.5 to 7.0, and the myosin is reprecipitated as before b y dilution to tL = 0.04. After the final precipitation the myosin gel is again dissolved b y the addition of solid KC1 to u = 0.5, and the solution is stored as concentrated as is possible at 0 ° in a flask with a trace of toluene on the stopper and containing the minimum of air space. Yield. Very approximately 100 ml. of 1% solution is obtained from 100 g. of minced muscle. Qp values at 25 ° of this preparation when fresh range from 2000 to 5000. L-Myosin constitutes about 40% of the total proteins of rabbit skeletal muscle, and consequently the ATPase activity per milligram of protein does not increase very much during the preparation. For example in one preparation the Qp values of the myosin after the first, second, and third precipitations were 3300, 4400, and 4700, respectively, measured at 25 ° . Prepared as above, myosin should liberate only one phosphate radical per molecule of A T P , but if on testing the preparation is shown to split a higher proportion of the A T P phosphate than this, adenylate kinase (myokinase) is present. This enzyme can be removed b y further reprecipitation. Reprecipitation of myosin can be carried out at least four times without the appearance of denaturation products, provided t h a t SUMMARY OF PURIFICATION PROCEDURE

(The volumes given were obtained from 100 g. of minced rabbit muscle)

Purification stage 1. Crude extract of muscle First precipitation 2. (Redissolved myosin 3. Actomyosin precipitation Second precipitation 4. Redissolved myosin Third precipitation Redissolved myosin

l

Total volume, (ml.)

pH

Ionic strength

230 3450 75 125 940 80 1000 90

6.5 6.5 6.6 6.6 6.8 6.8 6.8 7.0

0.60 0.04 0.50 0.30 0.04 0.50 0.04 0.50

586

ENZYMES IN PHOSPHATE METABOLISM

[94]

the total time does not exceed 5 days. The preparation usually contains 5-adenylic deaminase, which is not removed by repeated reprecipitations but which does not attack A T P or ADP.

Properties Within the last decade the question of the identity of myosin and adenosinetriphosphatase has frequently been investigated and often discussed. Claims of the separation of the enzyme from myosin have been made 13,'14 but have either been withdrawn or lacked conviction. More recently, exhaustive attempts at separation employing the butyl alcohol procedure have been unsuccessful, 9 and, in view of the work with sulfhydryl reagents and on the degradation of myosin by trypsin treatment, 1~ the facts strongly suggest that ATPase activity is a specialized function of the myosin molecule. P h y s i c a l Constants. A number of determinations of the molecular weight of rabbit myosin have been made, but the most reliable appears to be that made in Weber's laboratory. 6 By the sedimentation-diffusion method the molecular weight of L-myosin was 850,000. Dubuisson 18 gives the electrophoretic mobility of his/~-myosin (considered to be identical with L-myosin) prepared from rabbit muscle as - 2 . 9 X 10-5 cm.2/v./sec, for the ascending boundary. Kinetic studies by 0uellet et al., ~7 carried out at pH 7.0 and in the presence of 0.001 M CaC12, give a Michaelis constant for myosin ATPase (probably containing some actin) of 1.4 X 10-5 M at 24.6 °. Specificity. Myosin will split I T P and U T P in addition to ATP, and no doubt when tested the other nucleoside triphosphates will be found to be hydrolyzed. Although' I T P is split at a faster rate than A T P at higher substrate concentrations, the Michaelis constant is apparently lower for ATP. 18 It is claimed th at U T P is split 3 to 6 times as fast as ATP. TM Activators. The ATPase activity of myosin is profoundly affected by the presence of ions. If the protein actin is also present, these ionic effects are modified, and consequently the enzymic behavior of myosin in some cases differs from that of actomyosin. 2° 1, B. D. Polls and O. Meyerhof, J. Biol. Chem. 163, 339 (1946). 14R. K. Morton, Nature 166, 1092 (1950). 15E. Mihalyi and A. Szent-GySrgyi, J. Biol. Chem. 201~ 211 (1953). is M. Dubuisson, Biol. Revs. Cambridge Phil. Soc. 25, 46 (1950). 17L. Ouellet, K. J. Laidler, and M. F. Morales, Arch. Biochem. and Biophys. 39, 37 (1952). is W. F. H. M. Mommaerts and K. Seraidarian, J. Gen. Physiol. $0, 401 (1947). 19H. M. Kalckar, Science 119, 478 (1954). 99W. Hasselback, Z. Naturforsch. 7b, 163 (1952).

[94]

MYOSIN ADENOSINETRIPHOSPHATASE

587

MYOSIN. In the absence of salt other than ATP at approximately neutral pH, myosin has little ATPase activity. Ca ++ activates the enzyme, the exact course of the activation depending on the other salts present. Mg ++ inhibits the activity of pure myosin either in the presence or absence of other salts, and effectively counteracts the activation obtained with Ca ++. For example, with M g : C a ratios less than 1, t h e inhibition usually exceeds 90 %.1s Potassium chloride stimulates the efizymic activity, probably in a nonspecific way, both in the absence and presence of Ca ++, and although this effect is well established in the literature there is difference of opinion regarding the qualitative aspects. This is no doubt due to the varying ionic contributions of the buffers used by the various workers. At higher concentrations of KC1 (~ = > 0.2) the activation obtained with Ca ++ falls. ACTOMYOSIN. Activated by Ca ++ and in contrast to myosin, Mg++ will activate the enzyme when other ions are absent or in low concentration. In the presence of 0.1 M KC1, Mg ++ inhibits the ATPase of actomyosin. When actomyosin is present as intact myofibrils, its Mg++-activated ATPase, however, is much less sensitive to KC1, and with fresh preparations the enzyme is strongly activated by Mg ++ in 0.1 M I£C1.21 Inhibitors. Myosin contains appreciable amounts of cysteine, and with fresh preparations free sulfhydryl groups can be readily demonstrated by the nitroprusside test. Low concentrations of heavy metals such as Cu and Hg inhibit the enzyme. Sulfhydryl reagents vary in efficiency for inhibiting myosin ATPase; 2~ e.g., alkylating agents such as iodoacetate and iodoacetamide are not very efficient and need to be used at high concentrations (0.05 to 0.01 M) to produce any effect on the enzyme. On the other hand oxidizing agents such as iodosobenzoate, hydrogen peroxide, and iodine readily bring about inhibition. A most effective inhibitor is p-chloromercuribenzoate, which, in amounts equivalent to the myosin cysteine present, brings about complete inhibition. Cysteine will reverse partial inhibition induced by p-chloromercuribenzoate. (See Barron 2~ for summary of literature.) Ethylenediaminetetraacetic acid inhibits myosin ATPase activity in 0.05 M KC1 but strangely enough activates in 0.6 M KC1. ~4 Effect of pH. When activated by Ca ++ in borate buffer, myosin ATPase shows two optima. One is pronounced at pH 9.2, and the other much smaller at pH 6.5. Similar results are obtained with glycine buffer, but at the alkaline maximum much higher activities are obtained than 21 S. V. Perry, Biochem. J. 48, 257 (1951). ~2 K. Barley and S. V. Perry~ Biochim. et Biophys. Acta 1, 506 (1947). ~s E. S. G. Barron, Advances in Enzymol. 11, 201 (1951). ~4 E. T. Friess, Arch. Biochem. Biophys. 51, 17 (1954).

588

ENZYMES IN PHOSPHATE METABOLISM

[95]

with borate, and the reported values for the optimum v a r y from 9.0 to 9.5. In this range inactivation of the enzyme is appreciable, and values will be affected both by incubation time and temperature. In the presence of actin the activity-pH curve changes with the result t h a t two nearly equal maxima are obtained.

[95] M g - A c t i v a t e d

Muscle

ATPases

A T P + H~0 --~ A D P + P~

By W. WAYNE KIELLEY Assay Method

Reagents 0.02 M A T P solution, p H 7.0. 0.2 M histidine buffer, p H 6.8 to 7.0 (in 0.15 M KC1). 0.05 M MgC12. Enzyme. Dilute the stock enzyme solution so t h a t 0.1 ml. will hydrolyze 0.1 to 1.0 micromole of A T P under the conditions given below. 5 % perchloric acid (PCA). Reagents for determination of inorganic phosphorus (see Vol. I I I [147]).

Procedure. Tubes containing 0.1 ml. of A T P solution, 0.3 ml. of 0.2 M histidine buffer, 0.1 ml. of 0.05 M MgC12, 0.4 ml. of water, and 0.1 ml. of the diluted enzyme solution are incubated for 0 and 5 minutes at 38 °. The reaction is stopped b y adding 1.0 ml. of 5 % PCA. After centrifugation, aliquots of the supernatants are analyzed for inorganic phosphorus (see Vol. I I I [147]). Under these conditions the activity is a function only of enzyme concentration if not more than 1 micromole of the substrate is hydrolyzed. Definition of Unit and Specific Activity. Activity is expressed here in terms of micromoles of substrate hydrolyzed in 5 minutes under the conditions given above. Specific activity is expressed as micromoles of substrate hydrolyzed in 5 minutes per milligram of enzyme nitrogen (determined b y micro-Kjeldahl procedure--see Vol. I I I [145]). 1 Since the rather 1For protein nitrogen determination in fractions containing ammonium sulfate, a suitable aliquot of the enzyme solution was added to about a tenfold larger volume of 5% PCA, centrifuged, washed once with PCA, then taken up in 1.0 N NaOH and transferred to the digestion flask.

[95]

MG-ACTIVATED MUSCLE ATPASES

589

cumbersome Qp (microliters P per milligram of protein per hour) relationship has been used previously, 2 for comparative purposes these are also given in the table on page 590. Determination of Activity in Crude Muscle Homogenates. Attempts to assay unfractionated homogenates for this specific enzyme are complicated by the fact that the ATPase activity of actomyosin under some conditions is activated by Mg. Furthermore, since the isolated MgATPase may increase somewhat in activity with time, there is some uncertainty concerning the fraction of the total Mg-ATPase actually being determined. Purification Procedure The following procedure is that given by Kielley and Meyerhof 2 with only slight modification. Step 1. Preparation of Crude Extract. Chilled rabbit muscle (hind limb and back) is run through a meat chopper and then suspended in 5 vol. of cold extracting solution (0.1 M KC1, 0.04 M N a H C Q , 0.01 M Na~CO3, 0.001 M KCN) and agitated in a Waring blendor for about 1 minute. The suspension is allowed to stand for about 20 minutes and then centrifuged at 3000 X g for 15 minutes. These and all subsequent operations are carried out at approximately 5 °. The residue is resuspended in 5 vol. of the extracting solution and treated as before. The residue is then reextracted a second time. Step 2. Removal of Actomyosin. The combined extract is diluted with an equal volume of 0.001 M KCN or H20 and after standing for about one-half hour is centrifuged to remove the precipitated actomyosin. Step 3. Precipitation with Ammonium Sulfate. The enzyme is precipitated from the supernatant of step 2 by addition of ammonium sulfate-27 g. per 100 ml. of supernatant. After standing for about 1 hour, the precipitate is removed by centrifugation and washed once with 35% saturated ammonium sulfate. The precipitate is dissolved in a 1:1 dilution of the original extracting solution, and after clarifying at 10,000 X g (10 minutes) the ammonium sulfate precipitation is repeated. The precipitate is dissolved in 0.2 M KC1, 0.02 M NaHC03, 0.001 M KCN, the volume being about equal to the original weight of muscle. Step 4. Separation by Ultracentrifugation. After clarifying by centrifugation at 10,000 X g for about 15 minutes, the ATPase is sedimented by centrifugation at 70,000 X g for 1 hour. The precipitate is suspended in 0.2 M KC1, the volume being about one-fifth the previous volume. For resuspension an homogenizer is advantageous. This solution is centrifuged 2 W. W. Kielley and O. Meyerhof, J. Biol. Chem. 176, 591 (1948); 183, 391 (1950).

590

[95]

ENZYMES I N PHOSPHATE METABOLISM

at 10,000 X g for 15 minutes, and the precipitate is discarded. A tabular summary of the preparative procedure is given below. SUMMARY OF PREPARATIVE PROCEDURE

Fraction 1. 2. 3. 4. 5.

Crude extract Actomyosin-"free" extract 0.0-0.35 (NH,):SO4 fraction Ultracentrifuged fraction Maximum activity of ultracentrifuged fraction (3 days old)

Total activity, ~M. Pi released/ Specific activity, ~M. Pi released/ 5 min./100 g. 5 min./mg N muscle 4900 2230 4600 4040 7570

2.76 2.26 28.8 93.2 174

QP 124 101 1290 4175 7800

Properties Specificity. The enzyme is specific for the removal of the terminal phosphate of ATP. Activation and Inhibition. This ATPase is activated by Mg and to some extent by Mn. It is inhibited by Ca. The enzyme is also inhibited by F - and by p-chloromercuribenzoate. Effect of pH. The pH optimum for this ATPase is about 6.8 to 7.0; at pH 7.6 and 5.6 the activity is about 50% of that at the pH optimum. These conditions hold only for the Mg and ATP concentrations given above. Physical Nature of A TPase. The behavior of the enzyme on centrifugation indicates that it is associated with submicroscopic particles, and it is probable that many other enzyme activities are also present. The particles contain a high percentage of lipid material (30 to 40% of the dry weight), most of which is phospholipid. It has been observed that the lecithinase of Clostridium welchii, which is specific for the hydrolysis of choline-containing phospholipids (giving phosphoryl choline as a product) inactivates the ATPase, the degree of inactivation being proportional to the extent of phospholipid hydrolysis. Similar Preparations. The isolation of a magnesium-activated apyrase from insect muscle has been reported by Gilmour and Calaby 3 by a procedure similar to that given here. The insect muscle enzyme differs from that of mammalian muscle in (1) pH optimum (7.8 to 8.0)--although the conditions for assay were somewhat different from those described above, a D. Gilmour and J. H. Calaby, Arch. Biochem. and Biophys. 41, 83 (1952).

[96]

POTATO APYRASE

591

(2) in specificity--removing two phosphate groups from ATP but at different rates, and (3) in behavior to centrifugation at 20,000 X g--the insect muscle enzyme does not sediment in this gravitational field.

[96] P o t a t o A p y r a s e ATP ~- 2H20 --~ 5-AYIP ~ 2H3P04 By P. S. ]~RISHNAN

Assay Method Principle. The method consists in estimating the amount of PO, liberated from ATP under standard conditions. Reagents ATP solution. The Ba salt of ATP is converted into the Na salt by precipitation of the Ba as the sulfate 1 or by the use of an ion exchanger. 2 The resulting solution is neutralized with dilute NaOH. Stored frozen, the solution is stable for several weeks. At ice-cold temperature the solution can be stored for about two weeks. For the actual assay the solution is so diluted that each milliliter contains about 500 ~, of 10-minute P04. 0.1 M succinate buffer, pH 6.5. CaC12 solution, 0.5 %, adjusted to approximate neutrality. Enzyme. The stock solution of the enzyme is suitably diluted so as to liberate about 100 ~, of PO4 in the assay. Procedure. The various components are brought to the bath temperature of 30 °. Into a 15-ml. centrifuge tube 1 ml. of the buffer solution is added, followed by 1 ml. of the enzyme solution and 0.2 ml. of CaC12 solution. After mixing, 0.3 ml. of ATP solution is run in, the contents mixed well, and the extract time noted. At the end of 30 minutes 1 ml. of chilled 20 % (w/v) TCA is added, the tube shaken, and the contents diluted to about 7 ml. with water and chilled in an ice bath. A blank is simultaneously run, which differs from the experimental tube in that the ATP solution is added at the end, after the addition of TCA. After the tubes have been chilled for about 10 minutes they are centrifuged, preferably in the cold, and the residues washed once with 3-ml. portions of 2% (w/v) TCA, the wash liquid being added to the main solution in each case. The solutions are now analyzed for PO4. The esti1p. S. Krishnan and W. L. Nelson, Arch. Biochem. 19, 65 (1948). E. C. Slater, Biochem. J. 53, 157 (1953).

592

[96]

ENZYMES IN PHOSPHATE METABOLISM

mation is easily carried out b y direct development of color after addition of molybdate, H2S04, and reducing agent; but a more accurate value is obtained b y the isobutanol extraction method, 8 especially when the enzyme preparation is v e r y crude. The difference in P04 between the experimental and the blank runs gives the a m o u n t of P04 liberated from A T P b y apyrase. Definition of Unit and Specific Activity. One unit of the enzyme is defined as t h a t a m o u n t which in the standard test outlined above liberates 1 ~, of P04. The specific activity of the enzyme is expressed as units per milligram d r y weight or, better, per milligram of protein. Application of Assay Method to Crude Preparations. With partially purified enzyme preparations it is found t h a t the third phosphate group also is split off A T P .

Purification Procedure The following m e t h o d 4 is a modification of t h a t of Kalckar. 5 About 1 kg. of peeled p o t a t o is ground for 5 minutes in a Waring blendor with SUMMARY OF PURIFICATION PROCEDURE

Fraction 1. Potato extract 2. (NH4)2S04 fraction, 450 g./1. extract 3. Supernatant after dialysis 4. (NH4)2S04 fraction, as in 2 and 3 5. (NH4)2SO4fraction, 30-45 g./100 ml. solution and dialysis

Total Total volume, units, ml. thousands

Specific activity, Recovery, units/mg, dry wt. %

2120

13,740

57

100

355 800

15,630 9,200

1,613

114 67

155

5,555

1,914

40

1,005

66,600

7

1 1. of neutralized M / 1 0 0 K C N , pressed through cheesecloth, and the extract centrifuged in the cold. T h e enzyme is precipitated b y the addition, with mechanical stirring, of 450 g. of powdered (NH4)~S04 for every liter of solution. T h e precipitated material is filtered b y gravity through W h a t m a n filter paper, No. 1, and allowed to drain overnight in the cold. T h e solid material is taken up in water, dialyzed in the cold against running distilled water for 24 hours, centrifuged i n the cold, and the 8 V. Bajaj and P. S. Krishnan, Arch. Biochem. and Biophys. 47, 34 (1953). 4 p. S. Krishnan, Arch. Biochem. 20, 261 (1949). 5 H. M. Kalckar, J. Biol. Chem. 153, 355 (1944).

[97]

MITOCHONDRIAL ATPASE

593

supernatant reprecipitated with (NH4)2SO4 as above. The solid material is filtered, taken up in water, dialyzed, and centrifuged clear. To every 100 mh of this solution 30 g. of finely powdered (NH4)2SO4 is added, with stirring, and the precipitated material is discarded by filtration. To the filtrate 15 g. more of the solid is added, whereupon the enzyme is precipitated. The material is filtered, dialyzed in the cold after dissolving in a small volume of water, and centrifuged clear. The various steps are outlined in the table.

Properties The purified enzyme preparation splits two phosphate groups from ATP. The preparation is, however, still contaminated with traces of other phosphatases. 6 Lee and Eiler 7 have reported an apyrase preparation from potato which catalyzes the hydrolysis of both the labile phosphate groups of ATP at temperatures above 7 ° and of only the terminal group at temperatures lower than 7 °. 6 p. S. Krishnan, Arch. Biochem. 20, 272 (1949). 7 K. Lee and J. J. Eiler, Science 114, 393 (1951).

[97] Mitochondrial A T P a s e A T P + H20 --~ A D P + P~ By W. WAYNE KIELLEY

Assay Method Reagents

0.02 M ATP solution, pH 7.0. 0.20 M histidine buffer (in 0.15 M KC1), pH 7.5. 0.05 M MgC12. Enzyme. Dilute the stock enzyme solution so that 0.1 mh will hydrolyze 0.1 to 1.0 micromole of substrate under the conditions given below. Perchloric acid (PCA), 5 %. Reagents for determining inorganic phosphorus (see Voh III [147]). Procedure. Prepare tubes containing 0.25 ml. of the ATP solution, 0.25 mh of 0.2 M histidine buffer (pH 7.5), 0.1 ml. of 0.05 M MgC12, 0.3 ml. of water, and 0.1 mh of the diluted enzyme. The tubes are incubated at 28 ° for 0 and 5 minutes; the reaction is stopped by adding

594

ENZYMES IN PHOSPHATE METABOLISM

[97]

1.0 ml. of 5% PCA. After centrifugation, aliquots of the supernatants are analyzed for inorganic phosphorus (see Vol. III [147]). Under these conditions the reaction is a linear function of enzyme concentration when hydrolysis of the substrate does not exceed 1.0 micromole. Definition of Unit and Specific Activity. Activity is expressed in terms of micromoles of substrate hydrolyzed in 5 minutes under the conditions given above. Specific activity is expressed in micromoles of substrate hydrolyzed in 5 minutes per milligram of enzyme nitrogen (determined by micro-Kjeldahl procedure--see Vol. I I I [145]). Determination of Activity in Crude Homogenates. Mitochondria carefully prepared in isotonic sucrose (0.25 M) show very low or no ATPase activity, so that the application of this procedure to crude liver homogenates prepared as described above is of doubtful significance if maximum activity is desired. Other fractions of the cell also possess some ability to hydrolyze ATP, but whether any part of this activity is due to the same enzyme described here is not known.

Purification Procedure This procedure is taken from Kielley and Kielley. I

Step 1. Preparation of Mitochondria. Mitochondria are prepared from 6 to 7 g. of mouse liver by the isotonic sucrose fractionation procedure of Schneider: (see Vol. I [3]). Step 2. Disintegration of Mitochondria. The washed mitochondria are suspended in 20 ml. of 0.003 M K~HPO4 and treated in a chilled stainless steel micro-Waring blendor for 2 minutes (sonic vibration may also be employed). After centrifuging at 20,000 X g for 10 minutes, the supernatant is drawn off and the residue, suspended in 15 ml. of 0.003 M K2HP04, is treated as before. Step 3. Ultracentrifugal Fractionation. The combined supernatant is centrifuged at 110,000 X g for 30 minutes, the supernatant is poured off, and the pellets are suspended by homogenizing in 4.0 ml. of 0.003 M K2HP04. A tabular summary of the preparative procedure is given on page 595.

Properties

Substrate Specificity. When large amounts of enzyme are employed, ADP (or IDP) is slowly hydrolyzed. However, at enzyme concentrations comparable to those employed for activity measurement, only one phosphate is removed from ATP and ADP is not attacked. I T P is hydrolyzed at about one-third to one-half the rate for ATP. 1 W. W. Kielley and R. K. Kielley, J. Biol. Chem. 200, 213 (1953). W. C. Schneider, J. Biol. Chem. 176, 259 (1948).

[98]

INSECT ATPASE

595

Activation and Inhibition. The enzyme is activated b y Mg and is inhibited somewhat b y Ca. The enzyme is markedly inhibited b y ADP. At concentrations equal to t h a t of A T P in the incubation medium about 50 % inhibition is observed. p H Optimum. The p H optimum of the enzyme appears to be about 8.5. However, the p H - a c t i v i t y relationship is influenced b y the relative concentrations of A T P and Mg, and this optimum is observed for a molar ratio of A T P : M g of 2:1. At higher relative concentrations of Mg, the enzyme activity is increased at p H values below 8.5. Stability. The enzyme loses activity rapidly after preparation and m a y retain only about 50 % of its original activity after 24 hours of storage in the refrigerator. SUMMARY OF PREPARATIVE PROCEDURE a

Fraction Whole mitochondria in 0.003 M K_~HP04 (from 13 g. liver) Disintegrated mitochondria Supernatant after centrifugation at 20,000 X g Pellet from supernatant spun at 110,000 X g

Total activity, t~M. P released

Specificactivity, aM. P released/mg. N

415 1062

10.5 26.9

661

Not determined

556

114.0

W. W. Kielley and R. K. Kielley, J. Biol. Chem. 200, 213 (1953).

[98] Insect ATPase A T P -~ 2P + A M P B y DA~cY GILMOUR

This enzyme, which occurs in the mitochondria of insect muscle 1,2 and has been purified from whole muscle, ~ is a water-soluble, Mg-activated ATPase, which splits two phosphate groups from A T P .

Assay Method Activity is determined b y measuring orthophosphate released from 1B. Sacktor, J. Gen. Physiol. 36, 371 (1953). D. Gilmour, Australian J. Biol. Sci. 6, 586 (1953). s D. Gilmour and J. H. Calaby, Arch. Biochem. and Biophys. 41, 83 (1952).

596

ENZYMES IN PHOSPHATE METABOLISM

[98]

ATP. P is assayed by the Fiske and SubbaRow 4 method, using a Coleman Junior spectrophotometer to measure absorption at 700 mtt. The reaction mixture consists of 0.1 ml. of 0.16 M MgC12, 0.5 ml. of sodium-ATP containing 0.3 mg. of 7-minute P per milliliter (purified by passage through a column of Amberlite IR-100 resin), and 1.0 ml. of enzyme solution containing 5 to 20 ~, of ATPase and 150 ~, of GSH in 0.05 M sodium borate-NaOH buffer, pH 8.0. The reaction mixture is made up in the cold, substrate being added last. It is incubated for 5 minutes at 42 °, and the reaction is stopped by the addition of TCA to a final concentration of 8 %. Protein is removed by filtration (Whatman No. 44 papers), and P determined on the filtrate. Preformed P is measured in a blank tube to which TCA is added immediately after the addition of substrate. Assays are usually made at three different enzyme concentrations, activity being determined from the straight-line relationship between P evolved and enzyme concentration. Protein N is determined by nesslerization after sulfuric acid digestion of a washed TCA precipitate, using the Nessler reagent of ¥anselow. ~ Activity is expressed as Qp, as defined by Bailey. e Purification Procedure

Preparation of Muscle. The best source of the enzyme is the thoracic muscle of flying insects. The larger locusts or flies are convenient material. Thoraces are separated by cutting off the abdomen and pulling off the head in such a way that the thoracic section of the gut is withdrawn with it. The ventral side of the thorax is cut away, and the flight muscles exposed by spreading out the dorsal and lateral parts. Residual fat body is removed, and the flight muscle cut out with scissors or a knife. Step 1. Preparation of Crude Extract. The dissected muscle is dropped immediately into an ice-cold solution containing 0.5 M KC1 and 0.03 M KHCO3 (pH 8.0). When sufficient muscle has accumulated, the mixture is ground with sand in a cold mortar, stirred for li/~ hours in the cold with 10 vol. of the extractant, then centrifuged for 5 minutes at 2500 × g. The supernatant, which represents the crude extract, has a Qp in the presence of Mg of about 500. Step 2. Removal of Actomyosin. Actomyosin is precipitated by dialyzing the crude extract overnight in the cold against 15 vol. of 0.005 M borate buffer at pH 8.0. It is removed by centrifugation for 10 minutes at 3000 X g, washed once with water (pH adjusted to 8.0), and centrifuged again. The two supernatants are combined, given a final centrifu4 C. H. Fiske a n d Y. SubbaRow, J. Biol. Chem. 66, 375 (1925). 5 A. P. Vanselow, Ind. Eng. Chem., Anal. Ed. 12, 516 (1940). 6 K. Bailey, Biochem. J. 36, 121 (1942).

[98]

INSECT ATPASE

597

gation at 7000 X g to remove the last traces of actomyosin, and adjusted in volume to fifteen times the original weight of muscle by addition of 0.05 M borate buffer. This fraction retains 70 % of the ATPase originally present and has a Qp of about 2000. An inorganic pyrophosphatase is also present (Qp for the hydrolysis of sodium pyrophosphate, 200). Step 3. Fractionation with Ammonium Sulfate. Solid ammonium sulfate is added to a concentration of 35%, the pH being maintained at 8.0 with NaOH. After standing for 15 minutes in the cold the precipitate is partially consolidated by centrifugation, then separated on Whatman No. 42 paper in a Biichner funnel. The paper and attached protein are extracted with 0.05 M borate buffer. Paper and undissolved protein are removed by filtering again through Whatman No. 42 paper, and the volume of the filtrate is made up to fifteen times the original weight of muscle. This step removes most of the inorganic pyrophosphatase. About 30% of the ATPase is retained, and the Qr is increased to about 3000. SUMMARY OF PURIFICATION PROCEDURE

(All figures refer to the processing of 1 g. of insect muscle)

Fraction 1. KCI extract 2. After removal of actomyosin 3. (NH4)2SO4fraction, 0-35% 4. After treatment with alumina C~

Total Total Specific volume, Units/ml., units, Protein, activity, Recovery, ml. thousands thousands mg./ml. Qp % 10

7.5

75

15.0

500

--

15

3.6

54

1.8

2000

72

15

1.5

22.5

0.5

3000

30

16

1.0

16

0.25

4000

21

Step 4. Treatment with Alumina. The last trace of inorganic pyrophosphatase is removed by treatment with alumina C~. The best concentration of alumina, which is determined by trial, is usually about 1.7 rag. of A1203 per milliliter. After standing for 15 minutes in the cold, the alumina is centrifuged off. The Q, of the supernatant varies usually between 3000 and 5000. More rarely higher values, ranging up to 7000, are encountered. About 20% of the ATPase activity originally present in the muscle is retained. Properties

Specificity. Substrates, in order of effectiveness, are ATP > ITP >> ADP > IDP. Inorganic pyrophosphate and other organic phosphate and

598

ENZYMES I N PHOSPHATE METABOLISM

[99]

pyrophosphate esters are not split. At 42 ° ATP is hydrolyzed about ten times as fast as ADP. Activators and Inhibitors. The enzyme is optimally activated by Mg at 1 X 10-8 M. There is no activity if Mg is replaced by Na, K, or Ca. Ca inhibits in the presence of Mg. Glutathione is needed for full activity even in fresh enzyme preparations. The ATPase is inhibited by sulfhydryl reagents , p-chloromercuribenzoate producing 96% inhibition at 5 × 10-~ M, and ninhydrin 97% inhibition at 5 X 10-3 M. Iodoacetate is less effective (40% inhibition at

1 X 10-2 M). Effect of pH. The optimal pH range is 7.8 to 8.0. Effect of Temperature. The relationship between temperature and activity follows the Arrhenius equation between 0 ° and 20 °. The optimal temperature for 5-minute tests is 42 °. Activation energy for the hydrolysis of ATP is 29.6 kcal./mole; for the hydrolysis of ADP it is 16.4 kcal./mole. Activity in Relation to Substrate Concentration. The activity/log (S) curve is bell shaped, the optimal substrate concentrations being 1.6 × 10-a M for ATP and 1.4 X 10-8 M for ADP. KATe is 8.6 X 10-4 M; KADp is 3.3 X 10-3 M.

[99] Adenylate Kinase (Myokinase, A D P Phosphomutase) 2 ADP ~ ATP + AMP

By SIDNEY P. COLOWlCK Assay Method Principle. Since this reaction is accompanied by no readily detectable physical or chemical change (e.g., in light absorption, acidity, or labile phosphorus content) it is convenient to convert one of the reaction products to a derivative, the formation of which is readily detectable. For example, one may add an excess of any enzyme system which specifically removes the terminal phosphate from ATP (e.g., hexokinase or ATPase). In the presence of hexokinase and adenylate kinase, the following over-all reaction results: ADP + glucose -* G-6-P + AMP + H +

(1)

When hexokinase is present in excess, the rate of this reaction is proportional to the concentration of adenylate kinase. The resulting reaction may be followed manometrically by measuring CO2 liberation from a bicarbonate buffer, or chemically by measuring the disappearance of

[99]

ADENYLATE KINASE (MYOKINASE, ADP PHOSPttOMUTASE)

599

acid-labile phosphorus. These two procedures, which are essentially procedures for measuring hexokinase activity, are the ones originally used for the detection of myokinase. 1 These and other procedures for hexokinase assay, which should be readily applicable to the assay of adenylate kinase, are described by Crane s and will not be described in detail here. An alternative method is described below for the assay of adenylate kinase. It is based on the detection of A M P formation by the addition of the specific 5r-AMP deaminase of Schmidt. The preparation of the deaminase 3 and its use for the detection of 5-AMP formation 4 are described elsewhere in this treatise. A factor which interferes with the application of this procedure is the difference between the pH optima of the two enzymes involved. Whereas adenylate kinase is optimally active at pH 7.5, the deaminase is essentially inactive at this pH and shows a sharp optimum around pH 6. Although citrate ions shift the pH optimum of deaminase toward the neutral range, 3 they inhibit the activity of adenylate kinase because of its Mg ++ requirement. It is therefore advisable to carry out this assay in two steps, permitting the adenylate kinase reaction to proceed first, then stopping this reaction by addition of a neutral citrate buffer 3 or a strong KC1 solution, 5 and finally adding the deaminase to measure 5'-AMP. The chief advantage of this procedure is that it can be used not only for activity measurements but also for measuring other properties of the adenylate kinase system, such as the equilibrium constant or the effect of activators and inhibitors. It is obvious that certain of such measurements would be difficult or impossible with a one-stage hexokinase assay.

Reagents ADP-MgCI2-Tris mixture. Mix 1 ml. of 0.01 M Na-ADP (Vol. III [118]) with 8 ml. of 0.1 M Tris-HC1 buffer (Vol. I [16]) and 0.5 ml. of 0.1 M MgCI~. Adenylate kinase. Make appropriate dilution in cold H~0 (e.g., dilute crude muscle extract about 1:400). Citrate buffer. 0.1 M pH 6.4, prepared by addition of HC1 to trisodium citrate. Deaminase. Stock solution of fraction 3 (see Vol. II [68]) diluted to contain about 2000 units of deaminase per milliliter. 1 S. P. Colowick and H. M. Kalckar, J. Biol. Chem. 148~ 117 (1943). 2 R. K. Crane, Vol. I [33]. 3 See Vol. II [68]. 4 See Vol. I I I [121]. W. J. Bowen and T. D. Kerwin, Arch. Biochem. and Biophys. 49~ 149 (1954).

600

ENZYMES IN P H O S P H A T E

METABOLISM

[99]

Procedure. To 0.15 ml. of the ADP-MgCI.~-Tris mixture in a 3-ml. quartz cuvette, add 0.05 ml. of the diluted adenylate kinase. After 5 minutes at room temperature add 2.8 ml. of the citrate buffer to stop the reaction. Then add 0.03 ml. (60 units) of the deaminase preparation, and measure the optical density at 265 m~ exactly 15 seconds and 30 seconds after mixing. Then continue reading at 1-minute intervals until the rate of change in optical density falls to 0.001 or less per minute. Not more than 10 minutes should be required for complete deamination. To the total optical density change for the period from 15 seconds to the end of the reaction, add the change measured from 15 to 30 seconds, in order to correct approximately for the amount of reaction which occurred in the first 15 seconds after mixing. This cannot be measured directly because the deaminase itself contributes significantly to the initial absorption change at 265 m~. The total optical density change must not exceed 15 % of the actual reading at 265 mu prior to deaminase addition, since the maximum observable change with excess adenylate kinase is only 25% under the condition described here. The amount of 5'-AMP formed is, of course, proportional to the total optical density change; a reading change of 0.09 corresponds to 0.03 micromole of 5'-AS/[P under these conditions. Although the amount of AMP can also be estimated from the initial rate of the deaminase reaction 6 rather than from total optical density change, this procedure is not recommended here because of the possibility that inhibitors or activators of the deaminase might cause false estimates of AMP concentration. Definition of Unit. One unit of enzyme is defined here as that amount which causes the formation of 1 micromole of 5t-AMP per minute under the conditions of the assay. Protein may be determined nephelometrically after precipitation by trichloroacetic acid. 7 Specific activity is expressed as units per milligram of protein. Application of Assay Method to Crude Tissue Preparations. The onestage hexokinase assay is probably superior to the two-stage deaminase assay for this purpose. The presence of "apyrases," which may directly or indirectly result in dephosphorylation of ADP to form AMP, would lead to falsely high values for adenylate kinase in the deaminase assay but not in the hexokinase assay. The presence of deaminase in the crude extracts would of course invalidate the deaminase assay, but not the hexokinase assay. A potent ATPase in the crude extract could invalidate the hexokinase assay, but the excess of hexokinase added would in most cases be sufficient to "compete" successfully with the ATPase. 6 H. G. Albaum and R. Lipshitz, Arch. Biochem. 27, 102 (1950). T. Biicherp Biochim. et Biophys. Acta 1, 292 (1947).

[99]

ADENYLATEKINASE (MYOKINASE, ADP PHOSFHOMUTASE)

601

Purification Procedure The procedure outlined below is essentially t h a t originally described b y Colowick and Kalckar. ~ F u r t h e r purification by adsorption of impurities on alumina C~, followed b y fractionation with trichloroacetic acid, has been reported by Kalckar. 8 More recently, N o d a and K u b y have announced additional purification as the zinc salt2 However, the original procedure yields a preparation which is sufficiently active and free of interfering proteins for most purposes. Step 1. Preparation of Crude Extract. ~° R a b b i t skeletal muscle is cooled, ground, and extracted twice with 1 vol. of cold 0.03 N K O H 0.002 M Versene. The third extraction is with 0.5 vol. of 0.002 M Versene. Step 2. Acidification and Heating. The combined extracts are acidified with 0.05 volume of 2.0 N hydrochloric acid and heated as rapidly as possible to a temperature of 90 °. After 3 minutes at this temperature, the solution is cooled rapidly and neutralized to p H 6.0 to 6.5 with 2 N sodium hydroxide. A very large precipitate is formed which is removed b y filtration. The resulting filtrate shows about sixfold purification and serves as a convenient source of adenylate kinase for m a n y purposes. I t m a y be stored as a solution in the refrigerator for several weeks without appreciable loss in activity. Information on stability at - 1 5 ° is not available. SUMMARY OF PURIFICATIONPROCEDUREa

Fraction

Total protein~ mg.

Total units, ~M./min.

Specific activity, ~M./min./mg.

1. Crude extract (100 ml.) 2. Filtrate after heating in acid (ca. 100 ml.)

2370 320

2250 1500

0.82 4.68

The specific activity recorded here for the crude extract is about one-tenth of that originally observed. ~,c It should be noted, however, that the adenylate kinase may not be saturated with substrate under the conditions of the present assay, in which the ADP concentration is 8 X 10-~ M, as compared with 1.3 X 10-2 M in the original assay, c b S. P. Colowick and H. M. Kalckar, J. Biol. Chem. 148, 117 (1943). c H. M. Kalckar, J. Biol. Chem. 148, 127 (1943). 8 H. M. Kalckar, J. Biol. Chem. 148, 127 (1943). 9L. Noda and S. A. Kuby, Federation Proc. 14, 261 (1955). 10The use of alkaline Versene for extraction is primarily for the purpose of isolating 3-phosphoglyceraldehyde dehydrogenase (see Vol. I [60]). The preparation of adenylate kinase was a by-product in this particular case. In the original studies, distilled water was used for extraction.

602

ENZYMES IN PHOSPHATE METABOLISM

[99]

Step 3. Concentration by Salting Out. F o r convenience in storage, or for removal of nucleotides and other small molecules f r o m fraction 2, the a d e n y l a t e kinase m a y be salted out b y adding a m m o n i u m sulfate to 0.8 saturation. T h e resulting precipitate m a y be filtered off and stored in the cold as a paste or dissolved in the minimal volume of water. N o purification results f r o m this step.

Properties Stability. 1 T h e m o s t r e m a r k a b l e p r o p e r t y of the a d e n y l a t e kinase of muscle is its resistance t o w a r d acid and heat. I n 0.1 N hydrochloric acid at 100 ° , its half-life is almost 30 minutes. Distribution. E a r l y studies 1 on distribution were in error b e c a u s e it was not realized t h a t the stability of the enzyme varies with the source. When various tissues were assayed after boiling with 0.1 N HC1, it a p p e a r e d t h a t muscle was the m a j o r source, none of the e n z y m e being found elsewhere, except for small a m o u n t s in brain and heart. Hence the n a m e m y o k i n a s e was adopted. L a t e r it was shown t h a t when other tissues such as liver 11 and yeast 12 are assayed w i t h o u t boiling in acid, the e n z y m e is readily detected. I t was therefore suggested 13 t h a t the n a m e adenylate kinase would be more appropriate. A s y s t e m a t i c reinvestigation of distribution and stability of the e n z y m e would be desirable. I t is not y e t clear whether the adenylate kinase a c t i v i t y found in h e a r t , ' brain, I and spleen 14 after boiling in HC1 represents all the e n z y m e present in those tissues or just t h a t r e m n a n t which survives the acid t r e a t m e n t . Intracellular Distribution and Function. T h e adenylate kinase is present in the m i t o c h o n d r i a of liver ~5 and muscle tissue. ~6 T h e e n z y m e is p r e s u m a b l y necessary whenever A M P is to serve as a p h o s p h a t e acceptor or A D P as a p h o s p h a t e donor. Evidence for a specific role of the e n z y m e in the relaxation of contracted muscle has been reviewed b y Bailey. ~7 Specificity. Until recently, little had been known concerning the specificity of this e n z y m e except t h a t I D P would not serve as subs t r a t e in place of ADP.lS T h e recent studies of L i e b e r m a n et al., 19 S t r o m -

1: A. V. I/:otel'nikova, Chem. Abstr. 43, 6263 (1949). 12R. E. Trucco, R. Caputto, L. F. Leloir, and N. Mittleman, Arch. Biochem. 18, 139 (1948). 1~S. P. Colowick, in "The Enzymes" (Sumner and Myrb$ick, eds.), Vol. II, Part A, p. 148, Academic Press, New York, 1951. 14E. M. Uyeki, Federation Proc. 14, 295 (1955). 15p. Siekevitz and V. R. Potter, J. Biol. Chem. 200, 187 (1953). 16A. Kityakara and J. W. Harman, J. Exptl. Med. 97, 553 (1953). ~7K. Bailey in "The Proteins" (Neurath and Bailey, eds.), Vol. II, Part B, pp. 1053-5, Academic Press, New York, 1954. is A. Kleinzeller, Biochem. J. 36, 729 (1942). 19I. Lieberman, A. Kornberg, and E. S. Simms, J. Am. Chem. Soc. 76, 3608 (1954)

[99]

ADENYLATEKINASE (MYOKINASE, ADP PHOSPHOMUTASE)

603

inger et al. 2° and others (see in footnote 3 Strominger et al. 2°) have revealed the existence of enzyme systems in yeast and animal tissues which might be termed "nucleoside monophosphate kinases." According to Heppel and Strominger (personal communication) there may be two enzymes involved in liver, one of which is specific for A T P as phosphate donor: A T P + X M P --~ ADP + X D P and the other of which is specific for AMP as acceptor: X T P + AMP --* ADP + X D P In both cases, X may be adenosine, guanosine, uridine, or cytidine. However, according to Lieberman et al., 19 the muscle enzyme with which we are concerned here works only with adenine nucleotides. Activators and Inhibitors. The adenylate kinases of muscle 5,8 and liver 15 are Mg++-activated. Activating effects of Ca ++ have also been described. 5,15 Fluoride 15,~-23 citrate, and Calgon ~ are inhibitors by virtue of their metal-binding action. This is the basis of the finding 2~that fluoride prevents AMP, but not ADP, from functioning as a phosphate acceptor in respiring mitochondria. The adenylate kinase of muscle can be inactivated by warming with H:02 and reactivated by glutathione (GSH) or cysteine. 1 If the enzyme is not subjected to oxidants, glutathione and cysteine are without effect on the activity and need not be added to the assay system. The muscle enzyme is inactivated by commercial pepsin. p H O p t i m u m . Kalckar 8 reported that the muscle enzyme was maximally active at pH 7.5. Bowen and Kerwin 5 also report a value of about 7.5 for assays in the absence of 5/[g++ but find an optimum at pH 6 when Mg ++ is present. Since the latter figures were obtained by the deaminase " r a t e assay" instead of the "extent assay," the value found by Kalckar would appear to be the more reliable figure for the pH optimum of the enzyme. Equilibrium Constant. Kalckar s showed that the adenylate kinase reaction could be demonstrated in either direction. Difficulty was experienced by both Kalckar 8 and Bowen and Kerwin ~ in reaching the same equilibrium position from the two directions. Starting with ADP, all investigators ~,s,24 agree that somewhat more than half is utilized at 2oj. L. Strominger, L. A. Heppel, and E. S. Maxwell, Arch. Biochem. and Biophys. 52, 488 (1954). 2I S. S. Barkulis and A. L. Lehninger, J. Biol. Chem. 190, 339 (1951). 22A. V. Kotel'nikova, Chem. Abstr. 45, 198 (1951). 28E. C. Slater, Biochem. J. 53, 521 (1953). 24L. V. Eggleston and R. Hems, Biochem. J. 52, 156 (1952).

604

ENZYMES IN PHOSPHATE METABOLISM

[99]

equilibrium. Eggleston and H e m s ~4 report a constant of 0.444 for this reaction a t p H 7.4 and 25 ° with 0.01 M MgC12. According to Bowen and Kerwin, 5 the a p p a r e n t equilibrium constant increases significantly with M g ++ concentration, because of the f o r m a t i o n of a M g - A T P complex with a higher binding constant t h a n t h a t for the M g - A D P complex. The Adenylic Acid Effect. When the s u b s t r a t e for the reaction is A D P , there is a v e r y rapid falling off in the rate of the reaction with time, because of the a c c u m u l a t i o n of 5-AMP, which appears to be strongly inhibitory to the muscle enzyme. 1.8 A T P also appears to be s o m e w h a t inhibitory with the liver enzyme. 15 Slater 23 points out t h a t the 5 - A M P inhibition is readily observed even when a large excess of hexokinase and glucose is present, which m u s t certainly prevent appreciable back reaction of A M P with A T P in a homogeneous system. I t appears t h a t the A M P inhibition is therefore best interpreted as being due to a v e r y high affinity of A M P for the protein, relative to t h a t of A D P . 25 However, it m u s t be k e p t in mind t h a t A M P would not be expected to be an inhibitor of the e n z y m e when the latter is catalyzing the reaction of AS~[P and A T P . I t therefore appears t h a t Slater's ~3,26 use of A M P as an " i n h i b i t o r " of this e n z y m e in a particulate s y s t e m generating A T P oxidatively f r o m A D P could conceivably lead to erroneous results. The generated A T P m i g h t react with A M P within the particles preferentially to reaction with hexokinase and glucose present externally. This could account for the low values for a p p a r e n t A T P synthesis actually observed b y Slater in such a system. ~5Slater23 presents data indicating that ADP has a high affinity for the enzyme, but the conclusion does not appear to he warranted, since the AMP:ADP ratio was maintained constant as the ADP concentration was varied. From the consideration mentioned in footnote a in the accompanyfng table, as well as from the strong inhibitory effect of AMP, it would seem that ADP may actually have a rather low affinity for the enzyme. 26 E. C. Slater, Nature 166, 982 (1950).

[I00]

ATP~CREATINE TRANSPHOSPHORYLASE

605

[lC0] ATP-Creatine Transphosphorylase ATP -b Cr ~ ADP -b C r o P

By LAFAYETTE NODA, STEPHEN KUBY, and HENRY LARDY Assay Method

Principle. The formation of Cr--~P from ATP and Cr is followed by determination of the acid-molybdate labile P of C r o P . Reagents 60% HCIO4. 5 % (NH4)6MoTO24"4H~O. Reducing agent. 0.2 g. of recrystallized 1-amino-2-naphthol-4-sulfonic acid, 12 g. of NaHSO3, and 2.4 g. of Na2SO~ in distilled water to make 100 ml. 0.08 M creatine (soluble at room temperature but not at 3°). 0.10 M MgS04. 0.005 M ATP (Na+), pH 7. 0.40 M glycine (Na+), pH 9.0. A stock reaction mixture of creatine, MgS04, and glycine is prepared by mixing 3.0, 0.6, and 2.4 vol. of their respective solutions. This mixture may be stored at 3 °. For each experiment 2 vol. of the ATP solution is added to 6 vol. of stock mixture to form the reaction mixture.

Procedure. To each incubation tube, 8 ml. of the reaction mixture (containing ATP) is added, and, after equilibrating in a 30 ° bath, 2 ml. of the enzyme is added at its required dilution (1:5000 to 1 : 125,000, depending on the fraction and protein concentration; dilution made with 0.001 M glycine, pH 9, just before use). A 2.0-ml. zero-time .aliquot (removed within 20 seconds after addition of the enzyme) is pipetted directly into Evelyn tubes containing 1.0 ml. of molybdate solution, 0.8 ml. of perchloric acid, and distilled water to bring the total volume to 9.6 ml. Succeeding aliquots were similarly removed at desired times (usually 5, 10, and 15 minutes). No deproteinization is necessury since the amount of protein in the reaction mixture (0.2 ~, to 4 ~//ml.) is too low to interfere. After standing for exactly 30 minutes at room temperature (average about 23°), 0.4 ml. of reducing agent is added. After exactly 10 minutes for color development, the inorganic phosphate is determined with the Evelyn colorimeter (660-m# filter) or the Beckman B spectrophotometer

606

ENZYMES IN PHOSPHATE METABOLISM

[100]

(660 m~) equipped to handle Evelyn tubes. Suitable blanks and standards are also set up with each run. The zero-time aliquot is used to correct for any inorganic phosphate present in the ATP sample and the small amount of phosphate liberated from ATP by the acid-molybdate. C a l c u l a t i o n o f A c t i v i t y U n i t s . Throughout the concentration ranges of ATP, Cr, and Mg ++ that have been investigated, the enzymatic catalysis does not follow good zero-order or first-order kinetics. The enzymatic activity may be determined from the extrapolated initial velocity under conditions approaching maximal velocity (0.004 M ATP, 0.004 M MgSO4, 0.024 M creatine, 0.096 M glycine-NaOH, pH 9.0 buffer; 30°), since under these conditions the reaction is first order with respect to the enzyme. However, for routine fractionation work, determinations of the initial velocities proved to be somewhat tedious, and the more empirical but reliable method described here was devised for following the enzymatic activity. Under the conditions described below, the reaction follows an apparent second-order kinetics with respect to ATP. The empirical rate equadx

tion may be simply expressed as ~/ = kE[ATP]2[Cr]°; setting kE = k'; dx "'" dt -

x

k'[ATP]2' which integrates to k ' t - a ( a - x ) ' where a is the ini-

tial concentration of ATP, x the amount disappearing in time t (equal to the amount of Cr~-~P formed), and E the enzyme concentration. Using the concentration scale in micromoles per milliliter, the time scale in minx x utes, and the setting a = 1.0 ~M./ml., k't = 1- - -. x A plot of 1 - x vs. t should be linear with a slope proportional to k' (i.e., proportional to the enzyme concentration); this holds up to 40 to 50 % of equilibrium which is attained under these conditions when 80 % of the terminal ~ P of ATP has been transferred to Cr. One unit of enzyme is defined as that amount of enzyme per milliliter of reaction mixture which will catalyze the transphosphorylytic reaction between 0.001 M ATP and 0.024 M creatine in the presence of 0.006 M MgSO4, at pH 9.0 (glycine buffer) and 30 °, and yield an apparent second-order velocity constant, k ' (defined above), equal to 1.0 ml. (micromoles) -1 (minute) -1. The determination of k' must of course be restricted to the apparent second-order portion of the reaction (usually where k' is in the range 0.015 to 0.025). For routine purposes, three measurements are usually made within the apparent second-order range; the k' values are calculated individually, then averaged and the units of the enzyme preparation determined per milliliter. When familiarity is gained with the activity and protein concentration of each fraction, dilutions can be made so that a 2.0-ml. zero-time and 10-minute aliquot are sufficient.

[100]

ATP~CREATINE TRANSPHOSPHORYLASE

607

T h e a p p a r e n t l y anomalous enzyme kinetics result from a severe product inhibition of this highly reversible system, and an inhibition of the reaction (ca. 20%) at the ratio of M g + + / A T P = 6.0; the rate of change of the reaction curve with time tends to slope off rapidly, giving rise to the empirical rate expression described above.

Purification Procedure

Step 1. Preparation of Crude Extract. T h e b a c k and leg muscles of a rabbit are excised i m m e d i a t e l y after decapitation and thrust into ice. The muscles are passed through a chilled m e a t grinder and homogenized in a Waring blendor (3 minutes per batch) using 2 1. of cold 0.01 M KC1 per kilogram of ground muscle. The thick h o m o g e n a t e is gently stirred for 15 minutes in the cold room before draining and squeezing through cheesecloth. Step 2. Purification with Ethanol. Solid NH4C1 is added to fraction 1 to a concentration of 0.10 M, and the p H is brought to 9.0 with 5 M NH4OH. After stirring for 1/~ hour in an ice b a t h to allow complete precipitation of inorganic salts (primarily MgNH4P04), 1.5 vol. of cold ethanol is added. After stirring for 2.5 hours at 20 °, the denatured protein and salts are removed b y centrifugation for 1/~ hour at 1000 X g and the clear, pale yellow solution is retained. Step 3. Precipitation and Fractional Extraction. T o fraction 2 2.0 M MgSO4 (pH 8.5) is added, with stirring, to a final concentration of 0.03 M, and cold 9 5 % ethanol (1.5 times the volume of 2 M MgS04 used) is added. After stirring for 1/~ hour at 20 °, the precipitate is collected b y centrifugation. T h e precipitate is twice thoroughly resuspended and extracted at 0 ° with 0.07 M MgAc2, p H 9.0, in volumes equal to 6 % and 4 % of the volume of fraction 1. E a c h time the insoluble portion is separated at 2000 X g for 20 minutes. T h e exact volumes of 0.07 M MgAc2 used for extraction and of the combined extracts are noted for the purpose of calculating the a m o u n t of alcohol in the extract. ~ l The calculation, though purely empirical, has been found to be reliable and reproducible and is illustrated by the following example: The volume of combined 0.07 M MgAc~ extracts, 200 ml., minus the volume of 0.07 M MgAc2 used, 160 ml., is assumed to be 60 per cent (v/v) of 95% alcohol, and thus represents 40 X 0.60 = 24 ml. of 95% alcohol. As an approximation there is 200 - 24 = 176 ml. of aqueous 0.36 solution. The volume of 95% alcohol required is thus 176 X ~ - 24 -= 75 ml. of 95% alcohol to bring the extract to 36% (v/v) of 95% alcohol. The alcohol required to raise the concentration from 36 to 50%, neglecting the 36% alcohol precipitate, is also calculated on the basis of the original extract volume; i.e., the alcohol required for 50% concentration minus the alcohol for 36% is: 176 - (75 -{- 24) = 77 ml. of 95% alcohol. In the recrystallization step a similar calculation is made to determine the amount of alcohol and NH40H introduced from the first crystalline sediment.

608

ENZYMES IN PHOSPHATE METABOLISM

[100]

Step 4. Alcohol Fractionation. To fraction 3 (0 ° and p H 8), cold ethanol is slowly added with stirring to 36% (concentrations of alcohol are expressed in terms of volume per cent of 95% ethanol--assuming t h a t the volumes are additive). After ~ hour at 0 ° the precipitate is removed b y centrifugation at 2000 X g for 20 minutes. E t h a n o l is added to the clear supernatant solution to a final concentration of 50%, and after 1/~ hour at 0 ° the precipitate is collected. The precipitate is dissolved in 30 to 50 ml. of 0.05 M ammonium citrate, p H 9, and dialyzed against 1 1. of 0.05 M ammonium citrate for 5 to 6 hours with stirring and then against two changes of 14 1. of 1.7 X 10-3 M N H 4 O H at about 3 °. A trace of precipitate formed in the dialysis bag is centrifuged off to obtain a clear, colorless solution. The preparation at this stage is usually 85 to 95% pure (45 to 50 units/rag.), and the yields are good (70 to 85%); if lyophilization is desired, one should substitute 0.01 M glycine-NaOH buffer, p H 9.0, for the weakly ammoniacal solution used for the above dialysis. The lyophilized powder appears to be quite stable if kept in the refrigerator. However, lyophilization should not be a t t e m p t e d if crystals are desired. Crystallization and Recrystallization. T h e solution is diluted with 1.7 × 10 -3 M N H 4 O H to a protein concentration of 20 to 30 mg./ml, and while stirring efficiently at 0 ° is slowly brought to 56 % ethanol. The final concentration of N H 4 O H is brought to 3.0 X 10 -3 M b y the addition of 5 M NH4OH. The solution in a small E r l e n m e y e r flask is covered with Parafilm and is allowed to stand at - 1 0 ° with occasional opening and swirling of the flask. ~ Apparently crystallization is induced as the result of a slow loss Of NH~. However, if the N H , O H concentration drops too low, much amorphous precipitate will form which m a y be redissolved b y addition of a trace a m o u n t of 5 M NH4OH. Sometimes a small a m o u n t of amorphous denatured protein forms and must be centrifuged off before crystallization occurs. W i t h o u t seeding, crystallization m a y take two weeks. The protein crystallizes in the form of masses of large elongated needles. Seed crystals m a y be kept in the ammoniacal ethanol (tightly stoppered) for about three m o n t h s at - 1 0 °, after which t h e y begin to deteriorate. T h e crystals are collected at - 1 0 ° b y centrifugation for 1 hour at 2000 X g or preferably 1/~ hour at 15,000 X g and washed with 60% Crystallization is facilitated by the cautious addition of small aliquots, 0.5 -- 2 ml., of gaseous CO~ to the air space in the crystallizing flask (conveniently by needle and syringe from a Dewar of dry ice). Crystallization is allowed to proceed until the crystalline mass first tends to settle, leaving a clear solution above it. This usually required about 2 to 3 days after the first crystals appeared.

[100]

ATP-CREATINE TRANSPHOSPHORYLASE

609

E t O H containing 0.003 M N H 4 O H at - 10 ° and recentrifuged as before2 T h e crystals are then dissolved in 0.003 M N H 4 O H (20 to 30 rag. of protein per milliliter), a n y insoluble material is separated b y centrifugation, and the alcohol and N H 4 O H added to 5 6 % and 3 X 10 -3 M, respectively, taking into account the 6 0 % alcohol retained in the precipitate and the a m o u n t of N H 4 O H used for dissolution. 1 Recrystallization at - 10 ° is hastened b y seeding from the first crop. After complete crystallization (usually a b o u t 2 d a y s after seeding) the p r o d u c t is collected and washed, dissolved in 0.01 M glycine, p H 9.0, and dialyzed against the same buffer at ca. 3 ° . The protein is stable in concentrated solution (3 to 5 %) for several weeks in the refrigerator. If desired, it m a y be lyophilized or placed in the cold room at - 1 0 ° ; a 5 % protein solution will not readily freeze at this t e m p e r a t u r e . A typical purification is summarized in the table. SUMMARY OF PURIFICATION PROCEDURE

(Initially, 1.0 kg. of rabbit skeletal muscle)

Fraction

Total protein, mg.

1. 0.01 M KC1 ext. 40,000 2. 20°, 60% EtOH supernatant 9,050 3. 0.07 M MgAc2 ext. of MgSO~ ppt. 3,250 4. 0.07 M MgAc~, 36-50% EtOH 2,970 5. Crystals 2,480 6. Recrystallized 2,150

Total units

Units/ mg.

Purification

Yield, %

(189,000) a 179,000

(4.73) ~ 19.8

(1.0) 4.18

(100.0) 94.7

44.0 46.5 52.0 52.3

9.31 9.84 11.0 11.1

75.7 73.1 68.2 59.5

143,000 138,000 129,000 112,500

a Small correction was made for trace amount of ATPase activity by assaying in absence of creatine and for trace amount of myokinase activity by substituting ADP for ATP. Other fractions are free of these contaminants. Properties

Purity. T h e crystalline enzyme prepared b y the above procedure is homogeneous as determined b y electrophoresis in several different buffers at p H values of 5.5 to 8.9, b y sedimentation and b y solubility. Specificity. T h e enzyme is specific for the c o m p o n e n t s shown in the The volume of alcohol used for washing is about one-third to one-half the aqueous volume before addition of the alcohol. If crystallization is incomplete the mother liquor and washing are retained and allowed to stand at - 10° until further crystallization takes place (facilitated sometimes by the further cautious addition of gaseous CO~).

610

ENZYMES IN PHOSPHATE METABOLISM

[101]

initial equation. Neither ADP nor I T P can serve as donors of ~-~P; arginine or creatinine will not serve as acceptors of ~-~P. Activators. The enzyme has an absolute requirement for divalent cations such as Mg ++ or Mn ++. Ca ++ is about half as effective as Mg++. Ba ++ appears to be inactive; Zn ++ and Cu ++ inhibit. In the forward direction the optimum concentration of Mg ++ is equal to the concentration of ATP. In the reverse direction near maximum velocity is achieved by concentrations of Mg ++ equal to that of ADP, but the optimum concentrations of ~Ig ++ are somewhat higher. pH Optimum. The reaction velocity is greatest at pH 9 for the forward reaction and at pH 6 to 7 for the reverse direction. Michaelis constants measured at these respective optimum pH values and at 38 ° are as follows: ATP, 6 X 10-4; Cr, 1.9 X 10-2; ADP, 1 X 10-3; Cr~-~P, 5 X 10-3; all in moles X liter-L Turnover. Under optimum conditions, 1 mole of enzyme (80,000 g.) catalyzes the phosphorylation of 25,000 moles of Cr (forward direction) or 150,000 moles of ADP (reverse direction) per minute at 38 °.

[101] C o u p l i n g of P h o s p h o r y l a t i o n

with Oxidation

By F. EDMUND HUNTER, JR. The direct measurement of oxidative phosphorylation by determining the disappearance of inorganic phosphate or the formation of an organic phosphate compound is a very useful and widely applied procedure. However, if significant amounts of the organic phosphate formed are split by phosphatases or further metabolized with the release of inorganic phosphate, indirect assays must be used. Direct Assay

Principle. The enzyme system and substrate are incubated in a medium containing inorganic orthophosphate and a phosphate acceptor system. Oxygen consumption and the removal of inorganic phosphate are measured. Equipment. A Warburg apparatus or other suitable respirometer for measuring oxygen uptake. 1 W. W. Umbreit, in " M a n o m e t r i c Techniques and Tissue Metabolism" (Umbreit, Burris, and Stauffer, eds.), Chapter 1, Burgess Publishing Co., Minneapolis, 1949.

[101]

COUPLING OF PHOSPHORYLATION WITH OXIDATION

611

Reagents

Oxygen. Although oxygen may be used in the respirometer, air is usually satisfactory. 1 Medium. 2 For each component the final concentration in a 3.0-ml. reaction mixture will be indicated. In addition, a suggested volume and concentration of stock solution will be included in parentheses. Mixed medium can be prepared as convenient. If stock solutions are made essentially isotonic, any component may be omitted and the volume made up with isotonic KC1 or sucrose. 1. Potassium phosphate buffer, pH 7.4, 0.01 to 0.013 M (0.4 ml. of 0.1 M). Tris or glycylglycine buffer may be used in studies with lower phosphate levels. 2. Cytochrome c, 1.0 to 1.5 X 10-~ M (0.05 ml. of 1%). Routinely used by most workers, added cytochrome c is essential in many systems. 3. MgC12, 0.005 to 0.0075 M (0.2 ml. of 0.1 M). Must be added after the phosphate has been well diluted with other additions to avoid precipitation of magnesium phosphate compounds, especially if NaF is being used. 4. ATP-AMP. If an additional phosphate acceptor is used, only catalytic amounts of ATP, 0.001 to 0.002 M, pH 7.4 (0.05 ml. of 0.1 M), are needed. If AMP is to be the final phosphate acceptor, 0.005 to 0.01 M (0.25 ml. of 0.1 M) must be used. 3 5. Hexokinase. Yeast hexokinase, 4 approximately 20% pure, together with glucose is commonly used as a final phosphate acceptor system (0.05 ml. of 5 to 10 mg. of protein per milliliter, 20% pure). This provides a large excess of transferring activity. 6. Glucose, 0.02 M (0.2 ml. of 0.3 M). Sometimes creatine kinase 5 and creatine have been used in place of hexokinase and glucose. The lability of phosphocreatine and unfavorable position of the equilibrium with ATP are disadvantages. 7. NaF, 0.01 to 0.02 M (0.2 ml. of 0.15 M). This inhibitor is commonly used to reduce ATPase and phosphatase activity. It is essential with broken cell preparations but is less critical with intact, washed mitochondria. 2 8. Potassium malonate, pH 7.4.0.01 to 0.02 M (0.3 ml. of 0.1 M). 2j. H. Copenhaver, Jr., and H. A. Lardy, J. Biol. Chem. 195, 225 (1952). 3W. W. Kielley and R. K. Kielley, J. Biol. Chem. 191, 485 (1951). 4See Vol. I [32]. See Vol. II [100].

612

ENZYMES IN PHOSPHATE METABOLISM

[101]

This inhibitor is added only when it is desired to prevent oxidation beyond the succinate step. 9. Isotonic KC1 (0.15 M) or sucrose (0.25 M). Used to adjust final volume. Sucrose preferable for long incubations. 10. DPN or TPN (0.0002 to 0.0005 M). Essential in some systems. See later discussion. Substrate. 0.005 to 0.01 M, pH 7.4 (0.3 ml. of 0.1 M). Controls without substrate must be run with each experiment. Enzyme system. The amount should yield an oxygen consumption of 3 to 10 microatoms per 30 minutes. Homogenate representing 50 to 300 mg. or mitochondria representing 300 to 500 mg. of original tissue have been used (1.0 ml. of a suspension of mitochondria from 10 g. of tissue in 20 ml. of isotonic sucrose). Incubation Procedure. PREPARATION OF FLASKS. The flasks are kept in cracked ice while being prepared. All components except the hexokinase and glucose are placed in the main compartment of the vessel, with the enzyme preparation being added at the last moment. The hexokinase and glucose are placed in a side arm and tipped in after temperature equilibration. In this way the rather labile phosphorylating systems are never without the protective effects of substrate, ATP, and some oxidative activity. Moreover, the periods of net phosphate uptake and measured oxygen consumption correspond exactly. If the vessels have a second side arm, 0.3 to 0.5 ml. of 30 to 50 % perchloric acid or TCA should be placed there. TEMPERATURE. A temperature of 30 ° is commonly used. If the preparation deteriorates rapidly, temperatures as low as 15° may have advantages. EQUILIBRATION PERIOD. Short equilibration periods (5 minutes) are desirable, but must be demonstrated adequate by experiment. Control flasks should be deproteinized at the end of the equilibration period to give the true zero-time phosphate value. INCUBATION PERIOD. The incubation period is 15 to 30 minutes. STOPPING TEE REACTION. If possible, perchloric acid or TCA should be tipped in to stop the reaction. Otherwise immediate chilling and prompt deproteinization must be carried out. Mix thoroughly, and filter or centrifuge. Measurement of Phosphate Esterified. Inorganic phosphate is determined by any suitable procedure. 6 The ratio between inorganic phosphate removed and atoms of oxygen consumed is the P:O ratio. Under 6O. H. Lowry and J. A. Lopez, J. Biol. Chem. 162, 421 (1946); see also Vol. III [114].

[101]

COUPLING OF PHOSPHORYLATION WITH OXIDATION

613

certain circumstances, inorganic pyrophosphate may accumulate, and i n o r g a n i c p h o s p h a t e r e m o v e d is n o t e q u i v a l e n t t o o r g a n i c e s t e r f o r m e d . 7 When changes in phosphate are small, greater accuracy can be achieved TABLE I NUMBER OF PHOSPHORYLATIONS PER ATOM OF OXYGEN CONSUMED WITH DIFFERENT SUBSTRATES

Reaction studied

Number of oxidation steps undergone by substrate

~-Hydroxybutyrate -* acetoacetate D P N H --* D P N ~-Ketoglutarate -* succinate Succinate -~ fumaratef Pyruvate -~ acetate Pyruvate --* acetoacetate Citrate --* succinate Glutamate -~ succinate Malate -* (?) Oxalacetate --* (?) Pyruvate --* 3C02 W 2H~O Caprylate -* (?) Proline -* (?)

1 1 1 1 1 1 2 2 --5 ---

P: Oa Probable in Observed intact cell References 2.5 1.5 3.6 1.7 -2.6 2.6 2.6 2.4 2.1 2.5 1.6 2.1

3.0 3.0 4.0 2.0 4.0 3.0 3.5 3.5 --3.0 ---

b, c c b, d, e b, d, e b b b g g e, g h g

When more than one-step oxidation of the substrate occurs, the P: O ratio will be the average for the two or more steps. b j. H. Copenhaver, Jr., and H. A. Lardy, J. Biol. Chem. 195, 225 (1952). c A. L. Lehninger, J. Biol. Chem. 190, 345 (1951). d H. A. Krebs, A. Ruffo, M. Johnson, L. V. Eggleston, and R. Hems, Biochem. J. 54, 107 (1953). F. E. Hunter, Jr., in "Phosphorus Metabolism" (McElroy and Glass, eds.), Vol. 1, p. 297, The Johns Hopkins Press, Baltimore, 1951. / With succinate as substrate, the reaction seems largely confined to a single step in short experiments. o R. Cross, J. V. Taggart, G. A. Coco, and D. E. Green, J. Biol. Chem. 177, 655 (1949). h H. A. Lardy, in "Phosphorus Metabolism" (McElroy and Glass, eds.), Vol. 1, p. 387, The Johns Hopkins Press, Baltimore, 1951. by determining the G-6-P formed, s Occasionally ATP or creatine phosphate formation has been measured. The theoretical and experimental P : O r a t i o s o b t a i n e d w i t h v a r i o u s s u b s t r a t e s a r e s u m m a r i z e d in T a b l e I. 7 R. J. Cross, J. V. Taggart, G. A. Covo, and D. E. Green, J. Biol. Chem. 177, 655 (1949). 8 E. C. Slater, Biochem. J. 53, 521 (1953).

614

ENZYMES IN PHOSPHATE METABOLISM

[101]

Indirect A s s a y When direct assay cannot be applied, indirect methods have been used to estimate the phosphorylating activity. These assays are of several types. 1. Ability of an oxidation to maintain an ATP-organic phosphate pool at a constant level2 2. Rate of incorporation of p32 into organic phosphates, particularly ATP. 1° If critical conditions are met, quantitative calculations can be made from indirect assays. 1° Application of A s s a y to Crude E n z y m e Preparations

Qualitative detection of oxidative phosphorylation is easily made by noting the removal of some inorganic phosphate or the incorporation of p32 into ATP, but quantitative assay requires careful consideration of possible side reactions and losses which may occur. These are greater and less easy to control or evaluate with crude enzyme systems. Comparisons between tissues are difficult, for the losses due to phosphatases and other factors may be quite different. E n z y m e Preparations 11 Slices. Experiments are confined to indirect assays because of the limited permeability of cells to phosphate, ATP, and acceptor systems. Homogenates. In spite of phosphatase activity, considerable net uptake of inorganic phosphate may be obtained, so limited direct assays may be possible. Mitochondrial Suspensions. Preparations from liver, heart, and kidney have proved exceedingly useful for study of efficiency (P:O ratios) and some studies on mechanisms. Most of the citric acid cycle oxidations and accompanying phosphorylations of a cell occur in the mitochondria. 12 Isolated mitochondria show fairly high efficiency in conversion of substrate energy to ATP energy. ~,13 Even greater efficiency within the cell seems unlikely, but not impossible. The rate of oxidation in mitochondria is determined by pH and by the availability of phosphate acceptors. In addition, recent work suggests that other cellular components may influence the rate even in the presence of excess acceptor systems. ~4 9 V. R. Potter, G. G. Lyle, and W. C. Schneider, J. Biol. Chem. 190, 293 (1951). 10 H. A. Krebs, A. Ruffo, M. Johnson, L. V. Eggleston, and R. Hems, Biochem. J. 54,

107 (1953). xl See Vol. I [1, 2, 3]. 12W. C. Schneider, J. Histoehem. and Cytochem. 1, 212 (1953). 18F. E. Hunter, in "Phosphorus Metabolism" (McElroy and Glass, eds.), Vol. 1, p. 297, The Johns Hopkins Press, Baltimore, 1951. x4R. B. Johnson and W. W. Aekermann, J. Biol. Chem. 200, 263 (1953).

[101]

COUPLING OF PHOSPHORYLATION WITH OXIDATION

615

A g r e a t a d v a n t a g e of m i t o c h o n d r i a is t h e v e r y low A T P a s e a c t i v i t y . Modified Mitochondria. W a s h e d i n s o l u b l e r e s i d u e s / KC1 a g g r e g a t e d m i t o c h o u d r i a , a n d h y p o t o n i c a l l y t r e a t e d m i t o c h o n d r i a 1~ h a v e b e e n u s e d advantageously at times. However, such preparations show greater ATPase activity, cytochrome c and coenzyme requirements, and may s h o w less p h o s p h o r y l a t i o n . P r o p e r t i e s of t h e P h o s p h o r y l a t i n g S y s t e m s

Requirements. M g ++ is e s s e n t i a l for p h o s p h a t e i n c o r p o r a t i o n i n t o A T P a n d for f u r t h e r t r a n s f e r s . A D P ( A M P ) is e s s e n t i a l e i t h e r as a n a c c e p t o r s y s t e m or as a t r a n s f e r m e c h a n i s m t o a s e c o n d a r y a c c e p t o r s y s t e m . W h e t h e r c y t o c h r o m e c, D P N , T P N , etc., n e e d be a d d e d d e p e n d s on t h e e n z y m e p r e p a r a t i o n a n d s u b s t r a t e used. TABLE II EXAMPLES OF AGENTS ~TtnCH UNCOUPLE AEROBIC PHOSPHORYLATION

Approximate molar concentration required for

Uncoupling agent Gramicidin Dinitrocresol Usnic acid Dinitrophenol Mannitol hexanitrate Sodium azide Thiopental Aureomycin 2,4-Dichlorophenoxyacetic acid Quinacrine Arsenite Nitrite

50% uncoupling 4 2 6 1 3 2 4 5 5 2

X 10-7 X 10-6 X 10-6 X 10-5 X 10-~ X 10-4 X 10-4 X 10-4 X 10-4 X 10-8 -1 X 10-2

Essentially complete uncoupling 4 1 2 1 2 2 1

X 10-6 X 10-s X 10-5 X 10-4 X 10-4 X 10-a X 10-3 --5 X 10-3 1 X 10-~ --

The exact percentage uncoupling with a given conccntration of agent will vary with substrate and type of enzyme preparation. Phosphorylations occurring below the DPN level in the electron transport chain are not uncoupled by many of these agents.

Lability. T h e c o u p l i n g of p h o s p h o r y l a t i o n w i t h t h e s e o x i d a t i o n s is e a s i l y i n a c t i v a t e d . I n c u b a t i o n of t h e e n z y m e s y s t e m w i t h o u t s u b s t r a t e , e x p o s u r e t o low t o n i c i t y , t r e a t m e n t w i t h s u r f a c e a c t i v e a g e n t s , a n d m a n y o t h e r c o n d i t i o n s c a u s e a d e c r e a s e or c o m p l e t e loss of p h o s p h o r y l a t i o n . Uncoupling Phenomenon. A n u m b e r of c h e m i c a l a g e n t s , for e x a m p l e 2 , 4 - d i n i t r o p h e n o l , h a v e a v e r y s e l e c t i v e effect in e l i m i n a t i n g or u n c o u ~5A. L. Lehninger, J. Biol. Chem. 190, 345 (1951).

616

ENZYMES IN PHOSPHATE METABOLISM

[101]

pling phosphorylation with no inhibition of oxidation. The mechanism is not understood. A summary of a number of the uncoupling agents is given in Table II. Comments Qualitative detection of phosphorylation can be made without measuring oxygen consumption. Quantitatively the disappearance of substrate or formation of product may be substituted for and at times preferred to the measurement of oxygen consumption. The addition of few coenzymes is necessary with mitochondrial preparations, but in assaying altered mitochondria and crude tissue preparations the addition of cytochrome c, DPN, TPN, etc., is usually essential. When oxidative phosphorylation occurs, it does not prove that the phosphorylation is coupled with the first-step oxidation of the substrate. The oxidation may proceed through several steps of the cycle or even go to completion. Malonate is fairly effective for blocking at the succinate step. Direct measurement of products will determine how far oxidation is proceeding. The single-step oxidation of malate, isocitrate, and ~-hydroxybutyrate is exactly equivalent to the oxidation of D P N H or T P N H . However, with the oxidation of a-ketoglutarate to succinate and pyruvate to acetate, there is an additional phosphorylation below the D P N - T P N level.

[102]

PANTOTHENATE-SYNTHESIZING ENZYME

619

[102] P a n t o t h e n a t e - S y n t h e s i z i n g E n z y m e By G. DAVID NOVELLI

Assay Method Principle. The enzyme catalyzes the following reaction :1 Pantoate ~- ~-alanine -~ ATP --~ Pantothenate ~ AMP ~ PP In the presence of an excess of ATP the reaction proceeds to completion. The course of the reaction may be followed by measuring the formation of any of the products, but, since crude extracts are contaminated by ATPase, myokinase, and inorganic pyrophosphatase, the most accurate measure of the reaction is the microbiological assay of pantothenate. This assay procedure may be applied to resting cell suspensions, crude homogenates, or extracts. The method of isolation and the characterization of this enzyme was worked out by Dr. W. K. Maas, 2 and we are indebted to him for making available much unpublished information. Procedure. To a series of tubes containing, per milliliter, 10 uM. of ATP, 100 uM. of KC1, 20 ~M. of ~-alanine, 20 ~M. of K pantoate, 10 ~M. of MgS04, and 100 uM. of Tris buffer, pH 8.5, is added varying amounts of enzyme solution. The tubes are incubated at 25 ° for 30 minutes, and the reaction is stopped by heating in a boiling water bath for 3 minutes. The pantothenate is determined microbiologically with a pantothenate auxotroph of Escherichia coli as described by Maas and Davis2 Definition of Unit and Specific Activity. A unit of enzyme activity is defined as that amount resulting in the synthesis of 1 ~M. of pantothenate under the above conditions. Specific activity is expressed as units per milligram of protein. Protein is measured by the turbidimetric method of Bficher. 4

Purification Procedure Step 1. Preparation of Acetone-Dried Cells. Escherichia coli w, ACTCC 9637, is grown with aeration at 37 ° for 24 hours in a minimal medium enriched with yeast extract and hydrolyzed casein2 The cells are harvested by centrifugation with a Sharples centrifuge and washed twice with distilled water. An acetone powder of the cells is prepared by stirring the cells into 20 vol. of ice-cold acetone. The powder is collected on a Bfichner funnel, washed with 10 vol. of acetone, and then with 10 vol. of ether. w. K. Maas and G. D. Novelli, Arch. Biochem. and Biophys. 43, 236 (1953). 2W. K. Maas, J. Biol. Chem. 198, 23 (1952). 8W. K. Maas and B. D. Davis, J. Bacteriol. 60, 733 (1950). 4T. Biicher, Biochim. et Biophys. Acta 1, 292 (1947).

620

COENZYME AND VITAMIN METABOLISM

[102]

The powder is finally dried in a vaccum desiccator over P205 and paraffin chips. Step ~. Preparation of Dialyzed Extract. The acetone powder is extracted by suspending it in ten times its weight of 0.01 M phosphate buffer, pH 7.1. The suspension is allowed to stand with occasional stirring for 1 hour, after which the debris is removed by centrifugation. Equally active extracts have been obtained at room temperature and in the cold. The extract is dialyzed against 20 vol. of distilled water; the fluid is changed twice. Step 3. Precipitation with 0.6 Saturated Ammonium Sulfate. The extract from step 2 is brought to 0.6 saturation with solid ammonium sulfate. The precipitate is collected by centrifugation at 15,000 r.p.m, in the Servall angle head in the cold room. The supernatant is discarded. The precipitate is resuspended in one-fifth its original volume of 0.01 M phosphate buffer, pH 7.1. Step 4. Protamine Treatment. The solution from step 3 is treated with 2% protamine sulfate using 0.6 mg. of protamine sulfate per milligram of nucleic acid in the extract. (The nucleic acid is arbitrarily estimated by determining the turbidity produced by an aliquot of the extract when treated with 0.5 ml. of 2% protamine sulfate in 5.0 ml. of 0.01 M phosphate buffer at pH 6.0 and comparing with a standard curve prepared with yeast nucleic acid.) The precipitate is centrifuged off and discarded. Step 5. Fractionation with Ammonium Sulfate. The solution from step 4 is fractionated with solid ammonium sulfate, and the fraction precipitating between 0.25 and 0.5 saturation is collected on the centrifuge. The precipitate is dissolved in 0.01 M phosphate buffer, pH 7.1, using 0.3 vol. of the solution from step 4. Step 6. Second Protamine Treatment. The solution from step 5 is treated again with 2 % protamine sulfate, now using 2 mg. of protamine sulfate per milligram of nucleic acid. The precipitate is centrifuged off and discarded. SUMMARY OF PURIFICATION PROCEDURE Step

Specific activity

Total activity, units

Recovery, %

2 3 4 5 6 7 8

1.32 1.37

3870 3260 1810 1750 1500 83O 700

86 55 54 46 25 22

2.4 5.1 8.5

[102]

PANTOTHENATE-SYNTHESIZING ENZYME

621

Step 7. Fractionation with Ammonium Sulfate. The solution from step 6 is again fractionated with ammonium sulfate. The fraction precipitating between 0.3 and 0.46 saturation is collected and dissolved in a minimum volume of 0.01 M phosphate buffer, pH 7.1. Step 8. Treatment with Calcium Phosphate Gel. The solution is now treated with ~g vol. of calcium phosphate gel (26 mg. of solids per milliliter) at pH 6.0 in the cold. The precipitate is centrifuged off and discarded. The enzyme in the supernatant is then purified six-fold. The summary of the purification is given in the table.

Properties Stability. The enzyme is very stable. Storage in distilled water at 6 ° for a week or longer resulted in no detectable loss in activity. The acetone powder may be extracted at room temperature, and the enzyme may be subjected to prolonged dialysis against distilled water without loss of activity. pH Optimum. The enzyme is active over the pH range 7 to 9 with an optimum at pH 8.5. At pH 7.0 to 7.5 phosphate and Tris buffers yield identical activities. At pH 8 to 9 Tris and ammonium hydroxide yield identical activities. Substrate A~inities. The Michaelis constant as determined by the graphical method of Lineweaver and Burk for ~-alanine was 0.46, 0.57, 0.74, and 0.38, and for pantoate it was 1.52, 1.93, and 3.54. Although variable, these data indicate that the affinity for f~-alanine is several times greater than for pantoate. The enzyme is specific for pantoate, pantoyl lactone being inactive with the isolated enzyme, although the lactone has about one-third the activity of pantoate in resting ceils. ATP is the energy source and undergoes a depyrophosphorylation during the reaction. For optimal activity a high concentration approximately that of the substrates is required. Above optimal concentrations, ATP becomes inhibitory. A 50 % inhibition is seen when ATP is in sixfold excess over the substrates. Activators. The enzyme requires both monovalent and divalent cations. The monovalent cation requirement is satisfied by either K + or NH4 +. Ammonium ion is slightly better than potassium. Sodium ions are distinctly inhibitory even in the presence of K + or NH4 +. The requirement for these cations is fairly high, being of the order of 100 to 200 mM. The chlorides are as active as the sulfates. The divalent cation requirement is satisfied by either Mn ++ or Mg ++. Mg ++ is considerably superior to Mn ++. The optimum concentration is 10 mM., above which they become slightly inhibitory. Ca ++ and Zn ++ are not stimulatory and at 10-mM. concentration are slightly inhibitory.

622

COENZYME AND VITAMIN METABOLmM

[103]

Rate. The equilibrium of this reaction lies far to the side of synthesis. Within the limits of measurement the reaction proceeds to the complete conversion of the substrates. In crude extracts the rate is approximately 0.5 uM./mg./hr, at 25 °. Between 15 ° and 45 ° the rate increases three times for each 10 ° rise in temperature. During the 30-minute testing period the rate is constant. NOTE. The purified enzyme is relatively free from ATPase, myokinase, and inorganic pyrophosphatase. With the purified enzymes, rate studies are conveniently measured by running the reaction in Warburg vessels at pH 8.0 in a bicarbonate buffer and following the liberation of COs. COs is produced in a bicarbonate buffer because the over-all reaction results in a net production of acid. Also with the purified enzyme, the reaction may be followed by measuring the formation of A M P with 5-adenylic deaminase (see Vol. II [68]) or by following the production of inorganic pyrophosphate.

[103] T h i a m i n a s e

By AKIJI FUJITA I. Thiaminase from the Viscera of Clam (Meretrix meretrix) Pm.CHs'Th + q- B H ~ Pm'CHs'B q- Th q- H + (Thiamine)

(Base)

Assay Method Principle. The following method (Fujita et al. 1) is based on the fact that the enzymatic activity is interrupted by metaphosphoric acid and the remaining thiamine is determined fluorometrically by the thiochrome method after adsorption on Permutit (HennessyS). As the crude enzyme preparation contains the necessary base, its addition is usually not necessary.

Reagents 0.1 M citric acid-NaOH buffer, pH 5.5. 10% metaphosphoric acid. Enzyme. The viscera of clams are thoroughly ground with a small amount of water and sand, and the mixture is adjusted to pH 4.5 with 1 N HC1 and diluted tenfold. The mixture is kept at 30 ° for 1 A. Fujita, Y. Nose, S. Kozuka, T. Tashiro, K. Ueda, and S. Sakamoto, J. Biol. Chem. 196, 289 (1952). 2 D. J. Hennessy, Ind. Eng. Chem. Anal. Ed. 18, 216 (1941).

[103]

THIAMINASE

623

15 minutes with occasional stirring, and then centrifuged. The supernatant is used for the experiments after suitable dilution.

Procedure. The experimental samples contain enzyme solution (1.0 ml.), 0.02 M citrate buffer of pH 5.5 (2.0 ml.), thiamine (1 ~,), and water in a final volume of 10 ml. For full activity of the enzyme, aniline in a final concentration of 10-3 M is added; in all cases where specific activity was determined aniline was present. Suitable controls are carried out by omitting thiamine and with samples with heat-inactivated enzyme (20 minutes at 100°). After incubation at 50 ° for 1 hour, the samples are deproteinized with 5 ml. of 10% metaphosphoric acid, whereby the enzymatic activity is almost completely interrupted. The supernatant is adjusted to pH 5 with 1.0 N NaOH, and thiamine is determined, after its adsorption on Permutit, by a modification of the thiochrome method. Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which decomposes 1 ~ of thiamine in 1 hour under the above conditions, whereby the enzyme concentration is adjusted so that the decomposed thiamine attains more than 50%. Specific activity is expressed as units per milligram of protein. Protein is determined spectrophotometrically by measurement of optical density E (in a cell with a 1-cm. light path) at 280 m~. The protein concentration (mg./ml.) corresponds to 0.625 X E. Purification Procedure

For purification the adsorption by alumina C~ is found to be the most suitable. The adsorption by tricalcium phosphate and acetone precipitation are found also to be useful. Fractionation with ammonium sulfate (most suitable at 0.8 saturation), magnesium sulfate, or sodium sulfate (most suitable at 0.5 saturation) and precipitation with ethanol or methanol were found to be scarcely promising, the specific activity and the yield being low. The removal of nucleic acid by protamine sulfate was found not to be especially helpful. Step 1. Preparation of Crude Extract. Clam viscera is extracted with water as described under Reagents, Enzyme. Step 2. Precipitation with Acetone. To 40 ml. of the ice-cold supernatant of the centrifuged extract (1:10) is added 60 ml. of acetone which has been cooled by solid CO2. The acetone in the precipitate is removed by evacuation. The precipitate is dissolved in 40 ml. of 0.07 M ice-cold phosphate buffer, pH 6.5, and centrifuged. Step 3. Adsorption by Tricalcium Phosphate. Forty milliliters of icecold 15 mg./ml, tricalcium phosphate solution is added to the ice-cold

624

[103]

COENZYME AND VITAMIN METABOLISM

supernatant of step 2; the solution is adjusted to pH 6 and stirred for 15 minutes. After centrifugation in the cold, the supernatant fluid is discarded; the precipitate is washed with water and eluted with 40 ml. of 0.2 M Na~HPO~. Step ~. Adsorption by Alumina C~. To the supernatant fluid of step 3 is added 40 ml. of 6.0 mg./ml, alumina C~. The solution is adjusted to pH 7 and stirred for 15 minutes in the cold. It is centrifuged in the cold, the supernatant fluid is discarded, and the precipitate is washed with water. It is eluted with 40 ml. of 0.2 M Na:HP04 solution. This solution, when stored in the refrigerator in frozen state, can be kept for several days without detectable loss of activity. TABLE I SUMMARY OF PURIFICATION PROCEDURE FOR CLAM ENZYME

Fraction 1. Raw extract 2. Acetone precipitation 3. Eluate from Ca-phosphate 4. Eluate from Alumina C~

Total Specific volume, Total Protein, activity, Recovery, ml. Units/ml. units mg./ml, units/mg. % 40 40

54 52

2160 2080

5.1 0.91

10.6 57.1

100 96

40

36.5

1460

0.44

82.8

68

40

28.8

1150

0.11

262

53

The specific activity of this final solution can usually be attained with adsorption twice by alumina C, alone, without any other treatment. The purification procedure is summarized in Table I.

Properties Specificity. The shellfish enzyme is specific for thiamine, thiamine pyrophosphate, and thiamine derivatives with the 4-amino group of the pyrimidine moiety intact. The 2-position of the pyrimidine moiety or the 5-position of the thiazole moiety can be substituted (lVIurata3). It has no action on thiamine derivatives with a substituted 4-amino group of the pyrimidine moiety, thiothiamine, namely 3-[2'-methyl-4 taminopyrimidyl- (5')]-methyl-4-methyl-5-f~-hydroxyethylthiazole-2-thione (Murata3), thiamine disulfide of Zima,4 allithiamine, namely 2-(2'-methyl4'-aminopyrimidyl-5')-methylformamino-5-hydroxy-A 2-pentenyl- (3)-allyl 8 K. Murata, Bull. Cherr~ Soc. Japan 23, 37 (1950). 4 O. Zima and R. R. Williams, Bet. deut. chem. Ges. 78, 941 (1940).

[103]

THIAMINASE

625

disulfide of Fujiwara et al. 5 and Matsukawa et al. 6 and thiamine propyl disulfide of Matsukawa et al. (SakamotoT). Activators and Inhibitors. The activity of clam and crucian (Carassius carassius) enzyme is markedly activated by a series of aromatic amines, the amino group of which is directly attached to the benzene ring, as aniline and its derivatives as well as heterocyclic amines, such as pyridine, and quinoline, and further some sulfhydryl compounds in a final concentration of 10-3 M (Fujita ct al.1). The activation of crucian enzyme is qualitatively about the same as clam enzyme but quantitatively far less intensive. Sealock et al. s found that m-aminobenzyl-(3)-4-methylthiazolium salt increases the activity of carp enzyme. 2-Methyl-4-aminopyrimidine derivatives inhibit clam and crueian enzyme (Fujita et al.1). Sealock et al. s found that o-aminobenzyl-(3)-4-methylthiazolium salt is the most potent inhibitor to the carp enzyme, and the analogous 2-methyl compound is almost equally efficient, but the 2-4-dimethylthiazolium compound is distinctly less active. In experiments with crude clam and crucian enzyme, FeS04, Fe2(SO4)3, CuS04, and MnS04, in a final concentration of 10-3 M, showed more or less inhibitory action (Fujita et al.1). Effect of pH. The clam enzyme exhibits a sharp optimum for activity at pH 5.0 in citrate buffer (Fu]ita et al.~), the crude enzyme of P a p h i a philippinarum at pH 5 (Fujita et al2), that of Corbicula sandai at pH 5.5 (Kaminishi~°), and the crude crucian enzyme at 6.0 in phosphate buffer (Fujita et al.ll). Sealock et al. ~2 reported the optimum pH of the carp enzyme to be 9.1; Fabriani et al. 1~ reported 4.4. Reddy et al. 14 showed two optima of 3.6 and 6.5 for muscle enzyme. Tenmatay ~5 observed two optima of 3.6 and 9.0 for the quahog clam (Venus mercuriana). Effect of Temperature. The crude enzyme of P a p h i a philippinarum shows an optimum temperature of 60 ° (Fujita et al2), that of crucian enzyme is 43 to 45 ° (Fujita et al.~l). Sealock et al. ~2 found the optimum temperature for the carp enzyme to be 60 °. 5 M. Fujiwara and H. Watanabe, Proc. Japan Acad. 28, 156 (1952). 6 T. Matsukawa and S. Yurugi, Proc. Japan Acad. 28, 146 (1952). 7 S. Sakamoto, Vitamins (Japan) 7, 360 (1954). 8 R. R. Sealock and A. H. Livermore, J. Biol. Chem. 177, 153 (1949). 9 A. Fujita and I. Numata, Seikagaku 18, 63 (1944). l0 K. Kaminishi, Seikagaku 22, 45 (1950). 11 A. Fujita, S. Kozuka, K. Yamazaki, K. Kaminishi, and E. Hasegawa, Seikagaku 22, 205 (1950). 12R. R. Sealock, A. H. Livermore, and C. H. Evans, J. Am. Chem. Soc. 65, 935 (1943). 13 G. Fabriani, A. Fratoni, and M. A. Spadoni, Quaderni nutriz. 10, 98 (1947). ~4 K. K. Reddy, K. V. Giri, and R. Das, Enzymologia 12, 238 (1945). ~ A. L. Tenmatay, Thesis, Fordham University 1950; cited by J. D. Barnhurst and D. J. Hennessy, J. Am. Chem. Soc. 74, 353 (1952).

626

COENZYME AND VITAMIN METABOLISM

[103]

Heat Inactivation. T h e crude e n z y m e of Paphia philippinarum is ina c t i v a t e d b y heating at 90 ° for 15 minutes (Fujita et al2), t h a t of Corbicula sandai at 100 ° for 10 minutes (Kaminishil0), and crucian e n z y m e at 90 ° for 10 minutes (Fujita et al.11). Necessity of Oxygen. Oxygen is not necessary for the action. Velocity Constants. Sealock et al. 12 tested the carp e n z y m e and ass u m e d the reaction to be monomolecular; the reaction constants k =

co

log c

were found to be practically constant within 120 minutes, n a m e l y 2.3 X 10 -8. According to the view of F u j i t a et al. 18the reaction does not proceed m o n o molecularly b u t bimolecularly b y the base exchange reaction.

II. Thiaminase from the Culture Media of Bacillus thiaminolyticus Matsukawa et Misawa Assay Method Principle. T h e principle is the same as for clam enzyme, described above.

Reagents E n z y m e . A 3-day aerobic culture of B. thiaminolyticus on ordinary b r o t h y f r o m which thiamine has been largely removed b y kieselguhr, is centrifuged, and the s u p e r n a t a n t fluid is used as the enz y m e source. Other reagents are the same as for clam enzyme.

Procedure. T h e procedure is also the s a m e as for clam enzyme, except t h a t the e n z y m e solution is incubated at 30 ° for 1 hour. Purification Procedure F o r purification the adsorption b y alumina C~ is found to be the m o s t promising. The adsorption b y tricalcium p h o s p h a t e and acetone precipitation are also found to be useful. F r a c t i o n a t i o n with a m m o n i u m sulfate ( o p t i m u m : 0.5 saturation) showed relatively lower specific a c t i v i t y and is A. Fujita, Y. Nose, K. Ueda, and E. Hasegawa, J. Biol. Chem. 196, 297 (1952). ~7The culture media used for the study on thiaminase is the ordinary broth culture. It is adjusted to pH 4.5 and 3 g. of kieselguhr is added per 100 ml. and shaken vigorously, whereby the thiamine is largely removed. After centrifugation the supernatant is adjusted to pH 6.5 and sterilized. The bacilli are cultivated in this media at 37 ° for 3 days. At this time the enzymic activity becomes most potent; thereafter it decreases gradually [H. Saiki, T. Kishida, T. Tashiro, and M. Yamadori, Kitasato Arch. Exptl. Med. 28, 121 (1951)].

[103]

THIAMINASE

627

yield. Fractionation with sodium sulfate (optimum: 0.7 saturation) or magnesium sulfate and precipitation with ethanol or methanol were found to be scarcely applicable because of very low specific activity and yield. Step 1. Preparation of Crude Enzyme. Step 1 is the same as described under Reagents, Enzyme, for clam enzyme. Step 2. Adsorption by Alumina C~. To 40 ml. of the ice-cold crude enzyme is added 40 ml. of ice-cold 0.07 M phosphate buffer, pH 6, the pH being adjusted to 6. Forty milliliters of ice-cold alumina C~ (6.0 mg./ml.) is mixed in the cold for 15 minutes and centrifuged. The precipitate is washed with water and centrifuged. The precipitate is eluted with 40 ml. of 0.2 M Na2HPO4 in the cold and centrifuged. Step 3. Second Adsorption by Alumina C~. The eluate from the alumina is adjusted to pH 6, and 40 ml. of ice-cold alumina is mixed and stirred in the cold for 15 minutes. After centrifugation the supernatant fluid is discarded, and the precipitate is washed with water and centrifuged. The precipitate is eluted with 40 ml. of 0.2 M Na~HPO4. After centrifugation the supernatant fluid is used for the experiment. See Table II for a summary of the purification procedure. TABLE II SUMMARY OF PURIFICATION PROCEDURE FOR BACTERIAL ENZYME

Fraction 1. Raw extract 2. First eluate from alumina 3. Second eluate from alumina

Total Specific volume, Total Protein, activity, Recovery, ml. Units/ml. units mg./ml, units/mg. % 40

106

4240 9.05

11.7

100

40

74.8

2990 0.23

325

71

40

53.5

2140 0.066

806

51

Properties Specificity. The specificity is the same as for shellfish thiaminase (Murata, TM SakamotoT). The substrate specificities of the two enzymes are given in Table III. Activators and Inhibitors. The crude enzyme is also activated by aromatic amines, heterocyclic amines, and sulfhydryl compounds as shellfish enzyme, but the degree of activation by aniline derivatives is relatively low. That by heterocyclic amines and sulfhydryl compounds, however, is very remarkable. It is also activated by many pyrimidine is K. Murata and A. Ueba, J. Biochern. (Japan) 88~ 309 (1951).

628

COENZYME AND VITAMIN METABOLISM

[103]

d e r i v a t i v e s a n d sulfa d r u g s such as h o m o s u l f a n i l a m i d e , c o n t r a r y t o fish a n d shellfish e n z y m e ( F u j i t a et al.1). Effect of pH. T h e c r u d e e n z y m e e x h i b i t s a s h a r p o p t i m u m for a c t i v i t y , f a l l i n g t o o n e - h a l f of t h e o p t i m u m a t p H 4.5 a n d 6.5 (Tashiro19). Effect of Temperature. T h e c r u d e e n z y m e s h o w s a n o p t i m u m t e m p e r a t u r e a t 30 t o 37 ° , t h e a c t i v i t y f a l l i n g r e m a r k a b l y a t 20 ° a n d 50 ° (Tashiro19). Heat Inactivation. T h e c r u d e e n z y m e is c o m p l e t e l y i n a c t i v a t e d b y h e a t i n g a t 100 ° for 20 m i n u t e s . Necessity of Oxygen. O x y g e n is n o t n e c e s s a r y for its a c t i o n . TABLE I I I ~UBSTRATE SPECIFICITY OF THIAMINASE a

CH3

I

N(~=C--R1

C~C--R~

I ~ ®1

R2--C(~ C - - C H 2 - - - - N IIv It N(~)--CH

®/

+/ ~

/ CH--S Decomposition by

R1

R2

R~

OH NH--CH3 NH2 NH2 NH2 NIt2 NH~

CHs CH3 CH2OH Et CHs CH3 CH~

CH2CH:OH CHRCH~OH CH~CH~OH CH~CH~OH CH2CH20--Ac C00Et CH~CH~C1

Shellfish BMM b -+ + + + +

+ + + + +

Taken from K. Murata and A. Ueba, J. Biochem. (Japan) 38, 309 (1951). b BMM = Bacillus thiaminolyticus Matsukawa and Misawa. Murata [Bull. Chem. Soc. Japan 23, 37 (1950)] examined the substrate specificity of the enzyme and found that the amino group in the 4-position of pyrimidine is necessary for the enzymic action. If the amino group is substituted by another group, without any further change in the pyrimidine moiety, the decomposition does not take place at all. But, if the amino group of the pyrimidine moiety is intact, the methyl group in the 2-position of the pyrimidine and the side chain in the 5-position of the thiazole moiety can be exchanged without loss of activity. For the detection of the reaction the thioehrome method or diazo method was used. ~9 T. Tashiro, Seikagaku 23, 239 (1952).

[104]

FOLIC AClD CO~JUGASE

629

[1041 Folic Acid Conjugase

By HARRY P. BROQUIST Assay Method Principle. After t r e a t m e n t with folic acid conjugase, the sample is assayed for folic acid content b y microbiological procedures employing Streptococcus faecalis or LactobaciUus casei as described elsewhere. 1-~ Treatment of Sample with Folic Acid Conjugase. Folic acid in natural materials occurs commonly in the form of polyglutamates which contain two or more additional glutamic acid molecules joined in ~,-peptide linkages to the glutamate radicle of the parent molecule. These polyglutamates are termed " c o n j u g a t e s . " The folic acid conjugases m a y be described as a group of enzymes which act on conjugates of folic acid to release substances having "folic acid a c t i v i t y " for S. faecalis or L. casei. These enzymes are distributed quite widely in nature and m a y be divided into two groups: (1) the type present in chicken pancreas, having a p H optimum of 7.0 to 8.0, and (2) the type widely distributed in animal tissues, especially liver and kidney, with an optimum p H around 4.5. Recent evidence indicates that the folic acid activity of natural materials is due in part to citrovorum factor (CF). When yeast extract, a potent source of folic acid heptaglutamate, is treated with folic acid conjugase, the apparent CF content markedly increases, 4,5 from which it has been inferred t h a t CF, like PGA, exists in conjugated form. However, recent work of Silverman and Keresztesy s indicates that !0-formylfolic acid appears as an early product of the action of liver enzymes on bound folic acid, after which 10-formylfolic acid m a y be converted b y other liver enzymes to CF. Although the precise mechanism is not known whereby folic acid conjugates are converted to P G A or CF, it seems advisable to follow the present practice of liberating folic acid activity from its conjugates with crude tissues high in folic acid conjugase. Treatment of Sample with Chicken Pancreas. Homogenize 1 g. of sample with 100 ml. of 0.05 M phosphate buffer, p H 7.2. Autoclave for 1E. E. Snell, in "Vitamin Methods, Microbiological Methods in Vitamin Research," p. 327, Academic Press, New York, 1950. "Methods of Vitamin Assay," p. 231, Interscience Publishers, New York, 1951. 3E. C. Barton-Wright, "The Microbiological Assay of the Vitamin B-Complex and Amino Acids," p. 73, Pitman Publishing Corp., New York, 1952. 4 C. H. Hill and M. L. Scott, J. Biol. Chem. 196, 189 (1952). 50. P. Wieland, B. L. Hutchings, and J. H. Williams, Arch. Biochem. and Biophys. 40, 205 (1952). e M. Silverman and J. C. Keresztesy, Federation Proc. 12~ 268 (1953).

630

COENZYME AND VITAMIN METABOLISM

[104]

15 minutes at 15 pounds, cool to 37 °, and incubate with 20 mg. of desiccated chicken pancreas 7 under toluene for 24 hours at 37 °. After incubation autoclave the sample for 5 minutes, and then clarify it b y centrifugation or filtration. I t is then ready for microbiological assay. A blank to correct for the folic acid content of the chicken pancreas preparation should be included b y carrying out the above procedure in the absence of sample. Desiccated chicken pancreas can be obtained from Difco Laboratories, Detroit, Michigan, or it can be obtained b y preparing an acetone-dried powder of fresh chicken pancreas.

Alternative Procedures The conjugate-splitting enzyme from hog kidney m a y also be used. The procedure is similar to the m e t h o d just described; desiccated hog kidney 3 or a water extract of fresh hog kidney 1,2 is used. The p H optim u m is 4.5, b u t if a prolonged period of incubation is employed, destruction of C F m a y occur, since C F is extremely labile to mild acid. TakadiaFOLIC ACID (FA) AND CITROVORUM FACTOR (CF) CONTENT OF NATURAL MATERIALS BEFORE AND AFTER TREATMENT WITH FOLIC ACID CONJUGASE PREPARATIONS

Microbiological activity

Sample studied

Enzyme source

Before After enzyme enzyme treatment treatment References FA con(dry tent, ~ ~/g. weight)

Aqueous extract of plasmolyzed yeast Fleisehmann Type 3 yeast extract Difco yeast extract Difco yeast extract Liver Fraction L (Wilson) Liver Fraction L (Wilson)

Hog kidney Chicken pancreas

2.5

50

3.0 53.9 CF content, ~ (dry -y/g. weight) Hog kidney 0.7 59.7 Chicken pancreas 0.7 39.0 Hog kidney 6.1 15.0 Chicken pancreas 6.1 8.1

b c e e e e

FA content determined by microbiological assay with LactobaciUus casei. b O. D. Bird, B. Bressler, R. A. Brown, C. J. Campbell, and A. D. Emmet t, J. Biol. Chem. 159, 631 (1945). P. R. Burkholder, I. McVeigh, and K. Wilson, Arch. Biochem. 7, 287 (1945). CF content determined by microbiological assay with Leuconostoc citrovorum. *V. M. Doctor and J. R. Couch, J. Biol. Chem. 200, 223 (1953). P. R. Burkholder, I. McVeigh, and K. Wilson, Arch. Biochem. 7, 287 (1945).

[105]

d-BIOTIN OXIDASE

631

stase has been suggested for the release of folic acid from its conjugates, 8 but it has been found by some to give irregular results owing to hydrolysis of the folic acid conjugate in the takadiastase by conjugases present in the samples to be assayed. Several experiments giving typical data of the increase in folic acid or citrovorum factor activity from natural materials after treatment with two sources of folic acid conjugase are illustrated in the accompanying table. s V. H. Cheldelin, M. A. Eppright, E. E. Snell, and B. M. Guirard, Univ. Texas Publ. No. 4237, 32 (1942).

[105] d - B i o t i n O x i d a s e B y J. H.

QUASTEL

An enzyme capable of oxidizing d-biotin (Baxter and Quastel ~) with liberation of C02 is present in guinea pig kidney and liver. Rat liver and kidney are only about one-tenth as active as the guinea pig tissues. Slices of guinea pig brain and pigeon liver show no activity. So far, oxidation of d-biotin has been studied only in tissue slices, usually slices of guinea pig kidney cortex. No success has yet attended efforts to prepare a kidney homogenate that retains biotin oxidase activity; it is possible, however, that a mitochondrial preparation will be active.

Assay Method The substrate for the investigation made so far I has been d-biotin carboxyl-C 14. This substance is prepared ~by refluxing sodium cyanide-C 1. with d-3,4-(2'-ketoimidazolido)-2-(co-bromobutyl)thiophane, followed by hydrolysis of the resulting nitrile without prior isolation. The specific activity ranges from 10,000 to 200,000 c.p.m, per milligram in different preparations. The procedure in studying biotin oxidase activity is as follows. In the main compartments of Warburg manometric vessels are placed tissue slices, radiobiotin solution, Ringer-phosphate solution, and any other additions to a total volume of 3.0 or 3.2 ml. The center wells contain 0.2 ml. of 20 % NaOH solution, and the side arms 0.2 ml. of 8 N H,SO,. 1 R. M. Baxter and J. H. Quastel, J. Biol. Chem. 201, 751 (1953). 2 S. B. Baker, D. E. Douglas, and A. E. Seath, Nuclear Sci. Abstr. 5, 802, abstr. 5158

(1951).

632

COENZYME AND VITAMIN METABOLISM

[105]

The gas phase is air. The vessels are attached to the manometers and incubated at 37 ° usually for 3 hours. At the end of the incubation period, the acid is tipped into the main compartment. This stops further enzymatic activity and serves to drive off any CO2 trapped in the solution. After 20 minutes or more, the units are taken from the bath, and the alkali is removed from the center well. The carbonate present is precipitated as barium carbonate by addition of barium chloride after addition of sufficient sodium carbonate to give a precipitate of about 50 mg. After standing for several hours, usually overnight, the precipitate is filtered off in a weighed sintered glass crucible and assayed for radioactivity.

Properties The destruction of biotin, measured by the release of radioactive carbon dioxide, is accompanied by loss of growth-promoting activity for yeast. 1 The destruction that occurs aerobically is reduced by 90% on replacing air with nitrogen in the gas phase, and 80 to 90 % by the presence of sodium azide at a concentration of 0.01 M. The Michaelis constant (Kin) of the d-biotin-biotin oxidase system is approximately 6 × 10-~ M. d-Biotin oxidation by guinea pig kidney cortex is inhibited by sodium malonate and stimulated by sodium fumarate. It is also inhibited by the presence of a variety of fatty acids, the inhibitory effect increasing with length of the carbon chain. Of the four-carbon acids, n-butyrate, isobutyrate, and crotonate, n-butyrate is the most potent inhibitor, the inhibition being noncompetitive. The results are consistent with the view that biotin oxidation is accomplished by breakdown of the fatty acid component, the carboxyl group being removed as part of a 2-carbon fragment similar to that derived from fatty acids, and that this is subsequently oxidized to carbon dioxide and water via the citric acid cycle. d-Biotin oxidation is inhibited competitively by norbiotin, whose affinity for the enzyme involved is about one-tenth of that of d-biotin. Bis-homobiotin is an inhibitor of the enzyme, its affinity being about five times as great as that of d-biotin. Other biotin analogs that are inhibitors of d-biotin oxidation are desthiobiotin and/-biotin.'

[106]

PANTETHEINE KINASE

633

[106] Pantetheine Kinase B y G. DAVID N0VELLI

Assay Method The enzymatic phosphorylation of pantetheine to form 4'-phosphopantetheine is measured by converting the product to CoA by a second incubation with a protamine-treated extract of acetone-dried pigeon liver. 1 Such an extract is no longer able to carry out the phosphorylation of pantetheine, but it is still effective in synthesizing CoA from phosphopantetheine. This extract also contains the enzymes for the acetylation of sulfanilamide and thus the conversion of phosphopantetheine to CoA, and the quantitative measure of the latter may be carried out simultaneously. Reagents

Assay enzyme. A crude extract of pigeon liver acetone powder (prepared as described in Vol. I [101]) is treated with an equal volume of acid-washed Dowex-1 to remove CoA. The supernatant is treated two times with 1/.~0vol. of 2% protamine sulfate. After removal of the protamine precipitate, the supernatant is free of pantetheine kinase, although it can still effect the conversion of phosphopantetheine to CoA as well as the CoA-dependent acetylation of sulfanilamide. This enzyme is called PRS, protaminetreated supernatant. Pantetheine (stock solution containing 0.1 mM./ml, which must be in the reduced form). ATP (0.05 M) pH 7.0. MgC12 (0.1 M). Phosphate buffer (1 M), pH 7.2. Reagents for CoA assay s (see Vol. I [101] and Vol. III [132]). Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which results in the synthesis of one unit of phosphopantetheine, expressed in CoA units, in 30 minutes under the conditions described below. Specific activity is expressed as units per milligram of protein. Protein is determined by the turbidimetric method of Biicher2 Procedure. To a series of tubes are added 0.05 ml. of stock pantetheine (0.005 ~M), 0.10 ml. of ATP solution (5 ~M), 0.05 of MgC12 (5 ~M),

L. Levintow and G. D. Novelli, J. Biol. Chem. 207, 761 (1954). 2N. O. Kaplan and F. Lipmann, J. Biol. Chem. 174, 37 (1948). 3T. Biicher, Biochim. et Biophys. Acta 1, 292 (1947).

634

COENZYME AND VITAMIN METABOLISM

[106]

0.1 ml. of phosphate buffer, und enzyme containing between 0.5 and 2.0 units in a final volume of 0.5 ml. The incubation is carried out at 37 ° for 30 minutes, after which the tubes are immersed in a boiling water bath for 3 minutes to inactivate the enzymes. The tubes are cooled in an ice bath, and to each tube is added 0.1 ml. of cysteine HC1 ( M / l ) , 0.5 ml. of CoA assay mix, 2 0.1 ml. of M/1 Tris buffer, pH 8.0, and 0.15 ml. of assay enzyme PRS to a final volume of 1.5 ml. The tubes are further incubated at 37 ° for 90 minutes. During this period any phosphopantetheine formed in the first incubation is converted to CoA. Since the CoA assay mix contains ATP, Ac, and sulfanilamide, the sulfanilamide will be acetylated in proportion to the amount of CoA present. The reaction is stopped by adding 3.5 ml. of 5% TCA. The tubes are centrifuged, and residual sulfanilamide is determined on a 1.0-ml. aliquot by the method of Bratton and Marshall. 4 This value is compared with control tubes containing 0, 1, and 2 units of CoA. From these values the amount of CoA formed in the test samples can be calculated. This reflects the amount of phosphopantetheine formed by pantetheine kinase in the first incubation. A control tube with the assay enzyme PRS and pantetheine should be included to show that PRS is free from pantetheine kinase. Purification Procedure

Step 1. Preparation of Crude Extract. Acetone-dried pigeon liver powder is extracted with 10 parts of ice-cold 0.05 M KHC03 by stirring for 30 minutes at 5 °. The insoluble residue is removed by centrifuging in a Spinco preparative centrifuge at about 50,000 X g. The pH of the clear, red supernatant solution is usually 7.2 and contains about 41 mg. of protein per milliliter. If the supernatant is to be used to make PRS it is treated with an equal volume of acid-washed Dowex-1; otherwise fractionation is carried out without Dowex treatment. This supernatant should not be aged, since pantetheinekinase is largely destroyed by aging at this stage. Step ~. Protamine Treatment. To the supernatant from step 1 is added, dropwise, with stirring, ~ 0 vol. of 2% protamine sulfate. The precipitate is removed by a Servall centrifuge in the cold room at 5 °. The precipitate is discarded, and the supernatant is treated with ~ 0 vol. of protamine sulfate. The precipitate contains pantetheine kinase and is collected by centrifugation. The supernatant is saved to make PRS. Step 3. Elution from Protamine Sulfate. The gummy precipitate from the second protamine treatment is suspended in 0.05 M phosphate buffer, pH 7.0. Fifty milliliters of buffer is used for each 200 ml. of crude 4A. C. Bratton and E. K. Marshall, Jr., J. Biol. Chem. 128, 537 (1939).

[106]

PANTETHEINE KINASE

635

extract used in step 2. A Potter-Elvehjem homogenizer, employed manually as a mortar and pestle, is useful for dispersing the precipitate. The suspension is stirred for several minutes, centrifuged in the Servall centrifuge at 1~,000 r.p.m., and the precipitate discarded. Step 4. Adsorption and Elution from Calcium Phosphate Gel. The supernatant from step 3 is treated with 75 ml. of calcium phosphate gel (dry weight 29 mg./ml.) which adsorbs almost all the activity. Elution is achieved by a single treatment with 25 ml. of cold 0.2 M K2HPO4. The eluate is dialyzed for 12 hours against 0.04 M KC1 at 5 ° without loss of activity. Attempts to purify the enzyme by further employing ammonium sulfate or cold ethanol fraction invariably result in large losses of activity. A summary of the purification is presented in the table. SUMMARY OF PURIFICATION PROCEDURE

Fraction Original extract Extract of second protamine treatment Eluate from calcium phosphate gel

Protein, Specific T i m e s Recovery, mg./ml, activity purified % 42.8 13.7 10.7

2.8 28.5 41

10 15

80 50

Properties Specificity. The purified enzyme seems to be specific for pantetheine. It has no action on pantothenate or on pantethine unless the S--S group is reduced to --SH. Stability. The enzyme is largely inactivated after standing for 4 hours at 25 °. It is completely inactivated by heating to 50 ° for 10 minutes. The partly purified preparation very slowly loses activity when stored in the frozen state at - 1 0 °, appreciable loss being noted after two months. Effect of Ions. The reaction between pantetheine and ATP does not proceed in the absence of divalent cations. Mn ++ is about one-fourth more effective than Mg ++. Ca ++ is about half as effective as Mn ++, and Co++, Cu ++, and Fe ++ are inactive. The reaction also exhibits a requirement for phosphate ions. If phosphate is excluded, the reaction proceeds at only one-fourth to one-half the maximal rate. Effect of pH. With Mn ++ as activating ion the peak of maximal activity is near pH 6.5, with Mg ++ the peak is nearer pH 7.2. Substrate A.~nity. Maximal rates are observed with pantetheine concentrations of 2 X 10-4 M, and the Michaelis-Menton constant with re-

636

CO:ENZYM:E AND VITAMIN METABOLISM

[107]

spect to pantetheine is about 10-5. An ATP concentration of 6 )< 10-~ M is required for maximal activity, and the reaction rate diminishes again if the ATP concentration exceeds 3 X 10-~ M.

[107] T h i a m i n o k i n a s e Thiamine ~ ATP --~ T P P ~ A M P

By H. G. K. W:ESTENBRINK The phosphorylation of thiamine by ATP under the influence of protein precipitated by ammonium sulfate from Lebedew juice was discovered by Weil-Malherbe. 1

Assay Method

Principle. The method to be described was developed by SteynParv~. ~ The enzyme is incubated with thiamine and ATP in phosphate buffer at pH 7.0 in the presence of Mg ions. The reaction is stopped by boiling at pH 3, and the T P P formed is determined by the manometric method (decarboxylation of pyruvate under the influence of alkalinewashed dried brewer's yeast and TPP). Reagents 0.02 M ATP solution (potassium-salt). 0.1 M MgSOt solution. 0.1 M phosphate buffer, pH 7.0. 0.1 M phosphate buffer, pH 6.2. O.O5 N HCh Brewer's yeast dried at room temperature. 2-Methyl-4-amino-5-ethoxymethylpyrimidine ("pyrimidyl"), mg./ml. 10% KOH. 2.5% sodium pyruvate in 0.1 M phosphate buffer, pH 6.2. 0.2 M Na~HPO~ solution, pH about 10. 0.1 M MnCl~ solution. Standard solutions of T P P (0.2, 0.1, and 0.05 ~//ml.).

15

Procedure. The reaction mixture has a volume of 5 ml. and contains: thiamine-HC1, 5 mg.; 0.02 M K-ATP, 0.5 ml.; 0.1 M MgSO4, 0.5 ml.; enzyme solution; 0.1 M phosphate buffer, pH 7.0, to volume. 1 H. Weil-Malherbe, Biochem. J. 33, 1997 (1939). E. P. Steyn-Parv~, Biochim. et Biophys. Acta 8, 310 (1952).

[107]

THIAMINOKINASE

637

Incubate at 27 ° for an appropriate length of time (usually 60 minutes). Transfer 1 ml. of the reaction mixture into 5 ml. of boiling 0.05 N HC1 at the beginning and the end of the period of incubation, and continue the boiling for 1 minute to stop the reaction. After cooling adjust to pH 6.2 with 10% KOH and make up to 10 ml. with 0.1 M phosphate buffer, pH 6.2. Spin down the protein precipitate, and take an aliquot for determination of TPP. Comments. 1. The method described can also be applied to crude tissue preparations, homogenates, and cell suspensions. 2. The high amount of thiamine (5 mg.) is required only when crude enzyme preparations from baker's yeast are used, as they contain much phosphatase, which would decompose the TPP formed if not inhibited by a relatively high concentration of thiamine2 3. In Weil-Malherbe's experiments 1 the thiaminokinase is not separated from apocarboxylase. He therefore measures the C02 production from added pyruvate, during the formation of TPP by the thiaminokinase. It is obvious that this procedure cannot give exact measurement of thiaminokinase activity. The same applies to the procedure of NguyenVan-Thoai and Chevillard, 4 who omit to stop the thiaminokinase action before determining TPP. Determination of T P P ~ (range 0.05 to 0.2 ~/ml.). Two-tenths milliliter of a 2.5% solution of sodium pyruvate in 0.1 M phosphate buffer, pH 6.2, is placed in the side arm of a conical Warburg vessel (capacity 10 to 15 ml.). One milliliter of the solution containing a suitable amount of TPP and 0.5 ml. of a suspension of alkaline-washed yeast (both pH 6.2) are measured into the main compartment. After 10 minutes' equilibration in a water bath of 27 °, the pyruvate is tipped in. Readings of the COs evolved are taken 10, 20, and 30 minutes later. To obtain a standard curve for calculating results, each run must include besides the test solutions a blank and three solutions of known TPP content, viz., 0.05, 0.1, and 0.2 ~,/ml. Preparation of A l k a l i n e - W a s h e d Yeast. 3 Brewer's yeast is dried in a thin layer at room temperature. One gram of the dried yeast is suspended in 20 ml. of distilled water. An equal volume of 0.2 M Na2HP04 solution, which has previously been brought to a pH of about 10 with concentrated NaOH, is added. The final pH should then be about 8.5 and can be adjusted if necessary. The temperature should be 16 to 20 °. After 5 minutes of stirring the yeast is separated by centrifugation for 1 minute at 3000 r.p.m. The supernatant is discarded, and the yeast is rapidly washed three times on the centrifuge with 40 ml. of distilled water. The H. G. K. Westenbrink and E. P. Steyn-Parv6, Intern. Z. Vitaminforsch. 21, 461 (1950). Nguyen-Van-Thoaiand L. CheviUard, Bull. soc. chim. biol. $1, 204 (1949).

638

COENZYME AND VITAMIN METABOLISM

[107]

whole procedure must be carried out as rapidly as possible to minimize denaturation of the apocarboxylase and should not require more than 15 minutes in all. Then 1 ml. of 0.1 M MnC12 and 0.25 ml. of a " p y r i m i d y l " solution, containing 15 mg./ml., are added to the washed yeast, which is suspended to a volume of 6 ml. with 0.1 M phosphate buffer, pH 6.2. This amount of yeast suspension suffices for ten Warburg flasks. Comments. 1. Leuthardt and Nielsen 5 use apocarboxylase prepared according to Weil-Malherbe, 1 as they had difficulty in adequately removing T P P from brewer's yeast by alkaline washing. The former preparation is much more laborious, especially when purification is pursued so far that thiaminokinase is also removed. Therefore the use of alkalinewashed dried brewer's yeast is to be preferred. According to extensive experience in the author's laboratory this washing never fails, provided that the exact pH at which the yeast should be treated is determined in preliminary experiments. If the pH is too low, not all TPP is removed; if it is too high, the apocarboxylase is damaged. For a certain yeast the suitable pH may be somewhat higher or lower than 8.5. It may be necessary to redetermine this pH after the dried yeast has been stored for some time, as it sometimes tends to shift as the yeast ages. 2. Usually 0.2 rag. of thiamine is added to each gram of alkalinewashed yeast instead of 3.75 mg. of " p y r i m i d y l " in order to inhibit the yeast phosphatase. In this case the adding of thiamine should be avoided as the alkaline-washed yeast is not absolutely free of thiaminokinase. Purification Procedure The best material to prepare the enzyme from is fresh yeast. Preparations have also been made from rat liver 5 and dog liver, 4 but they will not be described here as the most potent samples obtained are much less active than those derived from yeast. Step 1. Preparation of Crude Extract. A. FROM BREWER'S YEAST.4 Two hundred grams of freshly pressed brewer's yeast is triturated with 20 to 30 g. of NaC1. After plasmolysis, 1 1. of 0.2 M phosphate solution, pH 10.1, is added. After 10 minutes the mixture, which has a pH of 9.4 to 9.6, is centrifuged. The supernatant is discarded, and the yeast residue is suspended in 200 ml. of distilled water. The suspension is kept at 37 ° for 2 to 3 hours and then centrifuged again. The supernatant contains the enzyme and can be further purified by fractionation.e 5 F. L e u t h a r d t and H. Nielsen, Helv. Chim. Acta 35, 1196 (1952). Crude and dialyzed extracts of brewer's yeast form a b o u t 8 -y of T P P per milligram of protein N in 60 minutes. Crude extracts of baker's yeast form 5 to 7 -y of T P P under the same conditions. Dialyzed baker's yeast extracts form from 22 to 30 -y.

[107]

TIIIAMINOKINASE

639

B. FROM BAKER'S YEAST.2 Fresh, pressed baker's yeast, as delivered by the factory, is washed three times with 10 vol. of distilled water. Portions of 10 g. of the washed yeast, contained in metal centrifuge tubes, are immersed in a mixture of acetone and solid CO2 (temperature approximately - 7 0 °) for 15 minutes and then thawed by dipping the tubes in cold water. This process of alternate freezing and thawing is repeated twice. Solid KC1 is then added to the liquefied yeast until a molarity of 0.5 is obtained, and the mixture is shaken in a water bath of 37 ° for 3 hours. After standing overnight in a refrigerator, the yeast macerate is centrifuged, and a slightly opalescent light brown fluid, with a good thiaminokinase activity, is obtained. Forty grams of fresh yeast yields about 25 ml. of extract. Comments. 1. The thiaminokinase activity of both crude extracts described above does not differ much. The extract from brewer's yeast contains more carboxylase, i.e., more preformed TPP. Lebedew juice is much less active. 2. If large amounts of baker's yeast are frozen at a time, the freezing time must be lengthened considerably, and the extracts are not so active as when small portions are frozen. Step 2. Fractionation with A m m o n i u m Sulfate. 2 During fractional pre~ cipitation of the yeast extract with saturated ammonium sulfate solution the bulk of the thiaminokinase comes down when the amount of ammonium sulfate solution in the mixture is raised from 45 to 60 vol. %. The increase of the activity per milligram of nitrogen as compared with the original extract is about tenfold. After the precipitate is dissolved in distilled water the enzyme is reprecipitated by fractionation with saturated ammonium sulfate solution, but it separates when the amount of ammonium sulfate in the mixture is raised from 55 to 65 vol. %. This precipitate is completely free of phosphatase. However, the activity per milligram of nitrogen is not higher than that of the first precipitate. Comments. 1. In the case of baker's yeast extracts the increase of activity per milligram of protein N as a consequence of ammonium sulfate precipitation depends in part on removal of unknown inhibitors (inorganic and organic), for the activity is increased about fivefold simply by dialysis of the crude extract against 0.02 M KC1. 2. According to Nguyen-Van-Thoai and Chevillard 4 the enzyme precipitates at somewhat lower ammonium sulfate concentrations, but from their communication it is not clear whether they have allowed for the lower solubility of ammonium sulfate in yeast extracts as compared to water and for the volume constriction occurring when solid ammonium sulfate is dissolved in water or aqueous solutions.

640

COENZYME AND VITAMIN METABOLISM

[108]

3. Salting-out analysis according to Derrien shows 2 that the ammonium sulfate precipitates of yeast extracts contain several protein components, incompletely separated in the salting-out diagrams, so that it will not be possible to purify the enzyme further by ammonium sulfate fractionation without incurring enormous losses.

Properties Activators and Inhibitors. 2 The baker's yeast enzyme is activated by Mg, Mn, and orthophosphate. Maximal activation with 5~g is attained at 2.10 -2 M. At a lower concentration level (2.10 -3 M) Mn is slightly superior to Mg as activator, but with increasing concentration the stimulation by Mn decreases to zero, whereas the influence of Mg increases steadily. For orthophosphate the optimal concentration is about 2.10 -3 M; strong inhibition is observed in M phosphate. The crude extracts from baker's yeast contain inhibitors, both inorganic and organic, which can be removed by dialysis. Effect of pH. The enzyme has a broad range of optimal action between pH 6 and 7.5. 3 Stability. The enzyme is much more stable in the crude or dialyzed extracts than after ammonium sulfate fractionation. 2

[108] F l a v o k i n a s e Riboflavin + ATP --* F M N + ADP

By EDNA B. KEARNEY

Assay Method Principle. The F M N formed during the enzymatic reaction is measured in a protein-free filtrate by an adaptation of the method of Burch et al. 1 The method 2 is based on the different distributions of F M N and riboflavin between benzyl alcohol and water. After extraction of the protein-free filtrate with benzyl alcohol, the flavin content of the water phase may be estimated spectrophotometrically at 450 m~ or fluorometrically, and the F M N content is calculated from a formula derived from the distribution coefficients of the flavins between benzyl alcohol and the aqueous solution (see Vol. I I I [141]). Reagents Riboflavin stock solution. 37.6 mg. of riboflavin is dissolved in 500 ml. of H20 (2 × 10-4 M); any undissolved material is ill1H. B. Burch, O. A. Bessey, and O. I-I. Lowry, J. Biol. Chem. 175, 457 (1948). 2 E. B. Kearney and S. Englard, J. Biol. Chem. 198, 821 (1951).

[108]

FLAVOKINASE

641

tered off, and the actual concentration is determined by light absorption at 450 m~, using the molar extinction coefficient, = 12.2 X 106 sq. cm. mole-~. The solution should be stored in the cold in a dark bottle, and the concentration should be redetermined occasionally if it is used over a period of months. Acid solutions of riboflavin are more stable but the solubility is no greater. More concentrated solutions may be made in alkali, but these must be used immediately, since the flavin deteriorates rapidly in alkaline solutions. 0.75 M Tris-HC1 buffer, pH 8.2 at 30 °. ATP, 3 MgSO4 at any convenient concentration. 17.5 % trichloroacetic acid. 2.4 M K2HP04. Benzyl alcohol. Merck reagent grade; need not be redistilled. The reagent is saturated with H20 at room temperature. Chloroform. Reagent is saturated with H20 at room temperature. Enzymatic Reaction. The enzyme is assayed in low-actinic test tubes in a 5.0-ml. reaction mixture containing 7.5 × 10-2 M Tris buffer, 1 X 10-~ M riboflavin, 1.0 × 10-3 M ATP, 1.0 X 10-3 M MgSO4, and 50 to 150 units of enzyme. (See definition below.) The mixture is incubated for 2 hours at 30 °, and the reaction is stopped by the addition of 2.0 ml. of trichloroacetic acid. Analysis. The solutions should be protected from light as much as possible throughout the following procedure. The reaction mixture obtained above is boiled for 10 minutes to hydrolyze any FAD which may have been formed, and the solution is cooled and filtered. A 5.0-ml. aliquot of the filtrate is neutralized with 1.25 ml. of 2.4 M K2HPOt, and the light absorption is measured at 450 m~. Five milliliters of the neutralized filtrate is then bubbled for 30 seconds in 20 × 150-mm. test tubes with 12.5 ml. of benzyl alcohol. After brief centrifugation, the benzyl alcohol layer is quantitatively removed, and the residual benzyl alcohol is removed from the aqueous phase by bubbling with 5.0 ml. of chloroform. The solution is again centrifuged, and the aqueous layer is pippetted into a cuvette for measurement of light absorption at 450 m~. Calculation. The flavin content of the initial 5.0-ml. reaction mixture is calculated from the light absorption of the unextracted sample as millimicromoles of total flavin per 5.0 ml. ; this is value A in the equation below. Value B is identically calculated from the light absorption after extraction. 8For critical experiments ATP should be freed from ADP and AMP. Methods for the purification of ATP are given in Vol. III [1] 7].

642

COENZYME AND VITAMIN METABOLISM

[108]

Millimicromoles of F M N per 5.0 ml. of reaction mixture = 1.25B - 0.125A 4 This value may be corrected for the slight loss of flavin in the deproteinization step by multiplying by the factor, millimicromoles riboflavin added/value A. This correction factor is very low except with crude enzymes, where the protein precipitate is bulky. Definition of Unit and Specific Activity. One unit is that amount of enzyme which catalyzes the synthesis of 1 millimicromole of FMN in 2 hours at 30 ° per 5.0 ml. of reaction mixture under the conditions of assay given above; specific activity is defined as units per milligram of protein. Protein concentration is determined by light absorption at 280 m~ at pH 7.0, and this value is related to the dry weight at various stages of purification. Applicability of Assay Method to Crude Preparations. The assay conditions described above are recommended only for the most highly purified enzyme preparations (steps 4 to 5 below). For less purified stages (steps 2 to 3), the final concentration of MgSO4 should be 3 × 10-4 M, since 1 X 10-3 M MgSO4 causes a slight inhibition. The presence of phosphatases and other hydrolytic enzymes in crude preparations (e.g., yeast autolyzate) complicates the assay; the effect of interfering enzymes is largely eliminated by the inclusion of 1 M K F in the reaction mixture.

Purification Procedure Preparations of flavokinase from brewer's yeast s and intestinal mucosa 5 have been described. The detailed procedure for the isolation of the enzyme from intestinal mucosa has not yet been reported, and the preparation has not yet been satisfactorily freed of phosphatase activity.5 The starting material is a thoroughly washed, low-temperature-dried beer yeast, Lot D-422, obtained from Anheuser-Busch, Inc. 4 The distribution coefficients of riboflavin and F M N between benzyl alcohol and the neutralized trichloroacetic acid filtrate are 3.6 and 0.044, respectively, corresponding to 10% and 90% remaining in the aqueous phase after extraction with benzyl alcohol. ~ Then, A = Riboflavin -{- F M N or B = 0.1 riboflavin + 0.9 F M N

Riboflavin -~ A -- F M N

Substituting for riboflavin in equation 2, B = 0.1A - 0.1 F M N + 0.9 F M N and FMN

B - 0.1A 0.8

S. Englard, Federation Proc. 11, 208 (1952).

1.25B -- 0.125A

(1) (2)

[10~]

FLAVOKINASE

643

Step 1. Autolysis. In essence, the conditions of autolysis determine the effectiveness of the entire purification procedure and should be adhered to as rigidly as possible. These conditions are so chosen as to give the most satisfactory compromise between yield and purity, with relatively little contamination by interfering enzymes. Five hundred grams of yeast is allowed to autolyze in 1 1. of water at 35 to 36 ° with effccient stirring. At the end of 2 hours at this temperature, 500 ml. of water is added, and the mixture is cooled rapidly to 30 °. Stirring is continued for 15 minutes, at which time the yeast is cooled to about 10° and centrifuged for 30 to 45 minutes at 4500 to 5000 r.p.m. Step 2. Ammonium Sulfate Precipitation. The supernatant fluid obtained above is cooled to 0 °, and the pH adjusted, if necessary, to pH 5.9 to 6.0. Solid ammonium sulfate is added to give 0.40 saturation (calculated as 71.5 g. per 100 ml. = 1.0 saturation), and the mixture is stirred for 45 minutes. The precipitate is collected by centrifugation for 45 to 60 minutes at 5000 r.p.m., redissolved in about 30 to 40 ml. of water, and dialyzed in the cold against running distilled water for 16 to 18 hours. The heavy precipitate formed during dialysis is centrifuged down at 10,000 r.p.m, for 30 minutes and discarded. Step 3. Precipitation at pH 5.0 and Adsorption of Impurities with Alumina. The dialyzed enzyme is diluted with water to give 8 to 10 rag. of protein per milliliter (E2s0 = 13.2 for 1% protein) and brought to pH 5.0 at 0 ° by the addition of 0.2 to 0.3 ml. of 1 N acetic acid. The precipitate formed is not centrifuged off. Aluminum hydroxide gel C~ ~ is added in the ratio of 60 to 80 mg. of gel per gram of protein. After 15 minutes of stirring, the mixture is centrifuged for 30 minutes at 10,000 r.p.m., and the gel is discarded. These two steps should remove about 45 to 50% of the protein present in the dialyzed solution. Step ~. Fractionation with Ammonium Sulfate at pH 6.0 and 7.2. The enzyme solution obtained in the previous step is adjusted to pH 6.0 at 0 ° with 0.4 to 0.5 ml. of 0.5 M K2HP04 and brought to 0.42 saturation with solid ammonium sulfate. The suspension is stirred for 30 minutes at 0 ° and then centrifuged for 15 minutes at 10,000 r.p.m. The precipitate is dissolved in 0.015 M phosphate buffer, pH 7.2, in half the initial volume (end of step 3), and the ammonium sulfate concentration is calculated, taking into consideration the volume of the precipitate, which is 0.42 saturated with respect to ammonium sulfate. The solution is centrifuged if not clear, and the ammonium sulfate concentration of the supernatant solution is raised again to 0.42 saturation with solid ammonium sulfate. The suspension is stirred for 15 minutes and then centrifuged for e For the preparation of alumina C~, see Vol. I [11].

644

COENZYME AND VITAMIN METABOLISM

[108]

10 minutes at 10,000 r.p.m. T h e precipitate is dissolved in a small volume of water and dialyzed for 2 to 3 hours against running distilled water at 0 °. The dialyzed enzyme is centrifuged free of any precipitate which forms and is lyophilized to dryness. Step 5. Differential Denaturation. Much of the protein present in the lyophilized powder will not go into solution at 0 ° in water or various buffers, and a continuous denaturation and precipitation of protein is apparent thereafter. The following procedure, however, results in a solution which remains water-clear for several hours at 0 ° and also effects considerable purification. The lyophilized powder is gently resuspended in water and 0.2 M succinate buffer, p H 6.0, is added to give a final molarity of 0.05 M and 5 to 6 mg. of enzyme per milliliter. The suspension is kept at 0 ° for 45 minutes, and the insoluble proteins are removed by 10 minutes of centrifugation at 18,000 r.p.m, at 0 °. Solutions of flavokinase at this stage of p u r i t y deteriorate rapidly and, therefore, the enzyme should not be dissolved until just before the assay. The most highly purified fraction still gives a linear reaction rate in the assay described for 1 hour but not for 2 hours. SUMMARY OF PURIFICATION PROCEDURE

Fraction 1. Centrifuged autolyzate 2. (NH4)~SO4ppt., 0.40, pH 6 3. pH 5.0 precipitation and A1 C~ adsorption 4. 0.42 (NH4)~SO~precipitation at pH 6.0 and 7.2 5. Differential denaturation

Total Total Specific volume, units, Protein, activity, ral. Units/ml. thousands mg./ml, units/mg. 770 82

52 450

40 37

90 15.4

0.57 30

128

260

37

4.9

55

15 296

1378 642

20 19

12.4 2.2

112 290

Properties Specificity. The purified enzyme acts on riboflavin, dichloroflavin [6,7dichloro-9-(D-l'-ribityl)isoalloxazine], and arabitylflavin [6,7-dimethyl-9(D-l'-arabityl)isoalloxazine], but not on isoriboflavin [5,6-dimethyl-9(D-l'-ribityl)isoalloxazine], 6-carbon alcohol derivatives of isoalloxazine such as galactoflavin, sorbitylflavin, and dulcitylflavin, alloxazine, or ring compounds of other than the isoalloxazine configuration. 7 None of these inhibits the phosphorylation of riboflavin. 7 E. B. Kearney, J. Biol. Chem. 194, 747 (1952).

[108]

FLAVOKINASE

645

Besides ATP, A D P can also act as a phosphate donor, but the maximal velocity in the presence of the latter is only half t h a t given by ATP. Although purified fractions still contain myokinase, 8 there is as yet no valid reason to believe t h a t A D P acts by virtue of prior conversion of A T P b y myokinase, since Km values for A T P and A D P are identical and since the V .... with A D P is much lower than with ATP. Kinetics. The enzyme follows a zero-order reaction under the assay conditions given above. At 30 °, the optimal p H range is 7.8 to 8.5. At p H 7.8, the apparent temperature optimum is 38 °. The enzyme is saturated b y 1 X 10-4 M riboflavin and half-saturated b y 1.0 X 10 -5 M. The Km for A T P as determined from the Lineweaver-Burk type of plot is 1.7 X 10-5 M, and t h a t for A D P is 1.6 × 10-5 M. Activators and Inhibitors. Mg ++, Zn ++, Co ++, and Mn ++ all activate the enzyme, but only Mg ++ exhibits fairly constant behavior. Relatively impure preparations are activated optimally b y 3 X 10-4 M 1Vig++, and the Km is 7.5 X 10-5 M. With the most highly purified preparations, the observed Km for Mg++ is 2.1 X 10-4 M; no inhibition is evident at high concentrations; and 9 X 10 -4 M Mg ++ is necessary to saturate the enzyme. The maximal activity in the presence of Mn ++ is low, although higher concentrations of this cation are noninhibitory. The extent of activation b y Co ++ is variable, although often much greater than b y Mg++; at high concentrations Co ++ is inhibitory. Activation b y Zn ++ is manifest only at certain stages of purification (end of steps 4 or 5 but not step 2); however, at these stages the activity in the presence of Zn ++ is far greater than in the presence of Mg ++. (Specific activity with 9 X 10 -4 M Zn++ = 352, with 1 X 10-3 M Mg ++ = 241.) The Km for Zn ++ is 9.0 X 10-5 M. Ca ++ does not produce activation but is inhibitory in the presence of other metals. 5-AMP is a competitive inhibitor of flavokinase (KI = 2.5 X 10 -5 M). Lumiflavin, when present in excess over riboflavin, inhibits phosphorylation of the latter. s S. Englard, J. Am. Chem. Soc. 75, 6048 (1953).

646

COENZYME AND VITAMIN METABOLISM

[109]

[109] Pyridoxal Kinase from Brewer's Yeast Pyridoxal + ATP --* Pyridoxal-5-phosphate + ADP

By I. C. GUNSALUS and W. E. RAZZELL

Assay Method Principle. Hurwitz 1 measures pyridoxal phosphate, formed by kinase action, by the activation of a crude tyrosine apodecarboxylase preparation from Streptococcus faecalis. ~ The assay is proportional to pyridoxal phosphate over the range of 1 to 10 muM. (1 to 10 units). After phosphorylation of pyridoxal by the specific kinase, ATP is removed by treatment with a potato apyrase 3 to prevent further phosphorylation by the kinase contained as an impurity in the decarboxylase preparation. Reagents 0.3 M phosphate buffer, pH 6.85. 0.05 M Na~ATP. 0.05 M pyridoxal hydrochloride, neutralized to pH 6.85. 0.01 M MgSO4. 0.1 M succinate buffer, pH 6.5. 0.045 M calcium chloride. 1 M sodium acetate, pH 5.5. 0.03 M tyrosine suspension, pH 5.5.

Enzymes Tyrosine apodecarboxylase (1500 #l. of COs per hour per milliliter; i.e., ~ ca. 10 mg. of dried cells). See text. Potato apyrase, 400 units/ml. Prepared according to Krishnan. ~

Procedure. Pyridoxal kinase activity is measured by adding 10 to 100 units in 1.2 ml. or less to a 13 × 100-mm. Pyrex test tube containing 0.2 ml. each of the phosphate buffer, sodium ATP, pyridoxal, and magnesium sulfate solutions, and enzyme plus water to 2 ml. Incubate for 2 hours at 30 °, and immerse in a boiling water bath for 3 minutes to stop the reaction; then dilute to 5 ml. and filter. ATP is removed with apyrase by adding 0.8 ml. or less of the diluted reaction mixture, containing about 1.6 ~M. of ATP, to a 13 × 100-mm. test tube containing the following: 0.5 ml. of succinate buffer, 0.2 ml. of calcium chloride, 0.5 ml. (200 units) of apyrase, and enzyme plus water 1 j . Hurwitz, J. Biol. Chem. 205, 935 (1953). 2 W. D. Bellamy a n d I. C. Gunsalus, J. Bacteriol. 50, 95 (1945). s p. S. Krishnan, Vol. I I [96].

[109]

PYRIDOXAL KINASE FROM BREWER'S YEAST

647

to 2 ml. Incubate for 30 minutes at 30 °, and stop the reaction by chilling it in an ice bath. Then dilute to 5 ml. with water. Pyridoxal Phosphate Assay. Transfer an aliquot containing 1 to 10 units of pyridoxal phosphate in not more than 1.8 ml. to the main compartment of a Warburg flask containing 0.2 ml. of acetate buffer (see reagents) plus 0.5 ml. (750 units) of tyrosine apodecarboxylase enzyme and water to 2.5 ml. Equilibrate for 10 minutes at 30 °, tip from the side arm 0.5 ml. of the tyrosine suspension, and measure the CO~ released during the first 10 minutes. Definition of Units. The pyridoxal kinase unit is 1 m~M. of pyridoxal phosphate formed in 2 hours at 30 ° in the protocol indicated. (Hurwitz's unit was defined at 33.5°; the unit is redefined at 30 ° to coincide with all other measurements in his publication.) The apyrase unit is the quantity of enzyme required to liberate 1 ~, of inorganic phosphate in 30 minutes at 30 ° in the presence of 300 ~ or more of ATP phosphorous. The tyrosine decarboxylase unit is the quantity of enzyme liberating 1 ~l. of C02 per 60 minutes at 30 ° in the protocol specified above. 4 The specific activity, in all cases, is the units per milligram of protein.

Preparation of Assay Enzymes Potato apyrase is prepared as described by Krishnan.3 Tyrosine apodecarboxylase can be prepared by growing Streptococcus faecalis, strain R, for 15 (___3) hours at 37 ° in the medium of Bellamy and Gunsalus. ~ The cell crop is harvested by centrifugation, washed once in a small volume of distilled water, resuspended in distilled water, and pipetted with stirring into 10 vol. of - 2 0 ° acetone. After 15 minutes the cells are collected on a Bfichner funnel, washed once with acetone, and once with cold ( - 2 0 °) ether. Tyrosine apodecarboxylase is prepared by suspending 20 mg./ml, of acetone powder in M/50 phosphate buffer, pH 5.5, followed by incubation for 20 hours at 37°. 5 The debris is removed by centrifugation, and the extract containing the tyrosine apodecarboxylase is lyophilized and stored in a desiccator at 0 °. The enzyme is stable indefinitely under these circumstances. Alternatively, the tyrosine apodecarboxylase can be extracted by the methods outlined in Vol. I [7]. For most purposes, acetone- or vacuum-dried cells are suitable for pyridoxal phosphate assay. 6,7 A water suspension of cells is lyophilized, 4 Tyrosine decarboxylase assay can be r u n at 37 °, with greater activity per u n i t of cells or extract. 5 5 H. M. R. Epps, Biochem. J . 38, 242 (1944). 6 W. W. U m b r e i t a n d I. C. Gunsalus, J. Biol. Chem. 179~ 279 (1949). 7 I. C. Gunsalus a n d W. W. Umbreit, J. Biol. Chem. 170, 415 (1947).

648

COENZYME AND VITAMIN METABOLISM

[109]

preferably in a petri dish containing not over 10 ml. of cell suspension, in a 250-mm. desiccator containing 2 to 3 pounds of Drierite by evacuation with a good vacuum pump. The Qco, (N) of such cells is approximately 2000, i.e., 200 units per milligram in the presence of excess pyridoxal phosphate, and approximately 5 units in the absence of this coenzyme. The cells contain an excess of pyridoxal kinase and are thus not suitable for pyridoxal phosphate assay unless devoid of either ATP or free pyridoxal.

Purification Procedure Hurwitz ~ used low-temperature-dried brewer's yeast (Anheuser Busch strain BSC) as a source of the kinase. Suspend 500 g. of dried yeast in 1 1. of water and stir for 2 hours at 35 °. Add 300 ml. of water, and cool to 30 ° with an additional 15-minute stirring. Then cool to 10° in an ice bath, and remove the cell debris by centrifugation. (This is essentially the autolysis procedure of Kearney and Englard. 8) All enzyme fractionations are carried out at 0 °. Precipitate the kinase by addition of 28 g. (0.4 saturation) of ammonium sulfate per 100 ml. of extract with stirring at 0 ° for 40 minutes. Remove the precipitated enzyme by centrifugation. Discard the supernatant, suspend the precipitated enzyme in 60 ml. of water, and dialyze against distilled water for 15 hours. Remove the precipitate by centrifugation, and discard. Adjust the clear supernatant to pH 5 with normal acetic acid, and stir for 30 minutes. Remove the precipitate by centrifugation, and add 1/~0 vol. of 2 M acetate buffer, pH 5, 0 ° (final concentration 0.067 M). Add ethanol cooled to - 10° slowly over a period of 15 minutes to a final concentration of 12% by volume. Recover the precipitate by centrifugation, and dry over Drierite at 0 ° in a vacuum desiccator. Owing to the removal of phosphatases, the recovery of kinase as compared to the autolyzed extract is 230% with a purification of 440-fold. The enzyme is stable at this stage and can be used for most experiments. The enzyme can be further purified by another ammonium sulfate fractionation and a heat step with a recovery of 40 to 50% (yield equal to activity measurable in autolyzate) at 1000-fold purification.

Properties The purified enzyme obtained by alcohol fractionation is stable at 30 ° in the presence of pyridoxal but is rapidly inactivated in the absence of the substrate. The kinase is specific for the substituted pyridine molecule; i.e., pyridoxine and pyridoxamine are phosphorylated at a rate approximately equal to that for pyridoxal. Other kinases, for glucose and ribos E. B. Kearney a n d S. Englard, J. Biol. Chem. 193, 821 (1951).

[110]

DEPHOSPHO-COA KINASE FROM PIGEON LIVER

649

flavine, for example, are also present in the alcohol-precipitated protein fraction, but their ratio compared to the pyridoxine phosphorylating enzyme varies with stage of purification, and they are thus considered to be impurities. The enzyme is also specific for ATP; I T P and inorganic phosphate neither phosphorylate nor inhibit. Adenine, adenosine, AMP, and ADP are inhibitors of the kinase. Fluoride, 0.1 M, is also inhibitory to the kinase. There is a rathei sharp pH optimum at 6.9, half-maximal activity being exhibited at pH 6.5 and 7.5.

[110] D e p h o s p h o - C o A K i n a s e f r o m P i g e o n L i v e r Mg+ ATP ~ D P C o A - ) CoA ~- ADP Enzyme By T. P. WANG Assay Method

Principle. Dephosphorylated coenzyme A (DPCoA), in contrast to CoA, is inactive in the routine phosphotransacetylase assay.l.2 The activity of DPCoA kinase is thus followed by the arsenolysis of acetyl phosphate by a phosphotransacetylase method in which the CoA formed from the phosphorylation of DPCoA is assayed. Reagents DPCoA. Prepared by dephosphorylation of CoA (see Vol. III [136]), 20 units. ATP (0.04 M). DPCoA kinase, 0.4 saturation, (NH4) 2SO~ fraction. Cysteine hydrochloride (0.1 M). MgCl2 (0.05 M). Tris buffer (0.5 M), pH 8.2. Phosphotransacetylase and reagents for arsenolysis of acetyl phosphate. Procedure. Twenty units of DPCoA, 2 micromoles of ATP, 5 micromoles of MgC12, 3 micromoles of cysteine HC1, 40 micromoles of Tris, and 0.05 rag. of enzyme are incubated at 37 ° for 30 minutes. The reaction mixture is then treated in a boiling water bath to stop the reaction and E. R. Stadtman, J. Biol. Chem. 196, 527 (1952); see Vol. I [98]. 2 If the level of phosphotransacetylase is increased 100 fold there is some activity with the DPCoA.

650

[I10]

CO:ENZYME AND VITAMIN METABOLISM

to denature any acetyl phosphatase that might be present in the enzyme preparation. It is necessary to observe this step, since the hydroxamic acid method for acetyl phosphate could not tell whether the disappearance of acetyl phosphate is obtained through the action of phosphotransacetylase or is the result of hydrolysis by acetyl phosphatase. The heated mixture is then assayed for its CoA content by the phosphotransacetylase method of Stadtman. 1 Definition of Units and Specific Activity. A unit of enzyme activity is defined as the amount of enzyme catalyzing the formation of 1 unit of CoA per 30 minutes under the above conditions. Specific activity is then the amount of CoA (in units) formed per milligram of protein per 30 minutes. The above procedure can be used for crude tissue preparations as well. Purification Procedure s

Steps 1 and 2. The source of the DPCoA kinase is the same pigeon liver acetone powder used in the preparation of D P N kinase. ~ Steps 1 and 2, extraction with K H C 0 3 and precipitation with protamine, are also the same as described for D P N kinase. The only difference is that the D P N kinase stays with the protamine precipitate whereas the DPCoA kinase activity remains in the supernatant (see Vol. II [111]). SUMMARY OF PURIFICATION PROCEDURE

Fraction KHCO3 extract Protamine supernatant Acetone fraction (NH4) 2S04 fraction, 0.4 saturation (NH4)2SO4 fraction, 0.6 "

Volume, ml.

Protein, mg./ml,

51 138 60 4 9

30.9 6.3 5.8 9.9 21.7

Specific Yield, activity units 34.1 34.3 69.7 153 74.3

53,900 29,600 24,200 6,050 14,500

Step 3. Acetone Precipitation. To the protamine supernatant, cooled to almost 0 ° in a salt-ice bath, is added slowly with stirring a stream of 150 ml. of cold acetone ( - 1 5 ° ) . The temperature of the solution is kept below - 3 ° after the addition of the first few milliliters of acetone. The precipitate formed is centrifuged at 18,000 r.p.m, for 10 minutes and dissolved in 50 ml. of Tris buffer, 1 M, pH 8.2. Part of the precipitate does not go into solution. The acetone fraction is then dialyzed against 6 of cold solution of 0.02 M KHCOa and 0.2 To KC1 for 6 hours. Some precipitate formed during the dialysis plus the undissolved material originally present is removed by centrifugation. a T. P. Wang and N . O . Kaplan, J . Biol. Chem. 206, 311 (1954) ; see also Vol. I I [111].

[110]

DEPHOSPHO-COA KINASE FROM PIGEON LIVER

651

Step ~. (NH4)2SO~ Fractionation. The acetone fraction is then fractionated with solid (NH4)2S04. Two protein fractions, precipitating at between 20 and 40% and 40 and 60% saturation with respect to (NH~)~SO~, are collected. The precipitates are dissolved in a small volume of 0.2 % KC1. The total yield is about 38% (both (NH4)~SO~ fractions combined), and purification is 4.5-fold (40% (NH4)2SO~ fraction). The procedures used in the purification of DPCoA kinase are given in the table.

Properties Specificity. Because of the lack of assay method, compounds structurally related to DPCoA such as deamino-DPCoA have not been tested with the DPCoA kinase. But among the phosphorylating agents tried, only ATP is active. Neither I T P nor ADP can be used to replace ATP. Metal Requirement. In addition to DPCoA, ATP, and enzyme, Mg ion is required for the phosphorylation of DPCoA to CoA. However, addition of fluoride ion (0.02 M) with or without inorganic phosphate (0.02 M) does not inhibit the reaction. Inhibitors. In contrast to D P N kinase, the DPCoA kinase is not inhibited to any significant extent by adenosine, 2'-, 3'-, and 5'-adenylic acids, ADP, ADPR, D P N H , DPN, and deamino-DPN. The enzyme is stable at - 15 ° for at least two months without losing activity. Repeated freezing and thawing also do not significantly alter the activity of the enzyme. The activity of DPCoA kinase can also be followed by means of the firefly bioluminescence system, since CoA but not DPCoA is capable of giving a secondary stimulation on the light production. ~ When DPCoA, cysteine, and the kinase are added to the firefly bioluminescence system consisting of luciferase, luciferin, ATP, Mg ion, and oxygen, the secondary light production is proportional to the activity of the kinase within certain limits. From experiments using this system, the Km for DPCoA has been estimated to be 3 X 10-~ M. The partially purified preparation DPCoA kinase requires cysteine for maximal activity. 4 W. D McElroy and J. Coulombre, in preparation.

652

COENZYME AND VITAMIN METABOLISM

[111]

[iii] D P N Kinase from Pigeon Liver ATP q- DPN

Mg ++

Enzyme

) TPN -f" ADP

By T. P. WANG Assay Method

Principle. The TPN-specific isocitric dehydrogenase from pig heart 1 which does not react with DPN is used for the assay of TPN. The amount of TPN formed under a specified condition serves, in turn, as a measurement of the DPN kinase activity. Reagents A T e (0.04 i ) . DPN (90 % purity). Dissolve 25 mg. in 1 ml. of water. DPN kinase. MgC12 (0.05 M). Tris buffer (0.05 M), pH 7.5. Isocitric dehydrogenase (0.5 to 0.6 saturation, (NH4)2SO~ fraction of pig heart extract). Isocitrate (0.05 M). Prepared by hydrolyzing isocitric lactone with acid and then neutralized with NaOH.

Procedure. One-tenth milliliter each of DPN, ATP, DPN kinase, MgCl~, and Tris buffer are incubated together at 37 ° for 60 minutes. One-half milliliter of water is then added to the mixture, and the diluted mixture is heated in boiling water for 2 minutes. After the coagulated protein has been removed by centrifugation, an aliquot of the clear supernatant is pipetted into a Beckman cuvette which contains enough Tris buffer, pH 7.5, to make the final concentration of the buffer around 0.05 M and the final volume 2.9 ml. Then 0.05 ml. of isocitrate is added with 0.1 ml. of the isocitric dehydrogenase, and readings are taken at 30-second intervals thereafter. In general, the reaction should be completed in about 2 minutes. The increase in reading at 340 mu indicates the formation of T P N H from TPN which is formed from the phosphorylation of DPN by ATP in the presence of DPN kinase. Definition of Unit and Specific Activity. Enzyme activity under the above conditions is expressed in micromoles of TPN formed per hour. The specific activity, defined as the unit of enzyme activity per hour per milligram of protein, 2 is used as the index of purity. 1A. Graffiinand S. Ochoa, Biochim. et Biophys. Acta 4, 205 (1950); see Vol. I [116]. 20. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

[111]

DPN KINASE FROM PIGEON LIVER

653

The same procedure can be used for crude tissue preparation, provided that a suitable amount of nicotinamide is present to inhibit the activity of DPNase. 3 Purification Procedure 4

Acetone powder of pigeon liver prepared according to Kaplan and LipmamP is used as the starting material for purification of DPN kinase. Step 1. Preparation of Crude Extract. Six grams of pigeon liver acetone powder is extracted with 70 ml. of ice-cold 0.02 M K H C Q . The extraction is facilitated by using a Tenbroeck homogenizer kept in an ice bath. (All subsequent operations are made at 0 to 5°.) The homogenate is then centrifuged at 18,000 r.p.m, for 15 minutes to remove the insoluble residue. The supernatant should be dark red in color. Step 2. Protamine Precipitation. A 0.2 % protamine sulfate solution in 0.04 M Tris buffer, pH 7.5, is added slowly with stirring to the above crude extract. The precipitate forms immediately during this addition. As soon as no further precipitation occurs, the addition of protamine solution is discontinued. In general, about 2 vol. of the protamine solution is required for 1 vol. of the crude extract. The protamine precipitare, collected by centrifugation at 4000 r.p.m, for 10 minutes, is extracted twice with 20 ml. each of an 0.2 M acetate buffer, pH 5.0. The two acetate extracts are combined, and the residue is discarded. Step 3. Adsorption on Alumina C~. 6 An equal volume of water is add e d to the acetate extract. To the diluted extract is gradually added 18.5 ml. of alumina C~ (dry weight 16.3 mg./ml.). After 10 minutes of stirring, the mixture is centrifuged at 4000 r.p.m, for 10 minutes. The supernatant is discarded. The alumina residue is then eluted three times with 18.5 ml. each time of 0.2 M phosphate buffer, pH 7.5, and discarded. Step 4. (NH4)2SO~ Precipitation. A solution of (NH~)2SO4, saturated at room temperature with a pH of 7.1, is added to the phosphate eluate to give 0.5 saturation. The precipitate, collected by centrifugation, is dissolved in 4.5 ml. of 0.02 M phosphate buffer, pH 7.5. The over-all yield of the enzyme from the acetate extract is 64%, and the purification from the crude extract is 46.5-fold. The low specific activity in the crude extract is probably due to the presence of inhibitors or competitive enzymes such as ATPase, DPNase, nucleotide pyrophosphatase, etc., in the crude extract. The steps in the purification of the DPN kinase are summarized in the table. P. Handler and J. R. Klein, J. Biol. Chem. 143, 49 (1942). 4 T. P. Wang and N. O. Kaplan, J. Biol. Chem. 206, 311 (1954). 5 N. O. Kaplan and F. Lipmann, J. Biol. Chem. 174, 37 (1948). 6 Alumina C~, see Vol. I [11].

654

COENZYME AND VITAMIN METABOLISM

[111]

SUMMXRY OF PURIFICXTION PROCEDURE

Fraction KHC08 extract Acetate extract Acetate eluate (NH4)2S04 fraction

Volume, ml.

Protein, rag./ml,

Specific activity

Yield, units

51 37 55 6.5

30.9 5.2 1.8 10.7

0. 0178 0. 469 0. 768 0. 828

28.2 a 90 75.6 57.7

a See text for explanation. This procedure has been used b y several workers besides the a u t h o r with the same or b e t t e r yield a n d / o r higher purification. Properties

Specificity. The D P N kinase from pigeon liver is specific for D P N with no activity on d e a m i n o - D P N , D P N H , and D P C o A . However, the enzyme will catalyze the phosphorylation of the 3-acetylpyridine analog of DPN.7 T h e requirement of A T P is also specific; neither A D P nor I T P can be used to replace A T P . The K~ for D P N is 6 × 10-4 M. Metal Requirements. Besides D P N and A T P , metal ions such as M g ++ or Mn++ are necessary for D P N kinase activity. T h e optimal concentration of M n ++ is about 1.8 X 10-3 M, and t h a t of Mg ++ a b o u t 0.9 X 10-2 M. At the optimal concentration of M n ++, the a m o u n t of T P N formed is about 25 to 30 % higher than t h a t formed at the optimal concentration of Mg ++. However, at higher concentration, M n ++ shows some inhibitory effect, whereas Mg ++ does not even at 1.8 X 10-2 M. Inhibitors. A group of compounds related to D P N have been found to have an inhibitory effect on D P N kinase. With the exception of dea m i n o - D P N , the more the compound resembles D P N , the greater inhibition it exhibits. For example, adenosine, 2-adenylic acid, and 3-adenylic acid are less inhibitory t h a n 5-adenylic acid, which, in turn, is not so inhibitory as A D P or A D P R . And among all the compounds tested, D P N H is the most p o t e n t inhibitor. E v e n at a concentration of 1.5 X 10-3 M, D P N H gives an inhibition of over 84% when the concentration of D P N is 2.5 X 10-8 M. The inhibition of D P N H on D P N kinase can be overcome b y the increase of D P N concentration. In other words, the inhibition of D P N H appears to be competitive. When D P N H is treated with a snake venom nucleotide pyrophosphatase, its inhibitory effect becomes greatly reduced to the level of t h a t of 5-adenylic acid. The reduced N M N seems to have no effect on D P N kinase at all. I t is interesting to note t h a t d e a m i n o - D P N has no inhibition on D P N kinase. Since d e a m i n o - D P N is a hypoxanthine derivative instead of an

[112]

NUCLEOTIDE PYROPHOSPHATASE

655

adenine compound, the amino group on the adenine ring m a y be responsible for the inhibitory effect of the adenine compounds. In this connection, it m a y be worth mentioning t h a t I T P , also a hypoxanthine derivatire, cannot be used as a phosphate donor. The presence of an amino group in the purine ring in both the phosphate donor and the phosphate acceptor seems essential for the activity of D P N kinase from pigeon liver. Other Properties. D P N kinase can be dialyzed against 0.02 M N a H C O s at 4 ° for at least 16 hours without losing any activity. I t is also stable toward freezing and thawing. Storage at - 1 5 ° for four months resulted in little loss of activity. The enzyme has been used successfully in preparing large quantities of T P N of high purity.7 7 T. P. Wang, N. O. Kaplan, and F. E. Stolzenbach, J. Biol. Chem. 211, 465 (1954).

[112] Nucleotide Pyrophosphatase 1 DPN TPN FAD ATP +

+ + + 2

H20 H20 H20 H20

--+ N M N + 5'-AMP --+ N M N + Adenosine-2',5'-diphosphate --+ Riboflavin Phosphate + 5'-AMP --* 5 ' - A M P + 2 P

By ARTHUR KORNBERQ Assay Method Principle. The method depends on the spectrophotometric determination of the removal of D P N . 2 In crude preparations the possible contributions to D P N removal b y other DPNases can be checked b y demonstrating the persistence of the pyridinium linkage 3 or the appearance of 5 ' - A M P ? Reagents

D P N (0.02 M), p H ca. 6. KH~PO4-K:HP04 (0.5 M), p H 7.0. Procedure. A mixture containing 0.1 ml. of D P N , 0.2 ml. of phosphate buffer, 1 to 5 enzyme units, and water to a final volume of 1.0 ml. was

1A. Kornberg and W. E. Pricer, Jr., J. Biol. Chem. 182, 763 (1950). 2 A. Kornberg, J. Biol. Chem. 182, 779 (1950). 3j. W. Huff and W. A. Perlzweig, J. Biol. Chem. 167, 157 (1947). H. M. Kalckar, J. Biol. Chem. 167, 445 (1947).

656

COENZYME A N D

VITAMIN METABOLISM

[112]

incubated for 20 minutes at 38 °. An aliquot of 0.10 ml. was pipetted directly into a cuvette containing (in 2.8 ml.) all the components needed for the reduction of D P N except the enzyme (i.e., alcohol dehydrogenase), and an initial reading was taken. After the addition of enzyme, reduction of D P N was complete in about 5 minutes. The concentration of D P N before and after incubation with nucleotide pyrophosphatase was based on the use of an extinction coefficient 5 of 6.22 × 106 cm.2/mole. Definition of Unit and Specific Activity. One unit of enzyme is defined as t h a t a m o u n t which causes the splitting of 1 micromole of substrate (i.e., D P N ) per hour. Specific activity is expressed as units per milligram of protein. Protein was determined by a b i u r e t procedure 6 for crude preparations and by ultraviolet light absorption 7 for purified preparations.

Purification Procedure

Step 1. Extraction. Two hundred grams of peeled Maine potatoes was extracted with 400 ml. of 0.40 saturated ammonium sulfate for 90 seconds in a Waring blendor. The extract was filtered on fluted papers at 2 °. F r o m 5 kg. of potatoes, 10 1. of filtrate was obtained. The use of Celite as a filter aid resulted in some loss of enzyme activity. The yield obtained b y extraction of potatoes with 2 vol. of water (aqueous extract, see the table) approximated t h a t of the a m m o n i u m sulfate fraction. Step 2. Ammonium Sulfate Fraotionation. T o 10 1. of filtrate was added 2 kg. of solid ammonium sulfate. Filtration was at 2 ° through a 50-cm. fluted paper with an arrangement for automatic refilling to permit collection of the precipitate on a single paper and completion of the filtration overnight. T h e brownish black precipitate was scraped from the paper and dissolved with water to a volume of 550 ml. The dark solution was dialyzed against running tap water (at 8 to 18 °) for 90 minutes in cellophane sacs. The volume after dialysis was 660 ml., and the p H 5.5 to 5.6. F o u r such batches of dialyzed ammonium sulfate fractions were combined (ammonium sulfate, see the table) and fractionated with ethanol. Step 3. Ethanol Fractionation. The dialyzed a m m o n i u m sulfate fraction was brought to p H 4.4 with acetic acid (61 ml. of 1 M). The solution was cooled to - 0 . 5 ° , and 95% ethanol was added with mechanical stirring. The t e m p e r a t u r e was maintained just above the freezing point during the early ethanol additions and at - 5 ° thereafter. The precipitates 5 B. L. Horecker a n d A. Kornberg, J. Biol. Chem. 176, 385 (1948). 6 T. E. Weichselbaum, Am. J. Clin. Path., Tech. Sect. 10, 40 (1946). 7 O. W a r b u r g a n d W. Christian, Biochem. Z. 310, 384 (1941-1942); see Vol. I I I [73].

[112]

NUCLEOTIDE PYROPHOSPHATASE

657

were centrifuged off at 0 ° and dissolved in water. Fraction 2 was refractionated as indicated (see the table). These two fractionations have been carried out four times with little variation from the results of the first trial. A t t e m p t s to standardize a third ethanol fractionation were unsuccessful. Fractions with high specific activity were obtained in good yield, but minor variations in temperature, time, and speed of ethanol addition influenced the a m o u n t of SUMMARY OF PURIFICATION PROCEDURE

Ethanol Volume Specific (95 %) of Total activity, added, fraction, activity, Yield, units/ ml. ml. units % mg. protein

Step Aqueous extract Ammonium sulfate Ethanol fraction I-1 Ethanol fraction 1-2 Ethanol fraction II-2a Ethanol fraction II-2b Ethanol fraction II-2c Ethanol fraction III-2c-1 Ethanol fraction III-2c-2 Ethanol fraction III-2c-3 Ethanol fraction III-2c-4• Ethanol fraction III-2c-5b Calcium phosphate, first adsorption Fractions 2c-3, 4, 5 Eluate 1 Eluate 2 Eluate 3 Calcium phosphate, second adsorption, eluate Fraction 2c-4 was the 2c-3 after standing at b Fraction 2c-5 was the 2c-4 after standing at

397 438 41 27 30 44 11 5.5

2750 1000 680 178 160 162 36 36 30 25 21

350,000 317,000 58,500 230,000 15,850 61,000 125,000 13,300 34,000 30,700 21,400 25,700

91 18 72 7 27 54 11 27 24 17 20

2.9 5.4 1.8 15.5 2.0 15.6 84.7 18.8 76.5 202 278 216

254 100 100 100

77,500 52,000 7,700 910

67 10 1

220 1625 1065 530

75

38,300

74

2200

precipitate which appeared in the supernatant of fraction - 1 0 ° for 3 hours. precipitate which appeared in the supernatant of fraction - 1 0 ° for 18 hours.

ethanol required. Accordingly, this fractionation was carried out by collecting several ethanol fractions and combining the best (see the table). Step 4. Calcium Phosphate Adsorption. The combined ethanol fractions (Nos. 2c-3, 4, and 5) (pH 4.4) were diluted with water to give a protein concentration of 1.5 mg./ml. Calcium phosphate gel 8 (202 ml., aged 2 months, dry weight 7.9 mg./ml.) was added, and the mixture was stirred mechanically for 5 minutes at room temperature. The precipitate 8 D. Keilin and E. F. Hartree, Proc. Roy. Soc. (London) B124~ 397 (1938).

658

COENZYME AND VITAMIN METABOLISM

[112]

was collected by centrifugation and washed four times with 100 ml. of 0.1 M potassium phosphate buffer, pH 7.4. The enzyme was eluted with three portions of 100 ml. of 0.20 saturated ammonium sulfate adjusted with ammonia water to pH 7.5. To concentrate eluates 1 and 2 to a small volume, 36 g. of solid ammonium sulfate was added to each. The precipitates, collected in a highspeed centrifuge, were dissolved in water to yield a protein concentration of 2 mg./ml. The yield in this step was 92%, and the specific activity was unaltered. A second calcium phosphate adsorption increased the specific activity to 2200 units/rag, with a yield of 80 %. Fifteen milliliters of the ammonium sulfate concea,trate of eluate 1 above were diluted to 300 ml., adsorbed with 15 ml. of calcium phosphate gel, washed three times with 150 ml. of 0.05 M potassium phosphate buffer, pH 7.0, and eluted with 75 ml. of 0.20 saturated ammonium sulfate, pH 7.5. The entire purification procedure resulted in a 750-fold purification with an over-all yield of 11%. The yield may be improved by combining and reprocessing some fractions of lower purity. The term "purified enzyme" in this report refers to a calcium phosphate eluate (or ammonium sulfate concentrate) with a specific activity of 1625 units or more per milligram and "crude enzyme" to the aqueous extract (see the table). Variations in Activity in Potatoes. The age and variety of potato influenced the nucleotide pyrophosphatase activity. Eleven varieties of Maine potatoes, which were harvested at the same time and stored under identical conditions prior to the initial assay, differed widely (3.3 to 21.9 units/ml.). Aging at 3 ° resulted in a variable increase in activity. Since the protein concentration was relatively uninfluenced by the variety of potato or its age, the specific activity reflected the enzyme activity. The low activity was not due to the presence of an inhibitor, since an extract of a potato of high activity (55 units/ml.) tested in the presence of an equal volume of an extract of a potato of low activity (3.3 units/ml.) was not inhibited. Stability. The enzyme is remarkably stable at 0 to 5 °. Preparations of varying purity (including the most purified) have shown no detectable loss of activity over a period of six years. However, some dilute solutions (50 ~, of protein per milliliter) lost 50 % of their activity in 5 days. There was no inactivation on incubation for 20 minutes at 38 ° at pH 3.2 in 0.1 M citrate buffer, or at pH 9.3 in 0.1 M glycine buffer. There was complete inactivation on incubation for 15 minutes at 38 ° at pH 12.5, or for 10 minutes at 38 ° at pH 1.4. The enzyme was not inactivated by freezing or by a 9-hour dialysis against running distilled water.

[112]

NUCLEOTIDE PYROPHOSPHATASE

659

Properties Effect of pH. As measured with DPN as substrate, there is a broad optimum between pH 6.5 and 8.5, and a decrease of activity of about 50% at pH 4:0 and 9.0. Enzyme inactivation did not contribute to this result, and a twofold increase in DPN concentration did not change the result. Specificity. Certain evidence suggests that the same enzyme splits DPNH, TPN, FAD, and ATP. This evidence is the relative constancy of ratios of these activities during the course of purification and kinetic data. There are weaker indications that thiamine pyrophosphate is a substrate. Park 9 has demonstrated that UDPG is very likely a substrate of nucleotide pyrophosphatase. The conclusion of Novelli et al. 1° that the enzyme from potatoes which splits CoA at the pyrophosphate bond is a different enzyme is based only on the fact that the pH optimum for this activity is much lower than that reported for DPN. Other Phosphatase Activities. The purified preparations still contained detectable amounts of phosphatase activity toward several phosphate esters including inorganic pyrophosphate, 5'-AMP, NMN, glucose-6-phosphate, and glycerophosphate. However, these activities were very low, splitting of inorganic pyrophosphate being only 3 % as active and the other activities being less than 1% of the rate for DPN splitting. There was no indication during the course of purification that any of these activities became stabilized with reference to DPN splitting, as was the case with adenyl pyrophosphate and thiamine pyrophosphate splitting. Michaelis constants determined for DPN, TPN, and ATP were as follows: 1.5 X 10-4 M, 3.0 X 10-3 M, and 2.0 X 10-3 M, respectively. Activators and Inhibitors. DPN splitting was not stimulated by Mg ++ or Ca ++ and was inhibited about 50% by NaF (0.1 M) only in the presence of phosphate (0.05 M). Somewhat different results were obtained with other nucleotides as substrates.

g J. T. Park, J. Biol. Chem. 194, 885 (1952). 10G. D. Novelli, F. J. Schmetz, Jr., and N. O. Kaplan, J. Biol. Chem. 206, 533 (1954).

660

COENZYME AND VITAMIN METABOLISM

[113]

[113] Animal Tissue D P N a s e (Pyridine Transglycosidase 1) N + R P P R A ~ H 2 0 -* N ~- R P P R A + H + N + R P P R A -]- X -~ X + R P P R A ~- N

By NATHAN O. KAPLAN

Assay Method 2 Principle. T w o m e t h o d s are used for following the cleavage of D P N . One is based on the cyanide reaction of D P N 2 This involves determining the D P N level b y cyanide before and after incubation with the enzyme. Cyanide reacts only with the q u a t e r n a r y nitrogen form of D P N and will not react with adenosine diphosphate ribose or free nicotinamide. A second m e t h o d involves following the splitting of D P N b y assaying with y e a s t alcohol dehydrogenase. T h e cyanide procedure is of value in t h a t it will signify the cleavage of the nicotinamide ribose link. B y the use of the alcohol dehydrogenase assay, it is not possible to ascertain whether splitting occurs at the nicotinamide glycosidic bond or at the p y r o p h o s p h a t e grouping. 3 Reagents Crystalline y e a s t alcohol dehydrogenase (Vol. I [79]).

M/1 KCN. 0.1 M phosphate, p H 7.2. 0.005 M D P N .

Determination of Enzymatic Activity. 2 T h e reaction consists of 0.3 ml. of the 0.1 M phosphate, 0.5 micromole of D P N , and e n z y m e (approxim a t e l y 1 unit) in a total volume of 0.6 ml. After incubation at 37 ° for 8 minutes, 3 ml. of the m o l a r cyanide is added, and the mixtures are read at 325 m~ (which is the absorption m a x i m u m of the D P N cyanide complex). F o r determination of the action with alcohol dehydrogenase, 3 ml. of a mixture containing 0.5 M ethanol and 0.02 M nicotinamide 4 in 0.1 M 1The name pyridine transglycosidase has been given to the enzyme, since the enzyme has been found not only to induce hydrolysis of DPN at the nicotinamide ribose linkage but also to promote a transfer of adenosine diphosphate ribose from one pyridine grouping to another. In the equation N -- nicotinamide, R ~- ribose, P = phosphate, A = adenine, and X -- a pyridine compound structurally related to nieotinamide. 2L. J. Zatman, N. O. Kaplan, and S. P. Colowick, J. Biol. Chem. 200, 197 (1953). a S. P. Colowick, N. O. Kap]an, and M. M. Ciotti, J. Biol. Chem. 191, 473 (1951). 4 The nicotinamide is added to prevent further cleavage of DPN at the end of the incubation period.

[113]

ANIMAL TISSUE DPNAS~

661

glycine NaOH buffer, pH 9.5, were added. Readings are taken at 340 mg before and after the addition of crystalline yeast alcohol dehydrogenase, a Definition of Unit. A unit of enzyme is that amount which will cleave 1 micromole of DPN per hour under the conditions described. Application to Crude Extracts. The enzyme is usually found in the insoluble fractions of tissue homogenates; the assay technique is applicable to whole homogenates. Purification Procedure As yet no success has been achieved in solubilizing the enzyme, and as a result no extensive purification of the enzyme has been made. However, some purification has been obtained by differential centrifugation. The preparation of the DPNase from beef spleen and pig brain will be described. These two enzymes, although carrying out an identical cleavage of DPN, differ with respect to their sensitivity to isonicotinic acid hydrazide and their activity in forming DPN analogs. Preparation of Spleen Enzyme. 6 Two hundred grams of fresh beef spleen was homogenized in a Waring blendor with ice and water for 4 minutes and made to 750 ml. with ice water and 15 ml. of M NaHCO3. The connective tissue was removed by centrifugation at 2000 r.p.m, for 10 minutes; the resulting red-brown opaque supernatant fluid can be stored in the deep-freeze for several weeks without more than 10 to 20 % loss in activity. A 100-ml. portion of this crude material was centrifuged at 18,000 r.p.m. 7 for 15 minutes, and the residue resuspended in 60 to 70 ml. of 0.02 M N a H C Q and respun at 3000 r.p.m, for 15 minutes. The supernatant fluid was then centrifuged at 18,000 r.p.m, for 15 minutes, and the residue, which is devoid of red pigment, resuspended in 80 ml. of 0.02 M N a H C Q . s The suspension was finally centrifuged at 18,000 r.p.m. for 1 minute and the opaque supernatant separated from the loose precipitate. The supernatant contains material of the highest specific activity, usually about 35 units/mg, of protein, and represents an approximately sevenfold purification from the crude homogenate. Protein was determined by the method of Lowry et al2 6 The splitting can also be followed manometrically, since acid is produced when D P N is cleaved a t the nicotinamide ribose link. 2 6 F r o m the procedure of Z a t m a n et al. 2 7 Carried out on an I n t e r n a t i o n a l centrifuge. 8 W h e n the enzyme is used in chromatographic experiments, distilled water is used instead of NaHCO3 for the final suspension of residue in order to avoid gas production on acidification of the column. 9 0 . H. Lowry, N. J. Rosebrough, A. L. Farr, a n d R. J. Randall, J. Biol. Chem. 195~ 265 (1951); see also Vol. I I I [73].

662

COENZYME AND VITAMIN METABOLISM

[113]

Further high-speed centrifugation always sedimented more of the enzyme, and attempts to render the enzyme soluble by heat, repeated freezing and thawing, salt, deoxycholate, and digitonin were unsuccessful. The highest specific obtained to date has been less than 1% of that of the DPNase purified from Neurospora (see Vol. II [114]). Preparation of Pig Brain Enzyme. TM Whole pig brain (150 g.) was blended with 500 ml. of ice water in a Waring blendor. After passing through cheesecloth, the homogenate was centrifuged for 20 minutes at 20,000 X g and the precipitate made up to 300 ml. with ice water. This was then centrifuged for 40 minutes at 20,000 × g, after which the precipitate was suspended in 150 ml. of ice water and treated for 15 minutes in the 10-kc. sonic oscillator (Raytheon). The suspension was finally centrifuged at 20,000 X g for 20 minutes, and the relatively small amount of precipitate discarded. The supernatant fluid is colloidal and contains nearly all the DPNase activity. This fluid contains about 20 units of enzyme per milliliter and can be stored at - 1 5 ° for at least a month without loss in activity.

Properties pH Optimum. The optimum for the beef spleen system is 7.2; the pig enzyme has roughly the same optimum. Specificity. Both the beef spleen and pig brain enzymes attack only the oxidized form of DPN, and not reduced DPN. Oxidized TPN is attacked by the beef spleen DPNase at 50% the rate of DPN; the pig brain enzyme also will cleave TPN. Deamino-DPN is split by the spleen enzyme at 23 % the rate of DPN. Nicotinamide riboside and mononucleotide are not hydrolyzed by either the spleen or brain enzymes. The pig brain enzyme will liberate the pyridine component from a number of DPN analogs. These include the 3-acetylpyridine, isonicotinic acid hydrazide (INH), marsilid, 1~ isonicotinamide, and ethyl nicotinate analogs of DPN. The beef spleen enzyme does not act on the isonicotinic acid hydrazide, marsilid, and isonicotinamide analogs but will split the 3-acetylpyridine analog. Inhibitors. Nicotinamide has been found to be a potent inhibitor of animal tissue DPNase. The spleen enzyme is inhibited 50 % by a nicotinamide concentration of 1.5 × 10-3 M. The inhibition has been shown to be of a noncompetitive nature. By the use of C14-1abeled nicotinamide, it has been possible to show that this inhibition involves an exchange reaction between free nicotinamide and the bound nicotinamide of DPN. TM 10 L. J. Zatman, N. O. Kaplan, S. P. Colowick, a n d M. M. Ciotti, J. Biol. Chem. 209~ 467 (1954). 11 Marsilid is the isopropyl derivative of I N H . 12 For a description of D P N prepared with labeled nicotinamide, see Vol. IV [34].

[113]

ANIMAL TISSUE DPNASE

663

I N H is a p o t e n t inhibitor of the beef spleen system. On the other hand, this same compound does not inhibit the cleavage of D P N b y the pig brain catalyst. T h e effect of I N H on D P N a s e s from various species was found to fall into two distinct categories, those which were strongly inhibited b y the compound and those which are not inhibited. The t e r m " I N H - s e n s i t i v e " enzyme has been used to refer to the beef spleen enzyme, which is inhibited, whereas the pig brain enzyme has been referred to as an " i n s e n s i t i v e " enzyme, because it is not inhibited b y the I N H.I3 A s u m m a r y of the effect of I N H on a n u m b e r of different D P N a s e s is given in the table. INH

SENSITIVITY OF VARIOUS ANIMAL TISSUE D P N A s E S ~

" I N H sensitive . . . . INH insensitive" Beef spleen Beef brain Lamb spleen Lamb brain Goat spleen Goat brain

Rat spleen Rat brain Mouse spleen Mouse brain Mouse lymphosarcoma Rabbit spleen

" I N H sensitive" " I N H insensitive" Pigeon brain Duck brain

Rabbit brain Horse spleen Horse brain Human spleen Human prostate Frog spleen

From L. J. Zatman, N. 0. Kaplan, S. P. Colowick, and M. M. Ciotti, J. Biol. Chem. 209, 453 (1954). I t has been found t h a t the " I N H - i n s e n s i t i v e " systems will f o r m the I N H analog of D P N v e r y readily. Although the " s e n s i t i v e " enzymes are strongly inhibited b y I N H , the analog is still formed b u r n t a b o u t 1 % the rate of the " i n s e n s i t i v e " systems. I°,I~ T o date the following D P N analogs have been synthesized with the pig brain enzyme as catalyst: I N H , I°,15 marsilid, 1° isonicotinamidel 1° 3-acetylpyridine, I6 and ethyl nicotinate. I7 Alivisatos and Woolley h a v e reported the synthesis of the 4-amino 5-carboxamido imidazole analogue of D P N f r o m D P N and the imidazole; this reaction is catalyzed b y the beef spleen system, is 1~L. J. Zatman, N. O. Kaplan, S. P. Colowiek, and M. M. Ciotti, J. Biol. Chem. 209, 453 (1954); Bull. Johns Hopkins Hosp. 91, 211 (1952). 14D. S. Goldman, J. Am. Chem. Soc. 76, 2841 (1954). 15L. J. Zatman, N. O. Kaplan, S. P. Colowiek, and M. M. Ciotti, J. Am. Chem. Soy. 75, 3293 (1954). 16N. O. Kaplan and M. M. Ciotti, J. Am. Chem. Soe. 76, 1713 (1954). 17N. O. Kaplan and M. M. Ciotti, in preparation. is S. G. A. Alivisatos and D. W. Woolley, J. Am. Chem. Soc. 77, 1065 (1955).

664

COENZYME AND VITAMIN METABOLISM

[114]

[114] N e u r o s p o r a D P N a s e + N R P P R A ~- H 2 0 --* N + R P P R A q- H +

B y NATHAN O. KAPLAN Assay Methods

T h e procedures used are identical with those described for the animal e n z y m e (Vol. I I [113]). T h e only difference is t h a t the incubation time is for 71/~ minutes at 37 °. Unit of Activity. One unit of a c t i v i t y is t h a t a m o u n t of e n z y m e which causes cleavage of 0.01 micromole of D P N u n d e r the conditions of t h e assay. Protein was determined b y the m e t h o d of L o w r y et al.1 Purification P r o c e d u r e T h e e n z y m e has been purified from m a t s grown in a zinc-deficient medium. ~ B y the inclusion of only a small a m o u n t of nitrate instead of the usual a m m o n i u m nitrate of the normal Fries medium, it has been possible to obtain m a t s of s o m e w h a t higher specific a c t i v i t y 2 The m e d i u m used for the growth of the mold is as shown in the table. M a k e up in triple-distilled water. Medium

G./1.

Medium

Sucrose KH2PO4 MgSO4.7H20 NaC1 CaC12 Biotin Na tetraborate

20 1 0.5 0.1 0.1 5 X 10-6 8.8 X 10-s

(NH4)sMo40~4 FeC13.6H20 CuCl: MnCI~.4H20 Na tartarate Na nitrate

G./1. 6.4 9.6 2.7 7.2

X 10-5 X 10-4 × 10-4 X 10-5 5.0 1.0

Step 1. Preparation of Crude Extracts. 2 T h e mycelia m a t s are collected in a Biichner funnel, and washed with triple-distilled water. F r o m 10 1. of m e d i u m 40 to 50 g. of m a t s (wet weight) is obtained. The m a t s are frozen for at least 1 to 3 hours, then homogenized in a Ten Brock glass homogenizer in three times their weight of 0.1 M p h o s p h a t e buffer at p H 7.5 and centrifuged in the Servall at 13,000 r.p.m, for 10 minutes 10. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951); see also Vol. I I I [73]. 2 A. Nason, N. O. Kaplan, and S. P. Colowick, J. Biol. Chem. 188, 397 (1951). A. Nason, N. O. Kaplan, and H. A. Oldewurtel, J. Biol. Chem. 201, 435 (1953).

[114]

NEUROSPORA DPNASE

665

at + 4 °. The supernatant, which is cell-free and turbid, is used as the enzyme source. N e a r l y all the enzyme is found in the extract. Step 2. Acidification. 4 The crude extract is adjusted to p H 5 with HC1 at 0 °. The resulting precipitate is discarded; the acid filtrate contains 80% of the initial activity, with a twofold increase in purity (see the summary). SUMMARY OF PURIFICATION PROCEDURE a

Recovery, % Total units Units/rag. protein units/mg, protein Crude extracts 1,600,000 pH 5 filtrate 1,260,000 pH 5, 60% acetone precipitate 609,000 pH 2.7, 60% acetone precipitate 480,000

2,650 4,980 25,800 85,600

-79 39 30

a 250 ml. of crude extract used in this experiment.

Step 3. Acetone Fractionation. Acetone is added to the acid filtrate of step 2 at 0 ° to a concentration of 35%. This precipitate contains little activity. The supernatant is then brought to 60% acetone, and the precipitate is dissolved in 0.1 M K~HPO4. This precipitate has an activity about five times t h a t found in the previous step. Step 4. pH 2.7 Acetone Precipitate. 5 The dissolved 60% precipitate from step 3 is brought to p H 2.7 at 0 ° b y adding normal HC1 dropwise. Acetone is then added to 60%. The resulting precipitate, spun at 0 °, is triturated with 5 ml. of 0.1 M phosphate buffer, p H 7.5, and the denatured protein is removed. The soluble protein represents a 30% yield with about 30-fold purification from the initial extract. I t is of interest to note t h a t to obtain the same specific activity from normal Neurospora mats 2 a 1500-fold purification would be required. Properties

Stability. The enzyme can be kept in the deep-freeze without loss of activity. I t slowly becomes inactivated at 4°; this is particularly true with very dilute solutions of the DPNase. Dialysis against a variety of buffers resulted in no loss of activity. Heating at 80 ° at p H 5 for 2 minutes completely destroys the activity of the enzyme. However, little loss occurs after heating at 55 ° for 2 minutes in the p H range 3 to 5. N. 0. Kaplan, S. P. Colowick, and A. Nason, J. Biol. Chem. 191, 473 (1951). 6 In the routine preparation of the enzyme, the final step has been omitted. This preparation has been used extensively for the determination of DPN (see Vol. III [128]).

666

COENZYME AND VITAMIN METABOLISM

[114]

Trichloroacetic acid only partially precipitates the enzyme; the precipitated activity can be recovered b y dissolving the precipitate in 0.1 M K2HPO4. Some of the enzyme is present in the trichloroacetic acid filtrate. The enzyme appears to be stable in such filtrates for a period of at least two weeks at 4 ° . p H Optimum. The enzyme operates over a v e r y broad p H range (3 to 9). The nature of the buffer does not influence the Neurospora DPNase, and citrate, phosphate, and acetate can be used interchangeably. Effect of Metal. Zinc, manganese, ferric, calcium, and magnesium ions in concentrations of 0.05 M have no effect on the activity of the enzyme. Fluoride, cysteine, Versene, and cyanide at 0.01 M do not inhibit the enzyme. Effect of D P N Concentration. The Km for D P N has been found to be approximately 5 X 10 -4 mole/1. Inhibition by Nicotinamide. Unlike the animal tissue DPNase, the Neurospora enzyme is quite insensitive to nicotinamide. Only at v e r y high concentrations (0.1 M) is the enzyme inhibited. The inhibition of nicotinamide is competitive in contrast to the noncompetitive inhibition observed with the beef spleen system. 6 Formation of D P N Analogs. The enzyme operates by a different mechanism from t h a t of the animal tissue DPNase, and it does not form D P N analogs. Specificity. The enzyme attacks both D P N and T P N . 4D e a m i n o - D P N 7 is split at a much slower rate t h a n is D P N . The various analogs of D P N (i.e., isonicotinie acid hydrazide, 3-acetylpyridine) are not cleaved b y the enzyme. A synthetic nucleotide, dinicotinamide ribose 5'-pyrophosphate, is split at about one-third the rate of D P N . s I t is interesting to note t h a t only one nicotinamide is removed from the compound b y the Neurospora enzyme. Reduced D P N or T P N , nicotinamide mononucleotide, and nicotinamide riboside are not hydrolyzed b y the enzyme. 9 Effect of Deficiencies on the Concentration of Enzyme. Neurospora mats grown on a zinc-deficient medium show an approximate 10- to 20-fold increase in the level of DPNase. Other metal deficiencies have little or no effect on the D P N a s e concentration. 2 A nitrogen deficiency also produces a v e r y marked elevation in enzyme. 3 Neurospora m u t a n t s grown on minimal levels of the specific nutrient also exhibit high DPNase.1 6L. J. Zatman, N. O. Kaplan, and S. P. Colowick, J. Biol. Chem. 200, 197 (1953). N. O. Kaplan, S. P. Colowick, and M. M. Ciotti, J. Biol. Chem. 194, 579 (1952). s L. Shuster, N. O. Kaplan, and F. E. Stolzenbach, J. Biol. Chem. in press. The alpha isomer of I)PN is also not split by the Neurospora enzyme [N. O. Kaplan, M. M. Ciotti, F. E. Stolzenbach, and N. R. Bachur, J. Am. Chem. Soc. 77, 815 (1955)].

[115]

DEPHOSPHO-COA PYROPHOSPHORYLASE

667

[115] Dephospho-CoA Pyrophosphorylase B y G. DAVID !X~OVELLI

Assay Method Principle. This enzyme catalyzes the reversible condensation between ATP and phosphopantetheine to yield dephospho-CoA and inorganic pyrophosphate. 1 The enzyme has not yet been separated from dephosphoCoA kinase (see Vol. II [110]); therefore, starting with ATP and phosphopantetheine the product of the over-all reaction is CoA. CoA is measured by means of the phosphotransacetylase 2 assay (see Vol. I [98]). Alternatively, the reaction may be measured by the determination of inorganic pyrophosphate. Crude preparations are usually contaminated with ATPase and inorganic pyrophosphatase, however, making such measurements less reliable. In more purified preparations inorganic pyrophosphatase is eliminated, but dephospho-CoA kinase is still present. The latter enzyme displaces the equilibrium in favor of CoA synthesis, and thus the inorganic pyrophosphate level is a direct measure of the condensation reaction. Reagents

4'-Phosphopantetheine, 0.02 M. ATP, 0.1 M. MgCI~, 0.1 M. Cysteine HC1, 0.1 M. Tris buffer (M/l), pH 7.7. Reagents for phosphotransacetylase assay for CoA 2 (see Vol. I [98] and Vol. III [132]). Dephospho-CoA pyrophosphorylase. Procedure. The enzyme activity is measured under the following conditions: 0.2 uM. of phosphopantetheine, 5 to 10 ~M. of ATP, 5.0 ~M. of Mg ++, 10.0 tiM. of cysteine, 50 tLM. of Tris buffer, pH 7.7, and enzyme sufficient to make 10 to 20 units of CoA when CoA is being measured or 1 to 2 uM. of CoA when pyrophosphate is being measured. Incubation is carried out at 37 ° for 60 minutes in a final volume of 1.0 ml. The reaction is stopped by boiling for 3 minutes in a final volume of 1.0 ml. The reaction is stopped by boiling for 3 minutes, and CoA is measured with the

1M. Hoagland and G. D. Novelli, J. Biol. Chem. 207, 767 (1954). E. R. Stadtman, G. D. Novelli, and F. Lipmann, J. Biol. Chem. 1911365 (1951).

668

COENZYME AND VITAMIN METABOLISM

[115]

phosphotransacetylase assay, or pyrophosphate by the time-color development method of Flynn et al. 3 Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which effects the synthesis of 1 unit of CoA or the liberation of 0.0025 ~M. of inorganic pyrophosphate in 1 hour at 37 ° under the conditions specified below. Specific activity is expressed as units of enzyme per milligram of protein. Protein is determined by the turbidimetric method of Biicher. 4 Purification Procedure

Step 1. Preparation of the Crude Extract. Fresh hog liver obtained from the slaughterhouse and kept in ice during transport to the laboratory is put at once through a chilled meat grinder and then blended in a Waring blendor for 45 seconds with an equal volume of cold 0.15 M KC1. This homogenate is filtered through cheesecloth and centrifuged in the preparative Spinco at 40,000 X g for 11/~ hours at 5 °. This process yields a red translucent supernatant from which most microsomes are removed. Higher speeds would be more satisfactory, but this is sacrificed in order to process larger volumes. The removal of particulate protein by this centrifugation procedure effects a fourfold purification. In addition the removal of nuclei eliminates a potent inhibitor. Step 2. Protamine Treatment. The supernatant from step 1 is diluted with 2 vol. of water and treated with 0.06 vol. of 2% protamine sulfate. The resulting precipitate is removed by centrifugation and discarded. To process large volumes the refrigerated Sharples operating at 50,000 r.p.m. is employed. This step removes some of the pantetheine kinase and a large amount of nucleic acid, the remaining microsomes, and inert protein. Step 3. Ammonium Sulfate Fractionation. The supernatant from step 2 is brought to pH 8.0 with Tris, and solid ammonium sulfate is added to 0.25 saturation. The precipitate is centrifuged off at 5 ° and discarded. The supernatant is raised to 0.38 saturation by the addition of solid ammonium sulfate. The resulting precipitate, which contains the bulk of the activity, is collected by centrifugation, and the supernatant is discarded. For large volumes, the precipitate may be conveniently collected in a single Sharples bowl. Step 4. Treatment with Calcium Phosphate Gel. The precipitate from step 3 is dissolved in water to give 50 rag. of protein per milliliter and is then treated with 2.5 vol. of calcium phosphate gel (dry weight, 36 mg./ml.). The gel with its adsorbed protein is quickly spun off and discarded, and the pH of the supernatant is restored to above pH 7 by the addition of 0.05 vol. of M Tris, pH 8.0. Finally the protein is cona R. M. Flynn, M. E. Jones, and F. Lipmann, J. Biol. Chem. 211, 791 (1954). 4 T. Biicher, Biochim. et Biophys. Acta 1, 292 (1947).

[115]

DEPHOSPHO-COA PYROPHOSPHORYL£SE

669

centrated by bringing the solution to 0.7 saturation with ammonium sulfate. The precipitate is collected and dissolved in a minimum volume of water. The purification procedure is summarized in the table. SUMMARY OF PURIFICATION PROCEDURE

Fraction Hog liver homogenate Hog liver supernatant Protamine supernatant Ammonium sulfate fractionation Calcium phosphate gel treatment Over-all

Specific activity

Purification, -fold

1.5 3.0 10.0 50 200 200

2 6.6 33.0 132 132

Recovery,% 600 50 80 40

Properties

Stability. The enzyme is quite stable when kept in the frozen state at - 1 0 °, no appreciable diminution of activity being noted in three months. pH Optimum. Dephospho-CoA pyrophosphorylase is active over the pH range from 6.5 to 8.5 with an optimum near pH 7.5. Mg Requirement. Mg ++ is required for activity of the enzyme. The optimum concentration appears to be around 2 × 10-3 M. Higher levels of Mg ++ appear to activate a latent inorganic pyrophosphatase. Rate. The rate of the over-all reaction, i.e., phosphopantetheine -~ dephospho-CoA--* CoA appears to be linear up to about 80% conversion of phosphopantetheine. It is necessary that the substrate be in the reduced form. The enzyme is totally inactive with the oxidized substrate. Cysteine or H~S have been found suitable in keeping the substrate reduced. Reaction Catalyzed. Dephospho-CoA pyrophosphorylase catalyzes the reversible reaction: Phosphopantetheine ~ ATP ~--Dephospho-CoA + P-P

(1)

Because the enzyme preparations are always contaminated with dephospho-CoA kinase, it has not been possible to measure the equilibrium of reaction 1. However, the presence of this contaminating enzyme makes possible the following dismutation. Dephospho-CoA ~ P-P ~--ATP + P-pantetheine Dephospho-CoA -b ATP--* CoA + ADP • Dephospho-CoA ~- P-P--~ CoA nu ADP ~ P-pantetheine

(sum)

(2) (3) (4)

This stoichiometry has been observed, indicating the reversibility of reaction 2.

670

COENZYME AND VITAMIN METABOLISM

[116]

[116] D P N P y r o p h o s p h o r y l a s e N M N -~- A T P . ~ D P N + P P B y ARTHUR KORNBERG

Assay Method Principle. The method is based on the initial rate of formation of D P N starting with a large excess of A T P and N M N . D P N is measured spectrophotometrically after its total reduction by the alcohol dehydrogenase system. 1 The extinction coefficient of 6.22 X 106 cm.2/mole at 340 m/~ ~ was used. Reagents ATP (0.02 M). N M N (0.05 i ) . Glycylglycine buffer (0.25 M), pH 7.4. MgC12 (0.15 M). Nicotinamide (2 M). Procedure. The incubation mixture contained 0.1 ml. of ATP, 0.05 ml. of N M N , 0.2 ml. of glycylglycine buffer, 0.1 ml. of MgC12, enzyme (1 unit or less), and water to make a final volume of 1.0 ml. To avoid interference by D P N nucleosidase, 0.1 ml. of nicotinamide was added in assays of crude liver fractions. After incubation at 38 ° for 20 minutes, 1.0 ml. of 10% trichloroacetic acid was added. The supernatant solution was neutralized with 2 N N a O H with the aid of an internal indicator (bromothymol blue or phenol red), and 1.0 ml. was pipetted into each of two absorption cells for D P N analysis. With the purified yeast and liver enzymes, the use of trichloroacetic acid was unnecessary and analyses were performed directly on aliquots of the incubation mixtures. Definition of Unit and Specific Activity. One unit of enzyme activity is defined as the amount causing the synthesis of 1 ~M. of D P N per hour, and specific activity as units per milligram of protein. Proportionality to enzyme concentration was observed in this test with crude as well as with purified preparations when 1 unit or less was present in the test system. Protein concentration was determined by a nephelometric method 3 with the Beckman spectrophotometer at 340 mt~. i A. Kornberg, J. Biol. Chem. 182, 779 (1950). 2 B. L. Horecker and A. Kornberg, J. Biol. Chem. 175~385 (1948). 8T. Biicher, Biochim. et Biophys. Aeta 1~ 292 (1947).

[116]

DPN PYROPHOSPHORYLASE

671

Purification Procedure T h e enzyme has been purified from both liver and yeast, but most extensively from the latter (see the table). Only the preparation of the liver enzyme is described here because the starting material is more uniform and more readily obtained and the most purified fraction, although only 5 % as pure (on a protein basis) as the purified yeast enzyme, is more stable and is adequate for equilibrium studies. Purification of Liver Enzyme. Homogenates of rat liver and brain carried out D P N synthesis from N M N and A T P in the presence of nicotinamide. A convenient source of the enzyme was hog liver, from which active, stable acetone powders were prepared. Fresh hog liver (100 g.) was homogenized in acetone ( - 1 0 °, 500 ml.) in a Waring blendor. The residue collected on a Biicher funnel were resuspended in cold acetone, filtered off, and dried at room temperature. Ten grams of powder was extracted with 100 ml. of 0.1 M Na2HPO~ for 10 minutes at room temperature. Subsequent operations were at 3 ° unless otherwise indicated. The residue was separated b y centrifugation and discarded. To the extract (see the table) was added 16 g. of ammonium sulfate; the precipitate was removed b y centrifugation and discarded. Eight grams of ammonium sulfate was added to the supernatant, and the precipitate collected b y centrifugation was dissolved in water to a volume of 40 ml. This fraction was reprecipitated b y adding 8 g. of ammonium sulfate, centrifuging, and dissolving the resulting precipitate in water (ammonium sulfate, see the table). This fraction was diluted with water (23 °) to 94 ml. and adsorbed on 9.4 ml. (75 mg.) of calcium phosphate gel 4 during a 5-minute period. The gel was washed three times with 36 ml. of cold 0.02 M phosphate buffer (pH 7.0) and eluted with 37 ml. of cold SUMMARY OF PURIFICATION PROCEDURES

Step Yeast Autolyzate Final step Liver Extract Ammonium sulfate Adsorption, ammonium sulfate

Volume of fraction, ml.

Total activity, units

Specific Over-all activity, yield, units/ % mg. protein

210 5

716 313

44

O. 24 453

80 13 4

202 163 57

81 28

0.11 0.71 12.4

D. Keilin and E. F. Hartree, Proc. Roy. Soc. (London) B124, 397 (1938).

672

COENZYME AND VITAMIN METABOLISM

[116]

0.5 M K2HPO4. To the clear eluate was added 6.3 g. of ammonium sulfate; the precipitate was collected by centrifugation and dissolved in water (adsorption, ammonium sulfate). This fraction was 100 times as active as the acetone powder extract on the basis of protein content and represented a yield of 28 %. The stability of this preparation was greater than that of the purified yeast enzyme; no significant loss of activity was detected during storage at 3 ° for one to two weeks.

Properties Specificity. N M N H and D P N H replace the corresponding oxidized nucleotides as formulated in the following equation: N M N H + ATP ~ D P N H + PP Nicotinamide nucleoside, ADP, TPN, FAD, orthophosphate, and metaphosphate are not substrates for this enzyme. Activators and Inhibitors. Mg ++ is required. The Michaelis constant is 5 × 10-4 M and 2 X 10-4 M for the yeast and liver enzymes, respectively. Stimulation by Mn ++ is relatively small. 2,4-Dinitrophenol (1 × 10-4 M) and fluoride (0.05 M) do not inhibit this enzyme. Substrate A~nities and Maximal Rates. Each substrate was tested in the presence of an optimal concentration of the corresponding reactant. The dissociation constants of the liver enzyme-substrate complexes calculated from the values were as follows, in moles per liter: 1.5 X 10-4 for N M N ; 4.6 X 10-4 for ATP; 0.83 × 10-4 for DPN; and 1.9 X 10-4 for PP. With use of the optimal substrate concentrations, values for the maximal reaction velocities were obtained. The ratio of V.... for DPN synthesis to V.... for DPN breakdown was 0.48 for the purified liver enzyme and 0.42 for the purified yeast enzyme. The ratio was 0.39 for the same yeast enzyme when tested after six weeks of storage, at which time it had lost 86 % of its activity.

[117]

FAD PYROPHOSPHORYLASE

673

[117] F A D P y r o p h o s p h o r y l a s e FM N -[- A T P ~ FAD -{- P P

By ARTHUR KORNBERG Assay Method Principle. The method I is based on the initial rate of formation of FAD, starting with A T P and FMN. The FAD is determined by the method of Warburg and Christian, 2 which involves the measurement of 02 consumption as a function of FAD concentration in the oxidation of DL-alanine by D-amino acid oxidase. ATP served not only as a substrate but also as a competitive inhibitor of FAD hydrolysis by the nucleotide pyrophosphatase present in the various enzyme fractions. Reagents

MgCI2 (0.15 M). ATP (0.02 M). F MN (2 X 10-4 M). KH:PO,-K2HP04 (0.25 M), pH 7.5. Procedure. The incubation mixture contained 0.05 ml. of MgCl~, 0.1 ml. of ATP, 0.1 ml. of FMN, 0.1 ml. of phosphate buffer, 3 to 10 units of enzyme, and water to a final volume of 1.0 ml. After incubation for 6 to 15 minutes at 37 °, the mixture was immersed in boiling water for 3 minutes, cooled, and centrifuged. An aliquot of the supernatant fluid was assayed for FAD by the D-amino acid oxidase test. Definition of Unit and Specific Activity. One unit of enzyme is defined as th at amount causing the synthesis of 1 millimicromole of FAD per hour and specific activity as units per milligram of protein. Protein concentration was determined by a nephelometric method ~ with the Beckman spectrophotometer at 340 m~.

Purification Procedure One hundred grams of dried beer yeast 4 was autolyzed with 300 ml. of 0.1 M sodium bicarbonate (saturated with a mixture of 95% N2 and 5% COs) for 24 hours at 23 °. All subsequent operations, including storage of solutions, were carried out at 3 °, unless otherwise specified. The 1A. W. Schrecker and A. Kornberg, J. Biol. Chem. 182, 795 (1950). 20. Warburg and W. Christian, Biochem. Z. 298, 150 (1938). 3T. Biicher, Biochim. et Biophys. Acta 1, 292 (1947). A. Kornberg, J. Biol. Chem. 182, 779 (1950).

674

[117]

COENZYME AND VITAMIN METABOLISM

mixture was centrifuged, and the supernatant (autolyzate, see the table) diluted with water to 324 ml. T h e precipitate obtained b y adding 108 g. of ammonium sulfate was centrifuged, dissolved in 60 ml. of water, and dialyzed against running, demineralized water for 1 hour. T h e dialyzed solution (fraction I) was diluted to 75 ml. with w a t e r and mixed with 75 ml. of 0.1 M sodium acetate buffer (pH 5.0). After 5 minutes, 12 ml. of 95% ethanol was added dropwise with mechanical stirring at 0 ° to - 1 °, and the precipitate collected b y centrifugation was discarded. T o the supernatant was added another 23 ml. of 95% ethanol at - 2 °. T h e precipitate was centrifuged and dissolved in 45 ml. of water and sufficient 0.1 N N a O H to give a nearly neutral solution which was then adjusted to p H 5.85 by cautious addition of 0.02 N acetic acid (fraction II). T o the solution was added 13.8 ml. of aluminum hydroxide gel C~ 5 (dry weight 15.5 g./1.). The suspension was centrifuged after 10 minutes, and the adsorbate washed with 13 ml. of 0.02 M sodium acetate buffer (pH 6.0) and eluted with three 14-ml. portions of 0.02 M phosphate buffer (pH 7.7). The combined eluates (fraction I I I ) were diluted to 48 ml. with water, and 1.0 ml. of 1.0 N acetic acid was added with mechanical stirring at 0 °, followed b y 6.9 ml. of 95% ethanol at - 1 ° to - 2 °. T h e precipitate was centrifuged and dissolved with 20 ml. of water and sufficient 0.1 N N a O H to give a nearly neutral solution which was then adjusted to p H 6.0 with 0.02 N acetic acid (fraction IV). I t was then treated with 21.4 ml. of calcium phosphate gel 8 (dry weight 8.2 g./1.) and centrifuged after 10 minutes. The adsorbate was washed with 17 ml. of 0.02 M sodium acetate buffer (pH 6.0) and eluted with four 4.3-ml. portions of 0.01 M phosphate buffer (pH 7.7). The combined eluates SUMMARY OF PURIFICATION PROCEDURE

Fraction Autolyzate, from 100 g. dried yeast I. Ammonium sulfate II. Ethanol III. Aluminum hydroxide gel eluate IV. Ethanol V. Calcium phosphate gel eluate

Volume, ml.

Total activity, units

162 66 48 41 26 17

4340 7070 4060 3480 3340 1970~

Specific activity, Yield, units/ % mg. protein 163 57 86 96 59

0.91 7.4 20.2 30.2 50.7 83.0

a In repeated preparations, the total activity of fraction V varied between 1600 and 2800 units and the specific activity between 51 and 86 units/mg. 5 R. Willst~itter and H. Kraut, Ber. 66, 1117 (1923). 6 D. Keilin and E. F. Hartree, Proc. Roy. Soc. (London) B124, 397 (1938).

[118]

UDPG PYROPHOSPHORYLASE FROM YEAST

675

(fraction V) were clear and colorless. The protein content varied between 1.3 and 1.9 mg./ml. This fraction represents an over-all yield of 45% and a 91-fold purification as compared to the autolyzate. The enzyme (fraction V), at 3 °, lost 20 to 30% of its activity in 4 days and 58% in 11 days. Since the most purified enzyme fractions contained high concentration of nucleotide pyrophosphatase and inorganic pyrophosphatase, specific inhibitors were employed to avoid their interference in balance studies of FAD synthesis and pyrophosphorolysis.

Properties pH Effect. At pH 6.0 the enzyme is 10%, and at pH 8.4 64%, as active as at pH 7.5. Specificity. ADP does not replace ATP, and metaphosphate and inorganic orthophosphate do not replace PP. With riboflavin in place of F M N and with increased amounts of enzyme, a small amount of FAD is synthesized, indicating the presence of a riboflavin kinase 7 in the preparation. Activators and Inhibitors. Mg ++ is required. The optimal concentration is around 1.5 X 10-s M. Higher concentrations are slightly inhibitory. Dissociation Constants. The values for FMN, ATP, and FAD are, respectively, 1.4 X 10-6 M, 1.2 X 10-5 M, and <5.3 X 10-e M. 7 E. B. Kearney and S. Englard, J. Biol. Chem. 193, 821 (1951).

[118] U D P G Pyrophosphorylase from Yeast 1 U D P G -~- PP ~ U T P -~- Glucose-l-P

By

AGNETE MUNcH-PETERSEN a n d HERMAN ~V[. KALCKAR

Assay Method Principle. The reaction from left to right may be followed by measuring the glucose-l-P formed. The latter is assayed by enzymatic conversion to glucose-6-P with excess phosphoglucomutase; the glucose-6-P is oxidized by T P N in the presence of excess glucose-6-P dehydrogenase (Zwischenferment) so that the rate of the pyrophosphorolysis may be measured in terms of T P N H formation spectrophotometrically at 340 m~. 1 A. Munch-Peterser., H. M. Kalckar, E. Cutolo, and E. E. B. Smith, Nature 172, 1036 (1953).

676

COENZYME AND VITAMIN METABOLISM

[118]

The actual conditions for assay of enzyme concentration will not be described here. Instead, the use of this enzyme for measurement of U D P G concentration by the above principle will be outlined. With minor modification, this procedure is, of course, also suitable as an enzyme assay.

Enzymatic Determination of UDPG by the Specific Pyrophosphorylase (H. M. Kalckar) E n z y m e s . Zwischenferment 2,3 which contains U D P G pyrophosphorylase and glucose-6-phosphate dehydrogenase, 1 or highly purified U D P G pyrophosphorylase (described below) plus purified glucose-6-phosphate dehydrogenase. 4 Purified phosphoglucomutase from muscle. 5 Indicator Substance. T P N (340 mu). Procedure. On addition of inorganic pyrophosphate and the enzymes, T P N is reduced, provided t h a t U D P G is present. An increase in density at 340 m~ of 0.630 6 corresponds to 0.1 micromole of U D P G . If phosphogluconate dehydrogenase is added, the corresponding AE340 is found to be 1.75 × 0.63 = 1.10. 7 The method permits one, therefore, to detect 0.005 micromole of U D P G . If Zwischenferment preparations are used, l0 mg. of an active preparation should be dissolved in 1 ml. of 0.1 M triethanolamine (TEA) buffer, p H 7.5. Ten to t w e n t y microliters (constriction pipet) of such a solution to 1 ml. of the above-mentioned buffer containing 0.0025 M magnesium chloride should give a suitable activity. For small a m o u n t s of U D P G (less than 0.05 ~M./ml.), it is advisable to add an a m o u n t of the pyrophosphorylase which in the indicator system gives a AE~40 of not more than 0.010 to 0.020 per minute. The freshly prepared highly purified U D P G pyrophosphorylase is very active, and as little as 1 ~g. of protein per milliliter would give (in the presence of phosphoglucomutase and glucose-6-phosphate dehydrogenase) a AE340 of 0.040 to 0.050 per minute. In this case only 0.5 ~g. of protein per milliliter of this enzyme should be used. Phosphvglucomutase (Najjar) should be used in excess (e.g., corresponding to a AE340 of about 0.20 to 1.00 per minute as assayed with glucose-l-phosphate and 6-ester dehydrogenase present) and be activated

O. Warburg and W. Christian, Biochem. Z. 254, 438 (1932); see VoL I [42]. s G. A. LePage and G. C. Mueller, J. Biol. Chem. 180, 975 (1949). 4A. Kornberg, J. Biol. Chem. 182, 805 (1950); see Vol. I [42]. 5 V. A. Najjar, J. Biol. Chem. 175, 281 (1948); see Vol. I [36]. 6 B. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1948). B. L. Horecker and P. Z. Smyrniotis, Biochim. et Biophys. Acta (Waxburg Vol.) 12, 98 (1953); see also Vol. III [19].

[118]

UDPG

PYROPHOSPHORYLASE

FROM

YEAST

677

in a small volume with a fresh neutral solution of cysteine. The mutasecysteine mixture is then transferred to the general mixture. If purified enzymes are used, glucose-6-phosphate dehydrogenase purified according to Kornberg 4 ~hould be used also in excess (e.g., hE340 per minute, about 0.20 to 1.00). T P N should be added in a good excess above the expected amount of U D P G ; an appropriate amount would be 0.2 to 0.3 ~M./ml. Inorganic pyrophosphate is usually added in amounts ranging from 0.2 to 0.8 ~M./ml. U D P G may also be determined by its function in the galactowaldenase reaction (see Vol. I [35] and ¥ol. III [143]) and by its oxidation by U D P G dehydrogenase (see Vol. III [143-A]). Purification Procedure (A. Munch-Petersen, unpublished) Twenty grams of dry brewer's yeast is extracted with 40 ml. of 2.2 % (NH4)2HPO4 for 18 hours in a shaking machine. This extract is precipitated with ammonium sulfate, and the fractions between 40 and 60% saturation are dissolved in 25 ml. of 0.013 M acetate buffer, pH 6.3. The solution is chilled to - 3 °, and 25 ml. of 50% ethanol, - 1 0 °, is added dropwise. The mixture, which has been kept in a freezing bath of - 10°, is spun at - 1 2 °. Addition of more ethanol yields a precipitate with some activity, but the first precipitate contains the bulk of the activity. The ethanol fractionation eliminates completely the dehydrogenases of 6-phosphoglucose and 6-phosphogluconate. The active ethanol fraction is dissolved in water and is precipitated with ammonium sulfate in the range between 45 and 60% saturation. When freshly prepared, this highly purified preparation shows an activity per microgram of protein per milliliter, as AE340, of 0.045 per minute at 22 °. The purest enzyme fractions so far obtained are relatively unstable; five days of storage at - 2 0 ° (with thawing and freezing twice) brought about a 50% reduction in activity. It would presumably be possible to retain the activity better by keeping concentrated solutions of the enzymes in several small microtubes or capillaries.

[119]

PYRIDINE NUCLEOTIDE TRANSHYDROGENASE

681

[119] Pyridine Nucleotide Transhydrogenase T P N H -k D P N - - * T P N + D P N H D P N H -k D e a m i n o - D P N - - * D P N ~- D e a m i n o - D P N H

(1) (2)

B y NATHAN O. KAPLAN Assay Method P r i n c i p l e . The usual assay for the enzyme involves the reduction of D P N by T P N H . The T P N is usually present in catalytic amounts and T P N H is generated by the TPN-specific isocitric dehydrogenase from pig heart. The reduced T P N in the presence of the transhydrogenase then reduces the D P N . Some transhydrogenases, such as the enzyme from hog brain, do not promote reaction 1 but will catalyze an exchange between the oxidized and reduced forms of D P N . 1 This type of transfer can be followed by the use of d e a m i n o - D P N as an oxidizing agent for reduced D P N (reaction 2). Assay methods for both reactions 1 and 2 will be described. Reaction 1. The procedure followed is essentially t h a t described by Colowick et al. 2 and can be summarized b y the following equations:

Isocitrate + T P N

pig heart enzyme ~ a-Ketoglutarate + CO2 -[- T P N H transhydrogenase

TPNH + DPN

) TPN + DPNH

Net reaction: Isocitrate -t- D P N --~ D P N H -t- a-Ketoglutarate ~- CO2 Reagents

D P N , 5 t~M./ml. T P N , 2 ~M./ml. D e a m i n o - D P N , 3 5 ~M./ml. Potassium phosphate (pH 7.5), 0.1 M. MgC12, 0.1 M. Sodium D-isocitrate, 0.05 M. Pig heart T P N isocitrate dehydrogenase.* 1N. O. Kaplan, S. P. Colowiek, L. J. Zatman, and M. M. Ciotti, J. Biol. Chem. 205, 31 (1953). S. P. Colowick, N. 0. Kaplan, E. F. Neufeld, and M. M. Ciotti, J. Biol. Chem. 195, 95 (1952). a N. O. Kaplan, S. P. Colowick, and M. M. Ciotti, J. Biol. Chem. 194, 579 (1952) ; see also Vol. III [129]. 4 See S. Oehoa, Vol. I [113].

682

RESPIRATORY ENZYMES

[119]

KCN, 1.0 M. Nicotinamide, 1.0 M. 2'-AMP, 0.04 M Na salt. 5'-AMP, 0.04 M Na salt. Procedure. The reaction mixture in a Beckman cuvette contains 0.10 micromole of TPN, 0.2 ml. of the pig heart enzyme, 0.1 ml. of MgC12, and phosphate to 2.6 ml. Ten micromoles of the isocitrate is added, and the change at 340 mt~ recorded. After the completion of the reaction, 1.0 micromole of DPN is added. The transhydrogenase preparation is then introduced, and the change again followed at 340 m~. This method can be used for crude extracts from yeast, animal, or bacterial sources. In crude preparations, however, it is important to carry out a control which does not include TPN. This is to rule out any contribution to the DPN reduction which might come from a DPN-linked isoeitrate dehydrogenase. Reaction 2. The procedure used is that described by Kaplan et al. 5 In this procedure reduced DPN is generated by the specific DPN isocitrie dehydrogenase from yeast; 6deamino-DPN is not reduced by this enzyme. Hence the transhydrogenase activity can be followed by the reduction of the deamino nucleotide. The sequence of reactions is summarized in the following equations :

yeast DPN isocitrate dehydrogenase ) a-Ketoglutarate + COs + D P N H transhydrogenase D P N H + Deamino-DPN ) DPN + Deamino-DPNH

Isocitrate + DPN +

Net reaction: Isocitrate + Deamino-DPN --~ a-Ketoglutarate + COs + Deamino-DPNH Procedure. The reaction mixture contains 0.15 micromole of DPN, 0.3 ml. of the yeast DPN isocitrate dehydrogenase, 0.1 ml. of M / I O MgC12, 3 micromoles of 5'-AMP, and 2.5 ml. of phosphate buffer. The DPN is reduced by the addition of 10 micromoles of isocitrate. One micromole of deamino-DPN is added, and then the transhydrogenase. The rate of the deamino nucleotide is followed by the change in absorption at 340 m~. Unit of Activity. A unit of activity is defined as the amount of transhydrogenase which will produce an optical-density change of 0.01 in

5N. O. Kaplan, S. P. Colowick,and E. F. Neufeld, J. Biol. Chem. 205, 1 (1953). s See A. Kornberg, Vol. I [118].

[119]

PYRIDINE NUCLEOTIDE TRANSHYDROGENASE

683

1 to 4 minutes after addition of the acceptor nucleotide (DPN in reaction 1, deamino-DPN in reaction 2). Application of Assay to Crude Extracts. The assay procedures can be used in both crude tissue and bacterial extracts. However, the addition of 3 micromoles of KCN should be included to prevent reoxidation of the reduced nucleotides. In the case of the animal preparations, it is essential to add 60 micromoles of nicotinamide to prevent splitting of the oxidized nucleotides by DPNase. The transhydrogenase from Pseudomonas fiuorescens is stimulated by the addition of 2'-AMP. 7 The rate of reaction 1 is increased some twofold by the mononucleotide, whereas reaction 2 is almost completely dependent on the presence of 2'-AMP. The addition of 10 micromoles of the nucleotide to the assay reaction is adequate for optimal activity. The animal enzymes are not influenced by 2'-AMP.

Purification of Pseudomonas Transhydrogenase The enzyme purification described below is essentially that described by Colowick et al. s Reaction 1 was used to assay the enzyme. Preparaiion of Extracts of Pseudomonas fiuorescens. After 2 days' growth at 30 ° on a medium containing 5.0 g. of NaNO2, 1.0 g. of KH2PO4, 0.5 g. of MgSO4-7H20, 5.0 g. of sodium citrate, 4.0 g. of yeast extract (Difco), H20 to 1 1., and N NaOH to pH 7, the cells were collected by a Sharples centrifuge. The yield was about to 2 to 3 g. of packed cells per liter of medium. The cells were frozen in a cold mortar (-15°), then powdered and ground with an equal weight of alumina powder (Alcoa A-301) accompanied by gradual addition of 5 vol. of cold 0.1 M phosphate buffer at pH 7.5. The suspension was centrifuged in the cold at 10,000 rpm for 40 minutes in a Servall angle, and the clear supernatant was stored frozen at - 1 5 °. In this state, the transhydrogenase retains its activity for many months. First Acetone Fractionation. Acetone, precooled to - 1 5 °, was added with stirring to 334 ml. of crude extract, while the temperature was maintained near the freezing point of the mixture, by means of a dry icealcohol bath. The precipitates obtained serially with 40, 43, 46, and 49 % acetone contained negligible amounts of the transhydrogenase and were discarded after centrifugation at - 5 °. The acetone concentration was raised to 52 %, and the precipitate, which contained nearly all the trans7 N. O. Kaplan, S. P. Colowick, E. F. Neufeld, and M. M. Ciotti, J. Biol. Chem. 205, 17 (1953). 8 Filter paper has been used in place of the calcium phosphate gel. 7 However, more recent experiments have indicated t h a t the gel is more suitable t h a n the filter paper. For the preparation of the gel see Vol. I [11].

684

RESPIRATORY ENZYMES

[119]

hydrogenase, was centrifuged at - 5 °. The supernatant fluid was discarded. The precipitate was extracted in the cold with 60 ml. of 0.1 M potassium phosphate, pH 7.5, and after centrifugation the residue was re-extracted three times with 30, 20, and 10 nil. of 0.1 M NaHCO3. The four extracts were combined. As can be seen from Table I, the combined solutions contain practically all the enzymes present in the crude extract, and the over-all purification is about sixfold. It is noteworthy that the enzyme is not readily dissolved after acetone precipitation and that the first material which goes into solution in phosphate buffer is not as high in specific activity as the material which comes out with subsequent additions of NaHCO3. First Adsorption. For 120 ml. of the combined solutions from the previous step, 37 ml. of calcium phosphate gel. s (9 months old, 16 mg. of dry weight per milliliter) was added. After 5 to 10 minutes at about 10 to 15°, the suspension was centrifuged in the cold. The supernatant fluid was treated with a second volume (37 ml.) of the gel as above, and then with 45-, 60-, 60-, and 60-ml. portions. Assay of the supernatant from the sixth gel showed that only a negligible amount of the transhydrogenase was present. Most of the activity was adsorbed on the fifth and sixth gels. The fifth gel was eluted at room temperature, first with 75 ml., then with 15 ml., of 0.1 M potassium phosphate (pH 7.5). The sixth gel was eluted at once with 20 ml., and then all three eluates were combined. The adsorption and elution procedure resulted in an almost quantitative recovery of the enzyme and a purification of approximately fivefold. Second Acetone Fractionation. The combined eluates were precipitated serially with increasing amounts of acetone. The material precipitating at 40 and 46% acetone was inactive. The fraction which precipitates with 60% acetone contains all the activity, higher acetone fractions being completely inactive for transhydrogenase activity. The 46 to 50 % acetone fraction was dissolved in 45 ml. of 0.1 M potassium phosphate (pH 7.5). The suspension was stirred occasionally at 0 ° for 30 minutes to ensure complete solution of the enzyme. This step resulted in doubling the activity, with a 70 % recovery of units. Second Adsorption. Forty-five milliliters of the second acetone fraction was treated stepwise with 18, 18, and 54 ml. of calcium phosphate gel, as described above. Most of the transhydrogenase was adsorbed on the third gel, which was then eluted twice with 18 ml. of 0.1 M potassium phosphate (pH 7.5). The two eluates were combined. This step gives a purification of threefold with a yield of 40%. A summary of the purification is presented in Table I.

[119]

P Y R I D I N E NUCLEOTIDE TRANSHYDROGENASE

685

TABLE I SUMMARY OF PURIFICATION OF TRANSHYDROGENASE FROM Pseudomonas

Fraction Crude extract 49-52% acetone ppt. Phosphate extract First NaHC03 extract Second NaHC03 extract Third NaHCOa extract Combined solutions Calcium phosphate Fifth gel, first eluate Fifth gel, second eluate Sixth gel, first eluate Combined eluates 46-50% acetone ppt. Calcium phosphate, third gel, combined eluates

Total volume, ml.

Total units

Total protein, mg.

334

41,700

1226

60 30 20 10 120

24,600 8,200 4,400 1,640 38,840

150 31.0 11.8 7.4 200

164 264 373 222 194

75 15 20 110 45

19,500 2,400 4,500 26,400 18,000

16.2 2.2 6.4 24.8 7.9

1200 1080 703 1063 2270

36

6,600

0.94

Units/mg. 34

7030

Properties Turnover Number. T h e t u r n o v e r of per 100,000 grams of purified enzyme under optimal conditions is 3000. Effect of Nucleotide Concentration. The concentration of D P N required to produce half-maximal activity is 7 X 10 -5 M ; the T P N level required for half-maximal activity is 5 X 10 -5 M. pH Optimum. The p H o p t i m u m of the Pseudomonas fluorescens transhydrogenase is a p p r o x i m a t e l y 7. Specificity. T h e e n z y m e is capable of p r o m o t i n g the following reactions :7 T P N H + D P N --* T P N + D P N H (1) D P N H + D e a m i n o - D P N --* D P N -t- D e a m i n o - D P N H (2) TPNH + Deamino-TPN-~ TPN + Deamino-TPNH (3) T P N H + N M N --~ T P N -t- N M N H (4) DPNH + NMN -* DPN + NMNH (5) Reaction 1 is reversible only to a very slight extent, and this reversibility is dependent on the use of low concentrations of T P N and phosphate. 2 ' - A M P induces a ready reversal of reaction 1; 2'-AS~IP also influences rcaction 1 in the forward direction, promoting the completeness of the oxidation of T P N H in the presence of phosphate. Reactions 2 and 5 are v e r y m a r k e d l y stimulated b y the mononucleotide. Reaction 3 is not

686

RESPIRATORY ENZYMES

[119]

influenced by 2'-AMP. The mononucleotide is also not essential for reaction 4. The Pseudomonas enzyme also catalyzes an exchange between oxidized and reduced forms of DPN; this reaction can be studied with C It-nicotinamide-labeled DPN. 1 Distribution. An enzyme similar to the Pseudomonas fluorescens transhydrogenase is present in Pseudomonas aeruginosa. ~ The enzyme has also been found in several Azotobacter strains2 The occurrence of the enzyme in E. coli has been reported by Mitsuhashi and Davis. ~° Purification of Pyridine Nucleotide Transhydrogenase from Beef Heart ~ Preparation of Particles. For every gram of fresh tissue, 10 ml. of 0.9% cold KC1 and 0.01 ml. of 1.0 M NaHC03 were added. Homogenization was carried out at 4 ° in a Waring blendor for 2 to 5 minutes. The homogenate was centrifuged for 4 minutes at 1500 r.p.m., and the insoluble material was discarded. The supernatant fluid was then centrifuged for 20 minutes at 16,000 r.p.m., and the particles obtained were suspended in 0.1 M phosphate (pH 7.0) in a volume equivalent to 1 ml. per gram of fresh tissue. High-speed centrifugation results in only a trace of activity in the supernatant, whereas nearly all the enzyme present in the whole homogenate could be recovered in the particles. Extraction of Beef Heart Particles. Warming with 10 % ethanol at 40 ° fails to dissolve the enzyme. Treatment with bile salts results in the solution of the particles, but the transhydrogenase activity is lost. No activity is found in either the extract or the homogenate of an acetone powder of the particles prepared by homogenizing a particle suspension with 20 vol. of cold acetone. Some success in extracting the enzyme can be achieved by treating the particles with digitonin. A 2 % digitonin solution is prepared by suspending 2 g. of digitonin in a few milliliters of water; about 20 ml. of 5 N NaOH is then added with vigorous shaking until the digitonin is dissolved. The solution is carried out with 5 N HC1 and diluted to 100 ml. with water, making the final NaC1 concentration approximately 1 M. The digitonin remains soluble under these conditions. For every milliliter of the particle suspension 1 ml. of the digitonin solution is added, and the mixture is allowed to stand for 20 minutes. The residual particles are then removed by centrifugation. From 50 to 75 % of the transhydrogenase activity is recovered in the supernatant solution. The digitonin extract cannot be frozen, and it loses 50% of the activity when kept at 4 ° for 24 hours. The instability of the extract makes it essential to carry out farther purification immediately. 9 NMN = nicotinamide mononucleotide. lOS. Mitsuhashi and B. D. Davis, Biochim. et Biophys. Acta 15, 54 (1954).

[119]

PYRIDINE NUCLEOTIDE TRANSHYDROGENASE

687

To 180 ml. of the above digitonin extract is added 90 ml. of calcium phosphate gel (12 to 5 mg. dry weight per milliliter), and the mixture is allowed to stand for 30 minutes at 4 ° . The gel treatment completely adsorbs the enzyme after it has been washed twice with 10-ml. portions of M K2HP04 and then eluted with ten 5-ml. portions of M KH2P04. The first three elutions with the primary phosphate contain very little of the enzyme; the enzyme can be almost completely recovered in the fourth to tenth elutions. The adsorption and elution procedures give approximately a tenfold purification. The active eluates should be brought to pH 6.7 and then stored, since under these conditions the transhydrogenase can be kept with little loss of activity in the deep-freeze for several weeks. Properties

pH Optimum. The pH optimum of the beef heart transhydrogenase is approximately 6 to 6.2. Specificity of Reaction. The enzyme will promote reactions 1 and 2 reversibly. It will not promote an exchange between T P N H and deaminoTPN. This indicates a marked difference in specificity between the beef heart and the bacterial transhydrogenase. The beef heart catalyst also promotes an exchange between oxidized and reduced forms of DPN, which can be studied with C14-nicotinamide-labeled DPN. 1 Effect of Inorganic Phosphate and 2'-AMP. These compounds have no influence on the beef heart reactions, in contrast to their action on the Pseudomonas transhydrogenase. Distribution. Table II gives the distribution of transhydrogenase activity in a number of animal tissues. The values were obtained by using the assay system of reaction 1. Although brain preparations seem not to promote reaction 1 to any extent, they readily promote reaction 2. TABLE II DISTRIBUTION OF TRANSHYDROGENASE (REACTION 1) IN ANIMAL TISSUES ~

Rabbit Hog Beef Pigeon

Kidney

Heart

Liver

Brain

Spleen

Limb muscle

50 196 90

46 256 480 12

73 256 0

20 0 0

0

24

Units are described in text.

Breast muscle

0 158

688

RESPIRATORY ENZYMES

[120] D P N H

[120]

C y t o c h r o m e c R e d u c t a s e (Animal)

D P N H -~ 2 Cytochrome c (Fe +++) --~ D P N + + 2 Cytochrome c (Fe ++) + H + B y HENRY R. MAHLER

Assay Method Principle. The reduction of ferricytochrome c by D P N H in the presence of the enzyme is measured spectrophotometrically at 550 m~. This assay method for the enzyme was developed by Edelhoch et al. 1 and somewhat modified by 1VIahler et al. 2 Reagents 0.3 M diol (2-amino-2-methyl-l,3-propanediol) buffer, pH 8.5. 0.3 M glycylglycine buffer, pH 8.5. 1% aqueous ferricytochrome c. 8 0.006 M D P N H 2 Enzyme solution containing 0.20 to 1.20 units/ml, in 0.02 M KHCOa. Procedure. Fill two cuvettes with 0.2 ml. of diol or glycylglycine buffer, 0.1 ml. of cytochrome c, and 0.1 ml. of D P N H solution. Add 2.6 ml. of water to the blank cell and 2.55 ml. of water to the experimental. Determine the optical density at 550 m~ (E550) of the experimental against the blank. At zero time add 0.05 ml. of the enzyme solution to the experimental cuvette, and determine the AEs~0 at 15-second intervals. The AE550 between 15 and 75 seconds constitutes the "initial rate." Definition of Unit and Specific Activity. The unit of enzyme activity is defined as that amount of enzyme which yields an "initial r a t e " of 1.00 per minute at 22 °. Specific activity is defined as the number of units per milligram of protein. Protein is determined by the biuret reaction.4 Application of Method to Crude Extracts. The method is applicable to the determination of reductase activity in homogenates or crude tissue extracts, provided that the following modifications and precautions are observed: (1) the turbidity introduced by the enzyme preparation should not be so great as to make accurate spectrophotometry impossible; (2) 1H. Edelhoch, 0. Hayaishi, andL. J. Teply, J. Biol. Chem. 197, 97 (1952). 2H. R. Mahler, N. K. Sarkar, L. P. Vernon, and R. A. Alberty, J. Biol. Chem. 199, 585 (1952). a Both eytochrome c and DPNH are obtainable commercially. For the preparation of these substances see Vol. II [133] and Vol. III [127]. 4A. G. Gornall, C. J. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949).

[120]

DPNH CYTOCHROME C REDUCTASE (ANIMAL)

689

the reaction is carried out in 0.05 M phosphate buffer, pH 7.4, in the presence of 0.001 M KCN. Purification Procedure 2

The method to be described has been repeated over one hundred times in the author's laboratory by a variety of workers. Although there is some variability in the activity of the first soluble extract, the same degree of purification has been reproducibly observed. The purification scheme makes use of the ease with which this enzyme can be separated from other proteins by means of ammonium sulfate fractionation at different hydrogen ion concentrations and in the presence or absence of phosphate ions. Step 1. Preparation of Lyophilized Extract. 4500 g. of chilled, diced, washed pig heart muscle is homogenized for 2 minutes in Waring blendors with 13.5 1. of 0.02 M K2HPO4. (All operations, except when otherwise indicated, are carried out at temperatures between 0 and 4°.) Cellular debris and some mitochondria are removed by centrifugation at 2000 × g for 30 minutes. The supernatant is brought to pH 5.4 by means of acetic acid, and the residue collected by centrifugation at 2000 × g. The sediment is washed by suspension in 2 1. of glass-distilled water and again centrifuged at 2000 X g for 25 minutes. The particles are then extracted with 700 ml. of 10% ethanol for 15 minutes at 42 to 44 °. The suspension is chilled rapidly, and insoluble material is removed by centrifugation for 20 minutes at 2000 X g. The yellow supernatant, containing the enzyme, is then lyophilized. The yield is 1.5 to 2.5 g. of dry, light tan powder of specific activity 20 to 35. Step 2. Acid Ammonium Sulfate Fractionation. Two grams of protein (3.5 to 4.0 g. of lyophilized powder) is dissolved in 300 ml. of distilled water, and 60 g. of solid (NH4)2SO4 is added slowly with thorough stirring. The preparation is allowed to stand for 20 minutes and is then centrifuged. The precipitate is discarded, and 90 g. of solid (NH4)2SO4 is added to the supernatant. The precipitate is centrifuged down and dissolved in 0.05 M phosphate buffer, pH 7.2, to a final volume of 75 ml. Step 3. Ammonium Sulfate Fractionation in Phosphate at pH 8.0. To the solution is added 19.5 g. of solid (NH4)2SO4. After standing for 15 minutes, the precipitate is collected by centrifugation and dissolved in 0.05 M phosphate, pH 7.2, to a final volume of 55 ml. Then 10.5 g. of solid (NH4)2SO4 is added slowly, the pH being maintained between 8.0 and 8.2 by means of concentrated ammonium hydroxide. The precipitate, collected by centrifugation, is discarded, and 3.0 g. of solid (NH4)~SO4 is added to the supernatant, the pH again being maintained between 8.0

690

RESPIRATORY ENZYMES

[19.0]

and 8.2. T h e solution is centrifuged and the precipitate dissolved in 15.0 ml. of H~O. (Fraction 6.) Step 4. Refractionation with Ammonium Sulfate. 3.25 g. of (NH4)2S04 is added to the solution. T h e precipitate formed is centrifuged out and dissolved in 15.0 ml. of H20. This solution shows a specific activity of 110 to 150 and is a very satisfactory enzyme for most purposes. B y the addition of 0.450 g. of (NH4)~S04 to the supernatant, a precipitate is formed which, when dissolved in 10.0 ml. of H20, shows specific activities from 140 to 180. Additional Purification. As can be seen from the table, recoveries are good up to and including the precipitation with neutral (NH4)2SO4 b u t drop rapidly thereafter. The yield of purified enzyme m a y be increased b y refractionation of the first precipitate obtained at p H 8.0 (step 3 above). SUMMARY OF PURIFICATION PROCEDURE

Fraction 1. 2. 3. 4. 5. 6. 7.

Homogenate Washed ppt., pH 5.4 Lyophilized extract Ppt. after acid (NH~)2SO4 Ppt. after neutral (NH~)2SO4 Ppt. after (NH4)~SO4at pH 8 Ppt. after final (NH4)2SO~

Specific activity, units/mg,

Total activity, units

Net protein, rag.

Recovery, %

0.377 a 1.18 ~ 30.0 48.2 118 152 195

45,200 ~ 36,700 a 30,000 28,000 25,250 12,160 7,880

120,000 31,000 1,000 560 214 80 42.8

-81 66 62 56 28 17.5

a Reductase measured in 0.05 M phosphate, pH 7.4, in the presence of 10-3 M cyanide. If a preparation shows lower specific activities than those indicated in the table, it can usually be further improved b y removal of some of the impurities b y means of calcium phosphate gel. 5 Conditions used were 0.5% protein solution and 3 to 5 mg. dry weight of gel per each 10 mg. of protein. Preparations with a specific activity of 180 to 190 could not be purified further, regardless of the m e t h o d tried. Such procedures as methanol, ethanol, or acetone ffactionation and adsorption on calcium phosphate and alumina C~ gels improve the absorption spectrum of the enzyme b y removing the last traces of persistent hematin impurities b u t are without effect on specific activity. Stability of Enzyme. T h e lyophilized extract is stable for several m o n t h s at - 1 2 °. T h e purified enzyme is best s t o r e d as a solution, 1% 5See Vol. I [11] for a description of the preparation of this gel.

[:19.0]

DPNH CYTOCHROME C REDUCTASE (ANIMAL)

691

in enzyme protein and 1 to 2 % with respect to albumin, in the frozen state at - 1 0 °. Such preparations show little loss of activity during several weeks of storage. At 0 ° they are stable for several hours; at 30 ° half the enzymatic activity is destroyed in 30 minutes.

Properties Physical Constants. Enzyme of specific activity 190 is electrophoretically homogeneous. In phosphate buffer, pH 7.1 and 0.05 molal, the mobility was - 2 . 5 X 10-5 cm. 2 volt -1 sec.-1; in glycylglycine buffer, pH 8.25 and ionic strength 0.05, it showed a mobility of - 3 . 3 X 10-~ cm. ~ volt -1 sec. -z. The sedimentation constant corrected to water at 20 ° was 5.1 to 5.3 at 59,780 r.p.m., and a protein concentration of 0.80% at pH 8.25. From physicochemical measurements the molecular weight was calculated to be 75,000 to 80,000; from flavin content (see below) a value of 78,000 was found, assuming one flavin per molecule; and from iron content, assuming four iron atoms per enzyme molecule (see below), the molecular weight could be determined as 80,000. Appearance and Absorption Spectrum. Solutions of highly purified DPNH-cytochrome c reductase with a specific activity of 175 or better are dark yellow, show a three-banded spectrum, characteristic of fiavoproteins, and do not fluoresce. Unless the last traces of a very persistent hemin impurity with a maximum at 412 mt~ are removed, the flavin spectrum is completely obscured. After removal of this impurity, the absorption spectrum of the enzyme shows maxima at 440, 355, and 275 m~. This is unusual, since in most other flavoproteins the two flavin maxima at 450 and 370 m~ are shifted about 5 to 10 mt~ toward longer wavelengths. The enzyme is easily reduced by chemical agents, such as dithionite or sodium borohydride. Reduction with borohydride is reversible, but some denaturation always accompanies dithionite reduction. A certain amount of residual absorption between 400 and 500 mtL is present after reduction. The reduced enzyme is easily oxidizable by air, but when anaerobicity is maintained the enzyme can be reduced by excess DPNH. Prosthetic Groups. The enzyme contains a flavin adenine nucleotide not identical with FAD (by paper chromatography and D-amino acid oxidase test) but giving the correct analysis for FAD (or a mixture of FMN and AMP). The best preparations contained 1.03 X 10-~ M flavin in a 0.80% protein solution (0.50% ribofavin by weight). Iron is also part of the prosthetic group. ~ Four preparations showing specific activities of 18.0, 34.0, 78.0, and 190 (corresponding to estimated enzymatic purities 6 H. R. Mahler and D. Elowe, J. Am. Chem. Soc. 75, 5769 (1953) ; J. Biol. Chem. 210, 165 (1954).

692

RESPIRATORY ENZYMES

[120]

of 9, 17, 40, and 95%) had iron contents of 0.250, 0.453, 1.05, and 2.67 7 of Fe per milligram of protein. Thus the ratio enzyme units per microgram of Fe is constant and equal to 73 + 2. A similar constancy obtains for iron:flavin ratios. I t equals 4.1 + 0.1. Samples of the enzyme show varying distributions of the metal between its two valence states. The substrate, D P N H , is capable of reducing all the iron to the ferrous form, but ferricytochrome c, even in considerable excess, does not convert all the iron initially present in the ferrous form to the ferric state. Thus not only the flavin but the iron as well is capable of being reduced by the substrate and oxidized by the acceptor. Kinetics and Turnover. 7 The Michaelis-Menten constants (Kin) and limiting velocity (V .... ) determined at an enzyme concentration of 10.9 M are, for D P N H , 1.9 × 10-5 M and 3.8 × 10-e M X min. -I, respectively; for cytochrome c, 1.2 X 10-4 M and 3.5 X 10-~ M × min. -1. The turnover number (moles of cytochrome c reduced per mole of enzyme) equals 13,000 at low enzyme concentrations. The activation energy for the enzymatic reaction has been determined as 12,600 in the range 0.0 to 21.0 °. Specificity. The enzyme is specific for D P N H . T P N H shows no activity. Besides cytochrome c, various dyes can be used as electron acceptors. Thus the enzyme shows diaphorase 8 action as well. The flavin part of the prosthetic group but not the iron is probably involved in the catalysis of dye reduction. Activators and Inhibitors. ANIONS. Citrate inhibits 85% at a concentration of 2 X 10-3 M. Pyrophosphate (after incubation with the enzyme for 2 minutes) also shows a similar degree of inhibition at comparable concentrations. Phosphate, vanadate, and perchlorate inhibit about 75% at 10-2 M. Arsenate, sulfate, and chloride are less effective inhibitors in the order mentioned, at this concentration, and C N - shows about 20% stimulation. Citrate and pyrophosphate inhibition is overcome competitively by cytochrome c, whereas phosphate inhibition is not. CATIONS. Cations also exert pronounced inhibitory action on enzymatic activity, divalent ions being most effective. To obtain 100% inhibition of reductase activity under standard assay conditions, mercuric ions at 10-4 M, cupric or zinc ions at 10-8 M, calcium, magnesium, or manganous ions at 10-2 M, and barium at 0.1 M are required. Sodium and potassium show only 40 % inhibition at 10-: M. L. P. Vernon, N. K. Sarkar, and H. R. Mahler, J. Biol. Chem. 199, 599 (1952). 8 See Vol. II [124] for a discussion of diaphorases and their relation to cytochrome reduetaees.

[121]

DPNH CYTOCHROME C REDUCTASE (BACTERIAL)

693

- - S H INHIBITORS.At a concentration of 10-4 M, p-chloromercuribenzoate shows 100% inhibition of both reductase and diaphorase activity. At the same concentration, arsenite, iodoacetate, and iodosobenzoate as well as organic arsenicals are completely ineffective. At 10-~ M arsenite inhibits 35 %, iodoacetate 38 %, and iodosobenzoate 100 %. FLAVINS AND FLAVIN ANALOGS. Atabrin (quinacrine) inhibits completely at 10-3 M, FAD at 10-4 M, and riboflavin at 10-3 M. FMN shows 50 % inhibition at 10-3 M. METAL-BINDING AGENTS. Most metal-binding agents not only do not inhibit at all but may even show slight stimulation of reductase activity. On incubation with o-phenanthroline (10-3 M) in the presence of D P N H (10-5 M), however, the activity with cytochrome e is lowered about 50 % whereas the capacity for dye reduction of the enzyme remains unimpaired. Similar results are obtained when the enzyme is dialyzed versus 5 X 10-4 M 8-hydroxyquinoline or is exposed to pH 4.0. Reductase activity of such preparations can be stimulated by preincubation of the enzyme with Fe +++ at 10-4 M. OTHER INHIBITORS. BAL9 and Antimycin 1° have been found to inhibit the reoxidation of D P N H by cytochrome c as carried out by mitochondrial preparations; these two inhibitors are completely inactive when tested with the soluble preparation described here. Deoxycorticosterone at a level of 1 mg./ml, when preincubated with the enzyme for 25 minutes inhibits 100%; cortisone under the same conditions is completely ineffective. pH Optimum. The initial rate of cytochrome c reduction is optimal at pH 8.5 in glycylglycine or diol. It is 60% of optimal at pH 7.5 and 80% at pH 9.5. 9 E. C. Slater, Biochem. J. 46, 484 (1950). ~0 V. R. Potter and A. E. Reif, J. Biol. Chem. 194, 287 (1952).

[121] D P N H C y t o c h r o m e c R e d u c t a s e (Bacterial) 2 Cytochrome e (Fe+++) ~ DPNH--~ DPN + 2 Cytochrome c (Fe++) -t- H +

By

ARNOLD F. BRODIE

Assay Method

Principle. The assay of this enzyme is based on a method originally used by Warburg to study the iron-containing respiratory enzyme and on the observation by Keilin I that reduced cytochrome c can be distini D. Keilin, Proc. Roy. Soc. (London) B98, 312 (1925).

694

RESPmATORY ZNZY~ES

[121]

guished from the oxidized state by its absorption spectrum. Essentially the spectrophotometric method involves following the formation of reduced cytochrome c by the increase in absorption at 550 m/~ or by following the disappearance of dihydrodiphosphopyridine nucleotide (DPNH) by the decrease in absorption at 340 m~. The method described below is similar to that first used by Haas et al. 2 for measuring the activity of TPN-linked reductase in liver. Reagents

D P N H solution (1.5 X 10-3 M). The reduction of this coenzyme (purity 0.65) is accomplished by a modification of the method of Gutcho and Stewart2 Two milliliters of a sodium hydrosulfite solution (2 mg. of Na2S~O4 per 1 ml. of 0.1 M dibasic sodium phosphate) is added to 15.5 mg. of DPN and placed in boiling water for 1 minute and then cooled immediately in an ice bath. The pH is adjusted to 7.4 with monobasic phosphate, and the volume is increased to 10 ml. The material is vigorously aerated until no further changes occur in the optical density at 340 m~. (See also Vol. I I I [126].) Enzymatieally reduced DPN prepared with yeast alcohol dehydrogenase can be used to provide a continuous source of D P N H and is useful in manometric experiments in which the oxygen consumption is measured. Cytochrome c solution (4 X 10-4 M). Pyrophosphate buffer (0.1 M), pH 8.0. Enzyme. Dilutions of the enzyme in either distilled water or 0.1 M phosphate buffer are used which will completely reduce (2.5 X 10-5 M) cytochrome c in 3 to 5 minutes. Procedure. The enzymatic activity is determined by following the rate of formation of ferrocytochrome c spectrophotometrically at 550 mp in the presence of reduced DPN. Before addition of the reductase, the system, consisting of buffer (1 ml. of 0.1 M pyrophosphate, pH 8.0), 500 of reduced DPN, and 0.2 ml. of cytochrome c (4 X 10-4 M), is incubated in a 3-ml. cuvette at 30 ° for 5 minutes. Readings at 550 mp are then taken at 30-second intervals before and after the mixing with 0.2 ml. of the enzyme. Definition of Unit and Specific Activity. One unit of enzyme activity is defined as that amount which causes an initial rate change in the optical density (AE550) of 1.0 per minute under the above conditions. The

2E. Haas, B. L. Horecker, and T. R. Hogness, J. Biol. Chem. 136, 747 (1940). s S. Gutcho and E. Stewart, Anal. Chem. 20, 1185 (1948).

[121]

DPNH CYTOCHROME C REDUCTASE (BACTERIAL)

695

specific activity is expressed as units per milligram of protein as defined by Horecker 4 for the yeast T P N reductase. Application of Assay Method to Crude Homogenate Preparations. Direct spectrophotometric analysis of crude extracts are difficult to ascertain owing to the opacity of the crude material. These preparations can be assayed for D P N H - o r TPNH-linked flavoproteins in general by means of indicators such as 2,3,5-triphenyltetrazolium chloride (TTZ) or neotetrazolium (NTZ). These tetrazolium salts have been shown to be specific for flavoproteins ~-7 and can be followed aerobically in a Beckman spectrophotometer or photoelectric colorimeter. The system consists of 1 ml. of 0.1 M pyrophosphate buffer, pH 8.0, 1.5 X 10-4 M N T Z or TTZ, 1 X 10-4 M D P N H , dilution of crude extracts, and water to a final volume of 3 ml. The tubes are incubated at 30 °, and the insoluble formazan formed is extracted with either acetone or pyridine and read at 530 m# for NTZ or 485 m# for TTZ. The formation of formazan is linear with respect to the D P N H concentration. With crude extracts, care must be taken to subtract the high endogenous activity from the complete system containing reduced DPN. Aeration of the crude extracts for 15 to 20 minutes will usually reduce the endogenous activity. The reduction of NTZ is approximately seventeen times slower than the reaction of the reductase with cytochrome c. It is best to use fresh extracts or homogenates when using the tetrazolium method as a crude assay, since aged preparations show an increased reduction of this dye. Although the assay system can be run aerobically at high pH, the formation of formazan is considerably increased under anaerobic conditions.

Purification Procedure Escherichia coli, strain ECFS, was used to obtain this enzyme, although it has been obtained by a similar procedure from other strains of E. coli and other organisms. Step 1. Preparation of Crude Extract. The organism is grown for 12 to 15 hours in peptone-yeast extract broth in 5-1. quantities and harvested by centrifugation. The cells are washed with distilled water and disintegrated by sonic vibration for 45 minutes at 9 kc. in a Raytheon magnetoconstrictor oscillator. This is followed by centrifugation at 13,000 r.p.m. (20,000 × g) for 45 minutes in a Servall angle centrifuge at 0 °, and the resulting clear yellow solution is pooled. 4B. L. Horecker, J. Biol. Chem. 183, 593 (1950). s A. F. Brodie and J. S. Gots, Science 116, 588 (1952). E. Kun, Proc. Soc. Exptl. Biol. Med. 78, 195 (1951). 7E. Shelton and W. C. Schneider,Anat. Record 112, 61 (1952).

696

RESPIRATORY ENZYMES

[121]

Step 2. Heat Treatment. This enzyme proved to be relatively heat stable. The yellow s u p e r n a t a n t solution is treated in a water bath for 10 minutes at 60 ° in order to remove the heat-labile proteins; the tube is immediately cooled in an ice bath and recentrifuged. T h e precipitate is discarded. Step 3. Fractionation with Ammonium Sulfate. T h e active s u p e r n a t a n t is then fractionated with a m m o n i u m sulfate at 0.2, 0.3, 0.4, and 0.55 saturation. T h e precipitate of the 0.4 to 0.55 saturation contains almost all the reductase activity. I t is collected and dialyzed against M/15 K H 2 P 0 4 at p H 6.0 for 12 hours with frequent changes. At this stage an active preparation is obtained which can be stored in a deep-freeze or lyophilized. Step 4. Treatment with Protamine Sulfate. F u r t h e r purification is obtained by precipitation of the nucleoproteins with protamine sulfate. The p H of the extract is adjusted to 6.5, and protamine sulfate solution (25 mg./ml.) is added dropwise until the spectrophotometric absorption at 260 m~ becomes less t h a n the absorption at 280 m~. The protamine precipitate is removed b y centrifugation before each reading, and 0.05 ml. of enzyme solution to be tested is added to 3 ml. of distilled water. After removal of the nucleoproteins the s u p e r n a t a n t is reprecipitated with ammonium sulfate and dialyzed. Active preparations can be obtained at this stage of purification and stored at - 5 ° for about two weeks. Step 5. Treatment with Tricalcium Phosphate. T h e dialyzed material from step 4 is adjusted to p H 8.0 to 9.0 and treated with four-month-old tricalcium phosphate (10 g./1.) prepared by the method of Sumner and O'Kane. 8 One milliliter of the tricalcium phosphate solution is added for TABLE I SUMMARY OF PURIFICATION PROCEDURE a

Fraction Sonic extractb Heat treatment (NH4)2SO4 fraction, 0.4-0.55 Protamine treatment Tricaleium phosphate eluate

Total Specific volume, Total Protein, activity, Recovery, ml. Units/ml. units mg./ml, units/mg. % 60 47 5 3.8 5

76 70.5

4550 3313

40.2 10.5

1.9 7.0

128 126

640 480

5.9 4.2

21.6 30.2

67

335

1.2

56

-73 14 10.5 7.3

a Data were compiled from different experiments and do not represent one fractionation experiment. Assay of activity obtained with neotetrazolium. s j. B. Sumner and D. O'Kane, Enzymologia 12~ 251 (1948).

[121]

DPNH CYTOCHROME C REDUCTASE (BACTERIAL)

697

every 10 rag. of protein and allowed to stand at room temperature f o r 10 minutes. The material is cooled and centrifuged at 0 °, and the supern a t a n t is readjusted to p H 6.8 and stored. B y this procedure a yellow solution was obtained with a 28- to 30-fold increase in activity. The best preparation had a specific activity of 58 units/rag. The enzyme can be stored at - 5 ° for about two to three weeks without great loss in activity. The purification steps are summarized in Table I.

Properties Specificity. The purified enzyme is specific for D P N H and is completely inactive in the presence of reduced T P N . The reductase will mediate the transfer of electrons from D P N H to cytochrome c at a greater rate than any other electron acceptor tested. I t has diaphorase-like properties in t h a t it will also transfer electrons to oxygen, tetrazolium salts, nitrofurans, 9 and Janus Green B. Bacterial reductases from different strains v a r y as to the types of acceptors and rates of reduction. The reductase from E. col~, strain 26, could not be demonstrated to link to oxygen. TABLE II RATES OF I~EACTION OF BACTERIAL AND YEAST REDUCTASE

Reaction with

Enzyme

DPNH k~~

Cytochrome c k2

Oxygen ks

Bacterial DPNH cytochrome c reductase b 14 × 104 740 X 104 1.7 X 104 Yeast TPNH cytochrome c reductase ~ -530,000 X 104 0.8 X 104 Old yellow enzymec -3 X 104 10 X 104

Neotetrazolium k4

44 X 104 ---

k = rate constant = liter X min. -1 X mole-1. bA. F. Brodie, J. Biol. Chem. 199, 835 (1952). E. Haas, C. J. ttarrer, and T. R. Hogness, J. Biol. Chem. 143, 341 (1942). The rate constant, k, for the reductase has been calculated b y the method described b y Haas et al.l° for TPN-linked cytochrome c reductase. These values are given in Table I I along with those given b y Haas et al. for comparison. Nature of the Prosthetic Group. The reductase has a typical flavoprorein spectrum exhibiting strong absorption in the ultraviolet, with a maxi9 The rate constant, k, for 5-nitro-2-furaldehyde semicarbazone was found to be 19 X 104 (A. F. Brodie and J. S. Gots, unpublished observations). 10E. Haas, C. J. Harrer, and T. R. Hogness, J. Biol. Chem. 143, 341 (1942).

698

RESPIRATORY ENZYMES

[121]

mum at 255 m#. In the visible range the oxidized enzyme has maxima at 370 and 460 m#. In the presence of excess D P N H , the enzyme, in the reduced state, loses its absorption at 460 m#. The reductase was treated according to the method of Warburg and Christian n in order to remove the flavins. By this method an apoenzyme was obtained which was inactive in the presence of ferricytochrome c and reduced D P N and which could be reactivated only in the presence of flavin adenine dinucleotide (FAD). Neither riboflavin nor flavin mononucleotide had any effect. Further evidence for the nature of the prosthetic group was provided by the ability of the heated reductase to reactivate the apoenzyme of pig kidney D-amino acid oxidase and by its similarity in R~ value to FAD on paper chromatograms. The flavin content of the bacterial reductase was also assayed by hydrolyzing the enzyme according to the method of Scott et al. 12 followed by fluorometric analysis. Enzymatic Activity. In the presence of excess D P N H the reduction follows a first-order reaction with respect to the cytochrome e concentration. The dissociation constant, K, of the protein component and flavin adenine dinucleotide is 82 × 10-9 mole 1.I This value is of the same order as has been calculated for other flavoproteins and indicates a high degree of stability for the reductase. The Michaelis constant as determined by the method of Lineweaver and Burk 1~ for the reductase and D P N H is 5.9 × 10-5 mole/1. Inhibitors. The reductase appears to require - - S H groups for its activity, since it is inhibited by low concentrations of p-chloromercuribenzoic acid (10-~ M). With this concentration, 81% inhibition was observed. Atabrin and acriflavine (10-3 M) caused 70 and 15% inhibition, respectively. Since these inhibitors have been shown by Hellerman et al.14 to be specific for flavoprotein, it is taken as further evidence for the nature of the prosthetic group. Cyanide, azide, urethan, and dinitrophenol (10-~ M) failed to show any inhibition. Antimycin A (1000 ~,) similarly had no effect. Effect of pH. The pH optimum of the bacterial reductase is 8.0. Phosphate does not appear to be necessary for enzymatic activity, as the enzyme shows similar activity in the presence of tris(hydroxymethyl)aminomethane buffer. Reduction of E. coli Hemochromogen. Experimentally the reductase can be shown to reduce an isolated hemochromogen from E. coli 15 as well ~1 O. Wavburg and W. Christian, Bioehem. Z. 298, 150 (1938).

~ M. L. Scott, F. W. Hill, L. C. Norris, and G. F. tIeuser, J. Biol. Chem. 165, 65 (1946). ~8H. Lineweaver and D. Burk, J. Am. Chem. Soe. 56, 658 (1934). ~4L. Hellerman, A. Lindsay, and M. R. Bovarnick, J. Biol. Chem. 163, 553 (1946). is A. F. Brodie and V. Vely, unpublished observations.

[122]

T P N H CYTOCHROME C REDUCTASE FROM YEAST

699

as mammalian cytochrome c. This hemochromogen appears to act physiologically as the terminal acceptor for the flavoprotein and can be reoxidized b y mammalian cytochrome oxidase although spectroscopically it differs from cytochrome c.

[122] T P N H

Cytochrome

T P N + -~ Zwischenferment ~

c Reductase

RCHO Glucose-6-phosphate

f r o m Y e a s t 1,2 ~- H~O

-~ TPN H ~ Zwischenferment ~ R C O O H W H + H + ~- T P N H ~ Reductase--* T P N + -~ Leuco Reduetase Leuco Reductase ~ 2 Ferricytochrome c --~ Reductase ~ 2 Ferrocytochrome c ~- 2H +

(1) (2) (3)

B y ERWZN HAAS

Assay Method Principle. In the enzyme system outlined above, T P N is rapidly reduced, whereas the oxidized cytochrome c does not react until the reductase is finally added. The reduction of ferricytochrome c is measured spectrophotometrically, and the concentrations of all components are so adjusted t h a t the rate of reduction is proportional to the reductase concentration. Each of the components of the system is sufficiently pure so t h a t no reaction proceeds in the absence of the cytochrome c reductase. Reagents

Glueose-6-phosphate. Good yields of the crystalline calcium 3 salt were obtained with extracts of top ale yeast, prepared at 0 °, as source of the phosphorylating enzymes. The b o t t o m yeasts which were available here and which had been autolyzed at 35 °, according to previous directions, were not satisfactory. The calcium salt (1.0 g.) is converted into the potassium salt with 3.15 ml. of M potassium oxalate (0.995 mole). Zwischenferment. The protein component of the T P N dehydrogenase was prepared according to the directions of Warburg and Christian. ~ This is a relatively impure product, b u t it is free of E. Haas, B. L. Horecker, and T. R. Hogness, J. Biol. Chem. 13G, 747 (1940). 2 E. Haas, C. J. Harrer, and T. R. Hogness, J. Biol. Chem. 143~ 341 (1942). 8 The calcium salt is available commercially. For methods of isolation or synthesis of this compound see Vol. III [19]. 40. Warburg and W. Christian, Biochem. Z. 254, 438 (1932).

700

RESPIRATORY ENZYMES

[122]

cytochrome reductase. The Zwischenferment obtained by the m e t h o d of N e g e l e i n a n d Gerischer, ~ d e s p i t e t h e f a c t t h a t its p u r i t y is fifty t i m e s as high, c o n t a i n s some of t h e r e d u c t a s e . T P N 6 was p r e p a r e d f r o m horse l i v e r i n a p u r i t y of 80 % b y a m o d i fication of W a r b u r g ' s p r o c e d u r e . 7 C y t o c h r o m e c. 8 P r o c e d u r e . T h e e x p e r i m e n t a l c o n d i t i o n s a n d t h e m e t h o d of calculat i o n for t h e a s s a y of c y t o c h r o m e r e d u c t a s e are s h o w n i n T a b l e I. All con-

TABLE I ~ I0 = light intensity (in arbitrary units) after passing through blank cell containing no cytochrome. I -- light intensity after passing through cell containing the cytochrome. I -- length of cell = 0.32 cm. X = 550 mtL. aox. (for CyFe +++) = 0.0956 X 108 cm2 X mole-1. a,ed. (for CyFe ++) = 0.281 X 108 era2 X mole-1. c = total cytochrome concentration = 5.4 X 10-8 mole/ml. (CyFe +++) = concentration of oxidized eytochrome e (mole/ml.). (CyFe+++) = 1/l log I o / I - ar,d. X c Otox

--

O~red.

Temperature, 25°; gas phase, air. 1.0 ml. of 0.025 M phosphate buffer, p i t 7.3. 0.90 rag. of potassium hexose monophosphate. 0.10 mg. of Zwischenferment. 0.20 mg. of TPN. 0.86 mg. of eytochrome e. Time min.

1o I-

(CyFe+++), moles/ml. X 108

0

1.47

5.40

1 2 3 4 5 6

1.62 1.74 1.87 1.99 2.10 2.23

4.70 4.14 3.62 3.16 2.76

2.36

log (CyFe +++) -{- 8 A log (CyFe +++)

0.732 Added 0.0O042mg. reductase 0.672 0.060 0.617 0.115 0.559 0.173 0.500 0.232 0.440 0.292 0.373 0.359

A log (CyFe+++), At rain. -1

0.060 0.058 0.058 0.058 0.058 0.060

" Reproduced from J . Biol. Chem. 136, 747 (1940). 5 E. Negelein and W. Gerischer, Biochem. Z. 284, 289 (1936); see Vol. I [42]. 6 TPN is commercially available. For methods of isolation see Vol. I I I [124]. 70. Warburg, W. Christian, and A. Griese, Biochem. Z. 282, 157 (1935). 8 Cytochrome c is available commercially. For methods of isolation see Vol. I I I [133].

[122]

TPNH CYTOCHROME C REDUCTASE FROM YEAST

701

stituents except the cytochrome reductase were added to the cell, and a few minutes were allowed for the reduction of TPN. The reduction of ferricytochrome c is measured spectrophotometrically at 1-minute intervals after the addition of the cytochrome reductase. Definition of Specific Activity. The rate of reduction of ferricytochrome e is proportional to the reductase concentration in the range between 0.2 and 1.0 % and the specific activity of the enzyme (W) can be expressed by W = A log (CyFe +++) [rain._ 1 × mg._l] At X mg. enzyme W is independent of the cytochrome c concentration; for the pure enzyme, 1. W has a value of 158 mg. X min." Purification Procedure

Source Material. Top ale yeast, Saccharomyces cerevisiae I, Hanson, is washed at 0 ° with tap water, pressed to remove excess of water, and dried at room temperature in a stream of air. Step 1. Extraction of Enzyme by Autolysis. Four kilograms of the dry yeast is suspended in 14 1. of water and kept for 33 hours at 20 °. The suspension is centrifuged, 6.9 1. of clear solution being obtained which contains most of the enzyme. The residue is washed with 6.5 1. of water, centrifuged off, and discarded. The supernatant solutions are combined to give 13.8 1. of extract which contains the enzyme. I t is easily denatured, and the following procedures therefore must be carried out at 0 °. Step 2. Fractionation with Ammonium Sulfate. For the precipitation of the enzyme, 4.2 kg. of solid ammonium sulfate (0.51 saturation) is added to the solution with stirring, followed by 220 ml. of 10 N acetic acid (pH 4.5) and centrifugation. The precipitate, which has a volume of 1.1 1., is suspended in 0.7 1. of water, and 3 1. of a solution, 0.31 saturated with ammonium sulfate, is added. The insoluble material is separated by centrifugation and discarded. From the supernatant solution (4 1.) the enzyme is precipitated by adding 470 g. of ammonium sulfate (0.51 saturation) and centrifugation. Step 3. Dialysis. The precipitate is dissolved in 500 ml. of water, and the solution is dialyzed for 17 hours and centrifuged for the removal of much inactive sediment. Step 4. Precipitation with Ethanol. The resulting solution (1200 ml.) is adjusted to pH 4.6 with a few milliliters of N KOH, and 600 ml. of cold 30% ethanol is added. After 45 minutes a fine white precipitate which contains the enzyme is collected by centrifugation. This precipitate is lyophilized.

702

RESPIRATORY ENZYMES

[122]

Step 5. Adsorption on Aluminum Hydroxide Gel. The enzyme preparation, about 5 g., is dissolved in 200 ml. of water and adjusted to pH 9 with 4 ml. of N KOH. C~-Aluminum hydroxide gel 9 is added in fractions (approximately 7 g. is required) and stirred until the supernatant solution just becomes colorless. Excess adsorbent should be avoided. The enzyme is eluted from the aluminum hydroxide by twice adding 120 ml. of a solution which is 0.64 saturated with (NH4)~S04 and contains 0.1 N NH4OH. The elution is continued with two 160-ml. portions of 0.40 saturated (NH4)~SO4 in 0.1 N (NH4)OH. The enzyme is precipitated from the combined eluates (560 ml.) by adding 90 g. of solid (NH4)2S04 (0.65 saturation) and 70 ml. of 2 M acetate buffer at pH 4.5. The precipitate, separated by centrifugation, is dissolved in 150 ml. of water, and the solution is adjusted to pH 9 with about 5 ml. of N ammonium hydroxide. Step 6. Adsorption on Calcium Phosphate. Tricalcium phosphate gel (about 17 g.) is added fractionally to the enzyme solution, stirred, and centrifuged until the supernatant becomes colorless. The precipitates are combined and washed with 400 ml. of water, and the enzyme is eluted with three 200-ml. portions of 0.2 M phosphate buffer, pH 6.1. From the combined eluates (600 ml.) the enzyme is precipitated by addition of 310 g. of (NH4)2S04 and eentrifugation and then redissolved in 70 ml. of 0.03 N NH4OH. This removes phosphate which interferes with the next purification step. TABLE II SUMMARY OF PURIFICATION PROCEDURE

Fraction 1. Extract of autolyzed yeast 2. (NH4)~S04fractionation, 0.31-0.51 saturation, pH 4.5 3. Dialysis 4. Fractionation with 10% Ethanol ~Adsorption on Al(OH)a 5. ~Elution with (NH4)zSO4 ~Adsorption on Ca~(P04): 6. t Elution with phosphate Adsorption on A1(OH)s 7. (Elution with (NH4)2SO~

Specific activity (W), Yield, 1 % min. X rag.

Purification

0.27 44 63 58

1.9 2.0 12.0

7.0X 7.4 × 44.5 ×

80

40.0

148 X

48

67.0

248 ×

33

155.0

574 X

9 R. Willst~tter and H. Kraut, Ber. 56, 1117 (1923).

[122]

703

TPNtt CYTOCttROME C REDUCTASE FROM YEAST

Step 7. Adsorption on AI(OH)3 Repeated. Portions of the aluminum hydroxide gel are again added to the enzyme solution and centrifuged until the supernatant liquid is colorless (about 3 g. of AI(OH)3 required). The enzyme is eluted by washing the adsorbent three times with 60 ml. of 0.64 saturated (NH~)2S04 in 0.1 N NH4OH. It is precipitated at pH 4.5 by increasing the ammonium sulfate concentration to 0.80 saturation and centrifugation. After adjustment of the suspension to about pH 9 with 0.30 ml. of 2 N NH4OH, it is lyophilized. No decrease in activity occurred during storage for two and one-half months at 0 °. Based on the titration with cytochrome c, a purity of the enzyme of 98% has been attained. The over-all purification in the seven steps of this isolation procedure is 600-fold; the yield of cytochrome reductase 2 %; its concentration about 600 mg./kg, of dry ale yeast.

Properties The enzyme can be split in acid solution and separated into F M N and a colorless protein; by combining the two, the active enzyme can be resynthesized. The dissociation constant at 25 ° is 1 X 10-9 mole X 1.-1. The velocity constants at 25 ° are: For oxidation by molecular oxygen: 8 × 10 3 (1. X mole -1 X min. -1) For oxidation b y cytochrome c: 5.3 X 100 (1. X mole -1 X min. -1) For reduction b y T P N H : 8.5 X 10 7 (1. X mole -1 X rain.-1)

The light absorption maxima are: Wavelength (m~) X 10-7 (cm. 2 X mole -~)

275 23

385 2.27

455 2.42

The molecular weight is 75,000. The enzyme is very unstable against acid reaction (pH 4.5) and elevated temperature (40°), and it is destroyed by dilute acetone, by dioxane and by dialysis against distilled water. It is inhibited by substituted phenols 2 and by atabrin. TM 10 E. Haas, J. Biol. Chem. 155, 321 (1944).

704

RESPIRATORY ENZYMES

[123] T P N H

Cytochrome c Reductase

[123]

(Liver)

T P N H -~ 2 Ferricytochrome c ~ T P N ~- 2 Ferroeytochrome c ~ H ÷

By B. L. HORECKER Assay Method Principle. The assay method, reagents, and procedure used are identical with those described for T P N H cytochrome c reductase from yeast. 1 Definition of Unit and Specific Activity. One unit of enzyme activity is defined as the quantity which produces a density change of 1.0 per minute, calculated from 30-second readings taken during the first 2 minutes after addition of the enzyme. Specific activity is the number of units per milligram of protein in the test. Protein is determined by the turbidimetric method of Bticher. ~ Purification Procedure The purification procedure is based on that described by Horecker.3 Preparation of Acetone Powder. Slice the livers of freshly slaughtered hogs into thin strips, and quickly chill in crushed ice. Homogenize 100-g. portions in Waring blendors with 500 ml. of acetone which has been cooled to -10% Filter with suction on large Bfichner funnels (18 to 24 cm.), using Whatman No. 1 paper. Strip the pad from the paper, again homogenize with cold acetone, filter, and dry at room temperature. To accelerate drying, break up the cake on wire screens until a fine powder sifts through. Store the powder at 2 °, and use within two weeks. Select only light livers; dark red livers contribute substances which interfere with the purification. All subsequent operations, unless otherwise specified, are carried out at 2 °. Step 1. Extraction of Acetone Powder. Suspend 50 g. of acetone powder in 750 ml. of 0.1 M Na2HP04, and shake gently for 10 minutes at 2 °. Centrifuge the suspension, and discard the supernatant solution. Homogenize the precipitate in a Potter-Elvejhem homogenizer 4 with small additions of 0.1 M Na2HP04 until about 50 ml. has been added and a smooth paste is obtained. Add the thick homogenate to 700 ml. of 0.1 M Na~HPO4, mix, and centrifuge for 5 minutes at 2500 r.p.m. (International size 2). Decant the supernatant solution through fine gauze. The suspension obtained can be kept for several days without loss of activity. IE. Haas, B. L. Horecker, and T. R. Hogness, J. Biol. Chem. 136, 747 (1940); see Vol. II [122]. T. Bficher, Biochim. et Biophys. Acta 1, 292 (1947). 8 B. L. Horecker, J. Biol. Chem. 183, 593 (1950). 4 V. R. Potter and C. A. Elvehjem, J. Biol. Chem. 114, 495 (1936).

[123]

TPNH CYTOCHROME C REDUCTASE (LIVER)

705

Step 2. Digestion with Trypsin and Ammonium Sulfate Fractionation. Pool the extracts from 300 g. of acetone powder (4.08 1.), add 1.32 g. of trypsin (Wilson, 1:300), and incubate for 30 minutes at 34 °. Cool the mixture to 2 °, and add 258 g. of ammonium sulfate per liter of solution. Centrifuge after 30 minutes, discard the precipitate, and add 342 g. of ammonium sulfate per liter of supernatant solution. Collect the precipitate on a Biichner funnel, dissolve it in 600 ml. of water, and dialyze the solution for 18 hours against 0.04 M sodium acetate, pH 7.0. Step 3. pH Precipitation. The pH range for optional precipitation varies considerably from preparation to preparation, and it is necessary to make a pilot run in which fractions collected at pH 5.3, 5.1, 4.9, and 4.7, successively, are dissolved and tested separately. The following, procedure is followed when optimal precipitation occurs between pH 5.3 and 4.9. Adjust the bulk of the dialyzed solution (540 ml.) to pH 5.3 with 39 ml. of 0.2 N acetic acid, and after 5 minutes at 2 ° centrifuge and discard the precipitate. Acidify the supernatant solution to pH 4.9 with 53 ml. of 0.2 N acetic acid, keep for 5 minutes, and centrifuge. Dissolve the precipitate in water with the aid of 11.4 ml. of 0.2 N NH4OH; the pH should be about 8.0. To the solution (153 ml.) add 95 g. of ammonium sulfate. Collect the precipitate by centrifugation, dissolve in water, and adjust to pH 8.0 with 4.0 ml. of 2 N NH4OH. Step 4. Calcium Phosphate Gel Adsorption and Elution. To the pit fraction (150 ml.) add 220 ml. of calcium phosphate gel 5 (1.93 g. of dry weight) which has been aged for three to six months. Centrifuge, discard the supernatant solution, and wash the gel twice with 60-ml. portions of 0.01 M phosphate buffer, pH 7.4. Elute the enzyme with four 60-ml. portions of 0.1 M K~HP04. Precipitate the enzyme by adding 167 g. of ammonium sulfate to the eluate (240 ml.). Centrifuge, dissolve the precipitate in 30 ml. of 0.15 M pyrophosphate buffer, pH 7.6, and lyophilize the solution. This yields 3.0 g. of powder containing 150 mg. of protein which can be stored in vacuo at 2 °. Step 5. Alkaline Ammonium Sulfate Fractionation. Dissolve the lyophilized powder in 200 ml. of water, and add 76 g. of ammonium sulfate together with 4.0 ml. of concentrated NH4OH to bring the pH to 8.0. Collect the precipitate by centrifugation, and dissolve it in 15 ml. of water. To the supernatant solution add 7.7 g. of ammonium sulfate, and collect and dissolve the precipitate as before. Again add 7.7 g. of ammonium sulfate to the supernatant solution. In this manner collect a total of six fractions, and analyze these separately. Combine the best fractions (usually the third, fourth, and fifth). 5D. Keilin and E. F. Hartree, Proc. Roy. Soc. (London) B124, 397 (1938).

706

RESPIRATORY ENZYMES

[123]

Step 6. Aluminum Hydroxide Gel Adsorption and Elution. Dilute the combined ammonium sulfate fractions (48 ml.) with 40 ml. of water, and add 40 ml. of aluminum hydroxide gel C~ 6 (aged six m o n t h s or longer, 456 rag. d r y weight). Centrifuge and wash the gel twice with 10-ml. portions of 0.025 M phosphate buffer, p H 7.2. Elute the enzyme with six 10-ml. portions of 0.1 M phosphate buffer, p H 7.2, and combine the eluates. Step 7. Further Purification. A small additional purification usually results from a repetition of steps 5 and 6. SUMMARY OF PURIFICATION PROCEDURE

Fraction 1. 2. 3. 4. 5. 6. 7.

Extract (NH~)2S04 fraction pH fraction Ca3(PO4)~eluate (NH4) 2SO4fraction AI(OH)3 eluate Repeat 5 -~ 6

Total Specific volume, Total Protein, activity, Recovery, ml. Units/ml. units mg./ml, units/mg. % 4080 540 150 240 48 60

2.5 16.0 39.6 22.0

77.0 43.9

10,200 22.7 8,660 9.4 5,910 6.2 5,280 0.92 3,690 1.10 2,630 0.31

0.11 1.71 6.4 24.0 70.0 140 140-160

-85 58 52 36 26

Properties

Specificity. D P N H cytochrome c reductase activity, which in the initial extract is about t w e n t y times as high as the T P N H activity, is almost completely removed b y the tryptic digestion and first a m m o n i u m sulfate fractionation. With the purified preparation the reaction with oxygen is very slow. The enzyme contains FAD. Effect of pH. The enzyme has optimum activity at about p H 7.8. Above p H 8.4 and below p H 7.5 there is a marked fall in activity. e it. Willstt~tter and H. Kraut, Bet. 56, 1117 (1923).

[124]

DIAPHORASES

[124]

707

Diaphorases

D P N H -~ A -~ H + --~ DPN + ~ AH2 B y HENRY R. MAHLER

Assay Method Principle. The methods originally developed by Green et al. 1,2 and yon Ruler et al. 3,4 involved MB reduction. Green and his co-workers generated the D P N H by the interaction of substrate, DPN, and a dehydrogenase and then used either the Thiinberg technique or manometric experiments; the Swedish investigators followed the disappearance of D P N H spectrophotometrically, using MB as the immediate and O~ as the final electron acceptor. The present method was developed by Edelhoch et al. 5 and Mahler et al. 6 and employs measurement of 2,6-dichlorophenolindophenol reduction at 600 mp. Reagenls

0.20 M Tris buffer, pH 7.5. 0.006 M DPNH. 7 0.0012 M 2,6-dichlorophenolindophenol. Enzyme. Dilute the enzyme to be tested with Tris or bicarbonate buffer until it contains 0.4 to 1.6 units/ml. (See definitions below.) Procedure. To two 3-ml. cuvettes add 0.3 mh of buffer, 0.1 ml. of DPNH, 0.1 ml. of dye, and 2.5 ml. of water. Determine the optical density at 600 mp (E~00). At zero time add 0.05 ml. of enzyme to one cuvette and 0.05 ml. of H20 to the other. Follow the change of E600 in both cuvettes at 30-second intervals for 3 minutes. Then subtract the changes observed in the blank from those observed in the experimental cuvette. 1 j. G. Dewan and D. E. Green, Biochem. J. 32, 626 (1938). H. S. Corran, D. E. Green, and F. B. Straub, Biochem. J. 33, 793 (1939). 3 H. yon Ruler and H. Hellstrom, Z. physiol. Chem. 256; 229 (1938). 4 E. Adler, H. yon Ruler, G. Gtinther, and R. Plass, Skand. Arch. Physiol. 82, 61 (1939). 5 H. Edelhoch, O. Hayaishi, and L. J. Teply, J. Biol. Chem. 197, 97 (1952). H. R. Mahler, N. K. Sarkar~ L. P. Vernon, and R. A. Alberty, J. Biol. Chem. 199, 585 (1952). 7 D P N H of 60 or 90% purity is now available commercially. For its preparation, see Vol. I I I [126]. In its stead D P N of the same concentration plus 0.05 ml. of absolute alcohol and 0.02 ml. of a 1 to 10 dilution of a crystalline alcohol dehydrogenase suspension (for the preparation of this enzyme see Vol. I [79]) may be used.

708

RESPIRATORY ENZYMES

[124]

Definition of Unit and Specific Activity. One enzyme unit is defined as that amount which causes an initial, corrected rate of change (AE600) of 1.00 per minute under the above conditions. Specific activity equals units per milligram. Protein is measured by the biuret reaction. 8 Application of Assay Method to Crude Tissue Preparations. The method as outlined is directly applicable to use with crude extracts or homogenates provided that (1) the turbidity introduced by the preparation is not so great as to make accurate spectrophotometry impossible and (2) the assay mixture is made 0.001 M with respect to CN-. Under these conditions, the method measures not only diaphorase, if any, but also dye reduction due to D P N H cytochrome reductase. 6 Purification Procedure The following procedure is essentially that developed by Straub for the preparation of soluble diaphorase from heart muscle. 9 Step 1. Preparation of Homogenate. Several pig hearts are freed of connective tissue and fat and are minced by means of an electric meat grinder with a finely perforated extrusion plate. The minced muscle is washed three times with 15 to 20 vol. of cold tap water. The mince is maintained in suspension by vigorous mechanical stirring for 20 minutes per wash. The bulk of the water is then squeezed out through cheesecloth, and 1660 g. of the washed mince is suspended in 2.5 1. of 0.02 M Na2HP04 and homogenized in Waring blendors for 90 seconds. Step 2. Precipitation at pH 4.6. The homogenate is suspended in 2.5 1. of deionized water, stirred, and centrifuged at 2000 X g for 20 minutes. Then 4.7 1. of the supernatant is mixed with 118 ml. of M acetate buffer, pH 4.6 (prepared by adding 0.5 mole of sodium acetate and 0.5 mole of glacial acetic acid to enough water to make 1.0 1.), and the mixture is centrifuged for 30 minutes at 2000 X g. Step 3. Alcohol Extraction. The residue is suspended in a solution prepared by the addition of 33 g. of (NH4)2SO4 and 50 ml. of absolute ethyl alcohol to 1660 ml. of deionized water. The temperature of the mixture is raised to 43 ° by means of a water bath and maintained at this level for 12 minutes. The preparation is centrifuged for 30 minutes at 2000 X g, and the yellow, opalescent supernatant is retained. Step 4. Treatment with Alumina. Into 1370 ml. of the supernatant is stirred 45 ml. of alumina C~1° (dry weight 20 to 25 mg./ml.). After 30 minutes the mixture is centrifuged at a speed sufficient to pack the gel well, and both the gel and the supernatant are saved. To the latter is 8 A. G. Gornall, C. J. Bardawill, and M. M. David, J. Biol. Chem. 17% 751 (1949). 9 F. B. Straub, Biochem. J. 33, I, 789 (1939). z0 See Vol. I [11] for preparation of alumina Cv.

[124]

DIAPHORASES

709

added an additional 22 g. of gel (sufficient to adsorb all the remaining yellow color), and the centrifugation is repeated. The two gel residues are combined, and the adsorbed enzyme is eluted by means of successive lots of 50 ml. each of 0.2 M Na2HPO4, until the eluate appears colorless. The combined eluates (240 ml.) are dialyzed until salt-free against running deionized water in the cold room. Step 5. Heat Treatment and First Ammonium Sulfate Fractionation. If a precipitate forms during dialysis it is dissolved by the addition of 2.8 g. of (NH4)2S04 to the 280 ml. of dialyzate. The temperature of the solution is raised to 60 ° by a water bath and is maintained at this value for 5 minutes. The solution is chilled and centrifuged, the precipitate being discarded. Then 76 g. of solid (NH,)2SO, is added to the solution, which is maintained at 0 ° for 1 hour. The mixture is centrifuged, the residue is discarded, and an additional 71 g. of (NH4)2SO4 is added to the solution. The mixture is centrifuged at high speed or filtered, and the supernatant is discarded. The residue is taken up in 30 ml. of distilled water. Step 6. Denaturation by Dialysis. The solution is dialyzed overnight against 1.5 1. of distilled water, and the precipitate which forms is discarded. The dialysis is then continued until the solution is salt-free. The precipitate which forms and contains the enzyme is dissolved in 0.02 M phosphate, pH 7.5. It is pure enough for most purposes but can be purified further by the following step. Step 7. Second Heat Treatment and Ammonium Sulfate Fractionation. The suspension formed at the end of step 6 is dissolved by the dropwise addition of 0.1% NH4OH, and the solution is heated to 62 to 65 ° and kept there for 5 minutes. The residue is removed by centrifugation, 10.9 g. of (NH4)~SO4 is added per 35 ml. of solution, and the mixture is again centrifuged. The precipitate is discarded, and an additional 4 g. of (NH4)2SO4 is added per 40 ml. of supernatant. The yellow precipitate is collected by filtration or high-speed centrifugation and dissolved in distilled water. The solution (8 ml.) is then thoroughly dialyzed against distilled water, and the first yellow precipitate which forms is discarded. To the solution is then added 0.5 ml. of 0.t M phosphate, pH 7.5, and this finM solution is stored in the frozen state. Test for Purity. After solubilization of the enzyme (step 3), the flavin content of the enzyme provides a useful guide to its purity. Flavin content is measured simply by determining the E4~0 of an enzyme solution E450 X 3.325 of known protein concentration; then mg. protein per ml. --- per cent riboflavin. Straub's purest enzyme contained 0.54% riboflavin. Corran et al. have shown that the flavin estimated by this procedure is equal to flavin determined enzymatically in a deproteinized extract. 2

RESPIRATORY ENZYMES

710

[124]

SUMMARY OF PURIFICATION PROCEDURE a,b

Fraction 1. Homogenate 4. Alumina eluate 5. (NH4)2SO4 fraction, 27-52% by weight 7. Final solution

Total volume, mh

Total units

Protein, mg./ml,

Specific activity, units/mg.

5000 240

17,00O 9,000

9.2 2.5

0.40 15.0

7,200 2,000

6.0 2.0

30 10

40 100

Recovery, % -53 42 11.7

F. B. Straub, Biochem. J. 38, I, 789 (1939). b H. R. Mahler, N. K. Sarkar, L. P. Vernon, and R. A. Alberty, J. Biol. Chem. 199, 585 (1952), and in preparation.

Properties Appearance and Absorption Spectrum. T h e purified e n z y m e is a yellow, greenish fluorescent flavoprotein. I t s s p e c t r u m shows absorption m a x i m a a t 272, 370, and 450 m~. F r o m the flavin content the m i n i m u m molecular weight (one flavin per molecule) can be calculated to be 70,000. Prosthetic Group. T h e prosthetic group of the e n z y m e has been identified as F A D . 2 T h e fluorescence of the solution and its absorption maxim u m at 450 m g disappears rapidly on incubation with excess D P N H . Specificity. T h e soluble, purified e n z y m e is specific for D P N H . T P N H is not oxidized b y dyes in the presence of enzyme, nor is the enzyme bleached b y T P N H . 11 This confirms the conclusions of A b r a h a m and Adler 12 and yon E u l e r et al., 3,18 reached on the basis of experiments with crude preparations. T h e reports b y some of the earlier investigators 2 of interaction between T P N H and the e n z y m e were p r o b a b l y due to the presence of e n z y m a t i c impurities in the crude glucose-6-phosphate dehydrogenase used to generate T P N H . T r a n s h y d r o g e n a s e 14 seems a likely c o n t a m i n a n t which m i g h t account for the results obtained. Nature of Electron Acceptor. T h e highly purified e n z y m e can interact with a wide v a r i e t y of dyes (A in the equation of the reaction) b u t is completely inert with c y t o c h r o m e c. T h e reports b y the C a m b r i d g e group on interaction of their p a r t i c u l a t e p r e p a r a t i o n with the c y t o c h r o m e syst e m 1,2 m a y be due to the fact t h a t these particles contained D P N H 11H. R. Mahlcr and D. Elowe, in preparation. 1~E. P. Abraham and E. Adler, Biochem. J. 84, 119 (1940). 1~H. yon Euler and G. Giinther, Naturwissenschaften 26, 676 (1938). 14See Vol. II [119] for a description of the function and mechanism of action of this enzyme.

[124]

DIAPHORASES

711

c y t o c h r o m e reductase 1~ and p r o b a b l y also bound c y t o c h r o m e c. W h e t h e r diaphorase as such even exists before solubilization and w h a t the electron acceptor is for this " n a t i v e " diaphorase are still m o o t questions. Kinetics and Turnover Number. Under the assay conditions the enzym a t i c reaction follows zero-order kinetics until b e t t e r t h a n 6 0 % of the dye has been reduced. Reaction rates are proportional to e n z y m e concentration over a considerable range. T h e t u r n o v e r n u m b e r as determined b y Corran et al. ~ approaches a limiting value of 8500 at low enzyme concentration. Other Sources of Enzyme. Diaphorase activity has been d e m o n s t r a t e d in particulate preparations and crude extracts of a large n u m b e r of animal tissues and organisms, 1-4,1~,~3,16 but S t r a u b ' s procedure described a b o v e is the only one devised so far for the isolation of a pure diaphorase. Other enzymes such as xanthine oxidase 17 and D P N H - c y t o c h r o m e reductase ~ isolated in a highly purified form show diaphorase activity, / however. TPNH-Diaphorase. I n analogy to the enzyme described here, a T P N H - d i a p h o r a s e m a y occur, b u t its existence has been b y no m e a n s proved. Since T P N H - c y t o c h r o m e reductases ~8 all show T P N H - d i a p h o rase (i.e., dye reduction) activity, ~9 the experiments of von Euler et al. 3,~3 and A b r a h a m and Adler 12 bearing on the existence of a separate T P N H diaphorase m u s t be critically re-examined. All their results m a y be explained b y simply ascribing the activities observed to the presence of T P N H - c y t o c h r o m e reductase in their preparations. A l t e r n a t i v e l y their observations m a y h a v e been due, at least in part, to the operation of t r a n s h y d r o g e n a s e plus D P N H - d i a p h o r a s e or D P N H - c y t o c h r o m e reductase. 15See Vol. II [120] for this preparation, also ref. 6 for a discussion of the relationship between this enzyme and diaphorase. 16D. E. Green and J. G. Dewan, Biochem. J. 33, 1200 (1938). 17See Vol. II [73] for the preparation of this enzyme; also see H. S. Corran, J. G. Dewan, A. H. Gordon, and D. E. Green, Biochem. J. 33, 1694 (1939). 18 E. Haas, B. L. Horecker, and T. R. Hogness, J. Biol. Chem. 136~ 747 (1940). 19 B. L. Horecker, J. Biol. Chem. 183~ 593 (1950) ; see also Vol. II [122, 123] for methods of preparation of this enzyme.

712

RESPIRATORY ENZYMES

[125]

[125] Triphosphopyridine and Diphosphopyridine Nucleotide Oxidases

By ERWIN HAAS I. Old Yellow Enzyme I

TPN q- Zwischenferment q- G-G-P -k H20 -~ T P N H -k H + q- Zwischenferment q- 6-PG T P N H -b Yellow E n z y m e - ~ TPN -k Leuco Enzyme Leuco Enzyme + 02 -~ Yellow Enzyme q- H~O2

(1) (2) (3)

The isolation of Warburg's old yellow enzyme has been of more than historical importance. It introduced the first of a new class of enzymes, the yellow enzymes, which since then have become of considerable importance. It made available for the first time an enzyme in which the catalytic activity could be understood according to simple chemical principles. It introduced the concept of a prosthetic group by demonstrating the reversible dissociation of the alloxazine nucleotide and its recombination with the enzyme protein. It established the close relationship between the yellow enzyme and vitamin B2 which led to a better understanding of the action of a vitamin. It permitted the first demonstration of the existence of a free radical of an enzyme, and finally it led to the discovery of another important class of biocatalysts, the pyridine nucleotides.

Assay Method Principle. The method is based on the manometric determination of the oxygen uptake in the following system: glucose-6-phosphateZwischenferment-TPN-yellow enzyme-molecular oxygen. Under the specified conditions the concentration of TPN is high, its reduction proceeds rapidly, and the concentration of the yellow enzyme will be ratedetermining in the consumption of oxygen. Catalase, present as an impurity in the Zwischenferment preparation, is inhibited by cyanide to prevent the decomposition of H~O~. Reagents Potassium glucose-6-phosphate (0.30 M). Two grams of the calcium salt s are suspended in 15 ml. of water and converted into 1o. Warburg and W. Christian, Biochem. Z. 266, 377 (1933); 298, 368 (1938). Crystalline calcium glucose-6-phosphate is available commercially;for methods of isolation or synthesis of this compound, see Vol. III [19].

[125]

PYRIDINE NUCLEOTIDE OXIDASES

713

the soluble potassium salt b y stirring for 30 minutes with 5 ml. of a solution containing 1.15 g. of p o t a s s i u m oxalate. Calcium oxalate is removed b y centrifugation, and the s u p e r n a t a n t solution is tested for the absence of calcium or oxalate. Zwischenferment2 This is the protein m o i e t y of the dehydrogenase which forms a dissociating complex with T P N ~ and results in the reduction of T P N b y G-6-P. 5 T h e purified Zwischenferment is labile in dilute solutions; it can be stabilized b y the addition of 20 to 30 % a m m o n i u m sulfate or b y lyophilization (see Vol. I [42]). T P N 5.6 is stable in aqueous solution. I t is stored at 0 ° to p r e v e n t bacterial growth. Procedure. T h e oxygen consumption is proportional to the concentration of the old yellow enzyme under the conditions outlined in T a b l e I. I t was measured after t e m p e r a t u r e equilibrium had been reached and T P N had been added to the main c o m p a r t m e n t .

TABLE I Temperature, 38°; gas phase, oxygen; center well, potassium hydroxide I

Side arm: TPN Main compartment: Water 0.3 M Potassium-G-6-P, pH 7.4 0.05 M KCN Zwischenferment Old yellow enzyme Microliters O: in 10 min. Microliters 02 in 20 min.

2

3

1.5 X 10-gmole

3.0 X 10-omole

0.1 mg. 2.0 0.2 0.2 0.2

ml. ml. ml. rag. 1 2

27 48

55 100

Purification P r o c e d u r e

Step 1. B o t t o m yeast is washed, dried, and extracted with w a t e r according to yon Lebedev. 1,7 Lead subacetate is added (400 ml. of liquor plumbi subacetic i D.A.B. 6 per liter of Lebedev juice), and the mixture is shaken vigorously after the addition of some octanol. T h e precipitate formed at 0 ° in 12 hours is separated b y centrifugation, and the excess

s E. Negelein and W. Gerischer, Biochem. Z. 284, 289 (1936). 4 E. Negelein and E. Haas, Biochem. Z. 282, 206 (1935). O. Warburg, W. Christian, and A. Griese, Biochern. Z. 282, 157 (1935). 6 TPN is obtainable commercially from various sources; for its isolation and purification, see Vol. I I I [124]. 7 A. yon Lebedev, Z. physiol. Chem. 73, 447 (1911).

714

RESPIRATORY ENZYMES

[125]

lead in the supernatant solution is precipitated by the addition of neutral phosphate buffer. To the supernatant solution, which contains the enzyme, is added half of its volume of acetone, and an inactive precipitate is centrifuged off and discarded after 24 hours at 0 °. The solution is saturated with COs and enough acetone is added (about half of its volume) to precipitate all the yellow pigment as a viscous oil. The acetone solution is decanted, the yellow enzyme dissolved in water, and a white, insoluble residue removed by centrifugation. The saturation of the aqueous solution with COs, followed by precipitation with acetone, is repeated twice, and the yellow enzyme thereafter is dissolved in water and precipitated with an equal volume of methanol. The precipitate is once more dissolved in water, precipitated with the threefold volume of methanol, washed on the Btichner funnel with absolute methanol, and lyophilized. To avoid losses of enzymatic activity it is essential to maintain a low temperature during the addition of acetone and methanol. Step 2. One hundred grams of the crude enzyme powder is suspended in 2000 ml. of a 1% NaC1 solution and shaken for 24 hours at 38 ° with 25 ml. of chloroform and 10 ml. of octanol. An inert precipitate is centrifuged off, and the solution is dialyzed and lyophilized. Step 3. The enzyme is redissolved and then subjected to electrophoresis for 14 hours at pH 4.53 which separates the polysaccharides, s Further purification results by precipitation at pH 5.2 with 0.67 saturated ammonium sulfate. This precipitation is repeated thrice after redissolving of the enzyme in acetate buffer, pH 5.2, and the enzyme finally is crystallized by the addition of 0.44 saturated (Ntt4)2S04 in acetate buffer and dialysis against 0.67 saturated (NH4)~SO4 and acetate buffer. TABLE II SUMMARY OF PURIFICATION PROCEDURE FOR TItE OLD YELLOW ENZYME

Step

1.

2.

3.

Procedure 'Extraction of autolyzed yeast Precipitation of proteins by lead subacetate Precipitation of proteins by 33 % acetone tPrecipitation of enzyme by 55 % acetone (three times) Precipitation of enzyme by 50% methanol .Precipitation of enzyme by 75% methanol Shaking at 38° with chloroform -{- octanol Electrophoresis at pit 4.5 Precipitation with 0.67 satd. (NH4)~S04 (four times) Crystallization from acetate--(NH4)2S04

s H. Theorell, Biochem. Z. 278, 263 (1935); 278, 291 (1935).

[125]

715

P Y R I D I N E NUCLEOTIDE OXIDASES

Properties The old yellow enzyme can be reduced b y T P N H and D P N H , the latter reaction taking place at a rate one-tenth as f a s t ) In the leuco form it can be reoxidized b y molecular oxygen or methylene blue, but not b y cytochrome c. The protein moiety of the yellow enzyme forms complexes with both F M N and T P N , and the free radical of F M N can be demonstrated to exist under certain conditions. 9 The molecular weight of the enzyme is 80,000; l° its isoelectric point is p H 5.25; its elementary composition has been analyzed s and its prosthetic ( F M N ) group has been split off reversibly. 1 The light absorption maxima of the old yellow enzyme are: Wavelength (m~)

tcm. l f~ X 10-~ [_m~-~'~eJ

275

380

465

28

2.26

2.42

The rate constants H of the old yellow enzyme at 25 ° are: For its oxidation by molecular oxygen: 1 × 106 For its reduction by TPNH: 60 X 106

1. min. X moles

1. min. X moles

The dissociation constanV 2 at 25 ° is 6 X 10-s mole X 1.-~. One kilogram of dry yeast (Schultheiss-Patzenhofer brand beer yeast) contains about 1.0 g. of the old yellow enzyme. 12 II. New YeUow Enzyme 1~ T P N W Zwischenferment W G-6-P ~ H20 -~ T P N H ~- H + -~ Zwischenferment ~- 6-PG T P N H -~ New Yellow E n z y m e - ~ T P N -~ Leuco E n z y m e Leuco E n z y m e -)- Methylene Blue --~ E n z y m e -~ Leuco Methylene Blue [Cu] Leuco Methylene Blue -~ O 2 - e Methylene Blue -]- I-I~O~

(1) (2) (3) (4)

Assay Method Principle. In the over-all reaction, as outlined, G-6-P is oxidized b y molecular oxygen, and the oxygen consumption, w h i c h is a function of the concentration of the new yellow enzyme, is measured manometri-

9 E. Haas, Biochem. Z. 290, 291 (1937). 10 R. A. Keckwick and K. O. Pedersen, Biochem. J. 30, 2201 (1936). 11E. Haas, Biochem. Z. 298, 378 (1938). 12E. Haas, B. L. Horeeker, and T. R. Hogness, J. Biol. Chem. 136, 747 (1940).

716

RESPIRATORY ENZYMES

[125]

cally. Both the leuco forms of the old and the new yellow enzyme are rapidly oxidized b y methylene blue, but the new yellow enzyme is reduced four times as fast b y T P N H , and it is oxidized seven times as slowly by molecular oxygen as the old yellow enzyme. This difference in activity permits a quantitative distinction of the two enzymes under the test conditions outlined in Table III. TABLE III Temperature, 25°; gas phase, oxygen; center well, potassium hydroxide 1

Side arm: 0.3 M potassium-G-6-P Main compartment: 0.5 M phosphate, pH 7.4 TPN Zwischenferment Methylene blue New yellow enzyme

2

3

0.20 mg. 1.5 X 10 -9 mole

0.20 mg.

0.1 ml.

2.9 ml. 0.1 rag. 0.06 rag. 1.5 X 10-9 mole

After addition of potassium G-6-P Microliters 02 in 5 rain. Microliters 02 in 10 min.

0.5 1.0

22 44

1.5 3

Reagents. The methylene blue preparations commercially available usually contain sufficient copper salts to catalyze the oxidation of the leuco form b y molecular oxygen. The other reagents required, G-6-P, T P N , and Zwischenferment, have been discussed previously. Procedure. The oxygen consumption is measured. In the absence of methylene blue it is negligible for the new yellow enzyme, but it becomes appreciable if the preparations contain the old yellow enzyme. This is determined in vessel 1 of Table III. Methylene blue is only slowly reduced b y T P N H , as shown by the slow rate of oxygen consumption in the absence of yellow enzyme (vessel 3). De~nition of Specific Activity. The specific activity of an enzyme preparation is determined from the oxygen consumption b y comparison with a standard preparation, and it is expressed as the fraction of enzyme, b y weight, contained in the enzyme preparation. Purification P r o c e d u r e

Starting Material. B o t t o m yeast, after completion of the brewing process, is washed, pressed in canvas bags, and dried at room t e m p e r a t u r e in a stream of air. The powdered yeast (32 kg.) is stirred with 96 1. of water

[125]

PYRIDINE NUCLEOTIDE OXIDASES

717

for 3 ~ hours at 36 °. After centrifugation, 40.5 1. of a crude extract is obtained containing the enzyme. Step 1. After cooling to 0 °, 1.73 1. of 5 N acetic acid (pH 4.5) and 15.4 kg. of solid ammonium sulfate (0.60 saturation) are added to the solution, and the precipitate, which contains the enzyme, is separated by centrifugation. The precipitate, with a volume of 13.7 1., is extracted with 13.7 I. of water which dissolves the enzyme but permits the separation of an insoluble, inactive residue by centrifugation. From the supernatant solution (22.5 1.) the enzyme is precipitated by the addition of 4.1 kg. of ammonium sulfate (0.6 saturation) and centrifugation. The precipitate is dissolved, diluted to a volume of 5.2 1. and dialyzed for 44 hours. The solution containing the e~zyme is clarified by centrifugation. Step 2. The enzyme is precipitated by the slow addition of 1.95 1. of ethanol to 7.5 1. of the enzyme solution at 0 ° and pH 4.5. It is collected by centrifugation after 18 hours at 0 ° and lyophilized. Step 3. The enzyme is redissolved in 3 1. of water by neutralization with 40 ml. of 2 N NaOH. An inactive precipitate, formed by acidification to pH 4.5 with acetic acid, is removed by centrifugation. To the supernatant solution is added 330 ml. of a saturated ammonium sulfate solution and 900 ml. of 2 N KOH (pH 10.0). The solution is rapidly warmed to 55 °, kept for 5 minutes at that temperature, and cooled immediately by immersion in ice water. A heavy precipitate of denatured protein is centrifuged off after neutralization of the solution with 340 ml. of 5 N acetic acid. Additional amounts of inactive protein can be precipitated and removed by centrifugation after acidification to pH 4.6 with 300 ml. of 5 N acetic acid and addition of 980 g. of ammonium sulfate (0.45 saturation). The enzyme finally is precipitated from the supernatant solution with 460 g. of ammonium sulfate (0.60 saturation). Step 4. The precipitate obtained in the preceding step is centrifuged at high speed to remove as much as possible of the adhering ammonium sulfate solution. It is then dissolved in 600 ml. of 0.01 M acetate buffer, pH 4.5. The enzyme is adsorbed by stirring for 10 minutes at 0 ° with 15 g. of aluminum oxide (Brockmann) and washed with water. For the elution of the enzyme from the aluminum oxide the suspension is stirred four times, each time for 5 minutes at 0 ° with 500 ml. of 0.02 M borate buffer, pH 10.2. The eluates are combined, acidified to pH 4.6 with about 40 ml. of 2 N acetic acid, and clarified by centrifugation. The enzyme is precipitated from the supernatant solution (2000 ml.) at 0 ° with 1.4 1. of ethanol and collected by high-speed centrifugation after 1 hour at 0 °. The precipitate is washed with 100 ml. of 0.003 N KOH (pH 6.8) and centrifuged. It is then dissolved at pH 8.5 in 110 ml. of water with 0.1 ml. of N NaOH. Acidification to pH 4.6 with 0.15 ml. of 2 N acetic acid re-

718

RESPIRATORY ENZYMES

[125]

sults in the precipitation of some additional inert protein which is discarded after high-speed centrifugation. T h e final solution contains 320 mg. of the new yellow enzyme. T h e over-all purification in this isolation procedure is ll00-fold, the yield 7 %. F r o m this value a concentration of 143 rag. of new yellow e n z y m e per kilogram of d r y y e a s t has been calculated. TABLE IV SUMMARY OF PURIFICATION PROCEDURE FOR THE NEw ~rELLOW ENZYME

Fraction Crude extract 1. (NH4)2SO, fractionation, 0.6-0.3-0.6 saturation 2. Ethanol fractionation /Denaturation at 55 °, pH 10 3. /(NH*)2SO~ fractionation, 0.45-0.60 tsaturation ~Adsorption on A120~ 4. ( Precipitation with ethanol

Recovery, %

Specific activity

Purification

0. 00091 62 67

0. 0073 0. 019

8× 21 X

40

0. 153

169 ×

40

1.0

1100 N

Properties T h e reduced f o r m of the e n z y m e can be oxidized b y molecular oxygen and m e t h y l e n e blue b u t not b y e y t o c h r o m e c. T h e oxygen consumption under the conditions outlined in T a b l e I I I can be doubled b y M / 1 0 0 H C N , p r e s u m a b l y b y inhibition of catalase present in the Zwischenf e r m e n t preparation. T h e new yellow e n z y m e can be split, b u t the prosthetic group is bound so firmly t h a t acidification to p H 0.4 is required for its dissociation. T h e active e n z y m e can be resynthesized f r o m the protein m o i e t y and the prosthetic group which is F A D . T h e protein of the new yellow e n z y m e is not identical with t h a t of the old yellow enzyme. A molecular weight of 60,000 has been calculated f r o m the F A D content of the new yellow enzyme; a value of 65,000 is indicated f r o m luminoflavin determinations. T h e velocity constants of the new yellow e n z y m e at 25 ° are: ll 1.

For its oxidation by molecular oxygen: 1.4 X 104 moles X min. 1. For its reduction by TPN-H:: 22 X 106 moles × rain. T h e dissociation constant at 25 ° 12 is 2.7 X 10 -s mole X 1.-1.

[126]

GLUTATHIONE REDUCTASE (PLANT)

719

The light absorption maxima of the new yellow enzyme are: Wavelength (ma) fl X'10-7

[em.2~ [~i~J

275

377

455

20

2.52

2.42

[126] Glutathione Reductase (Plant) GSSG -~- T P N H -~- H + -~ T P N + ÷ 2 GSH B y BIRGIT VENNESLAND

Assay Method Principle. The reaction m a y be followed b y measuring the disappearance of T P N H spectrophotometrically at 340 mu or b y measuring the appearance of GSH. The G S H m a y be measured enzymatically b y the glyoxalase test, 1 colorimetrically b y the nitroprusside reaction, 2 or titrimetrically with standard iodine or iodate. 3-6 T h e glyoxalase test is most specific for G S H but is also relatively cumbersome. Furthermore, high specificity is not necessarily desirable, since secondary reactions catalyzed b y crude extracts m a y cause the disappearance of G S H once it has been formed. The spectrophotometric test for disappearance of T P N H is convenient but requires relatively large amounts of T P N . Coupling with the glucose-6-phosphate dehydrogenase system, as described here, affords an assay which is both convenient and economical. 5 In this method, GSSG, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, and a catalytic a m o u n t of T P N are incubated with the reductase, and the G S H formed is determined iodometrically. The nitroprusside test m a y also be used to determine GSH and is particularly convenient for measuring smaller amounts than can be determined iodometrically. Such high sensitivity is seldom required, however. Reagents

GSSG, 6 10 mg./ml., adjusted to p H 7.0 to 7.4. T P N , 6 100 ~,/ml. A stock solution containing 1 mg./ml, can be stored for m a n y months at - 1 5 °. Dilute 1 to 10 before use. 1G. E. Woodward, J. Biol. Chem. 109, 1 (1935). A. Fujita and I. Numata, Biochem. Z. 300, 246 (1939). 3 A. Fuiita and I. Numata, Biochem. Z. 299, 249 (1938). 4L. W. Mapson and D. R. Goddard, Biochem. J. 49, 592 (1951). 5E. E. Conn and B. Vennesland, J. Biol. Chem. 192, 17 (1951). 6 All of these reagents are available commercially. It is not necessary that they be of high purity.

720

RESPIRATORY ENZYMES

[126]

0.05 M glucose-6-phosphate, 6 adjusted to pH 7.0 to 7.4. 0.5 M Tris-HC1 buffer, pH 7.4. Glucose-6-phosphate dehydrogenase. The preparation described by Kornberg 7 may be used. Dissolve enough of the lyophilized Kornberg preparation to give 1.25 mg. of dehydrogenase per milliliter of H20. The preparation must be tested for GSSG reductase activity by running the assay without added reductase. It a titer is obtained, it must be subtracted from all assay titers. Metaphosphoric acid. s Dissolve 5 g. in water to make a total volume of 25 ml. The solution should be clear. Store at 0 °, and make a fresh solution every week. 0.1 N K I O 3 . H I Q stock solution. Dilute 1 ml. to 100 ml. before use. 4 % KI. Dissolve 1 g. in 25 ml. of H~O. Make fresh daily. Starch. One gram of soluble starch is dissolved in 100 ml. of saturated NaC1.

Preparation of Plant Tissue for Assay. Plant glutathione reductase has not been highly purified. The assay procedure described has been developed for wheat germ extracts and has been applied only qualitatively to other plant sources2 Wheat germ 1° is extracted with 4 vol. of water for 30 minutes at room temperature. The solids are removed by centrifugation, and the extract is dialyzed against 0.025 M phosphate buffer, pH 7.4. Leaves and roots are ground, the juice is expressed through muslin, and the solids are removed by centrifugation. The juice is then dialyzed as above. High blank titers are obtained if dialysis is omitted. In most cases 0.2 to 1.0 ml. of the juice is used in the incubation mixture described below. If too large an amount of the juice is used, the test may be negative or very low. This is apparently due to enzymes destroying T P N 2 The addition of AMP to the test mixture often improves the results under these circumstances. Procedure. To a 15-ml. conical centrifuge tube, add 0.2 ml. of Tris buffer, 0.1 ml. of glucose-6-phosphate dehydrogenase, 0.1 ml. of glucose6-phosphate, 0.2 ml. of TPN, 0.2 ml. of GSSG, and sufficient water so t h a t the total volume after the addition of reductase is 2.7 ml. Bring this mixture and the reductase separately to 30 °, then mix the two solutions and incubate at 30 ° for 15 minutes. (Incubation under anaerobic conditions is not necessary.) Stop the reaction by adding 0.3 ml. of HPO3. Mix and centrifuge. Pour off the supernatant. Measure 2 ml. into A. Kornberg, J. Biol. Chem. 182, 805 (1950) ; see also Vol. I [42]. s Baker Chemicals HP03 is satisfactory. 0 D. G. Anderson, H. A. Stafford, E. E. Conn, and B. Venuesland, Plant Physiol. 9.7, 675 (1952). 10 Wheat germ S-50, provided by General Mills, Minneapolis, is a satisfactory source.

[126]

GL~TAT~IO~E

REDVCT~SE (PLANT)

721

a 50-ml. centrifuge tube immersed in a beaker of ice and water. Add 0.2 ml. of KI and two drops of starch. Titrate with iodate. A blank determination is run simultaneously by adding the metaphosphate to a separate tube of the incubation mixtures before the addition of reductase. The blank titer is subtracted from the titer of the incubation mixture. Precautions. The assay is of dubious quantitative value unless the titer is shown to increase linearly with the amount of enzyme employed. With wheat germ, titers above 4 ml. often show poor proportionality. With other enzyme sources, a linear relationship may not be obtained with titers above 2 ml. or less. Definition of Unit and Specific Activity. One unit of enzyme may be defined as the amount which gives a titration of 1 ml. of 0.001 N iodate (1 micromole GSH) under the conditions of the assay. The specific activity is defined as units of enzyme per milligram of protein. Protein may be determined according to Robinson and Hogden. 11

Properties Precipitation. The enzyme is readily soluble and can be precipitated by (NH4)2SO4. Most of the activity in a wheat germ extract can be obtained by collecting the protein fraction which precipitates between concentrations of 250 g. and 325 g. of (NH4)2SO4 per liter. 12 Heat Sensitivity. Forty-three per cent of the activity of the wheat germ extract is lost in 5 minutes at 50 °. Complete inactivation occurs in 1 minute at 100% pH Optimum. The pH optimum of wheat germ glutathione reductase in Tris buffer is 7.8. Specificity. The wheat germ enzyme is inactive with L-cystine, DLhomocystine, and the disulfides of aspartathione and of 5,-glutamylcysteine. The soluble enzyme in higher plants likewise is inactive with DPN. 5 Distribution. Glutathione reductase is very widely and perhaps almost universally distributed in higher plants. 9 Wheat germ, spinach leaf, parsley root, cantaloupe fruit, and cucumber fruit are particularly good sources of the enzyme. Uses. Glutathione reductase may conveniently be used to detect other TPN-linked dehydrogenases. 9 After the enzyme has been shown to be present in a particular extract, the presence in that extract of any dehydrogenase requiring TPN may be determined by measurement of the reduction of GSSG in the presence of TPN and the substrate of the enzyme for which the test is conducted. 11H. W. Robinson and C. G. Hogden, J. Biol. Chem. 135, 707 (1940). 12R. C. Barnett, H. A. Stafford, E. E. Conn, and B. Vennesland, Plant Physiol. 28, 115 (1953).

722

RESPIRATORY ENZYMES

[127]

A mixture of GSSG, TPN, and glutathione reductase can also serve as a Hill reagent, since GSH is formed and 02 is evolved when such a mixture is incubated with illuminated chloroplasts.l~ ~aD. D. Hendley and E. E. Conn, Arch. Biochem. and Biophys. 46, 454 (1953).

[127] G l u t a t h i o n e R e d u c t a s e ~ ( L i v e r a n d Y e a s t )

By E. RACKER

Assay Method Principle. In the presence of the enzyme hydrogen is transferred from T P N H to GSSG, and the reaction can be measured spectrophotometrically at 340 m~. Reagents 1 M potassium phosphate buffer, pH 7.6. 0.001 M TPNH. 1% solution of bovine serum albumin in 0.1 M phosphate buffer, pH 7.6. 2 % solution of oxidized glutathione. Enzyme. Dilute stock enzyme solution in 0.05 % solution of bovine serum albumin, pH 7.6, to obtain 200 to 2000 units of enzyme per milliliter. (See definition of unit below.)

Procedure. Into a final volume of 1 ml. the following solutions are placed into a microquartz cell with a 1-cm. light path: 0.05 ml. of phosphate buffer, 0.55 ml. of H20, 0.1 ml. of TPNH, 0.1 ml. of serum albumin, 0.1 ml. of diluted enzyme, and 0.1 ml. of GSSG. After mixing, density readings are taken at 340 mt~ in a Beckman spectrophotometer at 30-second intervals. Definition of Unit and Specific Activity. One unit of enzyme is defined as a change in log I o / I of 0.001 per minute under the above conditions. Specific activity is expressed as units of enzyme activity per milligram of protein. Protein concentration is determined spectrophotometrically. 2 With cruder preparations which contain considerable amounts of nucleic acid the biuret test s is used. Application of Assay Method to Crude Tissue Preparations. The enzyme can be measured spectrophotometrically in crude tissue preparaE. Racker, to be published. 20. Warburg and W. Christian, Biochem. Z. 810, 384 (1941). s H. W. Robinson and C. G. Hogdcn, J. Biol. Chem. 186, 727 (1940).

[127]

GLUTATHIONE REDUCTASE

723

tions, provided that the concentration of enzyme is high relative to oxidation of T P N H by side reactions in the absence of GSSG. Since crude extracts may contain GSSG, it is necessary to dialyze them free of residual GSSG before corrections for these side reactions can be applied.

Purification Procedure (Yeast Enzyme) Step 1. Preparation of yeast extracts from 300 g. of dried baker's yeast is carried out as described for the preparation of alcohol dehydrogenase (cf. Vol. 1 [79]). Step 2. Fractionation of the heated extract with acetone is also carried out as for alcohol dehydrogenase, but the first precipitate after the addition of 0.5 vol. of acetone is collected. This precipitate is extracted for 20 minutes with about 100 ml. of 0.01 M potassium phosphate, pH 7.4, and centrifuged. (These and the following steps are carried out at temperatures near 0 ° unless stated otherwise.) Step 3. The supernatant solution is adjusted to pH 5.9 with 0.2 N acetic acid, and for each 100 ml. of solution 20 ml. of 95 % ethyl alcohol is slowly added at - 3 ° (in an alcohol-dry ice bath) and centrifuged at - 3 ° in a refrigerated centrifuge (10 minutes at 15,000 X g). The precipitate is dissolved in 30 ml. of 0.05 M potassium phosphate buffer, pH 7.6. Step 4. Five ml. of 2% protamine sulfate (Nutritional Biochemical Corp.) is added, and the precipitate is centrifuged off. The supernatant solution is adjusted carefully to pH 5.5 with 0.1 N acetic acid. For each 10 ml. of solution 2 ml. of 95 % ethyl alcohol is added, the solution being kept at - 3 ° in an alcohol-dry ice bath. The mixture is then centrifuged at - 3 °, and the precipitate is dissolved in 10 ml. of H20. Step 5. The pH of the solution is adjusted to 5.6, and 0.2 vol. of calcium phosphate gel (20 mg./ml.) is added. The mixture is centrifuged. The gel is washed once with 5 ml. of H~O, then eluted four times at room temperature with 3 ml. of 0.05 M potassium phosphate, pH 7.6. Step 6. To the combined eluates 2.8 g. of solid dibasic ammonium phosphate is added per 10 ml. of solution, and the precipitate is centrifuged off. To the supernatant solution one additional gram of ammonium phosphate per 10 ml. is slowly added, and the mixture is allowed to stand in an ice bath for 1 hour. The crystalline material is collected by centrifuging at 16,000 × g for 20 minutes. A typical protocol, showing data on the specific activity of the various fraction and the yield obtained, are given in the table. It should be pointed out that some variations in the activity of crude extracts from different batches of yeast have been obtained, and it is essential to start with active extracts. Steps 1, 2, and 3 are quite repro-

724

RESPIRATORY ENZYMES

[127]

ducible, b u t in step 4 the a m o u n t of protamine which can be used without encountering severe losses in activity varies between 3 and 7 ml. of a 2 % solution. I t is essential, however, to remove most of the nucleic acid at this step, 4 and losses up to 25% are acceptable. Variation in step 5 m a y also be encountered, depending on the properties of the calcium phosphate gel. This, in fact, is the case in all procedures involving the use of aged gels. SUMMARY OF PURIFICATION OF YEAST GSSG REDUCTASE

Crude extract First acetone precipitate First alcohol precipitate Second alcohol precipitate Gel eluates First crystals

Units/per ml.

Specific activity

Total units

20,000 41,000 96,000 210,000 190,000 270,000

510 2,100 6,000 30,000 73,000 170,000

6,400,000 4,100,000 2,880,000 2,100,000 1,520,000 810,000

Properties

Purified preparations of glutathione reduetase from yeast react with both T P N H and D P N H , though much slower with the latter. The enzyme is stable if kept frozen at - 2 0 ° . One preparation of 35% p u r i t y (as compared to the best preparations obtained to date) which was stored in 0.05 M phosphate buffer lost about half its activity during a period of t w e n t y - t w o months of storage in the deep-freeze. However, in dilute solution rapid surface denaturation occurs, and the addition of serum albumin as a protecting agent is essential. For example, an enzyme preparation diluted in potassium phosphate buffer, p H 7.6, was found to contain 5600 units/ml., whereas when it was diluted in a solution containing buffer as well as 500 ~/ of bovine serum albumin per milliliter it assayed at 18,000 units/ml. When 1 mg. of serum albumin was also added to the test system in the Beckman cell the assay was 26,000 units/ml. The enzyme is unstable when heated at temperatures above 60 °, which leads to rapid inactivation. Although some losses occur during dialysis of the enzyme, no evidence for a coenzyme was obtained. Partial Purification of Liver Glutathione R e d u c t a s e

Frozen beef liver, stored in the deep-freeze at - 2 0 °, is used as starting material. Acetone-dried powder is prepared as described for aldehyde dehydrogenase (Vol. I [83]). T h e extraction of the enzyme with water 4The protamine solution is added in 1-ml. amounts until the supernatant solution has a 280/260 ratio of about 1.0.

[128]

PYRIDINE NUCLEOTIDE QUINONE REDUCTASE

725

and fractionation with alcohol is also carried out as for the aldehyde dehydrogenase except that the first alcohol precipitate is collected which contains most of the reductase activity. The second step of purification consists of iso'electric precipitations. The fraction is first adjusted to pH 6.5 to 6.6 and the precipitate is discarded. The supernatant solution is brought to pH 5.8 by the slow addition of 0.1 N acetic acid, keeping the mixture in an ice bath. After 20 minutes the precipitate is collected and dissolved with dilute alkali and adjusted to a final pH of 7.0. The precipitation of the enzyme between pH 6.5 and 5.8 is repeated several times without undue losses of enzyme activity. The over-all purification obtained is about twentyfold, and the specific activity obtained is about one-twentieth that of the yeast enzyme. Properties

Like the yeast enzyme, the liver enzyme reacts much more rapidly with TPN than with DPN. Glutathione reductase from liver can be precipitated at pH 5.8, whereas the yeast enzyme is completely soluble at this pH. The liver enzyme appears less sensitive to the inhibitory action of sodium chloride but is markedly inhibited by 0.05 M sodium azide. The enzyme preparations with the highest specific activity obtained so far show the typical absorption spectrum of a protein with a pronounced band at 410 m~, indicating the presence of a porphyrin. The possibility that this band might be due to an impurity cannot as yet be ruled out.

[128] P y r i d i n e N u c l e o t i d e Q u i n o n e R e d u c t a s e f r o m P e a S e e d s 1,2 D P N H + H + + p-Benzoquinone--* DPN + + Hydroquinone B y WALTER D. WOSILAIT and ALVIN NASON

Assay M e t h o d Principle. Quinone reductase activity is measured by following the oxidation of reduced pyridine nucleotide as indicated by the decrease in optical density at 340 m~. Concomitantly a slow nonenzymatic reduction of p-quinone by reduced pyridine takes place, and corrections for this contribution are made.

1W. D. Wosilait and A. Nason, J. Biol. Chem. 206, 255 (1954). 2 W. D. Wosilait, A. Nason, and A. J. Terrell, J. Biol. Chem. 206, 271 (1954).

726

RESPIRATORY ENZYMES

[[email protected]]

Hydroquinone is determined by a method already suggested2 It is based on the reduction by hydroquinone of ferric ions to ferrous ions which in turn react with added o-phenanthroline to form a red-colored complex, the intensity of which is proportional to the original amount of hydroquinone present.

Reagents 0.1 M potassium phosphate-HC1 buffer, pH 6.5 and 7.5. DPNH, enzymatically reduced 4 (6 ~M./ml.). p-Benzoquinone (3.0 × 10-3 M). Dissolve 32.4 mg. of freshly sublimed p-quinone in 100 ml. of distilled water and store at - 1 5 ° until ready for use. During use, each tube is wrapped with aluminum foil to prevent photochemical reduction of the p-quinone.5 After thawing, each tube of stock solution is used for not longer than 8 hours and then discarded. 0.01 M FeC13-6H20. 0.2 M sodium pyrophosphate. 0.01 M o-phenanthroline.

Procedure. STANDARD ASSAY. The reaction is started by the addition of 0.15 ml. (0.45 micromole) of p-quinone solution to a mixture containing 2.7 ml. of 0.1 M phosphate buffer, pH 6.5, 0.3 micromole of DPNH, and quinone reductase to give a final volume of 3.0 ml. in a cell having a 1-cm. light path. The decrease in optical density is measured at 340 m/~ at 30-second intervals. The nonenzymatic rate is determined spectrophotometrically as above, except that the enzyme is omitted from the reaction mixture. The enzymatic rate is corrected accordingly. HYDROQUINONE DETERMINATION. A reaction mixture containing 0.05 to 0.30 micromole of hydroquiaone in a 3.0-ml. volume is treated with 0.6 ml. of FeC13. After 5 minutes' incubation at 25 °, 0.25 ml. of sodium pyrophosphate is added to clarify the opalescent solution resulting from the excess FeC13. o-Phenanthroline (0.6 ml.) is added, the mixture is incubated for 30 minutes at 37 °, and the intensity of the red ferrous-ophenanthroline complex is determined in a Klett-Summerson colorimeter using a green filter (No. 54). In order to correct for hydroquinone formation resulting from photochemical reduction of quinone and interaction with protein, controls containing all the components of the reaction other than D P N H are used. In stoichiometric studies, large amounts of enzyme (170 units, see definition below) are used in order to minimize 8 W. D. Stephens, MetaUurgia 37p 333 (1948). 4 M. E. Pullman, S. P. Colowick, and N. O. Kaplan, J. Biol. Chem. 194~ 593 (1952). 5 F. Poup~!, Chem. Abstr. 41~ 7276 (1947).

[128]

PYRIDINE NUCLEOTIDE QUINONE REDUCTASE

727

the nonenzymatic contribution. Since the nonenzymatic reaction cannot be stopped by a suitable method, the amount of D P N H added to the system is kept limiting by providing an excess of p-quinone. Definition of Unit and Specific Activity. One unit of quinone reductase is defined as that amount of enzyme which results in a change in log I o / I of 0.001 per minute at 340 m~, calculated from the change between the 15- and 45-second readings under the above conditions, corrected for the nonenzymatic rate. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al. 6 Purification Procedure

All steps of the purification are carried out at 0 to 4°; centrifugations are performed at approximately 3000 X g. Cell-free extracts are prepared from dried pea seeds, Pisum sativum var. Laxton's Progress, which have been soaked in tap water at room temperature for about 20 hours and then blended with an approximately equal volume of cold 0.1 M phosphate buffer, pH 7.5, in a Waring blendor for 11/~ minutes at 4 °. The blend is pressed by hand through four layers of cheesecloth in order to remove the coarse material, and the fluid is then centrifuged for 20 minutes. The turbid supernatant solution is used as the crude extract as well as for further purification. Step 1. In the following example of a typical purification, 72 ml. of calcium phosphate geV (11 mg. of dry weight per milliliter), is added to 90 ml. of crude extract (fraction I). After standing for 10 minutes, followed by a 7-minute centrifugation, the supernatant is treated with 63 ml. of calcium phosphate gel as above, and the supernatant designated as fraction II. The activity in fraction II represents 97 % of the units of the crude starting material with a fourfold purification. Step 2. Fraction II is brought to 50 % saturation with solid ammonium sulfate. After standing for 15 minutes, followed by a 20-minute centrifugation, the supernatant solution is brought to 65% saturation with ammonium sulfate and treated as above. The precipitate is suspended in cold 0.1 M phosphate buffer, pH 7.5, brought to a volume of 10 ml., and designated as fraction III. Step 3. Fraction III is treated with two successive 1.0-ml. portions of alumina gel 8 (12.3 mg./ml.), for 10-minute periods, each followed by a 7-minute centrifugation. The supernatant solution designated as frac6 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951); see also Vol. I [73]. D. Keilin and E. F. Hartree, Proe. Roy. 8oc. (London) B124, 397 (1938). E. Bauer, in "Die Methoden der Fermentforsehung" (E. Bamann and K. Myrbi~ck, eds.), Vol. 2, p. 1426, George Thieme, Leipzig, 1941.

728

RESPIRATORY ENZYMES

[128]

tion IV is used as partially purified quinone reductase. The accompanying table, which gives a summary of this purification procedure, shows a 24-fold purification with a 30% recovery of the total activity in the crude starting material. The range in purification of fraction IV varies between 10- and 25-fold. The enzyme is most stable between pH 7.0 and 7.5, losing not more than 20% of its activity after 5 weeks at - 1 5 °. SUMMARY OF PURIFICATION PROCEDURE a

I. II. III. IV.

Fraction

Total units

Total protein, mg.

Crude extract Calcium phosphate supernatants 50-65% ppt., ammonium sulfate Alumina gel supernatants

850,000 550,000 262,000 244,000

7300 1460 148 88

Specific activity, units/rag. Recovery, protein % 116 380 1770 2780

65 31 29

W. D. Wosilait and A. Nason, J. Biol. Chem. 206, 255 (1954). In terms of turnover number, fraction IV catalyzes the oxidation of approximately 150 moles of D P N H per mole of protein per minute, assuming a molecular weight for the enzyme of 100,000. Properties Specificity. The enzyme is specific for D P N H and T P N H , but not deamino-DPNH, 4 as electron donors. As electron acceptors, p-quinone, toluquinone, p-xyloquinone, benzoquinone acetic acid, 1,4-naphthoquinone, and 1,2-naphthoquinone may serve, but not 2,6-dichloroquinone, 2,5-dichloroquinone, diacetyl, pyruvic acid, cytochrome c, or 2-methyl1,4 naphthoquinone. The latter compound is active as an enzymatic electron acceptor for menadione reductase2 An estimate of the dissociation constants (Kin) indicates that one-half maximal activity with D P N H is obtained at about 2.1 X 10-5, and with T P N H at about 0.8 X 10-5, in moles per liter. However, the maximal rate of activity achieved with D P N H was 65% greater than that with T P N H . The dissociation constants (K~) of the enzyme-p-quinone complex are 1.7 X 10-4 and 2.2 × 10-4 in moles per liter with D P N H and T P N H , respectively. Activators and Inhibitors. The inorganic ions Mg ++, MOO4--, B407--, Zn ++, Mn ++, Fe ++, or Fe +++, at 10-4 M concentration have no influence on the enzymatic or nonenzymatic rates. However, Cu ++ at 10-4 M concentration causes a 30% decrease in enzymatic activity when preincubated with the enzyme for 10 minutes. Cysteine and GSH at a final con9 w. D. Wosilait and A. Nason, J. Biol. Chem. 208, 785 (1954).

[129]

HYDROGENASE

729

centration of 10-3 M inhibit both the enzymatic and nonenzymatic reactions, presumably by reducing the p-quinone. IAA, p-chloromercuribenzoate, potassium cyanide, sodium azide, sodium arsenate, sodium fluoride, and ethylenediaminetetraacetate have no effect at 10-3 M final concentration on either the enzymatic or nonenzymatic reaction. A number of nitrophenols and related compounds have the following inhibitory effects at a final concentration of 10-4 M: 2,4-dinitrophenol, 95 %; 2,4-dinitroaniline, 0 %; 2,4,6-trinitrophenol, 96 %; o-nitrophenol, 45 %; p-nitrophenol, 75 %; 2-amino-4-nitrophenol, 88 %; 2,6-dinitro-4chlorophenol, 97 %; 2,6-dichloro-4-nitrophenol, 59 %; 2,4-dinitroresorcinol, 80 %; 2,4-dinitro-l-naphthol, 83 %. Effect of pH. The enzyme exhibits an optimum for activity at p H 6.5, the activity falling to one-half of its maximum at pH 5.0 and 7.5. The nonenzymatic rate is not affected by hydrogen ion concentration above pH 6.0; below that there is an increase in observed rate due to acid destruction of D P N H . Phosphate, acetate, and pyrophosphate buffers serve equally well. Glycylglycine was found to be unsatisfactory, owing presumably to a reaction with quinone. Products of the Reaction. D P N and hydroquinone have been identified as products of both the enzymatic and nonenzymatic reactions. There is a mole-for-mole relationship between the D P N H oxidized and the hydroquinone formed enzymatically. From the E'0 values at pH 7.0 of D P N H : DPN + and hydroquinone: quinone, - 0 . 2 8 and -t-0.30 volt, respectively, ~° a change in free energy of approximately -26,800 calories was calculated for the quinone reductase reaction. From this value the equilibrium constant for the reaction was calculated to be K = 102°. i0 L. Anderson and G. W. E. Plaut, in "Respiratory Enzymes" (H. A. Lardy, ed.), rev. ed., p. 84, Burgess Publishing Co., Minneapolis, 1949.

[129] H y d r o g e n a s e f r o m Clostridium Kluyveri H2 + X ~ XH2

By SEYMOUR •ORKES Assay Method Principle. Although the nature of the primary acceptor (X) is at present unknown, it has been demonstrated I that pyridine nucleotides can be reduced by molecular hydrogen. If DPN, pyruvate, and lactic dehyI s. Korkes, in "Phosphorus Metabolism" (W. D. McElroy and B. Glass, eds.), Vol. 2, p. 502, The Johns Hopkins Press, Baltimore, 1951.

730

RESPIRATORY ENZYMES

[129]

drogenase are added to the hydrogenase system, the over-all reaction Hz -}- p y r u v a t e ~---Lactate m a y be followed either m a n o m e t r i c a l l y b y m e a s u r e m e n t of hydrogen uptake, or colorimetrically b y m e a s u r e m e n t of p y r u v a t e disappearance. T h e latter procedure is m o r e suitable for routine assay and will be described in detail. F o r other assay methods, see Vol. I I [152]. Procedure. T h e reaction mixture contains the following (in micromoles): Tris buffer, p H 8 (100); D P N (0.5); p o t a s s i u m p y r u v a t e (2.5). I n addition, lactic dehydrogenase (2000 units), 2 a suitable source of cofactor, 8 and a p p r o p r i a t e a m o u n t s of e n z y m e are added. T h e t o t a l volume is 1 ml. T h e reaction is carried out in test tubes which are rapidly flushed for 1 m i n u t e with t a n k hydrogen or e v a c u a t e d and refilled with hydrogen four times in rapid succession. T h e tubes are i n c u b a t e d at 30 ° with agitation for 20 minutes, and the reaction is stopped b y addition of 4 ml. of 5 % trichloroacetic acid. Suitable aliquots are t a k e n for determination of p y r u v a t e b y the " t o t a l h y d r a z o n e s " procedure of F r i e d e m a n n and Haugen. 4 Results are calculated in t e r m s of micromoles of p y r u v a t e utilized, employing as blanks either zero time samples or samples i n c u b a t e d under helium. Definition of Unit and Specific Activity. One unit of e n z y m e is defined as t h a t a m o u n t catalyzing the disappearance of 1 micromole of p y r u v a t e per hour in the presence of s a t u r a t i n g a m o u n t s of cofactor. Specific activity is expressed as units per milligram of protein. Protein is determined b y the m e t h o d of L o w r y et al. 5

Purification Procedure Clostridium kluyveri 6 is employed as a source of this enzyme, based on the observation 7 t h a t cell-free p r e p a r a t i o n s of the organism catalyze the reduction of acetaldehyde b y molecular hydrogen. T h e organism is grown

2 See Vol. I [67]. 3 Thus far, the most satisfactory source of this factor has been boiled cell extracts of Cl. kluyveri. These are prepared by addition of 1 g. of dried cells to 20 ml. of boiling water, the temperature being maintained at 100° for 10 minutes, followed by eentrifugation. The residue is re-extracted with 10 ml. of boiling water, and the supernatant after centrifugation is combined with the first extract. This solution contains approximately 10 to 15 units of cofaetor (as defined in the text) per milliliter. 4 T. E. Friedemann and G. E. Haugen, J. Biol. Chem. 147, 415 (1943). s O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 198, 265 (1951). 6 H. A. Barker and S. M. Taha, J. Bacteriol. 43, 347 (1942). 7 E. R. Stadtman and H. A. Barker, J. Biol. Chem. 180, 1117 (1949).

[129]

HYDROGENASE

731

and harvested as described by Stadtman and Barker. 8 The dried cells are stored at - 15°. Preparation of Cell-Free Extracts. Individual lots of dry organisms vary widely in their activity and extractability. The extraction procedure given below describes the average behavior of such preparations. Ten grams of dried cells is ground in a mortar and added to an Erlenmeyer flask containing 150 ml. of 0.01 M potassium phosphate0.01 M cysteine solution, pH 7.0, which has been previously deaerated by evacuation and bubbling with hydrogen. The flash is flushed with hydrogen, stoppered, and incubated at room temperature for 4 hours. The suspension is agitated by means of a magnetic stirrer. After centrifugation at 15,000 X g for 20 minutes, the clear, yellow 9 supernatant fluid is transferred to a container which is alternatively evacuated and refilled with hydrogen. The residue is re-extracted in the same manner with 75 ml. of phosphate-cysteine solution, and the supernatant fluid obtained after centrifugation is combined with the initial extract. This may be stored at - 1 5 ° for several weeks without appreciable loss of activity. No attempt has been made at systematic fractionation of the preparation as yet. However, the addition of 0.1 vol. of 2 % protamine sulfate at pH 6.0 results in removal of large amounts of inert material in the precipitate. The enzyme may be concentrated by fractionation with solid ammonium sulfate. The fraction from 0.4 to 0.7 saturation contains the bulk of the activity. The average specific activity of such preparations in the assay described is 4.

Properties Twenty-four-hour dialysis of the extract against 0.01 M phosphate0.005 M cysteine, pH 7, under H~, results in essentially complete loss of activity. This activity is restored by addition of water extract of boiled cells2 Addition of increasing amounts of this cofactor results in a typical saturation curve. One unit of cofactor is defined as the quantity necessary to half-saturate the system. The cofactor may be concentrated and partially purified by adsorption on a charcoal column (Nuchar C, unground) at pH 2 and elution with a mixture of equal parts of 0.05 N NH3 and acetone. The cofactor is eluted in a fraction which precedes the elution of the flavin compounds present in the boiled juice. 8 E. R. Stadtman and H. A. Barker, J. Biol. Chem. 180, 1085 (1949); see Transacetylase, Vol. I [98]. 9 The color of the completely reduced preparation is a light amber. When oxidized the preparation darkens considerablyand may show a greenish hue.

732

RESPIRATORY ENZYMES

[130]

This fraction is evaporated to dryness and taken up in water. I t m a y be kept unfrozen at 5 ° . Indications t h a t more than one protein component m a y be involved in this process have been obtained. In several preparations, increasing the enzyme concentration has resulted in a parabolic increase in activity, even in the presence of saturating amounts of cofactor. D P N saturates the system described at 5 X 10-4 M. T h e reduction of T P N m a y be inferred b y the ability of hydrogen to reduce GSSG in the presence of yeast glutathione reductase upon addition of T P N . TM The saturating concentration for T P N in this latter system is also 5 × 10-4 M. The activity of the preparation is inhibited to 50% of the normal value in the presence of 10 -2 M arsenite. A m m o n i u m sulfate (0.15 saturated) also inhibits 50%, which necessitates dialysis (under hydrogen) prior to assay of a m m o n i u m sulfate fractions. lo See Vol. II [127].

[130] C y t o c h r o m e s

a, a l , a2, a n d a3

B y LUCILE SMITH

Occurrence and Properties of Cytochromes a, a~, a~, and a3 The cytochromes of types a, al, a2, and a3 are defined b y the spectral characteristics of the pigments and their carbon monoxide compounds. 1-~ Table I lists the peaks in the absorption spectra of the various compounds. 4,5 C y t o c h r o m e a3 is an oxidase, which is the terminal respiratory enzyme ~ in mammalian tissues, yeast, higher plants, a few bacteria, and a n u m b e r of other cells. T h e enzyme in mammalian tissues, yeast, and higher plants is part of the cytochrome c oxidase system which is oxidized b y molecular oxygen and then rapidly oxidizes reduced cytochrome c. The oxidase of bacteria does not oxidize mammalian cytochrome c. Cytochromes al and as are pigments which have been observed only in certain bacteria. In some bacteria only cytochrome al is present; other bacteria contain a mixture of cytochromes a~ and as. Cytochrome al has 1 B. Chance, J. Biol. Chem. 202, 397 (1953). D. Keilin and E. F. Hartree, Proc. Roy. Soc. (London) B127, 167 (1939). s A. Fujita and T. Kodama, Biochem. Z. 273, 186 (1934). B. Chance, J. Biol. Chem. 197, 567 (1952). L. Smith, Arch. Biochem. and Biophys. 50, 299 (1954).

[130]

CYTOCHROMES a, ai, a2, AND

733

aa

been shown to be the terminal respiratory e n z y m e of Acetobacter pasteurianum, but it is not k n o w n w h a t substance is oxidized b y this oxidase. Similar d a t a h a v e not been obtained with other organisms possessing a cytochrome with an a - b a n d in the absorption s p e c t r u m around 590 mt~. There is some evidence t h a t all of the pigments which h a v e been classified as cytochrome ai do not h a v e similar properties. The physiological role of c y t o c h r o m e a2 is unknown, and it does not seem to occur in the absence of c y t o c h r o m e al. N e i t h e r p i g m e n t will oxidize m a m m a l i a n c y t o c h r o m e c. ~,~ TABLE I POSITIONS OF PEAKS IN THE ABSORPTION SPECTRA OF REDUCED CYTOCHROMES aj al, a2~ AND a3 AND THEIR CARBON MONOXIDE COMPOUNDS a'b

Pigment in reduced form

Compound of CO with reduced pigment

Visible region,

Soret region,

Visible region,

m~

m~

mD

Soret region, m~

Cytoehrome

(a band)

(-~ band)

(a band)

('r band)

a al a2 a3

605 590 635 605

? 435-440 ? 445

None 590 650 590

None 427 430

a B. Chance, Nature 169, 215 (1950). b L. Smith, Arch. Biochem. and Biophys. 50, 299 (1954). C y t o c h r o m e a often, but not always, occurs together with c y t o c h r o m e a3 in cells. 2,7 I t s peaks in the absorption s p e c t r u m are suuerimposed upon those of cytochrome a3, but, unlike cytochrome a3, it does not react with carbon monoxide. T h e role of cytochrome a in the respiratory cycle has not been established. Assay Methods

Cylochrome a. T h e p e a k in the absorption s p e c t r u m at 605 m~ in the reduced f o r m which remains after a n y cytochrome a3 present has reacted with carbon monoxide represents the cytochrome a present. T h e extinction coefficients of c y t o c h r o m e a are not known, but Chance 8 assumes t h a t the values for verdoperoxidase m a y be used when the relative a m o u n t s of the cytochromes are assayed. Cytochrome ai. T h e presence of c y t o c h r o m e ai in intact cells of bacteria or in extracts m a y be t e n t a t i v e l y indicated b y the identification of

8 D. Keilin and C. H. Harpley, Biochem. J. 35, 685 (1941). 7L. Smith, Federation Prop. 12, 270 (1953). 8 B. Chance, Nature 169, 215 (1952).

734

RESPIRATORY

ENZYMES

[130]

an a b s o r p t i o n b a n d at a b o u t 590 m~ when the reduced form (anaerobic cells or e x t r a c t reduced with Na2S:O~) is e x a m i n e d w i t h a visual spectroscope. Positive identification of the p i g m e n t with the c y t o c h r o m e al of A . p a s t e u r i a n u m m u s t be m a d e b y p r e p a r a t i o n of the c a r b o n monoxide c o m p o u n d of the reduced c y t o c h r o m e and d e m o n s t r a t i o n of peaks in the a b s o r p t i o n s p e c t r u m at 590 a n d 427 m~. Since these peaks are v e r y close to those of the c a r b o n monoxide c o m p o u n d of c y t o c h r o m e a3, a v e r y a c c u r a t e s p e c t r o p h o t o m e t r i c i n s t r u m e n t is required. I n whole cells, where the l i g h t - s c a t t e r i n g p r o p e r t i e s interfere with s p e c t r o p h o t o m e t r i c studies of t h e a b s o r p t i o n spectra, this can be accomplished only b y m e a s u r i n g the p h o t o c h e m i c a l s p e c t r u m or the c a r b o n monoxide difference s p e c t r u m (difference b e t w e e n the c a r b o n monoxide c o m p o u n d a n d the reduced form of the p i g m e n t ) b y m e a n s of specially designed a p p a r a t u s such as t h a t described b y Chance. 1 W i t h e x t r a c t s which are n o t so t u r b i d , the c a r b o n monoxide difference s p e c t r u m m i g h t be m e a s u r e d b y d e t e r m i n i n g t h e s p e c t r u m of the c a r b o n monoxide c o m p o u n d a g a i n s t the reduced p i g m e n t as a b l a n k in a s p e c t r o p h o t o m e t e r ; d e t e r m i n a t i o n of the difference s p e c t r u m in this w a y will minimize interference due to light scattering. 8 F r o m the d a t a of t h e carbon monoxide difference s p e c t r u m , the m o l a r i t y of the p i g m e n t in Acetobacter p a s t e u r i a n u m can be c a l c u l a t e d from t h e value for Ae d e t e r m i n e d b y C h a n c e 9 a n d listed in T a b l e I I , p r o v i d e d t h a t it has been e s t a b l i s h e d TABLE II Ae VALUES FOR THE D I F F E R E N C E BETWEEN THE CARBON ~V[ONOXIDE COMPOUNDS OF CYTOCttROMES a l AND a3 AND THE REDUCED PIGMENTS AND FOR THE D I F F E R E N C E BETWEEN THE REDUCED AND OXIDIZED FORMS a

Cytochrome a~ Cytochrome at Heart muscle Yeast B. subtilis

Acetobacter pasteurianum

Ac carbon monoxide compound minus reduced

5e reduced minus oxidized

82 cm.-1 mM -1 from the peak at 430 mu to the trough at 445 mu 86 cm. -1 mM -1 from the peak at 430 mu to the trough at 445 mu 100 cm. -1 mM -~ from the peak at 430 mu to the trough at 445 mu 60 cm. -1 mM -1 from the peak at 427 mu to the trough at 440 mu

91 cm. -1 mM -1 at 445 mu

B. Chance, J. Biol. Chem. 202, 407 (1953). 9 B. Chance, J. Biol. Chem. 202, 407 (1953).

87 cm. -1 raM -~ at 445 m# 25 cm. -~ mM -~ at 445 mu 120 cm. -1 mM -1 at 427 m~

CYTOCHROMES a, al, as, AND a3

[130]

735

that there are no other pigments present which will react with carbon monoxide. I t should be pointed out that the values for the extinction coefficients given in Table. II may be different for the pigments in different species. For example, the values for the cytochrome a3 of B. subtilis are different from that of yeast and heart muscle, and they should be used with caution in other species. Cytochrome as. The presence of cytochrome as in bacteria or in extracts can be ascertained by the demonstration of an absorption peak at about 630 m~ in the reduced form, which shifts to 650 m~ on addition of carbon monoxide to the reduced bacteria or bacterial extracts. The difference spectra (reduced minus oxidized or carbon monoxide minus reduced) of the intact bacteria can be measured only in a specially designed spectrophotometer,8 but the difference spectra of less turbid extracts may be determined in an instrument such as a Beckmann spectrophotometer. No extinction coefficients have been determined for this pigment. Cylochrome a3. 1. ON THE BASIS OF SPECTRAL PROPERTIES. The enzyme is identified by the formation of a carbon monoxide compound of the reduced pigment with peaks in the absorption spectrum at 590 and 430 m~. Difference spectra of whole cells or cellular extracts can be measured as described above. The concentration of th'e enzyme can be calculated from difference spectrum measurements using the extinction coefficients listed in Table II for heart muscle, yeast, and B. subtilis. The values for B. subtilis are quite different from those of the other two. 2. ON THE BASIS OF ACTIVITY; OXIDATION OF MAMMALIAN CYTO-

CHROME C. The cytochrome a3 of mammalian tissues and yeast and higher plants is part of the cytochrome c oxidase system which will rapidly oxidize reduced mammalian cytochrome c. The cytochrome as of B. subtilis will not oxidize mammalian cytochrome c. Thus mammalian or yeast or plant cytochrome a3 can be assayed by measuring the rate of oxidation of reduced cytochrome c by cellular extracts. Whole cells cannot be used, since cytochrome c may not penetrate the cells. The assay method is as follows.l°-l~

Reagents Cytochrome c solution (90 ~M in 0.01 M P04 buffer, pH 7.0). The cytochrome c can be prepared by the method of Keilin and Hartree 1~and is available commercially. The cytochrome c must 10 H. G. Albaum, J. Tepperman, a n d O. Bodansky, J. Biol. Chem. 163, 641 (1946). 11 j . N. S t a n n a r d a n d B. L. Horecker, J. Biol. Chem. 172, 599 (1948). 1~ L. Smith, Arch. Biochem. and Biophys. §0, 285 (1954). 13 D. Keilin a n d E. F. Hartree, Biochem. J. $9, 289 (1945).

736

RESPIRATORY ENZYMES

[1~0]

show a very slow rate of autoxidation when reduced by H~ and Pd (see below). Palladium asbestos, 5%. Hydrogen and nitrogen gases, low in oxygen. 0.1 M phosphate buffer, pH 7.0. The ionic strength of the final assay mixture is optimal for the enzyme on heart muscle particles; the activity of the yeast enzyme is only slightly influenced by the ionic strength of the buffer. 12 Saturated K3Fe(CN)6. Enzyme. Dilute the tissue homogenate or fraction with the phosphate buffer to such an extent that the time required for complete reduction of the cytochrome c is between 3 and 10 minutes. Procedure. The rate of oxidation of reduced cytochrome c is determined by measuring spectrophotometrically the decrease in optical density at 550 m~ as the reduced cytochrome c is oxidized. It is best to reduce the cytochrome e with palladium and hydrogen in such a way that no H20~ is formed during the process. ~2H202 will slowly oxidize the cytochrome c and will lead to erroneous results if any cytochrome c peroxidase is present in the extract. No H202 is formed if the buffered cytochrome c solution in which palladium asbestos is suspended (about 0.2 g. per 10 ml. of cytochrome c solution) is gassed with nitrogen for 5 minutes, then with hydrogen for about 1 hour, and again with nitrogen for 5 minutes. The mixture is rapidly filtered and stored in a stoppered bottle. The reduced cytochrome c is very slowly autoxidized but will usually remain sufficiently reduced for a day or two. The extent of reduction can be tested by measuring the ratio of the optical density of the cytochrome c solution at 550 mr* to the optical density at 565 m~.14 If the ratio is less than 6, the proportion of reduced cytochrome c is too small for use in the cytochrome c oxidase assay. If H:O2 is absent, the rate of autoxidation of the cytochrome c is too slow to necessitate a blank run for the autoxidation. Mix 0.5 ml. of reduced cytochrome c (90 ~M) and 2.4 ml. of the phosphate buffer in a 3-ml. cuvette, and determine the optical density at 550 mt~ (water or buffer for a blank). Then stir in rapidly 0.1 ml. of ap14 The equation relating the ratio of optical densities a t 550 a n d 565 m~ to t h e fraction of eytochrome reduced is as follows:

OD~5o 0D6~5

19(x) + 9 --4.5(x) + 7.5

where x = fraction of cytochrome c reduced. The relationship is plotted in a paper b y K. G. Paul Arch. Biochem. 12, 441 (1947).

[130]

CYTOCHROMES a, al, a2, AND aa

737

propriately diluted enzyme, and take readings every 10 seconds (or as often as possible) for about 1 to 2 minutes. Add a small drop of the saturated K3Fe(CN)8, and determine the optical density of the completely oxidized cytochrome c. Calculation of Activity~ The cytochrome c oxidase activity is expressed as the first-order velocity constant for the oxidation; the reaction is first order with respect to the reduced cytochrome c. The value for the optical density of the totally oxidized cytochrome c (OD~) is subtracted from the values for optical density determined at definite time intervals during the oxidation; these values for O D - OD~ plotted against time should give a straight line when plotted on semilog paper (OD values on log scale). From this plot: k = log (OD - ODd)t, -- log (OD - ODd)t2 X 2.3 sec. -I t2 -- tl Purity of the enzyme may be indicated by relating the k to the dry weight. The assay method can be used with homogenates or extracts of cells, provided that the ratio of activity to turbidity is high. As long as the reduced cytochrome e is free of H202, cytochrome c peroxidase will not interfere. Washed particles or extracts free of substrates will have no eytochrome c reductase activity, and in broken cell suspensions of yeast the reductase activity resulting from endogenous substrate does not interfere with the measurement of the first-order velocity constant for the oxidase reaction, since the reductase reaction is zero order with respect to cytochrome c concentration over the range of cytochrome c concentrations used to calculate the k for the oxidase reaction, is Cytochrome c oxidase activity can also be determined manometrically ~5 by measuring the oxygen uptake in the presence of added cytochrome c and a reagent to reduce the cytochrome c as it is oxidized. Preparations

Cytochromes a, al, a2, and a3, like the other cytochromes, are attached to insoluble cellular particles. Cytochromes al and as have not been separated from the insoluble particles with which they are associated in the bacteria, and they have been studied only in the whole cells or in particulate suspensions. A suspension of heart muscle particles may be prepared le which contains cytochromes a and a~ and the rest of the cytochrome system plus succinic dehydrogenase and several other enzymes involved in respira15 E. C. Slater, Biochem. J. 44, 305 (1949). 18 D. Keilin and E. F. tIartree, Proc. Roy. Soc. (London) B129, 277 (1940).

738

RESPIRATORY ENZYMES

[130]

tion. This preparation has strong cytochrome c oxidase activity, and cytochrome c is also present, but, since the particles are well washed, there is no oxygen uptake unless substrates are added. Thus this preparation is useful either as a source of the cytochrome c oxidase system or in studying the reactions of the whole succinic acid oxidase system. The following method is essentially that of Keilin and Hartree, 16 except that the tissue is ground in a Waring blendor instead of in a mortar with sand. Heart Muscle Particle Suspension. 1. Fresh pig hearts are freed of visible fat and fibrous tissue, and the muscle is passed twice through a meat grinder. The minced muscle may be stored at - 2 0 ° for 10 days without loss of activity. The minced heart is washed with stirring in about 10 vol. of cool tap water (not above 20°), the washing being continued for about 4 hours. The water is squeezed out through cheesecloth, and the wash water is changed about six to eight times during this interval. 2. The mince is homogenized in a Waring blendor for 7 minutes with 0.02 M phosphate buffer, pH 7.4, using 500 ml. of buffer for 200 g. of unwashed heart. 3. The homogenate is centrifuged in an angle centrifuge at 2000 r.p.m. for 15 to 20 minutes in the cold. The cloudy supernatant fluid is decanted and saved. The precipitate is rehomogenized in the Waring blendor for 2 minutes with 300 ml. of phosphate buffer and centrifuged as before. The supernatant fluid is then added to that from the first centrifugation. Usually 500 to 600 ml. of suspension is obtained from 200 g. of heart. 4. The pH of the cold suspension (below 5 °) is reduced to 5.6 by the addition of cold 1 M acetic acid, and then the mixture is centrifuged at 5000 r.p.m, for 15 minutes. The clear reddish supernatant fluid is discarded, and the precipitate is washed once with ice water. The precipitate in ice water can be sedimented by centrifuging for 5 minutes at 2500 r.p.m. 5. The precipitate is suspended in about an equal volume of 0.25 M phosphate buffer, pH 7.4 (the final volume should be about 70 ml.). Any lumps of precipitate can be dispersed by shaking gently in a flask or by gentle treatment in a hand-operated ground-glass homogenizer. The preparation has about 16 mg. dry weight per milliliter, and the first-order velocity constant for the oxidation of reduced exogenous cytochrome c is around 0.14 sec. -1 at 25 ° when the preparation is diluted 600 times in the final assay mixture. In the presence of 7 mM succinate, the Qo, at 25 ° should be about 400. There is still a small amount of hemoglobin in the preparation which will not affect activity determinations but will interfere with spectrophotometric measurements, especially in the Soret region. To eliminate

[130]

CYTOCHROMES

a, a,, a2, AND a3

739

this,14 the hemoglobin can be oxidized to methemoglobin by the addition of nitrite (1 mg./ml.); then the nitrite is removed by precipitating the particles by centrifugation at 40,000 >< g and resuspending them in 0.15 M phosphate buffer, pH 7.0, with the aid of a hand-driven groundglass homogenizer. The washing should be repeated once. The heart muscle particle suspension should contain an active succinic oxidase system. This activity can be tested by (1) measuring the 02 uptake manometrically in the presence of succinate; 18 (2) measuring the half-time for reduction of the cytochromes after addition of substrate;17 (3) measuring the rate of oxidation of succinate by observing the formation of fumarate (increase in optical density at 250 m~).17 Purified Cytochrome Oxidase Preparation. A preparation derived from the heart muscle particles can be made 19 which contains cytochromes a plus a~, has very high cytochrome c oxidase activity, and has the advantage that it is optically clear and is free of cytochrome c. Thus it is also useful in testing for the activity of cytochrome c. First Stage Preparation. Heart muscle extract is prepared as described in steps 1 through 4 above, except that the phosphate buffer should be 0.1 M instead of 0.02 M and the final volume adjusted to 300 ml. Sodium cholate is added to the extract to make a final concentration of 0.8%. The extract is then digested with "1-300" trypsin preparation of the Nutritional Biochemicals Co. (0.2 mg./ml, of digest) for 1 to 2 hours at 4 °. At the end of this time there should be a considerable clarification observed in the cloudy extract when it is compared with a sample of undigested cholate extract. Solid ammonium sulfate is then added to make the digest 0.4 saturated, and the mixture is allowed to stand in the cold for several hours. The precipitate which forms is removed by centrifugation, and the supernatant fluid is decanted off and made up to 0.5 saturation with ammonium sulfate. The precipitate is collected by centrifugation (7000 >< g for 20 minutes is sufficient) and dissolved in 0.1 M Na2HPO4, using a volume of about 10 ml. for each 100 ml. of cholate extract digested. The whole procedure is carried out at 4 ° . Second Stage Preparation. The first stage preparation contains in addition to cytochromes a and as some pigment with an absorption peak in the reduced form at 560 m,. This latter pigment can be removed by diluting the first stage preparation with about 4 vol. of cold 0.1 M Na2HPO4 and centrifuging at 20,000 >< g for 1 hour. The sticky red precipitate is then dissolved in a mixture of 13 % ammonium sulfate plus 0.5 % sodium cholate. If the final solution is not absolutely clear, it is cen17 B. Chance, J. Biol. Chem. 197, 557 (1952). is D. Keilin a n d E. F. Hartree, Biochem. J. 44, 205 (1949). 1~ L. S m i t h a n d E. Stotz, J. Biol. Chem. 209, 819 (1954).

740

RESPIRATORY ENZYMES

[131]

trifuged hard for 15 to 20 minutes. Most of the cholate in the preparation can be removed by dialyzing it against cold 13 % ammonium sulfate. If there is insufficient digestion of the particulate mixture, the resulting preparation will become cloudy on standing. The preparation has very strong cytochrome c oxidase activity and can be stored in the frozen state for several months without measurable loss of oxidase activity. If it is diluted with a mixture of 13 % ammonium sulfate plus 0.5% sodium cholate or with 13% ammonium sulfate, the preparation remains optically clear. When diluted with water or buffer, cloudiness will develop with a loss of activity, unless the preparation is diluted as much as 100 times. When diluted 100 times with cold distilled water, a water-clear solution is obtained which will retain its cytochrome c oxidase activity unchanged for at least 24 hours, if kept in an ice bath.

[131] C y t o c h r o m e b ( M a m m a l s )

By

E L M E R STOTZ

Cytochrome b of mammalian tissue is a hemoprotein which in the reduced (ferrous) form has absorption bands centered at 564 mp (a), 530 mp (B), and 430 mp (7). l-a In heart muscle extracts which contain both an active succinic dehydrogenase and cytochrome oxidase, the addition of succinate causes reduction of cytochrome b and aeration causes its oxidation. ~ This hemoprotein does not appear to combine with carbon monoxide or cyanide. 4,5 Ball 6 estimated the oxidation-reduction potential of cytochrome b in heart muscle extracts to be - 0 . 0 4 (Eo' at pH 7.0). The proximity of this potential to that of the succinate-fumarate system, coupled with the belief that the cytochromes react in sequence in the succinoxidase system, has led to the assumption that succinate is the direct reductant of cytochrome b in the succinoxidase system; indeed, from studies with narcotics and other agents Keilin and H a r t r e d ,s have stated their belief that cytochrome b functions as a link between succinate and cytochrome c. Nevertheless, Chance, 9 from studies 1 D. 2 D. 3 D. 4 D. s D. 6 E. 7 D. 8 D. 9 B.

Keilin, Proc. Roy. Soc. (London) B98, 312 (1925). Keilin, Proc. Roy. Soc. (London) B100, 129 (1926). Keilin a n d E. F. Hartree, Proc. Roy. Soc. (London) B127, 167 (1930). Keilin, Proc. Roy. Soc. (London) B104, 206 (1929). Keilin a n d E. F. Hartree, Nature 141, 870 (1938). G. Ball, Biochem. Z. 295, 262 (1938). Keilin a n d E. F. Hartree, Proc. Roy. Soc. (London) B129, 277 (1940). Keilin a n d E. F. Hartree, Biochem. J. 44, 205 (1949). Chance, Nature 169, 215 (1952).

[131]

CYTOCHROME b

(MAMMALS)

741

on the rate of reduction of the cytochromes by succinate, concluded that cytochrome b does not form a link between succinate and cytochrome c. It is clear that elucidation of the interactions of cytochrome b awaits separation of the components of the succinoxidase system. In this direction, Clark et al.10 have described a preparation of cytochrome b which was free of cytochrome c but rich in succinic dehydrogenase; addition of succinate caused reduction of the cytochrome b and the latter catalyzed the reduction of cytochrome c by a succinic dehydrogenase preparation which could not otherwise reduce cytochrome e by addition of succinate. 11 This preparation, in conjunction with cytochrome c and the purified cytochrome oxidase of Smith and Stotz, 12 reconstituted a system which caused the rapid oxidation of succinate.10 Other reports on the purification of cytochrome b include those of Eichel et al. 13 and Hfibscher and Kiese. 14 The former method involves an extraction of heart muscle particles with 2 % deoxycholate, and the latter an ammonium sulfate fractionation of a cholate extract of heart muscle. Neither report provides an assay for cytochrome b, and, although the report of Kiese contains a spectrum which indicates good separation of cytochrome b from the other cytochromes, the method of preparation is not sufficiently detailed to permit ready duplication. Since the preparation of Clark et al. 1° resulted from purification of the factor linking succinate with cytochrome c, and this activity paralleled the intensity of the cytochrome b absorption spectrum, the assay and preparation employed by these investigators have been chosen for more detailed description.

Assay Method The method is based on the spectrophotometric determination of the rate of reduction of cytochrome c in the presence of succinate and an excess of succinic dehydrogenase. With the availability of a succinic dehydrogenase preparation which does not link directly with cytochrome'c, l~ it has been shown that the rate of cytochrome c reduction is proportional to a factor other than succinic dehydrogenase. 16 The assay method ~0H. W. Clark, H. A. Neufeld, C. Widmer~ and E. Stotz, J. Biol. Chem. 210, 851 (1954). 11C. Widmer, H. W. Clark, H. A. Neufeld, and E. Stotz, J. Biol. Chem. 210, 861 (1954). 12L. Smith and E. Stotz, J. Biol. Chem. 209, 819 (1954). 1~B. Eichel, W. W. Wainio, P. Person, and S. J. Cooperstein, J. Biol. Chem. 183, 89 (1950). 14G. Hiibscher and M. Kiese, Naturwissenschaften 22, 524 (1952). 16H. A. Neufeld, C. R. Scott, and E. Stotz, J. Biol. Chem. 210, 869 (1954). 16By definition such a factor can be called SC factor (succinate-cytochrome c), a term previously used by F. B. Straub [Z. physiol. Chem. 272, 219 (1942)].

742

RESPIRATORY ENZYMES

[131]

is not unlike that of Cooperstein et al. 17 for succinic dehydrogenase, which, at least in heart muscle extracts, appears to be critical for the factor linking succinic dehydrogenase with cytochrome c rather than for suceinic dehydrogenase itself. P r o c e d u r e . To a 3-ml. Beckman cuvette (10-mm. light path) are added the following: 1.0 ml. of 0.1 M phosphate buffer, pH 7.4; 0.2 ml. of 0.3 M sodium succinate; 0.2 ml. of neutralized 0.02 M potassium cyanide; 1.2 ml. of distilled water; 0.1 ml. of succinie dehydrogenase; TM and 0.1 ml. of the solution to be tested for its ability to link succinic dehydrogenase and cytochrome c (SC activity). To initiate the reaction 0.2 ml. of 2 X 10-4 M cytochrome c solution is added. Readings are taken at 550 mu every 30 seconds against a " b l a n k " solution containing all the above constituents except the cytochrome c and the factor being assayed. After an initial period of about 1 minute, the plot of optical density against time is essentially a straight line, and the slope of this line is proportional to the amount of SC factor employed. A unit of SC factor activity is defined as that amount which produces a change of 1.0 in optical density per minute at 28 °. Purification Procedure

A 400-g. portion of pig heart muscle, previously trimmed of fat, is ground twice and washed by constant stirring with 2 ~ 1. of distilled water for about 15 minutes. The muscle residue is allowed to settle, and, after the supernatant has been poured off, the residue is strained and squeezed through a layer of shroud cloth. This procedure is repeated twice more with water and once with 0.01 M (phosphate) buffer, pH 7.4. The pressed, washed muscle is divided into two equal portions, and each portion is homogenized for 5 minutes in the Waring blendor with 500 ml. of 0.1 M (phosphate) buffer, pH 7.4. The homogenates are centrifuged at 1800 X g for 25 minutes in the cold. The resulting supernatants are saved, and the two residues are each re-extracted two more times by homogenizing for 3 minutes in the blendor with 300-ml. portions of the phosphate buffer, followed by centrifugation as before. All the supernarants are then combined to give about 1900 ml. of phosphate extract with a purity of about 1.0 SC factor units/mg. This extract is stable for several days at 4 ° . 17S. J. Cooperstein, A. Lazarow, and N. Kuffess, J. Biol. Chem. 186, 129 (1950). 18In tests with heart muscle extracts or with any of the fractions described under Purification Procedure, it has been determined that succinic dehydrogenase is already present in excess and further addition of this enzyme is unnecessary. For the assay to be valid, however, it is necessary to prove that this is true, in which case it is necessary to employ a succinic dehydrogenase preparation which itself is unable to link with cytochrome c.

[131]

CYTOCHROME b

(MAMMALS)

743

All subsequent operations are carried out at 4 ° . Step 1. The p H of the phosphate extract is adjusted to 5.4 while the mixture is stirred mechanically in an ice bath and cold 1.0 M acetic acid is slowly added. The resulting mixture is centrifuged for 45 minutes at 1800 X g, and the supernatant fluid is discarded. The precipitate is resuspended in 500 ml. of 0.1 M phosphate buffer, p H 7.4, b y homogenizing for 30 seconds in the Waring blendor. The protein concentration of this solution is determined b y a sulfosalicylic acid turbidity method, 19 and the protein concentration is adjusted to 12 mg./ml. Step 2. To this mixture is added 1.25 ml. of 40% sodium cholate for every 100 ml. of solution, to yield a mixture containing 0.5% sodium cholate. T o every 100 ml. of resulting solution, 54 ml. of 0.1 M phosphate buffer, p H 7.4, saturated with ammonium sulfate, is added. The mixture is centrifuged for 30 minutes at 1800 × g. The supernatant fluid is saved and made up to 0.5 saturation with ammonium sulfate b y adding 30 ml. of the buffered ammonium sulfate solution to each 100 ml. After centrifugation as above, the precipitate is suspended in 0.1 M disodium phosphate to make a volume about one-eighth that of the extract used for ammonium sulfate fractionation. This solution (0.35 to 0.50 fraction) is allowed to stand overnight at 6 ° . Step 3. The precipitate present is discarded after centrifugation for 1 hour at 18,000 X g. The ammonium sulfate concentration of the supern a t a n t fluid is measured b y nesslerization after removal of the protein by tungstic acid 2° and then adjusted to 8.0% ammonium sulfate b y dilution with 0.1 M disodium phosphate. Step .~. To each 100 ml. of this solution is added 43 ml. of buffered ammonium sulfate solution, and the mixture is centrifuged for 15 minutes at 12,000 X g. The supernatant fluid is saved, and 16.7 ml. of buffered ammonium sulfate is added to each 100 ml. After centrifugation as above, the precipitate is resuspended in 0.1 M disodium phosphate to make a volume equal to one-eighth t h a t of the solution used for step 4. The solution obtained is a clear red solution which in SC factor activity is purified thirtyfold and concentrated sixtyfold over the heart 19An appropriate amount of protein solution (0.05 to 0.2 ml.) is diluted to 5.0 ml. with distilled water, 10 ml. of 3% sulfosalicyclie acid is added, and the turbidity read in a Klett colorimeter using the No. 44 blue filter. The protein content is compared with turbidities produced by known amounts of crystalline egg albumin under the same conditions. 2o Two-tenths milliliter of sample is diluted to 8.0 ml. with distilled water, followed by addition of 1.0 ml. of 10% sodium tungstate and 1.0 ml. of 1 N H2SO4. After mixing and centrifuging, an aliquot of the supernatant fluid is diluted to 45 ml. with distilled water, and 5.0 ml. of Nessler's solution is added. Colorimetric comparisons arc made against standard ammonium sulfate solutions.

744

RESPmATOnV ENZYMES

[132]

muscle extract used as the starting material. On spectrophotometric examination the solution may show a slight absorption peak at 553 m~ which is absent if the solution is treated with ferricyanide. Addition of dithionite produces a strong absorption band at 562 mt~ and a shoulder centered at 553 m~. Succinate causes only a partial reduction of the material absorbing at 562 mtL and complete reduction of the material absorbing at 553 mp. Addition of ascorbic acid selectively produces the 553-mu band with little change at 562 mtL. The preparation is rich in succinic dehydrogenase but apparently devoid of cytochrome c. Addition of succinate and cytochrome c to the preparation causes immediate reduction of the cytochrome c. It is concluded that cytochrome b is the predominant hemoprotein present in the preparation, but that at least one other hemoprotein, tentatively designated cytochrome 553, is also present. The role of the latter component in the linking of succinic dehydrogenase with cytochrome c is not yet clear. The positions of the absorption bands of the reduced components are as follows: Cytochromeb Cytochrome 553

562 m~ 553 mt~

530 mt~ 522 mtL

430 m~ 417 m~

[132] C y t o c h r o m e b G r o u p ( B a c t e r i a ) B y A. M.

PAPPENHEIMER,JR.

The heme-containing pigments which have been called cytochrome b represent a group of proteins which not only differ from one another in physicochemical properties but probably differ as well in their catalytic function in the chain of respiratory enzymes. Thus, although all members of the b group show a and ~ absorption bands in the visible spectrum and a strong ~, or Sorer band in the violet region, the exact position of these bands varies from one bacterial species or strain to another. In the table, the positions of the a, 2, and ~, bands for the cytochrome b components of certain representative bacterial species and for yeast are given. I t can be seen that they may be classified into groups according to the position of their a bands. 1. Cytochrome b with absorption maximum 562 to 565 mt~. 2. Cytochrome bl at 560 mu. 3. Cytochrome b2 of yeast at 556 mtL. 4. A component absorbing at 554 mt~ which may or may not belong to this group and which is characteristic of Acetobacter. 1 1L. Smith, Bacteriol Revs. 18, 106 (1954).

[132]

CYTOCttROME b GROUP (BACTERIA)

745

Finally, the recently discovered cytochrome e ~ with an a band at 552 to 553 m~ at liquid air temperature is present in Bacillus lichenformis as well as in yeast and various animal and insect tissues. Recent studies suggest t h a t cytochrome e should be considered with the b group2 DIFFERENCE SPECTRA OF CYTOCHROME b IN CERTAIN BACTERIAa

Position of maxima for reduced pigment, m~ Strain

a

-y

Type

Bacillus subtilis Staphylococcus albus Sarcina lutea

564 564 565 562

525 523 523 523

430 422 427 430

Aerobacter aerogenes Escherichia coli Azotobacter chroococcum Corynebacterium diphtheriae c

560 560 560 560

530 533 530 524

430 432 428 429

bl

Acetobacter pasteurianum Acetobacter suboxydans

554 554

523 525

428 422

?

Delft yeast a

557

528

424

b~

Baker's yeast

This table has been constructed from the data of Smith? Except for the bands of C. diphtheriae and of yeast, all values are for difference spectra, i.e., difference in extinction between oxidized and reduced pigments. bL. Smith, Bacteriol. Revs. 18, 106 (1954). A. M. Pappenheimer, Jr., and E. D. Hendee, J. Biol. Chem. 171, 701 (1947). d C. A. Appleby and R. K. Morton, Nature 173, 749 (1954). I n addition to the similarity in their absorption spectrum, the various members of the b group have certain other characteristics in common. Most of the cytochrome b's appear to be autoxidizable at an appreciable rate, although far more slowly than is cytochrome oxidase. T h e y do not combine with CO, H C N , or HN3, although all the group probably contain protoheme as their prosthetic moiety. Their oxidation potential lies below t h a t of cytochrome c, and in the few cases studied their position in the respiratory chain lies below t h a t of cytochrome c. Little is known of the function of the bacterial cytochrome b group. Cytochromes b and bl are both concerned in the oxidation of succinate. 4,5 2 D. Keilin and E. F. Hartree, Nature 164, 254 (1949). a A. M. Pappenheimer, Jr., and C. M. Williams, J. Biol. Chem. 209, 915 (1954). 4D. Keilin and C. H. Harpley, Biochem J. 35, 688 (t941). s A. M. Pappenheimer, Jr., and E. D. Hendee, J. Biol. Chem. 180, 597 (1949).

746

RESPIRATORY ENZYMES

[132]

Cytochrome b~ of yeast appears to be closely associated with lactic dehydrogenase activity (see Vol. I [68] for preparation and properties of yeast lactic dehydrogenase). The cytochrome bl of E. coli is not only involved in succinate oxidation 4 but may also be an integral part of the nitrate reductase system. 6 None of the cytochrome b group has been isolated in pure form as a soluble pigment with the possible exception of cytochrome b2 of yeast. 7,7~ All other members appear to be associated with large mitrochondrial and microsomal fragments. Preparation and Partial Purification of Cytochrome bl from Corynebacterium diphtheriae 5 Bacterial Cultures. The Toronto strain of Corynebacterium diphtheriae is grown in 5- to 10-1. lots in Povitsky bottles as a pellicle on the surface of Mueller and Miller's casein hydrolyzate medium s containing 1.5 % maltose. Just before inoculation 25 mg. of FeSO4"7H20 per liter is added as a sterile solution. The pH of the medium should be below 7.5 at the time of harvesting. After 6 to 7 days' growth at 34 °, the bacteria are collected by centrifugation and are washed three times with saline in the centrifuge. Extraction of Cytochrome bl from the Bacterial Cells. Washed cells from 5 1. of culture are suspended in 150 to 200 ml. of saline, and the thick suspension (6 to 8 mg. bacterial N per milliliter) is disrupted for 30 minutes in a 9000-cps sonic oscillator (Raytheon Corp., Waltham, Massachusetts) in 25-ml. charges. If a sonic oscillator is not available, the cells may be broken up by grinding. 9 Partial Purification of Cytochrome bl. The purification procedure by differential centrifugation is entirely empirical, and no rigid procedure can be outlined. The Qo, (succinate, KCN) and Qo~ (succinate, MB, KCN) were 146 and 4070 ~l./mg. N/hr., respectively, for a crude diphtherial extract containing 7.6 mg. N/ml. The purification of this particular lot was accomplished as follows: 180 ml. of extract was centrifuged for 30 minutes at 10,000 r.p.m, in a refrigerated International centrifuge to remove bacterial debris. The turbid supernatant was then centrifuged at 12,000 r.p.m. (ca. 18,000 X g) in a Servall centrifuge, Model SS-2, for 30 minutes. Then 6R. Sato and F. Egami, Bull. Chem. Soc. Japan 22, 137 (1949). 7 S. J. Bach, M. Dixon, and L. C. Zerfas, Biochem. J. 40, 229 (1946). 7~C. A. Appleby and R. K. Morton [Nature 173, 749 (1954)] have isolated the lactic dehydrogenase of yeast as a crystalline hemoprotein containing flavin. s j. H. Mueller and P. Miller, J. Immunol. 40, 21 (1941). 9 G. KalnitSky, M. F. Utter, and C. H. Werkman, J. Bacteriol. 49, 595 (1945).

[132]

CYTOCHROME b GROUt (BACTERIA)

747

25 ml. of fatty upper layer of low activity was removed and discarded. Next 130 ml. of a clear red layer containing more than 60% of the total succinic oxidase activity was removed from a small amount of sediment and centrifuged in the Servall at 16,000 r.p.m. (ca. 30,000 )< g) for 3 hours. Once again three layers were obtained: an inactive yellow upper layer, a clear reddish-brown layer with considerable activity, and a highly active dark wine-red transparent sediment at the bottom of the tube. The sediment was suspended in 0.02 M phosphate buffer, pH 7, and homogenized for 20 minutes in the sonic oscillator. Thirty-five milliliters of dark-red solution was obtained which was clarified by centrifugation in the Servall for 30 minutes at 12,000 r.p.m. This solution contained about 30% of the total original succinic oxidase activity. Its Qo2 (succinate, KCN) was 440 and Qo2 (succinate, MB, KCN) was 14,000 ;~l. O2/mg. N/hr. This represents a purification of 3- to 3.5-fold, on the basis of both hemin content and specific activity.

Properties The above preparation contained 3.9 mg. N/ml. and 1.09 ~, hemin per milligram of nitrogen. Solutions of the purified pigment are dark red in color, clear by transmitted light, but show a pronounced Tyndall effect. After reduction with dithionite or with succinate, the color changes to a more pinkish hue and intense absorption bands centered at 560 and 529 m~ become readily visible with a hand spectroscope. The Soret band of the oxidized pigment shows a maximum absorption at 415 m# which shifts to 429 m# on reduction. When examined in the ultracentrifuge, cytochrome bl is polydisperse, and no distinct sedimenting components can be identified. On standing, even in the cold, aggregation occurs, and within 1 or 2 days solutions become turbid without, however, appreciable loss in enzymatic activity. Thus, although cytochrome bl cannot be regarded as a truly soluble protein such as cytochrome c, freshly purified preparations, in contrast to similarly prepared material from mammalian tissues, yield clear solutions which can readily be studied in an ordinary Beckman spectrophotometer. The preparations can be dried from the frozen state or can be precipitated at pH 4.8 in the cold without loss of activity. However, reconstitution of dried or precipitated b~ in buffer always yields turbid solutions. When purified preparations are heated to 60 ° at pH 7.6, aggregation occurs and the solution rapidly becomes turbid. Actual coagulation does not occur until 75 °, at which temperature 75% of the succinic oxidase activity is lost in 30 minutes. Assay of Enzymatic Activity. Diphtherial cytochrome b~ preparations

748

RESPIRATORY ENZYME$

[132]

catalyze the oxidation of succinate to fumarate. Unlike the bl of E. coli, 4 the reaction is not affected by M/400 KCN. Since available evidence suggests that for the diphtherial system under these conditions autoxidation of bl is the rate-controlling step, manometric determination of succinie oxidase activity in the presence of cyanide can be used as a method of assay. The total volume in each Warburg vessel is 2.2 ml., including 0.2 ml. of 20% NaOH in the center well and 0.2 ml. of M / 5 sodium succinate in the side arm. Phosphate buffer, pH 7.8, to give a final concentration of M/IO, 0.1 ml. of M/20 KCN, and a suitable quantity of enzyme are placed in the vessel itself. Control vessels without substrate should always be included but usually show no oxygen uptake. Qo2 values may be calculated from the oxygen consumed during the first 30 minutes and expressed as microliters per milligram of nitrogen per hour. The enzyme shows a sharp pH optimum at 7.8. Other Methods of Assay. Since succinic oxidase activity as measured above has always proved proportional to succinic dehydrogenase activity, any method for measuring the latter (see Vol. I [121]) would appear suitable for assay of diphtherial cytochrome bl. Succinic dehydrogenase may be determined manometrically in the presence of methylene blue and cyanide or by the anaerobic ferricyanide method of Quastel and Wheatly. '° Both methods are more sensitive than the one described above, because the autoxidation of methylene blue is more rapid than that of cytochrome bl and because oxidation of reduced bl by either methylene blue or ferrieyanide is more rapid than its autoxidation by molecular oxygen. N~

Qco,(Suce., ferrieyanide) = 4Qo,(suec., MB, KCN) = 120Qo,(SUCC., KCN) Suecinate oxidation may also be determined spectrophotometrically by following the rate of reduction of the nonautoxidizable dye 2,6-dichlorophenolindophenol (Price and Thimann). H 10j. H. Quastel and A. H. M. Wheatley, Biochem. J. 32, 936 (1938). 11C. A. Price and K. V. Thimann, Arch. Biochem. and Biophys. 33, 170 (1951).

[133]

CYTOCHROME C (MAMMALS)

[133] Cytochrome

c

749

(Mammals)

By KARL-GusTAV PAUL Fe +++ (Cytochrome Oxidase) -t- Fe ++ (Cytochrome c) ~-Fe ++ (Cytochrome Oxidase) % Fe +++ (Cytochrome c) Fe +++ (Cytoehrome c) + R - ~ Fe ++ (Cytochrome c) + R

Assay Methods Spectrophotometric Method. The concentration of cytochrome c in a solution of unknown strength is determined, after suitable dilution with 0.05 to 0.15 M phosphate buffer of pH 6 to 8 and reduction with Na2S204, from the difference in light absorption between the a band (e550~ = 27.7 mM -1 X cm.-1) 1 and the minimum (e536,n~= 7.7 mM -1 X cm.-1) 1 between the a and ~ bands of the hemochromogen spectrum of reduced cytochrome c. Since the ~ band is very sharp, the readings have to be taken at every 0.5 m~ around 550 m~. The highest value is used. The above molar absorption coefficients have been found for several cytochrome e preparations obtained according to the method of Keilin and Hartree, 2 with Beckman DU spectrophotometers and with Na2S204 as reducing agent. Cytochrome c is, under certain conditions, e.g., extreme pH values and aging in solution, converted to a catalytically inactive form which is oxidizable by 02 even at neutral pH. k partial destruction of cytochrome c to the autoxidizable pigment cannot easily be detected by visual spectroscopy but can be determined according to Tsou. 8 This test should be made on every preparation prior to its use. The solution to be examined is suitably diluted with phosphate buffer of pH 7.3, final buffer concentration 0.05 to 0.15 M, and the optical density at 550 mu is determined after reduction with Na2S~O~. A current of CO is passed through the solution for 10 minutes, and the density at 550 mu is redetermined. The per cent of autoxidizable cytochrome c is given by 162(Drod.- D,ed+co)/D~d. where D~d. and D~od+coare the first and second readings. ~ 1 K. G. Paul, Acta Chem. Scand. 5, 389 (1951). 2 D. Keilin and E. F. Hartree, Biochem. J. 39, 289 (1945). C. L. Tsou, Biochem. J. 49, 362 (1951). 4 Tsou's method presupposes t~hat the molar absorptions of active and inactive cytochrome c are the same at 550 m~ after reduction. The latter gives, however, probably about 5 % higher absorption [D. L. Drabkin, J. Biol. Chem. 146, 605 (1942); E. Boeri, H. Baltscheffsky, R. K. Bonnichsen, and K. G. Paul, Acta Chem. Scand. 7, 831 (1953)].

750

RESPIRATORY ENZYMES

[133]

Manometric Method. The catalytic activities of different specimens of cytochrome c can be compared in the system O2/cytochrome oxidase/cytochrome c/succinate-succinic dehydrogenase. Succinic oxidase ( -- cytochrome oxidase ~- cytochrome c -F succinic dehydrogenase) preparations from kidney cortex are deficient in cytochrome c, which thus becomes the determining link. (For the preparation of succinic oxidase, ~,8 see Vol. I [121].) Reagents 0.4 M Na-succinate in water. 0.12 M phosphate buffer, pH 7.3. Kidney cortex preparation.

Procedure. Kidney cortex preparation (0.2 m l . ) + cytochrome c (<0.5 ml.) -F buffer to desired volume (3 to 3.3 ml.) are pipetted into the flask and 0.2 ml. of succinate into the side bulb. The vessels are equilibrated in the thermostat bath (39 °) for 20 minutes at a low rate of shaking. The succinate is poured into the main space and the shaking increased to 100 osc./min. The oxygen uptake between 5 and 15 minutes after the addition of succinate is a measure of the succinic oxidase activity. The kidney preparations may vary in activity, but by changing the amounts of kidney preparation per flask it is generally possible to find nearly linear proportionality between 02 uptake and the amount of added cytochrome c up to 2 × 10-8 mole of the latter. Good kidney preparations at suitable concentrations give a blank value, without added cytochrome c, of 5/~l. of 02 per 10 minutes and 25 to 30/~l. per 10 minutes with 2 X 10-s mole of cytochrome c. It is advisable to use equal amounts, in terms of cytochrome c-iron, of the specimens which are compared. Since the kidney preparations are unstable, the oxygen consumptions should be measured simultaneously. Cytochrome c which is originally present in kidney or heart muscle preparations is about one hundred times as active a catalyst as added cytochrome c. 6 Chance 7 studied spectrophotometrically the reactions in the succinic oxidase system from heart muscle without adding cytochrome c. Purification Procedures

A great number of preparation methods have been published. The extraction from the tissues of the basic cytochrome c (isoelectrie point 5 D. Keflin and E. F. Hartree, Proe. Roy. Sac. (London) B129, 277 (1940) ; Biochem. J. 41, 503 (1947). E. C. Slater, Biochem. J. 44, 305 (1949); 45, 1 (1949). B. Chance, Nature 169, 215 (1952).

[133]

CYTOCHROME C (MAMMALS)

751

10.65, 0 °) by means of an acid, first employed by Theorell 8 (H~S04), is used in most methods. The one described by Keilin and Hartree, 2 is at present commonly used. A fresh horse heart is rinsed from blood clots, fat, and tendons and minced. Titrated (0.145 M) trichloroacetic acid (TCA) is added with stirring, in all 1 1./kg. in three to four lots. After 3 to 4 hours the extract is squeezed out and adjusted to pH 7.3 with 10% N a O H cautiously added to avoid local excess. (NH4)2S04 is added (500 g./1.), and the precipitate is removed by filtration. Then 50 g. of salt per liter is added to the filtrate, which is left overnight at 0 to 5 °. After another filtration the filtrate is acidified with 20% TCA (25 ml./1.) cautiously added. Before this addition the cytochrome has been in the reduced, pink form. On acidification it is oxidized to the three-valent, brown-red form and precipitates. It is collected by centrifugation, washed with 300 ml. of saturated (NH4)2SO4 solution, suspended in a minimum of water, and dialyzed against 0.5% NaC19 until free from sulfate. The suspension is shaken with a few drops of chloroform, and the precipitate is filtered off. The iron content after this step is usually 0.34%, according to the authors, but eventually the (NH4)2SO4-TCA procedure has to be repeated after dilution to 1 1. The iron content is further increased by the following treatment. (NH4) 2SO4 is added until no more salt dissolves, and 1/6 vol. of ammonia (sp. gr. 0.88, not 0.91) is added drop by drop to pH 10 (as determined with a glass electrode). After standing overnight at 5 ° the solution is filtered, and the filtrate, which contains the cytochrome, is dialyzed against running tap water for 3 hours, finally against 0.5% KC12 The solution is concentrated by suspending the cellophane tube in a current of air (not by vacuum evaporation). After further dialysis against KC19 the preparation should contain 0.43 % Fe. The catalytic activities of cytochrome c with 0.34 and 0.43% Fe are the same. 2 The method has been used for the preparation of cytochrome c from beef (yield 125 mg. per heart), sheep, dog, and rat hearts. In the case of guinea pig heart it is preferable to replace TCA by 0.15 N H2SO4 for the extraction. The method gives poor yields from visceral organs. According to the author's experience the final Fe content is frequently slightly lower than 0.43 %. A contamination with myoglobin, occasionally seen, can be disclosed and determined in the following way. One volume of the dialyzed solution is slowly added to 10 vols. of ice-cold acetone, containing 1 ml. of concentrated HC1 per liter. After 10 minutes in the icebox the precipis H. Theorell, Biochem. Z. 279, 463 (1935). 9 The replacement of 0.5% NaC1 or KCI by 0.005% NH4OH will probably give a better removal of the traces of TCA which may be held back by eytochrome e.

752

RESPIRATORY ENZYMES

[133]

tate is filtered off. The m o t h e r liquor has a slightly brownish color, deriving from hemin, if myoglobin is present in the cytochrome c preparation. The mother liquor is filtered and evaporated i n vacuo, and the residue is dissolved in a few milliliters of (0.1 M N a O H : p y r i d i n e 3:1, v / v ) . After reduction with Na~S204 the solution gives a hemochromogen spectrum (a band at 557 m~, e = 34.8 mM. -1 × cm. -1) 10 if myoglobin has been present. T h e myoglobin is completely or nearly completely removed if the (NH4)~SO4-ammonia procedure is repeated. T h e iron content of the " 0 . 3 4 % Fe cytochrome c " can also be raised b y electrophoresis, 11 b y adsorption on cation exchangers,12.18 or b y heating. 3 Pal~us and Neilands 12 observed several fractions of cytochrome c when samples obtained according to the T C A m e t h o d were chromatographed on Amberlite IRC-50. Loftfield ~4 has recently developed the following procedure, which seems to offer definite advantages and employs very mild conditions. Pretreatment of R e s i n . Amberlite XR-64 (200 to 400 mesh) is suspended in a 5-1. beaker with distilled water, stirred, and decanted after 10 minutes. T h e washing is repeated at least five times to remove the finest particles. This resin, or resin recovered from columns, is heated on a steam b a t h with 2 M K O H for 6 hours and washed b y decantation several times. Dilute H2S04 is added to p H 1, and the resin is washed several times with water. Dilute N H 4 O H is added to p H 10, the resin is washed several times with water, filtered b y suction, stirred with acetone, filtered b y suction, and finally left in open air until no smell of ammonia can be traced. I t is then stirred in distilled water for 1 hour before it is used. T h e p H of the effluent should be 7 when a column is washed with distilled water. Preparation. F o u r beef hearts are rinsed and homogenized with 15 1. of ice water. 15 The p H is adjusted to 4.0 with 5 % H2SO4. The tissue is removed b y centrifugation after 2 hours, and the pulp is re-extracted with 10 I. of ice water. The combined extracts are adjusted to p H 7.0 with 5 % N H 4 0 H and left overnight. N e x t d a y the precipitate is rem o v e d b y filtration, and the filtrate is stirred gently with 70 g. of NH4Amberlite XR-64. After 2 hours the resin is collected on a v a c u u m filter. If necessary (check spectroscopically) the filtrate is stirred with another 10K. G. Paul, H. Theorell, and/~. ~$~keson,Acta Chem. Scand. 7, 1284 (1953). 11H. TheoreU and/~. ~keson, J. Am. Chem. Soc. 60, 1804 (1941). i~ S. Pal4us and J. B. Neilands, Acta Chem. Scand. 4, 1024 (1950). 13E. Margoliash, Nature 170, 1014 (1952). 14R. Loftfield, Acta Chem. Scand. to be published. 15Operations should as far as possible be made at 0 to 5°. Ordinary distilled water is used.

[133]

CYTOCHROME C (MAMMALS)

753

20-g. portion of resin, which is collected as before. The resin is washed on the funnel with 5-1. portions of water until the light absorption of the filtrate is negligible. The resin is converted to a 50-mm. column (about 500 mm. high) and extracted with NH4OH (pH 8). The cytochrome c (about 800 mg.) is collected in about 400 ml. The eluate is adjusted to pH 8.0, dialyzed for 6 to 8 hours against distilled water, and applied onto a 20 × 300-mm. column of fresh NH4-Amberlite XR-64. The column is washed with 4 to 5 I. of water and developed with NH4OH of pH 8.0. When the bright red band has migrated about three-fourths down, 'the column is cut on both sides of the band, and the cytochrome-free resin is cleaned away. The resin is washed with water and poured into a third tube (ca. 10 X 80 mm.), washed with water, and eluted with 0.5 M NH4OH. The cytochrome c (about 500 mg.) is obtained in about 8 ml. of effluent. Loftfield and Bonniehsen (personal communication) have successfully applied this method to a number of organs from various animals, even in microscale. Cytochrome c, obtained as above, is homogenous in the ultracentrifuge and electrophoresis apparatus as well as on the columns. It has an Fe content of 0.34 %. The molar absorption coefficients are not definitely determined but seem to be about 15% higher than those given above. The material is equally active in the manometric activity tests per microgram of Fe as the "0.34% Fe cytochrome c" obtained according to Keilin and Hartree. 2 The product appears completely in the reduced form. Boeri TM has shown that ferrocytochrome c is oxidizable by 02 in the presence of TCA at an acidity where it otherwise would be stable.

Properties Structure. Cytochrome c contains 1 mole of Fe in the prosthetic group per mole. In ferricytochrome at neutral pH the Fe atom is held by covalent bonds to the porphyrin ring and to two nitrogen groups in the protein moiety. T M At least one of these nitrogen atoms belongs to the imidazole ring of a histidine residue, and probably both. The porphyrin ring is linked to the protein with thioether bridges. 19 The splitting of them by salts of heavy metals gives an optically active isomer of hematohemin, ~° whereas reduction with sodium amalgam gives a mesoporphyrin. 21 The amino acid composition, titration curves, spectrum, and

~6E. Boeri and L. Tosi, Arch. Biochem. and Biophys. 52, 83 (1954). 17H. TheoreU, J. Am. Chem. Soc. 60, 1820 (1941). 18K. G. Paul, Acta Chem. Scand. 5, 379 (1951). ~9H. Theorell, Biochem. Z. 296, 242 (1938). 2oK. G. Paul, Acta Chem. Scand. 4, 239 (1950); 5, 389 (1951). 21H. E. Davenport, Nature 169, 75 (1952).

754

RESPIRATORY ENZYMES

[133]

magnetic properties of cytochrome c have been studied mainly on electrophoretically purified preparations. 11,18,22 Oxidation and Reduction. Catalytically active cytochrome c, in the reduced form, is nonenzymatically oxidized b y 05 below p H 4 and above p H 13. Fully oxidized cytochrome c is obtained when a solution is acidified with HC1 to p H 3 and neutralized with an equivalent a m o u n t of alkali when the band at 550 m~ has vanished completely. Complete oxidation can also be achieved b y a n u m b e r of inorganic salts, e.g., KaFe(CN)6, subsequently removed b y c h r o m a t o g r a p h y on a cation exchanger.~3 Preparations according to Keilin and Hartree 2 are slowly partially reduced when left at p H 8 to 9. C y t o c h r o m e c is conveniently reduced within the range p H 4 to 12 b y means of Pt-H2. 8 Ferricytochrome c is also reduced b y a n u m b e r of organic compounds (e.g., adrenaline, p-phenylenediamine, hydroquinone). In some cases their reducing ability is clearly connected to their ionization (e.g., thiols, ascorbic acid, uric acid). T h e oxidation-reduction potential of cytochrome c at p H 7 is around + 0 . 2 5 v. 23 I n f l u e n c e of p H . C y t o c h r o m e c seems to be infinitely stable within the range p H 1.7 to 12.3. 24 Destruction b y extreme p H values of an enzyme system, in which it participates, is therefore likely to hit the other enzymes before cytochrome c is damaged. Inhibitors. N o specific inhibitor for cytochrome c is known. CO and halogen ions do not combine with cytochrome c at physiological p i t . A ferrocytochrome c-CO compound exists below p H 4 and above p H 13. In ferricytochrome c the covalent bonds to the iron a t o m are replaced b y ionic bonds at low pH, which opens the way for the formation of ferricytochrome-anion compounds (in the presence of HC1).~2

%+ NH

/

÷/ C1-

Fe +

C1-

HN

%

With ferricytochrome c at p H 7.4 cyanide gives a compound containing CN: Fe 1:1 with an absorption band at 535 mu and a low general absorption in the red. 26 Because of its slow formation (half-time 60 minutes at p H 7.4, ~ E. Boeri, A. Ehrenberg, K. G. Paul, and tI. Theorell, Biochim. et Biophys. Aeta 12, 273 (1953). ~3R. Wurmser and S. Fflitti-Wurmser, J. chim. phys. $6, 81 (1938); E. Stotz, A. E. Sidwell, and T. R. Hogness, J. Biol. Chem. 124, 11 (1939); K. G. Paul, Arch. Binchem. 12, 441 (1947); F. L. Rodkey and E. G. Ball, J. Biol. Chem. 182, 17 (1951). ~4K. G. Paul, Acta Chem. Scan& 2, 430 (1948). z6 V. R. Potter, J. Biol. Chem. 137, 13 (1941); B. L. Horeeker and A. Komberg, J. Biol. Chem. 165, 11 (1946).

[134]

CYTOCHROME c (USTILAGO)

755

25 °, m M K C N ) the cyanide cytochrome complex will contribute but little to the effect of cyanide on the respiration. With ferricytochrome c azide ion rapidly forms a compound with absorption bands at 540 m~ and 570 m~. 26 Because of the large dissociation constant (0.15 M at 25 °) also the azide effect on cytochrome c exerts little influence on the respiration. ~ B. L. Horecker and J. N. Stannard, J. Biol. Chem. 172, 589 (1948).

[134] C y t o c h r o m e

c (Ustilago)

By J. B. NEILANDS

Assay Method Principle. For isolation purposes a suitable approximation of the concentration of the hemoprotein m a y be obtained from the relative strength of the absorption bands of the reduced compound as seen with the hand spectroscope. Procedure. The material to be examined is suspended or dissolved in 0.1 M phosphate buffer and a "knife-point" of solid sodium hydrosulfite is added. The relative concentration m a y be estimated b y comparing the density of the 550-mr, band with t h a t of a similarly treated standard solution of beef heart cytochrome c. The concentration should be at t h a t level where the/~ band, in the region of 520 m~, is just barely visible. A Zeiss band spectroscope is suitable for the above observations.

Purification Procedure The procedure is essentially the same as t h a t described in the original publication. 1 Step 1. Cultivation of Ustilago sphaerogena. This aerobic organism is cultured under conditions which will provide maximum cell yields. The medium contains 1.0% glucose and 1.8% Difco yeast extract. ~ The culture is started in 100-ml. amounts of this medium in ten 500-ml. Erlenmeyer flasks. T h e inoculum is grown on a gentle rocker in a 25 ° room. Meanwhile a fermentation unit 3 is filled with 200 1. of the same medium, and the t a n k and contents are subjected to 15 lb. of steam pressure for 1j. B. Neilands, J. Biol. Chem. 197, 701 (1952). 2 Although Ustilago sphaerogena may be propagated on almost any type of natural material, maximum cytochrome formation has been obtained only on Difco yeast extract; see also the article by P. W. Grimm and P. Allen, Plant Physiol. 29, 369 (1954). s j. j. Stefaniak, F. B. Galley, C. S. Brown, and M. J. Johnson, Ind. Eng. Chem. 38, 666 (1946).

756

RESPIRATORY ENZYMES

[134]

1 hour. After heavy growth appears in the Erlenmeyer flasks" (about 2 days), the contents are pooled and aseptically transferred to the cooled fermentation unit. Aeration is maintained at the maximum rate that will not cause loss of medium as foam going through the exhaust. Small amounts of Dow Antifoam A may be added if foaming becomes too serious. The temperature of the fermentation is maintained at 25 + 1°. After 24 hours the pH of the medium is taken at 2-hour intervals, and when it has risen to 7.5 to 8.0 there is no further growth. At this point, about 36 hours after inoculation, the fermentation may be considered complete. Step 2. Extraction. The tank is opened and 7 kg. of Filter-Cel is thoroughly mixed with the broth. The suspension is pumped through a filter press, and the cake is washed with liberal quantities of tap water. Enough water is added to the filter cake to give a fluid suspension, and N NaOH is added to bring the pH to approximately 10. The mixture is stirred mechanically, NaOH being added from time to time to maintain the pH at about 10. After 4 hours of continuous stirring, the mixture is again pumped through the filter press, and this time the clear, brownish extract is saved. Step 3. Fractionation with Ammonium Sulfate. The pH of the extract is reduced to 7 with syrupy phosphoric acid, and 487 g. of ammonium sulfate is added per liter of solution (about 75 % saturation). The material is left at 5° for 3 to 4 hours, after which a white precipitate can be removed by filtration through fluted papers. The filtrate is completely saturated with ammonium sulfate and allowed to stand at 5 ° overnight. The next morning, the cytochrome, which has precipitated, is filtered off and dialyzed against cold 0.027 % ammonium hydroxide until free from sulfate ion. This preparation is lyophilized and stored at 5 °. The yield is about 0.6 g. Step 4. Chromatography on Amberlite IRC-50. Commercial Amberlite IRC-50 is pulverized with a mortar and pestle until the particle size is reduced to 200 to 400 mesh. 4 The resin is washed on the centrifuge in succession with 5% sulfuric acid, 5 % sodium hydroxide, distilled water, and 0.1 M phosphate buffer, pH 7.0. A chromatography tube, 2.0 X 20 cm., is closed with a one-hole rubber stopper; a layer of glass wool and a layer of asbestos are placed over the stopper in order to prevent the particles of Amberlite from escaping from the tube. The resin, suspended in phosphate buffer, is poured into the tube to form a column about 10 cm.-high. The column is then washed with phosphate buffer until the effluent has a negligible optical density at 280 m~. 4 This resin may now be obtained in a fine particle size (Amberlite XE-97) from R o h m and Haas Co., Philadelphia.

[134]

CYTOCHROME C (USTILAGO)

757

The sample, about 0.6 g., is dissolved in the least amount of buffer and placed on the column. On washing with the same buffer, 0.1 M phosphate at pH 7.0, the cytochrome remains rather strongly adsorbed while a brownish, noncytochrome fraction migrates rapidly down the column and into the effluent. After the optical density at 280 m~ in the effluent has fallen to a value of less than 1.0 (1.00-cm. cell), the cytochrome is eluted with a saturated solution of ammonium acetate. The pigment is collected in a small volume and dialyzed against cold 0.027% ammonium hydroxide. The preparation, about 0.5 g., is then lyophilized and stored at 5° . A table summarizing the isolation procedure has not been included here. The method is extremely simple and, furthermore, the assay technique is not exactly quantitative. Weisel and Allen 5 found the dry cells of Ustilago sphaerogena to contain up to 1% cytochrome c. Since the above fermentation yields about 2 kg. of dry cells, the 0.5 g. of cytochrome represents only a few per cent of the total enzyme in the cells. Purity of Enzyme. No evidence of heterogeneity has been obtained when this preparation is examined in the Tiselius apparatus in the region of the isoelectric point. In these experiments a red filter (Corning No. 2424) is placed between the light source and the Tiselius cell, and the camera is loaded with red-sensitive spectroscopic film. A single, symmetrical boundary is observed when the material is analyzed in the oil turbine ultra centrifuge of Svedberg.

Properties Molecular Weight. The oil turbine ultracentrifuge gives a sedimentation constant, $20, of 1.4 X 10-13 second. The diffusion constant, D20, is calculated by the height-area method 8 to be 7.7 × 10-7 cm. 2 sec. -1. Assuming that the partial specific volume, ~, is 0.74 cm2 g.-1, these data give M = 18,000. Iron analyses by the method of Lorber 7 give the value of 0.28 % corresponding to a minimum molecular weight of 20,000. These figures must be considered preliminary, since the determination of molecular weight by the above methods can be open to large errors for low molecular weight proteins. Absorption Spectrum. The absorption spectrum of Ustilago cytochrome c is remarkably similar to that of the beef heart product. It shows the same tendency to become autoxidizable at extreme pH values. This means that the prosthetic group is derived from iron protoporphy5 p. Weisel and P. Allen, Abstracts, Meeting of the American Institute of Biological Sciences, Minneapolis, September, 1951. e L. G. Longsworth, Ann. N. Y. Acad. Sci. 41, 267 (1941). TL. Lorber, Biochem. Z. 181, 391 (1927).

758

RESPIRATORY ENZYMES

[135]

rin I X and further that the heme is probably attached to the protein part in exactly the same manner as in beef cytochrome c. Electrophoretic Mobility. Electrophoresis of Ustilago cytochrome c in the buffers used by Theorell s show the protein to be isoelectrie near pH 7. Catalytic Activity. Mole for mole, the Ustilago cytochrome c appears to be about as active as beef heart cytochrome c in the succinic dehydrogenase system of Pot t er 2 s H. Theorell, Biochem. Z. 28fi, 207 (1936). 9 V. R. Potter, in "Manometric Techniques" (Umbreit, Burris, and Stauffer, eds.), rev. ed., p. 213, Burgess Publishing Co., Minneapolis, 1949.

[135] Cytochrome c and Cytochrome c Peroxidase from

Pseudomonas fluorescens By HOWARD M. LENHOFF and NATHAN O. •APLAN I. Cytochrome c

Assay Method Principle. The cytochrome c obtained from Pseudomonas fluorescens ~ is identical in spectrum to animal cytochrome c, but it differs from animal cytochrome c in some of its biochemical and adsorptive properties. The Pseudomonas pigment has never been separated from the cytochrome peroxidase present in the extracts of Ps. fiuorescens, although it appears that these two components are not activities of the same protein. ~ This inseparability is disadvantageous in that it is difficult to study the reactivity of the cytochrome with electron donors and acceptors from other organisms owing to the interfering cytochrome peroxidase activity. On the other hand, it is advantageous in that the peroxidase allows one to assay for the presence of small amounts of the cytochrome c. Procedure. SPECTROPHOTOMETRICASSAY. The reduced pigment has an band at 550 m~, a smaller ~ band at 520 m~, and a large "r band at 415 m~ in the Soret region. The oxidized pigment absorbs slightly at 530 m~ and has its Soret band at 408 m~. These spectra are nearly identical to the spectra of the respective states of animal cytochrome c. The reduced animal cytochrome c has an extinction coefficient at 550 m~ of approximately 27.0 × 103 cm.2/mM., whereas that of the oxidized form is 9.0; the difference is 18.0 X 108 cm./mM. By determining H. M. Lenhoff and N. O. Kaplan, Nature 172, 730 (1953). 2 H. M. Lenhoff, Doctoral Thesis, Johns Hopkins University, Baltimore, 1955.

[135]

B A C T E R I A CYTOCHROME L C AND PEROXIDASE

759

the protein content and the change in optical density of the respective states of the bacterial pigment, and by assuming an approximate molecular weight of 14,000, 3 the percentage of the soluble pigment in 100 mg. of protein as bacterial cytochrome may be calculated. The percentage of soluble cytochrome c present in the bacterial extract may be calculated by the following formula: Dilution factor X E550 m~ X 14 × 100 Mg. protein/ml. X 18 = % of soluble protein which is Pseudomonas cytochrome pigment An average extract usually has 3.5 % of this cytochrome c. If the cytochrome is in the reduced state, it may be oxidized by the addition of H~O~ at a final concentration of 10-4 M or by the addition of a minute crystal of K~Fe(CN)8. The pigment is reduced chemically by the addition of a few crystals of Na~S~O4. ENZYMATIC ASSAY. Since the Pseudomonas cytochrome pigment is a substrate for the cytochrome peroxidase of this organism, a positive test for the peroxidase as given in the next section indicates the presence of small amounts of the cytochrome pigment.

Purification Procedure Although it appears that the Pseudomonas cytochrome pigment and cytochrome peroxidase are different proteins, ~ ig has never been possible to separate the two. Therefore, the pigment was purified in the same manner as described for the peroxidase (see below).

Properties During the past year several laboratories have reported a new bacterial cytochrome c present in the following organism: Pseudomonasfluorescens, 1 an unidentified halo4olerant bacteria, 4 Rhodopseudomonas spheroides, 5 and Azotobacter vinelandii. ~ The cytochrome c from the above organisms have features in common which distinguish them from the animal cytochrome c. Below are described some of the characteristics of the Pseudomonas cytochrome. Adsorption. The cytochrome from Pseudomonas fluorescens (and also from Rhodopseudomonas spheroides 6 and Azotobacter vinelandii e) has been 3 An approximate molecular weight of 14,000 was selected, as this figure represents an average of the molecular weights given for animal eytochrome c. 4 F. Egami, M. Itahashi, R. Sato, and T. Mori, J. Biochem. (Japan) 40~ 527 (1953). S. R. Elsden, M. D. Kamen, and L.P. Vernon, J. Am. Chem. Soc. 75~ 6347 (1953). T. G, G, Wilson and P, W, Wilson, Federation Pro¢. 18~ 322 (1954).

760

RESPIRATORY ENZYMES

[135]

shown not to be adsorbed on an Amberlite IRC-50 column, 1 whereas animal cytochrome c is readily adsorbedY The bacterial pigment probably differs from the animal one in the number and kind of charged groups available to the resinous exchange column. In order to check the possibility that some component in the bacterial preparation might be masking the possible adsorbing groups on the bacterial cytochrome, a mixture of animal and bacterial cytochrome c was incubated before being placed on the column. When the mixture was placed on the column, only the animal cytochrome c formed a sharp red band at the top of the column, whereas all the bacterial cytochrome (and nearly 100% of the cytoehrome peroxidase activity) passed through the column. Adaptive Formation. Cytochrome c has been shown to have the property of adaptively forming only when the cells are grown at a low oxygen tension; when Pseudomonas fluorescens is grown at a high oxygen tension (saturation with air), only a trace of the pigment forms. The cytochrome c content has been found to be as high as 4% of the soluble protein of the Pseudomonas extracts when the cells are grown at 1% oxygen2 Inactivity with TPN-Cytochrome c Reductase. The Pseudomonas cytochrome was not found to be reduced ~by the TPN-cytochrome c reductase obtained from liver, in contrast to animal cytochrome c which was reduced 8 by this enzyme. Spectrum. The absorption peaks are described in the section concerning the spectrophotometric assay procedure. Fluorescence. One other observation suggests another property which may be specific to this bacterial cytochrome. All dialyzed fractions of the bacterial extracts that contain the cytochrome fluoresce red when the extracts exhibit the reduced spectrum of the pigment. 2 On oxidation of the pigment by hydrogen peroxide, the red fluorescence disappears, whereas on the addition of a few crystals of Na2S204 it then reappears. All dialyzed fractions of the extract obtained from cells grown with aeration, and therefore having little cytochrome, and from cells grown in an iron-deficient medium with and without aeration, ~ do not exhibit this red fluorescence on reduction. On purification of the cytochrome c, this fluorescence is always associated with the cytochrome. It cannot be definitely said, however, whether the fluorescence is due to the cytochrome or to another nondialyzable component that goes along with the cytochrome on its purification. Animal cytochrome c does not fluoresce in this manner. 7 j. Neilands, J. Biol. Chem. 197, 701 (1952). 8 B. L. Horecker, J. Biol. Chem. 183, 593 (1950).

[135]

B A C T E R I A L C Y T O C H R O M E C AND P E R O X I D A S E

761

II. Cytochrome c Peroxidase 2 H + ~ H202 ~ 2 Cytochrome c (Fe ++) --~ 2 Cytochrome c (Fe +++) ~- 2 H~O Assay Method Principle. This method is based on the observation that a reduced dye, 2,6-dichlorobenzenoneindo-3'-chlorophenol, will chemically reduce the cytochrome c; the cytochrome is then oxidized by the added hydrogen peroxide mediated by means of the cytochrome peroxidase. Although the maximum change in absorption between the reduced and oxidized dye is at 660 m~, the reaction is measured spectrophotometrically at 575 m~, since the change is recorded more slowly at the latter wavelength. This is to facilitate recording the very rapid reaction. The most accurate results are obtained using a Beckman l~odel B spectrophotometer.9 Reagents

2,6-Dichlorobenzenoneindo-3'-chlorophenol (Eastman-Kodak) (0.001 M). Hydrogen peroxide (3 × 10-3 M). Orthophosphate buffer, pH 7.5 (0.1 M). Procedure. The reduced dye is prepared by dissolving 8.75 mg. of the oxidized dye in 25 ml. of water, filtering, and reducing with 1 ml. of a 0.2% suspension of 5% palladium-asbestos and by hydrogen gas according to the method of Smith and Stotz,l° The reduced dye is filtered free from the palladium-asbestos catalyst on Whatman No. 42 filter paper. It has been observed that the reduction procedure will also form H202 by reducing the dissolved oxygen present in the solution. The amount of endogenous H202 is diminished by bubbling prepurified N2 gas through the solution of oxidized dye before reduction with H202. A typical reaction mixture consists of 2.3 ml. of reduced dye (0.001 M), 0.1 ml. of 3 × 10-3 M H202 (giving a final molarity of 10-4 M), 0.01 ml. of crude extract, and enough 0.1 M orthophosphate buffer to bring the final volume of the reaction mixture in the cuvette to 3.0 ml. The reaction is started by the addition of the extract. In some cases the H202 concentration should be zero, as when determining the H~O~ saturation of the enzyme. The enzyme is then 9 In using this assay, one must be careful of other systems. If another nondialyzable cell componentwhich is reduced by the dye is in the tested fraction, and if this component can be oxidized by a peroxidatic mechanism, then the assay will also work with this non-cytochromesystem. 10L. Smith and E. Stotz, J. Biol. Chem. 179, 865 (1949).

762

RESPIRATORY ENZYMES

[136]

added to the reduced dye and buffer; the reaction proceeds for a short time until the endogenous H202 is utilized. When the reaction ceases, the known a m o u n t of H~O2 is added. Specific Activity. T h e specific activity is expressed as 0.01 change in optical density of the dye at 575 m~ per milligram of protein in 30 seconds.

Preparation and Purification Step 1. Pseudomonas fluorescens, strain 6009-2, is grown in a medium containing 5 g. of NaaC6HsOT"2H20, 5 g. of NaNO~, 1 g. of KH2P04, 0.5 g. of MgSO4-7H~O, and 4 g. of powdered yeast extract (Difco) per liter. The growth of the cells in 10 I. of medium in a 20-1. carboy gives a high cytochrome c content per cell. The cultures should not be aerated or agitated during growth. Two days' growth at 30 ° usually gives 5 g. of wet weight of cells per liter. T h e harvested cells should appear red in color. T h e cells are then washed with 0.9% NaC1, centrifuged, frozen, and ground in a cold m o r t a r with an equal weight of alumina powder (301-A). For each gram wet weight of cells, 5 ml. of cold orthophosp.hate buffer is slowly added to the cells and alumina during grinding. The homogenate is centrifuged at 25,000 X g in the cold for 30 minutes, or until the supernatant is particle-free; the length of time of centrifugation varies with the individual homogenates. The clear extract should be reddish brown in color. The extract is dialyzed overnight against cold 0.02 % KC1; if a n y precipitate forms, it is removed b y centrifugation. Step 2. Ammonium Sulfate. The extract is fractionated with solid (NH4) 2SO4, and the 40 to 80 % fraction is redissolved in orthophosphate buffer up to 40 % of its original volume. Step 3. Adsorption on Kaolin Column. Small aliquots of the 40 to 80 % (NH4)2S04 fraction are added to a chromatographic column, 1 × 15 cm., consisting of acid-treated kaolin (fuller's earth). 11 When on the column, the cytochrome peroxidase and both the oxidized and reduced form of the cytochrome move more slowly than the other protein components on the column. The pigment is best followed, however, when it is reduced b y the addition of a few crystals of Na2S:O4 before the fraction is added to the column. T h e peroxidase moves along 11The acid-treated kaolin was prepared by adding 1000 ml. of concentrated HC1 (sp. gr. 1.18) to 300 g. of kaolin. The mixture was boiled slowly for 12 hours over a period of 2 days. The HC1 was poured off, and the kaolin was washed with distilled water by decantation. The acid treatment and washing were repeated. The kaolin was then washed with water until the washings had a nearly neutral pH and the kaolin itself reacted acid on litmus paper. This is an adaptation of the method described by P. B. Hawk, B. L. Oser, and W. H. Summerson, "Practical Physiological Chemistry," p. 286, The Blakiston Co., Philadelphia, 1949.

[135]

B A C T E R I A L CYTOCHROME C AND P ER O X ID A S E

763

with the pigment. The proteins move very slowly, but this rate of flow ~is increased b y the application of air pressure b y means of a rubber pressure bulb. After the pigmented band moves 5 cm., the column is washed with orthophosphate buffer, in order to remove most of the contaminating proteins; the washing is stopped when the pigmented band moves to 10 cm. The column is eluted with 20 ml. of saturated ammonium acetate. Five fractions of approximately 3-ml. volume are collected; however, the number and volume of the fractions m a y vary. Nearly 100 % of the cytochrome peroxidase activity can be recovered with various degrees of purity, but usually the fourth fraction has the highest specific activity. PURIFICATION OF CYTOCHROME C PEROXIDASE FROM

Step Crude 40-80% (NH4)2SO4 Fourth ammonium acetate eluate from kaolin column

Pseudomonas fluorescens

Specific Units a X 10-3 Yield, % activity~ Purification 1180 1148

100 97

520 1045

1 2

84

7

6480

13

One unit is defined as a 0.01 change in optical density of the dye at 575 m~ in 30 seconds. b Defined in the text.

Properties pH Optimum. The enzyme has a p H optimum at p H 7.0. Most of the enzyme studies were carried out at p H 7.5; the enzyme is stable at this pH, and the reaction rate is slower and, therefore, easier to follow. Azide Inhibition. An azide concentration of 10-2 M does not appreciably inhibit the peroxidase at p H 7.5. However, at p H 5.5, 10-2 M azide will inhibit 95 %. These results indicate that hydrazoic acid will inhibit the enzyme, whereas azide ion does not. Other Inhibitors. Both cyanide and hydroxylamine were found to be p o t e n t inhibitors of cytochrome peroxidase. Fifty per cent inhibition was obtained with concentrations of 10-4 M cyanide and 3.2 × 10 -~ M hydroxylamine, respectively. Potassium ethyl xanthate, hydroxyquinoline, and carbon monoxide exhibited no appreciable inhibition. Hydrogen Peroxide as Substrate. During all purification processes, it was difficult to separate completely the cytochrome peroxidase from catalase. However, the K~ for H~O2 was determined b y inhibiting the catalase with 10-8 M azide at p H 7.5. At this p H the catalase is inhibited some 90%, whereas the peroxidase is not affected. The K~ for H202 in the presence of 10-3 M azide was found to be 5 X 10-e M.

764

RESPIRATORY ENZYMES

[136]

Distribution. Besides being present in Pseudomonas fluoreseens, appreciable cytochrome peroxidase activity was found in Azotobacter vinelandii, and the Poky mutant of Neurospora crassa. Assays of extracts of E. coli, BCG, Clostridium kluyveri, Neurospora crassa, and Azotobaeter agile demonstrated the presence of small amounts of cytochrome peroxidase activity only in the presence of 10-3 M sodium azide. The azide was added in order to inhibit the catalase; it had no significant effect on the peroxidase. Apparently, the ratio of catalase to cytochrome peroxidase was very high in these organisms, and the hydrogen peroxide was available to the peroxidase when the catalase was inhibited. Extracts of soybean leaves exhibited a very high specific activity. However, it cannot be definitely said whether the activity of the soybean extract was due to the cytochrome peroxidase, because it has not been possible to observe the cytochrome pigment in these extracts. The activity may be due to another peroxidase mediating the oxidation of the dye. In any case the activity exhibited by the soybean extract is due solely to a peroxidase, as it is completely inhibited by catalase.

[136] A s s a y of C a t a l a s e s a n d P e r o x i d a s e s Catalase: 2H~O~--~ 2H20 -~ 02 Catalase and Peroxidase: ROOH -~ AH~--~ H20 -~ ROH -{- A

(1) (2)

By BRITTON CHANCE and A. C. MAEHLY I. Catalase Assay by Disappearance of Peroxide A. Ultraviolet Spectrophotometry Principle. On the basis of the absorption curves for peroxide solutions (Lederle and Riechel), Chance and Herbert 2 devised a method for determining the activity of catalase by direct measurements of the decrease of light absorption in the region 230 to 250 mu caused by the decomposition of hydrogen peroxide by catalase. More detailed procedures have been published. 3.4 Range of Usefulness. This method is limited to the assay of catalase solutions that are pure enough to give negligible absorption at 230 to 1E. Lederle and A. Rieche, Ber. 62~ 2573 (1929). B. Chance and D. Herbert, Biochem. J. 46, 402 (1950). a R. F. Beers, Jr., and I. W. Sizer, J. Biol. Chem. 195~ 133 (1952). 4B. Chance, in "Methods of BiochemicalAnalysis" (Glick, ed.), p. 408, Interscience Publishers, New York, 1954.

[136]

765

A S S A Y OF CATALASES A N D P E R O X I D A S E S

250 m ~ i n t h e c o n c e n t r a t i o n s r e q u i r e d f o r t h e a s s a y . A s s a y s a c c o r d i n g t o e q u a t i o n 2 h a v e n o t b e e n s t u d i e d in d e t a i l b u t a r e a p p a r e n t l y p o s s i b l e . 5

Reagents 0.01 M p h o s p h a t e b u f f e r , p H 7.0. S t o c k s o l u t i o n of H~O2 (~-~1 M ) . S t o c k s o l u t i o n of c a t a l a s e (--~10 -4 M o r less).

Spectrophotometric Determination of Catalase Concentration. T h e m o l e c u l a r e x t i n c t i o n c o e f f i c i e n t s of t h e u s e f u l a b s o r p t i o n b a n d s of v a r i o u s t y p e s of c a t a l a s e s a r e p r e s e n t e d in T a b l e I a n d c a n b e u s e d f o r m e a s u r i n g TABLE I MOLECULAR WEIGHTS, EXTINCTION COEFFICIENTS, AND ki t VALUES (EQUATION 8) FOR CATALASES FROM VARIOUS SOURCES

Catalase source

Purified by

Human liver Horse liver Beef liver

Bonnichsen • Dounce and Frampton e Sumner and DounceS

Human erythrocytes Horse erythrocytes

Herbert and Pinsenti Bonnichsen*

Human kidney Bacteria

Bonnichsen a Herbert and Pinsent ~

~405,

klfj

Molecular weight

m M -1 × cm. -~

10 7 X M -1 X sec. -~

-225,000 a 225,000g 251,000 i 220,000~ (225,000 k) 250,0001 -232,000 ~

290o, b 340 ~ --

-3.0" 2.9 h

-378* 402' 366~. b 410 °

3.4 h 3.5" 6.6 ~ -5.3 °

R. K. Bonnichsen, Acta Chem. Scand. 1, 114 (1947). b These values are approximate since the molecular weight is estimated. c A. L. Dounce and O. D. Frampton, Science 89, 300 (1939). d K. Agner, Biochem. J. 32, 1702 (1938). e R. K. Bonnichsen, B. Chance, and H. Theorell, Acta Chem. Scand. 1, 685 (1947). / J. B. Sumner and A. L. Dounce, Science 86, 366 (1937). J. B. Sumner, A. L. Dounce, and O. D. Frampton, J. Biol. Chem. 136, 343 (1940). h A. C. Maehly, in "Methods in Biochemical Analysis" (Glick, ed.), p. 358, Interscience Publishers, New York, 1954. Calculated from published Kat.f. values. M. Shirakawa, J. Fac. Agr. Kyushu Univ. 9, 173 (1949). i D. Herbert and J. Pinsent, Biochem. J. 43, 203 (1948). k R. K. Bonnichsen, Arch. Biochem. 12, 83 (1947). l H. F. Deutsch, Acta Chem. Scand. 6, 1516 (1952). H. F. Deutsch, Acta Chem. Scan& 5, 815 (1951). D. Herbert and J. Pinsent, Biochem. J. 43, 193 (1948). ° B. Chance and D. Herbert, Biochem. J. 46, 402 (1950). 5 B. Chance, in "Investigation of Rates and Mechanisms of Reactions" (Friess and Weissberger, eds.), p. 637, Interscience Publishers, New York, 1953.

766

RESPIRATORY ENZYMES

[136]

the catalase concentration as is required for the computation of the specific activity as described below. Alternatively, the dry weight of enzyme used in the assay is divided by the molecular weight. Apparatus. Matched cuvettes, of 1-cm. optical path and 3-ml. volume (the cuvettes should be clean enough so that the " b l a n k " rate of peroxide decomposition is negligible); an ultraviolet spectrophotometer (Beckman, Unicam, Uvispek, etc.) having sufficient electrical stability so that the " d r i f t " of the optical density reading is less than 0.005 per minute; a micropipet delivering about 1 ~l. accurate to 2%. Procedure. At temperatures of 26 ° and less where convenient, the optical density of a 1:500 H~02 solution is measured in the region 230 to 250 m~ (this is x0). The apparatus is left set for the optical density reading (Beckman selector switch set at "1 "), and the shutter is closed. About 1 ~l. of catalase is placed as a drop on the end of a stirring rod, is rapidly stirred into the peroxide solution, and a stop watch is started. The cover for the cuvette holder is rapidly replaced, the shutter opened, and "tracking" begun (the null galvanometer of the spectrophotometer is kept continuously zeroed by a slow, continuous rotation of the optical density knob). The stop watch and the slowly changing optical density value are read every 10 seconds for the first 30 seconds to give the quantity x. Thereafter the optical density of the solution is measured in the usual manner by taking readings on both cuvettes at 50 and 70 seconds. Thus five readings of the quantity x are obtained. The catalase concentration is adjusted so that about half the peroxide is decomposed in about 30 seconds. If both the catalase and the spent hydrogen peroxide solutions absorb negligibly at 230 to 250 m~, as is usually the case, the end point of the reaction will correspond closely to zero optical density. If impure catalase is used or if the spent hydrogen peroxide solution contains absorbing substances, the end point of the reaction should be measured and subtracted from the optical densities read in the course of the reaction. If this proves inconvenient, an alternative form of calculation is provided below. Calculation of Results. Under these experimental conditions the kinetics of hydrogen peroxide disappearance closely follow first-order kinetics and the velocity constant (k) is calculated: k = 2i3--log Xo_

(3)

X

The units are see.-'. A fairly constant value of k is obtained with most catalases from the five determinations. If the catalase is extremely labile, the value of/c may be taken by extrapolation of the experimental values tot =0.

[136]

ASSAY OF CATALASES AND PEROXIDASES

767

An alternative form of equation 3 that is sometimes useful is 2.3 log xl k -- t~ - tl xg_

(4)

where t~ and t~ are the times corresponding to a pair of readings of the optical densities x~ and x~. The quantity k is used in two ways: 1. To calculate the specific activity of catalase (k~'). The quantity k~' is calculated from k by dividing the latter by the enzyme molarity, e, as determined spectrophotometrically. kl' = k/e M -1 × s e c . -1

(5)

The quantity k~' is related to the two principal steps of catalase action, 6 kl E + S ---+ES (6) k4J

ES -4- S ---+E + P

(7)

by the equation 1

kl'=

1------~

(8)

k~ + k4 -~ Representative values of kl' for various catalases are given in Table I. This method should be used to define the specific activity of a new catalase. If the value obtained for a known catalase is less than that given in Table I, the catalase (1) contains impurities that absorb light at the wavelengths used to determine the enzyme concentration spectrophotometrically, (2) contains a larger proportion of inactive hematin groups in the case of liver catalases, (3) represents a less active fraction of erythrocyte catalases. 2. To calculate the value of Kat.f. This quantity, although it involves a confusion of units, is still used for the comparison of catalase activity and purity. Kat.f. = 60k/2.3W (9) Here k is converted from sec. -~ to rain. -~ by multiplying by 60. Naperian logarithms are converted back to Briggsian by the factor 2.3. The term W is the grams of catalase in a final reaction mixture of 50-ml. volume and is 5o~ times the weight of catalase actually used in the method described here. Kat.f. is not suitable for comparing the activity of catalases of different molecular weights, and the specific activity kl' should be used. 6 B. Chance, D. S. Greenstein, and F. J. W. Roughton, Arch. Biochem. and Biophys. 37, 301 (1952).

768

RESPIRATORY ENZYMES

[136]

Accuracy of the Method. The results should be accurate to a few per cent and should agree to within that accuracy with the results obtained by rapid titration methods (see below). For bacterial catalase, Chance and Herbert 2 obtained kl' = 5.35 and 5.3 X 107 at 25 ° for rapid titrimetric and ultraviolet spectrophotometric assays of bacterial catalase. B. Permanganate Titration For those who have no ultraviolet spectrophotometer available or who wish to assay a very impure catalase, we include a brief summary of the titrimetric meth odJ -9

Reagents 0.25 N hydrogen peroxide. 0.01 M phosphate buffer, pH 7.0. Stock catalase solution diluted to about 1 ~M 0.01 N potassium permanganate. 2 % sulfuric acid solution. At a temperature of 20 ° or less when convenient, 15 ml. of 0.01 N hydrogen peroxide (1:25 in phosphate buffer) is placed in an Erlenmeyer flask, and 2.0 ml. of this solution is withdrawn and added to an excess of 2% sulfuric acid for titration with permanganate with an accuracy of 1.0%. Thus the quantity x0 is determined. Then 0.030 ml. of the catalase is pipetted onto a watch glass and is dropped into the peroxide solution which is being swirled rapidly. The stop watch is started at the same time. Samples of 2 ml. each are withdrawn at various times and are rapidly blown out (through a wide-tipped pipet) into separate flasks containing excess 2% sulfuric acid t hat is rapidly swirling at the time the sample is blown out. I t is possible to withdraw samples and blow them into the acid at 10, 20, 30, and 50 seconds after adding catalase. Permanganate titration of these solutions gives the values of x. The methods of calculation are the same as those described above. II. Method for Crude Cell Extracts Although it is now generally recognized that current manometric methods for catalase assay are completely unsuitable for accurate determination of catalase activity, 9,1° relative values of activity can be determined under conditions where other methods are inapplicable. 7 H. yon Euler and K. Josephson, Ann. 455, 1 (1927). 8 R. K. Bonnichsen, B. Chance, and H. Theorell, Acta Chem. Scan& 1, 685 (1947). 9 A. C. Maehly, in " M e t h o d s of Biochemical Analysis" (Glick, ed.), p. 358, Interscience Publishers, New York, 1954. lo K. Agner and It. Theorell, Arch. Biochem. 10, 321 (1946).

[136]

ASSAY OF CATALASES AND PEROXIDASES

769

Principle. Oxygen evolution caused by the decomposition of hydrogen peroxide is measured with the conventional manometric technique. Range of Usefulness. This is a method of last resort but may be used (1) when purification of the enzyme is difficult and the losses of such a procedure are not controlled; (2) when the cell extracts are too turbid to permit the use of the ultraviolet spectrophotometric method; (3) when the material contains substances that react with permanganate or interfere with polarographic methods. 4 Procedure. When a conventional manometric technique is used, the main compartment of the vessel should contain 3 ml. of 0.01 M phosphate buffer, pH 7.0, and 0.2 ml. of 0.2 M hydrogen peroxide. The catalase may be placed in the side arm but is preferably contained in a "dangling cup'11 or a magnetically released vessel'"- (Will Corporation, New York 12, N.Y.). Readings should be made at 1, 2, and 3 minutes after adding catalase. The temperature can be 20 ° or below, and the shaking rate 100 to 200 strokes per minutes. Calculation of Results. Since this method is recommended only for relative assays and not for the calculation of kl' or Kat.f., the results may best be expressed in values relative to those of some standard material such as liver, where the catalase content is known on the basis of purification procedures. III. Direct Spectrophotometric Assay of Catalase and Peroxidase in Cells and Tissues In biological materials that contain relatively large amounts of catalase, Chance ~ has shown that catalase may be assayed directly in the cells by means of sensitive spectrophotometric methods for the detection of changes of absorption that occur when an enzyme-substrate compound of catalase is formed or is decomposed. This method may ultimately replace the manometric one in many cases. It has also been possible to measure the formation of an enzyme-substrate compound of peroxidase in yeast cells by the addition of methyl hydrogen peroxide. I~ IV. Peroxidase Assay by Spectrophotometric Measurements of the Disappearance of Hydrogen Donor or the Appearance of Their Colored Oxidation Products

General Principles. Peroxidases catalyze the oxidation by peroxide of a wide variety of substances many of which have strong absorption bands 11 D. Keilin and E. F. Hartree, Proc. Roy. Soc. (London) Bl17, 1 (1935). 12 Lord Rothschild, J. Exptl. Biol. 26, 396 (1950). 1~ B. Chance, in " T h e Mechanism of Enzyme Action" (McElrey and Glass, eds.), p. 399, The Johns Hopkins Press, Baltimore. 1954.

770

RESPIRATORY ENZYMES

[136]

themselves or the oxidation products of which absorb strongly. 9 B u t in the peroxidases it is necessary to distinguish the two types of specific activity t h a t m a y be measured, depending on the experimental conditions: (1) the velocity constant for the formation of the enzyme-substrate complex: kl E + S --~ E S I (10) and (2) the velocity constant for the reaction of the secondary complex with the hydrogen donor molecule: k4t ESII -~ A H --* E -~ P (11) We shall describe methods t h a t approximate the measurement of each of these quantities or point out where current assay methods fail to measure either one. The use of guaiacol makes it possible to approximate kl or k4; pyrogallol gives no satisfactory approximation to k4; the mesidine test (Table III) has not been proved to measure k~ or k4. With the other peroxidases, lacto-, verdo-, and cytochrome c or yeast peroxidase, the assay conditions are not nearly so well worked out and the assay procedures m a y give only a mixture of the specific activities of the two ratedetermining steps in peroxidase action. A. T h e G u a i a c o l T e s t

P r i n c i p l e . 14 T h e rate of utilization of peroxide (dx/dt) to form the colored reaction product, 16 as measured at 470 m~, depends on the respective concentrations of substrate (peroxide) and donor (guaiacol) (equations 10 and 11) in the following way: dx d~ =

e 1

1

(12)

where e = concentration of enzyme. a0 = initial concentration of donor. x0 = initial concentration of substrate. If the conditions of the assay are chosen so t h a t k4ao >> klx0, equation 10 represents the rate-limiting step and we obtain kl: 14This method was developed by Mr. T. M. Devlin of this laboratory for the assay of cytochrome c peroxidase, and Devlin's method was used by Dr. P. George in 1953 during his visit here [see P. George, J. Biol. Chem. 201, 413 (1953)]. 15 A formulation of this reaction can be found elsewhere2

[136]

ASSAY OF CATALASES AND P E R O X I D A S E S

771

dx - - ~-- klxoe dt

(13a)

and 1 X dx x oe -dt

1 X Ax x oe At

(13b)

w h e r e A x / A t is t h e r a t e of s u b s t r a t e d i s a p p e a r a n c e o v e r t h e m e a s u r e d time interval. H o w e v e r , if t h e a s s a y is a d j u s t e d so t h a t klxo >2>k4ao, k4 c a n be o b t a i n e d , since in this case

dx

d--£ ~-" k4aoe

(14a)

and 1

A~5

k4 ~ -2- X - aoe At

(14b)

T h e e x p e r i m e n t a l p r o c e d u r e is t h e s a m e for b o t h m o d i f i c a t i o n s of t h e t e s t . Reagents 20 m M 10 m M 10 m M 40 m M --~10 -7

g u a i a c o l (0.22 ml. of g u a i a c o l in 100 ml. of w a t e r ) . p h o s p h a t e buffer, p H 7.0. h y d r o g e n p e r o x i d e s o l u t i o n (for kl d e t e r m i n a t i o n ) . h y d r o g e n p e r o x i d e s o l u t i o n (for k4 d e t e r m i n a t i o n ) . M p e r o x i d a s e solution.

Procedure. T h e v a l u e s b, c, d, a n d f d e p e n d on t h e c o n s t a n t t o be measured and are read from Table II. TABLE II AMOUNTS OF REAGENTS ADDED TO THE CUVETTE AND THE FINAL CONCENTRATION OF THESE REAGENTS FOR THE TWO MODIFICATIONS OF THE GUAIACOL ASSAY

Constant to be determined

Buffer Guaiacol [Guaiucol] H~02 stock [H202] Df ~ klxo k4a0

Units

Symbol used in text

kl

k4

ml. ml. M mM M cm. -1 ---

b c a0 d x0 f ---

1.0 2.0 1.3 X 10-2 10 3.3 X 10-5 0.2 300 4300

2.9 0.05 3.3 X 10-4 40 1.3 X 10-4 0.8 1170 110

Approximate final reading of optical density at 470 m~.

772

R~SPIRATORV ~NZVMES

[136]

A spectrophotometer or direct-reading colorimeter suitable for a wavelength region of 470 m~ m a y be used. The assay is carried out at room temperature. One of a pair of cuvettes of 1-cm. p a t h is filled with water, the other with b ml. of phosphate buffer, c ml. of guaiacol, and about 10-9 M peroxidase (see below). The initial optical density is read at 470 m~, and the optical density scale is offset to a value 0.050 greater t h a n this reading. The shutter is closed, and 10 td. of d m M hydrogen peroxide is added as a drop at the end of a stirring rod (the final concentration is x0). The solution is rapidly stirred, and a stop watch is started. The shutter is opened, and the time required (At) for the galvanometer to reach the null point is measured; it should be between 15 and 30 seconds. The end point of the reaction should correspond to an optical density of f. Determination of the Enzyme Concentration. The peroxidase concentration can be determined spectrophotometrically on the basis of the molecular extinction coefficients given on pp. 799, 812, 817. Alternatively, the dry weight of the enzyme used m a y be divided b y the molecular weight of the peroxidase (pp. 798, 808, 817) to give an effective concentration for an impure enzyme solution. Calculation of the Results. The hydrogen peroxide utilized in the formation of a tetraguaiacol solution t h a t gives an optical density of 0.05 4 × 0.050 at 470 m# is 26.6 m M -- 7.5 ~M, since 4 moles of H202 is required to form 1 mole of tetraguaiacol and since the latter has an extinction coefficient of e = 26.6 cm. -1 m M -1 at 470 m~. 9 This 7.5 ~M of H202 corresponds to Ax of equations 13b and 14b, and At (units sec.) to the measured time interval. Both x0 and e (units M. X 1.-~) can be measured spectrophotometrically (see above). F r o m equations 13b and 14b the rate constants k~ and k4, respectively, can thus be calculated: 1 Ax 7.5 X 10-8 1 .22 kl × . . . . (15) xoe At 3.3 X 10-6 X e At e At Experiments with pure horseradish peroxidase gave under the conditions of the test kl = 0.89 × 107 at 20 °, and 1.03 X 107 at 30 ° . T h e value of kl should be 0.9 × 107 at 25°. 1~ k4 =

Ax 7.5 X 1 0 - ~ 1 1 X -aoe At 3.3 X 10-~ X e At

__

10-2 e At

2.2 X

(16)

Actual experimental values were k4 = 2.2 X 10 s at 20 °, and 3.1 × 10 ~ at 30°; k4 should be 3.3 × 105 at 25 °.17 16B. Chance, Arch. Biochem. 22, 224 (1949). 1~B. Chance, Arch. Biochem. 24, 410 (1949).

[136]

ASSAY OF CATALASES AND PEROXIDASES

773

The value of h~ for lactoperoxidase, verdoperoxidase, and yeast peroxidase (cytochrome c peroxidase) is of the same order of magnitude as that for the horseradish enzyme, but experimental tests have not been made to determine whether this value can actually be obtained in the guaiacol test with any but the horseradish enzyme. Accuracy and Limitations of the Method. The results should be accurate to a few per cent. The main sources of error are caused by the use of this method with crude cell extracts. In some cases there are substances present that interfere with the formation of tetraguaiacol.17~ B. The Pyrogallol Test

Principle. The traditional test for peroxidase activity is the formation of purpurogallin from pyrogallol (Willstiitter and StolllS). This method was devised before the mechanism of peroxidase action was fully understood. The original experimental conditions seem not to be suitable for measuring k4 of equation 12, since klxo--~h~ao, and it was demonstrafed 17 that the enzymatic reaction proper was terminated (the enzyme-substrate compounds had disappeared) before all the end product (purpurogallin) had been formed. This shows that intermediate products must be involved and that the color formation is not really a direct measurement of enzyme kinetics. The assay will nevertheless be described for those who wish to correlate their data with those of the older literature. Reagents. All solutions should be made up in glass-distilled water, and rigidly cleaned vessels should be used. Pyrogallol (two times resublimed), 1.25 g. in 500 ml. (20 mM). Hydrogen peroxide, 12.5 rag. in 500 ml. (0.74 raM). Phosphate buffer, pH 7.0, 10 mM. Peroxidase, about 1 ~, in 500 ml. (5 X 10-11 M). Procedure. In one of the modified procedures the reagents are made up in a 500-ml. volume, and the reaction is allowed to proceed for 5 minutes after which time it is stopped by adding 5 ml. of 5 N H2SO4. The purpurogallin is extracted three times with ether, alcohol is added, and the solution is made up to a known volume. The concentration of purpurogallin is determined spectrophotometrically at 430 m~, where the extinction coefficient e = 2.47 cm. -I mM-L 19 A large number of varia17~I t m u s t be ascertained, therefore, t h a t no secondary reactions w i t h guaiacol or tetraguaiacol occur. Failure to take such reactions into account can give rise to misleading conclusions. Thus, P. George [J. Biol. Chem. 201, 413 (1953)l observed t h a t HOC1 reacts with guaiacol or its oxidation products directly. 18 R. Willsti~tter and A. Stoll, Ann. 416, 21 (1917). 19 A. C. Maehly, unpublished experiments.

774

RESPIRATORY ENZYMES

[136]

tions of this procedure h a v e been used, b u t as long as the concentration of the reactants is unchanged, the same result should be obtained. Calculation of Results. The activity of peroxidase is traditionally expressed b y the purpurogallin n u m b e r (PZ). PZ is the n u m b e r of milligrams of purpurogallin formed b y 1 mg. of e n z y m e under the conditions of the s t a n d a r d test. T h e milligrams of e n z y m e present are determined directly b y a d r y weight determination or spectrophotometrically.~° The value of PZ for pure peroxidase as determined b y the test is 1020 according to Theorell and Maehly, 23 and 1220 according to Keilin and Hartree. 24 T h e value of PZ reached with equation 12 and the k n o w n values of kl and k4 is 1500 (Chance17), which is s o m e w h a t in excess of t h a t actually realized under the assay conditions. TABLE III MISCELLANEOUS PEROXIDASE TESTS

Peroxidase Horseradish Yeast MyeloLacto-

Donor employed

Investigator

Cf. page

Mesidine Cytochrome c Uric acid Dihydroxyphenylalanine

Paul and Avi-Dor~ Altschul et al.b Agner ~ Polls and Shmukler ~

794 813

a K. G. Paul and Y. Avi-Dor, Acla Chem. Scand. 8, 649 (1954). b A. M. Altschul, R. Abrams, and T. R. Hogness, J. Biol. Chem. 136, 777 (1940). c K. Agner, personal communication. d B. D. Polls and H. W. Shmukler, J. Biol. Chem. 201, 475 (1953) L i m i t a t i o n s of the Method. Considerable care is necessary to obtain reproducible results in the purpurogallin test (see Reagents). I n addition, the test gives v e r y low values for lacto- and verdoperoxidase, owing to the fact t h a t these enzymes are not s a t u r a t e d with peroxide u n d e r the conditions of the test and are to some extent i n a c t i v a t e d b y the high H202 concentration. T h u s their respective PZ values of 71 (Theorell and /kkeson 25) and 41 (Agner 26) cannot be converted into a specific rate constant. Direct m e a s u r e m e n t s of k4 from the kinetics of an enzyme-subs t r a t e c o m p o u n d of lactoperoxidase in the presence of pyrogallol gives a

20The molecular weight of horseradish peroxidase is 40,200 according to Theorell and Ehrenberg, 21 and 39,800 according to Cecil and Ogston. 22 21 A. Ehrenberg, personal communication; cf. A. C. Maehly, Vol. II [143], p. 808. ~2R. Cecil and A. G. Ogston, Biochem. J. 49, 105 (1951). 23 H. Theorell and A. C. Maehly, Aeta Chem. Scan& 4, 422 (1950). 24 D. Keilin and E. F. Hartree, Biochem. J. 49~ 88 (1951). ~s H. Theorell and/~. ~keson, Arkiv Kemi, Mineral. Geol. 17B, No. 7 (1943) ~e K. Agner, Acta Physiol. Seand. 2, Suppl. 8 (1941).

[137]

LIVER CATALASE

775

value of 7 × 106, considerably greater t h a n the value for the horseradish enzyme. ~7

C. Other Peroxidase Assays Those working on the purification of peroxidases h a v e usually developed their own particular assay s y s t e m t h a t should p r o b a b l y be followed b y those who wish to duplicate the preparations. Table I I I lists some of these methods, which, however, do not clearly define a single reaction velocity constant. The guaiacol test is satisfactory for the enzymes listed in T a b l e I I I , although it has not y e t been proved t h a t accurate values of kl and k4 can be obtained in all cases. ~7B. Chance, J. Am. Chem. Soc. 72, 1577 (1950).

[137] L i v e r C a t a l a s e B y JAMES B. SUMNER and ALEXANDER L. DOUNCE

I. Introduction N e x t to urease, beef liver catalase is possibly the easiest enzyme to obtain in crystalline condition. The reasons for this are the unusual stability of this enzyme, its insolubility in w a t e r at its isoelectric point, and its relatively high concentration in beef liver. Since the p r e p a r a t i o n of crystalline catalase from beef liver b y S u m n e r and Dounce 1 crystalline catalases h a v e been obtained from a n u m b e r of other sources. These are: l a m b liver, 2 horse liver, 3 beef erythrocytes, 4 horse kidney and h u m a n liver, 5 guinea pig liver, 8 Micrococcus lysodeikticus, 7 and pig liver. 8 Recently a m e t h o d has been reported but not described for obtaining crystalline catalase from rat liver2 Methods for preparing catalase from the livers of animals other t h a n the ox are generally rather involved and for t h a t reason will not be described here. 1j. B. Sumner and A. L. Dounce, J. Biol. Chem. 121, 417 (1937). 2 A. L. Dounce, J. Biol. Chem. 143, 497 (1942). 3 A. L. Dounce and O. D. Frampton, Science 89, 300 (1939). 4 M. Laskowski and J. B. Sumner, Science 94, 615 (1941). 5 R. K. Bonnichsen, Acta Chem. Scand. 1, 114 (1947); Arch. Biochem. 12, 83 (1947). s R. K. Bonnichsen, Acta Chem. Scan& 2, 561 (1948). 7 D. Herbert and A. J. Pinsent, Nature 160, 125 (1947). 8 N. K. Sarkar and J. B. Sumner, Enzymologia 14, 280 (1951). g R. E. Greenfield and V. E. Price, Proe. Am. Assoc. Cancer Research 1, 21 (1953).

776

RESPIRATORY ENZYMES

[137]

II. M e t h o d of Sumner and Dounce I

Put beef liver through a household meat grinder four times, and mix 3O0-g. portions with 400-ml. portions of 35 % dioxane. 9a After stirring for 4 or 5 minutes, place on fluted filters (32 cm., Schleicher and Schuell, No. 595) and cover with watch glasses. Allow to filter into 500-ml. graduates overnight at room temperature. The next day add to every 100 ml. of filtrate 20 ml. of dioxane with stirring, and set the material in the ice chest. After 12 or more hours, filter and refilter in the ice chest until the solution is practically clear. Then precipitate the catalase by adding 10.2 ml. of dioxane to every 100 ml. of filtrate. Allow the material to stand in the ice chest overnight, and then filter off the precipitated catalase, refiltering if necessary. The residue on the filter paper must stand in the ice chest until all the liquid has drained through the filter paper. Then open up the filter paper and place it upon several dry filter papers. Serape the precipitate off with a spatula, and place it in a beaker. Depending on the yield of precipitate, stir with 5 to 15 ml. of water for each 300 g. of liver, and add a few drops of pancreatic amylase in order to digest the glycogen. Then filter the solution at room temperature, and extract the residue a second time. Crystals of catalase can be obtained from the combined filtrates in one of two ways: (1) Chill the combined filtrates, and add saturated ammonium sulfate cautiously until a haziness appears. The catalase crystallizes as fine needles almost immediately. Keep the material in the ice chest, and add ammonium sulfate until a good crop of crystals is obtained. However, it is not advisable to add a great excess of ammonium sulfate, since the catalase crystals, which are present as needles, are difficult to centrifuge down from solutions of high specific gravity. 2. Adjust the pH of the combined filtrates described above to approximately 5.7, the isoelectric point of catalase, and then dialyze for a day or two against a number of changes of distilled water. Catalase crystallizes out as very thin plates and occasionally as prisms. Observe the suspension microscopically, and stop the dialysis when appreciable numbers of spheroids appear. Catalase crystals may be stored suspended in ammonium sulfate solution for considerable time without appreciable loss in activity. The crystals can also be stored in a suspension in distilled water, but in this case they are apt to undergo gradual deterioration, owing to bacterial action, unless they have been crystallized from a sterile solution. 9~Dr. Roger Young of this laboratory has purified dioxane from such impurities as hydrochloric acid and ferric chloride by shaking each liter of dioxane with 20 ml. of saturated sodium hydroxide, separating the two liquids in a separatory funnel, and then filtering the dioxane layer through filter paper.

[137]

LIVER CATALASE

777

III. Method of Sarkar and Sumner s

Grind fresh beef liver in a meat grinder, putting the material through the grinder four times. To every kilogram of ground material add 1500 ml. of distilled water and mix well. Mix 1 vol. of 95 % alcohol with 1 vol. of c.p. chloroform, and add 480 vol. of this mixture to every 1000 vol. of the liver suspension. Shake violently for 30 seconds, and filter through 32-cm. Balston No. 12 folded filter papers at room temperature. When most of the liquid has filtered through, for every kilogram of liver pour about 500 ml. of distilled water on the residue to wash more catalase through. Cautiously bring the filtrate to pH 5.7 by adding glacial acetic acid, centrifuge, and remove protein impurity. Add 250 ml. of tricalcium phosphate suspension which is at about pH 5.7 (30 to 35 mg./ml.) to every liter of filtrate, and mix for 10 to 15 minutes. At once centrifuge down the adsorption complex, and discard the supernatant. Elute the catalase from the adsorption complex with 200 ml. of 0.1 M phosphate buffer of pH 8.0 (for about 2 kg. of liver used at the start), followed by centrifuging. Repeat this elution once more, using 200 ml. of the pH 8.0 phosphate buffer. To the combined eluates add solid ammonium sulfate, using 30 g. for every 100 ml., mix, and allow to stand in the ice chest overnight. Next day centrifuge the catalase down at high speed in a refrigerated centrifuge and discard the supernatant. Mix the sediment with just enough water to make a mushy suspension, transfer to a dialyzing parchment, and dialyze in the ice chest against several changes of distilled water. If a whitish or buff colored precipitate forms, it should be centrifuged down and discarded. The catalase will crystallize if it is from beef liver. This method will give satisfactory results starting with as little as 100 g. of beef liver. IV. Employment of the Tswett Column

Catalase can be purified by being adsorbed on tricalcium phosphate in a Tswett column at pH 5.7 and later by eluting with phosphate buffer of pI~ 8.0. However, since most preparations of tricalcium phosphate are rather impermeable to water, it is probably better to adsorb on Celite. V. Other Methods Kitagawa and Shirakawa obtained crystalline beef liver catalase by means of a method involving precipitations with acetone and ammonium sulfate. 1° 1oM. Kitagawa and M. Shirakawa, J. Agr. Chem. Soc. (Japan) 67, 794 (1941).

778

RESPIRATORY ENZYMES

[137]

Dounce ~ has prepared crystalline beef liver catalase, using dilute acetone. Mosimann, 11 Bonnichsen, 5 and Tauber and Petit 1~ have still other methods. The procedure of Herbert and Pinsent ~ for the crystallization of bacterial catalase has been mentioned already. Recently Brown 13 has claimed that he has succeeded in separating beef liver catalase into two components through a new procedure. VI. RecrystaUization of Catalase The original method described by Sumner and Dounce 1 advised dissolving the catalase crystals in water plus the least possible amount of 9.6% phosphate buffer of pH 7.4. However, if any considerable amount of ammonium sulfate is present, the catalase crystals will not dissolve. One can add a little solid sodium chloride and warm the preparation to 40 ° in order to dissolve the catalase. However, it is better to recrystallize catalase as follows: Centrifuge down the catalase crystals, and discard the supernatant liquid. Stir up the crystals with very little water, and add a few milliliters of 6 to 9 % phosphate buffer of pH 7.4 to 7.8. Place this suspension of catalase crystals in a dialyzing parchment, and dialyze in the ice chest against distilled water. As soon as most of the ammonium sulfate present has dialyzed away the crystals will dissolve, provided that enough water is present. The solution can then be centrifuged to remove any whitish protein impurity that may be present. Further dialysis will result in the precipitation of a part of the catalase as prisms, provided that the solution is sufficiently concentrated. In order to precipitate the catalase as plates it will be necessary to dialyze against changes of distilled water for several days. If the catalase solution is brought to pH 5.7 by adding acid potassium phosphate, dialysis will cause the catalase to precipitate as needles. If catalase has been crystallized originally by dialysis, recrystallization is extremely simple. ~4 The crystals are centrifuged down and dissolved in the minimal amount of sodium chloride solution at a final concentration of 10% NaC1. A volume of sodium chloride solution of about ten to twenty times the volume of the centrifuged crystalline precipitate usually suffices. Buffer is unnecessary, but reasonable care should be taken to keep the sodium chloride concentration correct. After the catalase is completely dissolved, as can be observed by the disappearance of the silkiness of the solution on swirling, amorphous conII W. Mosimann, Arch. Biochem. and Biophys. 3~ 487 (1951). 12H. Tauber and E. L. Petit, J. Biol. Chem. 195, 703 (1952). 13G. L. Brown, Biochem. J. 51, 569 (1952). 1~j. B. Sumner and A. L. Dounce~ J. Biol. Chem. 127~439 (1939).

[137]

LIVER CATALASE

779

taminating protein is centrifuged down with high speed, and the catalase is recrystallized b y dialysis for 24 hours against several changes of distilled water. The crystals appear as plates. If prisms are desired, the p H must be increased to 7.0 to 7.5 by the addition of M / 2 Na2HPO4 before dialysis. Catalase crystallizes as needles from ammonium sulfate; as extremely thin plates from distilled water on dialysis at or near the isoelectric point (pH 5.7); and as prisms when dialyzed from distilled water above the isoelectric point. All three crystalline forms are intraconvertable. The yield of crystals obtained b y dialysis is best when plates are obtained by dialyzing at the isoelectric point. Both prisms and plates are soluble in sodium chloride at a final concentration of 10%.

VII. Estimation of Catalase Activity In 1927 von Euler and Josephson ~5 described a method for the determination of catalase activity which in all probability is the best yet devised for solutions of pure or partially purified catalase. The method is as follows: Chill 50 ml. of approximately 0.01 N hydrogen peroxide 16 to 0 ° in a b a t h of chopped ice. The peroxide should be 0.0067 M in phosphate buffer of p H 6.8. Add 1 ml. of properly diluted catalase, mix rapidly, at once remove a 5-ml. sample, and immediately blow it into a small flask containing 5 ml. of 2 N sulfuric acid. Remove other samples in a similar manner at 3, 6, 9, and 12 minutes. Titrate the peroxide in the flasks with 0.005 N permanganate. Calculate the monomolecular velocity constants, using the formula: 1 A K =~logl0A -x where K = monomolecular velocity constant, t = time in minutes, A = milliliters of permanganate used at zero time, and A - x = milliliters of permanganate used at 3, 6, 9, and 12 minutes. Construct a graph using minutes plotted against K values. The K value for zero time is found b y extrapolation. This value represents the catalase units at the dilution employed in the analysis. T o obtain the p u r i t y or Katalasefahigkeit of the catalase the equation used is: K Kat.f . = g. catalase per ml. 15H. yon Euler and K. Josephson, Ann. 452, 158 (1927). le We find it best to prepare this by diluting 30% hydrogen peroxide which has been distilled in ~acuo. The redistilled peroxide must be kept in the ice chest.

780

R E S P I R A T O R Y ENZYMES

[137]

For crystalline catalases the K a t . f . value m a y range from 30,000 to 60,000, depending on the source of the catalase. In 1904 Jolles 17 described a m e t h o d for the estimation of catalase activity in which the unused hydrogen peroxide was determined b y adding potassium iodide and a drop of ammonium molybdate, followed by titration with thiosulfate in the presence of starch. This method is applicable to homogenates, where the permanganate m e t h o d is poor because of reduction of the permanganate b y material in the homogenate. T h e method, as modified b y Sumner and Dounce, TMis: Add 1 ml. of properly diluted catalase solution (e.g., h u m a n blood diluted 1 to 1000) to 50 ml. of ice-cold 0.01 N hydrogen peroxide in M / 1 5 0 phosphate buffer, p H 6.8. At once withdraw a 5.0-ml. aliquot, and blow this into 5 ml. of 10% sulfuric acid. Withdraw other samples after 3, 6, 9, and 12 minutes. Add to each sample 1 ml. of 5 % potassium iodide and 0.5 ml. of saturated molybdic acid solution. Mix well, and allow to stand for about 3 minutes. Add a few drops of soluble starch solution and titrate to the end point, using 0.005 N thiosulfate. The plotting of titration values against minutes of time and the calculation of K values and K a t . f . values are exactly as described for the procedure of yon Euler and Josephson. If it is desired to express the a m o u n t of catalase present, a catalase unit can be taken as the a m o u n t of catalase required to give a calculated K0 value of 1. TM This m e t h o d has been employed b y Dounce and Shanewise 2° for the estimation of catalase in rat liver and appears to be highly reliable. A similar m e t h o d has been described b y Balls and Hale. 21 (See also Schwartz et al. ~2) Bonnichsen, Chance, and Theorell 2a have modified the m e t h o d of yon Euler and Josephson b y employing much greater quantities of catalase and much shorter periods of digestion. Sarkar and Sumner s have claimed t h a t this modification is no improvement. Feinstein 24 has published a method for catalase activity wherein perborate is employed in place of hydrogen peroxide. Walker 25has determined catalase activity b y means of the polarograph. i~ A. Jolles, Mi~nch. Med. Wochschr. 51, 2083 (1904); quoted from It. yon Euler, Chemie der Enzyme, II Teil, 3 Abschnitt, p. 73. J. F. Bergman, Mfinchen, 1934. 18j. B. Sumner and A. L. Dounce, unpublished. 19E. Tria, J. Biol. Chem. 129, 377 (1939). ~0A. L. Dounce and A. B. Shanewise, Cancer Research 10, 103 (1950). 2~A. K. Balls and W. S. Hale, J. Assoc. O~c. Agr. Chemists 15, 483 (1932). 2~E. J. Katz, L. Holt, and S. Schwartz, Natl. Nuclear Energy Ser. Div. IV, 23, 283 (1951). ~3R. K. Bonniehsen, B. Chance, and H. Theorell, Acta Chem. Scand. 1, 685 (1947). 24R. N. Feinstein, J. Biol. Chem. 180, 1197 (1949). 2~B. S. Walker, Proc. Federation Am. Sac. Exptl. Biol. 1, 140 (1942).

[138]

BLOOD CATALASE

781

Perlman and Lipmann 2e have described a manometric method for catalase activity. However, Theorel127 states t h a t a t t e m p t s to estimate catalase monometrically have been made repeatedly, but without success. This statement is probably true if one is concerned solely with an investigation of the kinetics of catalase action, but from the standpoint of determining catalase concentrations it does not apply. Dounce 2s has devised a manometric method for catalase activity and a m a n o m e t e r to be employed in this method. Beers and Sizer 29 have described the determination of catalase by means of a manometric method. ~6G. E. Perlmann and F. Lipmann, Arch. Biochem. 7, 159 (1945). 27H. Theorcll, in "The Enzymes" (J. B. Sumner and K. Myrbgck, eds.), Vol. 2, Part 1, p. 397, Academic Press, New York, 1951. 2s A. L. Dounce, Natl. Nuclear Energy Ser. Div. VI, 1, 270 (1949). 29R. F. Beers, Jr., and I. W. Sizer, Science 117, 710 (1953).

[138] B l o o d C a t a l a s e B y ROGER BONNICHSEN

Assay Method Principle. M a n y methods have been devised for the determination of catalase. 1 The m e t h o d described below, developed b y Bonnichsen, Chance, and Theorell, 1 is based on the determination of the a m o u n t of H202 split b y the enzyme in 15 and 30 seconds at room temperature and extrapolating to zero time. The H202 is measured b y titration with permanganate. The rapid titration technique minimizes the effect of catalase destruction t h a t takes place during the reaction. The method is rapid and sufficiently accurate for most purposes. (For other methods, see Vol. II [136].) Reagents

0.01 M phosphate buffer, p H 7. H~O2 solution (0.7 ml. of Perhydrol to 50 ml. of water). 2 % sulfuric acid. 0.01 M permanganate solution. Enzyme. The catalase is diluted with phosphate buffer to about 300 ~g per ml. Procedure. Fifty ml. of buffer is pipetted into a 100-ml. Erlenmeyer flask, and 2 ml. of H202 solution is added. Two ml. is withdrawn and

1R. K. Bonnichsen, B. Chance, and H. Theorell, Acta Chem. Scan& 1, 685 (1947).

782

RESPIRATORY E N Z Y M E S

[138]

blown into a 25-ml. E r l e n m e y e r flask containing a few drops of 2 % sulfuric acid. The enzyme is conveniently added to the buffer solution on a small watch glass, and the time is noted. Fifteen and 30 seconds later another 2 ml. is withdrawn and blown into 2 % sulfuric acid. The a m o u n t of H~O2 in the three samples is determined with permanganate. Definition of Units and Specific Activity. T h e first order reaction constant can be used as a measure of the catalase activity. T h e a m o u n t of catalase in the solution can be calculated from the density of the Soret band at 405 m~. There is some u n c e r t a i n t y in the literature a b o u t the value of the react[0n constant for pure catalase. Bonnichsen, Chance, and Theorell 1 give a value of 3.5 × 107 1. × mole -I X sec. -1. Deutsch 2 gives a value as high as 6.6, which agrees with the value found b y Agner2 There is also a disagreement about the millimolar extinction at 405 m~. T h e values v a r y from 380 to 420 mM. -1 cm. -1 for pure catalase. ~,4 The millimolar extinction at 280 m~ is more constant, 280 mM. -1 cm. -1.1 Application of Assay Method to Crude Tissue Preparations. The abovementioned m e t h o d can be used. The first 2-ml. sample is in this case taken a few seconds after the addition of the enzyme, and this sample is used as zero value. For preparative purpose it is more convenient to use Kat.f. described b y von Euler and Josephson 5 to express the activity and the p u r i t y of the enzyme.

Kat.f . =

K g. of enzyme in the 50-ml. vessel

X0 1 where K = log10 - ~ t ' X0 = initial H202, and X = H~02 at t minutes. Purification P r o c e d u r e The following m e t h o d can be used to prepare catalase from horse and pig blood. Preparation from beef blood has been described b y Laskowski and Sumner, e from h u m a n blood b y H e r b e r t and Pinsent. 7 If the m e t h o d is applied to beef blood, precaution must be taken when the hemoglobin is denatured with alcohol-chloroform as this catalase is more sensitive t h a n t h a t from other animals. If h u m a n blood is used the adsorption procedure used b y H e r b e r t and Pinsent should be carried out after step 2. H. F. Deutsch, Acta Chem. Scand. 6, 1516 (1952). 3 K. Agner, Arkiv Kemi, Minerol. Geol. B17, No. 9 (1943). 4 H. Theorell and A. Ehrenberg, Arch. Biochem. and Biophys. 41, 443 (1952). 5 H. von Euler and K. Josephson, Ann. 452, 158 (1927). 6 M. Laskowski and J. B. Sumner, Science 94, 615 (1941). D. Herbert and J. Pinsent, Biochem. J . 45, 203 (1948).

[138]

BLOOD CATALASE

783

Step 1. The blood corpuscles are washed twice with 0.9% NaCl to remove the plasma. They are then laked by addition of twice the volume of distilled water. Step 2. Tsuchihashi's 8 method is used to remove the hemoglobin. To each liter of the solution is added 500 ml. of an alcohol-chloroform mixture (1 part of chloroform to 3 parts of ethanol), while the solution is stirred vigorously for about 30 minutes. The denatured hemoglobin is removed by centrifugation or filtration. The alcohol-chloroform is then evaporated in vacuo. It is convenient to concentrate the solution at this step. The volume should be about 50 ml. for each liter of blood used as starting material. The evaporated solution is dialyzed overnight against water. The spectrum of the catalase is now clearly visible in a hand spectroscope. The spectrum can from now on be used to follow the catalase concentration. The purity is usually about 10 %. Step 3. To the dialyzed solution is added 2 M acetate buffer, pH 4, until the pH of the whole solution is about 4.0. The solution is allowed to stand for several hours. A brownish precipitate that forms is centrifuged and discarded. To the solution is added phosphate buffer, pH 6, until the solution is 0.1 M with regard to phosphate. To each 100 ml. of solution is added 60 ml. of acetone at room temperature. The precipitate is centrifuged and discarded. Further addition of 50 ml. of acetone precipitates the enzyme which is dissolved in 0.01 M phosphate, pH 7. The purity is now about 30 %. Step 4. It is possible at this purity by repeated ammonium sulfate fractionations to get crystalline enzyme, as described by Deutsch. 2 The first precipitate on addition of the salt should be discarded. Several fractions of partly crystalline material will be obtained by the first fractionation. Each of these fractions can be recrystallized to get pure enzyme. Another way of getting crystalline materials is to dialyze the catalase for several days against large volumes of distilled water. The catalase will then precipitate in a semi-crystalline form together with some impurities. The precipitate is dissolved in weak ammonia, pH about 9 in the solution. The catalase will rapidly dissolve, leaving some colored impurities undissolved. This procedure is repeated, and this time the catalase will crystallize from water. If this procedure does not yield crystalline catalase, ammonium sulfate should be tried. To the dialyzed solution solid ammonium sulfate is added to about 38"% saturation. From there on small drops of a saturated ammonium sulfate solution are added until a faint turbidity appears. The solution is centrifuged, and the inactive precipitate discarded. Very small amounts of the salt are again added to the solution with stirring, and the catalase will begin to crystallize. After 8 M. Tsuchihashi, Biochem. Z. 140, 63 (1923).

784

RESPIRATORY ENZYMES

[139]

a few hours most of the catalase has crystallized and can be centrifuged. The procedure requires sgme practice in protein crystallization and has to be varied a little with each new batch. The catalase can also be crystallized from alcohol solution at 4 °. The catalase solution is made 0.02 M with respect to phosphate buffer, pH 6, and cooled to 0 °. Ninety per cent ethanol is added dropwise with stirring. When the ethanol is 15% (v/v), the solution is left standing in the cold. In a few hours the catalase crystallizes. Occasionally the ethanol concentration has to be increased to 20%. To get pure catalase the procedure is repeated two or three times. The yield is about 400 mg. of catalase from each liter of blood corpuscles.

Properties The catalase has a red color at neutral pH. The molecular weight is 225,000. I t contains 1.1% protohemin, which equals 4 heroins per molecule and 0.09 % iron. The nitrogen content is 16.8 %. The catalase spectrum in a 0.001 M phosphate buffer shows bands in the visible at 623, 583, 535, and 505 m~. The Soret band is situated at 405 m~. As mentioned above, the millimolar extinction of the Sorer band varies in different preparations from 380 to 420 mM. -1 cm. -1. This is probably due to damage to the bonds between the hemin and the protein in part of the catalase. The millimolar extinction of the protein band at 280 mp seems more constant, 280 mM. -I cm. -1. Deutsch and Ehrenberg 9 have measured the paramagnetic susceptibility of horse blood catalase prepared by Deutsch. 2 T hey found the susceptibility per iron atom to be constant and equal to 13,390 c.g.s.e.m.u, between pH 4.8 and 10.4. g H. F. Deutsch and A. Ehrenberg, Acta Chem. Scan& 6, 1552 (1952).

[139]

Catalase

from

Bacteria

(Micrococcus lysodeikticus)

2H202~2H20+02

By

DENIS HERBERT

Catalase has been isolated in a pure state from only one species of bacterium, Micrococcus lysodeikticus (Herbert and Pinsenti). It is not known whether the catalases of other bacteria are similar, but the protein constituents of the enzymes would be expected to differ. 1D. Herbert and J. Pinsent, Biochem. J. 43, 193 (1948).

[139]

CATALASEFROM BACTERIA (Micrococcus lysodeikticus)

785

Assay M e t h o d

Principle. Catalase is allowed to act on hydrogen peroxide under defined conditions, and the peroxide remaining after known time intervals is determined by iodometric titration. The original isolators 1 of the enzyme used the assay method of yon Euler and Josephson. 2 Bonnichsen et al. 3 have since pointed out the disadvantages of this method (enzyme destruction by H202, causing falling values of k), and the following procedure, based on their ideas, has been found to be an improvement. Procedure. Pipet 5.0 ml. of H202 (0.01 M) in phosphate buffer (0.01 M, pH 6.8) into each of five test tubes (6 × 1 inch), and stand them in a bath at 25 °. To one tube add 1.0 ml. of enzyme rapidly (blowout pipet), simultaneously starting a stop watch. Stop the reaction after ca. 15 seconds by adding ca. 2 ml. of N H2SO4 rapidly from a small cylinder. Repeat the procedure with three of the remaining tubes of H~O2 using reaction times of ca. 30, 45, and 60 seconds; with the fifth tube the initial H:O2 concentration is obtained by adding the H2SO4 before the enzyme. The H202 remaining in each tube is determined by adding 0.5 ml. of 10% KI, one drop of 1% ammonium molybdate, and, after the tubes have stood for 3 minutes, titrating the liberated iodine with 0.01 N sodium thiosulfate (starch-iodide indicator), the whole contents of each tube being titrated in situ. Calculation of Results. For each of the four time intervals calculate the observed first-order velocity constant as

(where So is the H202 concentration at zero time, and S is the concentration remaining after t seconds), and take the mean of the four values. They should agree to within 5%, and there should be no tendency for the values to fall with increasing reaction time. The value of ]Cobs.is a measure of the concentration of catalase in the reaction mixture. If w is the dry weight (mg.) of enzyme preparation used in each test, then its concentration in the reaction mixture is c = w / 6 (g./1.). The specific catalase activity of the preparation is k = kobs. (1. X g.-1 X sec. -1) C

The specific catalase activity of pure, crystalline M . lysodeikticus cata2 H. yon Euler and K. Josephson, Ann. 452, 158 (1927). s R. K. Bonnichsen, B. Chance, and H. Theorell, Acta Chem. Scand. 1, 685 (1947); see also Vol. II [136, 138].

786

RESPIRATORY ENZYMES

[139]

lase (k0) is 230 1. × g.-1 X sec. -1 at 25°. 4 Hence the purity of the enzyme preparation is k/230, and the concentration of pure catalase in the reaction mixture is $ = kob~./230 (mg./ml.). Remarks. The amount of enzyme taken for assay should be enough to give a time for disappearance of half the initial H~02 of 10 to 100 seconds. Very dilute solutions of catalase are somewhat unstable and should be diluted immediately before testing. Permanganate titration of the H20~ may be used with purified enzyme, but the iodometric titration is essential (to avoid blank reactions with protein) when assaying crude preparations or whole bacteria. Purification Procedure

Principle. An essential preliminary to purification is the extraction of enzyme from the bacterial cells, which is effected by lysing them with lysozyme. Catalase is stable to ethanol-chloroform, which may be used to remove nucleoprotein and other extraneous material; a subsequent step involves partitioning of the enzyme in the two-phase three-component system ethanol-(NH4)2SO4-H20. Other procedures are conventional. Step 1. Preparation and Lysis of the Bacterial Suspension. Micrococcus lysodeikticus (strain N.C.T.C. No. 2665) is grown on agar in large enameled trays 5 for 40 hours at 35 °, and the growth is suspended in 0.5% NaC15 to a density of 4% (on a bacterial dry weight basis). Crystalline lysozyme6 is added (1 mg./g, bacteria), and lysis is allowed to proceed for 2 hours at 30 °. Step 2. Ethanol-Chloroform Treatment. After lysis the cell suspension is transformed to a translucent jelly, owing to the liberation of intracellular nucleoprotein. It is stirred vigorously, 0.1 vol. of M acetate buffer (pH 5.6) added, and cooled to 0 °. Ethanol, precooled to - 1 0 °, is added slowly to a concentration of 33 % v/v. The gelatinous precipitate is centrifuged off, washed with an equal volume of 0.1 M acetate, pH 5.6, containing 33 % ethanol, and the washings added to the first supernatant. The combined supernatants are treated with 0.2 vol. of CHC13, shaken on a fast mechanical shaker for 15 minutes, and centrifuged. Two liquid layers separate with a thick layer of denatured protein at the interface. The top layer is siphoned off. Step 3. First Ethanol-(NH4)~S04 Partition. The top layer from step 2 is treated with ~ 0 vol. of M sodium acetate, and solid (NH4):SO4 is added (30 g. to each 100 ml.). On standing in large separating funnels 4B. Chance and D. Herbert, Biochem. J. 46, 402 (1950). 5See ref. 1 for details of growth media and optimum conditionsfor lysis with lysozyme. 6 G. Alderton and H. L. Fevold, J. Biol. Chem. 164, 1 (1946).

[139]

CATALASEFROM BACTERLk (Micrococcus lysodeikticus)

787

two liquid phases separate, the lower containing most of the (NH4)2SO4 and the upper most of the ethanol, some (NH4)2SO4, and water. The smaller top layer, which is pale brown and contains all the catalase, is separated. Step ~. Second Ethanol-(NH4)~SO~ Partition. The top layer from step 3 is treated with an equal volume of CHC13, shaken for 15 minutes, and centrifuged; the top layer is removed. (Some protein is removed by this treatment, but its main purpose is to reduce the ethanol concentration of the aqueous layer.) Solid (NH4)2S04 (23 g. to each 100 ml.) is added to the top layer, and two layers again separate. The top layer, which is much the smaller, is dark brown and contains all the catalase i it is removed in a separating funnel and dialyzed against several changes of 0.05 M acetate (pH 5.6). Step 5. First (NH~)2S04 Fractionation. The dialyzed solution from step 4 is roughly fractionated with (NH4)2SO4 by adding successive portions of the salt and centrifuged off the resulting precipitates. No attempt is made to standardize the procedure exactly, the aim being to divide the material into a series of roughly equal fractions; the precipitates are dissolved in 0.05 M acetate, pH 5.6, and assayed for catalase and total protein. In a typical experiment, three fractions precipitated betweea the levels 25 and 35% (w/v) (NH4)~S04 contained together 80% of the initial catalase and only 38 % of the total protein; these were combined for the next step. Step 6. Second (NH4) 2S04 Fractionation. The combined fractions from the last step, dissolved to a protein concentration of ca. 3 %, are refractionated by the dropwise additiou of 50% (w/v) (NH4)2S04 solution adjusted to pit 5.6 with NH4OH. Again the procedure is empirical, a succession of small fractions being taken at gradually increasing salt concentrations, the precipitates being separately assayed and the purest reserved for the next step. The color of the precipitates is a useful guide to fractiouation, those containing inert protein being visibly paler than the dark-brown precipitates containing the purest enzyme. Step 7. Crystallization. The purest fraction from the last step is treated with just enough 50 % (NH4)2SO4 to precipitate all the catalase. The precipitate is centrifuged down and redissolved by adding distilled water drop by drop, very slowly and with good stirring, until all but a trace has dissolved; this is centrifuged off. The supernatant is then saturated with amorphous catalase, nearly all of which crystallizes on standing for 24 hours at room temperature, leaving an almost colorless superna° tant. Recrystallization can be effected by repeating the procedure but is usually unnecessary, as the crystals first obtained are virtually pure enzyme.

788

[139]

RESPIRATORY ENZYMES SUMMARY OF PURIFICATION PROCEDURE

Stage 1. Lysed bacteria 2. Ethanol-chloroform treatment 3. First ethanol-(NH4)~S04 4. Second ethanol-(NH~)2S04 partition 5. First (NH4)2SO4fractionation 6. Second (NH4)~SO~ fractionation 7. Crystals

Total Total volume, protein, ml. g. 5085

203

Total catalase, g.

k I.g. sec.

Yield, %

1.94

22.2

100

7610 2760

29.5 8.9

1.60 1.46

12.5 37.8

83 75

600 61

5.4 2.1

1.14 0.90

49.5 99

59 47

63 --

0.74 0.37

0.49 0.37

151 230~

25 19

Expressed in the units of yon Euler and Josephson, ~ this specific activity of the pure enzyme corresponds to a Katalasefdhigkeit (Kat.f.) of 98,000. Properties

General. T h e pure e n z y m e crystallizes f r o m (NH4)2S04 as regular octahedra which are isotropic when viewed between crossed polaroids; it m a y also be crystallized b y exhaustive dialysis against distilled water, when similar crystals are formed. Strong solutions of the e n z y m e h a v e a brown-red color resembling t h a t of methemoglobin and are v e r y stable at alkaline or neutral p H values b u t readily d e n a t u r e d below p H 5. Prosthetic Group. Bacterial catalase solutions h a v e a characteristic absorption s p e c t r u m in the visible region, with three bands centered at 506, 545, and 631 m~ (in p h o s p h a t e buffer at p H 6.8). These are due to a h e m a t i n prosthetic group which m a y be split off the colorless protein c o m p o n e n t b y t r e a t m e n t with acetone-HC1 and has been identified as p r o t o h e m a t i n I X (identical with t h a t of hemoglobin). This catalase contains no biliverdin. T h e h e m a t i n content of the crystalline e n z y m e is 1.09%. T h e e n z y m e gives a typical hemochromogen s p e c t r u m on t r e a t m e n t with N a O H and Na2S204 and forms characteristic compounds with cyanide and azide.1 Physicochemical Properties. T h e crystalline e n z y m e is homogeneous in the ultracentrifuge and has a sedimentation constant of 11 X 10 -13. T h e h e m a t i n content of 1.09% corresponds to a molecular weight of 58,000 X n, where n is the n u m b e r of h e m a t i n groups per molecule. F r o m the sedimentation constant it can be deduced t h a t n = 4 and the molecular weight is 232,000.

[14~

PLANT CATALASE

[140]

789

P l a n t Catalase

By ARTHUR W. GALSTON Assay Method All assays for activity of fractions were made b y the Bonnichsen et al.1 modification of the von Euler and Josephson ~ technique. The relative p u r i t y of each fraction was expressed in terms of its Kat.f. 2 D r y weights of enzyme used, needed for calculation of Kat.f,, were obtained b y dialyzing 1.0 ml. of the preparation against 2 1. of distilled water (1 °) for 24 hours, then drying overnight at 105 ° on a previously tared watch glass and weighing. Purification Procedure 3 Freshly harvested spinach leaves are washed in tap water and stored in a cold room at W2 to 4 °. The leaves are transferred to large stainless steel vats, covered with commercial acetone previously chilled to - 1 5 °, and permitted to sit for at least 2 hours at this temperature. The cold, partially dehydrated leaves are now very brittle and are easily reduced to small pieces with a turmix (large Waring-type blendor). The blending is complete in 30 to 60 seconds, and the slurry is now gravity-filtered in the cold, the filtrate being discarded. The residue is washed several more times with portions of cold acetone, and the resulting gray-green material is permitted to dry overnight in a ventilated hood. The acetone powder is extracted repeatedly with ice-cold 0.1 M Na2HPO~, yielding a filtrate of Kat.f. approximately 50 and an inactive residue, which is discarded. The filtrate is half-saturated with solid (NH4)2SO~ and permitted to stand overnight in the cold. The precipitate, which contains all the activity, is redissolved in a small volume of 0.1 M Na~HPO4. This preparation, after clarification b y filtration or centrifugation, has a Kat.f. of about 180. Three successive fractionations with 20%, 7%, and 20% saturated (NH4)2SO4 are carried out, the precipitates being saved in each instance. The product has a Kat.f. of about 920, shows a faint absorption band at 630 m~ when viewed with a hand spectroscope, and gives absorption peaks at 275 m~, 330 m~, 405 m~, and 625 m~ in a Beckman spectrophotometer. The catalase solution is adjusted to p H 6.5 and made 0.1 M with respect to N a acetate b y addition of the solid salt. Cautious fractionation 1R. K. Bonnichsen, B. Chance, and H. Theorell, Acta Chem. Scand. 1, 685 (1947) ; see Vol. II [136, 138]. 2 H. yon Euler and K. Josephson, Ann. 452, 158 (1927). 3 A. W. Galston, R. K. Bonnichsen, and D. I. Arnon, Acta Chem. Scand. 5, 781 (1951).

790

RESPIRATORY ENZYMES

[140]

with s a t u r a t e d (NH4)2804, also adjusted to p H 6.5, yields several precipitates, v a r y i n g in Kat.f. f r o m 1200 to 10,000. T h e m o s t active preparations are dialyzed for 2 days in the cold against p H 7.14 p h o s p h a t e buffer of ionic s t r e n g t h 0.1 and t h e n subjected to p r e p a r a t i v e electrophoresis (18 milliamperes, 330 volts, 4 hours), yielding five fractions, three on the anodic side, one in the b o t t o m cell, and one f r o m the cathodic side. T h e Kat.f. and s p e c t r o p h o t o m e t r i c d a t a for these fractions, as well as Kat.f.'s of all other fractions in the p r e p a r a t o r y scheme, are shown in the table.

Kag.f.

VALUES AND SPECTROPHOTOMETRIC CHARACTERISTICS OF VARIOUS FRACTIONS OF THE SPINACH LEAF CATALASE PREPARATION

Fraction 1. Na2HP04 extract of acetone powder 2. Ppt. of fraction 1 insol, in 50% satd. (NH02SO4 redissolved in 0.1 M Na2HPO~ 3. Ppt. of fraction 2 insol, in 20% satd. (NH~)2S04 redissolved in 0.1 M Na2HPO4, sol. in 7% sat& (NH4)2S04 4. Ppt. of fraction 3 insol, in 20% satd. (NH4):SO~ redissolved in 0.1 M Na2HP04 5. Ppts. obtained from adjusting fraction 4 to pH 6.5, making 0.1 M with respect to Na acetate, then cautiously fractionating with solid (NH4)2SO4 6. Electrophoretic fractions of redissolved fraction 5 Cathodic Bottom cell Anodie No. 2 Anodic No. 1 Anodic (top cell)

Kat.f.

Ratio E2a0 ~

E4o5 m.w

50

--

180

--

425

--

920

--

10,100

--

23,600 9,020 12,100 7,320 2,620

1.54 2.07 2.07 2.37 2.54

T h e cathodic fraction of Kat.f. 23,600 yields needle-like crystals after concentration, dialysis against 0.1 M Na2HP04, and addition of solid (NI-I4) 2SO4 to a b o u t 12 % saturation. These needles, when redissolved in 0.1 M Na2HPO~, give a good catalase absorption s p e c t r u m (Fig. 1).

Properties T h e e n z y m e contains 0.049 % Fe, a p p r o x i m a t e l y half the value for a pure 4 - h e m a t i n catalase. T h e prosthetic group is p r o t o h e m a t i n , as shown b y p r e p a r a t i o n of a typical pyridine h e m o c h r o m o g e n on the acetoneHC1 s u p e r n a t a n t of an aliquot of the enzyme. T h e e n z y m e is inhibited b y K C N and NAN3, 50% inhibition being produced b y 5 X 10 -e M K C N and 2 × 10 -~ M NAN3. N a diethyldithioc a r b a m a t e is without effect on activity.

[141]

PEROXIDASE (LIVER)

791

The enzyme is completely inactivated by 10 minutes of incubation at 60 ° but is indefinitely stable at 1° between pH 5.3 and 8.9. The activity is optimal between pH 5.3 and 8.0, falling off quickly at more acid values and slowly at more alkaline values. 1"8!

f

1.6 1.4

4O5

1.2

~'

~ 1.0 ~ 0.8 0.6

0.4

~

510 545 ~t ~

620

0.2 I

I

I

220

300

400

I

~00

600

|!

700

Wavelength, m/s Fro. 1. The absorption spectrum of eleetrophore~ically prepared spinach leaf catalase. Kat.f. = 23,600. Concentration of the enzyme approximately 0.5 mg. per 1 ml.

[141] P e r o x i d a s e (Liver) By

MARGARET J . H U N T E R

Assay Methods Heme. Measurements at 405 m~ were performed on a Beckman spectrophotometer to establish the presence of heme-containing compounds and, in the fractionation procedures, 405/280 ratios were used as indices of purification. Catalase. The method described below was developed by Bonnichsen et al. 1 Slight modifications in technique were adopted. REAGENTS

0.01 M hydrogen peroxide. 0.01 N potassium permanganate. 2 % sulfuric acid. 1 R. K. Bonnichsen, B. Chance, a n d H. Theorell, Acta Chem. Scand. 1, 688 (1947).

792

RESPIRATORY ENZYMES

[141]

Enzyme diluted with 0.01 M phosphate buffer, pH 7, to approximately 0.02 %. PROCEDURE. The procedure was as in Bonnichsen's paper with the exceptions that the enzyme + peroxide was stirred with a magnetic stirrer and that the aliquots were removed from the mixture as quickly as possible, the time of reaction being taken as the time of half-emptying the pipet. All aliquots (usually four) were removed within 60 seconds of the addition of the catalase to the peroxide solution. D E F I N I T I O N OF UNIT AND SPECIFIC ACTIVITY X

2.3 l o g k = over-al] reaction constant = x0 ct where x0 = hydrogen peroxide concentration at t = 0, x = hydrogen peroxide concentration at t (sec.), and c = concentration of enzyme (moles). Duplicates gave agreement within 5 %. Peroxidase. As it proved impossible to remove the catalase from the peroxidase-active fraction and still maintain peroxidase activity, no accurate assay for peroxidase was possible. The presence of peroxidase activity was routinely demonstrated by the addition of 0.03 ml. of 0.02 M guaiacol to 3 ml. of solution to which 0.03 ml. of 0.02 M methyl hydrogen peroxide had previously been added. A red color developed. Crystalline beef liver catalase gave no reaction with guaiacol. Further proof of the existence of a heme-containing compound other then catalase was demonstrated by calculating c, the concentration of catalase, from Ely, measurements for catalase at 405 m~, and from hydrogen peroxide activity measurements. The latter figure was always much lower than the former (one-third to one-half). This was also shown by measurements at 425 m~ before and after the addition of 0.03 ml. of 0.02 N KCN when Ae425, the change in molar extinction at 425 m~, was always much less than for the crystalline catalase. 2 Purification Procedure

Step 1. Preparation of Crude Extract. Beef livers were perfused, frozen, and comminuted. The resultant frozen powders were fractionated in ethanol-water mixtures as described by Cohn et al.3 The a u t h o r wishes to express her t h a n k s to Dr. B r i t t o n Chance for his help on the spectrophotometric analyses. 3 E. J. Cohn, D. M. Surgenor, a n d M. J. H u n t e r , " E n z y m e s a n d E n z y m e Systems," p. 129, H a r v a r d University Press, Cambridge, 1951.

[141]

PEROXIDASE (LIVER)

793

All catalase and peroxidase activity was concentrated in fraction C~. This fraction contained 16 g. of dry protein per kilogram of fresh liver. The proteins of this fraction were insoluble in 9% ethanol, pH 5.8 acetate-phosphate buffer, F/2 0.02, at - 3 °, but were soluble in 0% ethanol, pH 5.8 acetate-phosphate buffer, F/2 0.15, at ~ 1 °. Step 2. A 10% solution of fraction C3 was dialyzed against pH 7.4 phosphate buffer, I'/2 0.01, at -t-1°, for 12 hours. The solution was then dialyzed against pH 6.2 phosphate buffer, F/2 0.05, at -[-1°, for 12 hours. A heavy, non-heme-containing precipitate was obtained. This was centrifuged and discarded. The brown supernatant (approximately 3 % protein) had a 405/280 ratio of 0.3 to 0.35. The supernatant was dialyzed against 10% ethanol, pH 7.1, F/2 0.1, at --3 °, and any precipitate so obtained was discarded. The alcohol concentration in the supernatant was raised to 15%, and the suspension was kept at - 5 ° for 12 hours. The suspension was then centrifuged at - 5 °, the precipitate washed with 15% ethanol, pH 7.1 phosphate buffer, F/2 0.1, at --5 °, and recentrifuged. The washed precipitate was dissolved in pH 7.5 phosphate buffer, F/2 0.1, at -l-1°, to give a 5% protein solution. The 405/280 ratio of this solution was 0.54. Spectrophotometric analyses of this fraction with cyanide and hydrogen peroxide activity measurements soon showed that another heme-containing protein was present as well as catalase. Cytochrome c was found absent by Warburg analyses, and the absence of reduced or oxidized hemoglobin, methemoglobin, and hematin was confirmed by various solubility and spectophotometric analyses. It was found, however, that this fraction had the ability to oxidize guaiacol after the addition of methyl hydrogen peroxide, and that sodium hydrosulfide caused the Soret band maximum to move from 405 m~ to 410 m~. The spectrum of catalase does not change in the presence of sodium hydrosulfite. Step 3. If the above fraction was adjusted to pH 4.2 with 0.1 N acetic acid at -[-1° and after 12 hours dialyzed against pH 7.1 phosphate buffer, I'/2 0.05, at ~-1 °, a brown heme-containing precipitate was formed. The supernatant was found to be 95 % pure catalase (405/280 = 0.92) which readily could be crystallized at its isoelectric point. The residue, which redissolved at pH 4.5, had a 405/280 ratio of 0.35 and showed about 75 % of one component by electrophoretic analysis at this pH. From its change in solubility characteristics it had obviously been denatured, and it gave no color reaction with methyl hydrogen peroxide and guaiacol, and did not react with hydrogen peroxide. After crystalline catalase had been subjected to pH 4.2 at 0 °, there was no observable change in its solubility behavior or activity to hydrogen peroxide. Many attempts were made to separate the catalase and guaiacol-oxi-

794

RESPIRATORY ENZYMES

[149. ]

dizing protein, without losing the activity of the latter. I n every case, however, when crystalline catalase was obtained, the solution lost its power to oxidize guaiacol and a heme-protein was precipitated at neutral p H ' s . This heme-protein m a y be similar to the catalase or the heme-prorein reported b y Brown. 4 4 G. L. Brown, Biochem. J. 51, 569 (1952).

[142] Myeloperoxidase 1 B y A. C. ~/[AEHLY Distribution. Myeloperoxidase (abbreviated M y P O ) has been isolated b y Agner 2 f r o m e m p y e m i c fluid, f r o m leucocytes of a p a t i e n t with empyemic leukemia, and f r o m cbloroleukemic infiltrates. M y P O seems to occur in all myelonic leucocytes, especially in the eosinophylic granules, b u t not in l y m p h o c y t e s . An historical review is found in Agner's paper. ~

Assay Methods T h e a c t i v i t y determination of peroxidases is discussed in Vol. I I [136]. Agner 4 r e c o m m e n d s a special assay for M y P O based on the oxidation of uric acid b y the peroxidase and H202. 4~ Principle. T h e disappearance of the ultraviolet absorption b a n d of uric acid at 290 m~ is measured spectrophotometrically during the course of the action of M y P O on this donor. Solutions 0.01 M uric acid (stable for several days). 0.07 M p h o s p h a t e buffer, p H 7.3. 15 m M h y d r o g e n peroxide (stable for weeks at 0°). Procedure. T o 2.0 ml. of buffer and 0.15 ml. of uric acid solution is added M y P O solution containing a b o u t 60 to 120 ~/of the enzyme. T h e 1 Originally called verdoperoxidase (VPO) by Agner. 2 The name referred to the color of the enzyme due to its prosthetic group, a green hemin. Since lactoperoxidase (Vol. I I [144]) also contains a green hemin, the newer name myeloperoxidase (MyPO) suggested by Theorell [Arkiv Kemi, Mineral. Geol. 17B~ No. 7 (1943)] is more appropriate, especially since the other known peroxidases carry names derived from their sources. MPO could be mistaken for milk peroxidase. 2 K. Agner, Acta Physiol. Scan& 2, Suppl. 8 (1941), M.D. thesis. a K. Agner, Advances in Enzymol. 3, 137 (1943). 4 K. Agner, in press (personal communication). 4~About the oxidation of uric acid by peroxidase cf. K. G. Paul and Y. Avi-Dor, Acta Chem. Scand. 8, 637 (1954).

[142]

MYELOPEROXIDASE

795

volume is brought to 3.0 ml. b y the addition of more buffer, and the optical density at 290 m~ is measured. 5 At to, 20 ~l. of H202 solution (micropipet) is stirred into the cuvette, and a stop watch is started. The optical density at 290 m~ is read at regular intervals for a period of 10 minutes, and the initial rate of oxidation obtained from the resulting plot is used for the calculation of the enzyme activity. Calculation of the Enzyme Concentration. AgneP found that, under the conditions of the test, 0.5 X 10-6 mole of uric acid is oxidized per minute b y 10 3" of M y P O per milliliter of assay solution. This figure, together with the concentrations of H202 and of uric acid used in the test, allows us to calculate the rate constant, k4 (see Vol. I I [136]), for the reaction of the M y P O - H 2 0 , complex with uric acid as a donor. An equation given b y Chance e is used for the computation.

dx k4=d[Xp

1 ....

Xa

(1)

where k4 = the second-order reaction velocity constant.

dx

d-t = the rate of disappearance of the substrate (H20~).

a = the concentration of the donor (uric acid). p .... = the steady-state concentration of the enzyme substrate complex. Since p .... has not been measured for this case it will be replaced b y p

....

=

f X e

(2)

where e = enzyme concentration. f = factor relating p .... and e (degree of " s a t u r a t i o n " of the enzyme with substrate in the steady state). Since 10 3' of M y P O (molecular weight assumed to be equal to the equivalent weight, see p. 798) oxidizes 0.5 X 10-~ mole of uric acid per minute and 4 moles of H202 is required to oxidize 1 mole of uric acid, 4 we get

dx = 0.19 mole sec. -1 for e -- 1.0 molar dt -

-

Using equations 1 and 2 and solving for f X k4, we obtain dx 1 f X ~:4 = ~- X X-----e ~

(3)

5 The increment of optical density due to the subsequent addition of H~O~ is only about 0.001 unit and needs not to be taken into account. B. Chance, Arch. Biochem. 24, 410 (1949).

796

RESPIRATORY ENZYMES

[142]

and inserting

dx - - = 0.19 mole sec. -1 dt e = 1.0 molar a = 0.5 X 10-3 molar we get, at p H 7.3 and ca. 25 °, f X k4 = 380 M -1 sec. -1 Using this value and solving equation 3 for e we arrive at

dx e = 5.3 X d--/

(4)

Under the conditions of the standard assay m e t h o d and using the extinction coefficient for uric acid [e = 12.2 _+ .5 cm. -1 mM-1] 6awe can express the enzyme concentration in terms of the decrease of optical density at 290 m~ and obtain dD e = 1.72 )< 10-~ X d~(5) where e = concentration of M y P O in the cuvette. dD d--/ = decrease of optical density (log~0 Io/I) per second. Preparation

T h e main problem in obtaining M y P O is the isolation of leucocytes in sufficient quantities. Agner 2 used h u m a n e m p y e m a as the starting material. I t contains M y P O in v e r y high concentrations (about 0.5 g./1.) but is difficult to obtain. In our laboratories ox blood was u s e d / w h i c h is easy to collect in large amounts b u t contains only very small concentrations of M y P O (about 5 mg./1, of blood). T h e isolation of the leucocytes is achieved b y repeated gentle centrifugation and removal of erythrocytes and of serum b y suction. A short outline of the procedure follows. TM The top layer of cells (about 50% leucocytes) is diluted with 4 parts of 0.01 M phosphate (pH 7.2) and homogenized in a Waring blendor for 20 seconds. The solution is incubated with trypsin for 3 hours at room t e m p e r a t u r e and an equal volume of ethanol is added in the cold. T h e enzyme is extracted from the precipitate with 0.1 M phosphate (pH 7.2) and fractionated with a m m o n i u m sulfate. T h e fraction of 40 to 60 % saturation contains most of the activity e~ E. S. Canellakis and P. P. Cohen, J. Biol. Chem. 213, 397 (1954). 7 Alternatively, packed human blood cells obtained from Sharp and Dohme (Philadelphia, Pa.) were used. 7~ W. J. Steele, in cooperation with L. Smith and A. C. Maehly, unpublished experiments.

[142]

MYELOPEROXIDASE

797

and is collected by centrifugation. Extraction with 5 % ammonium sulfate yields MyPO of about 50% purity. It can be further purified (to > 8 0 % purity) by electrophoresis, but this procedure involves sizable losses. In the following paragraphs the isolation and purification of MyPO from empyema according to Agner 2,3 is described. Crude MyPO from Empyema. The mixture of 1 1. of empyema, 2 1. of water, and 1 1. of ether is shaken with 1.6 kg. of ammonium sulfate, centrifuged, and after removal of the etherical and aqueous solutions by suction the semisolid middle layer is dissolved in water. About 3.6 1. of viscous opaque solution is obtained. Purification Procedure

Step 1. Purification by Barium Acetate. About 470 ml. of saturated barium acetate solution is added to the crude extract described above. 8 The heavy precipitate is removed by centrifugation, and the supernarant, A, is used for step 2. Step 2. Alcohol Fractionation. An equal volume of 95% EtOH 9 is stirred into solution A, and the precipitate is removed by centrifugation at high speed (Sharpies centrifuge). Next 0.27 vol. of EtOH is added (final concentration of EtOH about 65 vol. %), and the precipitate is centrifuged down and dissolved in about 170 ml. of water, solution B. The supernatant is discarded. Step 3. Ammonium Sulfate Fractionation. Solution B is incubated with an equal volume of saturated ammonium sulfate solution and centrifuged. The precipitate is discarded, and the supernatant is again treated with the same amount of ammonium sulfate solution as above. The green precipitate is dissolved in 60 ml. of water and gives a clear brownish green solution, C. Step 4. Second Barium Acetate Purification. Solution C is treated with saturated barium acetate solution as described in step 1. The precipitate is removed by centrifugation and discarded. Saturated ammonium sulfate solution is added to the supernatant to a final saturation of 60%, and the precipitate is collected by centrifugation and dissolved in water. The solution is dialyzed vs. distilled water, and if a precipitate forms in the dialysis sack the contents of the sack are centrifuged. The supernatant is saved, and the precipitate is dissolved in 1% NaC1 and again treated with barium acetate and ammonium sulfate. Step 5. Electrophoresis. Electrophoresis is carried out in a phosphate buffer of pH 6.8 and u = 0.1. The fraction moving toward the cathode 8 The proper amount must be determined by pilot experiments for each preparation. A large excess yields opalescent solutions after eentrifugation. 9 EtOH stands for ethanol.

798

RESPIRATORY ENZYMES

[142]

with a mobility of 2.0 × 10 -5 cm. ~ volt - I sec. -1 is collected. T h e p u r i t y is determined spectrophotometrically b y measuring RZ (Reinhcitszahl) or be at 637 or 475 m~ (see p. 801). A b o u t 160 rag. of purified M y P O is obtained. TABLE I SUMMARY OF PURIFICATION PROCEDURE

Fe, Volume gequiv, Crude extract Mter step 1 Mter step 2 After step 3 After step 5

3.6 1. -170 ml. 60 ml. --

8.5 7.4 5.2 4.3 2.9

MyPO, Total yield, mg. % 470 410 287 240 160

100 87 61 51 35

Physical Properties These d a t a were obtained b y Agner2 1. Molecular Weight. F r o m the iron content, an equivalent weight of 56,700 is obtained. Direct determinations of the molecular weight are not available. 2. Isoelectric Point. At p H 6.8, ionic strength 0.1, and 0 °, the electrophoretic mobility was found to be 2.0 X 10 -5 cm. ~ volt -J sec. -1 toward the anode. T h e isoelectric point lies at p H ~ 10. 3. Solubility. I n distilled water, M y P O is easily soluble. I n a m m o n i u m sulfate, it is soluble at < 50 % saturation, insoluble at > 65 % saturation. 4. pH Stability (for 10 minutes of t r e a t m e n t ) . At p H 13.8, 52 % of the a c t i v i t y was left ( N a O H ) ; at p H 12.8, 100% ( N a O H ) ; at p H 1.2, 0 % (HC1); with 0.66 N acetic acid, 100%. 5. Thermal Stability (after 10 minutes of t r e a t m e n t ) . At 60 °, 100% of the a c t i v i t y was left; at 70 °, 95 %; at 80 °, 94 %; at 90 °, 0 %. Chemical Analysis of Agner's MyPO. ~,3 Only the content of iron, copper, and nitrogen were determined quantitatively, with the following results: Element

Content, %

Number of atoms per gram equivalent of iron

Fe Cu N

0.0985 0.0010 17.15

1 <0.01 ~695

T h e copper m u s t thus be an i m p u r i t y and cannot be an integral p a r t of the molecule. T h e nitrogen content is considerably higher t h a n t h a t

[142]

MYELOPEROXIDASE

of h o r s e r a d i s h It

peroxidase

also surpasses

eral (~16%). The prosthetic

799

( 1 3 . 2 % l°) a n d of l a c t o p e r o x i d a s e

the value

considered

"normal"

(15.56%n).

for proteins

g r o u p c o n s i s t s of a g r e e n h e m i n

in gen-

of a s y e t u n k n o w n

structure.

Data on E n z y m e Inhibitors and Spectroscopic Data. T h e s p e c t r o p h o t o m e t r i c m e a s u r e m e n t s a n d t h e r e s u l t s of s t u d i e s w i t h i n h i b i t o r s a r e s u m m a r i z e d i n T a b l e I I . T h e d a t a a r e d u e t o A g n e r 3 ,3 TABLE II ACTIVITY AND SPECTRAL DATA OF M v P O DERIVATIVE8a Compound with ~

Optimal conditions

pH < 5

Activity c50 %,g vs. H~O2 ~ m M

a

--

Soret b a n d ~e

430

ef

69.0

Visible b a n d ~e

~f

(500)~ ? 570 10.7 625 6.5 690 3.2

Ox. -t- H202 Complex I [H202] < 50 m M --~4304 ? ? ? . . . . 455a ? 6254 ? Complex I I Ox. -]- H C N i 0.01 458 ? 634 ? Ox. ~ H~NOH i 1.4 460 ? 628 ? Red. pH > 5 i ? 475 65.2 637 16.7 Red. -b HN~ -i 0.3 460 ? 615 ? HF -i 10 Same spectrum as ox. M y P O CO and OH' Do not react as judged by lack of inhibition a n d the absence of spectral changes -

-

-

-

a K. Agner, Acta Physiol. Scand. 2, Suppl. 8 Ox. stands for oxidized MyPO, red. for its form; the other agents react with the form a stands for active, i for inactive. '~ The concentration of reagent t h a t leads to ~ is the wavelength in m~.

(1941). reduced form. H F reacts with either indicated. 50% inhibition.

I e = log~0 7/0 cm. --1 m M -1. The values are calculated from Agner's paper, where ,

the d a t a are plotted in terms of the absorption coefficient, /~ = I n ~

cm. ~

mole-1. g Weak band. h B. Chance, Advances in Enzymol. 12, 153 (1951). 10 H. Theorell, Arkiv Kemi, Mineral. Geol. 16A, No. 8 (1942). n B. D. Polls a n d H. W. Shmukler, J. Biol. Chem. 201, 475 (1953); see also Vol. I I [144].

800

[142]

R E S P I R A T O R Y ENZYMES

TABLE III DONORS USED IN DETOXICATION EXPERIMENTS DESCRIBED IN THE TEXT a Common name

Alternative name

D e t o x i c a t i n g effect

Aromatic amines Aniline o-Toluidine m-Toluidine p-Toluidinc Mesidine o-Anisidine o-Chloroaniline p-Chloroaniline o-Nitraailine m-Nitraniline p-Nitraniline A n t h r a n i l i c acid o-Phenylenediamine p-Phenylenediamine Benzidine o-Tolidine

-2-Methylaniline 3-Methylaniline 4-Methylaniline 2,4,6-Trimethylaniline 2-Methoxyaniline 2-Chloroaniline 4-Chloroaniline 1-Amino-2-nitrobenzene 1-Amino-3-nitrobenzene 1-Amino-4-nitrobenzene 2-Aminobenzoic acid 1,2-Diaminobenzene 1,4-Diaminobenzene 4, 4 ' - D i a m i n o b i p h e n y l 4,4'-Diamino-3,3'-dimethylbiphenyl

+ + + -+ + + --+ --

Other amines and amine derivatives Phenethylamine Tyramine Acetanilide Phenylurea N-Methylaniline N,N-Dimethylaniline

1-Amino-2-phenylethane p-(~-Aminoethyl)phenol N-Phenylacetamide ----

-+ -

Hydroxybenzene 1,2-Benzenediol 1,3-Benzenediol 1,4-Benzenediol /~-(p-Hydroxyphenyl)alanine /3- ( 3 , 4 - D i h y d r o x y p h e n y l ) a l a n i n e

+ + + ---

1,4-Cyclohexadienedione Benzopyrrol -2,3-Indolenedione a - A m i n o - 3 - i n d o l e p r o p i o n i c acid 4 - H y d r o x y - 2 - p y r r o l i d i n e c a r b o x y l i c acid a - A m i n o - 5 - i m i d a z o l e p r o p i o n i c acid 4-Imidazole~thylamine

+

Phenols Phenol Pyrocatechol Resorcinol Hydroquinone Tyrosine Dopa

Other donors Quinone Indole Indoleacetic acid Isatin Tryptophan Hydroxyproline Histidine Histamine

K. Agner, J. Exptl. Med. 92, 337 (1950).

---

[143]

PLhNT PEROXIDASE

801

Measurement of Enzyme Purity by Spectrophotometry. Two spectrophotometric tests are available for checking the degree of purification of MyPO obtained. One of them is based on the ratio of the heights of the bands in the Soret region and in the protein region. The ratio D43o/D27~ can be called RZ, as in the case of horseradish peroxidase (Vol. II [143]). Agner 2 found the RZ to be 0.79. The other test measures the increase of optical density on reduction of the fully oxidized peroxidase. This measurement is performed either at 637 m~, where ~red. - -

~ox. =

1.08

cm. -I

X

mM

-1

or at 475 m~, where ered. -- Cox. = 49.5 cm. -1 X mM -~ for the purified enzyme. Neither kinetic, magnetic, nor titration data on myeloperoxidase are at present available. Physiological Role. Although the physiological function of M y P 0 is by no means clearly established, it was shown that one function of the enzyme could very well be the detoxication of bacterial toxins. Experiments in this field were performed by Kojima and others, 12 but the most intensive research is again due to Agner. 13 He placed a cellophane bag containing solutions of MyPO and diphtheria toxin into a solution of 0.05 mM H202 and about 0.05 mM donor. Dialysis was allowed to proceed for 24 hours at pH 7.1 (phosphate buffer). The incubated toxin solutions were assayed by injection into guinea pigs. Agner found that certain donor substances are oxidized to products which destroy the toxic properties of diphtheria toxin (the immunological properties of the toxin are initially unaffected). Table III lists the substances which were tested for their antitoxic action under the experimental conditions described above. 12S. Kojima, J. Biochem. (Japan) 14, 95 (1931-32). is K. Agner, J. Exptl. Med. 92~ 337 (1950).

[143] P l a n t P e r o x i d a s e 1

By A. C. MAEHLY Distribution. Peroxidase has been found in most plant cells that were investigated and seems to be a normal component of such cells. Partial purification of the enzyme has been achieved from several sources, nora1For over-all reaction equation see p. 764.

802

RESPIRIkTORY ENZYMES

[143]

bly f r o m figs, 2 b u t the e n z y m e has not been isolated from plants in a pure state except in the case of horseradish peroxidase (henceforth design a t e d b y H R P ) . This e n z y m e has been p r e p a r e d b y Keilin and coworkers 8,4 and has been crystallized b y Theorell. 5 I t s enzymatic, physical, and chemical properties h a v e been studied extensively, b u t its physiological role is still largely unknown. Reaction M e c h a n i s m . T h e chemical reactions underlying the function of peroxidase are not known in detail, b u t m a n y properties of the intermediates and the kinetics of e n z y m e action are well understood, in cont r a s t to m o s t other enzymes. This is due to the work of Keilin and coworkers, 3,4 of George, 8-8 and especially of Chance, who published a great n u m b e r of papers and reviews on this subject2 -~2 According to Chance the reactions are in principle the following: F o r m a t i o n of green, active e n z y m e s u b s t r a t e c o m p o u n d I: kl H R P + H~O2 ~- C o m p l e x I (e) (x) k2

(1)

Transition of complex I to red, active complex I I :

k7 Complex I ~- A H --~ Complex I I -~- A (a) (p)

(2)

R e c o v e r y of e n z y m e b y reduction of complex I I : k~ Complex I I ~- A H --~ H R P ~ A ~ 2 H 2 0

(3)

T o a certain degree, spontaneous decomposition of complex I I and the J. B. Sumner and M. J. Howell, Enzymologia 1, 133 (1936). D. Keilin and T. Mann, Proc. Roy. Soc. (London) B122, 119 (1937). D. Keilin and E. F. Hartree, Biochem. J. 49, 88 (1951). s H. Theorell, Enzymologia 10, 250 (1942). 6 p. George, Advances in Catalysis 4, 367 (1953). 7 p. George, Biochem. J. 54, 267 (1953). s p. George, J. Biol. Chem. 9-01, 413 (1953). 9 B. Chance, Advances in Enzymol. 12, 153 (1951). i0 B. Chance, in "The Enzymes" (J. B. Sumner and K. Myrb~ck, eds.), Vol. 2, Part 1, p. 428, Academic Press, New York, 1951. 11B. Chance, in "Modern Trends in Biochemistry and Physiology" (E. S. G. Barron, ed.), p. 25, Academic Press, New York, 1952. 1~B. Chance, in "Investigation of Rates and Mechanisms of Reactions" (S. L. Friess and A. Weissberger, eds.), Vol. 8 of "Technique of Organic Chemistry," p. 627, Interscience, New York, 1953.

[143]

PLANT PEROXIDASE

803

formation of complex I I I from complex II and an excess of H20~ can occur: k3 Complex I I --* H R P Jr P Complex II ~ H~02--* Complex III, inactive

(4) (5)

The meaning of A and AH is explained in footnote 13; e is enzyme concentration; x is substrate concentration; p is concentration of the rate limiting enzyme substrate compound II; and a stands for the concentration of free donor. P means products without specifying their nature. Assay Methods. The general assay methods for peroxidases are described in Vol. l I [136]. A further detailed paper on activity determinations was published by Maehly and Chance. 14 For this reason, only a physical measurement of enzyme purity will be included in this chapter. The RZ Unit. In the later stages of purification and generally when purified H R P solutions are used a spectrophotometric measure of the purity is very convenient. The ratio of the optical density at 403 mg (due to the hemin group) to that at 275 mg (due to the protein) is determined. This ratio has been called RZ (for Reinheitszahl) and was found to be 3.04 for pure crystalline HRP. la I t is recommended that the purity of H R P be characterized in terms of RZ. The ratio Sorer band to protein band is a very convenient way to express the purity of hemoproteins in general. Preparation of Crystalline HRP. Theorell and co-workersT M as well as Keilin and co-workers a,4 have worked out preparative procedures for HRP. Theorell's group crystallized the enzyme several times, whereas Keilin's group, although not succeeding in this, claims to have obtained an even purer preparation as iudged by the molecular weight. 17 The following procedure has been successfully used several times in this laboratory. Steps 1 to 3 follow essentially Keilin's method; the others follow Theorell's procedure quite closely. The whole preparation, except for electrophoresis and alcohol fractionation, is carried out at room temperature. Step 1. Crude Extract from Horseradish Roots (Calculated for 100 kg.). The roots are either collected in the spring when they begin to sprout, la AH = donor, in the reduced form; A = donor, oxidized, cf. also p. 808. 14A. C. Maehly and B. Chance, in "Biochemical Analysis" (D. Glick, ed.), Vol. 1, p. 357, Interscience, New York, London, 1954. ~5H. Theorell and A. C. Maehly, Acta Chem. Scand. 4, 422 (1950). 16H. Theorell, Arkiv Kemi, Mineral. Geol. 16A, No. 2 (1942). 17Btt see footnote 41.

804

RESPIRATORY ENZYMES

[143]

or sprouting is achieved b y allowing the roots to lie in running w a t e r for several days. TM T h e roots, including the peroxidase-rich skin, are then minced mechanically. 19 This and the following operations should be carried out in the open air, and rubber gloves should be used ~° since irritating allylisothiocyanate is liberated. T h e mince is pressed out with a hydraulic press, and 30 1. of w a t e r is added to the d r y material. After standing and occasional stirring for several hours, the mass is pressed out again, and this operation is repeated once more. A b o u t 100 1. of extract is obtained. Step 2. First Ammonium Sulfate Fractionation. 2z T o 100 1. of extract 68 kg. of a m m o n i u m sulfate is added gradually and with vigorous mechanical stirring, which is continued for 1 hour after the last addition of salt. T h e solution is then left standing undisturbed overnight close to an aspirator. A solid layer collects at the top of the solution, and m o s t of the liquid is then r e m o v e d b y suction and discarded. T h e remaining suspension is washed into centrifuge cups with small a m o u n t s of saturated a m m o n i u m sulfate solution and gently centrifuged. T h e colorless solution is sucked off, and the remaining semiliquid paste (about 6 1.) is dialyzed vs. t a p w a t e r using h e a v y cellophane tubing. 22 After 1 day, the tubing is changed and dialysis continued until the solution is salt-free (2 to 3 days). ~3 T h e contents of the tubing are centrifuged to give a b o u t 10 1. of solution. N e x t 312 g. of a m m o n i u m sulfate per liter is added, and the solution is left standing overnight. T h e rather loose precipitate, a, is filtered off t h r o u g h large fluted filters. T o each liter of the filtrate 250 g. of a m m o nium sulfate is added. T h e precipitate, b, containing m o s t of the activity, sticks to the walls of the container when the s u p e r n a t a n t is poured off. Precipitates a and b are each suspended in a m i n i m u m a m o u n t of water, dialyzed salt-free and centrifuged, yielding solutions A and B. 18Daily a sample of about 50 g. is minced, pressed out, and the activity of the juice is measured. When the activity reaches its maximum, the whole batch of roots is used for the preparation. For Pennsylvania roots 3 days' sprouting gave the best results. 19 The firms delivering the roots usually have facilities to carry out the mincing operation. ~0If a good hood and gas mask are available, the operations may be carried out indoors. 21In this step ammonium sulfate "purum" can be used after pulverization. ~ Sometimes a filtration on Bfichner filters is necessary at this point to keep the volume of the suspension small. Kieselguhr (Celite) was found to absorb a fair amount of HRP and should be avoided. ~a Crude HRP preparations attack cellophane tubing. It is therefore safer not to use running water but to replace the dialysis water every few hours and to use mechanical stirring.

[143]

PLANT PEROXIDASE

805

Step 3. Purification with Calcium Phosphate Gel. Solution A (about 1 1.), containing a b o u t 15% of the total activity, needs further purification. T h e solution is treated with 100 g. of calcium p h o s p h a t e gel 24 per liter and, after 5 minutes standing, centrifuged. T h e s u p e r n a t a n t is t r e a t e d again in the same way, and the precipitates are discarded. T h e n 242 g. of a m m o n i u m sulfate per liter is added, and after several hours the precipitate is collected b y centrifugation and discarded. T h e s u p e r n a t a n t is treated with 205 g. of a m m o n i u m sulfate per liter, the precipitate centrifuged off, suspended in a little water, and dialyzed salt-free. T h e resulting solution is added to solution B of step 2 and the combined solutions (~-~4 1.) are the starting material for step 4. Step 4. Alcohol Fractionation. 25 T o each liter of solution from step 3, 4 1. of 95% E t O H 26 is added slowly and with mechanical stirring. T h e s u p e r n a t a n t is carefully decanted and discarded. T h e precipitate, which sticks to the walls of the container, is dissolved in 3 1. of w a t e r with the help of a rubber policeman, and 2 1. of 9 5 % E t O H is added as above. 27 Centrifugation yields solution C and a deposit which is stirred up with 500 ml. of 50% E t O H , centrifuged, and the s u p e r n a t a n t added to solution C ( ~ 5 1.). This solution is e v a p o r a t e d in vacuo and below 40 ° (cooling b y ice water or refrigerated brine) to a volume of 250 ml. F o u r volumes of 9 5 % E t O H is again added in the cold, and after centrifugation the deposit is dissolved in 150 ml. of water, yielding solution D. Step 5. Electrophoresis. Buffer solution is prepared b y adding N N a O H to 20 1. of 0.03 M Na2HPO4 until the p H is 10.3. Solution D is dialyzed against this buffer, and p r e p a r a t i v e electrophoresis is carried out in a large Tiselius apparatus, if necessary in batches. T h e H R P migrates slowly toward the anode; brown-colored impurities wander in the same direction b u t at m u c h higher rates. 28 A b o u t every 24 hours the impurities are removed b y sucking out the protein solution f r o m the top c o m p a r t ~ The gel is prepared as follows:~ To a 10-l. glass jar containing 1 I. of tap water (if the city water is of high quality) and 250 ml. of 0.6 M CaCl~ are added, with stirring, 250 ml. of 0.4 M Na3PO4, followed by N acetic acid until the pH is 7.3. The jar is then filled with water, the precipitate washed six times by decantation, centrifuged down, and suspended in water to give 400 ml. of a thick cream. ~5Step 4 should be performed at 0°, or ice-cooled solutions and vessels should be used. 26EtOH stands for ethanol. In Europe, the same alcohol is labeled 96 %. 27Sometimes it is advantageous to use more dilute solutions of the enzyme at this point, but the ratio 3 1. of solution to 2 1. of EtOH must be maintained. Pilot runs are recommended for this stage of the preparation. 28 Since all compounds migrate toward the anode, it is wisest to fill the protein solution into the bottom cell and into all cathodic cells, whereas all anodic cells are filled with buffer.

806

RESPIRATORY ENZYMES

[1~]

m e n t on the anode side and replacing it b y buffer. 29 F r o m time to time the protein solution is carefully moved back into its original position. When no more impurities can be removed (after 3 to 10 days), the electrophoresis is stopped. T h e contents of each cell c o m p a r t m e n t from each electrophoresis experiment are collected separately, and their RZ values are measured. Fractions with similar RZ values are combined, and those with RZ ~ 0.20 are discarded or added at step 2 of the next preparation. Step 6. Second Ammonium Sulfate Fractionation. ~° The progress of purification can now be followed spectrophotometrically b y determining the RZ. Activity tests need only be performed as an occasional check. The electrophoresis fraction with the lowest RZ is treated with 31 g. of ammonium sulfate per 100 ml. (50 % saturation), left standing for several hours, and centrifuged. The precipitate is set aside if its RZ > 0.20, and 13.5 g. of ammonium sulfate is added to every 100 ml. of the supern a t a n t (70 % saturation). After a few hours of standing, the suspension is centrifuged. The s u p e r n a t a n t and all loose particles are poured off completely and set aside if RZ > 0.20 after addition of water. T h e precipitate (as free from s u p e r n a t a n t as possible) is dissolved in the electrophoresis fraction which is next in purity. T h e procedure is repeated until the purest fraction is reached. The last precipitate is dissolved in a small a m o u n t of water and centrifuged to give solution E. All fractions below 50% and above 70% saturation having RZ > 0.20 are combined, brought to about 90% saturation with solid ammonium sulfate, and centrifuged. T h e precipitate is dissolved in little water and dialyzed. The resulting solution is refractionated, and the fraction from 50 to 70% saturation is added to solution E, which then is dialyzed salt-free. Step 7. Solubility Curve and Crystallization. Centrifuge tubes (15 ml.) are filled with aliquots of solution E, and saturated ammonium sulfate solution is added to each tube so t h a t the final degree of saturation ranges in steps from 50 to 70%. The tubes are well stoppered and left standing overnight. ~1 The next morning they are centrifuged, and the concentration of H R P (at 403 or 500 m~) and of total protein (at 275 m~) in the s u p e r n a t a n t determined spectrophotometrically and plotted as a function of per cent ammonium sulfate concentration. F r o m this graph, the values of ammonium sulfate saturation for " 1 0 % H R P precipita2g Sometimes a colored compound is seen migrating toward the cathode. This compound was called paraperoxidase by Theorel116and is regarded as an artifact of the preparation method. a0 From here on, analytical-grade ammonium sulfate should be used. 31If crystallization occurs in some of the tubes, the solubility values obtained for those samples cannot be relied on. The crystals should be saved for inoculation at a later stage.

[143]

PLANT PEROXIDASE

807

tion" and "90% HRP precipitation" are determined2 2 All samples are p o o l e d , d i a l y z e d salt-free, a n d a d d e d t o t h e r e s t of t h e s o l u t i o n . F o r c r y s t a l l i z a t i o n t h e c o n c e n t r a t i o n of H R P is a d j u s t e d t o a b o u t 0 . 5 % (--~0.1 r a M ) , a n d solid a m m o n i u m s u l f a t e is a d d e d t o give t h e l o w e r of t h e t w o s a t u r a t i o n v a l u e s d e t e r m i n e d b y t h e s o l u b i l i t y curve. A f t e r s t a n d i n g o v e r n i g h t t h e s o l u t i o n is c e n t r i f u g e d , t h e p r e c i p i t a t e , F, s e t aside, a n d t h e s u p e r n a t a n t t r e a t e d d r o p w i s e w i t h s a t u r a t e d a m m o n i u m s u l f a t e s o l u t i o n u n t i l c r y s t a l l i z a t i o n begins.3~ A f t e r t h e a p p e a r a n c e of t h e first c r y s t a l s t h e a d d i t i o n of a m m o n i u m s u l f a t e s o l u t i o n is cont i n u e d v e r y s l o w l y ( d u r i n g s e v e r a l h o u r s ) u n t i l t h e u p p e r v a l u e of t h e s a t u r a t i o n d e t e r m i n e d b y t h e c u r v e is r e a c h e d . A f t e r 24 h o u r s t h e c r y s tals are spun down and the mother liquor combined with precipitate F. T h e r e s u l t i n g s o l u t i o n s h o u l d b e p u r e e n o u g h for m o s t p u r p o s e s b u t can be p u r i f i e d f u r t h e r b y a s e c o n d or t h i r d f r a c t i o n a t i o n . TABLE I SUMMARY OF PURIFICATION PROCEDURE

(for 100 kg. of roots)

1. 2. 3. 4. 5. 6.

Fraction

Total volume, 1.

Concentration m g./ml,

Total amountj g.

Recovery, %

RZ a

Raw juice First (NH4)2S04 precipitate Before EtOH fractionation After EtOH fractionation Before crystallization Crystals

100 10 4 0.15 0.82 --

0.27 2.3 2.6 48 5.0 --

27.0 b 23.5 b 10.5 7.2 4.1 2.0

--100 68 39 19

---0.32 1.4 3.0 c

" The meaning of RZ is explained on p. 803. b Some oxidase activity is probably present. c Sometimes RZ values of only 2.8 were obtained. The value 3.0 is reached only after several recrystallizations. Specificity. T h e s u b s t r a t e s p e c i f i c i t y ( e q u a t i o n 1) of H R P is h i g h : o n l y H20~, M e O O H , 84 a n d E t O O H 35 c o m b i n e ecith H R P t o g i v e a c t i v e e n z y m e s u b s t r a t e c o m p l e x e s . 36,a7 33 These values for Swedish roots were ~ 5 3 % and 60 % saturation,~S and for Pennsylvania roots ~ 5 8 % and 62 % saturation (A. C. Maehly, unpublished data). 33 A silky shine is observed when a crystallizing suspension is swirled in strong light. Inoculation with crystalline HRP is helpful at this stage. 34 MeOOH stands for methyl hydrogen peroxide. 35 EtOOIt stands for ethyl hydrogen peroxide. 38 p . George~-S found that certain oxidizing agents form compounds with HRP which are apparently identical with those formed by the action of hydroperoxides. The mechanism of these oxidations is currently being investigated in several laboratories. This problem is, e.g., the subject of recent papers by R. R. Fergusson and B. Chance, Science, and J. Am. Chem. Soc, in press. ~ P. George suggested to replace the denotation "complex" by "compound."

808

RESPIRATORY ENZYMES

[143]

T h e s p e c i f i c i t y of t h e s e e n z y m e s u b s t r a t e c o m p l e x e s for h y d r o g e n d o n o r s ( e q u a t i o n s 2 a n d 3) is q u i t e low. S e v e r a l score s u b s t a n c e s a r e e a s i l y o x i d i z e d (see also T a b l e I V ) , m a i n l y p h e n o l s , a m i n o p h e n o l s , dia m i n e s , i n d o p h e n o l s , a s c o r b i c acid, l e u c o d y e s , a n d c e r t a i n a m i n o acids. 3s E x t e n s i v e lists of t h e s e c o m p o u n d s w e r e p u b l i s h e d b y K a s t l e a n d P o r c h 39 a n d b y Joslyn.40 P h y s i c a l P r o p e r t i e s . 1. Molecular Weight. T h e m o l e c u l a r w e i g h t is 40,200 a c c o r d i n g t o T h e o r e l l a n d E h r e n b e r g , 41 39,800 a c c o r d i n g t o Cecil a n d O g s t o n . 42 2. Isoelectric Point. T h e i s o e l e c t r i c p o i n t is 7.2. 43 3. Solubility. F i v e g r a m s is s o l u b l e in 100 ml. of w a t e r if t r a c e s of s a l t s a r e p r e s e n t . I n a m m o n i u m s u l f a t e t h e e n z y m e is s o l u b l e u p t o 58 % s a t u r a t i o n b u t i n s o l u b l e a b o v e 62 % s a t u r a t i o n . 4. p H Stability. ~4,45 T h e p H s t a b i l i t y is p H 5.5 t o 12 in t h e p r e s e n c e of fluoride, p H 4.5 t o 12 in t h e p r e s e n c e of o t h e r h a l o g e n ions, N3' a n d C N ' , a n d p H 3.5 t o 12 in t h e a b s e n c e of all ions l i s t e d a b o v e . 38 I. W. Sizer, Advances in Enzymol. 14, 129 (1953). 39 j. H. Kastle and M. B. Porch, J. Biol. Chem. 4, 301 (1908): ~0 M. A. Joslyn, Advances in Enzymol. 9, 613 (1949). 41 The value of 44,100 reported by Theorell [Arkiv Kemi, Mineral. Geol. 15B, No. 24 and 16A, No. 2 (1942)] was based on a sedimentation constant of s~0 -- 3.85 X 10 I3, determined in an oil turbine centrifuge. R. Cecil and A. G. Ogston [Biochem. J. 48, 592 (1948)] as well as S. Schulman [Arch. Biochem. and Biophys. 44, 230 (1953)] and several others have recently shown that the values for s20 obtained with ~his type of centrifuge are about 7-11% higher than those found with a Spinco ultracentrifuge. Most of this deviation was found to be due to the fact that the sample cell reaches a several degrees higher temperature than the environment during the run. A sample of crystalline and electrophoretically pure HRP prepared by K. G. Paul was recently used to determine the sedimentation constant. The determination was carried out by A. Ehrenberg at Professor Theorell's institute using a Spinco instrument. A single run at a protein concentration of 0.5% gave a value of s~0 = 3.59 X 1013 (personal communication from A. Ehrenberg). The deviation from the oil turbine value cited above is 7 % and thus in the range usually encountered on comparing data on the two types of instruments. The same value would be arrived at if it is assumed that the temperature of the oil turbine centrifuge cell was 2.8 ° higher than that assumed by Theorell in his earlier calculations. The new value for s2°0leads to a molecular weight for HRP of 40,200, when leaving all other magnitudes unchanged. Cecil and Ogston 42 found s~0 = 3.48 X 10 ia and arrived at a molecular weight of 39,800. 42 R. Cecil and A. G. Ogston, Biochem. J. 49, 105 (1951). 43 H. Theorell and A..~keson, Arkiv Kemi, Mineral. Geol. 17B, No. 7 (1943). 44 A. C. Maehly, in "Enzymes and Enzyme Systems" (J. T. Edsall, ed.), p. 81, Harvard University Press, Cambridge, Mass., 1951. 45 A. C. Maehly, Biochim. et Biophys. Acta 8, 1 (1952).

[143]

PLANT PEROXIDASE

809

5. Thermal Stability. If w a r m e d for 15 m i n u t e s t h e e n z y m e is s t a b l e u p to 63°. 15 A t r o o m t e m p e r a t u r e H R P is s t a b l e for weeks. 46 P a r t i a l rec o v e r y of a c t i v i t y a f t e r cooling of h e a t e d s o l u t i o n s h a s b e e n r e p o r t e d (e.g., b y B a c h a n d Wilensky47). 6. Titration. Acid base t i t r a t i o n curves were p u b l i s h e d b y Theorell. 471 M i c r o t i t r a t i o n t e c h n i q u e s h a v e b e e n a p p l i e d to H R P b y M a e h l y (in preparation). 7. Oxidation Reduction Potential. T h e redox p o t e n t i a l of H R P was r e c e n t l y d e t e r m i n e d b y H a r b u r y 47b a n d f o u n d t o be r e m a r k a b l y low, v a r y i n g f r o m E0' = - 0 . 2 0 7 0 a t p H 6.08 to E0' = - 0 . 2 7 8 7 a t p H 7.71. C h e m i c a l A n a l y s i s . Q u a n t i t a t i v e d a t a are a v a i l a b l e o n t h e c o n t e n t i n t h e following g r o u p s or e l e m e n t s : 43,4s ( T a b l e I I ) . TABLE II COMPOSITION OF HRP Element or group C H

O (by difference) N S Fe Protohemin Carbohydrates Arginine Histidine Lysine

Content, % 47.0 7.25

32.0 13.2 0.43 0.127 1.36 ~,b 1.30" 18.4 ~ 6.91 0.71 4.06

Groups per mole ---

-416 6 1 1 1 -18 2 12

Recently K. G. Paul, H. Thcorell, and /~..~keson [Acta Chem. Scan& 7, 1284 (1953)] have redetermined the extinction coefficient of pyridine ferroprotoporphyrin (new value e557 = 34.7 mM -1 X cm.-1). The figures for the hemin content listed in this paper are recalculated on the basis of the new value. Keilin and Hartree b have found a hemin content of 1.61% for their HRP preparation, but Paul et al. found 1.36% hemin for the same preparation when using ~7 = 34.7 mM -1 X cm.-1 in the pyridine hemochromogen test. b D. Keilin and E. F. Hartree, Biochem. J. 49, 88 (1951). c Determined by weighing the humin formed on acid hydrolysis. 48 Growth of bacteria or fungi in aqueous ttRP solutions is very rare even at room temperature. 4~ A. Bach and B. Wilensky, Biochem. Z. 226, 482 (1930). 471H. Theorell, Arkiv Kemi, Mineral. Geol. 16A, No. 14 (1943). 4 7 b H. A. Harbury, Dissertation, Johns Hopkins University, Baltimore, 1953. 48 H. Theorell, Arkiv Kemi, Mineral. Geol. 16A, No. 8 (1942).

810

RESPIRATORY ENZYMES

[143]

TABLE III R A T E AND EQUILIBRIUM CONSTANTS OF ENZYME SUBSTRATE COMPLEXES OF

HRP~ (pH 4.7; 25 to 30 °)

Substrate H202 MeOOH" EtOOH/

k~, b M -~ scc. -~ 0 . 9 X l0 T 1.5 X 106 3.6 X 106

k~ -~ k3, b sec. -~

kT, b,~ sec. -1

K, d M

0.10 0.15 --

3.5 3.9 3.8

10 -s 10 -7 --

B. Chance, Arch. Biochem. 2 2 , 2 2 4 (1949). b See equations 1 to 4 on p. 802. No added donor; varies from preparation to preparation due to endogenous donor. k2 + k3 d Apparent equilibrium constant for complex II; K = k~ ; see equations 1 and 4. M e 0 0 H stands for m e t h y l hydrogen peroxide. J E t 0 0 H stands for ethyl hydrogen peroxide. T A B L E IV VALUES OF k4 FOR THE REACTION OF H R P H20~ COMPLEX I I WITH VARIOUS DONORS a

(at 25 to 30 °) Donor type Phenols

Amines

Enediols

Others

Name p-Hydroxydiphenyl Hydroquinone IIydroquinone m o n o m e t h y l ether Catechol Catechol m o n o m e t h y l ether (guaiacol) Resoreinol Pyrogallol o-Phenylenediamine m-Phenylenediamine Aniline p-Aminobenzoic acid Reductone c Ascorbic acid Dihydroxymaleic acid Uric acid Leucomalachite green DPNH Nitrite

k4, M -1 sec. -1 8 3 2 2 2.5 3 3 5 1 7 1 1 2 2 2 3 3

X )< )< X X X X )< X X X X X X X X X 17

107 106 10 e 106 106 l05 105 107 106 104 103 106 104 104d 104 l0 s 10 ~

pH 7.0 7.0 7.0 7.0 6.7 b 7.0 6.7 b 7.0 7.0 7.0 7.0 4.2 4.7 4.0 7.0 4.7 7.0 7.0

B. Chance, in " T h e E n z y m e s " (J. B. Sumner and K. Myrbi~ck, eds.), Vol. 2, P a r t 1, p. 428, Academic Press, New York, 1951. b B. Chance, Arch. Biochem. 24, 410 (1949). c H. yon Euler and C. Martius, Svensk Kern. Tidskr. 45, 73 (1933). d Measured at 4 ° b y B. Chance, J . Biol. Chem. 197, 577 (1952).

[143]

PLANT PEROXIDASE

811

The following amino acids have also been shown to be present in H R P : 49 a l a n i n e , g l y c i n e , l e u c i n e , m e t h i o n i n e , p h e n y l a l a n i n e , p r o l i n e , s e r i n e , t h r e o n i n e , t y r o s i n e , v a l i n e , a s p a r t i c , g l u t a m i c , a n d c y s t e i c a c i d s . 5° Spectroscopic, Magnetic, and Kinetic Data on HRP and Its Derivatives. The spectroscopic and kinetic data on the enzyme substrate comp o u n d s a n d d e r i v a t i v e s of H R P a r e s u m m a r i z e d i n T a b l e s I I I t o V I . T h e m a g n e t i c s u s c e p t i b i l i t y of m a n y of t h e H R P c o m p o u n d s h a v e b e e n TABLE V THE SPECTRA OF THE ENZYME SUBSTRATE COMPLEXES OF H R P (wavelengths in m~) Substrate H~02 H~O~ H20~ H202 H20~ H20~ H20~ MeOOH~ MeOOH MeOOH EtOOH z

Compound I II II II III IIIf III I II IV I-IV

pH

X

e~

Ref.

k

~-

7.0 410 48 b --5.4 419 91 b . . . 7.0 418 95 b 527 8.5 9.0 418 93 b . . . 5.4 418 102 b . . . 7.0 416 106 b 546 9.9 9.0 416 116 b . . . 7.0 410 48 h i --4.7 419 91 h j 530 ? 7.0 410 ? j 557 k ? T h e spectra of the E t O O H c o m p l e x e s are of the M e O O H c o m p l e x e s . b

k

e~

Ref.

657 c ? d . 558 8 . 5 ~ b . . 583 8.6 d . 660 ? i 560 ? i 675 ? i the same as those

a T h e new value for the extinction coefficient of pyridine hemochromogen at 557 m~ found b y Paul et al. (see Table II, footnote a) was used for calculating the extinction coefficients in this a n d the following table. Theorell's* former value or H R P at 403 m~ becomes thus e~03 = 89.5 cm. -1 X m M -~. Keilin a n d Hartree ~ found e4o3 = 91.0. * H. Theorell, Enzymologia 10, 250 (1942). b B. Chance, Arch. Biochem. and Biophys. 41, 404 (1952). c Measured at a t e m p e r a t u r e of 4 °. d D. Keilin and E. F. Hartree, Biochem. J. 49, 88 (1951). ' Keilin a n d Hartree d found a lower E for this b a n d . f Complex I I I is best obtained from complex I I direetly, r a t h e r t h a n from the free enzyme. g M e O O H stands for m e t h y l hydrogen peroxide. h Recalculated according to the d a t a of B. C h a n c e ) B. Chance, Arch. Biochem. 9.1, 416 (1949). i B. Chance, in " T h e E n z y m e s " (J. B. Sumner a n d K. Myrbfiek, eds.), Vol. 2, P a r t 1, p. 428, Academic Press, New York, 1951. Weak b a n d . E t O O H stands for ethyl hydrogen peroxide.

49 A. C. Maehly a n d S. Pal6us, Acta Chem. Scan& 4, 508 (1950). 69 Oxidation product from cysteine a n d cystine.

812

RESP~R~ORr E ~ Z ~ E S

%

O~

dtl

I~

[143]

r~. v

%

% L-~

v

~fr

%

i>. •~

%

dll

0

Z 0 r~ 0

II

II

It

il

o

•F~~r2

,'-, X

XX

X

~

~g

~ Z ~

r~

M4

-~ ~ ~ ~4~m

~

©

o

t.

~ i ~ •

0

~. ~

°

[144]

LACTOPEROXIDASE

813

remeasured recently by Theorell and Ehrenberg, 51 using improved techniques of high sensitivity. A summary of the presently available data can be found in a paper by Chance and Fergusson. 5~ 61H. Theorell and A. Ehrenberg, Arch. Biochem. and Biophys. 41, 442 (1952). 52 B. Chance and R. R. Fergusson, in "The Mechanism of Enzyme Action" (W. D. McElroy and B. Glass, eds.), p. 389, The Johns Hopkins Press, Baltimore, 1954.

[144] L a c t o p e r o x i d a s e

By B. DAVID POLIS and H. W. SHMUKLER Assay Method The peroxidase concentrations of turbid milk fractions can be determined with a modification 1 of the purpurogallin test of Sumner and Gjessing. ~ With homogeneous solutions of the enzyme, a more convenient method results from the oxidation of dihydroxyphenylalanine (dopa) by peroxidase 1 and H~.O~ to a red derivative. Reactions are carried out in 1-cm. square cuvettes holding 3 ml. of solution. The reaction mixture is composed of 0.08 M phosphate buffer, pH 7.0, 16.67 × 10-4 M dopa, 2 X 10-4 M H202, and a suitable dilution of the enzyme. When the dopa is added as the last reagent, a zero-order reaction is obtained that can be determined by taking absorbency readings every 15 seconds at 475 m~ for 2 minutes. By adjusting the concentration of the lactoperoxidase to catalyze the utilization of 10% of the hydrogen peroxide per minute, the velocity constant obtained for the zero-order reaction may be expressed directly in terms of the turnover number of moles of hydrogen peroxide per milligram or mole of lactoperoxidase per minute. Protein concentrations may be determined by the optical density at 280 m~ or by the biuret reaction. A good approximation to the specific activity of the lactoperoxidase preparation is given by the ratio of the optical densities at wavelengths 412 and 280 m~.

Isolation Procedure The commercial skim milk used for the isolation of lactoperoxidase was processed most conveniently in 50-1. batches. A. The casein of 50 1. of milk is coagulated with rennet at room temperature and separated from the whey by straining through muslin bags. B. Salt Fractionation. (1) The whey protein is precipitated by the adB. D. Polis and H. Shmukler, J. Biol. Chem. 201, 475 (1953). J. B. Sumner and E. C. Gjessing, Arch. Biochem. 2, 291 (1943).

814

RESPIRATORY ENZYMES

[144]

dition of solid ammonium sulfate to a final concentration of 2.8 M, p H 6.0, and filtered overnight at 3 °. (2) T h e precipitate is redissolved in water to a protein concentration of 3%. Sodium t e t r a b o r a t e is added to 0.1 M, ammonium sulfate to 1.5 M, and the precipitate is filtered off. 3 (3) T h e salt concentration of the 1.5 M filtrate is increased to 1.9 M. The precipitate is filtered at 3 ° . (4) All the peroxidase in the 1.9 M filtrate is then precipitated at 2.5 M a m m o n i u m sulfate. (5) T h e precipitate of step 4 is redissolved in 0.1 M borax solution to a 2 % protein concentration, and ammonium sulfate is added to 1.9 M. The 1.9 M salt filtrate is then brought to 2.3 M. After standing for 1 hour at room temperature, a brown-green, sticky cake forms t h a t floats on the surface of the salt solution. This is filtered through glass wool, and the excess ammonium sulfate solution is removed from the precipitate b y kneading it into a ball. C. Chromatography. The ionic composition of the lactoperoxidase obtained b y salt fractionation is best controlled for c h r o m a t o g r a p h y b y adjusting the p H to neutrality with 0.5 M KH~P04 and then dialyzing against 20 col. of water for 2 hours with continuous stirring of the peroxidase within the membrane. In this way the ammonium sulfate concentration of the crude lactoperoxidase is reduced to about 0.02 M with only 10% loss in enzyme activity. The peroxidase is then diluted to a final concentration of 2 % protein in 0.1 M K2HP04, p H 9. C h r o m a t o g r a p h y is accomplished first on columns of calcium phosphate to about half-maximal purity. Final purification is attained with silica-Celite columns. 1. CALCIUM PHOSPHATE CHROMATOGRAPHY. Glass columns, 50 × 4 cm., are packed with 30 to 40 g. of calcium phosphate 4 contained between layers of glass wool on stainless steel screens. T h e columns must be packed in a m a n n e r t h a t prevents channeling and y e t permits swelling of the calcium phosphate particles without forming an impermeable block. This is accomplished b y tamping successive layers of a b o u t 10 g. of the adsorbent at a time to an approximate height of 4 cm. and then wetting the columns with 50 ml. of water. Flow rates of 1 to 3 ml./min. 3 See A. A. Green and W. L. Hughes, Vol. I [10], for calculation of (NH4)~SO4saturation. 4Stoichiometric concentrations of calcium chloride and disodium acid phosphate are combined in solutions made alkaline to phenolphthalein with ammonium hydroxide. The gel is washed free of chloride by decantation over a period of '~l~veek,~filtered, dried overnight at 70°, then ground in a mill to a powder fine enough to pass through a 90-mesh sieve.

[144]

LACTOPEROXIDASE

815

can then be maintained through these columns at pressures of 50 to 80 cm. of mercury. In the first chromatographic purification step, a 2 % protein solution in 0.1 M K2HPO4, pH 9, is filtered through successive 30-g. columns of calcium phosphate under such conditions that the peroxidase containing filtrate from one column is refiltered through a second column until the efituent contains no peroxidase. About three-quarters of the total protein passes through the columns and appears in the final filtrate. The columns are then washed with 0.1 M sodium tetraborate solution until the borate filtrate is protein-free. Subsequent elution of the columns with 0.5 M phosphate yields an effluent with a 25-fold increase in peroxidase purity. For further purification, the 0.5 M effluent is diluted to 0.1 M salt and 0.2 % protein concentration and again filtered through calcium phosphate columns. In this step, half the number of columns of the previous step is used. After the columns have been washed with borate, the peroxidase is again eluted with 0.5 M K:HPO4 to yield a fraction with hail maximal purity. 2. SILICA-CELITE CHROMATOGRAPHY. Final purification is accomplished by chromatography on silica-Celite columns. The combined eluares from the calcium phosphate columns are diluted to 0.1 M phosphate and passed through a column composed of a mixture of 2 parts of silicic acid to 1 part of Celite 5 at pressures of 50 to 80 cm. of mercury. With a ratio of 20 mg. of protein to 1 g. of adsorbent, approximately threequarters the length of a 30-g. column is saturated with enzyme. Both the lactoperoxidase and a red protein are adsorbed from 0.1 M solutions of K2HPO4. In contrast to the behavior on calcium phosphate, the red protein is held more firmly than the lactoperoxidase on silica-Celite, and as a result the lactoperoxidase is preferentially displaced with 0.5 M phosphate. The red protein remaining is eluted from the column with 1 M K2HP04, and the column may be regenerated by displacing the excess phosphate with water. Spectrophotometric analysis of the effluent aliquots permits the combination of the effluent from the column into fractions of comparable purity. Chromatography of the subfraction combinations concentrates the lactoperoxidase through steps of graded purity into the 0.5 M eluate with a ratio of optical densities D412/D28o equal to 0.9. D. Crystallization. The purified lactoperoxidase is obtained from the silica-Celite columns as a reddish-black solution in 0.5 M K:HPO4. This is precipitated completely by the addition of 4 M K2HPO4 at room tem5 Merck's reagent-grade silicic acid and Johns-Manville Celite analytical filter aid gave reproducible results without any special preparation.

816

RESPIRATORY ENZYMES

[144]

perature to a final concentration of 2.5 M and is filtered off with suction with the aid of 0.5 g. of Celite (analytical filter aid grade) per 10 ml. of solution. T h e enzyme then is eluted with 1 M K2HPO4, and sufficient 4 M K~HP04 is added to form a slight turbidity (2.2 M.). Approxim a t e l y 0.2 g. of Celite per 10 ml. of solution is added, and the precipitate is filtered off. The filtrate is then stored in a beaker covered with filter paper at 5 °. Crystals form after a few weeks. The crystalline enzyme is stable in the concentrated phosphate solution for over a year. SUMMARY OF ISOLATION PROCEDURE FROM

100 L. OF SKIM

MILK a

Specific activity

Fraction

Protein, Pyrogallol g. testb

Whey 715 2.3 M (NH4)2SO4 ppt. 207 0.5 M K~HPO4 eluate from Ca3(P04)~ First eluate 4.4 Second eluate 1.6 0.5 M K~HP04 eluate from sflica-Celite Fourth eluate 0.5 Crystallization from 2.2 M K~HP04 Second crop 0.25

D~12 Lactoperoxidase

D2s~-o yield, mg.

0. 049 1.09

2870 1850

58

0.19 0.42

974 764

122

0.9

500

122

0.9

250

27

B. D. Polis and H. Shmukler, J. Biol. Chem. 201,475 (1953). b Specific activity by the pyrogallol test is defined arbitrarily as the milligrams of purpurogallin formed in 20 seconds by 1 mg. of protein.

Properties In the presence of hydrogen peroxide, lactoperoxidase catalyses the oxidation of m a n y phenols and aromatic amines. In this respect, its behavior is similar to horseradish peroxidase, 6 with the exception t h a t pure lactoperoxidase has no apparent action on tyrosine. T h e enzyme binds phosphate strongly, causing a shift in the isoelectric point and also producing a threefold activation of dopa peroxidation. T h e reaction with dopa is especially interesting, since a competitive inhibition of the dopa oxidation is produced b y increasing concentrations of H20:. Lactoperoxidase exists in two forms t h a t have been designated A and B, differing primarily in electrophoretic mobility, spectrophotometric constants, and the rate of reaction with dopa. Lactoperoxidase B, obtained primarily from spring milk, is similar to the enzyme prepared 6j. B. Sumner and G. F. Somers, "Chemistry and Methods of Enzymes," 3rd ed., Academic Press, New York, 1952,

[145]

PLANT TYROSINASE (POLYPIIENOL OXIDASE)

817

by Theorell. 7.8 Crystalline lactoperoxidase A is a heme-protein containing 0.069% iron and 15.56% nitrogen. Its molecular weight is 82,000. The enzyme is isoelectric in 0.1-~ Veronal buffer at pH 8.0, in 0.1-~ phosphate buffer at pH 6.8. It is isoionic at pH 9.6. For pure preparations, the optical densities at 280 and 412 m~ are 1.541 and 1.390 at a concentration of 1.0 g./1. With dopa as a substrate, the turnover number of lactoperoxidase A (D412/D2so = 0.89) was 1500 moles of H~O~ per minute per mole of enzyme. The turnover number of lactoperoxidase B, (D412/D~so = 0.77) was 1013. 7 H. Theorell and ~. ,~keson, Arkiv Kemi, Mineral. Geol. 17B, No. 7, p. 1 (1943). H. Theorell and K. G. Paul, Arkiv Kemi, Mineral. Geol. 18A, No. 12, p. 10 (1944).

[145] P l a n t Tyrosinase (Polyphenol Oxidase)

By CHARLES 1%. DAWSON and RICHARD J. MAGEE Cresolase and Catecholase Activities The darkening of mushrooms, potatoes, apples, and many other plants and plant products on injury to the tissue is the result of the enzymatic oxidation of certain mo/aohydric and o-dihydric phenols. The enzyme responsible for these oxidations is called tyrosinase, for the phenolic amino acid, tyrosine, was the first experimental substrate. The purified enzyme is commonly prepared from the edible mushroom, PsaUiota campestris, and p-cresol and catechol have been most frequently employed as experimental substrates. Consequently, the two activities of the mushroom enzyme have come to be known as the "cresolase" and "catecholase" activities. The ratio of catecholase to cresolase activities (cat/cre ratio), as measured by the assay methods described below, is about 5:1 in crude extracts of the mushroom. In the course of purification this ratio is usually increased greatly in favor of the catecholase activity. Such "high catecholase" preparations of tyrosinase often contain little or no cresolase activity and appear to be very similar to the "polyphenoloxidase" and "catechol oxidase" described in the literature.l.~ It is also possible to prepare from the same starting material a purified enzyme possessing a cat/cre ratio comparable to or even lower than that of the original crude extract; such preparations have been termed "high cresolase" preparations of tyrosinase. No preparation has yet been obtained which possesses only cresolase activity. D. Keilin and T. Mann, Proc. Roy. Soc. (London) B125, 187 (1938). z F. Kubowitz, Biochem. Z. 292, 221 (1937).

818

RESPmXWORY ENZYMES

[145]

Assay of Cresolase Activity Principle. The method for measuring cresolase activity depends on the fact that, within a certain range of enzyme concentration, the rate of oxygen consumption during the oxidation of p-cresol is proportional to the amount of enzyme present. It must be noted that the oxidation of monophenols by tyrosinase does not set in immediately on mixing of the enzyme and substrate but is characterized by an induction period, the length of which may vary with the source and purity of the enzyme and with the presence of oxidizing or reducing agents2 An advantage of the assay method 4 described here is the fact that usually the induction phase of the reaction is over by the time the first reading is taken. Reagents p-Cresol solution (0.037 M). Dissolve 100 mg. of redistilled p-cresol in 25 ml. of water. Such solutions are sufficiently stable at room temperature to be used over a period of several weeks. 0.2 M Na2HP04--0.1 M citric acid buffer, pH 7.0. Gelatin solution. Add 750 mg. of gelatin to 150 ml. of water, and warm with stirring until the gelatin has dissolved. Add one crystal of thymol as preservative. Store at 5 °. This solution should be freshly made every week. Enzyme. Dilute the cold stock solution of enzyme with ice-cold water to obtain a dilution containing between 1 and 2.5 units of cresolase activity per milliliter. (See definition below.) Procedure. Manometers of the Warburg type are used with respirometer flasks of about 40-ml. volume. Prior to diluting the enzyme, prepare the reaction mixture by adding to each of the flasks 4.0 ml. of buffer, 1.0 ml. of p-cresol solution, 1.0 ml. of gelatin solution, and 3.0 ml. of water. Then dilute the enzyme, and add immediately 1.00-ml. aliquots of the enzyme dilution to the reaction mixtures. Attach the flasks at once to the manometers with the manometer stopcocks open, place in a thermostat at 25 __ 0.01 °, and shake at 120 oscillations per minute. After 10 minutes close the stopcocks and take readings at 5-minute intervals for 20 or 30 minutes, the extent of the period of linear rate. Activity measurements should be run in duplicate, along with a control containing the reaction mixture without enzyme. Definition of Unit and Specific Activity. One unit of cresolase activity is defined as that amount of enzyme which causes an oxygen uptake of 3 C. A. Bordner and J. M. Nelson, J. Am. Chem. Soc. 61, 1507 (1939). 4 M. F. Mallette and C. R. Dawson, J. Am. Chem. Soc. 69j 466 (1947).

PLANT TYROSINASE (POLYPHENOL OXIDASE)

[145]

819

10 ~l. per minute during the linear portion of the reaction. 5 Specific activity is expressed as units per milligram of dry weight. Dry weights are determined by the method of Mallette et al. e A useful spectrophotometric method is also available. ~

Assay of Catecholase Activity Principle. Although several useful colorimetric methods for the determination of eatecholase activity have been described, 1,8 the chronometric method,9 described below, is used in the writer's laboratory because it is less complicated by the possible side reactions of o-benzoquinone and because it permits the accurate evaluation of the rate of enzymatic oxidation of catechol during the period 15 to 150 seconds after the start of the reaction. Because the initial reaction course can be determined with greater precision, the chronometric method is superior to earlier manometric procedures. 5 The method involves several measurements of the time required for a given quantity of enzyme to produce from catechol certain amounts of o-benzoquinone. The amount of o-benzoquinone involved is that which is equivalent to a known amount of added ascorbic acid. As long as the ascorbic acid is present, no o-benzoquinone exists in the system (it is reduced as rapidly as formed). The end point, which is the first appearance of o-benzoquinone as indicated by a starch-iodide indicator, corresponds to the depletion of the ascorbie acid. If the measurement is run a number of times using different known amounts of ascorbic acid with a constant amount of enzyme and catechol, a series of end points is obtained which measures the production of o-benzoquinone as a function of time for that amount of enzyme. Reagents

H20 should be doubly distilled from an all-Pyrex apparatus to reduce copper content to less than 0.05 ~,/ml. Ascorbie acid solution (0.0057 M). Dissolve 100 rag. of ascorbic acid in 100 ml. of water containing 100 mg. of metaphosphoric acid (HPOa). This solution should be freshly made on the day of using. Catechol solution (0.182 M). Dissolve 1.00 g. of catechol in 50 ml. 5 M. Graubard and J. M. Nelson, J. Biol. Chem. 111, 757 (1935). 6 M. F. Mallette, S. Lewis, S. R. Ames, J. M. Nelson, and C. R. Dawson, Arch. Biochem. 16, 283 (1948). 7I. Z. Eiger and C. R. Dawson, Arch. Biochem. 21, 181 (1949). 8 j. D. Ponting and M. A. Joslyn, Arch. Biochem. 19, 47 (1948). W. H. Miller, M. F. Mallette, L. J. Roth, and C. R. Dawson, J. Am. Chem. Soc. 66, 514 (1944).

820

RESPIRATORY ENZYMES

[145]

of water. This solution should be freshly made on the day of using. 0.4 M Na2HP04--0.2 M citric acid buffer, pH 5.1. Starch-iodide indicator solution. To a mixture of 25 ml. of 10 % K I solution and 25 ml. of 2 N H~S04, add 0.5 g. of pyrogallol and 5 ml. of a 1% starch solution. This indicator solution can be used for several determinations. Enzyme. Dilute the cold stock solution of enzyme with ice-cold water so that 1 ml. of the dilution contains an amount of enzyme that will produce the end point in 40 to 60 seconds when 50 mg. of catechol and 3 mg. of ascorbic acid are used as described under Procedure. This dilution, which contains an enzyme concentration of about 30 units/ml., is kept in a small ice bath until the activity measurement is completed. Procedure. A 300-ml. round-bottom three-neck flask is clamped in a 25 ° thermostat. In one neck of the flask is placed a rubber stopper holding a glass capillary tube bent as a siphon so that through it the reaction mixture may be sampled dropwise (about 2 drops per second) into the starch-iodide indicator solution (see Fig. 1). Another neck of the flask

A,r

/Capillary siphon

s ' rer

thermostat

~ ~-- ~ ~ s~Starch'i°dide /t / ~=-*~ solution J ~/~Lamp

Fro. 1. A p p a r a t u s used in t h e chronometric m e t h o d for t h e m e a s u r e m e n t of catecholase activity.

contains a rubber stopper holding a glass tube through which air is bubbled to stir the contents of the flask at a medium rate. The center neck of the flask is open and is used for introducing the reagents which (except for the enzyme) are held until needed in separate flasks immersed in the same thermostat. The starch-iodide indicator solution is placed in a crystallizing dish illuminated from beneath through a white opalescent glass and is located close to the thermostat. This indicator solution should be mechanically stirred with a glass stirrer at a slow, constant rate.

[145]

PLANT TYROSINASE (POLYPHENOL OXIDASE)

821

The first step is the preparation of the proper enzyme dilution and the determination of the optimum concentration of catechol to be used. In the flask are placed 10 ml. of buffer and enough water so that the total volume will be 100 ml. after all the reagents have been added. The enzyme is diluted, and 1.0 ml. of the dilution is added to the reaction mixture through a small funnel, followed by 3 ml. of ascorbic acid solution and a known volume of rinse water. The siphon tube is then adjusted in position and the air bubbler inserted, the center neck of the flask being left open. The reaction is initiated by rapidly introducing 2.5 ml. of catechol solution from a small Erlenmeyer flask, and simultaneously the stopwatch is started. As soon as possible the siphon is put into operation by momentarily stoppering the center neck. The end point is indicated by the first fleeting appearance of a blue color at the point where the reaction mixture drops into the starch-iodide indicator solution. If the reaction time does not fall close to the desired 40 to 60 seconds, the enzyme should be rediluted. Once the proper dilution is obtained, the end point determination is repeated three or four times using the same amounts of enzyme and ascorbic acid but varying the catechol concentration until the quantity giving the shortest reaction time has been determined. Purified high catecholase preparations generally have an optimum catechol concentration of about 20 mg. per 100 ml., whereas crude preparations and high cresolase preparations generally show maximum activity at about 100 mg. of catechol. The second step is the actual rate measurement and the calculation of the enzyme activity. A reaction rate curve is obtained using the optimum amounts of enzyme and catechol and varying amounts of ascorbic acid, usually from 1 to 4 mg., so that the end points lie in the range of 20 to 100 seconds. The reciprocal of the milligrams of ascorbic acid used is then plotted against the reciprocal of the reaction time in seconds. The slope of this straight line is divided by 2.62 X 10-8 to give the "units of catecholase activity" present in the reaction. A utilization of 2.62 X 10-3 mole per second of ascorbic acid corresponds to the production of o-benzoquinone at the rate of 1.49 )< 10-s mole per second. Definition of Unit and Specific Activity. Prior to the development of the chronometric method, a unit of catecholase activity was defined as that amount of enzyme which causes an oxygen uptake of 10 ~1. per minute. TM This rate of oxygen uptake is equivalent to an o-benzoquinone production in the chronometric method of 1.49 X 10-8 mole per second. Specific activities are expressed as units per milligram of dry weight (see assay of cresolase activity). io D. C. Gregg and J. M. Nelson, J. Am. Chem. Soc. 62~ 2500 (1940).

822

RESPIRATORY ENZYMES

[145:

Purification P r o c e d u r e T h e procedure described below has been developed and used in the writer's l a b o r a t o r y during m a n y years of investigation on this e n z y m e I n this procedure the enzyme usually undergoes a b o u t a 15-fold increase in purity, and the final product, useful for most purposes, is a b o u t 30 ~( pure as judged b y the criteria of Mallette and Dawson. n As shown by the d a t a in Table I, the procedure through step 7 yields a high catechol. ase enzyme (cat/cre ratio 30 to 40). Suggestions for the preparation oJ high cresolase tyrosinase are given after step 7. TABLE I SUMMARY OF PURIFICATION PROCEDURE a

Fraction

Total Specific Units/ml., units, activity, Total thousands thousands Dry units/mg. volume, weight, Cat/ere ml. Cat Cre Cat Cre mg./ml. Cat Cre ratio

1. H20 extract 19,000 0.33 0.03 2. After acetone fractionation 5,200 1.07 0.13 3. (NH4),.SO, fraction 2,000 2.96 0.23 4. Dialyzed enzyme, after alumina treatment 2,400 1.26 0.07 5. After lead subacetate treatment 2,400 0.53 0.015 6. Eluate from alumina 210 8.00 - 7. Second lead subacetate fraction 55 7.60 0.22

6260 570 2.23

148 14.3

10

5570 675 3.85 5920 460 5.97

304 34.3 497 37.7

9 13

3020 168 3.68

343 19.0

18

1270 7.6 1680 - -

1.00 5.67

525 14.5 1410 - -

36 --

418 12.3

7.02

1080b 32

34

a Typical data taken from I. Z. Eiger and C. R. Dawson, Arch. Biochem. 21~ 181 (1949). b The decrease in specific activity from step 6 to 7 reported here is exceptional There is often a twofold improvement in specific activity at this stage. Procedures which yield essentially pure high catecholase and higI cresolase tyrosinase preparations have also been developed, b u t thest methods are considerably more laborious. T h e y have been described ii detail elsewhere. 6 Step 1. Preparation of Crude Extract. T h i r t y pounds (ten baskets) o: the c o m m o n mushroom, Psalliota campestris, is passed through a m e a grinder into 50 1. of acetone which has previously been chilled with dr~ ~ M. F. Mallette and C. R. Dawson, Arch. Biochem. 23, 29 (1949).

[145]

VLANT 'rYROSINASE (POLYPHENOL OXIDASE)

823

ice. T h e suspended pulp is collected b y suction filtration on filter p a p e r and pressed dry in a hydraulic press. T h e pressed pulp, which weighs 3 to 4 pounds, is frozen b y being placed in contact with d r y ice for at least 4 hours. I t m a y be stored in this frozen condition for several months, if desired. T h e frozen pulp is then broken up, suspended in a b o u t 20 1. of water, and allowed to stand overnight in the refrigerator a t a b o u t 5 ° in order to extract the enzyme. The suspension is filtered through cheesecloth, and the pulp wrapped in chain cloth and squeezed d r y under hydraulic pressure. Step ~. Fractionation with Acetone. An a m o u n t of acetone 1.5 times the volume of the filtrate from step 1 is added, and the resulting precipit a t e is filtered on Celite. 1~ The precipitate and Celite pad are suspended and stirred in 6 1. of cold water (5 °) to redissolve the protein. T h e solution is then refiltered through another Celite pad. Step 3. Fractionation with Ammonium Sulfate. Sufficient solid (NH4)~SO4 is dissolved in the cold solution from step 2 to m a k e the solution 0.35 M (0.6 saturation) in (NH~)2SO4. After stirring for a few minutes, the mixture is filtered on Celite, and the precipitated protein on the pad is washed with more cold 0.35 M (NH4)~S04. The precipitated protein is then redissolved b y stirring in a b o u t 2.4 1. of w a t e r as in step 2. Step 4. Partial Removal of Color with Alumina Gel. T o the solution from step 3 is added one-tenth its volume of a suspension of alumina gel. 18 After thorough mixing, the alumina and the adsorbed material, consisting mainly of dark-colored pigments, are filtered on Celite and discarded. T h e filtrate is dialyzed overnight against cold running w a t e r (15 to 20 hours). ~2Filtrations employing filter aids such as Celite (Johns-Manville No. 535) or infusorial earth (Fisher Scientific Co., white calcined powder) are carried out with suction in a Bfichner funnel using a quarter-inch pad of the filter aid on the filter paper. 13Alumina gel reagent (based on the method of Willst{itter14). Dissolve 170 g. of A12(SO4)3.18H20 in 2 1. of warm H20 (60°) in a 12-1. round-bottom flask, and to the solution add 2 1. of warm H20 containing 50 g. of (NH4)2S04. While swirling the flask, add slowly about 150 ml. of concentrated NH4OtI to produce a heavy precipitate of AI(OH)~. Fill the flask with hot tap water, stir, and, after allowing the precipitate to settle (30 to 40 minutes), add a little more concentrated NH4OH. When no further precipitate forms on the addition of NH4OH, siphon off the clear solution from above the precipitate, and refill the flask with warm H:O. Mix thoroughly and allow to settle. Usually about twelve washings with hot tap water, about four a day, are required to bring the pH of the washings to below 8.0. Then wash the AI(OH)3 with distilled H20 until the washings are at pH 7.0. Finally, make up the suspension to a volume of 2 1. with distilled H20. (Dry weight 0.144 g. per 10 ml. of the final suspension.) 14 R. Willstiitter, "Untersuchungen fiber Enzyme," p. 575. Springer, Berlin, 1928.

824

RESPIRATORY ENZYMES

[145]

Step 5. Further Removal of Color with Lead Subacetate Reagent. Lead subacetate reagent 15 is added dropwise to the dialyzate from step 4 until the solution is definitely cloudy, le Filtration on Celite produces a solution of lighter brown color. T h e precipitate is discarded. Step 6. Adsorption of Tyrosinase to Alumina. T h e filtrate from step 5 is treated with 0.2 its volume of alumina gel reagent. The alumina with the adsorbed crude enzyme is filtered on Celite in as small a funnel as feasible, and the filtrate is discarded. T h e tyrosinase is eluted at room t e m p e r a t u r e b y stirring the filter pad and gel in about 200 ml. of 0.2 M Na2HPO4 at room t e m p e r a t u r e for 30 minutes. T h e eluate is filtered through Celite and dialyzed against running tap water. Step 7. Fractionation with Lead Subacetate. A volume of cold acetone 0.1 times the total volume of the dialyzate is added, followed b y just enough lead subacetate reagent, added dropwise, to produce cloudiness.iS T h e resulting mixture is filtered and the filtrate similarly fractionated two to three more times with lead subacetate. N o acetone is added for these further fractionations, b u t each time just enough of the lead reagent is added to produce cloudiness. Each precipitate is dissolved with stirring in 30 to 60 ml. of 0.2 M NasHPO4 and the resulting solution refiltered on Celite. The filtrates are assayed for catecholase and cresolase activities. Fractions having the maximum activity and the desired c a t / cre ratio are usually combined. Such preparations are dialyzed against doubly distilled (copper-free) water at refrigerator temperature for 3 to 4 days before a copper assay is conducted or the specific activity determined. Preparation of High Cresolase Tyrosinase. The crude extract from step 1 is purified through step 4 as already described. If the resulting solution possesses a c a t / c r e ratio of about 15 or less, it is then subjected to the stepwise (NH4)2SO4 fractionation described below. If, however, the ratio is higher t h a n 15, it is usually helpful to fractionate four or five times at this point with lead acetate and acetone, as described in step 7. The resulting precipitates are dissolved in 0.2 M Na2HPO4, and the activity ratio of each fraction is determined. In this way it is possible to collect and combine the fractions having the lowest c a t / c r e ratios. 1~Lead subacetate reagent. Grind 420 g. of Pb(C~H30~)r3H~O and 140 g. of PbO together in a mortar, and mix with 1400 ml. of H20 in a glass-stoppered bottle. Shake thoroughly, allow to stand for a week at room temperature, and filter. Dilute one volume of the filtrate with 10 vol. of water. 16It is usually advisable to estimate the amount of lead subacetate reagent required by first adding it dropwise to a small aliquot of the dialyzate. Both pigment and enzyme are precipitated by this reagent. Therefore care must be taken not to precipitate too much of the enzyme. If not enough of the reagent is used, a colloidal dispersion results which passes through the filter.

PLANT TYROSINASE (POLYPHENOLOXIDASE)

[145]

825

The combined fractions are then dialyzed against cold water overnight and subjected to the stepwise (NH4)2S04 fractionation described in the next paragraph. The (NH4)2S04 fractionation is performed as follows: 22.5 g. of (NH4)2SO4 is dissolved in each 100 ml. of the dialyzed enzyme solution. Four additional fractions are then precipitated b y each time adding to the filtrate an a m o u n t of (NH4)2S04 equivalent to 7.6 g. for each 100 ml. of the original dialyzed enzyme solution. Each precipitate is collected separately b y filtration on Celite and redissolved in cold water. The cat/cre ratio decreases with increasing (NH4)2S04 concentration.

Properties Constitution of the Enzyme. I t appears likely t h a t mushroom tyrosinase as it exists in nature has a copper content of less than 0.1% and a molecular weight in excess of 200,000. However, highly purified enzymes (judged b y electrophoretic and ultracentrifuge criteria) possess the properties summarized in Table II. TABLE II PROPERTIES OF EXTENSIVELY PURIFIED HIGH CATECHOLASE AND HIGH CRESOLASE TYROSI NASEa

Units/mg. Units/7 Cu Type

Cu,b %

Cat

Cre

Cu in dry Molecular active weight Homoge-weight of frac- Cat/cre neity, active tion, Cat Cre ratio % fraction %

High catecholase 0.206 2130 48 4400 95 48 90-100% 100,000 0.25 High cresolase 0.028 856 536 237 149 1.6 75 -0.036 M. F. Mallette and C. R. Dawson, Arch. Biochem. 23, 29 (1949). b Copper analyses performed by the methods of 0. Warburg and H. A. :Krebs, Biochem. Z. 190, 143 (1927), and S. Ames and C. R. Dawson, Ind. Eng. Chem., Anal. Ed. 17, 249 (1945). The Priming Reaction in Monophenolase Action. Addition of small amounts of catechol to the monophenol-tyrosinase system removes the characteristic induction period. Oxidizing agents, such as potassium ferricyanide or laccase, greatly lengthen the induction period, whereas reducing agents such as ascorbic acid markedly shorten it. A theory of monophenolase action consistent with these facts has been proposed2 ,17 Specificity. Tyrosinase is the only enzyme known to catalyze the direct aerobic oxidation of monophenols. M a n y monophenols have been iv j M. Nelson and C. R. Dawson, Advances in Enzymol. 4, 99 (1944).

826

RESPIRATORY ENZYMES

[145]

investigated and found to be oxidized by this enzyme. Among those most commonly studied are tyrosine, phenol, p-cresol, 3,4-dimethylphenol, and 4-t-butylphenol. The corresponding o-dihydric phenols are also commonly used as experimental substrates. In addition, the enzymatic oxidation of adrenaline, pyrogallol, and numerous other substituted catechols 18 has been investigated. The tyrosinase oxidation of certain high molecular weight substrates, such as proteins 19 and tea tannins, has been reported. Inhibitors. Substances known to complex with copper, such as potassium cyanide, diethyldithiocarbamate, hydrogen sulfide, carbon monoxide, potassium ethyl xanthate, sodium azide, salicylaldoxime, p-aminobenzoic acid, sulfathiazole, thiouracils, thioureas, cysteine, glutathione, and BAL inhibit the enzyme. Certain of these agents have been found to inhibit the monophenolase and o-dihydric phenolase activities to about the same extent. 2° Reagents, such as iodoacetamide, p-chloromercuribenzoate, trivalent arsenic ion, and cupric ion, which react with free sulfhydryl groups do not inhibit polyphenoloxidase. 4-Nitrocatechol and 4-nitrophenol are competitive inhibitors. Stability. As a general rule, the cresolase activity of the enzyme is less stable than the catecholase activity. Conditions which cause protein denaturation (heating to 60 °, vigorous shaking) result in a serious loss of enzyme activities of both kinds, but an increase in cat/cre ratio. Purified tyrosinase solutions containing in excess of 1 mg. of enzyme per milliliter show little loss in activity over a period of several months if they are buffered at pH 7, inoculated with a few drops of toluene as antiseptic, and stored in the refrigerator. Highly diluted enzyme solutions, however, may exhibit a significant loss in activity within 15 to 20 minutes even at 5 °. Reaction Inactivation. One of the most characteristic features of the enzymatic oxidation of catechol by the purified enzyme is the marked inactivation of the enzyme that occurs during the reaction. 21,~2 Reaction inactivation of the enzyme during the oxidation of monophenols is much less pronounced. p H Optima. At pH below 5, tyrosinase rapidly loses activity. For the enzymatic oxidation of catechol the most satisfactory range of pH is 5.5 to 7. There is no marked optimum in this range. The oxidation of p-cresol shows an optimum in the region of pH 6 to 7, dependent to some extent on the state of purity of the enzyme. is M. L. Cushing, J. Am. Chem. Soc. 70, 1184 (1948). 19I. W. Sizer, J. Biol. Chem. 163, 145 (1946). ~0D. C. Gregg and J. M. Nelson, J. Am. Chem. Soc. 62, 2500 (1940). 9~I. Asimovand C. R. Dawson, J. Am. Chem. Soc. 72, 820 (1950). ~ L. L. Ingraham, J. Corse, and B. Makower, J. Am. Chem. Sac. 74, 2623 (1952).

[146]

MAMMALIAN TYROSINASE

827

Substrate Optima. The optimum concentrations of catechol and cresol are dependent on the cat/cre ratio of the tyrosinase preparation under examination, and on the structures of the phenol or catechol being used as substrate.

[146] M a m m a l i a n Tyrosinase

By AARON BUNSEN LERNER

Tyrosinase //%~, /

//

OH

\

//

OH

CH2CHCOOH I NH2

Tyrosine

NH

\\

\\

,~0

~0

C,H2~HCOOH NH2

Dopa

Melanin

Three methods are given for the assay of mammalian tyrosinase. Each has its merits, and the choice of assay depends in part on the type of material available for analysis.

1. Manometric Assay Method 1,2

Principle. The rate of oxygen utilization of dopa-tyrosinase mixtures in the conversion of dopa to melanin is measured with a Warburg type of respirometer in the usual manner. Dopa and not tyrosine is usually chosen as the substrate, because dopa is rapidly oxidized as soon as it comes in contact with oxygen and tyrosinase. With tyrosine, an induction period precedes the onset of the maximal rate of oxidation; however, if there is no objection to waiting for the induction period to be completed, tyrosine can be used as the substrate. Reagents Dihydroxyphenyl-L-alanine (dopa), 1.0 mg./ml. Add 5 mg. of dopa to 5.0 ml. of 0.1 M sodium phosphate buffer at pH 6.8. Heat the A. B. Lerner, T. B. Fitzpatrick, E. Calkins, and W. H. Summerson, J. Biol. Chem. 178, 185 (1949). A. B. Lerner and T. B. Fitzpatrick, Physiol. Rev. 30, 91 (1950).

828

RESPIRATORY ENZYMES

[146]

tube in boiling water, shaking occasionally, until the dopa dissolves--usually 1 to 5 minutes. The buffer should be made with water deionized by means of ion exchange resins or with doubledistilled water. The solution should be prepared freshly each day. 0.1 M sodium phosphate buffer, pH 6.8, is made with ion-free water. Enzyme. Tyrosinase obtained usually from melanomas or pigmented eye tissue diluted with the phosphate buffer so that 2 to 5 units of enzyme will be present in a 3-ml. mixture with dopa.

Procedure. The substrate, 1.0 mg. of dopa in 1 ml. of phosphate buffer, is added from the side arms of 15-ml. Warburg vessels to 2 ml. of the tyrosinase solution after 10 minutes of equilibration of the solutions at 38 °. Oxygen uptake is recorded every 10 minutes and plotted against time. From the slope of the curve, the rate of reaction can be determined. Definition of Unit and Specific Activity. One activity unit is the amount of enzyme required to catalyze the absorption of 1 gl. of oxygen per minute by 1 mg. of substrate when oxidation is proceeding at a maximal rate. 2. H i s t o c h e m i c a l A s s a y M e t h o d 3,4

Principle. Incubation of tyrosine or dopa with properly prepared fresh tissue containing melanocytes results in the formation and deposition of melanin granules in the cytoplasm of the melanocyte at the site of tyrosinase action in the cell. The amount of melanin granule formation visualized under the microscope gives an approximation of the quantity of enzyme present. The formation of melanin granules from dopa as the substrate is usually but not always specific, because dopa can be oxidized to melanin not only in the presence of tyrosinase but also in the presence of active oxidizing systems such as the cytochrome one. With tyrosine as substrate the reaction is specific, because only tyrosinase will catalyze the oxidation of tyrosine to dopa. The dopa in turn is oxidized and polymerized to melanin. Reagents L-Tyrosine or L-dopa (1.0 mg./ml.) in 0.1 M sodium phosphate buffer, pH 6.8. As described previously in the manometric assay method, tyrosine or dopa is dissolved in phosphate buffer made with ion free water. 0.1 M sodium phosphate buffer, pH 6.8, made with ion-free water. s T. B. Fitzpatrick, S. W. Becker, A. B. Lerner, and H. Montgomery, Science 112, 223 (1950).~ 4 A. B. Lerner,'and-T."B. Fitzpatrick, "Pigment Cell Growth," p. 319, Academic Press, New York, 1953.

[146]

MAMMALIAN TYROSINASE

829

Enzyme. Fresh tissue containing tyrosinase, such as melanomas, skin, or ciliary bodies, is cut into small pieces and placed in 5 to 30 ml. of buffer. Procedure. Fresh tissue is cut into slices 1 to 3 ram. thick and placed in a 5 % solution of formalin for 30 minutes at 5 °. The fixed tissue slices are washed once in 0.1 M phosphate buffer and placed in 5 to 30 ml. of freshly prepared tyrosine or dopa solution and allowed to remain for 12 to 15 hours at 5 °. The tissue slices are then reimmersed in fresh tyrosine or dopa solution and left in an incubator at 37 ° for 24 hours. As a control, one or two slices are treated just as described except that phosphate buffer without added tyrosine or dopa is used. Gross examination of the tissue slices at the end of the final incubation may reveal a darkening of the specimens incubated in tyrosine or dopa solutions but no color change in the controls. To prepare the slices for histologic examination, further fixation is done by immersing them in a 10% solution of formalin for 3 hours. Then they are dehydrated, cleared in toluene, embedded in paraffin, sectioned at 15 ~, and counterstained. Comparison of the amount of melanin granule formation in the cytoplasm of the melanocytes in the tissues incubated with tyrosine or dopa versus the controls give an approximation of the tyrosinase present. In heavily pigmented tissue, this histochemical method is of no value in estimating the presence of tyrosinase because usually it is not possible to differentiate between preformed melanin and that resulting from the incubation. For pigmented tissues, the following radioactive tyrosine method is used. 3. Radioactive Tyrosine Assay Method 4.5 Principle. Carbon-14-1abeled tyrosine on incubation with fresh tissue containing melanocytes is converted to radioactive melanin. By determining the quantity of melanin formed with a Geiger counter, an estimation is gained of the quantity of tyrosinase present. Reagents

C14-Labeled DL- or L-tyrosine (1.0 mg./1 ml.) in 0.1 M sodium phosphate buffer, pH 6.8, is prepared as described previously. Although DL-tyrosine can be used, it should be kept in mind that only the L-tyrosine will be converted to melanin. L-Tyrosine is readily obtained from the DL mixture, e 0.1 M sodium phosphate buffer, pH 6.8. Enzyme. Small pieces of tissue (5 to 50 mg.) are placed in a cold 5T. B. Fitzpatrick and A. Kukita, to be published. 6A. B. Lerner, J. Biol. Chem. 181, 281 (1949).

830

RESPIRATORY ENZYMES

[146]

mortar at 5 ° and by direct downward pressure are mashed with a cold pestle. The crushed moist tissue is placed on a slide exposed to air. It becomes dry in 3 to 5 minutes. The slide is immersed in 70% alcohol for 3 minutes, then removed from the solution and allowed to dry in air. Procedure. Each slide is incubated with 100 ~, of radioactive DL-tyrosine for 24 hours in the presence of 1000 units of penicillin. It is washed with running cold tap water for 5 hours, dried, and measured for radioactivity with a Geiger counter. Control slides show no radioactivity because only tyrosinase converts tyrosine to the insoluble product, melanin. It seems that because of either a lack of cofactors or inactivation during drying other enzymic processes which metabolize tyrosine are not functioning. As yet, no unit of activity has been proposed for results obtained by this method. The results are expressed in terms of number of counts per microgram of tissue on a dry-weight basis. Choice of Method and Properties of Tyrosinase. The choice of method of analysis depends on the concentration of enzyme available, the type of tissue to be analyzed, and the technical facilities at hand. When large quanti Lies of tyrosinase are available, as would be the case with a malignant melanoma or ciliary bodies, the manometric method is simple and rapid. The histochemical method is good for melanomas and skin when an approximation of tyrosinase activity within the cell is desired. The radioactive procedure is by far the most sensitive. Although localization of activity cannot be seen as in the histochemical procedure, it is excellent when working with small quantities of tissue and is essential for studying pigmented tissue. Mammalian tyrosinase is a relatively stable enzyme which requires copper for activity. It is located in the cytoplasm of melanocytes. In the conversion of tyrosine to melanin approximately five atoms of oxygen are used. Tyrosinase from different sources varies in activity. 7 The enzyme prepared from plants is obtained in colloidal solution. However, tyrosinase from mammalian tissue is retained on cytoplasmic particles. Plant tyrosinase is less specific in its action than mammalian tyrosinase. Some plant tyrosinases catalyze the oxidation of many phenol derivatives and o-dihydroxyphenyl compounds at a greater rate than the oxidation of tyrosine and dopa. With mammalian tyrosinase, tyrosine and dopa are oxidized at a much greater rate than any other substance structurally related to these amino acids. D-Tyrosine is not oxidized in the presence of mammalian tyrosinase. Unlike plant tyrosinase, mammalian tyrosin-

A. B. Lerner, Advances in Enzymol. 14~ 73 (1953).

[147]

ASCORBIC ACID OXIDASE

831

ase is not inactivated during the enzymic oxidation of a suitable substrate. Tyrosinase from grasshopper eggs occurs as a protyrosinase and must first be activated before it can exert any catalytic action on tyrosine or related derivatives. Usual activating agents are distilled water, sodium chloride, detergents, or changes in pH or temperature. Tyrosinase in human skin must also be activated before it can readily catalyze the oxidation of tyrosine to melanin. Irradiation of skin with ultraviolet light before excision can activate the enzyme. However, tyrosinase obtained from malignant melanomas appears to exist in an active state and requires no special treatment in order to function at maximal rate.

[147] Ascorbic Acid O x i d a s e Ascorbic Acid + ~/~O2~ Dehydroascorbic Acid -4- H~O B y CHARLES R. DAWSON and RICHARD J. ]VIAGEE

Assay Method Principle. The method 1,~ most generally used depends on the fact that, within a certain range of enzyme concentration, the rate of oxygen consumption during the oxidation of L-ascorbic acid is proportional to the amount of enzyme present. Reagents

H~O used in the reaction mixture and in the preparation of all reagents should be doubly distilled to reduce the copper content to less than 0.05 3,/ml. L-Ascorbic acid solution (0.028 M). Dissolve 250 mg. of L-ascorbic acid in 50 ml. of water containing 50 mg. of metaphosphoric acid. This solution should be freshly made every day. 0.2 M Na2HPO4--0.1 M citric acid buffer, pH 5.7. Gelatin solution. Add 750 rag. of gelatin to 150 ml. of water, and warm with stirring until the gelatin has dissolved. Add about 2 rag. of thymol as preservative, and store at 5 °. This solution should be freshly made every week. Enzyme. Dilute the stock solution of enzyme so as to obtain a solution containing between 1 and 2.5 units of enzyme per milliliter. (See definition below.) Usually this is best accomplished 1p. L. Lovett-Janison and J. M. Nelson, J. Am. Chem. Soc. 62, 1409 (1940). W. H. Powers, S. Lewis, and C. R. Dawson, J. Gen. Physiol. 27, 167 (1944).

832

RESPIRATORY ENZYMES

[147]

with a series of dilutions made with ice-cold water. The final dilution is made so as to contain 2 ml. of gelatin solution per 10 ml. of final volume. If a precipitate forms when the first subdilution is made, this solution is discarded, and a fresh dilution is made with ice-cold 0.1 M acetate buffer, pH 5.6.

Procedure. Manometers of the Warburg type are used with respirometer flasks of about 40-ml. volume. Prior to diluting the enzyme, prepare the reaction mixtures by adding to the main compartment of each flask 4.0 ml. of the phosphate-citrate buffer, 1.0 ml. of gelatin solution, 1.0 ml. of L-ascorbie acid solution, and 3.0 ml. of water. Then dilute the enzyme as described above, and add immediately 1.00-ml. aliquots to the side arms of the flasks. Place the flasks in a thermostat at 25 +_ 0.01 °, and equilibrate for 15 minutes with slow shaking. After initiating the reaction by tipping the enzyme from the side arms into the reaction mixtures, take readings every 2 minutes for at least 10 minutes while the flasks are shaking at about 120 oscillations per minute. Take the average of the three most constant consecutive values for the oxygen consumed in 2 minutes, and calculate the rate of oxygen uptake per minute. Activity measurements should be run in duplicate, along with a control containing the reaction mixture without enzyme. Definition of Unit and Specific Activity. One unit of ascorbic acid oxidase activity is defined as that amount of enzyme which causes an initial rate of oxygen uptake of 10 ~l. per minute. Specific activity is expressed as units per milligram of dry weight. Dry weights are determined by the technique of Mallette et al.3 with one modification. When samples of highly purified enzyme (purified at least up to step 5, Purification Procedure) are being measured, these are first dialyzed against cold 0.1 M acetate buffer, pH 5.7. A correction is therefore necessary for the dry weight of the buffer. Purification Procedure

Lovett-Janison and Nelson 1 attempted the preparation of highly purified ascorbic acid oxidase from eleven different plants and found the yellow summer squash, Cucurbita pepo condensa, to be the most satisfactory source. Procedures developed since that time have resulted in the preparation of solutions of enzyme which are homogeneous electrophoretically and in the ultracentrifuge. 2,4 The procedure described below is less time consuming and produces s M. F. Mallette, S. Lewis, S. R. Ames, J. M. Nelson, and C. R. Dawson, Arch. Biochem. 16, 283 (1948). 4 F. J. Dunn and C. R. Dawson, J. Biol. Chem. 189, 485 (1951).

[147]

ASCORBIC ACID OXIDASE

833

in much better yield an enzyme that is about 80 % pure as compared with the properties of homogeneous ascorbic acid oxidase. It has been recently developed in the writers' laboratory with the help of Mr. Stanley Lewis. Step 1. Preparation of Crude Extract. Four hundred pounds (10 bushels) of yellow squash are peeled. The rinds are minced to a fine pulp in a meat grinder, and the juice is squeezed through cheesecloth. The pulp is then wrapped in canvas and subjected to hydraulic pressure to remove all the juice. Step 2. Fractionation with Ammonium Sulfate. Enough solid Na2B40:10H20 is added to the juice to bring the pH to about 7.6. The crude juice is then treated with 1 M Ba(C2H~02)2 (10 ml./1, of juice) and made 1.6 M with respect to (NH4)2SO4 (0.3 saturation) by adding the solid salt at room temperature. The precipitate is allowed to settle overnight in the refrigerator so that the supernatant fluid is removable almost entirely by siphon, requiring centrifugation of only the settled material. The precipitate is discarded. The supernatant is treated with an amount of (NH4)2S04 equal to that previously added, and the resulting precipitate, after filtration, is either immediately treated according to step 3b or filtered on Celite 5 and stored at - 1 5 ° until needed (step 3a). Step 3. Refractionation with Ammonium Sulfate. (a) The frozen precipitate from step 2 is slurried in 10 1. of cold water and allowed to stand for 2 hours. The solution is then filtered, and the enzyme is reprecipitated from the filtrate by adding 4200 g. of (NH~)2S04. (b) The precipitate is filtered and redissolved by slurrying the pad and precipitate in 2.5 1. of cold water. After filtration, the resulting solution is dialyzed against several changes of distilled water in the refrigerator until a yellow precipitate forms (about 24 hours). This precipitate is discarded. Step 4. Adsorption of Enzyme to Alumina. To the solution is added one-fifth its volume of alumina gel reagent. 8 The alumina with its adsorbed protein is filtered immediately. The protein is eluted by placing the alumina and filter pad in a small amount of 0.2 M Na~HPO4 and stirring to a thick paste. The mixture is then diluted to about 800 ml. with 0.2 M Na~HP04, stirred for 30 minutes, and refiltered. The filtrate is dialyzed against several changes of water in the refrigerator. Step 5. Fractionation with Acetone. The dialyzed enzyme solution is treated with one-fifth its volume of cold acetone (5 °) and filtered immediately. The filtrate is treated with a second quantity of acetone equal to that used initially. The precipitate is removed by immediate filtration on a fresh filter pad. The process is repeated until about seven precipitates have been collected. A green to blue precipitate is obtained in the 5 All filtrations are c a r r i e d o u t u s i n g Celite as d e s c r i b e d in f o o t n o t e 12 in Vol. I I [145]. GSee f o o t n o t e 13 in Vol. I I [145].

834

RESPIRATORY ENZYMES

[147]

region of the fourth, fifth, or sixth acetone t r e a t m e n t s . T h e precipitates are redissolved s e p a r a t e l y in 150 to 200 ml. of 0.2 M N a ~ H P 0 4 and assayed for e n z y m e activity. T h e green or blue fractions i n v a r i a b l y contain the m o s t a c t i v i t y and are dialyzed against doubly distilled (copper-free) water. Usually during this dialysis, especially with more concentrated solutions, the e n z y m e precipitates as a blue a m o r p h o u s protein. This blue protein is redissolved in a small a m o u n t of 0.1 M acetate buffer and dialyzed against several changes of copper-free 0.1 M acetate buffer in preparation for the determination of copper content and specific activity. An overall yield of a b o u t 18 % (see the table) is obtained b y combining fractions of similar specific activity. SUMMARY OF PURIFICATION PROCEDURE

Fraction

Total volume, Units/ml., ml. thousands

1. Crude extract 50,0O0 0.05 2. (NH4)~SO4fraction . . . 3. Repreeipitation with (NH4) 2SO~ 3,000 0.26 4. Eluate from alumina 800 1.88 5. Acetone fractions 5 and 6 combined after dialysis 15.0 30.00

Total Specific units, activity, Recovery, thousands units/mg. %

2,500 .

1.2

--

. 780 545

110 290

31 22

450

1600

18

Properties

Constitution of the Enzyme. Ascorbic acid oxidase f r o m squash is a blue protein having a molecular weight of 150,000, a copper content of 0.25%, and properties of a globulin. 4 M a x i m a in its absorption spectra a p p e a r at 288 and 605 m~. H o m o g e n e o u s ascorbic acid oxidase has been shown to possess a specific a c t i v i t y of 2000 u n i t s / m g , and 750 u n i t s / 7 of copper. ~ Specificity. Purified ascorbic acid oxidase shows m a r k e d specificity for L-ascorbic acid. D-Ascorbic acid and a n u m b e r of other ene-diols similar in structure to ascorbic acid are also oxidized although m u c h more slowly. Monophenols are not oxidized alone or during the oxidation of ascorbic acid. Polyhydric phenols such as catechol and h y d r o q u i n o n e are not oxidized. Several studies concerning the specificity of ascorbic acid oxidase f r o m various sources h a v e a p p e a r e d in the literature. 8 7 The activity of the enzyme is more than one thousand times that of an equivalent amount of ionic copper. 8 For references, see C. R. Dawson and W. B. Tarpley in "The Enzymes" (J. B. Sumner and K. Myrbiick, eds.), Vol. II, Part 1, p. 492, Academic Press, New York, 1951.

[147]

ASCORBIC ACID OXIDASE

835

Activators and Inhibitors. Ascorbic acid oxidase is inhibited by cyanide, sodium sulfide, diethyldithiocarbamate, 8-hydroxyquinoline, and potassium ethyl xanthate. 8 It is not inhibited by ethylenediaminetetraacetate. ~ Recently, it has been found that the enzyme is inhibited by certain metallic ions including cupric ions. As a result, low concentrations of certain metal-complexing agents appear to activate the enzyme. 10.11 In higher concentrations, these agents inhibit the enzyme. Stability. The stability of solutions of ascorbic acid oxidase appears to depend on the source and degree of purity of the enzyme and the protein concentration of the solution. Experience indicates that in general the stability of the enzyme increases with increase in purity, i.e., increase in specific activity. Enzymes of maximum specific activity appear to be stable for long periods of time in concentrated solutions at refrigerator temperatures. Enzymes of specific activity in the region of 1400 to 1700 units/mg, usually undergo a slow denaturation even at refrigerator temperatures. Losses of 5 to 10 % of the activity per week are not uncommon. Highly diluted solutions of the purified enzyme, such as are required for activity measurements, undergo rapid loss of activity on standing. This effect is minimized by making the final dilution with a dilute solution of gelatin. ~ Reaction Inactivation. When the enzyme functions in the aerobic oxidation of ascorbic acid, it undergoes pronounced inactivation. 12,~3 The inactivation does not appear to be due to a rupture of the copper-toprotein bond. 14 Effects of p H and of Substrate Concentration. Solutions of ascorbic acid oxidase rapidly and irreversibly lose their activity at pH values below 4. The pH optimum for enzyme activity in citrate-phosphate buffer is about 5.6, regardless of the source and purity of the enzyme. The effect of substrate concentration appears to depend on the purity of the enzyme under investigation.2

9 Recently observed in these laboratories. l0 C. L. Gemmfll,J. Biol. Chem. 192, 749 (1951). H E. Frieden, Federation Proc. 11, 215 (1952). 1~H. G. Steinman and C. R. Dawson, J. Am. Chem. Soc. 64, 1212 (1942). 13W. H. Powers and C. R. Dawson, J. Gen. Physiol. 27, 182 (1944). 14C. R. Dawson,in "Copper Metabolism" (W. D. MeElroyand B. Glass, eds.), p. 18, The Johns Hopkins Press, Baltimore, 1950.

836

RESPIRATORY ENZYMES

[148]

[148] C a r b o n i c A n h y d r a s e ( P l a n t a n d A n i m a l ) By E. RoY WAYGOOD

CO2 ~- HsO ~- H2CO3~ H+ ~- HCO3When COs is dissolved in water it is slowly hydrated to carbonic acid which spontaneously ionizes according to the above equation. Hydration occurs in the pH range 6.5 to I0.0, whereas the reverse dehydration takes place in the pH range 5.5 to 7.5.1 This reversible reaction is catalyzed by many oxy-acid buffers, 2 including phosphate, cacodylate, Veronal, chromate, borate, selenite, and also by the enzyme carbonic anhydrase which catalyzes both phases of the reaction equally.', Assay Methods

Principle. There are two main methods for the determination of carbonic anhydrase activity. 1. Manometric Method. Activity may be determined by measuring the increased rate of COs output when carbonic acid, supplied in the form of a bicarbonate solution, is dehydrated by shaking with a buffer (usually phosphate, pH 6.6 to 6.8) and enzyme. The hydration of carbon dioxide is measured by the increased rate of COs uptake when gaseous COs is shaken with a buffer (usually Veronal, pH 8.0) and enzyme. 2. Colorimetric Method. When a solution of COs is mixed with an alkaline buffer, the pH drops rapidly, owing to the reaction COs-}OH---+ HC03- (above pH 8.0) accompanied and followed by the hydration of COs (below pH 10.0). The decrease in time taken for the buffer containing enzyme to drop to a specified pH is used as a measure of catalytic rate. The lower pH value is determined by a sharp change in the color of an appropriate indicator when the buffering capacity of the solution changes markedly. Procedure. (1) Manometric Procedure. Clark and Perrin 4 have suggested that Meldrum and Roughton's boat-manometric method 5 should be retained provisionally as the standard procedure for measuring enzyme activity. The procedure with elegant refinements which afford a more sensitive means of studying the kinetics of the enzyme under a 1F. J. W. Roughton and V. H. Booth, Biochem. J. 40, 309, 319 (1946); F. J. W. Roughton, Harvey Lectures, Series 39, p. 96 (1943-4). F. J. W. Roughton and V. H. Booth, Biochem. J. 32, 2049 (1938). 3 M. Kiese and A. B. Hastings, J. Biol. Chem. 132, 281 (1940). 4 A. M. Clark and D. D. Perrin, Biochem. J. 48, 495 (1945). 5 N. U. Meldrum and F. J. W. Roughton, J. Physiol. (London) 80, 113 (1933).

[148]

CARBONIC ANHYDRASE (PLANT AND ANIMAL)

837

wider range of conditions 1,2 has been described in considerable detail elsewhere. ~-7 However, for purely routine studies on the distribution or purification of the enzyme and the effects of inhibitors and activators, the use of the W a r b u r g a p p a r a t u s is recommended, since it comprises one of the standard items of equipment available in m a n y laboratories and suffers from no more serious disadvantages than does the b o a t apparatus. Nevertheless, for the accurate interpretation of d a t a derived from a n y manometric procedure measuring carbonic anhydrase activity, the operator m u s t be fully aware of the limitations imposed on the method b y the reaction system. These have been discussed in detail b y R o u g h t o n et al. 1,2,8 and b y Clark and Perrin. 4 Although a n u m b e r of investigators 9-1~ have described reliable methods using the W a r b u r g technique, the one described below, developed by K r e b s and Roughton, 14 is recommended because of its simplicity and the use of a low concentration of phosphate which becomes increasingly inhibitory toward the enzyme at high concentrations. KREBS-t~OUGHTONWARBURGTECHNIQUE. For experiments at 0 °, 2 ml. of 0.1 M N a 2 H P 0 4 and 0.1 M KH2PO4 in the proportion 3.2 are placed in the main c o m p a r t m e n t of a W a r b u r g flask with 0.2 ml. of water or enzyme. One milliliter of 0.1 M N a H C 0 3 is placed in the side arm. At zero time, after equilibration, the two solutions are mixed, shaken at 120 to 180 oscillations per minute, and pressure changes recorded at 30-second intervals over a period of 5 minutes or more. T h e pressure change due to the control (nonenzymically catalyzed reaction) as well as the reaction catalyzed b y small amounts of the enzyme is a linear function of time until a b o u t one-third (ca. 80 mm.) of the final pressure change has been attained. F o r experiments at 15 ° and above, 1 ml. of phosphate buffer and 1 ml. of 0.05 M N a H C 0 3 are used. DEFINITION OF UNIT AND SPECIFIC ACTIVITY. Pressure changes are converted to microliters of C02 b y the use of flask constants (kco,) in the conventional manner. ~ The increased C02 evolved in 30 seconds (i.e., 6 H. Van Goor, Enzyrnologia 13, 73 (1948). 7 F. J. W. Roughton and A. M. Clark, in "The Enzymes" (J. B. Sumner and K. Myrbtick, ed.), Vol. 1, Part 2, p. 1250, Academic Press, New York, 1951. s F. J. W. Roughton, J. Biol. Chem. 141, 129 (1941). 9 M. Leiner and G. Leiner, Biochem. Z. 311, 119 (1942). 10 W. C. Stadie, B. C. Riggs, and N. Haugaard, J. Biol. Chem. 161, 175 (1945). n M. D. Altschule and H. D. Lewis, J. Biol. Chem. 180, 657 (1949). 12E. R. Waygood and K. A. Clendenning, Can. J. Research C28, 673 (1950) ; Science 113, 177 (1951). 13R. U. Byerrum and E. H. Lucas, Plant Physiol. 27, 111 (1952). 14H. A. Krebs and F. J. W. Roughton, Biochem. J. 43, 550 (1948). 15W. W. Umbreit, R. H. Burris, and J. F. Stauffer, "Manometric Techniques and Tissue Metabolism," Burgess Publishing Co., Minneapolis, 1949.

838 ~

RESPIRATORY

ENZYMES

[148]

over-all rate - nonenzymic rate) in the region of 60 to 80 mm. of pressure can be used to calculate Qco, on a nitrogen or other appropriate basis. In the case of labile highly purified enzymes (see later), or when the effect of activators or inhibitors is being studied, it m a y be more accurate to express the enzymic activity as the difference between the true initial unimolecular velocity constants (k, - k,) of the enzymically and the nonenzymically catalyzed reactions, respectively, according to the methods of Mitchell et al. '8 and Clark and Perrin. 4,~7 F r o m the equation a of the first-order reaction kt = 2.303 log a - x' log (a - x) is plotted against t and the initial slope times 2.303 gives the value of the velocity constant. PRECAUTIONS. Phosphate has a strong catalytic effect on the dehydration of carbonic acid, ~ and the Q10 = 2.9 for this reaction differs markedly from the Q~0 = 1.4 for the enzymically catalyzed reaction (over-all r a t e - non-enzymic rate). ~8 Accordingly, in order to work within the range of strict proportionality between activity and enzyme concentration, the apparatus is limited to the use of enzyme concentrations which increase the nonenzymic activity up to sixfold at 0 o and twofold at 38 ° . If measurements are made at higher concentrations of enzyme, the reaction is limited b y the diffusion of COs and should be corrected b y applying the theoretical concepts and experimental treatments of Roughton. 8 According to Roughton el al., ~,7,8 in most cases, when diffusion is limiting the apparent rate m a y be corrected b y applyR,~Ro

ing the formula R - R~ - R~ where R0 is the apparent rate and Rm is the maximum observable rate in the presence of a large a m o u n t of enzyme. R~ is a function of liquid volume and the dimensions of the flask. As yet it has only been determined for the boat apparatus. When highly purified enzymes from blood are used, a proportion m a y be inactivated b y impurities or adsorption. Such enzyme preparations give a sigmoid relationship between activity and enzyme concentration and furthermore are subject to progressive inactivation during shaking. These errors m a y be overcome b y observing strict precautions in cleanliness of glassware, acid washing, use of doubly distilled water, or b y stabilizing the enzyme with 0.05 % peptone.l.4.~9 Or, on the other hand, since these errors result in deviations from the first-order reaction, during the latter part of the reaction time, the initial slope of the plot log (a -- x) le C. A. Mitchell, U. C. Pozzani, and R. W. Fessenden, J. Biol. Chem. 160, 283 (1945). 17A. M. Clark, Nature 168, 562 (1949). ~8F. J. W. Roughton, J. Physiol. (London) 107, 12P (1948). 19D. A. Scott and J. R. Mendive, J. Biol. Chem. la9, 661 (1941); 140, 445 (1941).

[148]

CARBONm ANHYDRASE (PLANT AND ANIMAL)

839

vs. t will give a true value for the velocity constant (k~) independent of the inactivation of the enzyme. 4,~7 Clark 4,~7 has made use of this method to distinguish between stabilizations and activations. By calculating the percentage decrease (D) in the value of k, during the first 100 seconds of reaction, the sensitivity of the preparation may be determined. In a similar way, the degree of stabilization (S) is calculated as 100(1 - d/d ~) where d and d ~are the values of D in the presence and the absence of stabilizing or activating agents, respectively. 2. Co!orimeoric Method. Colorimetrie techniques are usefu) for rapid tests but have a restricted range regarding the conditions under which enzyme activity can be investigated. Owing to the relatively large inhibitions of the enzyme by CO~= in the original method of Brinkman 2° and Philpot and Philpot, 2~ a more reliable technique using Veronal buffer has been developed by Roughton and Booth s and is being used successfully in other laboratories ~,s3 (C. A. Mawson, personal communication). CO2-VERONAL INDICATORMETHOD. Veronal buffer (3 ml. of 0.022 M Veronal in 0.022 M Na salt, pH 7.95), three drops of bromothymol blue, and 2.3 ml. of distilled water (or 0.3 ml. of enzyme and 2.0 ml. of water) are mixed in a 15-ml. stoppered weighing bottle and placed in ice water for 15 minutes. Five milliliters of ice-cold water saturated with COs (0.071 M) is added anaerobically from a long nozzled all-glass syringe. The time is observed for the pH to drop to 6.3, determined with the aid of a bromothymol standard at this pH. The solutions are mixed in less than 1 second without bubbling or loss of COs. The control time averages 90 seconds, compared to about 64 seconds in the presence of 14.3 p.p.m, of a crude chloroform preparation (Step 2, p. 841). DEFINITION OF UNIT. The enzyme unit E.U. -

to - -

t

t

, where t and

to

are time of reaction in the presence and the absence of catalyst, respectively. Since the velocity constant of the uncatalyzed reaction (k~) calculated from the experimental data was found to be 0.0022 mole/1./sec., which agrees well with the accepted value of 0.0021 at 0 °, the validity of the method is established. Accordingly, Roughton and Booth ~ calculated the rate of enzymic hydration of COs in moles per liter per second as follows, allowing a period of 1 second for mixing. R

--

Ro

Ro

to - -

t-

t

1

2~ R. Brinkman, J. Physiol. (London) 80, 171 (1933). 21 F. J. Philpot and J. St. L. Phflpot, Biochem. J. 80, 2191 (1936). 22 K. M. Wilbur and W. G. Anderson, J. Biol. Chem. 176, 147 (1948). .,3 E. R. Trethewie and A. J. Day, Australian J. Exptl. Biol. Med. Sc/. 27, 429 (1949).

840

RESPIRATORY ENZYMES

[148]

where R and R0 are the rates in the presence and the absence of catalyst, respectively. Ro = k~ [average COs] Then Rate = R - R 0 -

((tt 0- - t 1) ) ]c, [average COs]

since k~ = 0.00225 mole/k/see, and [average COs] = 0.0307 mole under the experimental conditions. Therefore, Rate of enzymic hydration of COs

_ (to -- t)

(t - 1) 6.91 X 10-5 mole/1./sec.

The figures for the all-liquid method are approximately 70 % of those expected from manometric data. The discrepancy of 30% may be put down to the inhibitory action of the indicator and experimental error. Wilbur and Anderson 22 have used an automatic syringe to introduce the COs, and the pH changes are measured electrometrically. RAPID-FLOW COLORIMETmCM~.THOD. All methods of assay heretofore discussed are subject to a mixing error and are restricted to the use of low concentration of enzyme owing to diffusion limitations. Accordingly, the development of the Hartridge-Roughton rapid-flow technique, which minimizes these errors and photoelectrically records the color change of a pH indicator, opens up a wider range of conditions under which the enzyme can be studied. Clark and Perrin 4 have used the rapid-mix, quickstop method of Chance. 24 A saturated solution of COs is rapidly mixed at room temperature with Veronal buffer, pH 8.6 and pK 8.0, in a modified Millikan 25 microapparatus. The flow through a capillary tube is stopped in milliseconds, and in the presence of phenol red the pH of the solution is recorded continuously as a function of time. Much higher temperatures and enzyme concentrations may be used in this technique. U s e of C a r b o n i c A n h y d r a s e as a B i o c h e m i c a l T o o l

In decarboxylation or other reactions where gaseous COs is produced, a question of some significance arises as to whether CO2 or HCOa- is the primary product. In systems rapidly producing COs into the gaseous phase, carbonic anhydrase will slow down the rate owing to the accelerated hydration of the COs. On the other hand, it is inferred that if HCO3is the primary product the rate would be initially accelerated. Krebs and Roughton ~4have shown that COs is the primary product of yeast carbox2~B. Chance, J. Franklin Inst. 229, 455 (1940). 25G. A. Millikan, Proc. Roy. Soc. (London) A155, 277 (1936).

[148]

CARBONIC ANHYDRASE (PLANT AND ANIMAL)

841

ylase and the urease reaction. Hansl and Waygood 26 have confirmed their findings and in addition have shown that C02 is the primary product of the plant pyruvic, glutamic, oxalacetic, and a-ketoglutaric decarboxylation systems. More recently, Conway and O'Malley ~7 have concluded from their theoretical and experimental treatments that HC03- may be produced concomitantly with CO2 in the latter two reactions. Source and Purification of the Enzyme Animal. Mammalian red blood cells are the richest source. Among other tissues, the pancreas, gastric mucosa, and kidney contain comparable amounts. The enzyme is absent from plasma and other body fluids with certain exceptions. 5,6 Many methods of purification have been described in the literature (see Van Goor 6) culminating in a crystalline preparation of ammonium carbonic anhydrase by Scott and Fisher. 2s In general, however, investigators have used one or all of three stages of purification, either lysed red cells or the crude chloroform preparation of Meldrum and Roughton 1,5 or highly purified enzymes prepared by the particular method of the investigator. Since many laboratories have successfully used the highly purified preparations of Keilin and Mann 29 and because of its relative simplicity the details of their procedure and those of the crude preparations are described. Step 1. Lysed Red Cells. The red blood cells of defibrinated ox blood are centrifuged from the serum and washed three times with an equal volume of 0.9 % NaC1. The washed cells are hemolyzed with an equal or half-volume of distilled water. Step 2. Crude Chloroform Preparation.1 To 10 ml. of lysed red cells are quickly added 8 ml. of 40 % ethanol and 4 ml. of chloroform. The mixture is stirred in a centrifuge tube for 3 minutes to a thin sludge and allowed to stand for 20 minutes. After centrifugation for 10 minutes at 3500 r.p.m., a three-phase system is formed consisting of a supernatant layer of enzyme solution, a central layer of denatured protein, and a bottom layer of chloroform. Step 3. Method I I of Keilin and Mann. 29 Dialysis of Alcohol-Chloroform Extract. The filtered enzyme solution is dialyzed for 24 hours against running water. Step 4. Total Precipitation with Ammonium Sulfate. The fluid is saturated with (NH4)2S04, and the precipitate after filtering on a Btichner 36 N. 37 E. 3s D. 39 D.

Hansl and E. R. Waygood, Can. J. Botany 30, 306 (1952). J. Conway and E. O'Malley, Biochem. J. 54, 154 (1953). A. Scott and A. M. Fisher, J. Biol. Chem. 144, 371 (1942) ; Nature 153, 711 (1944). Keilin and T. Mann, Biochem. J. 34, 1163 (1940).

842

RESPIRATORY ENZYMES

[148]

funnel is dissolved in a small a m o u n t of water, dialyzed against running tap water and centrifuged. Step 5. Fractional Precipitation with Ammonium Sulfate. T h e supern a t a n t fluid is saturated 4 5 % with respect to (NH4)2S04 and filtered. The filtrate is completely saturated with (NH4)2SO4 and, after filtering, the precipitate is dissolved in water and dialyzed. Step 6. Purification with Alumina C~ Gel. T h e solution is treated with three successive 5-ml. ( = 100 mg.) portions of alumina C~ gel at p H 6.8 and centrifuged, the cakes being discarded each time. Step 7. Fractional Precipitation with Ammonium Sulfate. The above solution is made 50 % saturated with (NH4)2SO4 and, after filtering, completely saturated. The precipitate is dissolved in water and dialyzed against distilled water until free from salt. SUMMARY OF PURIFICATION PROCEDURE a

Step

Total volume, ml.

Absolute E.U.

1 2-3 4 5 6-7

2000 2250 110 85 50

4,000,0002 1,488,000 1,100,000 765,000 500,000

M1./E.U.

Specific activity, E.U./mg.

Zn, %

Recovery, %

0.0005 0.0015 0.0001 0.0001 0.0001

14.3 384 588 830-1000 2220

0.C024 --0.15-0.17 0.33

-37 27 19.5 12

Method II [D. Keilin and T. Mann, Biochem. J. 34, 1163 (1940)]. b Calculated from Step 1, Method I [D. Keflin and T. Mann, Biochem. J. 34, 1163 (1940)]. Plant. Bradfield 8° and Waygood and Clendenning TM found the enzyme to be ~ocalized only in the cytoplasm of leaf tissue. The enzyme is absent from root tissue2 ° Sirois and Waygood (unpublished) recommend spinach (Spinacea oleracea) as a source of the enzyme, since it has the highest specific activity of all plants tested, b u t m a n y other sources are available, n o t a b l y leaves of New Zealand spinach (Tetragonia expansa), nast u r t i u m (Tropaeolum majus), lamb's quarters (Chenopodium album), and beet (Beta vulgaris). 12.13,30.31 Sibly and Wood 3~ have purified the plant enzyme b y the m e t h o d of Keilin and Mann, b u t the percentage recovery was low. We have found t h a t it is essential to protect the plant enzyme with cysteine at all stages during purification, and the following procedure is recommended. Midribs are removed from spinach leaves, and 200 g. is ground in a m o r t a r with 50 ml. of cysteine (final concentration 0.005 M). The brei is 30j. R. G. Bradfield, Nature 159, 467 (1947). sz p. M. Sibly and J. G. Wood, Australian J. Sci. Research B4, 500 (1951).

[148]

CARBONIC ANHYDRASE (PLANT AND ANIMAL)

843

pressed through nylon, and the crude juice is centrifuged for 20 minutes at 15,000 X g. The supernatant fluid (200 ml., A = 15 E.U./mg.) is shaken with 75 g. of Ca3(P04)2 and centrifuged. The cake is eluted with 0.2 M phosphate, pH 6.6, in 0.005 M cysteine. After an initial precipitation with 30 % alcohol, the enzyme is totally precipitated at a concentration of 75% ethanol. The white powder is completely soluble in water. The solution, when dialyzed against 0.005 M cysteine and lyophilized, yields a preparation containing 300 E.U./mg. and 0.05 % Zn with no other metals. The enzyme unit is that defined by Waygood and Clendenning. 12

Properties Specificity. Carbonic anhydrase is specific for the reaction CO2 + H20 ,~- HsCO3. It is doubtful if it catalyzes the reaction COs + O H - --* HC03- which predominates above pH 11.0 and is significant between pH 8 and 10.1,~,3,7 Since the latter occurs as a primary reaction succeeded by, or accompanying, the enzyme-catalyzed reaction, it may introduce errors in some colorimetric methods. There are numerous processes which are limited by the reaction COs + H20 ~---H~CO~, and their rates may be increased by the addition of enzyme, e.g., the deposition and solution of CaCO3, and the dissociation of carbamate into C02. 5,7 Stability. Crude chloroform preparations of the animal enzyme are stable for long periods in the dry state but are progressively inactivated in solution. Purer preparations are less stable. Plant carbonic anhydrase is stable in the dried form but deteriorates rapidly in solution. It is also much less stable during dialysis than the animal enzyme. Cysteine (0.005 to 0.01 M) protects the plant enzyme in solution and during dialysis. 30 Nature of the Enzymes. Carbonic anhydrase is a Zn-protein compound. ~9,32 Keilin and Mann's preparation ~9 and others contained 0.033% Zn, whereas Scott and Fisher's crystalline preparation 28 contained 0.02% Zn and had a molecular weight of 30,00023 The latter value corresponds to 1 atom of Zn per molecule. Our partially purified preparations of the plant enzyme contain 0.05 % Zn with no other metals. Sibly and Wood81 calculated ~hat their preparations of the plant enzyme contained 0.056 % Zn. The failure of dialysis to remove zinc from either animal 29 or plant carbonic anhydrase (Sirois and Waygood, unpublished) and the fact that Tupper et al. 34 have shown that Zn 6~ ions in the medium do not exchange a2 D. Keflin and T. Mann, Nature 144, 442 (1939). 33 M. L. Peterman and N~. ¥. ttakala, J. Biol. Chem, 14§, 701 (1942). 34 R. Tupper, R. W. E. Watts, and A. Wormalls, Bioehem. J. 50, 49 (1952).

844

RESPIRATORY ENZYMES

[148]

with the zinc moiety of the enzyme from blood indicates that the metal is firmly entrenched in the protein molecule. The evidence that we have been able to provide in favor of the view that plant carbonic anhydrase is a Zn-protein is as follows. The Zn content of the purified enzyme preparation increases proportionately with increasing specific activity during prolonged dialysis against cysteine. No other metals are present, but the possibility that Zn is present in a nonspecific protein is not excluded. Inhibition by cyanide and azide (see later) is also indicative of the metalloprotein nature of the enzyme in plants. The kinetics of the enzyme from blood have been studied in detail by Roughtou and Booth 1 using the refined boat method. Byerrum and Lucas 1~ give some kinetic data for the enzyme from plants. Since it is doubtful if all the necessary precautions were observed in their experiments, the results await confirmation. Effect of Enzyme Concentration. Provided that diffusion is not a limiting factor, there is a linear relationship between activity and concentration of crude preparations of the enzyme. When diffusion is limiting, the apparent rate may be corrected by Roughton's formula (see above). In the case of highly purified preparations a dilution effect may occur, giving a sigmoid relationship between activity and enzyme concentration. As pointed out earlier, this may be overcome by stabilizing the enzyme. Effect of Substrate Concentration. The value of the Michaelis constant (Kin) for the crude chloroform preparation is 0.009 M C02 (+0.001 M) at 0°. 1 The value is independent of pH, although Kiese ~5 has reported values at 1° of 0.0012 M at pH 7.4, rising to 0.0022 M at pH 9.3 for a highly purified enzyme. It is not certain whether Kiese took all the necessary precautions. Calculations from Leiner's data for a stabilized highly purified enzyme gives K~ = 0.0075 M + 20%. T M Turnover Number. The rapidity of the reaction is shown by calculations from the data of Roughton et al. 1,~ which gives a maximum turnover number of the order of 9.6 X 107 at 0 ° and pH 7.3 for the enzyme from blood. The highest turnover number previously recorded is 5 X 106 for catalase. The value of 1.8 × 108 for the velocity constant of combination of enzyme and substrate is also indicative of the rapid reaction and is of the same order as that for catalase. Effect of pH. The activity of the enzyme from blood is at a minimum at pH 6.5 and gradually increases to fivefold the activity at pH 10.0. There is evidence that the activity is increased below pH 6.0.1 The isoelectric point is at pH 5.3. 3~ 35M. Kiese, Biochem. Z. 307, 400 (1941). 36M. Leiner, Biochem. Z. 315, 31 (1943).

[148]

CARBONIC ANHYDRASE (PLANT AND ANIMAL)

845

Effect of Temperature. Recently corrected values give Q10 = 2.9 for the nonenzymic rate and Q10 - 1.4 for the enzymic rate (over-all rate nonenzymic rate). TMA plot of the log activity against l I T shows a linear relationship. Inhibitors. Heavy metal poisons--e.g., cyanide, azide, and sulfide-inhibit the activity of carbonic anhydrase from blood to the extent of 50% at concentrations ranging from 10`4 to 10-e M. 7 Whereas the activity of the plant enzyme is strongly inhibited by azide (70 to 90 % at 10-3 M), 12,3° cyanide, according to Bradfield 3° and Sibly and Wood, 31 only inhibits at much higher concentrations (65 to 75 % at 10-3 M; no inhibition at 10-3 M). However, Waygood and Clendenning ~2 have reported 50 to 75% inhibition of the activity of a crude and dialyzed enzyme preparation from Tradescantia fluminensis by 10-3 M HCN. These values have been confirmed by Sirois and Waygood (unpublished) using stabilized semipurified preparations from spinach leaves. The activity of a crude spinach preparation is inhibited 50% by 2.4 X 10-3 M HCN, whereas the activity of the purified enzyme is inhibited 50% by 1.25 X 10-3 M HCN. Nevertheless, the relative insensitivity of the plant enzyme to cyanide, compared with the enzyme from blood, constitutes one of the more important differences between the two enzymes. The markedly inhibitory effect of low concentrations of sulfanilamide and related substances containing the --S02NH2 group has been extensively investigated. 37-4° N-Substituted sulfonamides are ineffective.89 Thiophene-2-sulfonamide and p-sulfonamidobenzoic acid are eight to twelve times as effective as sulfanilamide which inhibits the activity of the enzyme from animals 50% at 10-7 M. 3s,39 A new and more specific inhibitor and less toxic drug, Diamox 6063 (2-acetylamino-l,3,4-thiadiazole-5-sulfonamide) has recently been reported. 4° In contrast, Bradfield a° and Sibly and Wood ~ found that sulfanilamide at a concentration of 10-5 M caused little or no inhibition of the activity of the plant enzyme from two different sources, thus indicating another important difference between the animal and the plant enzyme. The inhibitory effect of anions on the activity of the enzyme from blood in decreasing order I - > NO3- > Br- > C1- > acetate > SO4--. The effect of C1- is especially interesting, since it appears to cause inactivation by forming an inactive complex with the enzyme. The divalent ion COs-- also causes inhibition of enzyme activity, especially at high pH values.

37T. Mann and D. Keilin, Nature 146, 164 (1940). as H. W. Davenport, J. Biol. Chem. 168, 567 (1945). 39H. A. Krebs, Biochem. J. 48, 525 (1948). 40T. H. Maren, Trans. N. Y. Acad. Sci. 16, 53 (1952).

846

RESPIRATORY ENZYMES

[148]

Activators, Stabilizers and Protectors. Claims of activating substances reported in the literature are now suspect, owing to the questionability of the methods used. Clark and Perrin 4 have shown convincingly that apparent activation of the enzyme by certain substances, e.g., boiled horse plasma and glutathione, is due to the restoration of the activity of purified enzymes, lost by adsorption or the effect of impurities. No activation occurs when the enzymes are stabilized by 0.05 % peptone; thus these substances are acting as stabilizers. Indeed the stabilizing power of plasma obscured a weak inhibitory effect.4 Scott and Mendive 19 report a number of stabilizers of which horse serum (1:40) and 0.05% peptone are the most effective. We have used 0.025% gelatin to stabilize the plant enzyme. One of the most important differences between plant and animal carbonic anhydrase is the dependency of the former on free - - S H groups. Thus Bradfield 3° discovered that cysteine efficiently protected the plant carbonic anhydrase while standing or during dialysis. Furthermore, the enzyme was completely inhibited by 5 X 10-4 M p-mercuriochlorobenzoate, which has little effect on animal carbonic anhydrase. Sibly and Wood 31 have confirmed these findings and also demonstrated complete inhibition of activity by 5 X 10-3 M arsenite, another mercaptide-forming compound. The inhibition caused by both these substances could be reversed by cysteine o r glutathione. Iodoacetate does not cause inhibition, but the presence of sulfhydryl groups on plant carbonic anhydrase has been confirmed polarographically. 3~ The important papers of the Japanese workers, Kondo et al., 4~ on the kinetics of spinach carbonic anhydrase were not available to the author at the time of writing. They have reported the isolation of a metal-free electrophoretically homogeneous protein, stabilized by 0.1 M NaC1 having carbonic anhydrase activity. 41 K. Kondo, H. Chiba, and H. Kawai, Bull. Research Inst. Food Sci. Kyoto Univ. 8, 17-27 (1952); 13, 1-59 (1954). (English abstracts.)

[149]

DEHYDROASCORBIC REDUCTASE

847

[149] Dehydroascorbic Reductase D H A ~- 2 GSH--~ GS'SG ~ AA By M. A. JOSLYN

Assay Method Principle. The method used by Crook, I Crook and Morgan, ~ and Bukin 3 was based on measurement of ascorbic acid produced b y enzymecatalyzed reduction of dehydroascorbic acid (DHA) in the absence of oxygen. The method described below was developed b y Yamaguchi 4 and used for the determination of the distribution of dehydroascorbic acid reductase in the various tissues of the developing pea plant (Pisum sativum) and for assay of enzyme preparations from pea tissue. ~ I t is based on the determination of ascorbic acid produced b y reduction of dehydro-L-ascorbic acid in the presence of reduced glutathione in an evacuated Thunberg tube. The reduced ascorbic acid was determined by the m e t h o d of Loeffler and Ponting 6 from measurement of the concentration of unreduced 2,6-dichlorophenolindophenol in an E v e l y n photoelectric colorimeter with a 515-m~ filter. The reduction of dehydroascorbic acid is carried out at pH 6.3, 25 °, with 1.14 X 10-8 M dehydroascorbic acid, 2.28 X 10 -8 M glutathione, and 10 mg. of dry enzyme preparation in a total volume of 10 ml. of reaction mixture (sufficient to produce about 1 mg. of reduced ascorbic acid per 10 ml. of reaction mixture). Reagents

Dehydroascorbic acid. Crystalline dehydro-L-ascorbic acid was prepared b y the method of K e n y o n and Munro 7 from iodine-oxidized L-ascorbic acid in methanol. The hydriodic acid formed was removed b y precipitation with PbCOa. Excess lead was precipitated with H2S, the PbS removed. The residual methanol was removed by evaporation and the dehydroascorbic acid recrystallized from absolute ethanol at 0 °. Two milligrams of the dehydroascorbic acid were dissolved in 1 ml. of 0.1% acetic acid. E. M. Crook, Biochem. J. 35, 226 (1941). E. M. Crook and E. J. ~Iorgan, Biochem. J. 38, 10 (1944). 8V. N. Bukin, Biokhimiya St 60 (1943). 4 M. Yamaguchi, Ph.D. Thesis, University of California, Berkeley, 1950. 5 M. Yamaguchi and M. A. Joslyn, Plant Physiol. 26, 757 (1951); Arch. Biochem. and Biophys. 38, 451 (1952). s H. J. Loeffier and J. D. Ponting, Ind. Eng. Chem. Anal. Ed. 14~ 846 (1942). J. Kenyon and N. Munro, J. Chem. Soc. 158 (1948).

848

RESPIRA.TORY ENZYMES

[149]

Glutathione solution. Seven milligrams of pure reduced glutathione dissolved in 1 ml. of copper and iron-free redistilled water. 0.1 M phosphate buffer, pH 6.3 (SCrensen). 2,6-Dichlorophenolindophenol solution. Thirteen milligrams of pure reagent freshly dissolved in 1 1. of redistilled water, standardized so that 1 ml. of 1% metaphosphorie acid and 9 ml. of dye will give a 15-second reading of about 30 on the Evelyn photoelectric colorimeter with a 520"mt~ filter. 6 % metaphosphoric acid. Sixty grams of metaphosphoric acid dissolved in 900 ml. of water and diluted to 1 1. Store at 0 °, and dilute to 1% before use. 1% oxalic acid. Oxygen-free nitrogen. Procedure. Pipet 1 ml. of enzyme solution and 7 ml. of phosphate buffer into the main part of the Tam and Wilson 8 modified Thunberg tube, or 8 ml. of tissue extract at pH 6.3 (equivalent to 1 g. of tissue). Pipet 1 ml. of dehydroascorbic acid solution into the side arm. Pipet 1 ml. of glutathione solution into a glass tube, 2 mm. diameter X 25 mm., small enough to slide down the side arm; place some lanolin on the rim of this tube so that the solutions do not mix when the tube is inserted into the side arm or splash out during evacuation. Evacuate the Thunberg tube by oil pump, controlling foaming during evacuation. After the first evacuation relieve with oxygen-free nitrogen and evacuate again. Repeat the evacuation twice. Close the side arm, place the tube in constant temperature bath, and allow 15 minutes to reach bath temperature. After the tube and contents are brought to temperature, start the reaction by mixing the contents of the tube. Stop the reaction after 10 minutes at 25 ° or 5 minutes at 40 ° by quickly adding 40 ml. of 1% metaphosphoric acid (40 ml. of 1% oxalic acid may be used also, but this is not so satisfactory for crude preparations of enzyme or for tissue extract). Determine the amount of ascorbic acid present by pipetting 1 ml. of the resulting solution into an Evelyn colorimeter tube, adding 9 ml. of indophenol, and measuring the galvanometer deflection 15 seconds after mixing. Correct for nonenzymatic reduction of DHA by GSH at the same time by running a control Thunberg tube without added enzyme. The quantity of DHA reduced is proportional to time for the first 10 minutes at 25 °. The nonenzymatic reduction is appreciable at pH 6.9 [optimal for reduction by the enzyme in pea tissue (Joslyn and Yamaguchi~); Bukin 3 reported somewhat lower optimal pH for cabbage juice (6.7 to 6.8); and Crook and Morgan ~ also found this to be optimal for

8 R. K. Tam and P. W. Wilson, J. Bacteriol. 41, 529 (1941).

[149]

D E H Y D R O A S C O R BREDUCTASE IC

849

cauliflower preparations], but it is negligible at pH 6.3. For plant tissue juices and enzyme preparations of low activity, pH 6.3 is preferable to pH 6.8 for higher accuracy. Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount which causes the reduction of 1 mg. of dehydroascorbie acid in 10 minutes at 25 °. Specific activity is expressed as units per milligram of protein or per gram of dry weight of tissue. Enzyme activity is determined at pH 6.3 instead of the optimal pH of 6.9 used for purified enzyme to reduce correction for nonenzymatic reduction and at 25 ° instead of the optimal temperature of 40 ° because of rapid inactivation at 40 ° or above. The type and concentration of buffer markedly influence enzyme activity. "In pressed pea juice, activity was higher in orthophosphate buffer than in metaphosphate, citrate, or acetate buffer of the same concentration (0.1 M) at pH 6.3. This was also true for the purified enzyme. With the SOrensen orthophosphate buffer, increasing the concentration of phosphate from 10-4 M to 1 M resulted in increase in activity up to sharp optimum at 0.4 M. Metallic ions at low concentration (10-5 to 10-3 M) had no effect on activity but at 10-3 M and above produced appreciable inhibition (particularly true of Cu++). Purification Procedure The reductase is not widely distributed in plant tissue and is most active in the Cruciferae and Leguminosae. In the developing pea plant the enzyme is most active in the immature pea seed and in leaflets and stlpules, but activity in the petioles and tendrils and in stems and roots is low. As the pea seeds mature, the enzyme activity decreases; the activity in dry pea seeds is less than one-fourth that in the immature peas on~both the dry weight and protein basis. In the pods the activity decreases by about 50% on dry weight basis but remains constant on the protein basis. Plant tissue iuice is prepared by freezing the tissue with solid C02, macerating, and pressing at about 0 ° in a laboratory hydraulic press. To 100 ml. of the pressed juice at 0 °, add very slowly and with constant stirring 100 ml. of saturated (NH~)2S04, prepared by the method of Wildman and Bonner, 9 "adjusted to pH 6.8, and cooled to 0% Separate by filtration the inactive sediment formed on standing for 5 minutes, and discard. To 192 ml. of the resulting filtrate, add 288 ml. of cold saturated (NH4) 2SO4 to bring the solution to 80 % saturation. Separate the precipitate by centrifugation, redissolve in a small amount of 0.1 M phosphate buffer, and dialyze against phosphate buffer in a cellophane bag at 0 ° 9 S. G. Wfldman and J. Bonner, Arch. Biochem. 14, 381 (1947).

850

RESPIRATORY ENZYMES

[149]

for 8 hours. After this preliminary dialysis reprecipitate as above and dialyze again. Prolonged dialysis, particularly against water, decreases activity of enzyme. The final dialyzed preparation is preserved by lyophilization. The lyophilized preparation had an activity on a protein N basis over ten times that of the pressed juice. Tissue preparations are easily made by freezing the tissue, macerating in mortar, and expressing in a laboratory hydraulic press at 5000 p.s.i. This juice has reductase activity of that obtained from tissue ground at 0 ° in a Potter-Elvehjem 1° homogenizer. After centrifugation at 0 ° and filtration with diatomaceous filter aid, the pressed juice may be preserved by freezing and storage at - 1 0 to - 2 0 ° for several weeks with little loss in activity.

Properties The partially purified enzyme is more specific for the GSH component than the ascorbic acid component. Bukin a reported that thiolactic and thioglycolic acids could not replace GSH for the reductase in white cabbage. Crook 1 reported reduction of DHA by cysteine and thiolactic acid by the reductase in cauliflower. Yamaguchi and Joslyn ~ found that with cysteine and thioglycolate as hydrogen donors the nonenzymatic reduction was too rapid to permit accurate measurement of enzymatic reduction. Dehydro-D-araboascorbic acid and reductic acid (1,2,3-triketocyclopentane) are reduced but not so rapidly as DHA. The loss in activity of the reductase during dialysis, particularly against distilled water, was not due to loss of metallic catalysts (Mn ++ or Mg ++) or heat-stable coenzyme. The addition of Mn ++, Mg ++, or boiled juice did not restore activity. Addition of dihydrocozymase (coenzyme I) was without effect, contrary to results obtained by Bukin. 3 Optimal conditions for reduction of DHA for reductase in pea seeds were pH 6.9, 40 °, and D H A / G S H 2:1 (molar ratio). At constant DHA concentration, the reduction increased with increase in GSH at first rapidly up to 7 mg. per 10 ml., then more slowly. At constant GSH, the reduction of DHA increased rapidly up to 2 mg. per 10 ml. and then gradually decreased. At a D H A / G S H ratio of 2:1 (2:7 on weight basis), the quantity of ascorbic acid formed increased linearly with time at pH 6.3 and 25 ° for the first 10 minutes and then decreased. The reaction apparently was of zero order at first but became more complex later. 10 V. R. P o t t e r a n d C. A. E1vehjem, J. Biol. Chem. 114, 495 (1936).

[150]

CYPRIDINA AND FIREFLY LUCIFERASE

851

[150] Cypridina and Firefly Luciferase Cypridina (C. hilgendorfii) - LH2 + O2 ~ Light Firefly (Photinus pyralis) - LH2 + O2 + Mg++-+ ATP --~ Light B y WILLIAM D. MCELROY

Assay Method Luciferase is the enzyme which catalyzes the oxidation of luciferin (LH2) in the presence of molecular oxygen to yield visible radiation and unknown products. 1 Luciferin and luciferase differ, depending on the organism used. It is necessary, therefore, to specify the source of both substances. Since the intensity of the light emitted depends on both enzyme and substrate concentration, the quantitative determination of these substances can be made by measuring the light intensity by a suitable photocell arrangement. The Farrand photofluorometer is satisfactory. Qualitative estimations can be made with the naked eye. With the purified enzymes described below, light can be obtained when these are diluted l: 1000, which is still visible to the dark-adapted eye. Procedure for Cypridina. Dilute the stock enzyme 1:100 with 0.06 M KH2PQ'Na2HP04 buffer, pH 7.0, containing 0.01 M NaC1. Immediately prior to testing the luciferase preparation, 0.10 ml. of stock luciferin is placed in 10 ml. of phosphate-NaCl buffer in the reaction vessel of the light-measuring apparatus. Samples of the diluted enzyme are then added with sufficient buffer so that the final volume of the reaction mixture is 20 ml. Light emission is recorded until the reaction is complete. The firstorder velocity constants, k, are obtained from a semilog plot. Procedure for Firefly. The reaction mixture consists of 0.1 ml. of luciferase, 1 ml. of luciferin, 1 ml. of 0.01 M MgS04, and 1 ml. of 0.004 M ATP made up to a volume of 10 ml. with 0.05 M glycine buffer, pH 8.0. The reaction is initiated by adding ATP and recording the initial maximum light intensity, the latter being proportional to the enzyme concentration. Definition of Unit. One unit of enzyme is-defined as that amount which gives 1 unit of light in arbitrary values under the above stated conditions. Specific activity is expressed either as light units or as rate per milligram of protein. Protein is determined by the method of Lowry et al. 2 1 E. N. Harvey, "Bioluminescence." Academic Press, New York, 1952. -~O. H. Lowry, N. 0. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

852

RESPIRATORY ENZYMES

[150]

Cypridina Luciferase Purification Procedure

The following procedures are based on the original report of McElroy and Chase. 3 A crude active luciferase can be obtained from air-dried Cypridina by grinding the organisms first in a mortar and then in a Ten Brock tissue grinder. Starting with 20 g. of dried material, the first extraction is made with 100 ml. of ice-cold distilled water. After centrifugation at 3000 r.p.m, in an International refrigerated centrifuge for 15 minutes, the residue is re-extracted with an additional 50 ml. of water. The supernatants are combined and cooled to 10 °. The pH is rapidly adjusted to 4.5 with 1 N HC1, and the resulting precipitate is removed by centrifugation in the cold. After adjustment of the pH of the supernatant with NaOH to 7.5, cold acetone is added to 35 % saturation by volume. The temperature is kept between 0 and - 2 ° during this procedure. After 15 minutes at - 2 ° the precipitate is removed by centrifugation and discarded. Additional cold acetone is added to the supernatant to raise the saturation by volume to 55%. The mixture is kept at - 2 ° for 30 minutes. The resulting precipitate is removed by centrifugation and dissolved in 60 ml. of water (fraction 1). The pH is adjusted to 7.5, and solid (NH4)2S04 is added to 40% saturation. The pH is maintained at 7.5 with NaOH. After cooling at 0 ° for 15 minutes, the precipitate is removed by centrifugation and discarded. Solid (NH4)2SO4 is added to raise the per cent saturation to 65, while the pH is maintained at 7.5. After 15 minutes at 0 ° the precipitate is removed and dissolved in 15 ml. of water (fraction 2). Further purification is achieved by adsorbing onto and eluting from calcium phosphate gel. Five milliliters of calcium phosphate gel (1.6% by weight) is added to 15 ml. of fraction 2, and the pH is adjusted to 7.0. The mixture is stirred for 10 minutes at 5 °, after which the gel is removed by centrifugation in the cold. The pH of the supernatant is maintained at 7.0, and 60 ml. of calcium phosphate gel is added. After standing for 15 minutes, the gel is removed by centrifugation. All the luciferase is removed from the supernatant by the latter procedure. The enzyme is removed from the gel by washing three or four times with 25-ml. portions of 10% (NH4)2SO4 (pH 7.3). The combined eluates are again treated with (NH4)2S04, as described above, and the resulting precipitate, obtained with 40 to 65% (NH4)2SO4 saturation, is dissolved in 10 ml. of water (fraction 3). The activities of the various fractions are presented in Table I. 8 W. D. McElroy and A. M. Chase, J. Cellular Comp. Physiol. 38, 401 (1951).

[150]

CYPRIDINA AND FIREFLY LUCIFERASE

853

Preparation of the Substrate--Luciferin. A crude Cypridina luciferin prepared b y making a fresh hot-water extract can be used in assaying for the enzyme. Since Cypridina luciferin is autoxidizable, it is necessary to make up a fresh solution each time. A stable preparation can be prepared, and this has been described in detail b y Anderson. 4 TABLE I PURIFICATION OF

Cypridina LUCIFERASE a Rate constant

Fraction

Protein, mg./ml, k/ml. k X dilution

Crude extract 14.0 Fraction 1 2.4 (0.35-0.65 saturated acetone) Fraction 2 1.1 (0.40-0.65 saturated (NH4)2S04) Fraction 3 0.08 (calcium phosphateoelution)

Specific activity, rate/mg. protein

Recovery, %

1.56 0.81

156 162

11 68

100 46

4.46

446

405

32

1.00

100

1250

4.0

The preparation of the various fractions and the method of enzyme assay are described in the text. The k values were obtained from a plot of log (a - x) vs. time. In all cases, except for fraction 1, the enzyme preparations were diluted 100 times for assay. Fraction 1 was diluted 200 times.

Properties Specificity. Cypridina luciferase is known to catalyze only the luminescent oxidation of Cypridina luciferin. I t does not catalyze the oxidation of luciferins from other sources. Activators. N o requirements for the usual inorganic ions have been observed for the purified enzyme preparations. M a x i m u m activity of the enzyme depends on the presence of a high salt concentration. M a x i m u m activity is obtained with 0.01 M NaC1 and 0.06 M phosphate. pH and Temperature Effects. The enzyme has a rather sharp p H optim u m at 7.0 and a temperature optimum at approximately 25 °. A t 50 ° the enzyme loses approximately 50% of the original activity in 2 hours. Chase 5 has studied the temperature inactivation in some detail. The purified enzyme is very stable when kept in the frozen state, very little loss of activity occurring in five months under these conditions. I t has also been kept at refrigerator temperature for at least four weeks without appreciable loss of activity. 4 R. S. Anderson, J. Gen. Physiol. 19, 301 (1935). 6 A. M. Chase, J. Gen. Physiol. 33, 535 (1950).

854

RESPIRATORY ENZYMES

[150]

Firefly Lucifeiase Purification Procedure The following procedure is based on the original report of McElroy and Coulombre. 8 Five grams of the dried lanterns of Photinus pyralis is ground with sand and extracted three times with a total volume of 100 ml. of H20. The pH of the extract is adjusted to 8 with NaOH, and the solution is placed in the deep-freeze. After freezing and thawing, the inactive precipitate is removed by centrifugation. Twenty-five milliliters of a calcium phosphate gel (16.7 mg./ml.) is centrifuged, and the supernatant discarded. The extract is then thoroughly mixed with the gel, and the pH adjusted to 8. After 15 minutes the mixture is centrifuged and the gel discarded. The supernatant (preparation 2) is considerably more active than the crude extract. Ninety milliliters of the calcium phosphate gel is centrifuged and subsequently mixed with 90 ml. of preparation 2. The pH is maintained at 8. After 15 minutes the mixture is centrifuged and the supernatant discarded. In this latter step most of the luciferase is adsorbed onto the gel while the majority of the luciferin remains in the supernatant. To remove the residual luciferin as well as inactive protein, the gel is washed twice with cold alkaline water and then with a 2 % solution of (NH4)2SO4 at pH 8. Elution of the enzyme is obtained by washing the gel twice with a 7% solution of (NH4)2SO~ at pH 8 (preparation 3). The final volume of combined eluates of preparation 3 is 95 ml. Preparation 3 is then fractionated with (NH4)2SO4 in successive steps of 10% saturation up to 50% saturation and then in units of 2 to 3% saturation up to 65 %. The pH during this procedure is maintained at 8.0. The major part of the active enzyme is recovered between 57 and 65% (NH4)2S04 saturation. The latter precipitate is dissolved in 25 ml. of water (preparation 4), and the enzyme is readsorbed onto calcium phosphate gel as described above. The supernatant is discarded. The enzyme is eluted with 7% (NH4)2SO4 at pH 8 (preparation 5) and precipitated by adding solid (NH4)2SO4 to 70% saturation (pH 8). The precipitate is dissolved in 5 ml. of H20, and the pH is adjusted to 8 (preparation 6). A further treatment of preparation 6 with the low concentration of calcium phosphate gel removes some inert protein (preparation 7). The activity of the various fractions is summarized in Table II. In this procedure the enzyme was purified approximately seventy times on a protein basis with a total recovery of 15 %. In addition, the preparation is completely free of luciferin, and under these conditions no light is emitted on the addition of ATP. Preparation of the Substrate--Luciferin. 6 Most of the firefly luciferin 6 W. D. McElroy and J. Coulombre, J. Cellular Comp. Physiol. 39, 475 (1952).

[150]

CYPRIDINA AND FIREFLY LUCIFERASE

855

remains in the supernatant after the calcium phosphate gel treatment. The supernatant is adjusted to p H 3.5 and extracted two times with an equal volume of redistilled ethyl acetate. All the active luciferin passes into the ethyl acetate. The ethyl acetate is removed b y vacuum distillation, and the active luciferin is dissolved in a small volume of water. This crude preparation can be used for enzyme assay. F u r t h e r purification is TABLE II PURIFICATION OF FIREFLY LUCIFERASE

Preparation 1. 2. 3. 4. 5. 6. 7.

Crude extract Supernatant, first Ca3(PO,)~ gel Eluate, second Ca.~(PO4)2gel 57-65 % (NH4)2SO, precipitate Eluate, third Ca3(PO4)~gel 70 % (NH4)2S04 precipitate Supernatant, fourth Ca3(PO4)2 gel

Light units/ml., Protein, Specificactivity, volts mg./ml, volts/mg, protein 57 166 80 105 92 210 182

10.1 6.3 0.82 0.60 0.33 0. 627 0. 465

5.7 26 98 175 279

335 391

achieved by adsorbing the luciferin on an acid (2 N HC1)-treated Dowex 50 column (mesh size less than 80). The column is washed thoroughly with 2 N HC1 and finally water. The tuciferin is slowly developed on the column b y a weak solution of N H 4 0 H (1.5 %). The luciferin migrates down the column in a sharp band and is finally eluted. The luciferin can be readily followed on the column b y its brilliant yellow-green fluorescent band. The eluates containing the active luciferin are again extracted with ethyl acetate, and the latter is concentrated by vacuum distillation. The luciferin is finally concentrated in water. At neutral p H luciferin has two characteristic adsorption maxima, one major peak at 330 m~ and a secondary peak at 263 m~. The exciting wavelength for fluorescence corresponds to the adsorption peak at 330 m~. The concentrated luciferin is slightly yellow in alkaline solution b u t changes to a colorless solution in weak acid. In the former case the fluorescence on ultraviolet activation is an intense yellow-green, whereas in the latter case the fluorescence changes to a pale red. The luciferin can be maintained for several weeks without an appreciable loss of activity, either frozen in the aqueous solution or in the dried state. In aqueous solution at p H 3.5 and 100 ° complete inactivation occurs in 15 minutes and approximately 50 % loss of activity in 5 minutes. At p H 10 less than 5 % inactivation occurs in 20 minutes at 80 °. The inactive luciferin can be removed from the active b y extraction with ethyl acetate at p H 3.5. Under these conditions only the latter is removed from the aqueous phase.

856

RESPIRATORY ENZYMES

[151]

Properties Specificity. The only known function of firefly luciferase is that of catalyzing the luminescent oxidation of firefly luciferin. Oxidation of the latter depends, however, on the presence of ATP and Mg ++ ions. Activators and Inhibitors. Luciferin, Mg, and ATP are the only known requirements. Mn can replace Mg, but cobalt and iron are considerably less active. Calcium is inhibitory. No known phosphorylated compound will replace ATP. Among those which have been tested are ADP, UTP, ITP, CP, and AcP. 7 The enzyme is not affected by arsenate, cyanide, and azide, but it is strongly inhibited by pyrophosphate, various amines, copper, and p-chloromercuribenzoate, s Inhibition by the latter can be reversed by glutathione. Benzimidazole, benztriazole, and substituted derivatives inhibit the reaction by competing with luciferin (unpublished). pH and Temperature Effects. The pH optimum is at 7.8, and maximum light intensity is observed at 26 ° . The purified enzyme is stable for months in the frozen state but rapidly loses activity on repeated thawing and freezing. The enzyme is rapidly denatured by bubbling air through the solution or by exposing it to various surfaces such as shredded cellophane or glass beads. Activity is rapidly lost on dialysis and cannot be restored by the dialyzate or a crude boiled extract. Note added in proof: The firefly enzyme has recently been obtained in crystalline form (A. A. Green and W. D. McElroy, to be published). 7 W. D. McElroy, J. Biol. Chem. 191, 547 (1951). 8 W. D. McElroy, J. W. Hastings, J. Coulombre, and V. Sonnenfeld, Arch. Biochem. and Biophys. 46, 399 (1953).

[151]

BACTERIAL LUCIFERASE

857

[151] B a c t e r i a l L u c i f e r a s e O D P N H ~- F M N + RC

-{- Os -~ Light

\ H

or

O FMNH2 ~- RC

-[- O, --~ Light

\ H

By ARDA A. GREEN and WILLIA~ D. MCELROY Assay Method

Bacterial luciferase is a flavoprotein which catalyzes the oxidation of reduced D P N by numerous oxidants such as FMN, ferricyanide, quinones, and various dyes. In the presence of F M N and a long-chain fatty aldehyde the oxidation of D P N H is accompanied by light emission. ~Pyridine nucleotides are not essential for light emission, since chemically reduced F M N and aldehyde will support luminescence. The intensity of the light emitted depends on the concentration of these various components. Quantitative measurements can be made by a suitable photocell arrangement such as the Farrand photofluorometer. Qualitative estimations can be made with the naked eye. Procedure. The reaction mixture consists of 0.5 ml. of 0.1 M phosphate buffer, pH 6.8, 1.0 ml. of saturated dodecyl aldehyde, 0.2 ml. of riboflavin phosphate (2 X 10-4 M), 0.05 ml. of 1% bovine albumin, 0.2 ml. of D P N H (7.0 × 10-4 M for maximum luminescence), 0.05 or 0.1 ml. of diluted enzyme, and water to a total of 2.5 ml. The reaction is initiated with D P N H , and the light intensity recorded for various time periods. Definition of Unit. One unit of enzyme is defined as that amount which gives 1 unit of light in arbitrary values under the above stated conditions. Specific activity is expressed as light units per milligram of protein. Protein is determined by the method of Lowry et al3 Growth of the Organism. The salt-water bacterium Achromobacter fischeri was grown under forced aeration or by shaking in the following 1 W. D. McElroy and B. L. Strehler, Bacteriol Revs. 18, 177 (1954). 20. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193~ 265 (1951).

858

RESPIRATORY ENZYMES

[151]

medium: NaC1, 30 g.; Na2HP04, 5.3 g.; KH~P04, 2.1 g.; (NH4)~HPO4, 0.5 g.; MgSO4, 0.1 g.; glycerol, 3 ml.; peptone, 1 g.; and water, 1 1. The pH was adjusted to 7.1 to 7.3 with NaOH. At the peak of luminescence (15 to 20 hours) the cells were harvested by high-speed centrifugation. With good aeration approximately 4 g. (wet weight) of cells per liter is obtained. Purification Procedure

The following procedures are based in part on the original report of McElroy et al2 A crude bacterial extract is obtained by lysing the cells in distilled water (1 g. wet weight per 15 ml. of water). After thoroughly mixing in the cold, the debris and unlysed cells are removed by centrifugation at high speed in a Servall centrifuge. An active isoelectric precipitate is formed by adjusting the pH to 4.0 to 4.3 with HC1. The precipitate is separated by centrifugation in the cold, then suspended in water (one-tenth the volume used for lysing) and dissolved by the addition of N/IO NaOH to pH 6.8 to 7.0. This materially increases the volume and brings the final protein concentration to between 15 and 20 mg./ml. The centrifuged supernatant of the lysed cells contains about one-half of the total protein in the cells, and 85 to 90% of this protein is found in the isoelectric precipitate. Thus, the isoelectric precipitation is a concentration rather than a purification procedure, except for the removal of certain inhibitors, especially riboflavin. These inhibitors make it impossible to properly assay the initial extract. Ammonium sulfate precipitation has proved to be the only practical method of purification. To the solution of the isoelectric precipitate, fraction A, is added 0.1 vol. of Na4P~07 (0.2 M adjusted to pH 7 with HC1). Solid ammonium sulfate is then added with stirring, and the final pH is adjusted to 6.8. All pH values are read on the Beckman glass electrode pH meter without correction for salt concentration. Successive precipitates are separated by centrifugation in the high-speed Servall centrifuge, dissolved in 0.02 M pyrophosphate, and the pH readjusted to 6.8. The initial fractionation procedure yields four fractions. Fraction B. The precipitate obtained at 1.35 M ammonium sulfate (0.33 saturation) is voluminous and contains about 70% of the total protein. It is washed once with 1/~ vol. of 1.35 M ammonium sulfate and the precipitate discarded, since it contains a negligible amount of activity. Fraction C. The combined supernatants from the previous step are brought to 2.05 M ammonium sulfate (0.50 saturation). The precipitate contains about 5 % of the protein and 5 % of the activity. 3 W. D. McElroy, J. W. Hastings, V. Sonnenfeld, and J. Coulombre, J. Bacteriol. 67, 402 (1954).

[151]

BACTERIAL LUCIFERASE

859

Fraction D. The supernatant from fraction C is brought to 2.67 M ammonium sulfate (0.65 saturation). This fraction contains 15 to 20% of the total protein and 90 % of the activity. In fact, it may assay substantially higher total activity than fraction A, owing to assay in a more concentrated solution and to the removal of inhibitors. Fraction E. A fraction at 2.87 M ammonium sulfate contains so little protein and so little activity that it is not worth taking. Fraction D thus contains essentially all the luciferase with a specific activity which may vary from 1200 to 3000 light units per milligram of protein but is about five times the specific activity of fraction A. The variation is due largely to the particular strain of bacterium used. Besides protein impurity this preparation contains large amounts of nucleic acid. Attempts to remove this by protamine sulfate or manganese ion were unsuccessful. The fractionation was followed by optical density measurements at 280 and 260 mu. It was found that the nucleic acid tended to precipitate in the higher ammonium sulfate fractions. Thus, after repeated fractionation the worst fractions are almost pure nucleic acid and the best have a "280 to 260 ratio" of about 1.2. Fraction D is repeatedly refractionated in ammonium sulfate. The point of firstprecipitation depends on the concentration of the proteins and the character of the protein impurities but usually occurs around 2 M ammonium sulfate. Small increments of the solid salt are added so that the protein is divided into convenient amounts. All fractions are analyzed for protein, enzymatic activity, and ultraviolet absorption. Similar fractions are combined and refractionated. The table presents the results from a single run including the isoelectric precipitate, A, the first active ammonium sulFRACTIONATION OF LUCIFERASE BY AMMONIUM SULFATE

Fraction A D Dl D~ D3 D4 D5 86

Ml.

Total L.U. ~

186 26 2.5 5 9 8 3

986,000 1,639,000 54,000 210,000 864,000 384,000 3,000

Total protein, E280 b E ..... mg. L . U . / m g . protein ~ E . . . . /1000 L. U. mtL 2880 670 58 101 230 182 28

341 2420 925 2080 3760 2100 107 5200

0.705 0.712 0.89 0.78 0.79 0.74 0.57 1.24

9.8 1.41 1.7 1.04 0.62 1.75 11.8 0.29

260 260 265 265 263 260 260 275

L. U. = light units as defined in text. b E = optical density determined in a Beckman D U spectrophotometer in a 1-cm. cell.

860

RESPIRATORY ENZYMES

[151]

fate precipitate, D, and a series, D1 to Ds, derived from D. If the per cent protein and activity of the subfractions of D are calculated with the values for D taken as 100 %, 90 % of the protein and 93 % of the activity are recovered. Further fractionation may be accompanied by loss of activity. We have tried stabilizing the enzymes by the addition of cysteine, FMN, or the aldehyde without marked success. In fact, cysteine and the aldehyde, in the presence of the enzyme, form an inhibitor. In the last row of the table are given the characteristics of one of the best fractions we have obtained. About 70 % of this protein is in a symmetrical peak on electrophoresis, and activity seems to be correlated with this peak. The sedimentation constant is consistent with a molecular weight of about 100,000. Properties

Stability. Bacterial luciferase slowly loses activity even in the frozen state. It is rapidly inactivated at temperatures above 40 °. The enzyme can be dialyzed in the cold without great loss of activity with 0.02 M pyrophosphate buffer and 2 X 10-5 M FMN. Various attempts to demonstrate a metal requirement by dialysis against metal-free buffers have been unsuccessful. Effect of Inhibitors. The enzyme is particularly sensitive to various SH reagents, p-Chloromercuribenzoic acid at 4 X 10-e M inhibits light emission approximately 50%. This inhibition can be reversed by glutathione. Riboflavin is a potent inhibitor of luminescence (2 X 10 -e M gives approximately 50 % inhibition). It appears to compete with FMN. Cyanide and Versene likewise inhibit, as does copper, iron, and other heavy metals. Various quinones and ferricyanide inhibit light emission by removing reduced DPN. As indicated below, this inhibition is due to the rapid reduction of these compounds by D P N H in the presence of bacterial luciferase. Reduction of Dyes and FMN. In the absence of aldehyde bacterial luciferase catalyzes the reduction of methylene blue, various quinones, ferricyanide, and F M N by D P N H without light emission. In the presence of aldehyde there is a competition between light emission and the reduction of the various compounds." F M N does not appear to be required for the reduction of quinones and ferricyanide. 4 Requirements for Light Emission. Bacterial luciferase catalyzes a lightemitting reaction in the presence of oxygen, reduced FMN, and a longchain aldehyde. There is an absolute requirement for all these compo4 W. D. McElroy and A. A. Green, Arch. Biochem. and Biophys. §6t 240 (1955).

[152]

ASSAY AND PROPERTIES OF HYDROGENASES

861

nents, and all are utilized during the reaction. Cormier and Strehler 5 have shown that a number of long-chain aldehydes will function in the reaction. Dodecyl or tetradecyl aldehyde are excellent substrates. With F M N various reducing agents will support light emission. Reduced safranin T, indigotrisulfonate, and rosindulin GG are all effective. Strehler et al. e reported that reduced riboflavin would support luminescence in crude extracts. Our studies with the purified enzyme indicate, however, that F M N is an absolute requirement. Reduced D P N and T P N are both effective reducing agents and appear to be the natural substrates for light emission. Chemically reduced F M N is the most effective substrate for light emission and the evidence shows quite clearly that pyridine nucleotides are not required for luminescence. Kinetics. The kinetics of light emission starting with reduced F M N indicate that two F1VINH~ molecules combine with luciferase. It seems likely that a complex interaction between oxygen, two reduced F M N molecules, and aldehyde is necessary for luminescence. The concentration of reduced F M N which gives approximately one-half maximum light intensity is 2.5 X 10-8 M. The relationship between light intensity and enzyme concentration is proportional when reduced F M N is used but is nonlinear when D P N H and F M N is used. This latter effect appears to be due to the autoxidation of reduced F M N which is formed from D P N H and FMN. pH and Temperature Optimum. Light emission has been observed over a pH range of 5.5 to 8.5, with a peak at 6.8. At the latter pH the temperature optimum is approximately 27 °, which is remarkably similar to that observed in the intact bacterium. 5 M. J. Cormier and B. L. Strehler, J. Am. Chem. Soc. 75, 4864 (1953). 6 B. L. Strehler, E. N. Harvey, J. J. Cheng, and M. C. Cormier, Proc. Natl. Acad. Sci.

U.S. 40, 10 (1954).

[152] Assay and Properties of Hydrogenases By ANTHONY SAN PIETRO

Hydrogenase activity may be defined as the enzymatic activation of molecular hydrogen. The main methods which have been employed to assay hydrogenase activity, in cell-free preparations, are as follows: (a) The reduction of some substrate by molecular hydrogen. Various acceptors which have been used in this assay system include methylene

862

RESPIRATORY ENZYMES

[152]

blue 1 and other dyes, ~ ferricyanide, I,~ nitrate, 4,5 fumarate, 5 oxygen; 6 etc. (see also Vol. I I [129]). (b) The exchange reaction between molecular hydrogen and h e a v y water or between deuterium gas and normal water. 8,6-9 (c) The ortho-para hydrogen conversion reaction. 8 (d) Evolution of molecular hydrogen from a variety of substrates.m0.li The ability to catalyze any one of the above reactions has been used to demonstrate hydrogenase activity. However, catalysis of the exchange reaction has been generally assumed to be a fundamental p r o p e r t y of hydrogenase and is perhaps the most ideal assay system. 3,6,7,12 As noted b y Fisher et al. 6 this assay circumvents any difficulties connected with the activation of the reducible substance and directly measures the primary reaction catalyzed by hydrogenase. Unfortunately, the exchange reaction has not found widespread application probably owing to the lack of the necessary facilities. Most investigators have used the reduction of various acceptors b y molecular hydrogen as an assay for hydrogenase. As pointed out b y H y n d m a n et al. ~ interpretation of results from these studies is handicapped b y the necessity for intermediate electron carriers or additional enzymes for activation of the acceptors. The present chapter is concerned primarily with a description of those assay systems which make use of molecular hydrogen as substrate (methods (a), (b), and (c) above). For purposes of comparison, it will be assumed t h a t the source of the enzyme is Proteus vulgaris for each of the assays described.

Assay Methods 1. Exchange Reaction. s,8 P r i n c i p l e . The exchange reaction between molecular hydrogen and h e a v y water (H2 ~ H D O ~ H D W H20) results in the appearance of deuterium in the gas phase. The rate of the i H. Gest, J. Bacteriol. 63, 111 (1952). 2 W. K. Joklik, Australian J. Exptl. Biol. and Med. Sci. 28, 321 (1950). 8L. A. Hyndman, R. H. Burris, and P. W. Wilson, J. Bacteriol. 65, 522 (1953). 4 A. I. Krasna and D. Rittenberg, J. Bacteriol. 68, 53 (1954). H. Gest, in "Phosphorous Metabolism" (W. D. MeElroy and B. Glass, eds.), Vol. II, p. 522, The Johns Hopkins Press, Baltimore, Md., 1952. H. F. Fisher, A. I. Krasna, and D. Rittenberg, J. Biol. Chem. 209, 569 (1954). 7 W. Curtis and E. J. Ordal, J. Bacteriol. 68, 351 (1954). 8 A. I. Krasna and D. Rittenberg, J. Am. Chem. Soc. 76, 3015 (1954). 9 A. I. Krasna and D. Rittenberg, Proc. Natl. Acad. Sci. (U.S.) 40~ 225 (1954). 10It. I). Peck and H. Gest, Bacteriol. Proc. p. 117 (1955). n H. Gest, Bacteriol. Revs. 18~ 43 (1954). 1~L. Farkas and E. Fischer, J. Biol. Chem. 167, 787 (1947).

[152]

ASSAY AND PROPERTIES OF HYDROGENASES

863

reaction is followed b y removing samples of gas at various time intervals and analyzing for deuterium with a mass spectrometer. 13

Reagents 0.15 M phosphate buffer, p H 6.7. 99.5 per cent D20.14 Na2S204. Hydrogen gas rendered O~-free by passage over hot copper. Enzyme. Use an a m o u n t of enzyme to give a first order rate constant, k, of 1 X 10-3 per minute or a change in the deuterium concentration of the gas phase of 0.2 atom per cent excess per hour. 1~ For calculation of k see Definition of Activity below.

Procedure. The reaction is carried out in 30-ml. flasks fitted with a stopcock for evacuation, as illustrated in Fig. 2 of the paper by H o b e r m a n and Rittenberg. 16 Each flask contains enzyme (e.g. 0.5 ml. containing 1 mg. of total N), 0.5 ml. of 99.5% D20 and 0.15 M phosphate buffer, pH 6.7, to give a final volume of 5 ml. After cooling in ice for 10 minutes, approximately 1 mg. of solid Na2S204 is added 17 and the flasks are immediately evacuated b y a mechanical pump for an additional 10 minutes. The flasks are filled to approximately 40 cm. pressure with hydrogen, reevacuated, and refilled with hydrogen to between 30 and 60 cm. pressure. The flasks are shaken at room temperature on a r o t a r y shaker at 100 r.p.m, and aliquots of gas removed at intervals for analysis. Definition of Activity. The activity is defined in one of two ways; either as the value of dC/dt, in which C is the deuterium concentration in atom per cent excess of the gas phase and t is in minutes or as the value of the first order rate constant, k. This latter value is calculated from the slope of the curve of log (Coo - C) against time where C and C~¢ are the atom per cent excess deuterium in the gas phase at time t and at equilibrium, 18 See Vol. IV [21]. 14 H e a v y water m a y be obtained on allocation from the Atomic Energy Commission a n d purchased from the Stuart Oxygen Company, San Francisco, California. ~ The exchange reaction a n d the ortho-para hydrogen conversion reaction involve (1) solution of hydrogen in the medium a n d (2) activ~,tion of the dissolved hydrogen by the enzyme. At low enzyme concentration, the rate is proportional to the enzyme concentration a n d the rate limiting step is the activation of the dissolved hydrogen by the enzyme. At high enzyme concentration, the reverse is true, t h a t is, the rate is i n d e p e n d e n t of the enzyme concentration a n d the rate limiting step is the solution of hydrogen in the medium. Under the conditions described herein, the rate of the reaction is proportional to the enzyme concentration. ~6 H. H o b e r m a n a n d D. Rittenberg, J. Biol. Chem. 147, 211 (1943). 17 The a m o u n t of hydrosulfite necessary to activate a particular cell-free system seems to be dependent solely on the a m o u n t of oxygen dissolved in the water. 6

864

RESPIRATORY ENZYMES

[152]

respectively, and t is in minutes. The equilibrium value C~ found after 24 hours is 3.30 atom per cent excess deuterium and is identical to t h a t resulting from catalysis b y active platinum. Specific activity m a y be defined as the activity per rag. of total N. 2. Conversion Reaction. 8 Principle. The conversion reaction is defined as the reaction which converts parahydrogen to normal hydrogen. is The rate of the conversion reaction is determined b y measuring the concentration of parahydrogen in the gas phase at various time intervals according to the thermal conductivity m e t h o d of Bonhoeffer and Harteck. 19,2° This m e t h o d is based on the different thermal conductivities of the hydrogen modifications and involves the measurement of the resistance of a thermal conductivity cell containing the gas sample to be analyzed.

Reagents 0.15 M phosphate buffer, p H 6.7. Na2S20~. 50 to 60 per cent parahydrogen m a y be prepared b y adsorbing purified hydrogen on a degassed coconut charcoal catalyst at 60 to 70 ° K. TM The time necessary to establish spin equilibrium is dependent on the activity of the charcoal used; at the end of this time, the gas is desorbed. Enzyme. Use an a m o u n t of enzyme to give a first order rate constant, k', of 2 X 10 -3 per minute. ~5For calculation of k' see p. 865.

Procedure. The reaction is carried out in the same flasks as for the exchange reaction. ~s The procedure is identical to t h a t used for the exchange reaction with the exceptions t h a t no h e a v y water is added and the gas phase is parahydrogen. T h e flasks are shaken as previously described. Samples of gas are removed at various time intervals and analyzed for the concentration of parahydrogen in the following manner. The thermal conductivity cell is immersed in liquid nitrogen (77 ° K.) and its filament heated b y a direct current. The sample to be analyzed is introduced into the conductivity cell and the pressure adjusted to 30 ram. The resistance of the cell is measured b y making it one leg of a Wheatstone bridge. The bridge is balanced b y a slide wire resistance. is The term normal hydrogen denotes the equilibrium mixture at room temperature of ortho- and parahydrogen (25 per cent para and 75 per cent ortho). The term parahydrogen is used for hydrogen containing more than 25 per cent parahydrogen. A discussion of the various nuclear spin isomers of hydrogen can be found in the book by Farkas. 19 ~gA. Farkas, "Orthohydrogen, Parahydrogen and Heavy Hydrogen," Cambridge University Press, New York, 1935. s0 K. F. Bonhoeffer and P. Harteek, Z. physik. Chem. B4, 113 (1929).

[152]

ASSAY AND PROPERTIES OF HYDROGENASES

865

The reading of the slide wire, R, is proportional to the concentration of parahydrogen. Definition of Activity. The activity is defined as the value of the first order rate constant, k'. The value of this constant is calculated from the slope of the curve of log AR versus time, in minutes, where AR is the difference between the value of R when the conductivity cell is filled with the gas sample and when filled with normal hydrogen. Specific activity may be defined as the activity per rag. of total N. 3. Reduction of Methylene Blue. Principle. The method is based on the measurement of hydrogen uptake manometrically in the presence of methylene blue as the hydrogen acceptor.

Reagents 0.15 M phosphate buffer, pH 6.7. 0.05 M methylene blue solution. Fieser's solution. 21 Dissolve 20 g. of KOH in 100 ml. of water and add 2 g. of sodium anthraquinone-f~-sulfonate and 15 g. of Na2S204 to the warm solution with stirring until dissolved. The blood red solution is ready for use when it has cooled to room temperature. Hydrogen gas which has been purified by passage over hot copper to remove oxygen. Enzyme. Use an amount of enzyme to give a hydrogen uptake of about 500 ~l. per hour.

Procedure. Each Warburg cup contains enzyme (e.g. 0.25 ml. containing 0.5 rag. of total N) and 0.15 M phosphate buffer, pH 6.7, in the main compartment, 0.2 ml. of methylene blue in the side arm, and 0.2 ml. of Fieser's solution in the center well with filter paper, total liquid volume 3.0 ml. The vessels are flushed with hydrogen for 5 minutes, closed and equilibrated for 1 hour. After equilibration, the reaction is started by tipping in the methylene blue from the side arm. Definition of Specific Activity. The specific activity, QH,(N), is generally defined as the rate of hydrogen uptake, in td. per hour, per rag. of total N. Preparation and Properties ~2,2~

1. Proteus vulgaris. 4,6 Growth Medium. Proteus vulgaris is grown at 37 ° for twelve to twenty hours in 15 liters of broth of the following 21 A. I. Vogel, " A Textbook of Practical Organic Chemistry," Longmans, Green and Co., New York, 1948. 22 See Vol. II [129] for description of hydrogenase of Clostridium kluyveri. 28 The oxidation and evolution of molecular hydrogen by microorganisms has recently been reviewed by Gest. 11

866

RESPIRATORr ENZYMES

[152]

composition: Na2HPO4, 7.93 g. ; KH~PO4, 1.47 g. ; glucose, 5.0 g.; nutrient broth (Difco), 10.0 g.; NaC1, 5.0 g.; casamino acids (Difco), 5.0 g.; FeSO4.7H20, 0.005 g.; and H20, 1 liter. The bacteria are harvested by centrifugation in a Sharples supercentrifuge and washed twice with water. The cell paste may be stored for several weeks in the cold without appreciable deterioration. Preparation of Cell-free Extract. 6 g. of the bacterial paste is diluted to 25 ml. with water and treated for 20 minutes in a Raytheon 9 kc. oscillator. The cell debris is removed by centrifugation at 13--20,000 )< g. The extract is generally stored in the cold under hydrogen. The activity appears to reside in the particulate elements of the cell and may be sedimented by centrifugation at higher speeds (Krasna and Rittenberg, personal communication). Nitrogen values, as determined by the Kjeldahl procedure, are in the range of 3 to 4 mg. of total N per ml. Properties. The properties described below are based on experiments with intact cells (prior to 1954) and with cell-free extracts (1954 et seq.). It has been suggested 16 that the enzyme is an iron-porphyrin-protein complex which is active only in the reduced (ferrous) form. The evidence presented in support of this conclusion is: (a) The active enzyme is inhibited by carbon monoxide and the inhibition is partially reversed by light. With equal volumes of hydrogen and carbon monoxide, 80% inhibition occurs in the dark and 55% in the presence of light; (b) The exchange activity is depressed when the cells are preincubated in the presence of oxygen. Complete inactivation occurs when the bacterial suspension is shaken with oxygen for 24 hours. This inactivation by oxygen is reversible and the enzyme can be reactivated by incubation under hydrogen or by the addition of glucose, pyruvate, formate, succinate, 24 fumarate or sodium hydrosulfite; (c) The addition of cyanide anaerobically to the actively exchanging system in 10-2 M concentration is without effect. When cyanide is added to the system under aerobic conditions in 10-3 M concentration, the enzyme is completely and irreversibly inactivated. Oxygen inhibits hydrogenase by two different mechanisms; namely, oxidation and oxygenation. 6 The oxygenated enzyme is inactive and can be reactivated by any process which removes oxygen; for example, addition of hydrosulfite, 17 or glucose oxidase and glucose, or physically by degassing. 6 Hydrosulfite is to be preferred since it cannot only deoxygenate the enzyme, but also reduce it. Neither thioglycolate nor hypophosphite at a concentration of 10-2 M causes any appreciable activation of the oxygenated enzyme. 8 ~In the experiments of Farkas and Fischer,1~ the addition of succinate had no restoring effect on the partially inactivated system.

[152]

ASSAY AND P R O P E R T I E S OF HYDROGENASES

867

Heavy suspensions of P. vulgaris fail to reduce methylene blue under hydrogen when the system is made 2% with urethane; whereas the exchange reaction is not inhibited under these conditions.18 When added to the exchanging system (which is operating under anaerobic conditions), the following compounds are without effect on the rate of the exchange reaction: 1% fluoride, 2% urethane, l0 -3 M iodoacetate, 10-2 M K2S2Os and 0.08 M malonate.16 Aerobic incubation with Ag+ results in complete inhibition of the exchange reaction at a concentration of 10-2 M, 85% inhibition at 10-3 M and is without effect at 10-4 M.IG The pH optimum for both the exchange reaction 16 and the reduction of nitrate by molecular hydrogen 4 is approximately 7. For the reduction of nitrate by molecular hydrogen, the system is saturated at a concentration of 0.02 % nitrate. The reduction of one mole of nitrate is accompanied by the uptake of close to one mole of hydrogen. Nitrite is not reduced further by this system in the presence or absence of benzyl viologen. Both nitrate and nitrite inhibit the deuterium exchange reaction. 4 In each case, the inhibition is 27% with an inhibitor concentration of 10-~ 31. The rate of hydrogen oxidation with fumarate is increased by the addition of fumarate to the growth medium. 1~ However, the level of the exchange activity is unaffected by the presence or absence of fumarate during growth. The rate of the exchange reaction is retarded by the addition of fumarate. The effect is related to the fumarate reduction activity of the cells; the higher the hydrogenation activity toward fumarate, the lower is the rate of exchange in the presence of fumarate. The enzyme is completely inhibited by high concentrations of nitric oxide. 9 At nitric oxide concentrations of 2 × 10-3, 8 × 10-3 and 1 × 10-1 vol. per cent in the gas phase, the per cent inhibition is 87, 93 and 97, respectively. With 1 vol. per cent or more, the inhibition is not reversible even by the addition of sodium hydrosulfite. With inhibitor concentrations of less than 1 vol. per cent, the inhibition could be partly reversed by removal of the nitric oxide-hydrogen mixture and its replacement with hydrogen. Nitrous oxide is not an inhibitor of hydrogenase. 2. Escherichia coli. The preparation described is essentially that reported by Gest. Growth Medium. E. coli, strain B, is grown in deep stationary culture for 8 to 24 hours at 37 ° in a medium of the following composition: Difco peptone, 5 g. ; glucose, 10 g. ; Difco beef extract, 3 g. ; NaC1, 5 g. ; distilled water, 1 liter; pH adjusted to 7.5. Preparation. 12 g. (wet weight) of 8-hour-old cells are washed with

868

RESPIRATORY ENZYMES

[152]

25 ml. of water and ground in a mortar by hand with 30 g. of alumina (Alcoa A-303). After extracting the paste for 20 minutes with 60 ml. of water and removing the alumina, etc., by centrifugation, the brown colored extract is treated with ~ 0 vol. of 1 M MnC12 which precipitates a considerable amount of nucleoprotein. The precipitate is removed and the hydrogenase precipitated by 50% saturation with (NH,)2SO4. This precipitate is dissolved in water and the residual insoluble particles removed by centrifugation. The highest specific activity reported, at this stage of purification, is 30,000 ~l. hydrogen per hour per mg. protein N at 37 ° with methylene blue as the acceptor. The enzyme is generally stored at 5 ° under hydrogen. Properties. The activity of the cell-free enzyme is markedly depressed by oxygen and this inactivation is partially reversed by prolonged incubation with H2.1,~,25 The ability of various reducing agents to reactivate the enzyme varies with the " n a t u r e " of the enzyme preparation. Ferrous sulfate, thioglycolic acid, GSH, ammonium polysulfide and sodium hydrosulfide, individually, at a concentration of 10-3 M, rapidly activates the ammonium sulfate-precipitated enzyme to the same degree as prolonged exposure to H2. 2 With the pre-aerated crude extract, incubation under hydrogen for only 5 minutes in the presence of Na2S204 causes a comparable stimulation; however, reducing agents other than Na2S204 are without effect under these conditions. 2 Cyanide at a concentration of 10-2 M inhibits aerated preparations about 9 % whereas it causes a 47 % inhibition after two hours anaerobic incubation. 28 Carbon monoxide inhibits the enzyme and the inhibition is not reversible by light. 36 A somewhat constant inhibition of about 70 % is obtained when the carbon monoxide/hydrogen ratio is varied from 9:1 to 1 : 1. 38 The above-mentioned results with cyanide and carbon monoxide are different from those found for Proteus vulgaris. In contrast to the particulate preparations from anaerobically grown cells, the soluble enzyme reduces methylene blue (and a few oxidationreduction indicators of the type of methylene blue) but shows no activity with nitrate, fumarate, ferricyanide, or oxygen. 2,5 Cell-free preparations which show high activities with methylene blue do not catalyze the reduction of cytochrome c or pyridine nucleotides ~,2 (cf. Vol. II [129]). The enzyme is not inhibited by 1,10-phenanthroline, a,a'-dipyridyl, or diethyldithiocarbamate in concentrations up to 0.01 M. The hydrogenase of E . coli shows almost maximal activity over a broad pH range (5.5-8.5). 3 ~5K. J. C. Back, J. Lascelles, and J. L. Still, Australian J. Sci. Research 9, 25 (1946). ~ W. K. Joklik, Australian J. Exptl. Biol. and Med. ,.~ci. 28, 332 (1950).

[152]

ASSAY AND PROPERTIES OF HYDROGENASES

869

3. Desulfovibrio desul]uricans. This organism, which exhibits unusually high hydrogenase activity, has been used by Sadana and Jagannathan ~7as starting material for the preparation of the most active hydrogenase fraction described to date. Preparation. 27 "Hydrogenase was extracted from acetone-dried bacteria (specific activity 5 X 104 td. per hour per mg. N) with 0.2 M phosphate buffer, pH 6.4, and centrifuged at 18,000 × g for 2 hours. The supernatant was heated at 60 ° for 10 minutes and centrifuged to remove the denatured proteins. The supernatant, which contains hydrogenase, was then adjusted to pH 4.5 and the small precipitate formed was removed by centrifugation." Hydrogenase was then precipitated by adjustment of the pH to 4.0 and redissolved in 0.1 M Tris buffer at pH 7.0. The solution was adjusted to pH 5.0 with acetic acid, heated at 50 ° for 2 minutes, and centrifuged to give a water clear pale pink solution.28 The recovery was about 90% and the enzyme had a specific activity of 2.5 X 108 ~l. per hour per mg. N at 34 ° with methylene blue as the acceptor. Properties. Like other hydrogenase preparations, this enzyme is sensitive to oxygen and activated by sulfhydryl compounds. The purified enzyme could be stored in the frozen state without loss of activity. It could be precipitated by (NH4)2S04 between 70 and 100% saturation at pH 5.5 and was not sedimented by centrifugation for one hour at 80,000 X g. The activity with riboflavin-5'-phosphate as hydrogen acceptor was less than 5 % of that obtained with methylene blue. 4. Clostridium pasteurianum. 29 Preparation. Cell-free extracts are prepared by subjecting the organism to sonic vibrations. The purification of the enzyme involves the following procedures: separation of the particulate fraction by centrifugation, removal of impurities with protamine sulfate, followed by zinc hydroxide gel and finally fractional precipitation with (NH4)2S04. Properties. The enzyme is not sedimented by centrifugation at 144,000 X g for 30 minutes and has a specific activity of 7.5 X 105 ~I. per hour per mg. protein N with methylene blue as the acceptor. Treatment of the enzyme with ammonium sulfate, followed by dialysis, reduces the activity with either methylene blue or cytochrome c to a very low level. The activity with methylene blue can be restored by the addition of FAD; it is necessary to add both FAD and Mo to restore cytochrome c activity. The presence of phosphate is required only for the metal catalyzed reaction. 27 j . C. Sadana a n d V. J a g a n n a t h a n , Biochim. et Biophys. Acta 14, 287 (1954). 28 This information was kindly furnished b y Dr. Sadana. 22 A. L. Shug, P. W. Wilson, D. E. Green, a n d It. R. Mahler, J. Am. Chem. Soc. 76, 3355 (1954).

870

RESPIRATORY ENZYMES

[152]

T h e difference spectrum of the purified enzyme reveals maxima at 390 m~ and 450 m~ and resembles t h a t of riboflavin. F A D in boiled extracts is demonstrable b y spectroscopic and enzymatic tests. Participation of the flavin in the action of the enzyme is indicated b y the negligible difference in spectrum between the "oxidized e n z y m e " and the " r e d u c e d e n z y m e " in an evacuated cuvette. This observation is interpreted to mean t h a t the interaction of hydrogen with flavin is reversible; at low pressures of hydrogen reduced flavin is oxidized to H2 gas and oxidized flavin. 5. Other Sources. Hydrogenase has been reported in cell-free preparation from a variety of other bacteria; A zotobaeter vinelandii,3 A zolobaeler agile, 3° Micrococeus aerogenes, 7 Rhodospirillum rubrum, l Hydrogenomonas ruhlandii, 3~ Micrococcus lactilyticus, 32 and Hydrogenomonas facilis. ~3 Addendum: Sadana and J a g a n n a t h a n (Biochim. et Biophys. Acta, in press) have been able to demonstrate an activation of the hydrogenase of Desulfovibrio desulfuricans by ferrous chloride, with an enzyme purified by a modified procedure, which includes acetone precipitation and adsorption on calcium phosphate gel. T h e y have found in addition t h a t the enzyme is cyanide-sensitive. 2s

a0 M. Green and P. W. Wilson, J. Bacteriol. 65, 511 (1953). a~L. Packer and W. Vishniac, Biochim. et Biophys. Acta 17~ 153 (1955). 3 : H. Whiteley and E. J. Ordal, Bacteriol. Proc. p. 117 (1955). 3~D. E. Atkinson and B. A. McFadden, J. Biol. Chem. 210, 885 (1954).

List of Abbreviations (Selected from Volumes I and II) A

Ac, acetate ACF, anhydrocitrovorum factor Ac-SCoA, acetyl coenzyme A ADP, adenosine diphosphate ADPR, adenosine diphosphate ribose AMe, S-adenosylmethionine 2'-AMP, 2'-adenylic acid (a adenylic acid) 3'-AMP, 3'-adenylic acid (b adenylic acid) 5'-AMP-5'-adenylic acid (muscle adenylic acid) AR, adenosine ATP, adenosine triphosphate ATPase, adenosine triphosphatase B

BAL, British anti-lewisite BCG, bacillus of Calmette and Guerin (strain of M. tuberculosis) C CDR, cytosine deoxyriboside CF, citrovorum factor CHOFAH4, Nl°-formyltetrahydrofolic acid CoA, coenzyme A CoASH, coenzyme A, reduced

Cr, creatine CR, cytidine CTP, cytidine triphosphate D

DAP, dihydroxyacetone phosphate DFP, diisopropyl fluorophosphate DNA, deoxyribonucleic acid DNase, deoxyribonuclease DNP, 2,4-dinitrophenol DP-enzymes, diisopropyl phosphate enzymes DPN, diphosphopyridine nucleotide DPNase, DPN nucleosidase DPNH, diphosphopyridine nucleotide, reduced DPCoA, dephospho-coenzyme A E

EDTA, ethylenediaminetetraacetate (Versene) F

FAD, flavin adenine dinucleotide FAH4, tetrahydrofolic acid FAH4CHO, Nl°-formyltetrahydrofolic acid FDP, fructose-l,6-diphosphate FMN, flavin mononucleotide 871

872

LIST OF ABBREVIATIONS

F-l-P, fructose-l-phosphate F-6-P, fructose-6-phosphate G

GA, glyceraldehyde Gal-I-P, galactose-l-phosphate GAP, glyceraldehyde-3-phosphate GPC, glycerophosphorylcholine GPE, glycerophosphorylethanolamine G-l-P, glucose-l-phosphate G-6-P, glucose-6-phosphate GR, guanosine GSH, glutathione GSSG, glutathione, oxidized H

HxR, inosine

I, 5 (4) amino-4(5)-imidazolecarboxamide IAA, iodoacetate IDP, inosine diphosphate 5'-IMP, 5'-inosinic acid INH, isonicotinic acid hydrazide IR, 5 (4)-amino-4(5)-imidazolccarboxamide riboside IRMP, 5 (4)-amino-4 (5)-imidazolecarboxamide ribotide ITP, inosine triphosphate K

KG, a-ketoglutarate L LTPP, lipothiamide pyrophosphate

M

MB, methylene blue ]~[-6-P, mannose-6-phosphate N

NS/IeN, N 1-methylnicotinamide NMN, nicotinamide mononucleotide NR, nicotinamide riboside NTZ, neotetrazolium O OAA, oxalacetate P PEP, phosphoenolpyruvic acid 3-PGA, 3-phosphoglyceric acid PGA-P, 1,3-diphosphoglyceric acid PNPA, p-nitrophenol acetate P, orthophosphate, inorganic PP, pyrophosphate, inorganic PuR, purine riboside PyR, pyrimidine riboside R

RNA, ribonucleic acid RNase, ribonuclease R-l-P, ribose-l-phosphate R-5-P, ribose-5-phosphate T TCA, trichloroacetic acid THAM, tris(hydroxymethyl) aminomethane TPNH, triphosphopyridine nucleotide, reduced

LIST OF ABBREVIATIONS

TPP, thiamine pyrophosphate TPN, triphosphopyridine nucleotide Tris, tris(hydroxymethyl) aminomethane TTZ, 2,3,5-triphenyltetrazolium

UDPGal, uridinediphosphogalacrose UDR, uracil deoxyriboside UR, uridine UTP, uridine triphosphate X

U

UDPG, uridinediphosphoglucose

873

XR, xanthosine

Author Index T h e numbers in parentheses are footnote numbers and are inserted to enable the reader to locate a cross reference w h e n the author's n a m e does not appear at the point of reference in the text.

A Aas, K., 140, 153 Abbott, L. D., 328 Abderhalden, E., 97 Abraham, E. P., 121, 123, 710, 711(12) Abrams, R., 774 Abul-Fadl, M. A. M., 527 Ackermann, W. W., 614 Adams, E., 84, 87, 92, 97, 101, 102, 104(15, 19) Adelberg, E. A., 250, 253(16) Adler, E., 221, 224(2), 707, 710, 711(4, 12) Aggeler, P. M., 149 Agner, K., 765, 768, 774, 782, 794, 795, 796, 797, 798, 799, 800, 801 Aihara, D., 232 ~keson, ,~., 752, 754(11), 774, 808, 809, 811,817 Alais, C., 77 Albaum, H. G., 600, 735 Alberty, R. A., 25, 688, 707, 708(6), 710(6), 711(6) Albrecht, G. S., 79, 80(9) Alderton, G., 786 Alexander, B., 347 Alexander, H. E., 433 Alicino, J. F., 120 Alivisatos, S. G. A., 663 Alkjaersig, N., 144, 145 Allen, F. W., 433 Allen, M. B., 421, 422(1) Allen, P., 755, 757 Allen, R. J. L., 582 Allfrey, V., 437, 445(2), 447(2) Allgdn, L. G., 442 Almy, L. H., 315 Altschul, A. M., 774 Altschule, M. D., 837 Ambros, O., 64 Ambrose, J. A., 9 Ames, S. R., 819, 822(6), 825, 832 Anderson, A. E., 25 Anderson, D. G., 720, 721(9) Anderson, L., 415, 729 Anderson, R. S., 853

Anderson, W. G., 839, 840 Andrews, E. B., 162 Anfinsen, C. B., 224, 225(6), 429, 435(13) Anslow, W. P., Jr., 364, 367(14) Anson, M. L., 3, 34, 55, 77, 79, 80(1), 81 Appleby, C. A., 745, 746 Archibald, R. IV[., 351,357, 358, 359, 365, 368, 375 Armstrong, A. R., 328 Armstrong, M. D., 311, 312(3) Arnon, D. I., 789 Aschaffenburg, R., 533 Asenjo, C. F., 56/57, 63 Asimov, I., 826 Astrup, J., 158, 162, 165, 166 Atkinson, D. E., 870 Audrain, L., 37, 53 Auerbach, G., 97 Avi-Dor, Y., 774, 794 Awapara, J., 178 Ayengar, P., 215, 217(13) Azarkh, R. M., 318 B

Bach, A., 809 Bach, S. J., 746 Bachur, N. R., 666 Back, K. J. C., 868 Bagdy, D. D., 160 Bagot, E. A., 371 Bailey, K., 160, 583, 584, 587, 596, 602 Bajaj, V., 577, 579(13), 580(13), 592 Baker, S. B., 631 Balcazar, M. R., 56/57, 62, 64 Ball, E. G., 449, 482, 483, 485, 740, 754 Balls, A. K., 13, 14, 23, 24, 35, 37, 56/57, 58, 59, 60, 61, 62, 63, 780 Baltscheffsky, H., 749 Bamann, E., 579, 581 Bancroft, F. W., 140 Bard, R., 417 Bardawill, C. J., 357, 360(6), 365(6), 688, 7O8 Bardos, T. J., 519 Barger, F. L., 519 Barker, H. A., 730, 731 875

876

AUTHOR INDEX

Barkulis, S. S., 603 Barnes, B. A., 450, 452(16) Barnes, F. W., Jr., 178 Barnett, R. C., 721 Barnhurst, J. D., 625 Barron, E. S. G., 434, 587 Barsky, J., 396 Bartlett, G. R., 203 Barton-Wright, E. C., 629, 630(3) Bartz, Q. M., 7 Baudet, P., 76 Bauer, E., 727 Baum, H., 489 Baxter, R. M., 631, 632(1) Beadle, G. W., 168, 235, 320 Bean, R. C., 37, 48(8), 51, 53 Becker, S. W., 828 Beers, R. F., Jr., 764, 781 Behrens, O. K., 93, 12 3 Beinert, H., 485 Bellamy, W. D., 646, 647 Bender, A. E., 200, 202(3), 211 Bendich, A., 148, 149 Bentley, R., 556 Bergeim, O., 316 Berger, J., 89, 93(6), 108 Bergeret, B., 333 Bergmann, M., 21, 66, 84, 89(5), 93(5), 100, 101(14), 102, 114, 399 Bergold, V. G., 35 Bern, H. A., 523 Bernheim, F., 202 Bernheimer, A. W., 447 Bernstein, P., 22 Berridge, N. J., 69, 70(2), 74, 75, 76(11), 77(11) Bertoye, A., 166 Bessey, O. A., 640 Bessman, M. J., 524 Bettelheim, F. R., 8, 160 Bier, M., 32, 35, 37, 5.1, 53 Binkley, F., 311, 312(3), 318, 320, 322(22) Bird, O. D., 630 Birnbaum, S. M., 107, 108(21), 109(21), 112(2), 114, 115, 117(1), 118(1, 10), 119(9), 382, 399 Blanehard, M., 209, 210(9), 211(9) Blaschko, H., 177, 191, 196, 197, 390, 393 Blau, K., 21 Bloch, K., 342, 343(2, 3, 4), 344

Block, R. J., 457 Bloom, E. S., 510, 519(10) Bodansky, O., 447, 735 Boeri, E., 749, 753, 754 Boeseken, J., 290 BSttger, I., 442 Bolomey, R. A., 433 Bonhoeffer, K. F., 864 Bonner, C. D., 528 Bonner, D. M., 253 Bonner, J., 849 Bonnichsen, R. K., 749, 765, 768, 770(9), 772(9), 775, 778, 780, 781, 782, 785, 789, 791 Booth, V. H., 836, 837(1, 2), 838(1, 2), 839, 841(1), 843(1, 2), 844(1), 845(1) Bordner, C. A., 818, 825(3) Borek, E., 267, 268(1), 269(1), 338, 341(7) Borsook, H., 260, 347, 348, 415 Bovarnick, M. R., 203, 698 Bowen, W. J., 599, 603, 604 Bradfield, J. R. G., 842, 843(30), 845, 846 Brahinsky, R. A., 36, 40, 50, 51 Brand, E., 311, 435 Brandenberger, H., 178 Bratton, A. C., 406, 407, 634 Braun-Men~ndez, E., 124, 136 Braunshtein, A. E., 318, 348 Brenner, M., 21 Bressler, B., 630 Brinkhaus, K. M., 140 Brinkman, R., 839 Brodie, A. F., 695, 697, 698 BrSmel, H., 200, 201, 204(2), 212 Brown, C. S., 755 Brown, D. M., 9, 17(17), 24, 59, 83, 89, 91(8), 93(8), 433, 568 Brown, G. L., 778, 794 Brown, H., 486 Brown, K. D., 9, 18(14), 25, 436, 447 Brown, R. A., 630 Brown, R. K., 450, 452(16) Brown, W. T., 203 Buchanan, J. M., 448, 453(9), 502, 503, 504, 509, 512, 514, 517(7, 17), 519 Biicher, T., 290, 291(7), 335, 404, 483, 600, 602(7), 619, 633, 668, 670, 673, 704 Bueding, E., 406

AUTHOR INDEX Bukin, V. N., 847, 848, 850 Bull, H. B., 35 Bullock, I. H., 160 Bumpus, F. M., 136 Burch, H. B., 640 Burk, D., 698 Burkholder, P. R., 630 Burris, R. H., 837, 862, 870(3) Burton, H. S., 123 Burton, K., 203, 204(17), 211 Butler, G. C., 549, 561, 562, 563 Buzzell, A., 35 Byerrum, R. U., 837, 842(13), 844(13) C Cahill, G. F., ~311 Calaby, J. H., 590, 595 Calkins, E., 827 Cammarata, P. S., 172, 178, 179(4), 180(4), 182(9), 289 Campbell, C. J., 630 Campbell, E. W., 149 Canellakis, E. S., 796 Cannan, R. K., 51, 167 Cantoni, G. L., 254, 255(3), 256(2), 257, 259, 260(4), 262 Caputto, R., 499, 602 Carlisle, C. H., 435 Carpenter, D. C., 56/57, 61 Carpenter, F. H., 433 Carroll, R. T., 160, 161(50) Carter, C. E., 111, 382, 433, 434, 442, 461 Carter, J. R., 145 Castafieda, M., 56/57, 62, 64 Cavalieri, L. F., 442 Cecil, R., 774, 808 Ceriotti, G., 434 Chain, E., 121 Chamberlin, H. A., 523, 528(4) Chance, B., 732, 733, 734, 735(8), 739, 740, 750, 764, 765, 767, 768, 769, 770(9), 772, 774, 775, 780, 781, 782, 785, 786, 789, 791, 795, 799, 802, 803, 807, 810, 811, 812, 813, 840 Chantrenne, H., 347, 348(6), 349(6) Charalampous, F. C., 448, 453(9), 512 Chargaff, E., 148, 149, 320, 322(23), 433, 441, 445, 446(35), 447 Chase, A. M., 852, 853

877

Chatagner, F., 333 Cheldelin, V. H., 631 Chen, S. Y., 302 Cheng, J. J., 861 Cherbuliez, E., 76 Chevillard, L., 637, 638(4), 639 Chiba, H., 846 Chinard, F. P., 376 Christian, W., 203, 213, 247, 255, 257, 260(5), 290, 302, 305, 308, 407, 416, 656, 673, 676, 698, 699, 700, 712, 713, 715(1), 722 Christman, J. F., 320, 322(26) Chung, C. W., 422 Ciminera, J. L., 166 Ciocalt~u, V., 3, 55, 329, 559 Ciotti, M. M., 308, 310(27), 475, 476(1), 477(1), 660, 662, 663, 666, 681, 683, 684(7, see 8), 685(7), 686(1, 2), 687(1) Clark, A. M., 836, 837, 838, 839, 843(7), 844(7), 846 Clark, D. G. C., 166 Clark, H. W., 741 Clark, V. M., 501 Clark, W. G., 198, 199(9) Clendenning, K. A., 837, 842, 843, 845 Cliffton, E. E., 166 Cohen, G. H., 218 Cohen, P. P., 170, 172, 176(1), 177(1), 178, 179(4), 180(4), 182(9), 289, 341, 350, 351(2), 354, 355, 796 Cohen, S. S., 149 Cohen-Bazire, G., 233, 237 Cohn, E. J., 450, 452(16), 792 Cohn, W. E., 433, 524, 540 Collier, H. B., 37, 38(13), 54(13) Collinson, E., 434 Colowick, S. P., 233, 237(3), 308, 310(27), 311, 411, 471, 475, 476(1), 477(1), 498, 599, 601, 602, 603(1), 604(1), 660, 661(2), 662, 663, 664, 665, 666, 681, 682, 683, 684(7, see 8), 685(7), 686(1, 2, 5), 687(1), 726, 728 Conn, E. E., 719, 720, 721, 722 Connors, W. M., 76, 77(15), 298, 299 Consden, R., 172 Conway, E. J., 22, 472, 841 Cook, R. P., 387, 388(5) Cooke, R., 472

878

AUTHOR INDEX

Coon, R. W., 151 Cooper, E. J., 443 Cooperstein, S. J., 741, 742 Copenhaver, J. H., Jr., 611, 613, 614(2) Cori, C. F., 583 Cormier, 1V[.J., 861 Corran, H. S., 707, 709(2), 710(2), 711 Corse, J., 826 Cosulich, D. B., 519 Couch, J. R., 630 Coulombre, J., 651, 854, 856; 858 Covo, G. A., 613, 615(7) Crandall, D. I., 292, 293, 294, 295(15), 299(12) Crane, R. K., 599 Crewther, W. G., 32 Crook, E. M., 847, 848 Cross, R., 613, 615(7) Cubiles, R., 433, 436, 523, 524, 526, 528(4, 13) Cunningham, L. W., Jr., 20, 35 Curtis, W., 862, 870(7) Cushing, M. L., 826 Cutolo, E., 675, 676(1) Czaky, T. Z., 417 D

Dabrowska, W., 443 Dainton, F. S., 434 Dainty, M., 580, 581(5, 6) Dalgliesh, C. E., 249, 508 Damodaran, M., 388 Darling, S., 158, 162 Dart, R. M., 528 Das, N. B., 221, 224(2) Das, R., 625 Davenport, H. E., 753 Davenport, H. W., 845 David, M. M., 357, 360(6), 365(6), 688, 7O8 Davidson, J. N., 486, 489 Davie, E. W., 19, 26, 27(12) Davis, A. R., 56/57, 62 Davis, B. D., 300, 301, 303, 305, 307(3), 309(5, 14), 311(25a), 380, 619, 686 Davis, N. C., 84, 87(4), 92, 97, 98, 99(10), 100(10), 101, 103, 104(17), 105(16, 17, 19) Davis, W. B., 63

Davis, W. W., 136 Davison, M. M., 529 Dawson, C. R., 818, 819, 822, 825, 826, 831, 832, 834, 835 Day, A. J., 839 De Baun, R. M., 76, 77(15) de Duve, C., 542 Dekker, C. A., 433 del Campillo, A., 303 del Capella de Fern~indez, M., 56/57, 63 Della Monica, E. S., 534, 535, 538, 557 Delory, G. E., 562 Delwiche, C. C., 267 Delwiche, E. A., 315, 316(4), 317(4) De Maria, G., 80, 81(11) Dent, C. E., 192 de Robertis, E., 157 Derouaux, G., 450, 452(16) Derow, M. A., 529 Desnuelle, P., 8, 9(7), 14(7), 16(7), 23, 26, 36(13), 315, 318(3) Desreux, V., 4 Detolle, P., 166 Deutsch, H. F., 37, 49, 51, 53, 765, 782, 783, 784 Devlin, T. M., 770 Dewan, J. G., 221, 707, 710(1), 711 Dickman, S., 434 Dietrich, L. S., 195 Dillon, R. T., 115 Dische, Z., 438 Dixon, M., 18, 482, 484(1), 746 Doctor, V. M., 630 Dogson, K. S., 324, 325, 326, 327, 328, 332 Doherty, D. G., 21 Dole, V. P., 446 Dolin, M. I., 213 Doty, D. M., 559 Doughty, C. C., 377, 378(16) Douglas, D. E., 631 Dounce, A. L., 379, 483, 765, 775, 776, 778, 780, 781 Drabkin, D. L., 749 Dreskin, O. H., 150 Dreyfuss, J.-C., 161 Driscoll, P. E., 84, 87(2) Dubnoff, J. W., 347, 348, 357 Dubos, R. J., 427, 432, 434(4) Dubuisson, M., 586 Ducay, E. D., 37, 48(8), 51, 53

AUTHOR INDEX Duckert, F., 155 Duke, A. J., 51, 53 Dunn, F. J., 832, 834(4) Dunn, M. S., 33 Durand, M. C., 432, 433(22) Durrum, E. L., 457 Duthie, E. S., 49, 122 du Vigneaud, V., 93 Dyckerhoff, H., 97 l] Eakin, R. E., 514 Edelhoch, H., 688, 707 Edlbacher, S., 202, 203 Edman, P., 136 Edsall, J. T., 164, 584 Edson, N. L., 170 Egami, F., 417, 746, 759 Egan, R., 25 Eggleston, L. V., 603, 604, 613, 614 Ehrenberg, A., 754, 774, 782, 784, 808, 813 Eichel, B., 741 Eiger, I. Z., 819, 822 Eiler, J. J., 593 Eirich, F., 35, 53 Eiscnberg, M. A., 19 Elam, D. W., 7 Elkins, E., 80 Ellfolk, N., 386, 387, 388, 390(10) Elliott, W. H., 264, 337, 338(2), 339(2) Ellis, D., 84, 85, 86, 87(3) Ellis, W. J., 56/57, 64 Ells, V. R., 435 Elowe, D., 691, 710 Elsden, S. R., 759 Elvchjcm, C. A., 704, 850 Emmett, A. D., 630 Engel, F. L., 370 Engel, M. G., 370 Engelhardt, V. A., 534 Englard, S., 640, 642, 645, 648, 675 Eppright, M. A., 631 Epps, H. M. R., 187, 188, 189, 647 Epstein, E., 157 Errera, M., 382 Evans, C. H., 625, 626(12) Evans, H. J., 408, 411, 414, 415(2) Evans, W. C., 273

879 F

Fabre, C., 8, 23, 26, 36(13) Fabriani, G., 625 Fager, J., 442 Fahcy, J. L., 153, 157 Fankuchen, I., 24, 435 Farkas, L., 862, 864, 866, 867(12) Farr, A. L., 234, 320, 401, 409, 412, 417, 428, 438, 454, 469, 472, 476, 490, 492(3), 494(3), 498, 547, 552, 566, 652, 661, 664, 666(1), 727, 730, 851, 857 Fasciolo, J. C., 136 Fearon, W. R., 486 Feigl, F., 406, 407 Feinstein, R. N., 780 Feldman, L. I., 171, 172, 173(11), 175(11), 176(11), 177(11), 178 Fellig, J., 434 Ferguson, J. H., 154, 159, 164, 166 Fergusson, J. D., 523, 528(5) Fergusson, R. R., 807, 813 Fermi, C., 64 Ferry, J. D., 160 Fessenden, R. W., 838 Fevold, H. L., 786 Fiale, S., 162 Filitti-Wurmser, S., 754 Fischer, E., 862, 866, 867(12) Fischer, F. G., 442 Fischer, H. A., 577, 579(4, 10), 580(4), 581, 582(8) Fischmann, J., 523, 528(4) Fisher, A. M., 841, 843 Fisher, H. F., 862, 863(6), 865(6), 866(6) Fisher, J., 186, 187(1), 188(1), 189(1) Fishman, W. H., 528 Fiske, C. H., 530, 534, 540, 547, 551, 571, 577, 582, 596 FitzGerald, P. L., 433 Fitzpatrick, T. B., 827, 828, 829 Flaks, J. G., 509, 514, 517(7, 17) Florey, H. W., 121 Florey, 5/[. E., 121 Flynn, J. E., 151 Flynn, R. M., 668 Fodar, P. J., 114 Folin, O., 3, 55, 260, 329, 559 Folley, S. J., 539

880

AUTHOR INDEX

Fones, W. S., 117, 118 Fontaine, T. D., 56/57, 63 Ford, J. H., 120 Forro, F., Jr., 35 Fouts, J. R., 396 Fowden, L., 178 Fowler, D. I., 192 Fraenkel-Conrat, H. L., 37, 48, 51, 53 Frampton, O. D., 765, 775 Frankenthal, L., 580, 581(3), 582(3) Fraser, P. E., 170, 382 Fratoni, A., 625 Frederieq, E., 37, 49, 51, 53 Freedman, R. I., 443 Fried, M., 21, 65 Friedemann, T. E., 170, 316, 730 Frieden, E., 835 Friedkin, M., 448, 449, 456 Friess, E. T., 587 Frisch-Niggemeyer, W., 442 Fromageot, C., 315, 318(3), 333 Fruton, J. S., 9, 21, 64, 65, 66, 68, 84, 85, 86, 87(2, 3), 100, 114 Fu, S.-C. J., 117, 118(10), 399 Fuchs, H., 253 Fujita, A., 622, 625, 626, 628(1), 7!9, 732 Fujiwara, M., 625 G Gailey, F. B., 755 Gale, E. F., 170, 171, 173(3, 4, 13), 174(3, 4), 176(3, 4), 177(13), 182, 187, 189, 241, 320, 322(24) Galston, A. W., 789 Garkovi, P. G., 94 Garner, R. L., 427 Garrison, L., 123 GavarrSn, F. F., 56/57, 62, 64 Gay, H., 441 Gehrig, R. F., 375, 376, 378(14) Gemmill, C. L., 835 George, P., 770, 773, 802, 807 Gerber, C., 64 Gerischer, W., 700, 713 Gest, H., 862, 865, 867, 868(1, 5), 870(1) Gibbs, R. J., 51, 53 Gilbert, J. B., 114 Gilbert, L. M., 432, 441, 442 Gillespie, J. M., 450, 452(16)

Gilmour, D., 590, 595 Gilvarg, C., 301 Giri, K. V., 625 Gjessing, E. C., 813 Gladner, J. A., 8, 9(9), 12, 19, 20, 21, 509, 517(7) Glendening, M. B., 149 Goddard, D. R., 719 Goebel, W. F., 434 Goldblatt, H., 124, 125(1), 126(4), 133(2), 134(5, 8), 135 Goldman, D. S., 663 Goldthwait, D. A., 509 Gollan, F., 134 Gomez, C. G., 171, 173(16), 175(16) Gomori, G., 292, 544, 546(4) Gordon, A. H., 172, 711 Gordon, M., 233, 470 Gorini, L., 32, 37, 53 Gornall, A. G., 357, 360(6), 365(6), 688, 7O8 Gots, J. S., 519, 695, 697 Graffiin, A., 652 Graham, W. R., 533 Grassmann, W., 64, 83, 88, 93, 97, 100, 105, 107, 115, 384 Graubard, M., 819 Grauer, A., 580, 582(4) Greco, A. E., 400, 436, 447, 568 Green, A. A., 136, 814, 856, 860 Green, D. E., 170, 172, 174(5), 175(5), 209, 210(9), 211(9), 225, 226(1), 485, 559, 613, 615(7), 707, 709(2), 710(1, 2), 711, 869 Green, M., 870 Green, N. M., 20, 32, 33, 35, 39, 40, 50, 51, 53, 78 Green, R. H., 559 Greenbaum, L. M., 524 Greenberg, D. M., 56/57, 62, 63(11), 289 Greenberg, G. R., 509, 510, 512, 517(11, 13), 518(11), 519(11) Greenfield, R. E., 775 Greenstein, D. S., 767 Greenstein, J. P., 107, 108(21), 109(2!), 110, 111, 112(2), 114(1, 2), 115, 117(1), 118(1), 119(9), 298, 299, 300(20), 318, 319, 380, 382, 383(2), 384(2), 399, 434, 442 Gregg, D. C., 821, 826

AUTHOR INDEX Gr~goire, J., 442 Griese, A., 700, 713 Grimm, P. W., 755 Grisolia, S., 350, 351(2), 355 Grob, D., 49 Grossowicz, N., 267, 268(1), 269(1), 338, 339, 341(7) Grunert, R. R., 302 Guba, F., 584 Gtinther, G., 221, 224(2), 707, 710, 711(4, 13) Guest, M. M., 158 Guevara-Rojas, A., 134 Guirard, B. M., 631 Gulland, J. M., 540, 561 Gunsalus, C. F., 275, 276, 277, 278(8) Gunsalus, I. C., 170, 171, 173(11), 175(11), 176(la, 11, 17), 177(la, 11), 178, 182, 183(18), 212, 215(1), 216, 236, 237(11), 238, 239(2), 241(2), 275, 276, 277, 278(8), 303, 319, 320, 322(19), 324, 646, 647 Gurd, F. R. N., 450, 452(16) Gutcho, S., 694 Gutfreund, H., 24, 35 Gutfreund, K., 160 Gutman, A. B., 328, 523, 528(6-9) Gutman, E. B., 328, 523, 528(6-9) Gutmann, H. R., 64 H

Haas, E., 124, 125, 126(4), 133(2), 134(5, 8), 135, 694, 697, 699, 703, 704, 711, 713, 715, 718(11, 12) Haehn, H., 388 Hakala, N. V., 843, 844(33) Hale, W. S., 63, 780 Halkerston, I. D. K., 330 Halliday, D., 317 Halvorson, H. O., 213, 215(6) Hamilton, P., 115 Hampil, B., 512, 518(15) Hand, D. B., 379 Handler, P., 202, 653 Hankinson, C. L., 76 Hansl, N., 841 Hanson, H. T., 83, 94, 95, 96 Happold, F. C., 238, 242 Harbury, H. A., 809

881

Harman, J. W., 602 Harpley, C. H., 733, 745, 746(4), 748(4) Harrer, C. J., 697, 699, 703(2) Harteck, P., 864 Hartley, B. S., 24 Hartman, S. C., 509, 519 Hartree, E. F., 202, 203(6), 235, 303, 405, 455, 482, 485, 487, 489, 657, 671, 674, 705, 727, 732, 733, 735, 737, 738, 739, 740, 745, 749, 750, 751(2), 753, 754, 769, 774, 802, 809, 811, 812 Harvey, E. N., 851, 861 Hasegawa, E., 625, 626 Hashimoto, K., 281, 282(11) Haskins, F. A., 167, 470 Hasselback, W., 586 Hastings, A. B., 836, 843(3) Hastings, J. W., 856, 858 Hatch, B., 442 Haugaard, N., 837 Haugcn, G. E., 170, 316, 730 Hawk, P. B., 316, 762 Hayaishi, O., 229, 245, 249, 250, 251, 252, 281, 490, 492(2), 688, 707 Hayano, M., 354 Hayflick, L., 166 Hearn, W. R., 65, 68 Heatley, N. G., 121 Hecht, L., 433, 524, 526(13), 528(13) Heise, R., 195 Hellerman, L., 203, 698 Hellstrom, H., 707, 710(3), 711(3) Helmer, O. iV[., 134, 136 Hems, R., 603, 604, 613, 614 Hendee, E. D., 745, 746(5) Hendley, D. D., 722 Heneage, P., 197 Hennessy, D. J., 622, 625 Henry, R. J., 120, 123(7), 124(7) Henry, S. S., 178 Henstell, H. H., 443 Heppel, L. A., 404, 405, 449, 453, 462, 463(18), 482, 484(4), 547, 548, 566, 568, 569, 575, 576, 603 Herbert, D., 764, 765, 768, 775, 778, 782~ 784, 785(1), 786, 788(1) Herbert, F. G., 527 Herbst, R. M., 290 Hern~ndez, A., 62 Herriott, R. M., 4, 5(3), 7, 13

882

AUTHOR INDEX

Hers, H. G., 542 Hess, W. C., 311 Hesselvik, L., 51 Heumiiller, E., 579, 581 Heuser, G. F., 698 Heyde, W., 83, 88, 93, 97, 100, 105, 107, 115 Hill, C. H., 629 Hill, F. W., 698 Hilmoe, R. J., 404, 405, 453, 462, 463(18), 547, 548, 566, 568, 569, 575, 576 Hilton, S., 72, 74 Himwich, W. A., 336, 337(5) Hinman, R. L., 241 Hird, F. J. R., 289 Hirs, C. H. W., 9, 12, 435, 436(54) Hirschmann, D. J., 53 Hoagland, M., 667 Hoberman, H., 863, 864(16), 866(16), 867(16) Hoff-JCrgensen, E., 464 Hoffmann-Ostenhof, O., 442 Hogden, C. G., 66, 213, 721, 722 Hogeboom, G. H., 168, 486 Hogness, T. R., 694, 697, 699, 703(2), 704, 711, 715, 718(12), 754, 774 Holden, M., 136 Holmberg, C. G., 486, 489 Holmes, B., 434 Holt, L., 780 Holter, H., 77 Holtz, P., 195 Homburger, F., 528 Hoogerheide, J. C., 217, 218(4) Hoover, S. R., 58 Horecker, B. L., 449, 482, 484(4), 497, 656, 670, 676, 694, 695, 699, 704, 711, 715, 718(12), 735, 754, 755, 760 Horn, F., 376 Horowitz, N. H., 211 Hotchkiss, R. D., 458 Houghton, J. A., 66 Housewright, R. D., 120, 123(7), 124(7), 171, 173(16), 175(16), 176 Howard, A., 441 Howell, M. J., 802 Huber, H., 580 Hudson, P. B., 529 Hiibscher, G., 489, 741

Huff, J. W., 257, 454, 655 Huggins, C., 528 Hughes, D. E., 219, 382 Hughes, W. L., 814 Hultquist, M. E., 519 Hunter, F. E., 613, 614 Hunter, M. J., 792 Hurst, R. 0., 549, 561, 562, 563 Hurwitz, J., 646 Hussey, C. V., 157 Hutchings, B. L., 629 Hyndman, L. A., 862, 870(3)

Ichihara, T., 388 Ingelmann, B., 577, 578(5), 579(5, 6), 58O(5, 6) Ingraham, J. L., 284 Ingraham, L. L., 826 Ingram, V. M., 25, 68 Irving, G. W., Jr., 56/57, 63 Itahashi, M., 759 Izumiya, N., 118, 399

Jackson, E. M., 540, 561 Jacobs, G., 436, 447 Jacobsen, C. F., 8, 15, 16 Jacobsohn, K. P., 388, 577, 579(3) Jaenicke, L., 510, 517(11), 518(11), 519(11) Jaffa, W. G., 56/57, 63, 64 Jagannathan, V., 869 Jakoby, W. B., 253 Jang, R., 13 Jansen, E. F., 11, 12, 13, 14, 23j 24, 35, 56/57, 61 Jeffreys, C., 35 Jelinek, V. C., 120 Jendrassik, L., 254, 577 Jennings, M. A., 121 Johnson, C. A., 134 Johnson, M., 613, 614 Johnson, M. J., 89, 93(6), 97, 108, 755 Johnson, R. B., 614 Johnson, S. A., 149 Johnston, R. B., 21, 64, 342, 343(3) Joklik, W. K., 862, 868

AUTHOR INDEX Jolles, A., 780 Jolliffe, N., 406 Jones, M. E., 65, 68, 668 Jones, W., 427 Josephson, K., 768, 779, 782, 785, 788, 789 Joslyn, M. A., 808, 819, 847, 848, 850 Jul6n, C., 163 Jungner, G., 442 Jungner, I., 442 K

Kahnt, F. W., 450, 452(16) Kalckar, H. M., 448, 449, 456, 458, 469, 470, 473, 474, 480, 481(2), 482, 485, 486, 498, 501, 586, 592, 599, 601, 602(1), 603, 604(1, 8), 655, 675, 676(1) Kallio, R. E., 318 Kalnitsky, G., 459, 746 Kamehora, T., 281, 282(11) Kamen, M. D., 759 Kamin, H., 203 Kaminishi, K., 625, 626(10, 11) Kapeller-Adler, R., 394 Kaplan, A, 578 Kaplan, N. O., 233, 237(3), 308, 310(27), 311, 411, 475, 476, 477, 552, 553(4), 554, 633, 634(2), 650, 653, 655, 659, 660, 661(2), 662, 663, 664, 665, 666, 681, 682, 683, 684(7, see 8), 685(7), 686(1, 2, 5), 687(1), 726, 728, 758, 759(1), 760(1) Kassell, B., 311 Kastle, J. H., 808 Katz, E. J., 780 Katz, S., 160 Katz, Y. J., 124, 125(1), 134, 135 Kaufman, S., 8, 15, 21, 22(40), 23(40), 24, 34, 36(29), 90 Kawai, H., 846 Kay, H. D., 533, 539 Kazal, L. A., 36, 40, 50, 51 Kazenko, A., 9, 17(12), 18(13) Kearney, E. B., 171, 172(15), 203, 205, 207(2), 208(2), 209, 333, 640, 642 (2), 644, 648, 675 Keckwick, R. A., 715 Keil, H. L., 73

883

Keilin, D., 102, 202, 203(6), 235, 303, 405, 455, 482, 485, 487, 489, 657, 671,674, 693, 698, 705, 727, 732, 733, 735, 737, 738, 739, 740, 745, 746(4), 748(4), 749, 750, 751(2), 753, 754, 769, 774, 802, 809, 811, 812, 817, 819(1), 841, 843, 845 Keith, C. K., 9, 18(13) Kenney, J. C., 512, 518(15) Kenyon, J., 847 Keresztesy, J. C., 629 Kerwin, T. D., 599, 603, 604 Kielley, R. K., 350, 594, 595, 611 Kielley, W. W., 589, 594, 595, 611 Kies, M. W., 56/57, 62 Kiese, M., 741, 836, 843(3), 844 Kilby, E. F., 24 Kimmel, J. R., 59 King, E. J., 328, 428, 429(10), 527, 559, 562 Kingsley, R. B., 115, 117(1), 118(1), 119(9) Kintner, E. P., 528 Kirberger, E., 290, 291(7) Kirchheimer, W. F., 396 Kishida, T., 626 Kitagawa, IVI., 777 Kitasato, T., 577 Kityakara, A., 602 Kjeldgaard, N. 0., 449, 485 Klausmeyer, R., 417 Kleczkowski, A., 435 Klein, J. R., 202, 203, 653 Klein, P. D., 157 Kleiner, I. S., 73 Kleinzeller, A., 580, 581(5, 6), 602 Klenow~ H., 448, 449, 458, 482, 485 Kline, D. L., 164 Knivett, V. A., 376, 377(11), 378(11) Knox, W. E., 242, 244, 246(3), 247, 249 (5), 250(13), 252, 253(13), 287 Kocholaty, W., 217, 218(4) Kodama, T., 732 Koerner, J. F., 561, 565 Kohlstaedt, K. G., 134 Ko]ima, S., 801 Koller, F., 155 Kondo, K., 846 Korkes, S., 303, 494, 729 Korn, E. D., 448, 453(9), 504, 508, 512

884

AUTHOR INDEX

Kornberg, A., 448, 453, 454, 473, 490, 492(2), 493, 496, 497, 498, 501, 502 (2), 503(2), 504(2), 516, 550, 551, 602, 655, 656, 670, 673, 676, 677, 682, 720, 754 Korzenovsky, M., 376, 377(13), 378(13) Kotake, Y., 249 Kotel'nikova, A. V., 602, 603 Kozloff, L. M., 436 Kozuka, S., 622, 625, 626(11), 628(1) Krasna, A. I., 862, 863(6), 864(8), 865 (4, 6), 866(6), 867(4, 9) Kraut, H., 455, 484, 548, 567, 576, 674, 702, 706 Krebs, H. A., 170, 178, 182(14), 200, 202 (3), 203(8), 204(8), 211(1), 613, 614, 825, 837, 840, 845 Kreshaw, B. B., 37, 48(7), 53 Krishnan, P. S., 577, 579(13), 580(13), 591, 592, 593, 646, 647 Krueger, R., 347 Krumey, F., 579, 581, 582(9) Kubacki, V., 9, 25 Kubowitz, F., 817 Kuby, S. A., 601 Kukita, A., 829 Kun, E., 695 Kunitz, M., 4, 5(3), 8, 9(1), 11(4), 12(1, 4), 14(3), 16, 17, 19, 24, 25, 26, 27(4), 28(4, 8), 29, 30(4), 31, 32(8), 33, 34, 35, 36, 37, 38, 40(4), 44, 46, 49, 50, 51, 53, 427, 429, 431, 433(2, 5), 434 (2), 435(2), 437, 438, 439, 441, 442, 443(3), 462, 571, 574, 576 Kurfess, N., 742 Kurnick, N. B., 437 Kutscher, W., 523, 527, 528(2), 529 L Lacombe, G., 375, 376(3) La Du, B. N., Jr., 289 Lagrain, A., 434 Laidler, K. J., 586 Laine, T., 387 Lajtha, A., 269, 270(7), 271(7) Laki, K., 159 Lamanna, C., 433 Lamfrom, H., 124, 125, 126(4), 133(2), 134(5, 8)

Lampen, J. O., 448, 458, 461, 478, 479(2), 480(2) Lamy, F., 146 Lanchantin, G. F., 162 Landwehr, G., 347 Lansford, M., 519 Lardy, H. A., 611, 613, 614(2) Larrieu, YI. J., 147 Lascelles, J., 868 Laskowski, M., 9, 17(12, 17), 18(13, 14), 19(20), 20(20), 24, 25, 37, 38, 46(6), 47, 48, 49(12, 14), 50, 51, 53, 54(14), 436, 437, 442(4), 443, 447, 775, 782 Laskowski, M., Jr., 37, 46(6), 47, 48, 49 (14), 50, 51, 53, 54(14) Laufer, S., 62, 63, 64 Laurell, C.-B., 140 Lawrence, A. S. C., 580, 581(5, 6) Lawrence, J. M., 316, 317(7) Lazarow, A., 742 Lea, D., 434 Leadbetter, W. F., 528 Leanza, W. J., 192 Lederle, E., 764 Ledou×, L., 434 Lee, K., 593 Lee, M., 117, 118 Legge, J. W., 169 Lehmann-Echternacht, H., 442 Lehninger, A. L., 301, 302(7), 556, 603, 613, 615 Leidy, G., 433 Lein, J., 238 Leiner, G., 837 Leiner, M., 837, 844 Leitch, R. H., 72 Leloir, L. F., 124, 136, 170, 172(5), 174(5), 175(5), 602 LelVIay-Knox, iV[., 287 Lemberg, R., 169 Lenhoff, H. M., 758, 759(1, 2), 760(1, 2) Lennox, F. G., 56/57, 64 Leone, E., 486, 489 Leopold, H., 388 LePage, G. A., 676 Lerner, A. B., 827, 828, 829, 830 Lerner, F., 528 Leuthardt, F., 115, 178, 182(12), 347, 348, 638

AUTHOR INDEX

Leuthardt, F. M., 114, 319, 380, 382, 383 (2), 384(2) Levenberg, B., 509, 517(7), 519 Lever, W. F., 450, 452(16) Levintow, L., 115, 117(1), 118(1), 341, 633 Levy, G. B., 120, 122 Levy, R. S., 523 Lewis, C. J., 53 Lewis, H. A., 124, 125(1), 134, 135 Lewis, H. D., 837 Lewis, J. H., 154, 164, 166 Lewis, J. I. M., 332 Lewis, S., 819, 822(6), 831, 832, 835(2) Li, S.-O., 77 Lichstein, H. C., 170, 176(1, la), 177(1, la), 320, 322(25, 26) Lichtenstein, N., 341 Lieberman, I., 493, 496, 501, 502(2), 503 (2), 504(2), 602 Linderstr0m-Lang, K., 89, 115, 526 Lindsay, A., 203, 698 Lineweaver, H., 37, 53, 56/57, 59, 60, 698 Lipmann, F., 65, 268, 338, 555, 556, 633, 634(2), 653, 667, 668, 781 Lipschitz, R., 600 Little, H. N., 400, 402 Little, J. A., 561, 562 Liu, C. H., 450, 452(16) Livermore, A. H., 625, 626(12) Loaeza, F., 62 Loeffier, H. J., 847 Loeliger, A., 155 Loftfield, R., 752 Logan, M. A., 375, 376(4), 378(4) Lohmann, K., 254, 577 Lominski, I., 376 London, M., 529 Longsworth, L. G., 51, 757 Loomis, E. C., 143 Loomis, W. D., 264, 266(2), 267 Lopez, J. A., 577, 579(11), 612 Lorand, L., 145 Lorber, L., 757 Lorenz, L., 49 Loring, H. S., 433, 458 Louhivuori, A., 389 Love, S. H., 519 Lovelace, F. E., 56/57, 61 Lovett-Janison, P. L., 831, 832

885

Lowry, O. H., 234, 320, 401, 409, 412, 417, 428, 438, 454, 469, 472, 476, 490, 492, 494, 498, 547, 552, 566, 577, 579(11), 612, 640, 652, 661, 664, 666(1), 727, 730, 851, 857 Lucas, E. H., 837, 842(13), 844(13) Liidtke, K., 195 Lundquist, I., 527 Lustig, H., 356 Lyle, G. G., 614 Lyubimova, M. N., 534 M

Maas, W. K., 619 McBride, T. J., 160, 161(50) McCann, S. F., 38 MeCarty, M., 433, 438, 441(8), 442(8), 443(8), 444(8), 446, 447 McClaughry, R. I., 147, 157 McCubbin, J. W., 136 McDonald, M. R., 26, 29, 429, 432, 434, 435, 444 McElroy, W. D., 476, 477, 651, 852, 854, 856, 857, 858, 860 McFadden, B. A., 870 MacFadyen, D. A., 115, 428, 565 MeGilvery, R. W., 350, 543, 544, 545, 546(1) McGinty, D. A., 157 McIlwain, H., 459, 479 McInnes, D. A., 51 McLaren, A. D., 53 McMeekin, T. L., 74 MeNutt, W. S., 468 McVeigh, I., 630 Maehly, A. C., 765, 768, 773, 774, 796, 803, 807, 808, 809(15), 811, 812 Magasanik, B., 172, 176(21), 433 Mahler, H. R., 489, 688, 689(2), 691, 692, 707, 708(6), 710, 711(6), 869 Makower, B., 826 Mallette, M. F., 433, 818, 819, 822, 825, 832 Malmgren, H., 577, 578(5), 579(5, 6, 7), 580(5, 6) Mamelak, R., 217, 218(3), 219, 220 Mandl, I., 580, 582(4) Mann, T., 102, 578, 579(15), 580(15), 802, 812, 817, 819(1), 841, 843, 845

886

AUTHOR INDEX

Manson, E. E. D., 123 Manson, L. A., 448 Mapson, L. W., 719 Maren, T. H., 845 Margoliash, E., 752, 754(13) Maritz, A., 207, 208(5) Markham, R., 433, 434, 466, 526, 568 Mars, P. H., 47, 50, 51, 53 Marsh, W. H., 508 Marshak, A., 442 Marshall, E. K., Jr., 406, 407, 634 Martin, A. J. P., 172, 435 Martin, J. B., 559 Martius, C., 810 Martland, M., 540 Massart, L., 434 Mathies, J. C., 560 Matsukawa, T., 625 Matsuoka, H., 281, 282(11) Mattenheimer, H., 77 Matter, M., 155 Maver, M. E., 400, 436, 447, 568 Mawson, C. A., 839 Maxwell, E. S., 603 May, M., 519 May, S. C., Jr., 13 Mayr, O., 384 Mehl, J. W., 221 Mehler, A. H., 228, 242, 244, 246(3), 247, 249(5) Meister, A., 110, 114, 170, 171(2), 172 (12), 173(12, 14), 174(14), 176(2, 12), 177(12), 178, 182, 188, 189(10), 289, 298, 299, 341, 380, 381(3, see 6), 382 (3, see 6), 385(14) Mejbaum, W., 506 Mela, P., 269, 270(7), 271(7) Meldrum, N. U., 836, 837(5), 841, 843(5) Mellander, O., 526 Mendive, J. R., 838, 846 Metzler, D. E., 177, 320, 322 Meyerhof, O., 578, 586, 589 Miall, M., 580, 581(5, 6) Michaelis, L., 226 Michelson, C., 264, 266(2) Mihalyi, E., 586 Miller, A., 269 Miller, I. L., 250, 253(16) Miller, K. D., 162 Miller, L., 66

Miller, P., 746 Miller, W. H., 819 Miller, Z. B., 436 Millikan, G. A, 840 Milstein, S. W., 427, 428(6) Milstone, J. H., 140, 156 Mims, V., 190, 191(1), 192(1) Mingioli, E. S., 303, 305, 309(14), 380 Mirsky, A. E., 437, 445(2), 447(2) Mishuck, E., 35, 53 Mitchell, C. A., 838 Mitchell, H. K., 167, 233, 238, 470, 476, 477 Mitchell, M. B., 167 Mitsuhashi, S:, 305, 307, 311(25a), 686 Mitsui, H., 417 Mittelman, D., 450, 452(16) Mittelman, N., 602 Miura, Y., 434 Miyaji, T., 442 Mocquot, C., 77 Mommaerts, W. F. H. M., 584, 586, 587 (18) Monod, J., 233, 237 Montgomery, H., 828 Moore, D. H., 148 Moore, S., 79, 435, 436(54) Morales, M. F., 586 Morell, D. B., 485 Morgan, E. J., 847, 848 Mori, T., 759 Morrison, P. R., 160 Morrison, R. B., 376 Morton, R. K., 112, 530, 532, 535, 537, 538(9), 539, 557, 558, 559(1, 2), 560, 586, 745, 746 Mosimann, W., 778 Mouton, R. F., 450, 452(16) Mtiller, A. F., 178, 182(12) Mueller, G. C., 676 Miiller, H. R., 21 Mueller, J. H., 746 Mfillertz, S., 164 Mullen, J. E., 533 Munch-Petersen, A., 675, 676(1), 677 Mufioz, J. M., 124, 136 Munro, N., 847 Muntz, J. A., 434 Murata, K., 624, 627, 628 Murphy, R. C., 162

AUTHOR INDEX

Murray, C. W., 37, 53 Mycek, M. J., 21, 64 N

Najjar, V. A., 186, 187(1), 188(1), 189(1), 421, 422(1), 676 Nakamura, Y., 434 Narrod, S. A., 215, 217(12) Nason, A., 233, 237(3), 408, 411,414, 415 (2, 7), 664, 665, 666(2, 3, 4), 725, 728 Needham, D. M., 580, 581(5, 6) Needham, J., 580, 581(5, 6) Negelein, E., 200, 201, 204(2), 212, 700, 713 Neidle, A., 268 Neilands, J. B., 752, 755, 750 Nelson, J. M., 818, 819, 821,822(6), 825, 826, 831, 832 Nelson, N., 457 Nelson, W. L., 591 Nemchinskaya, V. L., 442 Neuberg, C., 356, 577, 579(3, 4, 10), 580, 581, 582(3, 4, 8) Neuberger, A., 249, 508 Neufeld, E. F., 308, 310(27), 311, 411, 681, 682, 683, 684(7, see 8), 685(7), 686(2, 5) Neufeld, H. A., 741 Neuman, R. E., 98, 100(12) Neurath, H., 8, 9(9), 11, 12, 15, 19, 20, 21, 22(40), 23(40), 26, 27(12), 32, 33, 34, 35, 36(29), 77, 78, 80(2), 81(11) Newton, B. L., 166 Nguyen-Van-Thoai, 637, 638(4), 639 Nicholas, D. J. D., 414, 415(7) Nielsen, H., 347, 348, 638 Nienburg, H., 97 Nikiforuk, G., 471 Nisrnan, B., 218, 220 Nisonoff, A., 178 Nitschmann, H., 77 Nocito, V., 170, 172(5), 174(5), 175(5) 209, 210(9), 211, 225, 226(1) Noda, L., 601 Nokayama, T., 249 Nord, F. F., 32, 35, 37, 51, 53 Norris, E. R., 7, 480, 482(1) Norris, L. C., 698

887

Northrop, J. H., 4, 5(3), 7, 8, 9(1), 12(1), 16, 24, 25, 26, 27(4), 28(4), 29, 30(4), 35, 36, 37, 38, 46, 53, 58, 429, 439 Nose, Y., 622, 626, 628(1) Novelli, G. D., 619, 633, 659, 667 Numata, I., 625, 626(9), 719 Nutting, M. D. F., 13 O

Oehoa, S., 303, 652, 681 O'Dell, B. L., 510, 519(10) Oginsky, E. L., 375, 376, 378(14) Ogston, A. G., 774, 808 Ohlmeyer, P., 527 O'Kane, D., 696 Oleott, H. S., 37, 48(8), 51, 53 Oldewurtel, It. A., 664, 666(3) Olitzky, P. K., 434 Olivard, J., 177 Olsen, N. S., 203 Olson, J. A., 224, 225(6) O'Malley, E., 841 Ordal, E. J., 862, 870 Osato, R. L., 66 Oser, B. L., 762 Otey, M. C., 382, 448 Oullet, L., 586 Overend, W. G., 432, 441, 442(17) Owades, P., 269 Owren, P. A., 140, 145, 146, 151,153, 154, 160

Packer, L., 870 Page, E. W., 195 Page, I. H., 136 Palade, G. E., 168 Pal~us, S., 752, 811 Palmer, K. J., 24 Pany, J., 523, 527(3), 529 Pappas, A., 344, 351, 356, 358(1), 515 Pappenheimer, A. M., Jr., 745, 746(5) Park, J. T., 659 Parker, R. P., 519 Parks, R. E., Jr., 22, 23(45) Parrish, R. G., 584 Paseyro, P., 157 Patwardhan, V. N., 178

888

AUTHOR INDEX

Paul, K. G., 736, 749, 752, 753, 754, 774, 794, 808, 809, 811, 817 Paulsen, M. M. P., 150 Peabody, R. A., 509, 519 Peanasky, R. J., 37, 49(12), 50 Peck, It. D., 862 Pedersen, K. O., 715 Peeters, G., 434 Peiree, J. D., 134 Pele, S. R., 441 Perlmann, G. E., 526, 781 Perlzweig, W. A., 454, 655 Permin, P. IV[., 166 Perrin, D. D., 836, 837(4), 838(4), 839, 846 Perry, S. V., 583, 587 Person, P., 741 Peterjohn, H. R., 461 Peterman, M. L., 843, 844(33) Petit, E. L., 778 Petrack, B., 356, 359, 360(10), 364, 365 (2), 367(14) Pfiffner, J. J., 510, 519(10) Pfister, K., 192 Pfister, R. W., 21 Phillips, P. H., 302 Philpot, F. J., 839 Philpot, J. St. L., 839 Pillard, E., 35 Pinsent, J., 765, 775, 778, 782, 784, 785 (1), 786, 788(1) Plantl, A. A., 136 Plass, R., 707, 711(4) Plaut, G. W. E., 22, 23(45), 415, 729 Plesner, P., 448 Ploeser, J. M., 458 Pogell, B. M., 543, 544, 545, 546(1) Poilroux, M., 8, 9(7), 14(7), 16(7) Polglase, W. J., 92 Polls, B. D., 586, 774, 799, 813, 816 Pollock, M. R., 120, 122(8), 123 Ponting, J. D., 819, 847 Porch, 1V[.B., 808 Porter, I. A., 376 Porter, R. R., 435 Portzehl, H., 583, 584, 586(6) Potter, V. R., 602, 603(15), 604(15), 614, 693, 704, 754, 758 Poup~, F., 726 Powers, W. H., 831, 832(2), 835

Pozzani, U. C., 838 Praetorius, E., 489 Price, C. A., 748 Price, V. E., 110, 114, 448, 775 Pricer, W. E., Jr., 473, 498, 516, 550, 551, 655 Pucher, G. W., 267 Pullman, M. E., 726, 728 Putnam, F. W., 77, 80(2)

Q Quastel, J. H., 217, 218(3), 219, 220, 387, 388(4), 631, 632(1), 748 Quick, A. J., 140, 149, 157 R

Racker, E., 358, 407~ 722 Rall, T. W., 301, 302(7) Randall, R. J., 234, 320, 401, 409, 412, 417, 428, 438, 454, 469, 472, 476, 490, 492(3), 494(3), 498, 547, 552, 566, 652, 661, 664, 666(1), 727, 730, 851, 857 Rao, K. R., 109, 114(1), 117, 119(9) Ratner, S., 209, 210(9), 211(9), 225, 226 (1), 344, 351, 356, 358(1), 359, 360 (10), 364, 365(2), 367(14), 515 Raub, A., 194 Ravdin, R. G., 292, 293, 299(12) Ravel, J. M., 514, 519 Ravin, H. A., 22 Raynaud, M., 218 Reddy, K. K., 625 Reid, J., 311, 312(3) Reif, A. E., 693 Reinhart, H. L., 528 Reis, J., 546 Reissig, J. L., 319, 320, 321(18), 322 Reissig, M., 157 Richardson, E., 124, 125(1), 134, 135 Rieche, A., 764 Riggs, B. C., 837 Rittenberg, D., 21, 862, 863, 864(8, 16)~ 865(4, 6), 866(6, 16), 867(4, 9, 16) Ro, K., 489 Roberts, D., 448 Roberts, E., 215, 217(13) Roberts, I. S., 580, 581(3), 582(3)

AUTHOR INDEX Robinson, D., 328 Robinson, D. S., 107, 108, 109, 112, 114

(2) Robinson, H. W., 66, 213, 721, 722 Robison, R., 540 Roche, J., 375, 376(3) Rodkey, F. L., 754 Roholt, 0. A., Jr., 371 Roll, P. M., 433 Rosebrough, N. J., 234, 320, 401, 409, 412, 417, 428, 438, 454, 469, 472, 476, 490, 492(3), 494(3), 498, 547, 552, 566, 652, 661, 664, 666(1), 727, 730, 851, 857 Rosenthal, N., 150 Rosenthal, R. L., 150 Ross, H. E., 341 Roth, B., 519 Roth, J. S., 427, 428(6), 434, 436 Roth, L. J., 819 Rothen, A., 435 Rothschild, Lord, 769 Roughton, F. J. W., 767, 836, 837, 838, 839, 840, 841, 843(1, 2, 5, 7), 844(1, 7), 845(I, 18) Roush, A., 480, 482(1) Rovery, M., 8, 9(7), 14(7), 16(7), 23, 26, 36 Rowen, J. W., 448, 453, 454 Rowsell, E. V., 172, 289 Roy, A. B., 326, 328, 330, 331, 332(6, 16) Rudman, D., 171, 172, 173(12), 176(12), 177(12), 380, 381(3, see 6), 382(3, see 6) Rtifenacht, K., 21 Ruffo, A., 613, 614 Russell, J. A., 375, 376(2) S

Sable, H. Z., 478, 479(2), 480(2) Sacktor, B., 595 Sadana, J. C., 869 Saenz, A. C., 434 Saffran, M., 501, 504(1) Saiki, H., 626 Sailer, E., 21 Saito, H., 388 Sakami, W., 517 Sakamoto, S., 622, 625, 627(7), 628(1)

889

Salamon, I. I., 305 Salazar, W., 62 Samuels, P. J., 342 Sanders, A. G., 121 Sarkar, N. K., 688, 689(2), 692, 707, 708 (6), 710(6), 711(6), 775, 777, 780 Sato, R., 746, 759 Saunders, J. P., 336, 337(5) Scarano, E., 262, 501,502, 503(8), 504(1) Schachter, D., 348, 349(9), 350(9) Sch~ffner, A., 579, 581, 582(9) Schaffer, N. K., 13 Schales, O., 136, 182, 188, 190, 191(1, 3), 192(1, 3), 193(2), 195, 196, 198, 199(6) Schales, S. S., 136, 182, 190, 191(1, 3), 192(1, 3), 193(2), 195, 198 Schepartz, B., 295 Schlamowitz, M., 427 Schieich, H., 114 Schmetz, F. J., Jr., 659 Sehmid, J., 147 Schmid, K., 450, 452(16) Schmidt, G., 433, 436, 469, 470, 471, 474, 523, 524, 526, 528(4, 13), 533 Schmidt, G. C., 375, 376(4), 378(4) Schmitz, A., 49 Schneider, C. L., 149 Schneider, W. C., 168, 350, 486, 594, 614, 695 Sch5berl, A., 311, 312(3) Seholefield, P. G., 203 Schott, H. F., 198, 199(9) Schou, M., 269, 339 Schramm, G., 584 Schrecker, A. W., 673 Schubert, M. P., 226 Sehuler, W., 489 Schulman, S., 808 Schutze, M., 508 Schwander, H., 77 Schwartz, S., 780 Schwarzenbach, G. M., 193 Schwert, G. W., 8, 11, 15, 19, 21, 22(40), 23(40), 24, 34, 36, 78, 80(5, see 11), 81(11) Scott, C. R., 741 Scott, D. A., 838, 841, 843, 846 Scott, E. W., 403 Scott, M. L., 629, 698

890

AUTHOR I N D E X

Scouloudi, H., 435 Scudi, J. V., 120 Seale, B., 178 Sealock, R. R., 625, 626(12) Seath, A. E., 631 Seegers, W. H., 141, 143, 144, 145, 147, 151, 153, 156, 157, 158, 162 Seller, A., 160 Sekine, T., 376 Seligman, A. M., 22, 347 Sera, Y., 229, 232 Seraidarian, K., 433, 524, 526(13), 528 (13), 586, 587(18) Seraidarian, M., 433, 524, 526(13), 527, 528(13) Sevag, M. G., 317, 435 Shanewise, A. B., 780 Shapiro, B., 555, 556(2) Shapiro, H. S., 441 Shapot, V. S., 442 Shatas, R., 578 Shelton, E., 695 Shemin, D., 290 Shen, S.-C., 580, 581(6) Sheppard, E., 53 Shinn, M. B., 403 Shinowara, G. Y., 528 Shipley, R. E., 134 Shirakawa, IV[., 765, 777 Shive, W., 514, 519 Shmukler, H. W., 774, 799, 813, 816 Shug, A. L., 869 Shugar, D., 435 Shulman, S., 160 Shupe, R. E., 9, 18(14) Shuster, L., 475, 477, 552, 553(4), 554, 666 Sibly, P. M., 842, 843(31), 845, 846 Sidwell, A. E., 754 Siekevitz, P., 602, 603(15), 604(15) Silverman, M., 629 Simms, E. S., 501, 502(2), 503(2), 504(2), 602 Simms, H., 431 Singer, T. P., 171, 172(15), 203, 205, 207(2), 208(2), 209, 333, 434 Sinsheimer, R. L., 561, 565 Sistrom, W. R., 282 Sizer, I. W., 764, 781, 808, 826 Slade, H. D., 376, 377(12), 378(12, 16) Slamp, W. C., 377, 378(16)

Slatcr, E. C., 560, 591, 603, 604, 613, 693, 737, 750 Sleeper, B. P., 274 Sloane-Stanley, G. H., 197 Slonim, N. B., 89, 92(9) Smathers, W. M., 149 Smith, C. L., 443 Smith, E. E. B., 675, 676(1) Smith, E. L., 9, 17(17), 21, 24, 59, 78, 83, 84, 87(4), 89, 91(8), 92(9), 93(5, 7, 8, 13), 94, 95, 96, 97, 98, 99(10), 100(10, 12), 101(14), 102, 103, 104 (15, 17), 105(16, 17), 107(20), 108, 100(22, 23, 24), 118, 134, 399 Smith, H. P., 140, 157 Smith, J. D., 433, 466~ 526 Smith, J. M., Jr., 519 Smith, J. N., 328 Smith, K. M., 434 Smith, L., 732, 733, 735, 736(12), 739, 741, 744, 745, 761,796 Smith, V. A., 84, 87(2) Smolens, J., 435 Smyrniotis, P. Z., 676 Smythe, C. V., 315, 316, 317(7) Snell, C., 411 Snell, E. E., 177, 320, 322, 468, 629, 630(1), 631 Snell, F. D., 411 Snell, N. S., 53 Snellman, O., 163 Snoke, J. E., 21, 22(40), 23(40), 34, 36(29), 342, 343(4), 344 Snyder, H. R., 241 Soares, M., 388 Sober, H. A., 171, 173(14), 174(14), 178, 182, 188, 189(10), 382 Soda, T., 330 SSrbo, B. H., 334, 335(1), 336, 337 Somers, G. F., 816 Somogyi, M., 457 Sonnenfeld, V., 856, 858 Soulier, J. P., 147 Sourkes, T., 197 Spackman, D. H., 89: 91(8), 92, 93(8, 13), 104(19) Spadoni, M. A., 625 Speck, J. F., 337, 338(1), 339 Spencer, B., 324, 325, 326, 327(2), 328, 332 Spicer, D. S., 36, 40, 50, 51

AUTHOR INDEX Spizizen, J., 512, 518(15) Spray, R. S., 233 Sprinson, D. B., 21, 320, 322(23) Sprissler, G. P., 7 Sreenivasan, A., 289 Sri Ram, J., 37 Stadie, W. C., 203, 837 Stadtman, E. R., 649, 650, 667, 730, 731 Stafford, H. A., 720, 721 Stage, A., 166 Stanier, R. Y., 245, 249, 250, 251, 252, 273, 274, 275, 276, 277, 278(8), 282, 284 Stanley-Brown, M., 140 Stanly, A. R., 233 Stannard, J. N., 735, 755 Stauffer, J. F., 837 Steele, W. J., 796 Stefaniak, J. J., 755 Stefanini, M., 149 Stein, W. H., 79, 435, 436(54) Steiner, R. F., 35, 53 Steinman, H. G., 835 Stephens, W. D., 726 Stephenson, M., 320, 322(24) Sterndorff, I., 166 Stewart, B. T., 213, 215(6) Stewart, E., 694 Steyn-Parvd, E. P., 636, 637, 639(2), 640(2) Stickland, L. H., 217, 218(1), 220 Still, J. L., 868 Stitch, S. R., 330 Stoll, A., 773 Stolzenbach, F. E., 475, 655, 666 Stotz, E., 298, 299, 300(20), 739, 741, 754, 761 Stout, B. K., 73 Straub, F. B., 584, 707, 708, 709(2), 710, 711(2), 741 Strecker, It. J., 224, 494 Strehler, B. L., 857, 861 Strickler, N., 433, 524, 526(13), 528(13) Strominger, J. L., 603 Struyvenberg, A., 242 Stumpf, P. K., 264, 266(2), 267 Sturtevant, J. M., 53 SubbaRow, Y., 530, 534, 540, 5471 551, 571, 577, 582, 596 Subramanian, S. S., 388

891

Suda, M., 281, 282, 292, 295 Sujishi, K., 295 Sullivan, M. X., 311 Sullivan, R. A., 76, 77(15) Sumiki, Y., 388 Summerson, W. H., 13, 762, 827 Sumner, J. B., 378, 379, 483, 577, 696, 765, 775, 776, 777, 778, 780, 782, 802 813, 816 Surgenor, D. M., 450, 452(16), 792 Sutherland, G. L., 519 Swanson, M. A., 542 Swenson, T. L., 37 Sylvdn, B., 163 Sz£ra, S., 160 Szent-GySrgyi, A., 484, 583, 586 T Tabor, H., 228, 229 Taggart, J. V., 348, 349(9), 350(9), 613, 615(7) Taha, S. M., 730 Takeda, Y., 292, 295 Takeuchi, M., 229, 232 Talaley, P., 528 Tallan, H. H., 68 Tam, R. K., 848 Tamm, C., 441 Tanaka, T., 295 Taniguchi, S., 417 Taquini, A. C., 136 Tarpley, W. B., 834, 835(8) Tashiro, T., 622, 626, 628 Tatum, E. L., 168, 235, 320 Tauber, H., 21, 37, 48, 53, 55, 56/57, 62, 63, 64, 73, 778 Taylor, E. S., 187, 189 Tenmatay, A. L., 625 Teply, L. J., 688, 707 Tepperman, J., 735 Terminiello, L., 37, 51, 53 Terrell, A. J., 725 Thannhauser, S. J., 433, 436, 474, 524, 526, 528(13), 533 Thayer, P. S., 211 TheoreU, H., 482, 714, 715(8), 751, 752, 753, 754, 758, 765, 768, 770(9), 772(9), 774, 780, 781, 782, 785, 789, 791,794, 799, 802, 803, 807(16), 808, 809, 811, 812, 813, 817

892

AUTHOR INDEX

Thimann, K. V., 748 Thomas, J., 325, 327(2) Thomas, L., 21 Thomas, R., 432, 433(22) Thompson, C. B., 241 Thompson, R. H. S., 427 Thompson, R. R., 56/57, 62 Thorne, C. B., 171, 173(16), 175(16), 176 Thornley, B. D., 72, 74 Tice, S. V., 170, 171(2), 173(14), 174(14), 176(2), 178, 182, 188, 189(10), 382 Tietze, F., 28, 35 Tinoco, I., Jr., 160 Tiselius, A., 470 Tissieres, A., 167 Toeantins, L. M., 160, 161(50) Todd, A. R., 433 Tones, L. N., 528 Tonhazy, N. E., 170, 174(7), 178 Torriani, A.-M., 122 Tosi, L., 753 Toyoda, J., 417 Trano, Y., 197 Trautmann, M. L., 443 Trethewie, E. R., 839 Tria, E., 780 Troll, W., 167 Trucco, R. E., 602 Tsao, T. C., 584, 586(9) Tsou, C. L., 749, 752(3) Tsuchida, M., 250, 274 Tsuchihashi, M., 783 Tsuda, N., 388 Tulpule, P. G., 178 Tupper, R., 843 Tuttle, L. C., 65, 268, 338, 556 Twigg, G. H., 120 Tytell, A. A., 375, 376(4), 378(4)

Uroma, E., 450, 452(16) Utter, F. M., 459, 746 Uyeki, E. M., 602

Vallee, B. L., 78 van Creveld, S., 150 Vandenbelt, J. M., 143, 510, 519(10) Vandendriessche, L., 432 van der Burg, B., 73 van der Scheer, A. F., 73 Van Goor, H., 837, 841 Vanhoucke, A., 434 van Orden, L. S., 396 Vanselow, A. P., 596 Van Slyke, D. D., 115, 358, 365, 368 Varin, R., 77 Vaughan, J., 66 Vely, V., 698 Vennesland, B., 719, 720, 721 Vercauteren, R., 442 Vernon, L. P., 688, 689(2), 692, 707, 708(6), 710(6), 711(6), 759 Verwey, W. F., 166 Vickery, H. B., 267 Vignos, P. J., Jr., 262 Vinet, G., 218, 220 Virtanen, A. I., 387, 388, 389 Vishniac, W., 870 Vlitos, A. J., 330 Vogel, A. I., 865 Volkin, E., 433, 524, 540 yon Euler, H., 221, 224(2), 707, 710, 711(3, 4, 13), 768, 779, 780, 782, 785, 788, 789, 810 von Lebedev, A., 713 von Schoenebeck, O., 97

U

W

Uber, F. M., 435 Ueba, A., 627, 628 Ueda, K., 622, 626, 628(1) Umbarger, H. E., 172, 176(21) Umbreit, W. W., 170, 174(7), 176(la), 177(la, 11), 178, 182, 183(18), 216, 236, 237(11), 238, 239(2), 241(2), 320, 322(25), 324, 610, 611(1), 647, 837

Waelsch, H., 267, 268(1), 269(1), 270(7), 271(7), 338, 339, 341(7) Wagner, A., 311, 312(3) Wagner, W. H., 273 Wainfan, E., 267, 268(1), 269(1), 338, 341 (7) Wainio, W. W., 741 Wakerlin, G. E., 134 Waley, S. G., 21

V

AUTHOR INDEX Walker, B. S., 780 Walker, J. B., 367, 376 Walser, A., 202 Walti, A., 56/57, 61 Wang, T. P., 458, 475, 477, 478, 479(2), 480(2), 554, 650, 653, 655 Warburg, O., 203, 213, 247, 255, 257, 260(5), 290, 302, 305, 308, 407, 416, 656, 673, 676, 698, 699, 700, 712, 713, 715(1), 722, 825 Ware, A. G., 141, 143, 151, 153, 156, 158, 162 Warner, E. D., 140, 145 Warner, R. C., 526 Wasserman, A. E., 166 Watanabe, H., 625 Watts, R. W. E., 843 Waugh, D. F., 146 Waygood, E. R., 837, 841, 842, 843, 845 Webb, M., 432, 441, 442(17), 443, 444, 447(31) Weber, H. H., 583, 584, 586(6) Weichselbaum, T. E., 255, 257, 260(6), 656 Weil-Malherbe, H., 559, 636, 637, 638 Weisel, P., 757 Weiss, U., 301, 305, 309(5) Werkheiser, W. C., 457 Werkman, C. H., 376, 377(13), 378(13), 459, 746 Werle, E., 194 Werner, A. E., 369 Wertheimer, E., 555, 556(2) Westenbrink, H. G. K., 637 Wheatley, A. H. M., 748 Whitby, L. G., 203 White, N. G., 170, 174(7), 178 White, S. G., 149 Whiteley, H., 870 Whitfeld, P. R., 568 Widmer, C., 741 Wieland, O. P., 629 Wiggans, D. S., 68 Wilbur, K. M., 839, 840 Wilcox, P. E., 19 Wildman, S. G., 849 Wilensky, B., 809 Williams, C. M., 745 Williams, J. H., 629

893

Williams, J. N., Jr., 289 Williams, R. R., 624 Williams, R. T., 328 Williams, V. R., 322 Williams, W. J., 502, 503, 504(9) Williamson, D. H., 382 Willstiitter, R., 64, 455, 484, 548, 567, 576, 674, 702, 706, 773, 823, 833(13, see 6), 834(4) Wilson, K., 630 Wilson, P. W., 759, 848, 862, 869, 870 Wilson, T. G. G., 759 Winitz, M., 68 Winniek, T., 56/57, 62, 63(11) Winzler, R. J., 457 Wise, W. S., 120 Wiss, O., 202, 203, 249, 250, 253 WSrner, A., 523, 527, 528(2) Wolberg, H., 523 Wood, J. G., 842, 843(31), 845, 846 Wood, W. A., 171, 176(17), 212, 215(1), 217(12), 236, 237(11), 238, 239(2), 241(2), 319, 320, 322(19), 324 Woods, D. D., 217, 218(2), 219 Woodward, C., 74, 75 Woodward, G. E., 343, 428, 719 Woolf, B., 387, 388(4, 5) Woolley, D. W., 663 Work, E., 39, 40, 50, 51, 53, 202 Wormalls, A., 843 Wosilait, W. D., 725, 728 Wright, R. D., 37, 48(7), 53 Wroblewski, F., 447 Wu, F. C., 9, 19(20), 20(20), 37 Wurmser, R., 754 Y

Yamada, T., 417 Yamadori, M., 626 Yamaguchi, M., 847, 848, 850 Yamazaki, K., 625, 626(11) Yanari, S., 342, 343(4), 344 Yaniv, H., 301 Yanofsky, C., 236, 238, 319, 320, 321(18), 322(19), 323(32) Yefimochkina, E. F., 348 Yoshida, A., 579 Young, R., 776

894

AUTHOR INDEX

Yudkin, W. H., 114 Yurugi, S., 625 Z Zahler, P., 77 Zamenhof, S., 433, 441, 445, 446(35), 447 Zapp, J. A., 203 Zatman, L. J., 660, 661(2), 662, 663, 666, 681, 686(1), 687(1)

Zeller, E. A., 207, 208(5), 390, 393, 394, 395(9), 396, 550 Zerfas, L. C., 746 Zima, O., 624 Zittle, C. A., 382, 384(7), 434, 441, 534, 535, 538, 557 ZSllner, N., 433, 434, 524, 526(13), 528(13) Zweig, G., 457

Subject Index Note on use of indexes for Volumes I and I I . Under the name of a given enzyme, inclusive pages are listed for the chapter in which it is described. Under this heading, the assay method, purification procedure and properties are not ordinarily listed, since these regularly appear within a few pages of each other and in the order mentioned. Only when unusual topics are covered, or when the organization of the material is more complex than usual, do sub-entries other than source appear under the name of the enzyme. Instead, all items of interest within each chapter have been listed under their own names. Thus, the names of individual sources, substrates, eoenzymes, activators and inhibitors of the enzymes are listed as major entries. The index may therefore be used to ascertain (1) the various enzymes which arc obtainable from a given bacterial, plant or animal source, (2) the various enzymes which are activated (or inhibited) by a given metal ion, eoenzyme, or other agent, and (3) the various fates of a given substrate. Other useful lists include one for crystalline enzymes and one for adaptive enzymes. Enzymes marked by asterisk (*) are covered in detail in Volume I.

Acetylation, enzymes for, in liver extracts~ 633 Acacia, Acetyldehydroalanine, fibrinoplastic action of, 159-160 as substrate for dehydropeptidase II, presence of calcium in, 156 109 use in two-stage prothrombin assay, 141 Acetyl-L-glutamate, Accelerin, see Serum Ac-globulin role in citrulline synthesis, 355 Acetaldehyde, p-Acetylphenylsulfate, formation by nitroethane oxidase, 400 as substrate for arylsulfatases, 327 Acetal phosphatides, Acetyl-L-phenylalanine ethyl ester, presence in thromboplastin, 150 as substrate for trypsin and ehymoAcetate, trypsin, 23 activation of 5'-AMP deaminase by, Acetyl phosphatase, 472 action at phosphorus-oxygen bond, formation by reduction of glycine, 217 556 inhibition of erythrocyte carbonic from animal tissues and bacteria, 555anhydrase by, 845 556 5-(Acetic acid)-hydantoinase, interference in acetyl-phosphate assay, cyclization of ureidosuecinic acid by, 650 496-497 Acetyl phosphate, Acetoacetate, CoA assay by arsenolysis of, 649 conversion of tyrosine to, 287-300 hydrolysis of, 555-556 formation by fumarylacetoacetate 3-Acetylpyridine analog of D P N , 654, hydrolase, 298-300 662, 663 Acetobacter sp., phosphorylation of by D P N kinase, eytochrome al as terminal respiratory 654 enzyme of, 732, 733, 734 3-Acetylpyruvate, eytoehrome component absorbing at action of hydrolase on, 299 554 mu in, 744-745 N-Acetyl-L-tyrosine ethyl ester, Acetonitrile, as substrate for trypsin and chymoinhibition of aspartase by, 388 trypsins, 23 Aeetyl amino acids, Ae-globulin, see Plasma Ac-globulin and action of acylases on, 116, 118, 119 Serum Ac-globulin 895 A

896

SVBJEC~ INDEX

8-C ~4-Adenine, Achromobacter fischeri, incorporation into nueleotide fraction, growth of, 857-858 501-502 luciferase from, 857-861 mechanism of hydrolytic nueleosidase Acid-soluble deoxypentose compounds, action studied with, 464 release by DNase, 437 Adenine deoxyriboside, "Acid-soluble phosphorus," R! values for, 466 measurement in RNase assay, 427 in transdeoxyribosidase reaction, 464, Acridine, inhibition of RNase by, 434 468 Acriflavine, bioassay of, 464 inhibition of DPNH cytochrome c reAdenosine (AR), ductase by, 698 inhibition of DPN kinase by, 654 Actin, of inosine cleavage by, 463 effect of on pH curves for myosin of pyridoxal kinase by, 649 ATPase, 588 nucleosidase action on, 459, 463 modification of ion effects on myosin Adenosine-3'-benzylphosphate, ATPase by, 586-587 3'-adenylic acid formation from, by Actomyosin, intestinal diesterase, 570 ATPase activity of, 589 Adenosine compounds, activation by Mg, 589 assay of with deaminase, 474-475 ion effects on ATPase activity of, 587 direct deamination of, 475 removal from Mg-activated ATPases, resistance of 2'-derivatives of to 589, 596 deaminase, 477 from myosin, 584 Adenosine deaminase, N-Acyl amino acids, see also under anonspecific, Amino acids, from takadiastase, 475-478 as substrates for carboxypeptidase, 78 affinities of various substrates for, Acyl phosphates, 478 as probable substrates for alkaline specific, phosphatase, 538 from calf intestinal nmcosa, 473-475 Acylpyruvase, assay of 3'-nucleotidase with, 551 probable identity with fumarylacetoseparation of from phosphatase, acetate hydrolase, 298 473, 474 Adaptive enzyme(s), see also Aromatic Adenosinediphosphate (ADP), rings, cleavage of, activation energy for hydrolysis of, 598 hydroxylamine reductase from Neuroactivation of 3,-glutamyltransferases spora crassa as, 411-417 by, 266, 272 kynureninase, bacterial, as, 253 deaminase action (non-specific) on, nitrate reductase from Neurospora as, 477, 478 411, 415 inhibition of D-amino acid oxidase by, nitrogen gas forming systems of Ps. 203 stutzeri and B. subtilis as, 423 of DPN kinase by, 654 tryptophan peroxidase of liver as, 244 of glutamine synthetase by, 339, 342 246, 253 of GSH formation by, 343 of Pseudomonas as, 245 of liver mitochondrial ATPase by, Adenine, 595 as acceptor of deoxyriboside group, 468 of pyridoxal kinase by, 649 inhibition of pyridoxal kinase by, 649 insect muscle ATPase action on, 597 R / v a l u e for, 466 liver mitochondrial ATPase action on, spectrophotometric assay of, 458 594 synthesis of nucleotides from, 501-504

SUBJECT INDEX myokinase action on, 598 " 5 " nucleotidase action on, 549 in oxidative phosphorylation assay, 615 as phosphate acceptor in citrullinase reaction, 377, 378 phosphorylation of by phosphocreatine, 605 in phosphorylation of riboflavin, 645 spectrophotometric assay via glucose6-phosphate dehydrogenase, 497 Adenosine-2',5'-diphosphate, cleavage of by potato adenosine-5phosphatase, 550 resistance of to "5" nueleotidases of seminal plasma and snake venom, 549, 550 TPN conversion to, by nucleotide pyrophosphatase, 655 Adenosine diphosphate phosphomutase, see Adenylate kinase Adenosine diphosphatc ribose (ADPR), action of nonspecific deaminase on, 477, 478 formation and transfer of by animal tissue DPNase, 660 formation of by Neurospora DPNase, 664 inhibition of DPN kinase by, 654 Adenosine monophosphate, see 2'-,3'- and 5'-Adenylic acids Adenosine-5-phosphatase, see also 5'Nucleotidase, from potato, 550 Adenosine phosphates, inhibition of nucleotide synthesis by, 504 Adenosine phosphokinase (adenosine kinase), in baker's yeast, 498 from brewer's yeast, 497-500 specificity of, 499, 500 in liver and kidney, 499 Adenosine triphosphatase (ATPase), in E. coli, 619 effect on arginine synthesis, 358, 359 on assay of adenylate kinase, 598 on glutamine synthetase reaction, 338, 339 on GSH synthesis, 343

897

in liver, 542, 593-595, 615, 653, 667 microsomes, 542 mitochondria, 593-595, 615 activity of intact versus modified, 594, 595, 615 from muscle, 588-591 possible identity of with myosin, 586 Adenosine triphosphatase, Mg-activated, from muscle (insect), 590-591, 595-598 comparison with mammalian enzyme, 590-591 occurrence in muscle mitochondria, 595 from muscle (rabbit), 588-591 simulation of by actomyosin, 589 Adenosine triphosphate (ATP), activation energy for hydrolysis of, 598 activation of ~-glutamyltransferase (brain) by, 272 complex of with Mg, 604 as component of firefly luciferase system, 651, 851, 854, 856 specificity for, 856 formation of by adenylate kinase, 598 by citrullinase, 376-377 with muscle enzyme fraction and phosphoglycerate, 515 by oxidative phosphorylation, 613 in formylation of glycinamide ribotide, 505, 510 of 5-IRMP, 519 of tetrahydrofolic acid, 517 hydrolysis of by apyrase, 591 by deaminase (non-specific), 477, 478 by myosin, 586 by 5-nucleotidase, 549 by nucleotide pyrophosphatase, 655, 659 by prostatic phosphatase, 525 incorporation of p~2 into as measure of oxidative phosphorylation, 614 inhibition of adenylate kinase by, 604 of arginine synthesis by, 356 of citrulline synthesis by, 355 of FAD hydrolysis by, 673 of pantothenate-synthesizingenzyme by, 621 phosphorylation of adenosine by, 497 of dephosphooCoA by, 649, 651 of DPN by, 652, 654

898

SUBJECT INDEX

of nucleoside monophosphates by, 6O3 of pantetheine by, 633, 635 of pyridoxal by, 646, 649 of riboflavin by, 640, 645 of thiamine by, 636 speetrophotometric estimation via glucose-6-phosphate dehydrogenase, 497 in synthesis of active methionine, 254 of amino-imidazolecarboxamideribotide, 514-515 of arginine, 356-357, 364 of citrulline, 350 of dephospho-CoA, 667-669 of DPN from NMN, 670 of DPNH from NMNH, 672 of FAD from FMN, 673 of glutamine, 337 of glutathione, 342 of glycinamide ribotide, 504, 509-512 of hippuric acid, 346, 348, 349 of IMP, 505, 518-519 of nucleotide from adenine and R-5-P, 501-504 of pantothenate, 619 of 5-phosphoribosyl pyrophosphate, 5O4 Adenosine triphosphate-creatine transphosphorylase (creatine kinase), from rabbit muscle, 605-610 crystallization of, 608-609 kinetics of, anomalous nature of, 606-607 S-Adenosylmethionine (AMe), methylation of guanidinoacetic acid by, 260 of nicotinamide by, 257 as product of methionine-activating enzyme, 254 Adenylate kinase (myokinase, ADP phosphomutase), distribution of, 602 in E. coli, 619 in flavokinase preparations, 645 nomenclature, basis for, 602 from rabbit muscle, 598-604 as byproduct of 3-phosphoglyceraldehyde dehydrogenase preparation, 601

removal of from myosin, 585 role in muscle relaxation, 602 Adenylic acid (s), bone phosphatase action on 2'-, 3r-, and 5'-, 540, 541 potato phosphatase action on 2'- and 3'-, 550 suppression of at high pH, 550 prostatic phosphatase action on 2'-, 3'and 5'-, 524-525 kinetics of, 525 2'-Adenylic acid (2'-AMP) (yeast adenylic acid, isomer A), activation of Pseudomonas transhydrogenase by, 683, 685-686 dependence on nature of substrate, 685-686 cleavage by non-specific phosphatases, 552 inhibition of DPN kinase by, 654 3'-Adenylic acid (3'-AMP) (yeast adenylic acid, isomer B), action of nonspecific deaminase on, 477, 478 cleavage by non-specific phosphatases, 552 inhibition of DPN kinase by, 654 paper chromatography of, 516 as substrate for 3'-nucleotidase, 551553 5'-Adenylic acid (5'-AMP, adenosine5'-phosphate), deaminase action on, non-specific, 477, 478 specific, 469 in glutathione reductase assay, 720 inhibition of adenylate kinase by, 604 of D-amino acid oxidase by, 203 of DPN kinase by, 654 of flavokinase by, 645 of pyridoxal kinase by, 649 in isocitric dehydrogenase system (DPN-linked) of yeast, 682 molecular extinction of, 469 in oxidative phosphorylation assay, 611, 615 paper chromatography of, 516 phosphatase (AMPase) for in microsomes, 542

SUBJECT I N D E X

as phosphate acceptor in citrullinase reaction, 377, 379 in nucleoside monophosphate kinase reaction, 603 in respiring mitochondria, 603 fluoride effect on, 603 as product of adenylate kinase action, 598 of apyrase action, in insect, 590-591, 595-598 in potato, 591 of nucleotide pyrophosphatase action, 655 of pantothenate synthesizing system, 619, 622 of thiaminokinase action, 636 as substrate for " 5 " nucleotidases: 547-549, 550, 561 synthesis of from adenine and R-5-P, 501-504 from adenosine, 497 5'-Adenylic acid deaminase (Schmidt's deaminase), in assay of adenylate kinase, 599 of AMP derivatives, 473 of pantothenate-synthesizing enzyme, 622 from rabbit muscle, 469-473 as byproduct of preparation of other muscle enzymes, 470 presence of in purified myosin, 586 Adenyl pyrophosphate, see Adenosine triphosphate Adenylyl uridylic acid, as substrate for spleen phosphodiesterase, 568 Adrenaline, see Ephinephrine Aerobacter aerogenes, cytochrome bl in, 745 5-dehydroquinase in, 305-307 5-dehydroshikimic reductase in, 304 7-glutamyltransferase (GTF) in, 269 nucleotide transhydrogenase in, 308 quinic dehydrogcnase from, 307-311 transaminase in, 173-174 tryptophan synthetase formation in, 237 Aerobic phosphorylation, see Phosphorylation, oxidative

899

A gkistrodon piscivorus (water moccasin), 5'-nucleotidase in venom of, 561 separation of from phosphodiestero ase, 563, 564 phosphodiesterase activity of venom of, 565 Agmatine, action of diamine oxidase on, 396 L-Alaninamide, hydrolysis by amidase, 399 by leucine aminopeptidase, 92 Alanine, D-amino acid oxidase and, 171, 176, 199, 200, 613 formation by kynureninase, 249 inhibition of alkaline phosphatase by, 538 in peptide B from fibrinogen, 160 transaminase for valine and, 176 transamination with pyridoxamine phosphate in C. welchii, 173 ~-Alanine, enzymatic conversion to pantothenate, 619 formation by aspartic acid decarboxylase, 188 Alanine racemase, in Bacillus spores, 215 from S. faecalis, 212-215 L-Alanyl dipeptides, action of acylase I on, 118 ~-Alanyl dipeptides, resistance of to glycylglycine dipeptidase, 109 L-Alanylglycylglycine, as substrate for aminotripeptidase, 87 ~-Alanyl-L-histidine (carnosine), activity of carnosinase on isomers of, 93, 94, 96 carbobenzoxy derivative of, resistance to carnosinase, 96 presence in muscle, 93 Albumin, serum, as protective agent in enzyme assays, 346, 483, 492, 724 Alcohol(s), inhibition of prostatic phosphatase by, 527 *Alcohol dehydrogenase, yeast, DPN assay with, 660, 670

900

SUBJECT INDEX

DPNH generation with, 694, 707 nitroaryl reductase assay with, 407408, 411 Aldehyde(s), formation of by amine oxidase~ 390 by diamine oxidase, 394 long chain aliphatic, as component of bacterial luciferase system, 857, 860, 861 combination of with cysteine to form inhibitor, 860 as substrate for xanthine oxidase, 484 *Aldehyde oxidase, reduction of dinitrobenzene by, 409 Alkaptonurics, homogentisate oxidase and, 294 Alkylsulfatases, 324, 330 Allantoin, formation of by uricase, 485 D-Allocystathionine, as substrate for cleavage enzyme~ 312 L-Alloisoleucinamide, hydrolysis of by leucine aminopeptidase, 92 Alloxazine nucleotide, see also Flavin nucleotides, reversible dissociation from old yellow enzyme, 712 Allylisothiocyanate, from horseradish, 801-813 Alum, in clarification of rennet, 73 Alumina gel, preparation of, 823 Amberlite IRC-50, chromatography of Ustilago eytochrome c with, 756-757 Amberlite XR-64, purification of cytochrome c with, 752 Amidase, action on amino acid amides, 397 chymotrypsins as, 22-23 trypsin as, 34-36 Amides, resistance to action of iminodipeptidase, 100 as substrates for leucine aminopeptidase, 91 Amine oxidase, see also Diamine oxidase and Monoamine oxidase, from steer plasma, 390-393 Amines, activation of thiaminase by, 625, 627

determination of non-acetylatable, diazotizable, 574 inhibition of diamine oxidase by, 396 of firefly luciferase by, 856 of ~-glutamyltransferase (bacterial) by, 269 oxidation of by horseradish peroxidase complex II, 810 by lactoperoxidase, 816 by myelopcroxidase, 800 role in detoxication of diphtheria toxins, 800 a-Amino acid(s), activation of D-amino acid oxidase by, 202 N-aeyl derivatives of, hydrolysis by aminoaeylases, 113, 115, 117-118 effect of variation of acyl group, 118 amides and esters of as substrates for trypsin, 32, 34-36 inhibition of ~-glutamyltransferase (bacterial) by, 269 of ~-glutamyltransferase (brain) by, 272 of Neurespora kynurenir,ase by, 253 liberation of by amidase, 397 oxidation by peroxidase complex II, 8O8 presence of in horseradish peroxidase, 811 as product of aminotripeptidase action, 83 reduction of, 217-220 resolution of by means of aminoacylases, 119 substances competing with, in D-amino acid oxidase reaction, 203 N-substituted, as substrates for Damino acid oxidase, 202 transamination among aliphatic, 171 among aromatic, 171 among D-forms of, 171, 175-176 Amino acid(s), aromatic, amides and esters of as substrates for chymotrypsins, 21 inhibition of carboxypeptidase by D-form of, 79 Amino acid acylase I (dehydropeptidase II, soluble acylase I), from hog kidney, 109-114, 115-119

SUBJECT INDEX

action as acylase, 115-119 heat stability of, 117 specificity of, 117-118 action as dehydropeptidase, 109-114 preparation of, 116-117 Amino acid acylase II (aspartic acid acylase), from hog kidney, 115-119 preparation of, 117, 119 specificity for acyl aspartic acids, 119 Amino acid acylase III, evidence for, 118 Amino acid amidase, from hog kidney, 397-400 Amino acid amides, participation in transaminase reactions, 170 role of manganous ion in destruction of, 398 Amino acid decarboxylases, from animals, 195-199 3,4-dihydroxyphenylalanine (dopa) decarboxylase, 195-199 other amino acid decarboxylases, 199 from bacteria, 185-189, see also under names of individual amino acids listed below, arginine, 187 aspartic acid, 188 glutamic acid, 186-187 histidine, 187 lysine, 188-189 ornithine, 189 tyrosine, 188 inhibition of by carbonyl reagents, 241 from plants, 190-194 glutamic acid decarboxylase, 190194 other amino acid decarboxylases, 194 D-Amino acid oxidase, assay of alanine racemase with, 212 assay and identification of FAD with, 200, 673, 698 occurrence with glycine oxidase, 227 requirement for FAD, 227 from pig kidney, 171, 212 in measurement of transaminations involving D-alanine, 176 from sheep kidney, 199-204

901

L-Amino acid oxidase(s), detection of, 179 in Neurospora crassa, 211 in rabbit liver, 213 interference in racemase assay, 213 from rat kidney, 209-211 in rat liver, 209 from snake venom, 205-209 in tissue slices, apparent presence of, 211 Amino acid racemases, 212-217, see also under names of individual enzymes, alanine racemase, 212-215 glutamic acid racemase, 215-217 Amino acid reductases, from Cl. sporogenes, 217-220 evidence of separate enzymes for, 220 other sources of, 218 Aminoacrylic. acid, as probable intermediate in tryptophanase reaction, 238 Aminoacylase(s), see also Amino acid acylase I and Amino acid acylase II, resolution of racemic a-amino acids by, 119 2-Aminoadenosine (2,6-diamino-9-~-Dribofuranosylpurine), phosphorylation of, 497 ~-Aminoadipic acid, as substrate for glutamine synthetase, 341 p-Aminobenzoic acid, estimation of, 350 inhibition of tyrosinase by, 826 oxidation of by peroxidase complex II, 810 as product of aromatic biosynthesis, 300 m-Aminobenzyl-(3)-4-methylthiazolium salt, activation of thiaminase by, 625 o-Aminobenzyl-(3)-4-methylthiazolium salt, and related compounds as inhibitors of thiaminase, 625 DL-~-Amino-n-butyramide, hydrolysis by leucine aminopeptidase, 92

902

SUBJECT INDEX

a-Aminobutyric acid, transaminase for valine and, 176 -r-Aminobutyric acid, formation by glutamie acid deearboxylase, 182, 186 L-a-Aminobutyryl-L-histidine, activity of earnosinase on, 96 L-Aminocaproie acid, as substrate for ~-amino acid oxidase, 211 p-Aminodimethylaniline, reaction with sulfide to form methylene blue, 315 Aminodinitrotoluene, reduction of trinltrotoluene to, 406 p-Aminohippurie acid, enzymatic synthesis of, 350 estimation of, 350 3-Amino-4-hydroxydichloroarsine, inhibition of aspartase by, 388 m-Amino-p-hydroxyphenylarsenoxide, inhibition of amino acid reductases by, 220 5 (4)-Amino-4(5)-imidazoleearboxamide, as acceptor of deoxyriboside group, 468 conversion to DPN analog by spleen DPNase, 663 paper chromatography of, 513 R/values for, 466 5 (4)-Amino-4(5)-imidazoleearboxamide riboside, enzymatic synthesis of, 448, 453 conversion to ribotide by yeast enzyme, 514-516 isolation of from E. coli cultures, 512514 paper chromatography of, 513, 516 5 (4)-Amino-4-(5)-imidazolecarboxamide ribotide, characterization by spectrum, 515 isolation of by ion exchange chromatography, 514-516 molecular extinction coefficient of, 516 paper chromatography of, 516 as precursor of inosine-5'-phosphate (5'-IMP), 505, 518-519 of riboside in sulfa-treated E. coli, 512 preparation from inosinie acid (IMP), 514

recovery from sulfa-treated E. coli, 514 5-Amino imidazole ribotide, basis for structure of, 519 conversion to 5-amino-4-imidazolecarboxamide ribotide, 505 2-Amino-2-methyl-l,3-prepanediol, glutamic dehydrogenase and, 224 2-Amino-4-nitrophenol, inhibition of quinone reductase by, 729 p-Amino-ornithuric acid, enzymatic synthesis of, 350 Aminopeptidases, 83-93 aminotripeptidase (tripeptidase), 8387 leucine aminopeptidase, 88-93 Aminophenols, oxidation by peroxidase complex II, 808 a-Aminosulfonic acids, aliphatic, inhibition of L-amino acid oxidase by, 208 Aminotripeptidase(s) (tripeptidase), 8387 from calf thymus, 84, 85-86 from horse erythrocytes, 84-85 leueine aminopeptidase and metalactivated dipeptidases as contaminants in, 87 presence in prolinase preparations, 97 solubility in ammonium sulfate solutions, 84 in swine intestinal mueosa, 84 table of specificity of thymus and erythroeyte enzymes, 87 Amino-unsaturated acid, as probable product of desulfhydrases, 315 ~-Aminovaleric acid, formation by reduction of ornithine or proline, 217 L-a-Aminovaleric acid, as substrate for L-amino acid oxidase, 211 Ammonia, citrulline synthesized from COs, ornithine and, 350 formation from adenosine, 473 from adenosine compounds, 475 by 5'-adenylic acid deaminase, 469 by amino oxidase, 390

SUBJECT INDEX

by amino acid amidase, 397-400 by arginine desimidase, 374 by asparaginase, 383 by aspartase, 386 by eathepsin C, 64 by citrullinase, 374 by cystathionine cleavage, 311-314 by bacterial enzyme, 314 by liver enzyme, 311 by eysteine desulfhydrase, 315-318 by cytosine nucleoside deaminase, 478 by dehydropeptidases, 110, 111 by diamine oxidase, 394 by exocystine desulfhydrase, 319 by glutaminase, 380 by glyeine oxidase, 225 by guanase, 480 by histidase, 228 by hydroxylamine reduetasc, 416 by leueine aminopeptidasc, 88 by reduction of glycine, 217 by L serine (L-threonine) dehydrase, 319 322 by tryptophanase, 238 in glutamie dehydrogenase reaction, 220 in glutamine synthetase reaction, 337 in 5-glutamyltransferase reaction, 263, 267, 269 inhibition of v-glutamyltransferase (brain) by, 272 measurement of, 204, 316, 397 for assay of L-amino acid oxidase, 204 Ammonium chloride, inhibition of nitroethane oxidase by, 402 Ammonium ion, activation of pantothenate-synthesizing enzyme by, 621 of tryptophanase by, 242 effect of on RNase, 433 inhibition of mammalian L-amino acid oxidase by, 211 Ammonium polysulfide, see Sulfide Ammonium sulfate, effect of on protein solubility in aqueous acetone, 132 equation for changes in saturation of, 571

903

inhibition of arginine-synthesizing system by, 359 of hydrogenase by, 732 measurement of concentration of by conductivity, 391 nomogram for concentration of, 18 Amylamine, amine oxidase action on, 393 *Amylase, in preparation of crystalline liver eatalase, 776 Anana sativa, see Pineapple Ai~giotonase, 135, 136 Angiotonin (hypertensin), bioassay of, 136 formation by renin, 124, 135 preparation of, 135 136 ultraviolet absorption of, 136 Anhydroleueovorin (anhydrocitrovorum factor) (ACF), chemical intereonversion of N ~°-formyltetrahydrofolic acid, and leucovorin, 518, 519 in formylation of IRMP to IMP, 519 spectrophotometric measurement of, 518 Aniline, as base in thiaminase reaction, 623 detoxication of diphtheria toxin by myeloperoxidase in presence of, 800 oxidation by peroxidase complex II, 810 Aniline citrate, decarboxylation of oxalacetate with, 170-171, 174-175 preparation of solution of, 174 Anions, inhibition of L-amino acid oxidase by bi- and trivalent, 208 of DPNH cytochrome c reduetase by, 692 of erythrocyte carbonic anhydrase by, 845 monovalent, stabilization of purine nueleosidase by, 460 o-Anisidine (2-methoxyaniline), detoxication of diphtheria toxin by myeloperoxidase and, 800

904

SUBJECT INDEX

Anthranilic acid, absorption maximum for, 250 formation by kynureninase, 249 inhibition of kynurenine formamidase by, 246, 249 as precursor of catechol, 273 tryptophan peroxidase and, 244 Antifibrinolysin,from ox lung, 165 Antifoam agent, 756 Antihemophilic factor (AHF, PTC), activation by thrombin, 157 assay of, 147-148 concentration in oxalated horse plasma, 147 as heat labile factor in thromboplastin, 139, 147-148 purification from plasma, 149 stability of, 150 Antimycin, inhibition of DPNH cytochrome c reductase activity of mitochondria by, 693 Antipenicillinase serum, effect on penicillinase activity, 123 Antirenin, neutralization of pressor effect of renin by, 134 production in human subjects, 129, 134 Antisera, inhibition of pancreatic and streptococcal DNases by respective, 447 Antithrombin, 158, 162, 163 in defibrinated plasma, 162 heparin cofactor and, 163 Antithromboplastin(s), 160-162 lipid antithromboplastin from brain, 161 protein antithromboplastin from muscle, 161-162 Apocarboxylase, preparation for thiamine pyrophosphate assay, 637, 638 Apurinic acid, disintegration by Mg ++, 441 resistance to DNase action, 441 Apyrase(s), see also Adenosine triphosphatase, from insect muscle, 590-591, 595-598

interference with adenylate l~inase assay by, 600 from potato, 591-593, 646 removal of ATP by, 646 temperature effect on nature of products, 593 L-Arabinose, paper chromatography of, 513 Arabitylflavin, phosphorylation by flavokinase, 644 Arachain, protease from peanut, 57, 63 Arachis hypogen, see Peanut Arginase, in assay of arginine-synthesizing system, 356-357, 359, 360, 364-365 from beef liver, 357, 358 effect of heat on, 370 from horse liver, 368-374 physical-chemical tests of purity of, 373-374 seasonal variation in content of, 371 L-Arginine, action of arginase on, 368 activation of DNase by, 442 content in horseradish peroxidase, 809 enzymatic synthesis of, 356-367 condensing enzyme system for, 359-364 liver enzyme, 360-362 yeast enzyme, 360, 362-364 over-all reaction, 356-359 from kidney, liver and yeast, 357, 358-359 splitting enzyme for, 364-367 preparation from beef liver or pig kidney, 365-367 hydrolysis of to eitrulline and NHs by desimidase, 3741 inhibition of splitting enzyme for arginine synthesis by, 367 pK of a-amino group, 368 reduction of, 217 stabilization of arginase by, 373 as substrate for L-amino acid oxidase, 2O8 Arginine decarboxylase, from E. coli, 187 resolution of, 189

SUBJECT INDEX in measurement of transaminase reactions, 171 Arginine desimidase, distribution of, 376 from yeast, 375-376 Arginine dihydrolase system~ 374-378 arginine desimidase component of, 374, 375-376 citrullinase component of, 374, 376378 L-Argininosuccinic acid, as intermediate in synthesis of arginine, 359, 364-365, 367 Aromatic biosynthesis, bacterial enzymes for, 300-309 5-dehydroquinase, 305-307 5-dehydroshikimic reductase, 301304 quinic dehydrogenase, 307-309 scheme for, 300 Aromatic rings, cleavage of, in aerobic bacteria, 273-287 enzymes in scheme for, benzaldehyde dehydrogenases (TPN- and DPN-linked), 280-281 benzoylformic carboxylase, 273, 278-280 lactonizing and lactone-splitting enzymes, 282-284 L(~)-mandelic acid dehydrogenase, 273, 274, 277-278 mandelic acid racemase, 273, 274, 276-277 protocatechuic acid oxidase, 284287 pyrocatechase, 273, 274, 281-282 reaction sequences for, 273 Arsenate, activation of -y-glutamyltransferase by, 264, 267, 272 inhibition of alkaline phosphatase by, 538 of DNase by, 442 of D P N H cytochrome e reductase by, 692 of glucose-6-phosphatase by, 542 of metaphosphatase by, 579 of purine nucleosidase by, 460

905

replacement of phosphate, Mg ++ and ADP requirements of citrullinase by, 377, 378 stabilization of pyrimidine nucleosidase by, 459 Arsenicals, inhibition of aspartase by, 388 Arsenious oxide, inhibition of eysteine desulfhydrase by, 317 Arsenite, see also Arsenious oxide, effect of on metaphosphatases, 579 inhibition of amino acid reductases by, 220 of arginine desimidase by, 376 of D P N H cytochrome c reductase by, 693 of hydrogenase by, 732 of plant carbonic anhydrase by, 846 reversal by cysteine or glutathione, 846 as uncoupling agent, 615 Arsenolysis, CoA assay by, 649 Arsenoxides, organic, inhibition of amino acid reductases by, 220 Arylsulfatases, from marine mollusks, 332 from ox liver, 330-332 preparation of arylsulfatase A, 330-332 of arylsulfatase B, 332 from takadiastase, 328

Ascaris, digestion of by plant proteinases, 55 trypsin inhibitor from, 37, 54 Asclepain, protease from mllkweed~ 56, 61-62 amorphous form of, from leaves, 62 activation by sulfite, 62 crystallization of, from latex, 62

Asclepias speciosa, mexicana, syriaca, see Milkweed Ascorbate, activation of homogentisate oxidase by, 292 of p-hydroxyphenylpyruvate enolketo tautomerase by, 292, 295 of tyrosine-oxidizing system by, 287, 288, 289

906

SU-B J E C T I N D E X

in catecholase assay, 819 cytochrome c reduction by, 754 determination of, 847-848 effect on absorption bands of cytochrome b, 744 formation by dehydroascorbic reductase, 847-850 oxidation by peroxidase complex II, 808, 810 L-Ascorbate, as substrate for ascorbic acid oxidase, 831-835 comparison of with D-form and other ene-diols, 834 Ascorbic acid oxidase, from yellow squash, 831-835 absorption spectrum of, 834 reaction inactivation as characteristic of, 835 L-Asparaginase, from guinea pig serum, 383-384 in yeast, 384 Asparagine, comparison of rate of cleavage of D- and IMsomers by asparaginase, 384 ~-keto aeid-mediated deamidation of, 382 transamination with aliphatic keto acids, 171 Asparagine transaminase, distinction from asparaginase, 382 Aspartase, 386-390 determination of L-aspartic acid with, 389, 390 in protein hydrolysates, 390 from propionic acid bacteria, 386-387, 388 from Ps. fluorescens, 387 Aspartie acid, action of D-serine (D-threonine) dehydrase on, 324 N-acyl derivatives of, as substrates for acylase II, 119 arginine synthesis from, 356-357, 364 as component of transaminase system, 180 conversion to fumaric acid and ammonia, 386 formation by asparaginase, 383

inhibition of glutamic dehydrogenase by, 224 measurement by chloramine-T method, 170-171 quantitative determination by aspartase, 389, 390 in protein hydrolysates, 390 as C-terminal residue, effect of on iminopeptidase, 100 transaminase reactions involving, 170, 172, 174, 175, 176, 179-182 measurement of formation of oxalacetate in, 174, 175, 179-182 Aspartic acid acylase, see Amino acid acylase II Aspartic acid decarboxylase, from C1. welehii, 182, 188 measurement of transaminase reactions with, 171 L-Aspartic diamidc, hydrolysis by leucine aminopeptidase, 92 ~-L-Aspartyl-L-histidine, activity of carnosinase on, 96 AspergiUus sp., metaphosphatase in, 577, 578, 579

Aspergillus niger, glucose oxidase in, inhibition by nitrate, 579 growth of, 578

Aspergillus oryzae, commercial takadiastase and clarase from, 579 triphosphatase in takadiastase from, 580, 582 Atabrine, see Quinacrine Aureomycin, as uncoupling agent, 615 Auric salts, inactivation of renin by, 134 8-Azaguanine, deamination by guanase, 482 enzymatic synthesis of riboside and deoxyriboside of, 448 Azide, compound of catalase with, 788 of ferricytochrome c with, 755 of horseradish peroxidase with, 812 and cytochrome b, lack of reaction between, 745

SUBJECT INDEX determination of cytochrome c peroxidase with, 764 effect on activity and spectrum of myeloperoxidase, 799 inhibition of D-amino acid oxidasc by, 203 effect of pH on, 203 of aspartase by, 388 of d-biotin oxidation by, 632 of carbonic anhydrase by, 844, 845 of catalase by, 764 of cytochrome c peroxidase by, 763 pH dependence of, 763 of homogentisate oxidase by, 295 of hydroxylamine reductase by, 419 of liver glutathione reductase by, 725 of metaphosphatase by, 579 of nitrate reductase by, 415 of nitroaryl reductase by, 410 of nitroethane oxidase by, 402 of spinach catalase by, 790 of tryptophan peroxidase by, 246 of tyrosinase by, 826 as uncoupling agent, 615 Azotobacter spp. transhydrogenase in, 686 Azotobacter agile, hydrogenase in, 870 Azotobacter chroScoccum, cytochrome bl in, 745 Azotobacter vinelandii, cytochrome c in, 759 cytochrome c peroxidase in, 764 glutamic-oxalacetic transaminase in, 184 hydrogenase in, 870 B

Bacillus cereus, penicillinase from, 120-124 growth medium for, 122 Bacillus lichenformis, cytochrome e in, 745 Bacillus subtilis, action of lysozyme on, 421 cytochrome a3 in, 734 cytochrome b in, 745 enzymes for nitrogen gas formation from, 420-423 growth of thermophilic strain of, 421

907

protease from, 80 transaminase in, 173, 176 Bacillus thiaminolyticus, growth of, 626 thiaminase from culture medium of, 626-628 Bacteria, see also under names of individual species, catalase in, 765 cytochromes a, as and a3 in, 732 cytochrome b group in, 744-748 dcsulfhydrases in, 318 desulfinases in, 333 press for extraction of, 219 L-serine (L-threonine) dehydrase in, 320 transaminases in, 170-177 distribution of, 172, 173-4, 176 Bacterium cadaveris, lysine decarboxylase from, 188-189 Barbital, see Veronal Barbiturase, from Mycobacterium, 492-493 Barbituric acid, hydrolysis to urea and malonic acid, 492 Barium carbonate, use in purification of prothrombin, 144-145 Barium ion, inhibition of DPNH cytochrome c reductase by, 692 Barley, 3'-nucleotide phosphatase in, 524 phosphatase in, 540 Beet (Beta vulgaris), carbonic anhydrase in leaf of, 842 Benadryl, inhibition of amine oxidase by, 393 Bentonite, in purification of soybean trypsin inhibitor, 41 Benzaldehyde, formation by benzoylformie carboxylase, 273, 277, 278-280 by amine oxidase, 390 molar extinction coefficient for, 390 oxidation of, 273, 277 pyridine nucleotide requirement for, 277

908

SUBJECT INDEX

Benzaldehyde dehydrogenases (TPN- and DPN-linked), from Ps. fluorescens, 273, 280-281 Benzidine (4, 4'-diaminobiphenyl), detoxication of diphtheria toxin by myeloperoxidase and, 800 sulfate determination with, 324-326 Benzimidazole (s), acceleration of RNase action by, 434 competition with firefly luciferin, 856 Benzoic acid, S-benzoyl-CoA formation from, 346 conversion to hippuric acid, 346 and derivatives, inhibition of D-amino acid oxidase by, 203 formation by benzaldehyde dehydrogenase, 273, 280, 281 inhibition of venom L-amino acid oxidase by, 208 o-Benzoquinone, detection of in chronometric assay of tyrosinase, 819 p-Benzoquinone (p-quinone, quinone), photochemical reduction of, 726 reaction with glycyglycine, 729 reduction by cysteine and GSH, 728 by DPNH, 725, 728 Benzoquinone acetic acid, inhibition of homogentisate oxidase by, 294 reduction by DPNH, 728 L-(~-Benzoyl)-alanine, as substrate for liver kynureninase, 253 a-Benzoylargininamide, as substrate for trypsin, 36 Benzoyl-L-arginine ethyl ester, as substrate for trypsin, 36 a-Benzoyl-L-arginine methyl ester, cross reactivity with trypsin and chymotrypsins, 21 as substrate for trypsin, 36 S-Benzoyl-CoA, condensation with glycine, 346, 349 speetrophotometric measurement of, 349-350 as intermediate in hippuric acid synthesis, 346 Benzoylformic acid, conversion to benzaldehyde, 273, 277, 278-280

formation by mandelic acid dei~ydrogenase, 273, 277 Benzoylformic carboxylase, from Ps. fluorescens, 273, 278-280 a-Benzoyl-L-lysinamide, as substrate for trypsin, 36 Benztriazole, competition with firefly luciferin, 856 Benzyl alcohol, distribution of riboflavin and F M N between water and, 642 Benzylamine, molar extinction coefficient for, 390 as substrate for amine oxidase, 390, 393 a-Benzyl-i-argininamide, as substrate for assay of papain, 59 2-Benzylglyoxaline, inhibition of penicillinase by, 123 Benzylpenicillin, as substrate for penicillinase, 120-121 Benzylviologen, reduced, oxidation by amino acids, 217, 218 Beryllium ion, inhibition of alkaline phosphatase by, 538 Beta vulgaris, see Beet Bicarbonate, in carbonic anhydrase assay, 837 distinction from CO2 as product of enzymatic decarboxylations, 840841 in over-all system for I M P synthesis, 506 Bioluminescence, enzyme systems for, 851-861 Biotin, L-amino acid oxidase and, 211 Biotin analogs, inhibition of d-biotin oxidation by /-biotin and, 632 d-Biotin carboxyl-C 14, oxidation by liver slices, 631, 632 preparation of, 631 d-Biotin oxidase, in kidney and liver slices, 631-632 Bis-p-nitrophenylphosphate, hydrolysis by prostatic phosphatase, 524

SUBJECT INDEX Bisulfite, inhibition of hydroxylamine reductase by, 419 Blood clotting, 139-166, see also under names of individual components, antifibrinolysin, 165 antithrombin, 162 antithromboplastins, 160-162 cofactor of heparin, 163 convertin (SPCA, Factor VII, cothromboplastin), 155-156 fibrinogen, 158-160 fibrinolytic activators from tissues, 166 hypothesis of, 139-140 plasma Ac-globulin (proaccelerin, Factor V), 151-153 pro-convertin (SPCA precursor), 153154 pro-fibrinolysln (plasminogen), 163165 prothrombin, 140-146 serum Ac-globulin (accelerin), 153 thrombin, 156-158 thromboplastin (thrombokinase), 146151 Blood plasma, see Plasma Boiled juice (kochsaft), from pig heart, as source of flavin, 407, 416 Bone, alkaline phosphatase from, 539-541 phosphodiesterase in, 540 Borate, catalysis of CO~ hydration by, 836 complex of with a-hydroxy acids, 290 with carbohydrates, 291 inhibition of alkaline phosphatase by, 538 of ATPase (myosin) by, 588 of DNase by, 442 of "5" nucleotidase of seminal plasma by, 549 stabilization of enol form of p-hydroxyphenyl pyruvate by, 290 Borohydride, sodium, reduction of flavin component of cytochrome c reductase by, 691 Bottom yeast, see Yeast, brewer's Brain, adenylate kinase (myokinase) in, 602

909

DPNase (pyridine transglycosidase) from, 662 I)PN pyrophosphorylase in, 671 glutamic acid decarboxylase in, 199 glutamic-oxalacetic transaminase activity of, 184 L-glutaminase in, 382 glutamine synthetase from, 339, 341342 ~-glutamyltransferase (Mn-dependent) from, 269-271 lipid antithromboplastin from, 161 pyridine nucleotide transhydrogenase in, 681, 687 thromboplastin from, 139, 149 Brewer's yeast, see Yeast, brewer's British anti-lewisite (BAL) (2,3-dimercaptopropanol), activation of aspartase by, 388 of citrullinase by, 378 of guanidinoacetate methylpherase by, 263 effect on metal content of uricase, 489 inhibition of DPNH cytochrome c reductase activity of mitochondria by, 693 of tyrosinase by, 826 Bromelia pinguin, see Maya plant Bromelin, protease from pineapple, 56, 62-63 as meat tenderizer, 55 Bromoacetyl amino acids, action of acylase I on, 118 n-Butanol, extraction of aspartase from bacteria with, 387 extraction of kidney with borate and, 486, 487 Butylamine, amine oxidase action on, 393 4-t-Butylphenol, oxidation by tyrosinase, 826 n-Butyrate, inhibition of d-biotin oxidation by, 632 Butyryl phosphate, hydrolysis of, 556 C Cabbage, dehydroascorbic acid reductase in, 848, 85O

910

SUBJECT INDEX

Caeodylate, catalysis of CO2 hydration by, 836 Cadaverine (1,5-diaminopentane), as substrate for diamine oxidase, 396 Cadmium ion, activation of iminodipeptidase by, 100 inhibition of aminotripeptidase by, 87 of aspartase by, 388 of prolidase by, 105 Calcium carbonate, carbonic anhydrasc effect on deposition and solution of, 843 Calcium chloride, in assay of potato apyrase, 591, 646 of prothrombin, 140 of rennin by milk-clotting, 69 Calcium ions, activation of actomyosin ATPase by, 587 of adenylate kinase by, 603 of alkaline phosphatase by, 538 of ATP-creatine transphosphorylase by, 610 of metaphosphatase by, 579 of myosin ATPase by, 587 of pantetheine kinase by, 635 of proconvertin by, 154 of prothrombin by, 145 of thromboplastin by, 150 of triphosphatases by, 581 effect on isoelectrie point of trypsin, 35 on stability of trypsin, 32 on trypsinogen transformation, 26 inhibition of condensing system for arginine synthesis by, 364 of D P N H eytochrome c reductase by, 692 of firefly luciferase by, 856 of flavokinase by, 645 of glutamine synthetase by, 342 of glycyl-L-leucine dipeptidase by, 107 of inorganic pyrophosphatase by, 575 of insect muscle ATPase by, 598 of liver mitochondrial ATPase by, 595 of Mg-Activated ATPase by, 590 of " 5 " nucleotidase of seminal plasma by, 549

antagonism by magnesium ion, 549 of pantothenate-synthesizing enzyme by, 621 of penicillinase by, 123 of RNase by, 433 of thrombin activity by, 160 role in blood clotting mechanism, 152, 154, 158, 160 Calcium phosphate, chromatography of lactoperoxidase on, 814 of liver catalase on, 777 preparation for chromatography, 814 preparation of gel of, 214, 572, 805 Calgon, inhibition of adenylate kinase by, 603 Canavanine, formation of canavaninosucclnic acid from, 367 Cancerous tissue, dehydropeptidases in hepatic, 114 triphosphatase in, 580, 581, 582 Cantaloupe fruit, glutathione reductase in, 721 Caper spurge (Euphorbia lathyris, cerifera), protease (euphorbain) from latex of, 57, 64 Capryl alcohol, inhibition of apparent L-amino acid oxidase by, 211 Caprylate, P : 0 ratio during oxidation of, 613 Carassius carassius (crucian), thiaminase in, 625 Carbamate, carbonic anhydrase effect on dissociation of, 843 Carbamyl-n-glutam ate, role in citrulline synthesis, 350-355 Carbitol acetate (diethylene glycol monoethyl ether acetate), fractionation of arginase with, 373 Carbobenzoxyglycyl-L-amino acids, as substrates for carboxy peptidase, 78, 79 Carbobenzoxy-L-phenylalanine, as substrate for a-chymotrypsincatalyzed oxygen exchange reaction, 21

SUBJECT INDEX Carbobenzoxy-L-tyrosine ethyl ester, as substrate for trypsin and chymotrypsin, 23 Carbohydrates, content of in horseradish peroxidase, 809 presence of in thromboplastin, 150 Carbon dioxide, bicarbonate formation from hydroxyl ion and, 843 catalysis of reversible hydration of, 836-841, 843-846 by carbonic anhydrase, 836-841, 843-846 by oxy-acid buffers, 836, 838 citrulline synthesized from ornithine, ammonia and, 350 distinction from HC03- as product of enzymatic decarboxylations, 840, 841 formation by citrullinase, 374 by uricase, 485 velocity constant for uncatalyzed hydration of, 839, 840 Carbonate ion, inhibition of alkaline phosphatase by, 538 of carbonic anhydrase by, 839, 845 Carbonic acid, see Carbon dioxide Carbonic anhydrase, 836-846 assay methods for, 836-840 colorimetrie procedures for, 836, 339-840 CO2-Veronal indicator method, 839-840 rapid-flow method, 840 Krebs-Roughton manometric procedure, 836-839 limitation by COs diffusion rate, 838, 844 distinction between enzymatic COs and HC03- production by use of, 840-841 from erythrocytes, 838, 841-842, 843-846 crystalline preparation of, 841 inactivation during shaking, 838 prevention by peptone, 838 Keilin and Mann method for purification of, 841-842

911

zinc content of, 842, 843 sources of, 841, 842 from spinach, 836-840, 842-846 cyanide and sulfonamide insensitivity of, 845 essential sulfhydryl groups in, 846 evidence for zinc-protein nature of, 844 isolation as metal-free homogeneous protein, 846 zinc content of, 843 in Tradescantia fluminensis, 845 Carbon monoxide, complex with ferrocytochrome c, 754 compound with horseradish peroxidase, 812 detection of inactive form of cytochrome c by, 749 effects on spectra of cytochrome a group, 733, 734 inhibition of hydrogenase by, 866, 868 light-reversal of in P. vulgaris, 866 distinction from E. coli enzyme, 868 of tryptophan peroxidase by, 246 reversal by light, 246 of tyrosinase by, 826 non-reactivity with cytochrome b, 745 Carbonyl reagents, see also under names of individual reagents, inhibition of L-amino acid oxidase by, 2O8 of bacterial transaminases, by, 177 of diamine oxidase by, 396 of pyridoxal phosphate-requiring enzymes by, 241, 253 *Carboxylase (a-keto acid), in squash, 192, 193 inhibition by phenol, 192 in yeast, C02 versus HCO3- as product of, 840 Carboxyl groups, liberation by peptidases, estimation by Grassmann and Heyde procedure, 83, 88, 93-94, 97, 100, 105-106, 107-108 Carboxylic acids, inhibition of carboxypeptidase by aromatic and heteroeyclic, 79

912

SUBJECT INDEX

,-y-Carboxymethyl-Aa-butenolide, absorption spectrum of, 282 as lactone intermediate in/~-ketoadipic acid formation, 273, 282, 284 ~-Carboxymuconic acid, conversion of to B-ketoadipic acid, 273, 285 formation by protocatechuic acid oxidase, 273, 284-287 molar extinction coefficients for, 285 ~-Carboxymueonic acid decarboxylase, separation from protocatechuic acid oxidase, 286 Carboxypeptidase, from beef pancreas, 77-83, 118 acylases I and I I I compared with, t18 crystallization of, 82-83 effect on chymotrypsins, 8 failure to activate procarboxypeptidase, 80 formation from zymogen and pancreatic exudate, 81 physical properties of, 78 presence of zinc in, 78 resistance to DFP, 78 stability of, 78 substrates for, 78 strt~ctural analogs of as inhibitQrs, 79 Carica papaya, see Papaya Carnosinase, sources of, 94 from swine kidney, 93-96 Carnosine, see/~-Alanyl-L-histidine Carp, thiaminase in, 625 Carrots, glutamic acid decarboxylase in, 194 Casein, action of prostatic phosphatase on aform of, 526-527 inhibition by f~-casein, 527 proteolysis by rennin, 77 as substrate for trypsin and chymotrypsins, 19, 33-34 Catalase(s), assay of, direct spectrophotometric method for intact cells, 769

iodometric titration method, 785, 789 manometric method, 768-769, 781 perborate as oxidizing agent for, 780 permanganate titration method for, 768, 779, 781-782, 791-792 polarographic method, 780 ultraviolet spectrophotometric method for, 764-768 calculations for specific activity of, 767, 785 from liver, 775-781, 791-794 crystallization of, 776-779, 793 isoelectric point of, 776 separation into two components, 778 from M. lysodeikticus, 784-788 absorption bands for, 788 molecular weight of, 788 in Ps. fluorescens, 764 association with cytochrome c peroxidase, 764 from red blood cells, 781-784 absorption bands for, 784 crystallization of, 783-784 extinction coefficients for, 782, 784 molecular weight of, 784 paramagnetic susceptibility of, 784 sensitivity to alcohol-chloroform treatment, 782 from spinach leaves, 789-791 absorption bands of, 789 crystallization of, 790 role in assay of alanine racemase, 212, 213 of D-amino acid oxidase, 199 of L-amino acid oxidase, 199 of diamine oxidase, 394 of glycine oxidase, 225 of tryptophan peroxidase, 244, 246 of xanthine oxidase, 483 separation from D-amino acid oxidase, 2O0 from L-amino acid oxidase, 209 from various sources, 209, 765, 775 crystallization of, 775 molecular weights, extinction coefficients and specific activities of, 765 in Zwisehenferment preparations, 712, 718 inhibition by cyanide, 712

SUBJECT INDEX

Catechol, oxidation b y peroxidase complex II, 810 by pyrocatechase, 281-282 by tyrosinase, 819 Catechol monomethyl ether, see also Guaiacol, oxidation by peroxidase complex II, 810 Catecholase (catechol oxidase, polyphenol oxidase), see also Tyrosinase, tyrosinase as, 817, 819-821 assay of, 819-821 Catechols, substituted, oxidation by tyrosinase, 826 Cathepsin C, from beef spteen, 64-68 action on esters, 68 hydrolytic action of, 64 transamidation by, 65, 68 insoluble polymeric peptides as products of~ 68 pH dependence of, 68 Cations, divalent, inhibition of aminotripeptidase by, 87 of D P N H cytochrome c reductase by, 692 pantothenate synthesis requiremen~ for monovalent and, 621 Cauliflower, dehydroascorbic reductase from, 849, 85O Cclite, chromatography of liver catalase on, 777 Cellulose column, separation of venom phosphodiesterase from monoesterase by, 562-563 Cephalosporin P, inactivation by crude penicillinase, 123 Cetyltrimethylammonium bromide, inhibition of aspartic acid decarboxylase by, 188 reversal by pyruvate, 188 Charcoal, preparation of activated, 532 Chelate compound(s), formation by cobalt in glycylglycine dipeptidase action, 109 by manganese ion in prolidase reaction, 105

913

Chenopodium album, see Lamb's quarters Chloralosane, effect on renin action, 134 Chloramine-T, measurement of aspartate with, 170171

ChloreIla pyrenoidosa, arginine desimidase in, 376 Chloride, activation of 5'-AMP deaminase by, 472 effect on I~amino acid oxidase by, 208 inhibition of D P N H cytochrome c reductase by, 692 of erythrocyte carbonic anhydrase by, 845 stabilization of metal-free carbonic anhydrase by, 846 Chloroacetyl-amino acids, action of acylases on, 117, 118, 119 of dehydropeptidases I and II on, 113 of liver peptidase on, 114 Chloroaeetyldehydroalanine, preparation of, 110 as substrate for dehydropeptidase II, 109, 110 for liver dehydropeptidase, 114 Chloroacetyldehydroamino acids, action of dehydropeptidases I and I I on, 113 Chloroacetyldehydroleucine, as intermediate in synthesis of glycyldehydroleucine, 110 Chloroacetyl-L-glutamate, role in citrulline synthesis, 355 Chloroacetyl-L-phenylalanine, as substrate for carboxypeptidase, 78 Chloroaniline, detoxication of diphtheria toxin by myeloperoxidase and o and p forms of, 800 3-Chloro-4, 4'-diaminodiphenyl sulfone, effect on prothrombin activation, 145 Chloroform, activation of profibrinolysin by, 165 use in denaturation of hemoglobin, 102-103 p-Chloromercuribenzoate, activation and inactivation of RNase by, 434

914

SUBJECT INDEX

inhibition of aspartase by, 388 of bacterial luciferase by, 860 of D P N H cytochrome c reductase by, 693, 698 of firefly luciferase by, 856 reversal by glutathione, 856 of glutamic dehydrogenase by, 224 of glutamine synthetase by, 342 of homogentisate oxidase by, 295 of hydroxylaminc rcductase by, 419 irreversibility of, 419 of p-hydroxyphenylpyruvate enolketo tautomerase by, 292 of insect muscle ATPase by, 598 of Mg-activated ATPase by, 590 of myosin ATPase by, 587 of nitrate reductase by, 415 of plant carbonic anhydrase by, 846 reversal by cysteine or glutathione, 846 of prolidase by, 105 of tryptophan synthetase by, 237 Chloroplasts, Hill reaction with glutathione reductase system and, 722 dl-Chloropropionyl-amino acids, action of acylase I on, 118 Cholate, in purification of cytoehrome oxidase, 739 solubilization of eytochrome b with, 743 Cholesterol, presence of in heparin eofaetor, 163 in thromboplastin, 150 Chondrosulfatases, 324 Chromate, catalysis of C02 hydration by, 836 Chromatography, column, purification of barbiturase by, 493 of eatalase on calcium phosphate or celite by, 777 of eytoehrome e (bacterial) on kaolin by, 762 of dihydroSrotic dehydrogenase by, 496 of lactoperoxidase on calcium phosphate by, 814 of lactoperoxidase on silica celite by, 815

Chromatography, paper, of purine and pyrimidine bases and nucleosides, 457 for qualitative measurements of transaminations, 171, 172, 173, 175 separation of deoxyribosides by, 465, 466 Chymotrypsin (s), 8-26 action of on amides and esters, 21 cross-reactivity with trypsin on a synthetic substrate, 21 determination of activity of, 19-26 colorimetric test for amidase activity, 22-23 manometric assay of esterase activity, 23-26 milk clotting test, 20 potentiometric determination of esterase activity of, 23 substrates for, 23 spectrophotometric method of Kunitz, 19-20 modification of, 20 effect of carboxypeptidase on, 8 isoelectric points of, 25 molecular weights of, 24 optical factors for, 19 standard activity curves for, 20 terminal groups of, 8 a-Chymotrypsin, see also Diisopropyl phosphate a-chymotrypsin, action of on carbobenzoxy-L-phenylalanine (oxygen exchange), 21 as contaminant of a-chymotrypsinogen, 11-12 crystallization of, 12-13 formation of from a-chymotrypsinogen, 8, 12 hydrolysis of C - - C bond by, 21 transformation into fl- and -y-chymotrypsins, 8-14 transpeptidation and, 21 ~-Chymotrypsin, crystallization from pancreas, 14-15 7-Chymotrypsin, crystallization of from pancreas, 14 ~-Chymotrypsin, see also Diisopropyl phosphate ~-chymotryFrin, formation of from a-chymotrypsinogen, 8, 15-16

SUBJECT I N D E X

7r-Chymotrypsin, formation of from a-chymotrypsinogen, 8 Chymotrypsin B, comparison with a-chymotrypsin, 9 crystallization from pancreas, 9, 18 formation from chymotrypsinogen B, 9, 18 a-Chymotrypsinogen, chromatographic purification of, 12 conversion to ~-chymotrypsin, 8, 12 to ~r- and ~-chymotrypsins, 8, 15, 16 crystallization of, 8, 10, l l mechanism of activation by trypsin and by chymotrypsin, 8-9 Chymotrypsinogen B, activation to chymotrypsin B, 9, 18 crystallization of, 17, 18 deoxyribonuclease and, 10, 17 Chymotrypsinogen(s), 8-26 as byproduct of DNase preparation, 439 isoelectric points of, 25 molecular weights of, 24 Ciliary bodies, tyrosinase from, 829, 830 Citrate, activation of 5'-AMP deaminase by, 469, 472 of cystathionine cleavage enzyme by, 313 of prostatic phosphatase by, 557 of prothrombin by, 145 inhibition by triaminodiphenyl sulfones, 145 stimulation by 3-chloro-4,4'diaminodiphenyl sulfone, 145 effect on pH optimum of 5'-AMP deaminase, 473, 599 inhibition of acetyl phosphatase by, 556 of adenosine kinase by, 500 of adenylate kinase by, 599, 603 of aspartase by, 388, 390 of carnosinase by Mn ++ plus, 96 of DNase by, 442, 447 of D P N H eytochrome e reductase by, 692 competition with cytochrome c, 692

915

of iminodipeptidase by, 100 of leucine aminopeptidase by, 93 of phosphatases by, 313 of plasma Ae-globulin by, 152 of prolidase by, 105 P : O ratio during oxidation of, 613 Citrovorum factor (CF), see also Leucovorin, effect of folic acid conjugase on content of, in natural materials, 629 formation from 10-formylfolic acid by liver enzymes, 629 lability to acid, 630 Citrullinase (citrulline ureidase), in bacteria, 374, 376-378 ATP generation by, 376-377 distribution of, 378 Citrulline, action of citrullinase on, 374 arginine synthesis from, 356-357, 364 formation by arginine desimidase, 374 role of heavy metals in colorimetric determination of, 351,359 as substrate for L-amino acid oxidase, 2O8 Citr ulline-synthesizing system, from rat liver, 350-355 enzyme E1 for synthesis of "Intermediate," 350, 355 enzyme E~ for conversion of "Intermediate" and ornithine to citrulline, 350, 355 preparation of "Intermediate," 352353 Clam, see Meretrix meretrix, Venus mercuriana Clarase (Takamine), metaphosphatase in, 579 CIostridia, amino acid reductases in, 218 Clostridium kluyveri, cytochrome c peroxidase in, 764 hydrogenase from, 729-732 Clostridium pasteurianum, hydrogenase from, 869-870 Clostridium perfringens, arginine desimidase in, 375, 376 citrullinase in, 378 Clostridium septicum, ornithine decarboxylase from, 189

916

SUBJECT I N D E X

Clostridium welchii, aspartic acid decarboxylase from, 182, 188 glutamic-oxalacetic transaminase activity of, 84 L-glutaminase in, 382 growth of, 187, 188 histidine decarboxylase from, 187 inactivation of ATPase by lecithinase from, 590 transaminase in, 173, 176, 182 for pyridoxamine phosphatealanine system, 173 Clot-dissolving system, mode of action of, 140 Clotting, see Blood clotting and Milk clotting Cobaltous ion, activation of acylase I by, 119 dependence on nature of substrate, 119 activation of arginine desimidase by, 376 of citrullinase by, 378 of 3,-glutamyltransferase (brain) by, 272 of glycylglycine dipeptidase by, 107, 109 mechanism of, 109 of inorganic pyrophosphatase by, 575 of metaphosphatase by, 579 of triphosphatase by, 581 effect of on firefly luciferase system, 856 on flavokinase, 645 inhibition of aspartase by, 388 of 5-dehydroquinase by, 307 of fructose diphosphatase by, 546 of RNase by, 434 of D-serine (D-threonine) dehydrase by, 324 of splitting enzyme for arginine synthesis by, 367 of tryptophan synthetase by, 237 of uricase by, 489 Coenzymes, see also under names of individual coenzymes, metabolism of vitamins and, 619-677 requirement by modified mltochondria, 615

Coenzyme A, cleavage by potato enzyme, 659 effect on firefly bioluminescence system, 651 formation in liver extracts, from dephospho CoA, 649-651 from phosphopantetheine and ATP, 633, 667, 669 measurement of, 633, 649-650, 667-668 reagents for, 633 3'-nucleotidase action on, 553 removal of with Dowex-1, 349, 633 role in hippuric acid synthesis, 346, 348, 349 Colostrum, trypsin inhibitor from, 37, 46-48, 50, 51, 52 Convertin [serum prothrombin conversion accelerator (SPCA), Factor VII, cothromboplastin], complexes of with thromboplustin and plasma Ac-globulin, 151 purification of from plasma, 155-156 from serum, 156 role in clotting mechanism, 139-140, 150, 155, 156 Copper, see also Cupric ion, catalysis of leuco MB oxidation by, 716 complexing agents for, effect on ascorbic acid oxidase, 835 on tyrosinase, 826 content in ascorbic acid oxidase, 834 comparison with activity of ionic copper, 834 content in tyrosinase from mushroom, 825 in uricase, 489 inhibition of bacterial luciferase by, 860 of firefly luciferase by, 856 of glycine oxidase by, 227 of nitroethane oxidase by, 402 of D-serine (D-threonine) dehydrase by, 324 of tryptophan synthetase by, 237 of uricase by, 489 requirement for mammalian tyrosinase activity, 830 Corbicula sandal, thiaminase in, 625

SUBJECT INDEX Cortisone, effect on tryptophan peroxidase activity of liver, 246 Corynebacterium diphtheriae, cytochrome bl in, 745 growth of, 746 Cothromboplastin, see Convertin Coupled phosphorylation, see Phosphorylation, oxidative Cream, xanthine oxidase from, 483 Creatine, enzymatic phosphorylation of, 605 in oxidative phosphorylation assay, 611 synthesis by guanidinoacetate methylpherase, 260 Creatine kinase, see Adenosine triphosphate-creatine transphosphorylase Creatine phosphate, see Phosphocreatine p-Cresol, as substrate for tyrosinase, 818, 826 Cresolase, see also Tyrosinase, Monophenolase, tyrosinase as, 817, 818-819 assay of, 818-819 Crotalus atrox venom, "5" nucleotidase action of, 550 Crotonate, inhibition of d-biotin oxidation by, 632 Crucian, see Carassius carassius Cruciferae, dehydroascorbic reductase in, 849 Crystallization, of asclepain (protease) from latex of milkweed, 62 of ATP-creatine transphosphorylase, 608-609 of carbonic anhydrase from erythrocytes, 841 of carboxypeptidase from pancreas, 82-83 of catalase from beef liver, 776-779, 793 from M. lysodeikticus, 787 from red blood cells, 783-784 from spinach leaves, 790 of chymopapain (protease) from latex of papaya, 61 of a-chymotrypsin from pancreas, 1213

917

of ~o and ~-chymotrypsins from pancreas, 14-15 of ehymotrypsin B from pancreas, 9, 18 of chymotrypsinogen B from pancreas, 17-18 of cystathionine cleavage enzyme from pig liver, 312 of DNase from beef pancreas, 440--441 of DP-a-chymotrypsin, 14 of DP-~-chymotrypsin, 8, 16 of ficin (protease) from latex of fig, 61 of firefly luciferase, 856 of L-glutamic dehydrogenase from liver, 223-224 of glutathione reductase from yeast, 723 of inorganic pyrophosphatase from yeast, 573-574 of lactic dehydrogenase (cytochrome b2) from yeast, 746 of mexieain (protease) from latex of cuaguayote, 62 of old yellow enzyme, 714 of papain (protease) from latex of papaya, 59, 60 of pepsin from commercial pepsin, 5 of pepsinogen from swine mucosa, 6 of peroxidase from horseradish, 806-807 from milk, 815-816 of rennin from commercial rennet, 75-76 of rhodanese, 334-337 of ribonuclease from pancreas, 430-432 of trypsin from pancreas, 26, 29-31 of trypsin complex with trypsin inhibitor from colostrum, 47-48 from lima bean, 48-49 from pancreas, 38-39 from soybean, 44-46 of trypsin inhibitor from pancreas, 36, 38-40 from soybean, 42-44 of trypsinogen from beef pancreas, 26, 27-29 of urease, 378-379 Crystal violet, inhibition of glutamine synthetase by, 342

918

SUBJECT INDEX

Cuaguayote (Pileus mexicanus), crystalline protease (mexicain) from latex of, 56, 62 Cucumber fruit, glutathione reductase in, 721 Cucurbita pepo condensa, see Squash, yellow Cupric ion, see also Copper, activation of -y-glutamyltransferase (bacterial) by, 269 of p-hydroxyphenlpyruvate enolketo tautomerase by, 291 inhibition of arginine desimidase by, 376 of ascorbie acid oxidase by, 835 of ATP-creatine transphosphorylase by, 610 of dehydroaseorbie reductase by, 849 of 5-dehydroquinase by, 307 of DNase by, 442 of DPNH eytochrome e reductase by, 692 of fructose diphosphatase by, 546 of myosin ATPase by, 587 of nitrate reductase by, 415 of organic nitrate reductase by, 405 of quinone reductase by, 728 of renin by, 134 of RNase by, 434 of thiaminase by, 625 of tryptophan peroxidase by, 246 Cyanide, activation of guanidinoacetate methylpherase by, 263 of papain by, 59 in assay of cytochrome bl, 746-748 of cytochrome c reductase, 689 of diaphorase, 708 compound of catalase with, 788 of ferricytochrome c with, 754-755 of peroxidase with, 812 and cytochromc b, lack of reaction between, 745 effect of on activity and spectrum of myeloperoxidase, 799 on metaphosphatase, 579 on 3'-nucleotidase, 554 inhibition of alkaline phosphatases by, 527, 533

of amine oxidase by, 393 of apparent L-amino acid oxidase by, 211 of ascorbic acid oxidase by, 835 of aspartase by, 388 of bacterial luciferase by, 860 of carbonic anhydrase by, 844, 845 of carnosinase by, 96 of catalase by, 712, 718, 790 of cysteine desulfhydrase by, 317 of cytochrome bl of E. coli by, 748 distinction from diphtherial cytochrome bl, 748 of cytochrome c degradation by, 169 of cytochrome c peroxidase by, 763 of 5-dehydroquinase by, 307 of diamine oxidase by, 396 of DPNH oxidase by, 308, 683 of enzymatic nitrogen gas formation by, 423 of histidase by, 231 of homogentisate oxidase by, 292 of hydrogenases by, 866, 868, 870 dependence on aerobic conditions in P. vulgaris, 866 on anaerobic conditions in E. coli, 868 of hydroxylamine reductase by, 419 of kynureninase by, 253 of nitrate reductase by, 415 of nitroaryl reductase by, 410 of nitroethane oxidase by, 402 of rhodanese by, 337 of D-serine (D-threonine) dehydrase by, 324 of tryptophanase by, 241 of tryptophan peroxidase by, 246 of tryptophan synthetase by, 237 of tyrosinase by, 826 of uricase by, 489 of xanthine oxidase by, 484 relation to metal content, 484-485 removal of molybdenum from nitrate reductase with, 415 as substrate for rhodanese, 334-337 Cyanide-C 14, in preparation of radiobiotin, 631 Cyclic nucleotides, see under Mononucleotides, Dinucleotides, Pyrimidine ribose nucleotidcs

SUBJECT INDEX

Cypridina hilgendorfii, luciferase and luciferin from, 851-853 Cyst, athionine, preparation of L- and D-allo-isomers of from ~-homocysteine, 311,312 Cystathionine cleavage enzyme(s), 311314 from bacteria, 314 from liver, 311-314 Cysteic acid, decarboxylation in mammalian tissues, 199 Cysteine, see also Thiol compounds, Mercapto compounds, activation of adenylate kinase by, 603 of 5'-AMP deaminase by, 472 of eathepsin C by, 68 of dephospho-CoA kinase by, 649, 651 of dihydroSrotic dehydrogenase by, 496 of fructosediphosphatase (purified) by, 546 of guanidinoacetate methylpherase by, 263 of p-hydroxyphenylpyruvate enolketo tautomerase by, 292 of myosin ATPase by, 587 of nitrate reductase by, 415 of papain by, 59 of phosphoglucomutase by, 676677 activation and stabilization of tryptophanase by, 241 in assay system for hippuric acid synthesis, 349 effect on nitroaryl reductase, 410 on 3'-nucleotidase, 554 formation by cleavage of cystathionine, 311, 312 glutathione synthesis from, 342 inhibition of alkaline phosphatases by, 527, 538 of aminotripeptidase of thymus by, 87 of carnosinase by, 96 of fructose diphosphatase by, 546 of hippuric acid formation by, 350 of n-serine (D-threonine) dehydrase by, 324

919

of tryptophan synthetase by, 237 of tyrosinase by, 826 phosphopantetheine maintenance by, 669 presence in myosin, 587 protection of carbonic anhydrase (plant) with, 8A2, 843, 846 of hydrogenase by, 218 of nitrate reductase by, 414 reduction of dehydroascorbie acid by, 850 of, nitrate esters by, 405 of p-quinone by, 728-729 as substrate for L-amino acid oxidase, 2O8 Cysteine desulfhydrase, distribution of, 318 from liver, 315-318 Cysteine sulfinate--glutamate transaminase, paper chromatography for detection of, 172 Cysteine sulfinic acid, conversion to ~-suliinyl pyruvic acid by transaminase, 333 Cystine, action of cysteine desulfhydrase on, 317 inhibition of homogentisate oxidase by, 295 reversal by mercaptoacetie acid, 295 Cytidine (cytosine riboside) (CR), deamination of, 478 molecular extinction of, 478 nueleosidase action on, 459 R/values for, 466 Cytidine-3'-benzylphosphate, 3'-cytidylic acid formation from by spleen diesterase, 568 Cytidine triphosphate (CTP), as phosphate donor to AMP, 603 3'-Cytidylic acid (3'-CMP), 3r-nucleotidase action on, 553 5'-Cytidylic acid (5'-CMP), as acceptor in nucleoside monophosphate kinase reaction, 603 "5" nucleotidase action on, 549 Cytidylic acid derivatives, 2'- and 3'-cytidylic acid formation from, 568

920

SUBJECT INDEX

Cytochrome a, assay of with CO, 733 extinction coefficient for, 733 in heart particles, 737, 739 occurrence with cytochrome a3, 733 in oxidation of hematin peptide, 169 in purified cytochrome oxidase preparation from heart particles, 739 Cytochrome al, in bacteria, 732 particulate nature of, 737 as terminal respiratory enzyme of Acetobacter pasteurianum, 732733, 734 extinction coefficients for CO compound and reduced form, 734 Cytochrome a2, in bacteria, 732, 735 occurrence with cytochrome al, 733 particulate nature of, 737 Cytochrome a.~ (cytochrome oxidase), assay of, 735-737 by cytochrome oxidase activity, 735-737 by spectral properties, 735 i~ B. subtilis, 734, 735 failure of to oxidize mammalian cytochrome c, 735 in cytochrome c assay, 750 distribution of, 732 extinction coefficients for CO compound and reduced form in different sources, 734 in heart particles, 734, 737-740 oxidation by molecular oxygen, 732 oxidation of bacterial hemochromogen by mammalian, 699 of cytochrome b by~ 740 of reduced cytochrome c by, 732 in purified preparation from heart particles, 739 as terminal respiratory enzyme, 732 Cytochrome a group (a, al, a2, a3), 732740 absorption spectra of reduced and CO compounds, 733 assay methods for, 733-737 preparations containing, 737-740 heart muscle particle suspension, 738-740

special aPparatus for distinguishing CO compounds of reduced components, 734, 735 Cytochrome b, in baker's yeast and bacteria, 744, 745 in oxidation of hematin pcptide, 169 from pig heart, 740-744 absorption bands of ferrous form, 740, 744 comparison with bands of cytochrome 5532 744 assay of, 741-742 as link between succinate and cytochrome c (SC activity), 740741,742, 745 oxidation by cytochrome oxidase, 740 oxidation-reduction potential of, 740 purification of, 741, 742-744 Cytochrome bl, 744, 745, 746-748 from Corynebacterium diphtheriae, 746-748 absorption bands of, 747 assay of enzyme activity of, 747748 autoxidation of, 748 hemin content of, 747 methylene blue or ferricyanide oxidation of, 748 reduction of, 747 nitrate reduction and, 746 in succinate oxidation, 745, 746 Cytoehrome b2, in yeast, 744, 745, 746 association with lactic dehydrogenase activity, 746 Cytochrome b group (b, bl, b2), in bacteria, 744-748 absorption bands of, 744, 745 variation with species and strain, 744, 745 autoxidizability of, 745 non-reactivity with iron reagents, 745 oxidation potential of, 745 position in respiratory'chain, 745 protoheme as prosthetic moiety of, 745

SUBJECT INDEX Cytochrome c (Ferri- and Ferrocytochrome e), absence from purified cytochrome oxidase preparations, 739 bacterial sources of, 759 from heart, 749-755 assay methods for, 749-750 manometric, 750 spectrophotometric, 749 bound form of in particulate preparations, 710, 738 combination of oxidized form with azide, 755 with cyanide, 754-755 with cytochrome c reductase, 692 of reduced form with CO, 754 competition with citrate and pyrophosphate for cytochrome reductase, 692 cytochrome b as possible link between succinate and, 740-741, 742 cytochrome b assay by reduction of, 741-742 difference in activity of added and endogenous, 750 electrophoretically purified preparations of, 754 enzymatic degradation of, 167-169 enzymatic reduction by L-a-glycerophosphate, 559 equation for degree of reduction of, 736 extinction coefficients for oxidized and reduced, 700 failure of bacterial eytochromes al and as to oxidize mammalian, 733 fractionation by chromatography, 752 histidine residue involved in iron binding by, 753 inactive autoxidizable form of, 749 detection by CO method, 749 iron content of, 751, 752 iron in prosthetic group of, 753 isoelectric point of, 750-751 isolation of oxidized form by TCA method, 751-752 of reduced form by resin method, 752-753

921

microscale application of, 753 magnetic properties of, 754 molar absorption coefficients for, 749, 753 myoglobin as contaminant of, 751752 oxidation by acidification, 751, 754 by alkalinization, 754 by ferricyanide, 737, 754 by H~O2, 736 oxidation reduction potential of, 754 in oxidative phosphorylation assay, 611, 615, 616 peroxidase assay with, 774 preparation of H~O2-free, 736 presence in various species, 751 reduction, by chemical agents, 754 by D P N H cytochrome reductases, 692, 698-699 by T P N H cytochrome c reductase system, 703 by hydrogenase system, Mo, FAD and phosphate requirements for, 869-870 by succinate and cytochrome b, 744 by xanthine oxidase, 482 requirement by modified mitochondria, 615 spectrophotometrie measurement of reduction, 688 spectrum of reduced, 749 stability of, 754 structure compared with Ustilago cytochrome c, 758 from Ps. fluorescens, 758-760 absorption bands for oxidized and reduced, 758 adaptive formation of, 760 association of pigment with cytochrome c peroxidase, 758, 759 comparison with animal pigment, 758 inactivity with liver TPN cytochrome e reductase, 760 red fluorescence of reduced form, 760 from Ustilago sphaerogena, 755-758 absorption spectrum of, 757

922

SUBJECT INDEX

assay with Zeiss band spectroscope, 755 autoxidizability of, 757 chromatography of, 756--757 iron content of, 757 isoelectric point of, 758 prosthetic group of, 757-758 purity of, 757 Cytoehrome c oxidase, see Cytochrome aa

Cytochrome c peroxidase, distribution of, 764 effect on assay of cytochrome a~, 737 from Ps. fluorescens, 760-764 Cytochrome c reductase, see also DPNH and TPNH eytochrome c reductases, effect on assay of cytoehrome a3, 737 Cytochrome e, distribution of, 745 as member of cytochrome b group, 745 Cytochrome component, absorbing at 554 m# in Acetobacter, 744, 745 at 553 m~ in eytochrome b preparations from heart, 744 Cytochrmne o×idase, see Cytoehrome a3 Cytochrome system, 732-764, see also under names of individual components, dopa oxidation to melanin by, 828 in heart particles, 737-740 role in oxidation of mandelic acid, 277 Cytosine, as acceptor of deoxyriboside group, 468 Rs values for, 466 Cytosine compounds, absorption change on deamination of, 478 Cytosine deaminase, spectrophotometric determination of cytosine with, 458 Cytosine deoxyriboside (CDR), bioassay of, 464 comparison with cytidine in rates of deamination, 479 deamination of, 478 RI values for, 466 in transdeoxyribosidase reaction, 464468

Cytosine nucleoside deaminase, from E. coli, 478-480 D

Deaminodiphosphopyridine nucleotide (Deamino-DPN), as coenzyme for L-glutamic dehydrogenase, 224 lack of DPN kinase inhibition by, 654 Neurospora DPNase action on, 666 transhydrogenase reaction with DPNH, 681-687 Deaminotriphosphepyridine nucleotide (Deamino-TPN), transhydrogenase reaction with TPNH, 685 Deeamethylenediamine, amine oxidase action on, 393 Dehydrases, 319-324 D-serine (D-threonine) dehydrase, 322324 n-serine (5-thrconine) dehydrase, 319322 Dehydroalanine, peptides of, specific absorption bands of, 110 Dehydro-D-araboaseorbic acid, as substrate for dehydroascorbic reductase, 850 Dehydro->aseorbic acid, preparation of, 847 as product of aseorbie acid oxidase, 831 reduction by thiol compounds, 847-85C enzymatic, 847-850 non-enzymatic, 848-849, 850 Dehydroaseordic reductase, in cabbage juice, 848, 850 m cauliflower, 849, 850 distribution of, 84 from pea plant, 847-850 Dehydropeptidase (s), assay by ammonia determination, 110 111 of kidney, 109-114 of liver, 114 possible role in exocystine desulfhydrase action, 319 preparation of substrates for, 110 terminology of, 114 Dehydropeptidase I (solubilized aminopeptidase),

SUBJECT INDEX from particulate fraction of hog kidney, 107, 108, 109-114 action on glyeyMeucine isomers, 107 comparison with glycylglycine dipeptidase of muscle, 108 purification of, 112-113 Dehydropeptidase II (soluble acylase I), see Amino acid acylase I Dehydropeptide(s), amino acyl, as substrates for dehydropeptidase I, 109 dehydroalanyl, as specific substrates for dehydropeptidase II, 113 as probable product of exocystine desulfhydrase action, 319 resistance to prolidase, 105 5-Dehydroquinase, distribution of, 307 from E. coli, 305-307 effect on quinic dehydrogenase assay, 308 separation from 5-dehydroshikimie reductase of E. coli, 306 5-Dehydroquinic acid, formation of by quinie dehydrogenase, 307-311 as intermediate in aromatic biosynthesis, 300, 301 5-Dehydroshikimie acid, accumulation of in E. coli mutant, 307 bioassay of, 308, 309 as intermediate in aromatic biosynthesis, 300, 301 molar extinction coefficient of, 305 as product of 5-dehydroquinase action, 305-307 5-Dehydroshikimic reductase, in A. aerogenes, 308 distribution of, 304 from E. coli, 301-304 Deoxycholate, extraction of cytochrome b with, 74 Deoxycorticosterone, inhibition of D P N H cytochrome e reductase by, 693 Deoxygenase, see Glucose oxidase Deoxyoligonucleotide, as substrate for phosphodiesterase and 5'-nucleotidase, 561

923

Deoxyribonuclease(s) (DNase), from baker's yeast, 445-446 chymotrypsinogen B and, 10, 17 from group A hemolytic streptococci, 446-447 inhibition of pancreatic and streptococcal, by respective antisera, 447 in mouse leukemic tissues, 447 from pancreas, 438-443 crystallization of, 440-441 denaturation by salt, 443 physical-chemical properties of, 443 in serum, 447 from spleen, 444-445 from thymus, 443-444 activation by acid treatment, 444 Deoxyribonuclease inhibitor, as byproduct of DNase preparation from yeast, 446 in tissues, 443 Deoxyribonucleic acid (DNA), deoxyoligonucleotide formation from by DNase, 561 denatured, hydrolysis by DNase, 441 depolymerization of by sonic treatment, 274 DNase for removal of from Pseudomonas extracts, 274 inhibition of RNase by,'434 hydrolysis of by~DNase,r 437, 441, 442 physical and chemical changes during, 437, 442 products of, 441 resistance to prostatic phosphatase, 526 source of, 437 Deoxyribose-l-phosphate, elimination as intermediate in transdeoxyribosidase reaction, 468 preparation from guanine deoxyriboside, 449 Deoxyriboside (s), of 8-azaguanine, enzymatic synthesis of, 448 chromatography of and RI values for, 465-466 of hypoxanthine and guanine, action of phosphorylase on~ 448 quantitative bioassay of with thermobacterium acidophilus, 464

924

SUBJECT I N D E X

Deoxyxanthosine, phosphorolysis of, 448 Dephospho-CoA, formation from ATP and phosphopantetheine, 667, 669 Dephospho-CoA kinase, assay by firefly bioluminescence system, 651 in dephospho-CoA pyrophosphorylase preparations, 667, 669 from pigeon liver, 649-651 Dephospho-CoA pyrophosphorylase, from hog liver, 667-669 Desthiobiotin, inhibition of d-biotin oxidation by, 632 Desulfhydrase(s), activity in liver tissue, 313 cysteine desulfhydrase, 315-318 exocystine desulfhydrase, 319 homocysteine desulfhydrase, 318 from P. morgani,.318 Desulfinases, in animal tissues, 333 in bacteria, 333

Desulfovibrio desulfuricans, hydrogenase from, 869, 870 Deuterium oxide, see Heavy water Deuteron and electron bombardment, for molecular weight of DNase, 443 Diacetylmonoxime, in colorimetrie determination of citrulline, 351, 359 Dialyzer, Kunitz and Simms, 431 Dialyzing membranes, treatment of with glutathione, 414 Diamine oxidase (histaminase), from hog kidney, 394-396 Diamines, oxidation by peroxidase complex II, 8O8 Diaminopimelic acid, D-amino acid oxidase and, 202 Diamino purine, see 2-Aminoadenosine Diamox 6063 (2-acetylamino-l,3,4thiodiazole-5-sulfonamide), inhibition of erythrocyte carbonic anhydrase by, 845 Diaphorase, action of D P N H cytoehrome c reductases as, 692, 697

DPNH-linked, from pig heart, 707711 TPNH-linked, questionable existence of, 711 Diazo-coupling method, nitroaniline assay by, 406 Diazomethane (CH2N2), compound with horseradish peroxidase, 812 Dibenamine, inhibition of amine oxidase by, 393 Dichloroflavin, phosphorylation by flavokinase, 644 2,6-Dichlorobenzenoneindo-3'-chlorophenol, in assay of cytochrome c peroxidase, 761-762 preparation of reduced form, 761 cytochrome c reduction by reduced form of, 761 2,6-Dichloro-4-nitrophenol, inhibition of quinone reductase by, 729 2,6-Dichlorophenolindophenol, in diaphorase assay, 707 measurement of ascorbic acid with, 847, 848 reduction of by L(-k)-mandelie acid dehydrogenase, 278 succinate oxidation measured with, 748 2,4-Dichlorophenoxyacetic acid, as uncoupling agent, 615 Dichloroquinone chlorimide, color reaction for uric acid, 486 Dicoumarol, effect of on clotting mechanism, 140, 154 Di (dinitrophenyl) phosphate, hydrolysis of by 3'-nucleotide phosphatase of barley, 524 Diethyldithiocarbamate, activation of uricase by, 489 inhibition of ascorbic acid oxidase by, 835 of hydroxylamine reductase by, 419 of tyrosinase by, 826 Digitonin, solubilization of beef heart transhydrogenase by, 686-687 DihydroSrotic acid, hydrolysis by dihydroSrotase, 496

SUBJECT INDEX DihydroSrotic dehydrogenase, from Zymobacterium oroticum, 493-496 Dihydroxymaleic acid, oxidation by peroxidase complex II,, 810 o-Dihydroxyphenyl compounds, oxidation by plant versus mammalian tyrosinase, 830 3, 4-Dihydroxyphenyl-L-alanine (dopa), decarboxylation in higher plants, 194 hydrogen peroxide inhibition of peroxidation of, 816 oxidation by mammalian tyrosinase, 827-831 plant tyrosinase compared with, 830 oxidation to melanin by cytochrome system, 828 peroxidase assay with, 774, 813 3,4-Dihydroxyphenylalanine (dopa) decarboxylase from guinea pig or rabbit kidney, 195, 199 from liver, 199 2,5-Dihydroxyphenylpyruvate, oxidation of, 288 Diisopropyl fluorophosphate (DFP), reaction of with a-chymotrypsin, 13 with ~-chymotrypsin, 16 with trypsin and chymotrypsins, 14 Diisopropyl phosphate a-chymotrypsin (DP-a-chymotrypsin), preparation of, 13-14 crystallization of, 14 Diisopropyl phosphate ~-chymotrypsin (DP-~-chymotrypsin), crystallization of, 8, 16 Diisopropyl phosphate trypsin (DPtrypsin), molecular weight of, 35 optical factor for, 33 Diketo acids, action of hydrolase on, 298-299 2,3-Dimercaptopropanol, see British anti-lewisite p-Dimethylaminobenzaldehyde, determination of indole with, 234 Dimethylg]ycine, as buffer, 226 3,4-Dimethylphenol, oxidation by tyrosinase, 826

925

4,6-Dimethyltryptophan, as substrate for tryptophanase, 242 Dinicotinamide ribose-5'-pyrophosphate, cleavage by Neurospora DPNase, 666 m-Dinitrobenzene, as substrate for nitroaryl reductase, 406-411 p-Dinitrobenzene, reduction of by Neurospora, 410 3,5-Dinitrobenzoic acid, reduction by Neurospora, 410 Dinitrocresol, as uncoupling agent, 615 2,4-Dinitrophenol and related compounds, inhibition of quinone reductase by, 729 reduction by Neurospora, 410 as uncoupling agent, 615 Dinitrophenylhydrazine, in colorimetric measurement of pyrurate, 174-175 2,4-Dinitrophenylhydrazone, of a-ketoglutarate, 170 Dinucleotides and related compounds, as substrates for spleen pbosphodiesterase, 568 Diol (2-amino-2-m ethyl- 1,3-propanediol) buffer, 688 Dipeptidases, carnosinase, 93-96 glycylglycine dipeptidase, 107-109 glycyl-I~leucine dipeptidase, 105-107 iminodipeptidase (prolinase), 97-100 prolidase (imidodipeptidase), 100-105 Dipeptidases, metal activated, inhibition by Versene, 87 Dipeptide(s), containing normal peptide bond, resistance to prolidase, 104 I~-histidine-containing, as substrates for carnosinase, 93, 96 lacking peptide hydrogen, as substrates for prolidase, 104 as product of aminotripeptidase action, 83 z-proline or hydroxy-L-proline-containing, as substrates for iminodipeptidase, 97 as substrates for dehydropeptidase I (solubilized aminopeptidase), 113 for leucine aminopeptidase, 91

026

SUBJECT INDEX

Dipeptide amides, as substrates for leucine aminopeptidase, 92 Diphenylamine procedure, for deoxypentose, in DNase assay, 438 Diphenylphosphate, resistance to bone phosphatase activity, 539-541 as substrate for phosphodiesterase, 561, 564

Sym-Diphenylpyrophosphate, conversion to monophenylphosphate by venom diesterase fractions, 565 3",5'-Diphosphoadenosine, action of nonspecifie deaminase on, 477, 478 Diphosphopyridine nucleotidase (DPN nucleosidase) (DPNase), from animal tissues (pyridine transglycosidase), 653, 660-663, 683 from brain, 662 inhibition of by nicotinamide, 662, 670, 683 in pigeon liver, 653 species variation in sensitivity to INH, 663 from spleen, 661-662 from Neurospora, 664-666 in potato, 655 Diphosphopyridine nucleotide (DPN, cozymase, coenzyme I), see also under DPN, action of nonspecific deaminase on, 477, 478 analogs of formed by brain DPNase, 663 cleavage by Neurospora DPNase, 664, 666 by nucleotide pyrophosphatase, 655, 659 as coenzyme for amino acid reductases, 220 for benzaldehyde dehydrogenase, 280-281 for dihydro5rotie dehydrogenase, 493, 496 for I,-glutamic dehydrogenase, 220 for quinic dehydrogenase, 307-311 for quinone reductase, 725

measurement by alcohol dehydrogenase, 660, 670 by cyanide reactivn, 660 measurement of pyridinium linkage in, 655 in nitroaryl reductase assay, 407 in oxidative phosphorylation assay, 612, 615, 616 reduction by molecular hydrogen, 729, 732 as substrate for cleavage and group transfer by animal DPNase, 660 synthesis of from NMN and ATP, 670 transhydrogenase reaction of with deamino-DPNH or TPNH, 681687 with NMNH, 685 Diphosphopyridine nucleotide, C l~-nicotinamide-labeled, Pseudomonus transhydrogenase exchange reaction studied with, 686 Diphosphopyridine nueleotide, reduced (DPNH), acid destruction of, 729 cleavage by potato nucleotide pyrophosphatase, 659 by snake venom nucleotide pyrophosphatase, 654 compared with TPNH in GSSG reductase system of liver, 725 of yeast, 724 dehydroascorbic reductase and, 850 extinction coefficient of, 670 as hydrogen donor in bacterial luciferase system, 857, 860, 861 inhibition of DPN kinase by, 654 Km value for combination with cytochrome c reductase, 692 methods for preparation of, 694 oxidation of by old yellow enzyme, 715 by peroxidase complex II, 810 P:O ratio during oxidation of, 613, 616 reduction of flavin component of cytochrome c reductase by, 691 of hydroxylamine to NH3 by, 416, 418 of iron component of cytochrome c reductase by, 692

SUBJECT INDEX of nitroaryl compounds by, 406, 408, 410 of quinones by, 725-729 role in nitrogen gas formation from nitrite, 422 as substrate for diaphorase, 707 synthesis from N M N H and ATP, 672 transhydrogenase reaction with deamino-DPN or TPN, 681-687 with NMN, 685 Diphosphopyridine nucleotide'analogs, cleavage by animal DPNase, 662 failure of Neurospora DPNase to form or cleave, 666 Diphosphopyridine nucleotide kinase (DPN kinase), from pigeon liver, 652-655 Diphtheria toxin, detoxication of by myeloperoxidase plus donor substances, 801 Diplococcus pneumoniae, aspartase in, 388 Dipotassium-2-hydroxy-5-nitrophenylsulfate, as substrate for arylsulfatascs, 328 a,a'-Dipyridyl, activation of uricase by, 489 inhibition of homogentisate oxidase by, 295 of hydroxylamine reductase by, 419 Disulfide bond, as possible active group in rhodanese, 337 Dithionite (hydrosulfite), activation of hydrogenases by, 866 decomposition of hematin peptide by, 169 deoxygenation with, for hydrogenase assays, 863, 866, 868 effect on absorption bands of cytochrome b, 744 inhibition of heparin cofactor by, 163 reduction of cytochrome bl by, 747 of cytochrome c by, 483, 749, 759 of flavin component of cytochrome e reductase by, 691 of horseradish peroxidase by, 812 Dithizone, activation of uricase by, 489

927

I.-Djenkolic acid, cleavage of, 313 Dodecyl aldehyde, as component of bacterial luciferase system, 857, 861 Dodecylamine, amine oxidase action on, 393 Dopa, see 3,4-Dihydroxyphenyl-Lalanine Dowex-1, removal of CoA with, 349, 633 DPN nucleosidase, see Diphosphopyridine nucleotidase DPN pyrophosphorylase, in brain, 671 from. hog liver, 671-672 rates of DPN synthesis and breakdown by, 612 from yeast, 671 D P N H cytochrome e reductase, from heart, 688-693 diaphorase activity of, 708, 711 from E. coli, 693-699 rate constants compared with yeast T P N H enzyme and old yellow enzyme, 697 in mitochondria, BAL and antimycin sensitivity of, 693 in particulate preparations, 710 separation from T P N H enzyme of liver, 706 D P N H oxidase, in A. aerogenes, 308 DP-derivatives, see under Diisopropyl phosphate Dyes (oxidation-reduction indicators), reduction by l)-amino acid oxidase, 204 by bacterial luciferasc system, 857, 86O by cytochrome c reductase, 692 by hydrogenase system, 862, 868 Dyes, reduced, as electron donors to F M N in bacterial luciferase system, 861 E

Egg white, trypsin inhibitor from, 37, 49 Electrophoresis, purification of L-amino acid oxidase by, 207

928

SUBJECT INDEX

of horseradish peroxidase by, 805 of myeloperoxidase by, 797-798 separation of polysaccharide from old yellow enzyme by, 714 Empyema, myeloperoxidase from, 797-798 Ene-diols, oxidation by ascorbic acid oxidase, 834 by peroxidase complex II, 810 Enol-keto tautomerase, for p-hydroxyphenylpyruvate from liver, 289-292 Enterokinase, from swine duodenum, 31-32 determination of activity of, 32 Entropy of activation, for DNase, 443 Enzyme-substrate complexes, in carbonic anhydrase reaction, 844 rate constant for formation of, 844 in peroxidase reaction, 770, 801-813 magnetic susceptibility of, 811, 813 oxidation of various hydrogen donors by, 808 rate and equilibrium constants for formation of, 810 spectra of, 811 Epinephrine (adrenaline), cytochrome c reduction by, 754 oxidation of by tyrosinase, 826 Erythritol tetranitrate, as substrate for organic nitrate reductase, 405 Erythrocytes, acid phosphatase in, 527 aminotripeptidase from, 84-85 carbonic anhydrase from, 838, 841842 catalase from, 209, 765, 775, 781-784 prolidase from, 101, 102-103, 105 RNase in, 436 uric acid riboside (UAR) in, 459 Escherichia coli, acetyl phosphatase from, 556 adenylate kinase in, 619 arginine decarboxylase from, 187 aspartase in, 388 ATPase in, 619 eytochrome bl in, 745, 746

in suecinate oxidation and nitrate reduction, 746 cytochrome c peroxidase in, 764 cytosine nucleoside deaminase from, 478, 480 5-dehydroquinase from, 305-307 5-dehydroshikimie reductase from, 301-304 D P N H cytochrome e reductase from, 693-699 glutamic acid decarboxylase from, 182, 183, 186, 187-216 growth medium for, 183 glutamic-oxalacetic transaminase activity of, 184 L-glutaminase from, 380, 382 7-glutamyltransferase (GTF) in, 269 glutathione-synthesizing system in, 342 growth of, 186, 187, 239, 380-381, 479, 512, 695, 867 hydrogenase from, 867, 868 inorganic pyrophosphatase in, 619 isolation of 5-amino-4-imidazolecarboxamide riboside from cultures of, 505, 512, 514 lysine decarboxylase from, 186-187 pantothenate auxothroph of, 619 pantothenate-synthesizing enzyme from, 619, 622 pyrimidine nucleoside phosphorylase in, 480 D-serine (D-threonine) dehydrase from, 322, 323 L-serine (L-threonine) dehydrase in, 320 transaminase from, 172, 173, 174, 176, 177 transhydrogenase in, 686 tryptophanase from, 238-242 tryptophan synthetase in, 234 Esterase activity, of cathespin C, 68 of trypsin, 36 of trypsin and chymotrypsins, 23 Ethanol, activation of ,-glutamyltransferase (bacterial) by, 269 solubilization of diaphorase by, 708 of DPNH cytoehrome c reductase of heart by, 689

SUBJECT INDEX

929

of DNase by, 442 of glutamic dehydrogenase by, 224 of penicillinase by, 123 of renin by, 134 of splitting enzyme for arginine synthesis by, 367 of thiaminase by, 625 reduction by hydroquinone, 726 Ferricyanide, effect of on induction period of monopheno!ase, 825 inhibition of aspartase by, 388 oxidation of cytoehrome b by, 748 of cytochrome e by, 737, 759 reduction of by bacterial luciferase system, 857, 860 by hydrogenase system, 862 Ferricytochrome c (CyFe+++), see Cytochrome c Ferrous ion, see also Iron, activation of fructose diphosphatase by, 546 of homogentisate oxidase by, 294, 295 of hydrogenases by, 868, 870 of p-hydroxyphenylpyruvate enolketo tautomerase by, 291 of metaphosphatase by, 579 of pyrocatechase by, 282 of triphosphatases by, 581 inhibition of 5-dehydroquinase by, 307 of dissociable amino acid decarboxylases by, 189 of DNase by, 442 of leucine aminopeptidase by, 93 of splitting enzyme for arginine synthesis by, 367 of thiaminase by, 625 Fibrin, mechanism of formation of, 140 urea resistant clot of, 158, 160 Fibrinogen, Fat, neutral, changes in end groups on conversion to presence of in heparin eofactor, 163 fibrin, 160 in thromboplastin, 150 concentration of in oxalated horse F a t t y acids, plasma, 147 inhibition of D-amino acid oxidase by, purification of, from blood, 158, 159 203 from fraction I (Armour), 159 of d-biotin oxidation by, 632 role in clotting mechanism, 140, 158Ferric ion, see also Iron, 160 inhibition of 5-dehydroquinase by, 307

Ether, effect on renin action, 134 plasmolysis of top yeast by, 581 Ethylenediamine, action of diamine oxidase on, 396 inhibition of histidase by, 231 Ethylenediaminet etraacetate (Versene), activation of cystathionine cleavage enzyme by, 313 of uricase by, 489 effect on myosin ATPase, 587 inhibition of aspartase by, 388 of bacterial luciferase by, 860 of histidase by, 231 of leucine aminopeptidase by, 87, 93 of metal-activated dipeptidases by, 87 of prolidase by, 105 of tryptophan synthetase by, 237 protection of hydroxylamine reductase by, 419 of nitrate reductase by, 414 Ethyl nicotinate, conversion to and release from DPN analog by brain DPNase, 662, 663 Ethyl-5-phenyl-3-ketovalerate, hydrolysis of C - - C bond of by a-chymotrypsin, 21 Euphorbain, protease from latex of caper spurge, 57, 64 Euphorbia lathyris, cerifera, see Caper spurge Exocystine desulfhydrase, from liver, 319 Eye tissue, pigmented, tyrosinase from, 828

930

SUBJECT INDEX

inhibition of DPNH eytochrome c two-stage prothrombin assay with, 141, reductase by, 693 142 as prosthetic group of D-amino acid Fibrinolysin (plasmin), oxidase, 199, 200, 227 activators and inhibitors of, 165 separation of from, 200 formation of by activators of proof L-amino acid oxidase of N. crassa, fibrinolysin, 165 211 role in clot-dissolving, 140 of L-amino acid oxidase of snake streptokinase-activated, 165 venom, 205 Fibrinolytic activators, of diaphorase, 710 fibrinokinase from heart, 165 reduction of by DPNH, 710 fibrinolysokinase from lung, 165 of DPNH cytochrome c reductase of urine activator, 165 bacteria, 698 Fibrinoplastic substances, of glycine oxidase, 227 acacia as example of, 159 of hydrogenase, 869, 870 Ficin, of new yellow enzyme, 718 protease from latex of fig, 56, 61 reversible removal of, 718 crystallization of, 61 of nitrate reductase, 415 Fieser's solution, preparation of, 865 of nitroaryl reductase, 407, 410 Fig (Ficus carica, glabrata, doliara), of TPNH cytochrome c reductase crystalline protease (ficin) from, 56, 61 of liver, 706 peroxidase in, 801-802 of xanthine oxidase, 485 Firefly (Photinus pyralis), protection of L-amino acid oxidase by, luciferase and luciferin from t 851, 8542O8 856 source of, 201 Firefly bioluminescence system, see also substance competing with in D-amino Luciferase, acid oxidase reaction, 203 CoA effect on, 651 synthesis of from FMN and ATP, 673 Flavin(s), see also under Alloxazine and Flavin adenine dinucleotide pyrophosRiboflavin, phorylase, from brewer's yeast, in boiled juice of Cl. kluyveri, 731 673-675 from boiled pig heart extract, 407, 416 Flavin adenine nucleotide, yeast lactic dehydrogenase containing as component of DPNH cytochrome e cytochrome b2 and, 746 reductase of heart, 691-692 Flavin adenine dinucleotide (FAD), involvementin diaphorase action, 692 activation of hydroxylamine reductase non-identity with FAD, 691 by, 419 Flavin mononucleotide (FMN, riboof nitrogen gas-forming enzyme by, flavin-5'-phosphate), 423 as absolute requirement for bacterial boiled pig heart as source of, 408 luciferase system, 861 cleavage of by pyrophosphorylase, 673in assay of nitrate reductase, 412, 415 675 complex with protein moiety of old complex of with quinine, 203 yellow enzyme, 715 determination of by D-amino acid free radical of, 715 oxidase, 673, 698 distribution coefficient (benzyl alcohol: water) for, 642 hydrolysis of by boiling, 641 by nucleotide pyrophosphatase, 655, FAD synthesis from ATP and, 673 formation of by flavokinase, 640, 641 659 ATP as competitive inhibitor of, inhibition of DPNH cytochrome c reduetase by, 693 673

SUBJECT INDEX

as prosthetic group of mammalian L-amino acid oxidase, 210 of old yellow enzyme, reversible removal of, 715 of TPNH cytochrome c reductase of yeast, 703 reduction by DPNIt in bacterial luciferase system, 857, 860 by hydrogenase system, 869 stimulation of nitrogen gas formation from nitric oxide by, 423 Flavin mononucleotide, reduced (FMNH~), autoxidation of, 861 as hydrogen donor in bacterial lueiferase system, 857 Flavokinase, from brewer's yeast, 640-645 in intestinal mucosa, 642 Flavoproteins, absorption maxima of, 691, 703, 710, 715, 719 I~-amino acid oxidases as, 208, 210, 211 atabrine and aeriflavine as specific inhibitors of, 698 DPNH- or TPNH-linked, assay by means of T2Z or NTZ, 695 Fluoride, compound with horseradish peroxidase, 812 effect on activity and spectrum of myeloperoxidase, 799 on glueose-6-phosphatase, 542 inhibition of aeetyl phosphatase by, 556 of acid prostatic phosphatase by, 527 of adenylate kinase by, 603 of aminodipeptidase, 100 of Y-AMP deaminase by, 472 effect of 5'-AMP concentration on, 472 of pH on, 473 of arginine-synthesizingsystem by, 359, 364 of citrullinase by, 378 independence of Mg ++, 378 of DNase by, 442, 447 of fructose diphosphatase by, 546 of glutamine synthetase by, 342 of -/-glutamyltransferase by, 267 of metaphosphatase by, 579

931

of methionine-activating enzyme by, 256 of Mg-activated ATPase by, 590 of nitroethane oxidase by, 402 of "5" nucleotidase of seminal plasma by, 549 of nucleotide pyrophosphatase by, phosphate requirement for, 659 of prolidase by, 105 of pyridoxal kinase by, 649 Fluoroaeetyl amino acids, action of acylase I on, 118 Folio acid eoniugase, from chicken pancreas, 629-630 in hog kidney, 629, 630 sources of, 629 in takadiastase, 630, 631 Folio acid de ivative(s), see also Leueovorin, Tetrahydrofolie acid, Anhydroleucovorin, removal by Dowex-l, 509 as requirement foJ~fo:n~ylglycinamide ribotide synthesis, 509 Folio acid heptaglutamate, effect of folio acid conjugase on apparent CF content of, 629 Folin-Cioealteu phenol reagent, in assay of hydroxytyramine, 195 of pepsin, 3-4 of proteinases, 55 Formaldehyde, inhibition of ItNase by, 434 Formamido compounds, aromatic, as substrates for kynurenine fo:mamidase, 2=19 Formate, activation of hydrogenase system by, 866 formation from formylkynurenine, 246 C 14-Formate, in measurement of formylation of 5-IRMP, 519 of formylglycinamide ribotide synthesis, 509-510 of purine synthesis, 505, 507 of tetrahydrofolate formylase reaction, 517 formazan, as insoluble product of reduction of TTZ or NTZ, 695

932

SUBJECT INDEX

Formyl amino acids, action of acylase I on, 118 Formylanthranilic acid, as substrate for kynurenine formamidase, 246, 249 Formylase, see Kynurenine formamidase Formylation, stable cofactor(s) for, 517 tetrahydrofolic acid as cofactor for, 516, 517 Nl°-Formylfolic acid, as intermediate in conversion of bound folic acid to CF, 629 as product of oxidation of tetrahydro compound, 518 Formyl-~glutamate, role in citrulline synthesis, 355 Formylglycinamide ribotide, biosynthesis of, 509-512 requirement of folic acid derivative for, 509 conversion to 5-amino imidazole ribotide, 505 isolation of, 511, 512 Formylkynurenine, hydrolysis of~ 246 as product of tryptophan peroxidase reaction, 242 N 10-Formyltetrahydrofolic acid (CHOFAH4), chemical synthesis of from N ~°formylfolie acid, 519 conversion to NS-N~°-imidazolium derivative (ACF), 518 as formyl donor to 5-IRMP, 518, 519 to glycinamide ribotide, 518 oxidation of to Nl°-formylfolic acid, 518 as product of reaction of FAH4 with formate, 517 with serine, 518 spectrophotometrie measurement of, 518 Fraction I (Armour), purification of fibrinogen from, 159 Fraction I I I of plasma, profibrinolysin from, 164-165 Fraction IV (Armour), as source of antihemophilic factor, 149

Free radical, yellow enzyme in form of, 712 Fructose-l,6-diphosphatase, from rabbit liver, 543-546 Fructose-l,6-diphosphate (FDP), resistance toward prostatic phosphatase, 525 Fructose-l-phosphate (F-I-P), hydrolysis by fructose diphosphatase, 545-546 Fructose-6-phosphate, formation of by fructose-l,6-diphosphatase, 543, 546 unknown product accompanying, 546 Fuller's earth, see Kaolin *Fumarase, in assay of splitting enzyme for arginine synthesis, 364-365, 367 Fumarate, activation of d-biotin oxidation by, 632 inhibition of aspartase by, 390 of cytochrome c degradation by, 169 of hydrogenase exchange reaction by, 867 of splitting enzyme for arginine synthesis by, 367 as product of arginine-synthesizing system, 356 of aspartase action, 386 of fumarylacetoacetate hydrolase, 298-300 of tyrosine oxidation, 287 reduction of by hydrogcnase system, R62, 867 effect of fumarate in growth medium on, 867 spectrophotometric measurement of~ 739 Fumarylacetoacetate, conversion to fumarate and aceto° acetate, 298-300 conversion of maleylacetoacetate to, 295-298 molar extinction coefficient of, 296, 299 Fumarylacetoacetate hydrolase, in assay of maleylacetoacetate isomerase, 296

SUBJECT INDEX from liver, 293, 294, 297, 298, 300 separation from homogentisate oxidase, 293, 294 from maleylacetoacetate isomerase, 297-298 Furfurylamine, amine oxidase action on, 393 G Galactose, presence of in fibrinogen and fibrin, 160 *Galactowaldenase, UDPG determination with, 677 Gastric mucosa, carbonic anhydrase in, 841 pepsin and pepsinogen from swine, 3-7 from various species, 7 Gelatin, protective action on pcnicillinase, 123 stabilization of carbonic anhydrase by, 846

Glomerella cingulata, tryptophan synthetase in, 234 Glucoascorbic acid, in oxidation of p-hydroxyphenyl pyruvate, 289 Glucosamine, presence in fibrinogen and fibrin, 160 Glucose, as acceptor for transphosphorylation by phosphatases, 533, 556 activation of -y-glutamyltransferase (bacterial) by, 269 of hydrogenase system by, 866 determination of by yeast fermentation, 330 formation of by glucose-6-phosphatase, 541-543 kinase for in pyridoxal kinase preparations, 648-649, see also Hexokinase, in oxidative phosphorylation assay, 611 *Glucose dehydrogenase, generation of D P N H by, 493, 494 Glucose-l,6-diphosphate (G-1,6-P2), ribose- 1,5-diphosphate formation from, 503

933

*Glucose oxidase (notatin, deoxygenase), deoxygenation for hydrogenase assays by use of glucose and, 866 inhibition by nitrate, 579 peroxide generation by, 244 Glucose-6-phosphatase, affinity for G-6-P, 560 from rat liver, 541-543 sources of, 542 Glucose-l-phosphate (G-I-P), enzymatic assay of, 675 UDPG synthesis from UTP and, 675 Glucose-6-phosphate (G-6-P, Robison ester), determination of formation during oxidative phosphorylation, 613 enzymatic assay of, 675 as hydrogen donor in nitrogen gas formation, 420, 422 TPN as cofactor for, 422 preparation of crystalline calcium salt, 699 as product of transphosphorylation by phosphatases, 556 enzymatic measurement of, 560 prostatic phosphatase action on, 525 as substrate for alkaline phosphatase, 533 *Glucose-6-phosphate dehydrogenase (Zwischenferment), assay of ATP with, 497 of glucose 1-P with phosphoglucomutase and, 675, 676, 677 cytochrome reductase-free preparations of, 699-700 lyophilized preparation of, 720 in preparation of ribose-l,5-diphosphate, 504 removal by ethanol fractionation, 677 stabilization of, 713 TPNH generation by, 699, 712, 715, 719 Glucose-6-sulfate, as substrate for glucosulfatases 330 Glucosulfatases, 324, 330 Glutamate, action of D-serine (D-threonine dehydrase on, 324 chromatographic determination of, 289

934

SUBJECT INDEX

as component of transaminase system, 176, 180 determination of in protein hydrolysates, 192 in fibrinogen, 160 formation of by glutaminase, 380 formation of in tyrosine-glutamic acid transamination, 289 incorporation into glutathione, 342, 343 manometric estimation of, 182 P:O ratio during oxidation of, 613 requirement for in citrulline synthesis, 355 role in tyrosine-oxidizing system, 287 as substrate for L-glutamic dehydrogenase, 220-225 as substrate for glutamine synthetase, 337 as C-terminal residue, effect on iminodipeptidase, 100 transaminations involving, 170-177 with aspartic acid, 170, 171, 172, 174, 175 with cysteine-sulfinate, 172 D-Glutamate, assay of in presence of L-isomer, 175176 inhibition of glutamic dehydrogenase by, 224 resistance to D-amino acid oxidase, 2O2 as substrate for glutamo-transferasc, 341 Glutamic acid decarboxylase, assay of ~glutamic acid with, in glutamic acid raeemase system, 215 in protein hydrolyzates, 192 in transaminase tests, 170, 173-174, 176, 182-183, 289 bacterial sources of, C1. welchii, 182 E. coli, 182-183, 186-187, 216 in brain, 199 plant sources of, 182, 190-194 carrots, 194 squash, 182, 190-194 unsuitability in transaminase assay, 182

product of, C02 versus HCO3- as, 841 Glutamic acid racemase, from L. arabinosus, 215-217 L-Glutamic dehydrogenase, from liver, 220-225 i~Glutaminase, from animal tissues (glutaminase I), 382 distribution of, 382 from E. coli, 380-382 Glutamine, deamidation of in presence of a-keto acids, 382 formation from glutamic acid and ammonia, 337 formation of glutamyl hydroxamic acid from, 263, 267 hydrolysis of by glutaminase, 380 inhibition of glutamic dehydrogenase by, 224 in synthesis of 5-amino imidazole ribotide, 505 of glycinamide ribotide, 504, 509512 of inosinic acid, 506 transamination with aliphatic keto acids in animal tissues, 172 Glutamine (CONI~H2), ~.-glutamyltransferase reaction for preparation of, 267 Glutamine synthetase, association with ~,-glutamyltransferases, 264, 267, 272, 341 in liver, 339, 341-342 from pea seeds, 337-342 from sheep brain, 339, 341, 342 in Staph. aureus, 342 Glutamine transaminase, 382 Glutamohydroxamic acid, see 7-Glutamylhydroxamic acid Glutamotransferase, see -y-Glutamyltransferase L--/-Glutamyl-~-cysteine, as intermediate in formation of glutathione, 342 v-Glutamylhydroxamic acid (glutamohydroxamic acid, GHA), assay of glutamine synthetase by formation of, 337-338 colorimetrie determination of, 267, 268

SUBJECT INDEX

as product of 3,-glutamyltransferase reaction, 263, 267 synthetic, 268 3,-Glutamyltransferases (glutamotransferases, GTF), metal and nucleotide dependent, association with glutamine synthetase activity, 264, 267, 272, 341 from brain, 270-271 from liver, 271 from peas, 263-266 from P. vulgaris, 271-272 from pumpkin (PGT), 263-264, 266-267 metal and nucleotide independent, occurrence in bacteria, 269 preparation from P. vulgaris, 268269 Glutathione (GSH), see also Mercapto compounds, Thiol compounds, activation of adenylate kinase by, 603 of alanine racemase by, 213, 215 of amino acid reduetases by, 220 of cathepsin C by, 68 of eitrullinase by, 378 of guanidinoacetate methylpherase by, 260, 263 of histidase by, 231 of hydrogenase by, 868 of insect muscle ATPase by, 598 of lucifcrase (bacterial) by, 860 of nitrate reductase by, 415 of pyrocatechase by, 282 of thio ether cleavage enzyme by, 314 of tryptophanase by, 241 of tryptophan synthetase by, 237 as coenzyme for maleylaeetoacetate isomcrase, 295, 296, 298 effect on 3'-nucleotidase, 554 formation by reduction of GSSG, 719 hydrolytic enzymes for in liver, 343 inhibition of hippuric acid formation by, 350 of metaphosphatase by, 579 of tyrosinase by, 826 measurement by various methods, 719 reduction of dehydroascorbic acid by, 849, 850 of nitrate esters by, 403-406

935

enzymatic, 403-406 non-enzymatic, 405, 406 of p-quinone by, 728-729 requirement for, in methionineo activating reaction, 254, 255, 256 role in blood clotting mechanism, 160 stabilization of carbonic anhydrase by, 846 of nitrate reductase by, 414 of tryptophanase by, 241 of tryptophan synthetase by, 237 treatment of dialyzing membranes with, 414 Glutathione, oxidized (GSSG), reduction of by molecular hydrogen, 732 by TPNH, 719, 722 Glutathione reductase, in assay of 5-dehydroshikimic reductase, 301-302 from beef liver, 724-725 evidence for pgrphyrin in, 725 from higher plants, 719-722 distribution of, 721 in Itill reaction, 721 reduction of GSSG with hydrogenase and, 732 from yeast, 723-724 crystallization of, 723 Glutathione-synthesizing system, in E. coli extracts, 342 from pigeon liver, 342-346 L--y-glutamyl-L-cysteineas intermediate in, 342 enzyme for formation of, 342 glutathione synthetase for utilization of, 342-346 Glutathione synthetase, from brewer's yeast, 345-346 distribution of, 343 from pigeon liver, 344-345 Glycerol, as acceptor for transphosphorylation by phosphatases, 556 activation of ~-glutamyltransferase (bacterial) by, 269 extraction of brewer's yeast with, 581 Glycerol trinitrate, see Nitroglycerine

936

SUBJECT I N D E X

Glycerophosphatase, in microsomes, 542 Glycerophosphates, prostatic phosphatase action on aand ~-, 525 fl-Glycerophosphate, as substrate for alkaline phospharases, 530-533, 538, 539 DL-a-Glycerophosphate, as product of transphosphorylation by phosphatases, 556 L-a-Glycerophosphate, enzymatic measurement by reduction of ferricytochrome c, 559 *L-a-Glycerophosphate dehydrogenase, in insoluble particles of rabbit muscle, 559 Glycinamide, hydrolysis by leucine aminopeptidase, 92 Glycinamide ribotide, formylation of, 505, 509-512 isolation of, 511-512 Glycine, determination of, 347 in fibrin, 160 inhibition of alkaline phosphatase by, 538 of histidase by, 231 oxidation of, 225-227 as precursor of 5-amino-4-imidazolecarboxamide ribotide, 512 as product of aminotripeptidase action, 87 of formylation of FAH4 by serine, 518 reduction of, 217 resistance to D-amino acid oxidase, 202 stabilization of arginase by, 373 of renin by, 134 in synthesis of glutathione, 342, 343 of glycinamide ribotide, 504, 509-512 estimation with glycine-l-C TM, 509-511 of hippuric acid, 346 of inosinie acid, 506 Glycine buffer, high values for ATPase in, 587 Glycine oxidase, from pig kidney, 225-227

*Glycolytic enzymes, in liver, 343 from rabbit muscle, 358 regeneration of ATP with, 351, 356, 357 Glyeylallohydroxy-L-proline, as substrate for prolidase, 104 Glycyl-amino acids, action of acylases on, 118, 119 of dehydropeptidases I and I I on, 113 Glyeyldehydroamino acids, action of dehydropeptidases on, 109, 110, 113, l l 4 Glycylglyeine, action of acylase I on, 118 derivatives of, resistance to glycylglycine dipeptidase, 109 probable reaction with quinone, 729 as product of aminotripeptidase action, 83 Glycylglycine dipeptidase, 107-109 from human uterine tissue, 108-109 presence in purified iminodipeptidase, 99 from rat muscle, 108 comparison with dehydropeptidase I of swine kidney, 108 sources of, 108 stability of, 109 from swine kidney, 108, 109 Glycylglycylglycine (triglycine), as substrate for aminotripeptidase, 83-84, 87 Glycylglycyl-L-leucine, as substrate for aminotripeptidase, 83, 87 comparison with D-form, 87 Glycylglyeyl-~-proline, as substrate for aminotripeptidase, 87 Glycyl-L-histidinamide, activity of carnosinase on, 96 Glycyl-L-histidine, activity of carnosinase on, 96 Glycylhydroxy-L-proline, as substrate for prolidase, 104 Glycyl-L-leucinamide, and carbobcnzoxy derivative of, resistance to glycyl-L-leucine dipeptidase, 107 as substrate for leucine aminopeptidase, 92

SUBJECT INDEX Glycyl-n-leucine, carbobenzoxy derivative of, resistance to glycyl-~leucine dipeptidase, 107 as product of aminotripeptidase action, 83 as substrate for dehydropeptidase I, 107 comparison with D-isomer, 107, 113 Glyeyl-L-leucine dipeptidase, from human uterine tissue and swine intestinal mucosa, 105-107 specificity of, comparison with dehydropeptidase I, 107 Glycyl-L-leucylglycine, as substrate for aminotripeptidase, 87 comparison with D4orm, 87 Glycyl-L-phenylalaninamide (GPA), action of amidase on, 399 as substrate for cathepsin C, 64-65, 68 hydrolysis reaction, 64 transamidation reaction with hydroxylamine, 65 Glycyl-L-phenylalaninamide acetate, preparation of, 66 Glycyl-L-phenylalanine ethyl ester, as substrate for cathepsin C, 68 Glycyl-L-proline, resistance of carbobenzoxy derivative of to prolidase, 104-105 as substrate for prolidase, 100, 104 Glycyl-u-prolylglycine, comparison with other tripeptides in aminotripeptidase reaction, 87 Glycylsarcosine, as substrate for prolidase, 104 Glycyl-I,-tyrosinamide, as substrate for cathepsin C, 68 Glycyl-L-tyrosine ethyl ester, as substrate for cathepsin C, 68 *Glyoxalase, assay of glutathione with, 343, 719 Glyoxylic acid, as product of glycine oxidase reaction, 225 reaction with hydrogen peroxide, 225 Gramieidin, as uncoupling agent, 615 Grasshopper eggs, tyrosinase occurrence as protyrosinase in, 831

937

Grassmann and Heyde procedure, assay of peptidases by, 83, 88, 93-94, 97, 100, 105-106, 107-108 Guaiacol, see also Catechol monomethyl ether, assay of peroxidase with, 770-773, 792 Guanase, from rat liver, 480-482 removal of guanine with, 449 spectrophotometric determination of guanine with, 458, 481-482 Guanidines, inhibition of diamine oxidase by, 396 Guanidinoacetic acid (GA), methylation of, 260, 263 stoichiometry of reaction, 263 Guanidinoacetate methylpherase, from pig liver, 260-263 Guanine, as acceptor of deoxyriboside group, 468 conversion to xanthine, 480-482 shift in spectrum during, 480 RI values for, 466 spectrophotometric assay of, 458, 481482 Guanine deoxyriboside, bioassay of, 464 phosphorolysis of, 448 R/values for, 466 in transdeoxyribosidase reaction, 464468 Guanosine (guanine riboside) (GR), inhibition of inosine cleavage by, 463 nucleosidase action on, 459, 463 phosphorolysis of by spleen enzyme, 448 by yeast enzyme, 453 Guanosine triphosphate (GTP), as phosphate donor to AMP, 603 3'-Guanylic acid (3'-GMP), 3'-nucleotidase action on, 553 5'-Guanylie acid (GMP), as acceptor in nucleoside monophosphate kinase reaction, 603 H

Heart, adenylate kinase (myokinase) in, 602 cytochrome a in particles from, 737, 739

938

SUBJECT INDEX

cytochrome a3 in particles from, 734, 737, 740 cytochrome b from, 740-744 cytochrome c from, 749-755 in particles from, 738 cytochrome c oxidase activity in particles from, 738 cytochrome component absorbing at 553 m~ in, 744 diaphorase (DPNH-linked) from, 707711 D P N H cytochrome c reductase from, 688, 693 fibrinokinase from, 165 glutamic-oxalacetic transaminase activity of, 184 isocitric dehydrogenase in, 681 nitroaryl reductase in, 406 oxidative phosphorylation in mitochondria from, 614 preparation of particle suspensions from, 737, 740 hemoglobin removal from, 738-739 pyridine nucleotide transhydrogenase from, 686, 687 species variation in, 687 succinie dehydrogenase in, 737 succinic oxidase system in, 738, 750 Heat of activation, for DNase, 443 Heavy metals, inhibition of D-amino acid oxidase by, 202 of bacterial luciferase by, 860 Heavy water (D20), in hydrogenase assay by exchange reaction, 863 Helvolic acid, inactivation by crude penicillinase, 123 Hematin, content in bacterial catalase, 788 Hematin compounds, removal from cytoehrome c reductase by adsorption with gels, 690, 691 Hematin peptide, formation from cytochrome c, 167169 Hematohemin, formation of from cytoehrome c, 753

Iteme, spectrophotometric assay of, 791 Heme-protein, as byproduct of catalase crystallization from liver, 794 Hemin, see also Protoporphyrin, content of in cytochrome bl, 747 content of in horseradish peroxidase, 809 green, as prosthetic group of myeloperoxidase, 799 as impurity in D P N H cytochrome c reductase of heart, 691 Hemochromogen, from E. coli, as acceptor for D P N H cytochrome reductase of bacteria, 698-699 reoxidation by mammalian cytochrome oxidase, 699 extinction coefficient for pyridine ferroprotoporphyrin, 809 preparation of from catalase, 788, 790 Hemoglobin, in assay of pepsin, 3-4 denaturation by Tsuchihashi procedure, 102 proteolysis by rennin, 77 removal by alcohol-chloroform treatment, 783 removal of from heart particles1 738-739 solubility of in ammonium sulfate solutions, 84 Hemoglobin digestion method, for determination of trypsin, 34 Hemophilic plasma reagent, in assay of antihemophilic factor, 148 Hemoproteins, see Cytochromes Heparin, cofactor of from mast cells, 163 inhibition of RNase by, 434 of thrombin by, 158 precipitation of by protamine, 163 Heptylamine, amine oxidase action on, 393 ttexametaphosphate, as substrate for metaphosphatase, 577-578

SUBJECT INDEX Hexamethylenediamine, action of diamine oxidase on, 396 *Hexokinase, in assay of adenylate kinase, 598-599, 6O0 of ATP, 497 coupling of citrullinase reaction with, 377 in oxidative phosphorylation assay, 611 *Hexose isomerase, see Phosphohexoisomerase Hexosephosphate, inhibition of acetyl phosphatase by, 556 Hexylamine: amine oxidase action on, 393 Hill reaction, glutathione reductase in, 721 Hippuric acid, enzymatic synthesis of in kidney cortex extracts, 348-349 in liver, 346-350 Hippuricase, see also Amino acid acylase I, probable identity with acylase I, 115 a-Hippuryl-L-lysinamide (HLA), as substrate for trypsin, 36 Histaminase, see Diamine oxidase Histamine, formation of from histidine in higher plants, 194 oxidative deamination of, 394 Histidase, in liver, 229 from Ps. fluoresceus, 228-231 Histidine, activation of DNase by, 442 as component of dipeptides hydrolyzed by carnosinase, 93, 96 content of in horseradish peroxidase, 8O9 conversion to urocanic acid by histidase, 228 decarboxylation of in higher plants, 194 in mammalian tissues, 199 as substrate for L-amino acid oxidase, 2O8

939

Histidine decarboxylase, from Cl. welchii, 187 in measurement of transaminase reactions, 171 Histozyme, see also Amino acid acylase I, probable identity with acylase I, 115

bis-Homobiotin, inhibition of d-biotin oxidation by, 632 Homocysteine~ action of cysteine desulfhydrase on, 317 determination by modification of Brand method for, 314 formation from eystathionine, 314 in over-all system for I M P synthesis, 5O6 Homocysteine desulfhydrase, from liver, 318 Homogenates, tissue, oxidative phosphorylation in, 610-616 Homogentisate, conversion of to maleylacetoacetate, 292-295 oxidation of, 288 Homogentisate oxidase, distribution of, 294 from liver, 292-295 spectrophotometric method for assay of, 295 Homosulfanilamide, activation of bacterial thiaminase by, 628 amine oxidase action on, 393 Homotryptophan, inhibition of tryptophanase by, 241 Horse nettle (Solanum elaeagnifolium), protease (solanain) from, 57, 63-64 Horseradish, allylisothiocyanate from, 804 peroxidase from, 801-813, see also under Peroxidase Hura crepitans, see Jabillo plant Hurain, protease from sap of jabillo plant, 57, 64 Hyaluronic acid, inhibition of acetyl phosphatase by, 556 Hydrazine, in glutamine synthetase reaction, 341 inhibition of bacterial transaminases by, 177

940

SUBJECT I N D E X

of tryptophanase by, 241 as substrate for ~.-glutamyltra~lsferase, 269 Hydrazoie acid, see Azide Hydrochloric acid, compound with horseradish peroxidase, 812 Hydrocyanic acid, see Cyanide Hydrogen gas, absorption of in amino acid reduetase reaction, 217 role of hydrogenase in, 218 activation of hydrogenases by, 866, 868 O~-free, 863-865 ortho and parahydrogen conten~ of at equilibrium, 864 different thermal conduetivities of isomers, 864 reduction of pyridine nucleotides by, 729 of various substances by, 861 reversible combination of with flavoproteln, 870 Hydrogenase(s), 729-732, 861-870 in amino acid reductase reaction, prevention of air inactivation of, 218 assay of, 729-730, 862-865 by conversion of parahydrogen, 864-865 by evolution of hydrogen from substrates, 862 by exchange reaction, 862, 864 by reduction of methylene blue (manometric method), 865 of pyruvate via DPN, 729-730 preparation and properties of, 730-732, 865-870 from Cl. kluyveri, 730-732 cofactor for, 730, 731 purification of, 731, 732 from Cl. pasteurianum, 869, 870 characterization as Mo-FADprotein, 869, 870 spectrum of, 870 from Desulfovibrio sulfuricans, 869, 87O from E. coli, 867, 868 from Proteus vulgaris, 865, 867 characterization as iron-porphyrin-protein complex, 866

variation in inhibitor effects with nature of assay, 867 sources of, 870 specific activity of preparations of, 868

Hydrogenomonas facilis, hydrogenase in, 870

Hydrogenomonas ruhlandii, hydrogenase in, 870 Hydrogen peroxide, formation of by amine oxidase, 390 by amino acid oxidases, 199, 204 by diamine oxidase, 394 by glycine oxidase reaction, 225 by leuco methylene blue oxidation, 715 by old yellow enzyme, 712 by uricase, 485 by xanthine oxidase, 482 inhibition of adenylate kinase by, 603 of amino acid reductase by, 219 of myosin ATFase by, 587 oxidation of cytochrome c (mammalian) by, 736 of cytochrome e (Ps. fluorescens) by, 759, 761 of a-keto acids by, 204 of tryptophan by, 242, 246 as substrate for horseradish peroxidase, 807, 810, 811 see also Enzyme-substrate complexes for lactoperoxidase, 816 Hydrogen sulfide, see also Sulfide, determination of, 315-316 formation by eysteine desulfhydrase, 315-319 inhibition of alkaline phosphatase by, 527 of D-amino acid oxidase by, 203 phosphopantetheine maintenance by, 669 Hydroquinone, determination of, 726 formation of by quinone reductase, 725 reduction of cytochrome c by, 754 of peroxidase complex I I by, 810 Hydroquinone monomethyl ether, oxidation of by peroxidase complex II, 810

SUBJECT INDEX Hydrosulfite, see Dithionite Hydroxamic acid method, acetyl phosphate analysis by, 650 p-Hydroxyaeetophenone, assay as organic product of arylsulfatases, 327-328 a-Hydroxy acids, complex formation with borate, 290 L-a-Hydroxy acids, as substrates for L-amino acid oxidase, 211 Hydroxyanthranilic acid, tryptophan peroxidase and, 244 p-Hydroxybenzoic acid, as precursor of protocatechuic acid, 273 as product of aromatic biosynthesis, 300 p-Hydroxybenzoyl formic acid, decarboxylation of, 280 /~-Hydroxybutyrate, P:O ratio during oxidation of, 613, 616 p-Hydroxydiphenyl, oxidation by peroxidase complex II, 810 fl-Hydroxyglutamic acid, as substrate for glutamic acid decarboxylase of plants, 192 7-Hydroxyindole, as substrate for tryptophan synthetase, 236 L-Hydroxykynurenine, as substrate for liver kynureninase, 253 Hydroxylamine, effect on activity and spectrum of myeloperoxidase, 799 inhibition of amine oxidase by, 393 of arginine desimidase by, 376 of bacterial transaminase by, 177 of cytochrome c peroxidase by, 763 of 5-dehydroquinase by, 307 of dopa decarboxylase by, 198 of glutamic acid decarboxylase by, 193 of glutamic dehydrogenase by, 224 of -y-glutamyltransferase (brain) by, 272 of kynureninase by, 253 of nitroethane oxidase by, 402

941

of D-serine (D-threonine) dehydrase by, 324 of tryptophanase by, 241 of tryptophan synthetase by, 237 replacement of NH3 in glutamine synthetase reaction by, 338 role in ~-glutamyltransferase reaction, 263, 267, 269 in transamidation by eathepsin C, 65 Hydroxylamine reductase, as adaptive enzyme, 411, 417 in Bacillus organisms, 417 from Neurospora crassa, 416-419 presence in nitroaryl reduetase preparations, 410, 411 turnover number of, 418 Hydroxylaminodinitrotoluene, reduction of trinitrotoluene to, 406 Hydroxyl radicals, inhibition of RNase by, 434 p-Hydroxyphenylpyruvate, ascorbate requirement for oxidation of, 288 determination of, 289 enol form of, light absorption of, 289 stabilization of, 290 keto form as product of tyrosineglutamic acid transamination, 289 m-Hydroxyphenylpyruvate, enol-keto tautomerase action on, 291 p-Hydroxyphenylpyruvate enol-keto tautomerase, distribution of, 291 from liver, 289-292 Hydroxyphenylserine, decarboxylation of in mammalian tissues, 199 Hydroxy-L-prolinamide, hydrolysis by leucine aminopeptidase, 92 Hydroxy-L-prolylglycine, as substrate for iminodipeptidase, 97 Hydroxy-L-prolylglycylglycine, as substrate for aminotripeptidase, 87 8-Hydroxyquinoline, inhibition of ascorbic acid oxidase by, 835 of D P N H cytochrome c reductase by, 693

942

SUBJECT INDEX

of nitrate reductase by, 415 of D-serine (D-threonine) dehydrase by, 324 of tryptophan synthetase by, 237 Hydroxytyramine, biological and colorimetric determinations of, 195 formation of from 3,4-dihydroxyphenyl-L-alanine in higher plants, 194 Hypertensin, see Angiotonin Hypertensinogen, see Renin substrate Hypoxanthine, as acceptor of deoxyriboside group, 468 incorporation of C ~4formate into, 505-509 isolation and degradation of, 507-508 apparatus for, 508 molecular extinction coefficient of, 507 oxidation to uric acid, 448, 449, 482, 484 RI values for, 466 spectrophotometric assay of, 458 as substrate for nucleoside phosphorylase, 448 synthesis of IMP from R-5-P and, 503 Hypoxanthine deoxyriboside, phosphorolysis of, 448 R! values for, 466 in transdeoxyribosidase reaction, 464468 bioassay of, 464 Hypoxanthine riboside, see Inosine

Imidazole, inhibition of xanthine oxidase by, 485 Imidodipeptidase, see Prolidase Iminodipeptidase (prolinase), from swine kidney, 97-100 pH optimum of, dependence of on purity, 97 Indigodisulfonate, in assay of diamine oxidase, 394 Indigotrisulfonate, as electron donor to FMN in bacterial luciferase system, 861

Indole (benzopyrrol), colorimetric measurement of, 233-234, 238-239 detoxication of diphtheria toxin by myeloperoxidase and, 800 formation of by tryptophanase in E. coli, 238 inhibition of tryptophanase by, 241 of tryptophan synthetase formation by, 237 L-tryptophan synthesis from, 233 Indophenols, oxidation by peroxidase complex II, 8O8 Induced enzymes, see Adaptive enzymes Induction, see Sequential induction Infusion broth, protective action on penicillinase, 123 Inorganic phosphate, see Phosphate, inorganic Inorganic pyrophosphatase, see Pyrophosphatase, inorganic Inosine (Hypoxanthine riboside, HxR), formation from adenosine, 473 inhibition of nicotinamide riboside phosphorolysis by, 453, 455, 456 nucleosidase action on, 459, 463 phosphorolysis of, by spleen enzyme, 448 by yeast enzyme, 453 RI values for, 466, 513 Inosine compounds, formation of by deaminase, 475 Inosine diphosphate (IDP), action of insect muscle ATPase on, 597 of liver mitochondrial ATPase on, 594 Inosine-5'-phosphate, see 5'-Inosinic acid Inosine triphosphate (ITP), action of insect muscle ATPase on, 597 of liver mitochondrial ATPase on, 594 of myosin on, 586 3'-Inosinic acid (3'-IMP), 3'-nucleotidase action on, 553 5'-Inosinic acid (inosine-5'-monophosphate, Y-IMP), formation of from 5'-AMP, 469

SUBJECT INDEX

molecular extinction coefficient of, 469 " 5 " nucleotidase action on, 549 paper chromatography of, 516 preparation of aminoimidazolecarboxamide ribo~ide from, 514 scheme for de novo biosynthesis of, 504-5O5 synthesis of from R-5-P and hypoxanthine, 503 Insect muscle, see Muscle Insect tissues, see also under names of individual species, cytochrome e in, 745 Intestinal mucosa, adenosine deaminase from, 473-475 aminotripeptidase in, 84 flavokinase in, 642 glycyl-L-leucine dipeptidase from, 105107 leucine aminopeptidase in, 89 phosphodiesterase from, 569-570 phosphomonoesterase from, 473-475, 530 533, 539 prolidase in, 102, 105 pyrophosphatase in, 475 Intestine, glucose-6-phosphatase in, 542 kynurenine formamidase in, 248 Iodine, inhibition of alkaline phosphatase by, 538 of aspartase by, 388 of erythrocyte carbonic anhydrase by, 845 of myosin ATPase by, 587 Iodoacetamide, inhibition of aspartase by, 388 of 5-dehydroquinase by, 307 of myosin ATPase by, 587 of prolidase by, 105 protection against by manganese ion, 105 of RNase by, 434 resistance of leucine aminopeptidase to, 93 Iodoacetate, inhibition of L-amino acid oxidases by, 208, 211 of DPNH cytochrome c reductase by, 693

943

of p-hydroxyphenylpyruvate enolketo tautomerase by, 292 of insect muscle ATPase by, 598 of myosin ATPase by, 587 of RNase by, 434 Iodoacetyl amino acids, action of acylase I on, 118 Iodosobenzoate, inhibition of aspartase by, 388 of DPNH cytochromc c reductase by, 693 of myosin ATPase by, 587 Ionophoresis, of purine and pyrimidine bases and nucleosides, 457 Iron, see also under Ferric ion and Ferrous ion, as component of DPNH cytochrome c reductase of heart, 691, 692 valence state of, 692 effect of DPNH and ferricytochrome c on, 692 content of in catalase from blood, 784 from spinach, 790 content of in lactoperoxidase A, 817 in myeloperoxidase, 798 in uriease preparations, 489 firefly luciferase system and, 856 inhibition of bacterial luciferase by, 860

of uricase by, 489 in prosthetic group of cytochrome c, 753 Iron enzymes, derivatives of cytochrome c as, 167-169 Iron-porphyrin-protein, hydrogenase of P. vulgaris as, 866 Iron protoporphyrin, as prosthetic group of Ustilago cytochrome c, 757-758 Isoalloxazine," derivatives of, as substrates for flavokinase, 644 D-Isoascorbic acid, in oxidation of p-hydroxyphenylpyruvate, 289 Isobutyrate, inhibition of d-biotin oxidation by, 632 Isocitrate, P:O ratio during oxidation of, 616

944

SUBJECT INDEX

*Isocitric dehydrogenase, DPN-linked, 'in purified yeast fraction, transhydrogenase assayed with, 682 *Isocitric dehydrogenase, TPN-linked, from pig heart, TPN assay with, 652 TPNH generation with, 681 TPNH preparation with, 411 Isocitric lactone, as source of isocitrate, 652 L-Isoglutamine, hydrolysis by leucine aminopeptidase, 92 L-Isoleucinamide, hydrolysis by leucine aminopeptidase, 92 L-Isoleucine, as substrate for L-amino acid oxidase, 211 transamination with valine in microorganisms and animal tissues, 171, 176 Isonicotinamide, reversible conversion to D P N analog by animal DPNases, 662, 663 1-Isonicotinyl-hydrazine (isoniazid) (isonicotinic acid hydrazide) (INH), inhibition of amine oxidase by, 393 of diamine oxidase by, 396 reversible conversion of to DPN analog by animal DPNases, 662, 663 species variation in sensitivity of animal DPNases to, 663 1-Isonicotinyl-2-isopropylhydrazine (iproniazid) (marsilid) (isopropyl INH), inhibition of amine oxidase by, 393 reversible conversion of to D P N analog by animal DPNases, 662, 663

Jabillo plant (Hura crepitans), protease (hurain) from sap of, 57, 64 Jack bean meal, urease from, 378-379

Janus Green B, as electron acceptor for D P N H eytochrome c reductase of bacteria, 697 K

Kaolin, cytochrome c (bacterial) purification by chromatography on, 762 preparation of acid-treated, 762 Kat. f . ( Katalasef dhigkeit) ,

catalase activity and purity expressed as, 767, 779-780, 782, 788, 789, 790, 791 a-Keto acid(s), activation of aspartic acid decarboxylase by, 188, 189 measurement of, for assay of L-amino acid oxidase, 204 oxidation by hydrogen peroxide, 204 a-Keto acid-~-amidase, from rat liver, 382, 384-385 *a-Keto acid decarboxylases, see Carboxylase f~-Ketoadipic acid, aromatic ring cleavage and, 273 formation of from t~-carboxymuconic acid, 273, 285 from ~-carboxymethyl-A=-butenolide, 273, 282-284 ~-Ketobutyrate, formation by cleavage of cystathionine, 311 by dehydrase action on threonine, 319-322 by desulfhydrase action on homocysteine, 318 ~-Ketoglutaramate, as intermediate in deamidation of glutamine, 382 as substrate for a-keto aeid-~-amidase, 384 a-Ketoglutarate, in glutamic dehydrogenase reaction, 220 measurement of as succinate with succinoxidase, 170 as 2,4-dinitrophenylhydrazone, 170 P: O ratio during oxidation of, 613, 616 requirement for in tyrosine-oxidizing system, 287, 288

SUBJECT INDEX

transaminase measurement with, 170, 172-175, 179-182 transamination with L-tyrosine, 289 with various amino donors, false glutamate values due to, 193 *a-Ketoglutaric decarboxylation system, COs versus HC0s as product of, 841 Ketone reagents, inhibition of cysteine desulfhydrase by, 317 Ketosteroid, as product of steroid sulfatase, 330 a-Ketosuccinamate, as substrate for a-keto acid-~-amidase, 384 Kidney, acetyl phosphatase from, 556 adenosine kinase in, 499 amino acid amidase from, 397-400 D-amino acid oxidase from, 171, 199204, 212 L-amino acid oxidase from, 209-211 arginine-synthesizing system from, 357, 358-359, 365-367 argininosuccinate-splitting enzyme as component of, 365-367 d-biotin oxidase in slices of, 631-632 carbonic anhydrase in, 841 catalase in, 765, 775 cysteine desulfhydrase in, 381 decarboxylases for various amino acids in, 199 diamine oxidase (histaminase) from, 394-396 3,4-dihydroxyphenylalanine (dopa) decarboxylase from, 195-199 folio acid conjugase in, 629-630 glucose-6-phosphatase in, 542 glutamic-oxalacetic transaminase activity of, 181 L-glutaminase in, 382 glycine oxidase from, 225-227 hippuric acid synthesis in, 348-349 kynurenine formamidase in, 248 oxidative phosphorylation in mitochondria from, 614 peptidases from, amino acid acylase I (soluble acylase I, dehydropeptidase II), 109-114, 116-119

945

amino acid acylase II, 117, 119 carnosinase, 93-96 dehydropeptidase I (solubilized aminopeptidase), 109-114 glycylglycine dipeptidase, 108, 109 iminodipeptidase, 97-100 leucine aminopeptidase, 88-93, 98 prolidase, 98, 101, 103-104, 105 purification of by common initial procedure, 98 renin from, 124-135 RNase in, 436 succinic oxidase in cortex of, 750 transhydrogenase in, 687 triphosphatase in, 580, 581 uricase from, 485-489 King-Armstrong unit, for phosphatase values in serum, 528 Kochsaft, see Boiled juice Kojic acid, inhibition of D-amino acid oxidase by, 203 Kynurenic acid, absorption maximum for, 250 tryptophan peroxidase and, 244 Kynureninase, distribution of, 253 from Ps. fluorescens, 249-253 from rat liver, 244, 249-253 L-Kynurenine, conversion to anthranilic acid and L-alanine, 249 determination of, 242 extinction coefficients of, 250 formation by formamidase, 242, 246 Kynurenine formamidase (formylase), distribution of, 248 in Neurospora crassa, 253 from rat liver, 246-249 Kynurenine transaminase, formation of kynurenic acid by, 250 in liver homogenates, 244 L Laccase, effect on induction period of monophenolase, 825 Lactate, activation of 5'-AMP deaminase by, 472

946

SUBJECT INDEX

L-Lactic acid, as substrate for L-amino acid oxidase, 211 *Lactic dehydrogenase, pyruvate reduction with hydrogenase and, 729-730 in yeast, as crystalline hemoprotein (cytochrome b2) containing flavin, 746 LactobaciUus arabinosus, gtutamic acid racemase from, 215-217 growth medium for, 216 Lactobacillus casei, in assay for "folic acid activity," 629 Lactobacillus delbriickii, acetyl phosphatase from, 556 nucleoside transdeoxyribosidase in, 467 LactobaciUus fermenti, arginine desimidase in, 376 Lactobacillus helz,eticus, aspartase in, 388 growth of, 467-468 nucleoside transdeoxyribosidase from, 467-468 in preparation of medium for propionic acid bacteria, 386 Laclobacillus ~rlesenteroides, arginine desimidase in, 376 Lactobacillus pentosus, growth of, 459 hydrolytic nucleosidases from, 456-461 Lactone-splitting enzyme, from Ps. fluorescens, 273, 282-284 Lactonizing enzyme, for cis,cis-muconic acid from Ps. fluorescens, 273, 282-284 Lactoperoxidase A, see also Peroxidase from milk, chemical and physical properties of, 817 nitrogen content of, 799 preparation of crystalline, 813-817 Lactoperoxidase B, from spring milk, 816-817 turnover number of, 817 Lamb's quarters (Chenopodium album), carbonic anhydrase in leaf of, 842 Lanthionine, meso- and L-forms as substrates for cleavage enzyme, 312, 313

Latex, proteolytic enzymes from, see Proteolytic enzymes, plant Lead ion, activation of metaphosphatase by, 579 inhibition of aspartase by, 388 of leueine aminopeptidase by, 93 of prolidase by, 105 Lead subacetate reagent, preparation of, 824 Lead sulfide, colloidal, measurement of light scattered by, 315 Leaf tissue, carbonic anhydrase in, 842 Lebedew juice, phosphorylation of thiamine by protein from, 636 as source of old yellow enzyme, 713 Lecithin, presence of in heparin cofactor, 163 *Lecithinase, of Cl. welchii, inactivation of Mgactivated ATPase by, 590 Leguminosae, dehydroascorbic reductase in, 849 L-Leucinamide, action of dehydropeptidases I and II on, 113 as substrate for amidase, 399 for leucine aminopeptidase, 88, 89, 91, 92 comparison with D-isomer, 91 L-Leucine, inhibition of D-amino acid oxidase by, 203 as product of aminotripeptidase action, 87 as substrate for L-amino acid oxidases, 205, 208, 209, 211 transaminase for, 176 Leucine aminopeptidase (leucylpeptidase), action on amides and peptides~ 92 distribution of, 93 inhibition of by Versene, 87 substrates for, 89, 91-92 in swine intestinal mucosa, 89 from swine kidney, 88-93, 98 Leucobenzylviologen,see Benzylviologen, reduced

SUBJECT INDEX

Leucocytes (W.B.C.), myeloperoxidase from, 245, 794-800 Leucodyes~ oxidation by amino acids in Cl. sporogenes, 218 by peroxidase complex II, 808, 810 Leucovorin, see also Citrovorum factor, in formylation of glycinamide ribotide, 510 I,-Leucyl-D-alanine, comparison with I~form in leucine aminopeptidase reaction, 92 L-Leucylglycine, as substrate for leucine aminopeptidase and other peptidases, 89, 91 comparison of with D-isomer, 91 L-Leucylglycylglycine, as substrate for aminotripeptidase, 83, 87 comparison with D-form, 87 as substrate for leucine aminopeptidase and other peptidases, 83, 89 L-Leucyl-D-amino acids, hindrance of leucine aminopeptidase action by D-residues of, 92 Leucyl peptidase, see Leucine aminopeptidase Leukemic tissues, mouse, DNase in, 447 Lima bean, protease from, 63 trypsin inhibitor from, 37, 48-49, 51, 53 Lipoprotein~ nucleoside phosphorylase as f~-, 452 thromboplastin as, 150 Littorina littorea (mollusk), arylsulfatases in, 332 Liver, acetone powder preparation from, 509, 704 activation of "1-C unit" as folic acid derivative in extracts of, 505, 516518 adenosine kinase in, 499 adenylate kinase (myokinase) in, 602, 603, 604 amine oxidase from, 393 L-amino acid oxidase in, 209, 213 arginase from, 357-358, 368-374

947

arginine-synthesizing system from, 357, 358-359 condensing enzyme, 360-362 splitting enzyme, 365-367 ATPase in extract of, 653 from mitochondria of, 593-595 d-biotin oxidase in slices of, 631-632 boiled extract of, 506 carnosinase in, 94 catalase from, 765, 775-781 citrovorum factor formation from 10formylfolic acid by enzyme from, 629 citrulline-synthesizing system from, 350-355 CoA formation in extract of, 633 cystathionine cleavage enzyme from, 311-314 cysteine desulfhydrase from, 315-318 decarboxylases for various amino acids in, 199 dehydropeptidases in, 114 changes of in malignancy, 114 dephospho-CoA kinase from, 649-651 dephospho-CoA pyrophosphorylase from, 667-669 desulfhydrase activity of, 313 dopa decarboxylase from, 199 DPN kinase from, 652-655 DPN pyrophosphorylase from, 671672 DPNasc in, 653 exoeysiine desulfhydrase from, 319 folio acid eonjugase in, 629 fruetose-l,6-diphosphatase from, 543546 fumarylaeetoaeetate hydrolasc from, 293-294, 297, 298-300 glueose-6-phosphatase from, 541-543 L-glutamie dehydrogenase from, 220225 glutamic-oxalacetic transaminase activity of, 184 L-glutaminase in, 382 glutamine synthetase from, 339, 341342 7-glutamyltransferase (Mn-dependent) from, 271 glutathione reduetase from, 724, 725

948

SUBJECT I N D E X

glutathione-synthesizing system from, 342-346 glycolytic enzymes in, 343 guanase from, 480-482 guanidinoacetate methylpherase from, 260-263 hippuric acid synthesis by enzyme from, 346-350 histidase in, 229 homocysteine desulfhydrase from, 318 homogeniisate oxidase from, 292-295 inorganic pyrophosphatase in, 362 a-keto acid-a-amidase from, 382, 384385 kynureninase from, 249-253 kynurcnine formamidase (formylase) from, 246-249 maleylaeetoacetate isomerase from, 293-294, 295-298 metaphosphatases from, 579 methionine-activating enzyme (MAE) from, 254-256 myokinase in, 362 nicotinamide methylpherase from, 257260 nicotinamide riboside phosphorylase from, 453, 454-456 nitroaryl reductase in, 406 nucleoside phosphorylase from, 453 nucleotide pyrophosphatase in, 653 nucleotide synthesis from R-5-P and adenine in extracts of, 501-504 nucleotide synthesis de novo in extracts of, 504-519 organic nitrate reductase from, 403-406 oxidative phosphorylation in mitochondria from, 614 pantetheine kinase from, 633-636 peroxidase from, 791-794 rhodancse from, 334-337 RNase in, 436 synthesis of formylglycinamide ribotide and glycinamide ribotide in extracts of, 505, 509-512 tetrahydrofolate formylase in, 517 thymidine phosphorylase from, 453 as TPN source, 700 TPNH cytochrome c reductase from, 704-706

transformylation of active "1-C u n i t " to form I M P in extracts of, 505 transhydrogenase in, 687 L-tryptophan peroxidase from, 242-246 uricase from, 489 urocanase in, 232 Liver fraction L, folic acid and CF content of, 630 effect of folic acid conjugase on, 630 Luciferase, bacterial (Achromobacter flscheri), 857861 identification as flavoprotein, 857, 860-861 kinetics of, 861 from Cypridina, 851-853 from firefly, 851, 854-856 assay of, 851 CoA assay with, 651 crystallization of, 856 Luciferin (LH~), from Cypridina, preparation of, 853 from firefly, absorption maxima of, 855 CoA assay with, 651 competition of with bcnzimidazole and benztriazole, 856 fluorescence of, exciting wave length for, 855 preparation of, 854-855 role in luciferase system, 856 Lumiflavin, inhibition of flavokinase by, 645 molecular weight of new yellow enzyme by determination of, 718 Lung, antifibrinolysin from, 165 fibrinolysokinase from, 166 prolidase in, 105 thromboplastin from, 139, 148-149 Lymphosarcoma, I N H sensitivity of DPNase from mouse, 663

L-Lysinamide, hydrolysis by leucine aminopeptidase, 92

Lysine, activation of DNase by, 442 content in horseradish pcroxidase, 809

SUBJECT INDEX

Lysine decarboxylase, from Bact. cadaveris or E. coli, 188189 resolution of, 189 in measurement of transaminase reactions, 171 L-Lysine ethyl ester, as substrate for trypsin, 36 *Lysozyme, extraction of B. subtilis with, 421 of M. Lysodeikticus with, 786 M

MacFadyen's reagent, use of in RNase assay, 428 Maclura pomifera Raf., see Osage orange Magnesium chloride, activation of RNase by high concentrations of, 433-434 in conversion of aminoimidazolecarboxamide riboside to ribotide, 515 in enzymatic assay of UDPG, 676 in isocitric dehydrogenase system, 682 in oxidative phosphorylation, 611 in synthesis of glycinamide ribotide, 510 of inosinic acid (IMP), 506 Magnesium hydroxide, preparation of and use in purification of prothrombin, 143, 144 Magnesium ion, activation of acetyl phosphatase by, 556 of adenosine kinase by, 500 of adenylate kinase by, 599, 603 of alkaline phosphatase by, 533, 538 of amino acid amidase by, 399 of ATPase of actomyosin by, 587,589 of insect muscle by, 598 of liver mitochondria by: 595 in muscle particles by, 588-591 of ATP-creatine transphosphorylase by, 610 relation of amount required ~o nucleotide concentrations, 610 of bone phosphatase by, 540 of eitrullinase by, 377, 378

949

of citrulline synthesis by, 355 of dephospho-CoA kinase by, 649, 651 of dephospho-CoA pyrophosphorylase by, 667, 669 of dihydroSrotic dehydrogenase by, 496 of DNase (pancreatic) by, 442 relation to DNA concentration, 442 of DPN kinase by, 652, 654 of DPN pyrophosphorylase by~ 672 of FAD pyrophosphorylase by, 675 of firefly luciferase by, 651,856 of flavokinase by, 645 of fructose diphosphatase by, 546 of glutamine synthetase by, 341 of 7-glutamyltransferases by, 266, 272 of inorganic pyrophosphatase by, 575 antagonism by calcium, eobaltous, and manganous ions, 575 of lactonizing enzyme for cis,cismuconic acid by, 284 of latent inorganic pyrophosphatase by, 669 of leucine aminopeptidase by, 88, 93 time required fol:, 88, 93 of metaphosphatase by, 579 of " 5 " nucleotidase of seminal plasma by, 549 of oxidative phosphorylation system by, 615 of pantetheine kinase by, 633, 635 of pantothenate-synthesizing enzyme by, 621 of system for nucleotide synthesis by, 504 of tetrahydrofolate formylase by, 517 of thiaminokinase by, 636, 640 in assay system for hippuric acid synthesis, 348, 349 complex of with ATP, 604 effect on adenylate kinase equilibrium, 604 on apurinic acid, 441

950

SLrBJECT I N D E X

on triphosphatases, 581 inhibition of alkaline phosphatase (crude) by, 530, 535 of arginine desimidase by, 376 of ATPase (myosin) by, 587 of DNase (pancreatic) by high concentrations of, 442 of D P N H cytochrome c reductase by, 692 of RNase by low concentrations of, 433, 434 requirement for in methionine-activating reaction, 254, 256 Magnesium oligonucleotide, as substrate for phosphodiesterase and 5r-nucleotidase, 561 Magnesium sulfate, in pyridoxal kinase assay, 646 Magnetic susceptibility, of horseradish peroxidase compounds, 811, 813 Malate, as hydrogen donor in nitrogen gas formation, 420, 422 DPN as cofactor for, 422 1): 0 ratio during oxidation of, 613, 616 Maleate, solubilizing effect of on manganous ion, 368, 371-372 Maleic acid, dissociation constant of, 372 Maleylacetoacetate, conversion to fumarylacetoacetate, 295-298 extinction coefficients of, 296 oxidation of homogentisic acid to, 292-295 Maleylacetoacetate isomerase, from liver, 293-294, 295-298 separation from fumarylacetoacetate hydrolase, 297 from homogentisate oxidase, 293294 Mal0nate, blocking of succinate step in oxidative phosphorylation by, 511, 616 inhibition of aspartase by, 390 of d-biotin oxidation by, 632 of cytochrome c degradation by, 169

as product of barbiturase action, 492 Mandelic acid, inhibition of L-amino acid oxidase by, 2O8 Mandelic acid-C140OH, racemization of, 277 L(4-)-Mandelic acid dehydrogenase, from Ps. fluorescens, 273, 274, 277-278 assay of mandelic acid racemase with, 276 Mandelic acid racemase, from Ps. fluorescens, 273, 274, 276-277 Manganous chloride, removal of nucleic acid by, 303 Manganous ion, activation of adenosine kinase by, 500 of alkaline phosphatase by, 538 of amino acid amidase by, 397, 399 of arginase by preincubation with, 369 of ATPase of muscle particles by, 590 of ATP-creatine transphosphorylase by, 610 of carnosinase by, 94, 95, 96 time required for, 94, 95 of citrullinase by, 378 of DPN kinase by, 654 of DPN pyrophosphorylase by, 672 of flavokinase by, 645 of fructose diphosphatase (purified) by, 546 of glutamine synthetase by, 341 of ~,-glutamyltransferases by, 266, 272 of glycylglycine dipeptidase by, 107, 109 of glycyl-L-leucine dipeptidase by, 105, 106, 107 time course of, 106, 107 of iminodipeptidase by, 100 of inorganic pyrophosphatase by, 575 of lactonizing enzyme for cis,cismuconic acid by, 283, 284 of leucine aminopeptidase by, 88, 93 time required for, 88, 93 of luciferase (firefly) by, 856 of metaphosphatase by, 579

SUBJECT INDEX

of pantetheine kinase by, 635 of pantothenate-synthesizing enzyme by, 621 of prolidase by, 100-101, 103, 105 mechanism for, 105 time required for, 101, 103 of thiaminokinase by, 640 binding agents for, 100 effect on amino acid amides, 398 on pH optimum of carnosinase, 96 inhibition of condensing system for arginine synthesis by, 364 of DPNH cytoehrome c reductase by, 692 of fructose diphosphatase (crude) by, 546 of RNase by, 433 of thiaminase by, 625 of uricase by, 489 solubilizing effect of maleate on, 368, 371-372 stabilization of carnosinase by, 94, 96 of iminodipeptidase by, 98, 100 Mannitol hexanitrate, as uncoupling agent, 615 Mannose, presence in fibrinogen and fibrin, 160 Marsilid, see 1-Isonicotinyl-2-isopropylhydrazine Mast cells, heparin cofactor from, 163 Maya plant (Bromelia pinguin), protease (pinguinain) from fruit of, 56, 63 Melanin, formation of by cytochrome system, 828 by mammalian tyrosinase, 827-831 Melanocytes, histochemical assay of tyrosinase in, 828-829, 830 cytoplasmic location of, 830 radioactive tyrosine method for assay of tyrosinase in, 829-830 Melanoma, tyrosinase from, 828-831 Menadione reductase, 2-methyl-l,4-naphthoquinoneas electron acceptor for, 728

951

Mercapto compounds, see also under names of individual compounds, activation and stabilization of tryptophanase by, 241 ~-Mercaptoethanol, activation of cathepsin C by, 68 ~-Mercaptovaline, see Penicillam~ne Mercurials, organic, inhibition of citrullinase by, 378 p-Mercurichlorobenzoate, see p-Chloromercuribenzoate Mercuric ion, inhibition of aminotripeptidase by, 87 of 5'-AMP deaminase by, 472 of arginine desimidase by, 376 of aspartase by, 388 of citrullinase by, 378 of DPNH cytochrome c reductase by 692 of glutanfic dehydrogenase by, 224 of homogentisate oxidase by, 2.(t5 reversal by mercaptoacetic acid, 295 of p-hydroxyphenylpyruvate enolketo tautomerase by, 292 reversal by cysteine, 292 of leucine aminopeptidase by, 93 of metaphosphatase by, 579 of myosin ATPase by 587 of prolidase by, 105 of RNase by, 434 Mercuripapain, crystallization of, 59-61 Merelrix meretrix (clam), thiaminase from viscera of, 622-626 Merthiolate, as preservative for phosphodiesterase, 564 Mescaline, amine oxidase action on, 393 Mesidine, peroxidase assay with, 774 Mesoporphyrin, formation of from cytochrome c, 753 Metal-chelating substances, activation of D-amino acid oxidase by, 202 effect of on ascorbic acid oxidase, 835 inhibition of alkaline phosphatase by, 538

952

SUBJECT INDEX

Metallic ions, see also under names of individual metals, inhibition of dehydroascorbic reductase by, 849 Metalloenzymes, see under names of individual metals Metals.heavy, see also under names of individual metals, in colorimetric determination of citrulline, 351, 359 Metaphosphatase (s), 577-580 assay procedure for low molecular weight substrates, 577 for high molecular weight substrates, 578 change in pH optimum with source and substrate, 579-580 molecular weight of A. niger enzyme, 580 occurrence of, 577, 579 Metaphosphates, inhibition of ~-glutamyltransferase (brain) by, 272 Metaphosphates, high molecular weight, depolymerization of by metaphosphatase, 577, 578 assay by viscosity change, 578 Metaphosphates, low molecular weight, orthophosphate formation from by metaphosphatase, 577-578 Methanol, activation of ~-glutamyltransferase (bacterial) by, 269 Methemoglobin, hemoglobin conversion to, 739 Methionine, cleavage of, 313 conversion to S-adenosylmethionine, 254 stimulation of tryptophan synthetase formation by, 237 as substrate for L-amino acid oxidase, 208, 211 Methionine-activating enzyme, from rabbit liver, 254-256 separation of into two protein fractions, 256 Methionine sulfoxide, inhibition of glutamine synthetase by, 342

Methyl acceptor systems, 257-263 guanidinoacetate methylpherase, 260-263 nicotinamide methylpherase, 257-260 S-Methylcysteine, cleavage of, 313 5-Methylcytosine, as acceptor of deoxyriboside group, 468 RI values for, 466 Methyl hydrogen peroxide (MeOOH), for peroxidase assay (direct) in yeast cells, 769 for peroxidase assay with guaiacol, 792 as substrate for horseradish peroxidase, 897, 810, 811 see also Enzyme-substrate complexes 2-Methyl-l,4-napthoquinone, as electron acceptor for menadione reductase, 728 Methylene blue (MB), copper salts in commercial preparations of, 716 as electron acceptor for D-amino acid oxidase, 204 for bacterial luciferase system, 860 for cytochrome bl, 746-748 for diaphorase system, 707 for hydrogenase systems, 862, 865, 868, 869 FAD requirement for, 869 for new yellow enzyme, 715, 716, 718 for old yellow enzyme, 715 for xanthine oxidase, 482 formation of from sulfide and p-aminodimethylaniline, 315 Methylene blue, leuco form, oxidation of by molecular oxygen, 715, 716, 748 catalysis by copper salts, 716 Methylene blue-sulfate ester, in assay of alkylsulfatases, 330 a-Methylglutamic acid, as substrate for glutamine synthetase, 341 a-Methylhistamine (imidazoleisopropylamine), resistance to histaminase, 396

SUBJECT INDEX

6-Methylindole, as substrate for tryptophan synthetase, 236 N-Methyl-L-leucine, as substrate for ~-amino acid oxidase, 211 N 1-Methylnicotinamide (NMeN), determination of, 257 synthesis of by nicotinamide methylpherase, 257 3-Methyl-4,6,4'-triaminodiphenyl sulfone, use in preparation of thrombin, 157 6-Methyltryptophan, as substrate for tryptophanase, 242 Mexicain, protease from latex of cuaguayote, 56, 62 crystallization of, 62 Mickle electric shaker, 468, 495 Micrococcus spp., hydrogenase in, 870 Micrococcus aureus, aspartase in, 388 Micrococcus lysodeikticus, catalase from, 775, 784-788 crystallization of, 787 growth and lysis of, 786 Microsomes, AMPase in, 542 ATPase in, 542 glucose-6-phosphatase in, 542 glucose-6-phosphatase as indicator for presence of, 542-543 glycerophosphatase in, 542 hexose isomerase in, 542 Milk, alkaline phosphatase from~ 533-539 peroxidase (lactoperoxidase) from, 245~ 813-817 xanthine oxidase from, 482-485 Milk clotting, assay of rennin by, 69 effect of CaCI~ in, 69 assay of various proteinases by, 56-59 Milkweed (Asclepias speciosa, mexicana, syriaca), crystalline protease (asclepain) from latex of, 56, 61-62

953

Mitochondria, adenylate kinase (myokinase) in muscle and liver, 602 ATPase in insect muscle, 595 ATPase from mouse liver, 593-595 low activity of in intact, 615 disintegration of, 594 DPNH cytochrome c reductase activity in, 693 glucose-6-phosphatase content of, variation with method of preparation, 543 hippuric acid-forming enzymes in, 348 oxidative phosphorylation in, 610-616 oxidative phosphorylation in modified, 615, 616 from poky strain of Neurospora, 168 uricase from liver, 489 Moccasin venom (A gkistrodon piscivorus ) , ~amino acid oxidasc from, 205-209 Molds, metaphosphatase in, 577 Molecular weight determinations, deviations due to use of different ultracentrifuges, 808 Mollusks, see Patella vulgata and Littorina littorea Molybdate, inhibition of glueose-6-phosphatase by, 542 Molybdenum, as constituent of nitrate reductase, 415 cyanide in removal of, 415 requirement of for reduction of cytochrome c by hydrogenase system, 869 Monoamine oxidase, from liver, 393 Monoesterase, see Phosphatase Monomethylamine, as product of ssrcosine oxidation, 225 N-Monomethylglycine, see Sarcosine Mononucleotides, see also Nucleotides, and under names of individual nucleotides, alkyl esters of as substrates for intestinal di'esterase, 570 for spleen phosphodiesterase, 568 inhibition of RNase by, 428, 434

954

SUBJECT INDEX

2'-Mononucleotides, formation from cyclic structures by spleen phosphodiesterase, 568 3'-Mononucleotides, formation from diesters by spleen enzyme, 568 from diesters or cyclic structures by intestinal diesterase, 570 5'-Mononucleotides, formation from ribo- and deoxyribopolynucleotides by venom diesterase, 561 Monophenolase (cresolase), induction period for, 825, 827 effects of various agents on length of, 825 Monophenols, see Phenols Monophenylphosphate, alkaline phosphatase action on, 533, 534, 538, 539 as donor for transphosphorylation, 539, 556 formation of from sym-diphenylpyrophosphate, 565 prostatic phosphatase action on, 524 kinetics of, 525 Morphine, effect of on renin action, 134 cic,cis-Muconic acid, absorption spectrum of, 282 formation of by pyrocatechase, 281282 lactonizing enzyme for, 273, 282-284 Muscle, acetyl phosphatase from, 555-556 adenylate kinase (myokinase) from, 598-604 5'-adenylic acid deaminase from, 469473 apyrase (ATPase), Mg-activated, from insect, 590-591 ATPase from insect, 595-598 ATPase, Mg-activated, from particles of, 588-591 ATPase (myosin) from, 582-588 ATP-creatine transphosphorylase from, 605-610 L-a-glycerophosphate dehydrogenase in insoluble particles from, 559 glycolyzing enzymes from, 351, 358

glycylglycine dipeptidase from, 108 nucleotide synthesis from R-5-P and adenine in extracts of, 501-504 prolidase in, 105 protein antithromboplastin from, 161-162 transhydrogenase in pigeon breast and rabbit, 687 triphosphatase in, 580, 581 "Muscle adenylic acid," see 5'Adenylic acid Muscle enzyme fraction, ATP generation with phosphoglyccrate and, 515 Mushroom, tyrosinase (polyphenol oxidase) from, 822-827 Mussel, thiaminase in, 625

Mycobacterium, aromatic oxidations in, 273 barbiturase from, 492-493 uracil-thymine oxidase from, 490-491 growth of cells for, 490-491 Mycobacterium tuberculosis, strain BCG, eytochrome e peroxidase in, 764 Myeloperoxidase (verdoperoxidase), see Peroxidase from leucocytes Myoglobin, test for in cytochrome c preparations, 751-752 Myokinase, see also Adenylate kinase, in adenosine kinase assay, 497 in arginine condensing system from liver, 362 assay of ADP with hexokinase and, 497 Myosin, ATPase activity of, 582 as specialized function of myosin molecule, 586 triphosphatase in, 580 L-Myosin, impurities in "crystalline" myosin and, 583-584 Myrosulfatases, 324 N

Naphthoquinones, reduction of by DPNH, 728

SUBJECT INDEX

a-Naphthylamine, nitrite assay with, 400 a-Naphthylethylenediamine, nitroaniline assay with, 407 N-(1-Naphthyl)ethylenediamine, nitrite determination with, 403, 412 Narcotics, inhibition of prostatic phosphatase by, 527 Nasturtium (Tropaeolum majus), carbonic anhydrase in leaf of, 842 Nembutal, effect on renin action, 134 Neotetrazolium, in assay of flavoproteins, 695 Nessler method, interference with by caprylic alcohol, 111 Nessler reagent, of Vanselow, 596

Neurospora, L-amino acid oxidase in, 211 cytochrome c destroying system in poky strain of, 168 cytochrome e peroxidase in poky strain of, 764 DPNase from, 664-666 effect of deficiencies on enzyme concentration, 666 growth medium for, Zn-deficient, 664 growth of, 168, 235, 401,417 hydroxylamine reductase from, 416419 kynureninase in, 253 kynurenine formamidase in, 253 nitrate reductase from, 411-415 nitroaryl reductase from, 406-411 independence of nitrogen source, 409, 411 nitroethane oxidase from, 400-402 D-serine (D-threonine) dehydrase from, 322-324 L-serine (L-threonine) dehydrase from, 319-322 tryptophan synthetase from, 233-238 Nickel(ous) ion, activation of arginine desimidase by, 376 inhibition of aspartase by, 388 of DNase by, 442

955

of fructose diphosphatase by, 546 of RNase by, 434 of uricase by, 489 Nicotinamide, formation of by animal tissue DPNase, 660 by Neurospora DPNase, 664 by nicotinamide riboside phosphorylase, 454 inhibition of animal tissue DPNase (DPN nucleosidase) by, 662, 670, 683 of Neurospora DPNase by, 666 methylation of, 257 Nicotinamide, C14-1abeled, exchange with bound nicotinamide of DPN, 662 Nicotinamide methylpherase, from rat liver, 257-260 5'-Nicotinamide mononucleotide (NMN), DPN formation from, 670 formation by nucleotide pyrophosphatase, 655 "5" nucleotidase action on, 549, 550 transhydrogenase reaction with DPNH or TPNH, 685 Nieotinamide mononucleotide, reduced (NMNH), DPNH synthesis from, 672 formation from DPNH by snake venom enzyme, 654 Nicotinamide riboside (NR), fluorimetric method for, 454 nucleosidase action on, 463 synthesis of by purine nucleoside phosphorylase, 448 Nicotinamide riboside phosphorylase, from beef liver, 453, 454-456 possible identity with nucleoside phosphorylase, 448, 453, 455 Ninhydrin, inhibition of insect muscle ATPase by, 598 of RNase by, 434 Ninhydrin-CO~ analysis, Van Slyke, assay of aminoacylases by, 115 Ninhydrin method, colorimetric, assay of carboxypeptidase by, 79 of glycylglyeine dipeptidase by, 107

956

SUBJECT I N D E X

Nitrate, inhibition of erythrocyte carbonic anhydrase by, 845 of glucose oxidase of A. niger by, 579 of hydrogenase exchange reaction by, 867 reduction of by hydrogenase system, 862, 867 stoichiometry of, 867 requirement of in growth medium for production of nitrogen gas-forming enzymes, 423 Nitrate esters, reduction of by eysteine, non-enzymatic, 405 by glutathione, 403-406 enzymatic, 403-406 non-enzymatic, 405, 406 Nitrate reductase, as adaptive enzyme, 415 mechanism of action of, 415 from Neurospora, 411-415 in soybean leaves, 414-415 comparison of TPNH and DPNH in, 414, 415 turnover number of, 413 Nitrate reductase, organic, from hog liver, 403-406 Nitric oxide gas, alkaline sulfite as trapping agent for, 420, 422 conversion to nitrogen dioxide by oxygen, 421 formation from nitrite and conversion to nitrogen gas, 420-423 inhibition of hydrogenase by, 867 Nitrite, determination of, 403 enzymatic conversion to nitric oxide and to nitrogen gas, 420-423 formation of by nitrate reductase, 411 by nitroethane oxidase, 400 measurement of, 400 by organic nitrate reductase, 403, 405 hemoglobin oxidation by, 739 inhibition of hydrogenase exchange reaction by, 867 oxidation of by peroxidase complex II, 810

as uncoupling agent, 615 Nitrite reductase, as adaptive enzyme, 411 presence in hydroxylamine reductase preparations, 418 in nitrate reductase preparations, 412 cyanide inhibition of, 412 in nitroaryl reductase preparations, 410, 411 m-Nitroaniline, formation from m-dinitrobenzene, 406 Nitroaryl reductase, distribution of, 409 in liver, 406 from Neurospora crassa, 406-411 in pig heart, 406 p-Nitrobenzoic acid, reduction by Neurospora, 410 4-Nitrocatechol, competitive inhibition of tyrosinase by, 826 Nitroethane oxidase, from Neurospora crassa, 400-402 Nitrofurans, as electron acceptors for DPNH cytochrome c reductase of bacteria, 697 Nitrogen, effect of deficiency of on Neurospora DPNase content, 666 Nitrogen dioxide, formation from nitrogen oxide, 421 Nitrogen gas, enzymatic formation of from nitrite and nitric oxide, 420-423 adaptive nature of enzymes for, 423 particulate nature of reducing system for, 422 Nitroglycerine (glycerol trinitrate), as substrate for organic nitrate reductase, 403, 405 Nitrophenol(s), inhibition of quinone reductase by, 729 p-Nitrophenol (4-Nitrophenol), competitive inhibition of tyrosinase by, 826 liberation from bis (p-nitrophenyl) phosphate by phosphodiesterase, 561

SUBJECT INDEX

m-Nitrophenylhydroxylamine, formation from m-dinitrobenzene, 406 p-Nitrophenyl phosphate, as substrate for alkaline phosphatase, 533 Nitrophenylphosphates, as possible substrates for clinical determination of phosphatase, 528 bis(p-Nitrophenyl) phosphate, as substrate for phosphodiesterase, 561 Nitropropane, action of nitroethane oxidase on 1- and 2-isomers of, 402 f3-Nitropropionic acid, action of nitroethane oxidase on, 402 Nitroprusside reaction, g|utathione measurement with, 719 Nonprotein nitrogen (NPN), method for assay of proteinases, 58 Norbiotin, inhibition of d-biotin oxidation by, 632 DL-Norleucinamide, hydrolysis by leucine aminopeptidase, 92 L-Norleucine, as substrate for L-amino acid oxidase, 208 transaminase for, 176 DL-Norvalinamide, hydrolysis by leucine aminopeptidase, 92 L-Norvaline, as substrate for x-amino acid oxidase, 208 transaminase for, 176 *Notatin, see Glucose oxidase Nucleic acid(s), enzymes for metabolism of, 427-519 inhibition of acetyl phosphatase by, 556 precipitation of aspartase as complex with, 387 protein fractionation with, 340, 498 Nucleosidases, hydrolytic, from baker's yeast, 461-464 purine nucleosidase, 462-464 uridine nucleosidase, 461-462 evidence for separate purine and pyrimidine nucleosedases, 460 from L. pentosus, 456-461

957

mechanism of action of, 460 Nucleoside monophosphate kinases, in yeast and animal tissues, 603 Nucleoside phosphorylase, purine, see also Thymidine phosphorylase, from beef liver, 453 from calf spleen, 448-453 differential spectrophotometry of purine compounds with, 449 from yeast, 453 Nucleoside transdeoxyribosidase, from bacteria, 464-468 from Lactobacillus helveticus, 467, 468 sources of, 467 Nucleoside triphosphates, hydrolysis of by myosin, 586 3'-Nucleotidase (3'-nucleotide phosphatase), hydrolysis of di(dinitrophenyl) phosphate by barley enzyme, 524 from rye grass, 551-555 factor responsible for heat lability of, 554-555 5'-Nucleotidase, see also Adenosine-5phosphatase, apparent activation of diesterase by, 561 from potato, 550 from seminal plasma, 547-549 from snake venom, 549-550, 561 Nucleotide(s), see also Mononucleotides and Dinucleotides, bone phosphatase action on, 539 inhibition of 3'-nucleotidase by 2'- and 5'-, 554 as substrate for prostatic phosphatase, 524 synthesis from R-5-P and adenine, 501504 enzyme for phosphorylating R-5-P, 502 heat lability of, 504 nucleotide-forming enzyme, 502 heat stability of, 504 Nucleotide (s), purine, de novo synthesis of, 504-519 activation of "1-carbon unit" as folic acid derivative in pigeon liver extract, 516-518

958

SUBJECT INDEX

conversion of 5-amino-4-imidazoleearboxamide riboside to ribotide by yeast enzyme, 514-516 isolation of 5-amino-4-imidazoleearboxamide from E. coli cultures, 512-514 over-all system for in pigeon liver extracts, 505-509 synthesis of formylglycinamide ribotide and glycinamide ribotide in pigeon liver extracts, 509-512 tentative scheme for, 504-505 transformylation of "1-carbon u n i t " to form IMP, 518-519 Nucleotide pyrophosphatase, ATP inhibition of FAD hydrolysis by, 673 as contaminant of FAD pyrophosphorylase, 675 D P N H cleavage by snake venom enzyme, 654 in pigeon liver, 653 from potato, 656-659 other phosphatase activities in, 659 variation with age and variety, 658 Nncleotide transhydrogenase, see also Pyridine nucleotide transhydrogenase, in A. aerogenes, 308 role in coupling shikimic acid reduction with quinic acid oxidation, 309, 310 equilibrium constant for, 311 O Oat seedlings, glutamic-oxalacetic transaminase activity of, 184 Octanoyl phosphate, hydrolysis of, 556 Oligonucleetide, see Deoxyoligonucleotide, Magnesium oligonucleotide "One-carbon unit" (" 1-C unit"), in final step of I M P synthesis, 505, 518-519 in formylation of glycinamide ribotide, 505 Organic nitrate reductase, see Nitrate reductase, organic

~-Ornithine, citrulline synthesis from, 350-355 colorimetric assay for p-amino benzoyl derivatives of, 350 colorimetric method for, 376 formation of by arginase, 356, 368 by citrullinase, 374 inhibition of citrullinase by, 378 oxidation of by L-amino acid oxidase, 208 reduction of, 217, 219, 220 stabilization of arginase by, 373 Ornithine decarboxylase, citrullinase and, 378 from Cl. septicum, 189 resolution of, 189 measurement of transaminase reactions with, 171 Orotic acid, incorporation of into nucleotides, 504 reduction of by DPNH, 493 loss of ultraviolet absorption during, 493 Ortho-para hydrogen conversion, catalysis by hydrogenase, 862, 864865 Orthophosphate, see Phosphate, inorganic Orthophosphomonoesters, as substrates for alkaline phosphatase, 533, 538 Osage orange (Maclura pomifera Raf.), protease (pomiferin) from latex of, 57, 64 Ovalbumin, conversion of component A1 to As by prostatic phosphatase, 526 Ovomucoid, as trypsin inhibitor, 37, 49, 51, 53 Oxalacetate, as component of transaminase system, 170-171 measurement of formation of, 171, 174-175, 179-182 by decarboxylation with aniline citrate, 171, 174-175 by determination of pyruvic acid after decarboxy|atiou, 171, 175 by spectrophotometric method, 171, 175, 179-182

SUBJECT INDEX

non-enzymatic decarboxylation of, 180 P: 0 ratio during oxidation of, 613 *0xalaeetic decarboxylation system, COs versus ttCOa- as product of, 841 Oxalate, activation of prothrombin by, 145 inhibition of acetyl phosphatase by, 556 of aspartase by, 388, 390 Oxidases, see under names of substrates for individual oxidases Oxidation-reduction indicators, see Dyes Oxidative phosphorylation, see Phosphorylation, oxidative Oxy-acid buffers, catalysis of COs hydration by, 836 Oxygen, catalase assay by manometric determination of, 769 as electron acceptor for DPNH cytochrome c reductase of bacteria, 697 for new yellow enzyme, 716, 718 for old yellow enzyme, 715, 716 for TPNH cytochrome c reductase system of yeast, 703 inhibition of amino acid reductase by, 219 of hydrogenases by, 866, 868, 869 mechanism of, 866 reactivation by hydrogen or other reducing substances, 866, 868 reduction by hydrogenase system, 862 requirement for, in firefly luciferase system, 651 1a Palmityl phosphate, hydrolysis of; 556 Pancreas,

carbonic anhydrase in, 841 carboxypeptidase and procarboxypeptidase from, 77-83 chymotrypsinogen and chymotrypsins from, 8-26 cysteine desulfhydrase in, 318 DNase from, 438-443 folic acid conjugase from, 629-630 RNase from, 427-436

959

trypsin and trypsinogen from, 26-36 trypsin inhibitor of Kazal from, 36, 40, 50, 51, 52 of Kunitz and Northrop from, 36, 38-40, 50, 51, 52 Pantetheine kinase, from pigeon liver, 633-636 Pantoate, enzymatic conversion to pantothenate, 619 Pantothenate-synthesizing enzyme, from E. coli, 619-622 assay of by chemical method, 622 by manometric method, 622 by microbiological method, 619 Papa[n, protease from latex of papaya, 56, 59, 60 activation of by cyanide, 59 by cysteine, 59 crystallization of, 59, 60 inactivation of penicillinase by, 123 use as meat tenderizer, 55 Papaya (Carica papaya), latex of as source of chymopapain, 56, 61 of papain, 56, 59, 60

Paphia philippinarum, thiaminase in, 625 Parahydrogen, conversion to normal hydrogen by hydrogenase, 864-865 Parsley root, glutathione reduetase in, 721 Patella vulgata (mollusk), arylsulfatase in, 332 steroid sulfatase in, 332 Peanut (Arachis hypogen), protease (arachain) from, 57, 63 Pea plant (Pisum sativum), dehydroaseorbic reductase from, 847850 distribution in plant and changes in development, 849 Peas (Pea seeds), 5-dehydroshikimie reductase and 5-dehydroquinase in, 304, 307 glutamine synthetase from, 337-342 ~-glutamyltransferase from, 263-266 pyridine nucleotide quinone reductase from, 725-729

960

SUBJECT INDEX

Penicillamine (/~-mercaptovaline), inhibition of penicillinase by, 123 Penicillin, as antibiotic in tyrosinase assay, 830 assay of, 120-121 Staph. aureus in, 121 inhibition of RNase by, 434 Penicillinase, from B. cereus, 120-124 as antigen in rabbits, 123 heat stability of, 123 Penicillium notatum,

aspartase in, 388 Penicillium sp.,

metaphosphatasc in, 577, 579 Penicilloic acid, as product of penicillinase reaction, 120 Pentacyanoammine ferroate, assay of m-nitrophenylhydroxylamine with, 406, 407 Pentothal, see Thiopental Pepsin, from commercial preparations of swine pepsin, 3-7 crystallization of, 5 inactivation of adenylate kinase by, 603 of RNase by, 434 inhibitor of from pepsinogen, 7 sources of, 7 Pepsinogen, from swine mucosa, 3-7 crystallization of, 6 from various species, 7 Peptidases, see Aminoacylases, Aminopeptidases, Carboxypeptidases, Dehydropeptidases, Dipeptidases Peptides, formation of by cathepsin C, 68 formation from fibrinogcn, 160 hydrolysis of, Grassmann and Heyde procedure for estimation of, 83, 88, 93-94, 97, 100, 105-106, 107-108 Peptides, synthetic, as substrates for proteolytic enzymes, 21 Peptone, protection of carbonic anhydrase by, 838, 846

Perborate, as oxidizing agent in catalase reaction, 78O Perchlorate, inhibition of D P N H cytochrome c reductase by, 692 Periodic acid, inhibition of RNase by, 434 Permanganate, assay of catalase by titration of H202 with, 768, 779, 781-782, 791-792 oxidation of a-ketoglutarate with, 170 Permutit, deaminase purification with, 476 separation of hydroxytyramine from dopa with, 195 Peroxidase(s), assay of, 769-775 by direct measurement of enzymesubstrate compound in intact cells, 769 by guaiacol test, 770-773, 792 by pyrogallol test, 773-775 by various tests, 774, 794 interpretation in terms of rate constants for formation and utilization of enzyme-substrate complex, 770, 775 from beef liver, 791-794 catalase association with, 793-794 conversion to inactive heme-protein, 794 distribution in plants, 801-802 from horseradish, 770, 801-813 absorption bands of, 803 activity, dissociation constants and spectra of derivatives of, 812 assay of, 803 chemical analysis of, 809, 811 compounds (complexes) formed with oxidizing agents~ 807 with substrates, 770, 801-813 see also Enzyme-substrate complexes with various reagents, 812 crystallization of, 806-807 nitrogen content of, 799 oxidation reduction potential of, 809 physical properties of, 808-809

SUBJECT INDEX

rate constant for formation of complex I, 773 reaction mechanism for, 770, 802 formation of complexes I and II, 8O2 of complex III, 803 RZ unit as measure of purity of, 803 solubility curve described for, 806 specificity of, 807-808 spectroscopic, magnetic and kinetic data on, 810, 811-813 titration curves for, 809 from leucocytes (myeloperoxidase, MyPO) (verdoperoxidase), 794801 assay of purity by activity measurements (uric acid method), 794796 by spectrophotometric measurements, 801 physiological role of in detoxication, 800, 801 preparation from empyema, 797-798 from ox leucocytes, 796-797 properties of, 798-799, 801 rate constant for formation of complex I, 773 sources of, 794 from milk (lactoperoxidase), 813-817 assay of, 813 crystallization of, 816-817 extinction coefficients for, 817 isoelectric point of, 817 phosphate effect on, 816, 817 optical density ratios for assay of, 813 rate constant for formation of complex I, 773 turnover number of, 817 from yeast, rate constant for formation of complex I, 773 Peroxide, see also Hydrogen peroxide, Methyl peroxide, Ethyl peroxide, inhibition of homogentisate oxidase by, 295 o-Phenanthroline, activation of uricase by, 489 complex of with ferrous ions, 726

961

inhibition of DPNH cytochrome c reductase by, 693 of hydroxylamine reductase by, 419 of nitrate reductase by, 415 Phenol(s), detoxication of diphtheria toxin by myeloperoxidase and, 800 inhibition of squash carboxylase (a-keto acid) by, 192 liberation of from diphenyl phosphate by phosphodiesterase, 561,564 oxidation of by lactoperoxidase, 816 by peroxidase complex II, 808, 810 by tyrosinase, 825-826, 830 comparison of plant and mammalian enzymes in, 830 as precursor of catechol, 273 as product of transphosphorylation by phosphatases, 556 measurement of, 559 substituted, inhibition of TPNH cytochrome c reductase of yeast by, 703 Phenol color method, for assay of proteinases, 55-58 Phenolic ethereal sulfates, as substrates for arylsulfatases, 328 Phenolphthalein phosphate, in assay of serum phosphatase, 528 inhibitory effect of, 528 L-Phenylalaninamide, action of amidase on, 399 hydrolysis of by leucine aminopeptidase, 92 L-Phenylalanine, decarboxylase action on various hydroxy derivatives of, i97 decarboxylation of in mammalian tissues, 199 inhibition of D-amino acid oxidase by, 203 as product of aromatic biosynthesis, 300 stimulation of tryptophan synthetase formation by, 237 as substrate for L-amino acid oxidase, 208 transaminase for, 176 L-Phenylalanine ethyl ester, as substrate for trypsin and chymotrypsin, 23, 25

962

SUBJECT INDEX

use in manometric assay of chymotrypsin, 25 ~Phenylalanylhydroxy-L-proline, as substrate for prolidase, 104 Phenylarsine oxide, inhibition of aspartase by, 388 Phenylenediamine, cytochrome c reduction by p-form of, 754 oxidation of o- and p-forms of by peroxidase complex II, 810 L-Phenylglycolie acid, as substrate for i-amino acid oxidase, 211 Phenylhydrazine, inhibition of cysteine desulfhydrase by, 317 Phenylisocyanate, inhibition of RNase by, 434 L-Phenyllactic acid, as substrate for L-amino acid oxidase, 211 Phenyl phosphate, see Monophenyl phosphate f~-Phenylpropionic acid, inhibition of carboxypeptidase by, 79 Phenylpyruvate, enol-keto tautomerase action on, 291 Phosphatase, see also Phosphomonoesterase and Phosphodiesterase, effect on oxidative phosphorylation in tissue homogenates, 614 inhibition of by citrate and Versene, 313 of yeast enzyme by "pyrimidyl" and thiamine, 638 lung thromboplastin and, 150 rennin as, 77 Phosphate, inorganic (orthophosphate), activation of dehydroascorbic reductase by~ 849 of dopa peroxidation by, 816 competitive inhibition of by H20~, 816 of ~-glutamyltransferase by, 267, 272 of p-hydroxyphenylpyruvate enolketo tautomerase by, 292 of pantetheine kinase by, 635 of prothrombin by, 145 binding of by lactoperoxidase, 816

catalysis of COs hydration by, 836, 838 effect on thiaminokinase, 640 enzymes for metabolism of, 523-616 formation of by acid and alkaline phosphatases, 523-541 by apyrsse, 591 by ATPases, 582, 588, 593, 595 by fructose-l,6-diphosphatase, 543 by glucose-6-phosphatase, 541-543 by inorganic pyrophosphatase, 570 by metaphosphatase, 577, 578 by methionine-activating system, 254 by "5" nucleotidases, 546-550 by nucleotide pyrophosphatase action on ATP, 655 by nucleotide synthesizing system, 501 by triphosphatase, 580 inhibition of acetyl phosphatase by, 556 of alkaline phosphatase by, 473-475, 533, 538 of L-amino acid oxidase by, 208 of 5'-AMP deaminase by, 472 effect of 5'-AMP concentration on, 472 of arsenate-catalyzed citrullinase reaction by, 378 of arylsulfatases by, 328 of carnosinase by Mn ++ plus, 96 of D P N H cytochrome c reductase by, 692 of iminodipeptidase by, 100 of purine nucleosidases by, 460, 463-464 of xanthine oxidase by, 485 isobutanol extraction method in measurement of, 592 requirement of, for activation of glycyl-L-leucine dipeptidase by zinc, 107 for fluoride inhibition of nucleotide pyrophosphatase, 659 for reduction of cytochrome e by hydrogenase system, 870 role in nicotinamide riboside phosphorylase reaction, 454

SUBJECT X~DEX

in nueleoside phosphorylase reaction, 448 in Pseudomonas transhydrogenase reaction, 685 stabilization of pyrimidine nucleosidase by, 459 uptake of during citrullinase reaction, 376, 378 replacement of by arsenate, 377, 378 during oxidative phosphorylation, 610 Phosphatides, presence of in thromboplastin, 150 Phosphoamidase, rennin as, 77 Phosphoamides, as substrates for alkaline phosphatase, 533, 538 Phosphocholine, see Phosphorylcholine Phosphocreatine, determination of, 605 formation during oxidative phosphorylation, 613 phosphomonoesterase-eatalyzed transfer of phosphate to glucose from, 533 phosphorylation of ADP by, 605 as substrate for alkaline phosphatase, 533, 538 Phosphodiesterase (s), in bone, 540 separation from monoesterase, 540 from calf intestine, 569-570 RNase as, 433 from snake venom, 561-565 from spleen, 565-569 fractions free of activity against cyclic nueleotides, 568 Phosphoenolpyruvate, as substrate for alkaline phosphatase, 538 *Phosphoglucomutase, in assay of glucose-l-P, 675, 676 formation of R-I,5-P2 with, 503 *6-Phosphogluconate dehydrogenase, in glucose-6-phosphate assay, 676 removal by ethanol fractionation, 677 6-Phosphoglucose, see Glucose-6phosphate

963

3-Phosphoglyceric acid (3-PGA), regeneration of ATP by, in various biosynthetic processes, 343, 351, 356, 357, 506, 510, 5157 517 *Phosphohexoisomerase (hexose isomerase), in microsomes, 542 Phosphomonoesterases, group-specific, 523-541 sources of, 540 comparison with activity of bone enzyme, 540 substrate-specific, 541-555 transphosphorylation by acid and alkaline, 556-561 Phosphomonoesterase, acid, distribution of in animal tissues, 523 in erythrocytes, 527 differentiation from serum phosphatase of prostatic origin, 527528 in preputial glands of rats, 523 from prostate gland, 523-530 action of on high-molecular phosphorus compounds, 525-527 nucleic acids, 526 phosphoproteins, 526-527 dependence of pH optimum on substrate, 527 effect of hormones on enzyme concentration, 523 inhibitors of, 527-528 K~ values for, 527 limitations as analytical tool, 525, 526 purification of, 529-530 species variation in, 523 specificity of, 524-525 titration method for detecting diesterase action of, 525 in seminal vesicles of guinea pigs, 523 in serum, 524, 527, 528 ~tartrate inhibition as test for prostatic origin of, 528 Phosphomonoesterase, alkaline, from bone, 539-541 from intestine, 473-475, 530-533, 539 inhibition of by phosphate, 473-475 separation of from adenosine deaminase, 473-474

964

SUBJECT

from diesterase, 569-570 transferase activity of, 533 from milk, 533-539 transphosphorylation activity of, 539 from various sources, 539, 560 comparison of, 539 extraction of with n-butanol, 560 transphosphorylation by, 561 4~-Phosphopantetheine, conversion to CoA by pigeon liver extract, 633 to dephospho-CoA, 667, 669 formation by pantetheine kinase, 633 maintenance of in reduced form, 669 Phosphopeptone, as substrate for phosphatase action of rennin, 77 Phosphoproteins, see Casein, Ovalbumin Phosphopyruvate, role in adenosine kinase assay, 497 5-Phosphoribosyl pyrophosphate, as intermediate in nucleotide synthesis from adenine and R-5-P, 501 in nucleotide synthesis de nova, 504 Phosphorus 82, incorporation into ATP as measure of oxidative phosphorylation, 614 Phosphorus: oxygen ratio (P: O ratio), definition of, 612 table for value with different substrates, 613 Phosphorylation, oxidative, in homogenates and mitochondria, 610-616 assay systems for, 610-614 direct, 610-613 indirect, 614 protective effects of substrate, ATP and oxidative activity on, 612 P:O ratios with different substrates, 613 requirements for and lability of, 615 uncoupling phenomenon in, 615-616 in tissue slices, 614 Phosphoryl choline (phosphocholine), a s product of Cl. welchii lecithinase action, 590

INDEX

prostatic phosphatase action on, 525 high pH optimum for, 527 Pho sphorylethanolamine, prostatic phosphatase action on, 525 Phosphorylserine, prostatic phosphatase action on, 525 Phosphotransacetylase, in assay of dephospho-CoA kinase, 649 Phosphotungstic acid, color reaction for uric acid, 486 Photinus pyralis, see Firefly Pileus mexicanus, see Cuaguayote Pineapple (Anana sativa), protease (bromelin) from, 56, 62-63 Pinguinain, protease from fruit of maya plant, 56, 63 Pisum sativum, see Pea plant Pituitary, prolidase in, 105 Plants, see also under names of individual species, cytochrome a3 in, 732 triphosphatase in, 580 Plasma, Ac-globulin from, 151-152 amine oxidase from, 390-393 antihemophilic factor from, 149 antithrombin in defibrinated, 162 convertin from, 155-156 fibrinogen from, 158-160 preparation of from horse blood for assay of antihemophilic factor, 147 proconvertin from, 153-154 prothrombin-free, 140 use in prothrombin assay~ 140 prothrombin from~ 140-146 stabilization of carbonic anhydrase by, 846 thrombin from, 156-158 thromboplastin, heat-labile component of (AHF, PTC), in, 139 trypsin inhibitor from, 37, 49-54 Plasma Ac-globulin (proaccelerin, Factor V), activation of by thrombin, 157 complexes of with thromboplastin and convertin, 151

SUBJECT INDEX concentration of in bovine and human plasma, 152 in oxalated horse plasma, 147 preparation of serum Ac-globulin from, 153 use of thrombin for, 152, 153 purification of from ox blood, 151-152 role in clotting mechanism, 139, 150, 152 Plasma thromboplastin antecedent (PTA), adsorption of by BaS04, 150 role in clotting mechanism, 139 Plasma thromboplastin component (PTC), see Antihemophilic factor Plasmin, see Fibrinolysin Plasminogen, see Profibrinolysin Plastic matting (Neotex), use in collection o[ pancreatic juice, 81 Platelet factor (heat stable factor of thromboplastin), assay of, 147-148 purification of, 150 Platelet reagent, use in assay of antihemophilic factor, 148 Platelets, accelerin in, 139 lysis of by thrombin, 157 thromboplastin in, 139 Platinic salts, inactivation of renin by, 134 P: 0 ratio, see Phosphorous: oxygen ratio Poky mutant, see Neurospora Polarograph, catalase assay by use of, 780 Polynucleotides, hydrolysis of by spleen phosphodiesterase, 555-556 by venom phosphodiesterase, 561,564 Polypeptide(s), as component of heparin cofactor, 163 as substrates for leucine aminopeptidase, 91 Polyphenoloxidase, see Tyrosinase Polyribopho sphate, degradation of by RNase, 433 Polysaccharides, separation from old yellow enzyme by electrophoresis, 714

965

Pomiferin, protease from latex of Osage orange, 57, 64 Porphyrin, see also Hemin, Hematin, Protoporphyrin, possible presence in liver glutathione reductase, 725 Potassium chloride, effect of on myosin ATPase, 587 inhibition of nitroethane oxidase by, 402 of xanthine oxidase by, 485 in oxidative phosphorylation assays, 612 Potassium ethyl xanthate, inhibition of ascorbic acid oxidase by, 835 of hydroxylamine reductase by, 419 of nitrate reductase by, 415 of tyrosinase by, 826 Potassium ion, activation of pantothenate-synthesizing enzyme by, 621 of tryptophanase by, 242 inhibition of DPNH cytochrome c reductase by; 692 Potassium nitrate, inhibitionof nitroethane oxidase by, 402 Potato, adenosine-5-phosphatase from, 550 apyrase from, 591-593, 646 diphosphopyridine nucleotidases in, 655 glutamic-oxalacetic transaminase activity of root, stem and leaf from, 184 nucleotide pyrophosphatase from, 655659 phosphatase in, 540 triphosphatase from, 581, 582 Preputial glands, acid phosphatase in rat, 523 Pressor substance, see Angiotonin Proaccelerin, see Plasma Ac-globulin Procarboxypeptidase, from beef pancreas, 77-83 activation of by trypsin, 80 electrophoretic mobility of, 79-80 purification of, 79-80 resistance to activation by other proteases, 80

966

SUBJECT I N D E X

Proconvertin (SPCA precursor), concentration of in oxalated horse plasma, 147 effect of dicoumarol on synthesis of, 140, 154 purification of from human plasma, 153-154 role in clotting mechanism, 140, 154 Profibrin, in fibrinogen solutions, 160 Profibrinolysin (plasminogen), 140, 163-165 from Fraction I I I of plasma, 164-165 role in clot dissolving, 140 Prolidase (imidodipeptidase), from equine erythrocytes, 101, 102103, 105 in intestinal mucosa, 102, 105 from kidney, 98, 101, 103-104, 105 mechanism cf action of, 105 presence of in purified iminodipeptidase, 99 L-Prolinamide, amidase action on, 399 leucine aminopeptidase action on, 92 Prolinase, see Iminodipeptidase ~Proline, P:O ratio during oxidation of, 613 as product of aminotripeptidase action: 87 reduction of D- or, 217, 219, 220 as substrate for L-amino acid oxidase, 211 ~-Prolylglycine, as substrate for iminodipeptidase, 97 ~Prolylglycylglyeine, as substrate for aminotripeptidase, 83, 87, 97 in crude prolinase preparations, 97 L-Prolyl-L-proline, as substrate for prolidase, 104 Propionic acid bacteria, aspartase from, 386-387, 388 growth of, 386--387 Propionyl amino acids, action of acylase I on, 118 Propionyl-L-glutamate, role in citrulline synthesis, 355 Propionyl phosphate, hydrolysis of, 556

Propylamine, amine oxidase action on, 393 Propylenediamine, action of diamine oxldase on, 396 Prorennin, activation of to rennin, 72, 73 Prostate gland, acid phosphomonoesterase from, 523530, 540 elevated acid phosphomonoesterase in serum following carcinoma of, 524 INH insensitivity of DPNase from human, 663 ribonuclease in, 526, 529 Protamine, activation of profibrinolysin by, 165 fractionation of barbiturase with, 492-493 of dihydro6rotic dehydrogenase with, 495 of protein with, 344 inhibition of enzymatic degradation of cytochrome c by, 169 precipitation of heparin by, 163 of nucleic acid by, 213, 214, 275, etc. preparation of solution for, 214 variability in effectiveness for, 275 of pantethcine kinase from liver extract by, 633 Protease, see Proteolytic enzymes Proteinases, see Proteolytie enzymes Protein(s), determination of concentration of by optical method, 19 by turbidimetric method, 743 enzymes for metabolism of, 3-423 oxidation of by tyrosinase, 826 Proteolysis, by mitochondria from poky strain of Neurospora, 168 Proteolytic activity, removal of during RNase purification, 430 Proteolytic enzymes, animal, see under names of individual enzymes Proteolytic enzymes, plant, 54-64, see also under names of individual enzymes,

SUBJECT INDEX

determination of activity of, 55-59 milk clotting method, 58-59 nonprotein nitrogen method, 58 phenol color method, 55-58 heat resistance of, 54 isolation of arachain from peanut, 57, 63 of asclepain from milkweed, 56, 61-62 of bromelin from pineapple, 56~ 62-63 of chymopapain from papaya, 56, 61 of euphorbain from caper spurge, 57, 64 of ficin from fig, 56, 61 of hurain from jabillo, 57, 64 of mexicain from cuaguayote, 56, 62 of papain from papaya, 56, 59, 60 of pinguinain from maya, 56, 63 of pomiferin from osage orange, 57, 64 of solanain from horsenettle, 57, 63-64 of soyin from soya bean, 56, 63 of tabernamontain from

Tabernamontana grandiflora, 56, 63 milk clotting activity of, 54 non-SH enzymes, 54, 63-64 optimum pH for, 54 SH enzymes, 54, 59-63 table of properties of, 56-57 technological and medical uses of, 55 various other sources of, 63, 64

Proteus, transaminase in, 173-174

Proteus morganii, cystathionine cleavage enzyme from, 314 desulfhydrases from, 318

Proteus vulgaris, aspartase in, 388 -r-glutamyltransferase from, 268-269 -r-glutamyltransfer~se (Mn-dependent) from, 271-272 growth medium for, 268, 865-866 hydrogenase from, 865-866 hydrolysis of ~-glutamyl hydroxamic acid by extracts of, 339

967

metaphosphatase in, 577, 579 Prothrombin, 140-146 activation of, 146 assay of, one-stage, 140-141 two-stage, 141-143 concentration of in oxalated horse plasma, 147 as contaminant of proconvertin, 154 preparation of thrombin from, 157 purification of from plasma, 143-145 simplified method for, 145 role in clotting mechanism, 139, 152, 156 Protocatechuic acid, molar extinction coefficients for, 285 spontaneous oxidation of, 287 Protocatechuic acid oxidase, from Ps. fluorescens, 273, 284-287 Protohematin, as prosthetic group of spinach catalase, 790 Protohematin IX, as prosthetic group of catalase from M. lysodeikticus, 788 Protohemin, see also Hemin, content of in horseradish peroxidase, 809 in catalase, 784 Protoporphyrin, extinction coefficient for pyridine hemochromogen of ferro-, 809 Protyrosinase, activating agents for in grasshopper eggs, 831 Psalliota compestris, see Mushroom

Pseudomonas, citrullinase in, 378 tryptophan peroxidase in, 245

Pseudomonas aeruginosa, arginine desimidase in, 376 aspartase in, 388 transhydrogenase in, 686

Pseudomonas fluorescens, aspartase from, 387, 388 determination of L-aspartic acid with, 389 benzaldehyde dehydrogenase from, 273, 280-281 benzoylformic carboxylase from, 273, 278-280

968

SUBJECT I N D E X

cytochrome c from, 758-760 cytochrome c peroxidase from, 761-764 cytochrome system in particulate fraction from, 275, 277 growth of, 229, 251,387, 762 histidase from, 228-231 kynureninase from, 249-253 lactone-splitting enzyme from, 273, 282-284 lactonizing enzyme for cis,cismuconic acid from, 273, 282-284 L(W)-mandelic acid dehydrogenase from, 273, 274, 277-278 mandelic acid racemase from, 273, 274, 276-277 particulate fraction of, electron transport system of, 275 L(~)-mandelic dehydrogenase in, 275 protocatechuic acid oxidase from, 273, 284-287 pyridine nucleotide transhydrogenase from, 681-686 pyrocatechase from, 273, 274, 281-282 strain differences in aromatic oxidations by, 273 transaminase in, 172-173 urocanase from, 231-233 Pseudomonas pyocyaneus, see also Pseudomonas aeruginosa, aspartase in, 388 Pseudomonas stutzeri, enzymes for nitrogen gas formation from, 420-423 growth of, 421 Pteroyglutamic acid, see Folic acid Pumpkin, 7-glutamyltransferase (PGT) from, 263-264, 266-267 protease in, 64 Purines, as aeeeptors of deoxyriboside group, 468 chromatographic and ionophoretic separation from nucleosides, 457 as products of nucleosidase action, 456 spectrophotometric methods for determination of, 457-458 synthesis of, method for determining with C14-formate, 505-507

Purine nucleosidase, see Nucleosidases, hydrolytic Purine nucleoside phosphorylase, see Nucleoside phosphorylase, purine Purine ribosides (PuR), synthetic, nucleosidase action on, 463 Purpurogallin, extinction coefficient for, 773 formation of from pyrogallol by peroxidase, 773-775 Purpurogallin number, peroxidase activity exprcssed by, 774 Purpurogallin test, in assay of peroxidase, 813, 816 Putrescir~e (1,4-diaminobutane), as substrate for diamine oxidase, 396 Pyribenzamine, inhibition of amine oxidase by, 393 Pyridine, activation of thiaminase by, 625 Pyridine hemochromogen, see also Itemochromogen, hematin peptide and, 169 Pyridine nucleotide oxidases, 712-719 new yellow enzyme, 715-719 old yellow enzyme, 712-715 Pyridine nucleotide quinone reductase, from pea seeds, 725-729 Pyridine nucleotide transhydrogenase, 681-687, see also Nucleotide transhydrogenase, in brain, 681-687 specificity of hog enzyme for exchange reaction, 681 distribution of, 686, 687 in animal tissues, 687 in bacteria, 686 from heart, 686-687 from Ps. fluorescens, 681-686 role in apparent reaction of TPNH with diaphorase, 710, 711 in yeast, 682 Pyridine nueleotides, see also Di- and Triphosphopyridine nucleotide, discovery of, 712 reduction of by molecular hydrogen, 729 requirement for benzaldehyde oxidation, 277

SUBJECT INDEX

Pyridine transglycosidase, see Diphosphopyridine nucleotidase Pyridoxal, phosphorylation of by A.TP, 646 stabilization of pyridoxal kinase by, 648 Pyridoxal kinase, from brewer's yeast, 646-649 Pyridoxal phosphate (pyridoxal-5phosphate) (B6alP), ammonium salt of, 233 assay of, 647 calcium salt of, 212 as coenzyme for various systems, amino acid dcearboxylases, 189, 193, 198 effect of coenzyme on kinetics, 193, 198 cysteine and homocysteine desulfhydrases, 318 kynureninase, 250, 253 racemases, 213, 217 alanine racemase, 213 glutamic racemase, 217 D-serine (D-threonine) dehydrase, 323-324 L-serine (L-threonine) dehydrase, 320, 322 transaminases, 170, 172-175, 177, 179-182, 289 tryptophanase, 238, 241 demonstration of with pyridoxinerequiring mutant, 237 stabilizing effect of coenzyme, 237 tryptophan synthetase, 236-237 combination of with carbonyl reagents, 241 content of in D-amino acid oxidase preparation used for racemase assays, 213 formation of, 646 protection of by citrate and Versene, 313 Pyridoxamine, inhibition of amine oxidase by, 393 pyridoxal kinase action on, 648 Pyridoxamine phosphate, transamination with cysteine-sulfinatc in Cl. welchii, 173

969

Pyridoxine, pyridoxal kinase action on, 648 Pyrimidine(s), as aceeptors of deoxyriboside group, 468 chromatographic and ionophoretic separation from nucleosides, 457 distinction from nucleosides by light absorption in alkali, 458, 461 enzymatic methods for, 458 as products of nucleosidase action, 456 Pyrimidine derivatives, activation of thiaminase by, 627-628 inhibition of thiaminase by, 625 Pyrimidine nucleoside phosphorylase, see also Thymidine phosphorylase, distinction from purine nucleoside phosphorylase, 448 in E. coli, 480 Pyrimidine oxidase, see Uracil-thymine oxidase Pyrimidine ribose nucleotides, cyclic (2/3'-monohydrogen phosphate esters of nucleosides), digestion by RNase, 433 Pyrimidine riboside 3'-phosphates, secondary phosphate esters of as substrates for RNase, 433 "Pyrimidyl" (2-methyl-4-amino-5ethoxymethylpyrimidine), in assay of thiaminokinase, 636 inhibition of yeast phosphatase by, 638 Pyrocatcchase, from Ps. fluorescens, 273, 281-282 Pyrocatechol (1,2-bcnzenediol), detoxication of diphtheria toxin by myeloperoxidase and, 800 Pyrogallol, in assay of peroxidase, 773-775 oxidation of by peroxidase complex II, 810 by tyrosinase, 826 Pyrogallol test, see Purpurogallin test Pyrophosphatasej inorganic, as contaminant of FAD pyrophosphorylase, 675 distinction from metaphosphatase by heat, 579 from triphosphatase by heat, 581

970

SUBJECT INDEX

in E. coli, 619 in liver, 362, 667, 669 activation of latent enzyme by magnesium ion, 669 in methionine-activating reaction, role of, 256 from yeast, 570-576 purification by method of Kunitz, 571-575 alternative procedure, 576 crystallization of, 573-574 Pyrophosphatase, organic, in intestinal mucosa, 475 role in determination of bound adenosine, 475 in snake venom, relation to diesterase, 565 Pyrophosphate, inorganic, activation of D-amino acid oxidase by, 202 formation and utilization of by reversible pyrophosphorolysis of dephospho-CoA, 667, 669 of DPN, 670 of DPNH, 672 of FAD, 673 of UDPG, 675, 676 formation of by mononucleotide synthesizing system, 501 by oxidative phosphorylation, 613 by pantothenate-synthesizing system, 619, 622 by triphosphatase, 580, 582 inhibition of aeetyl phosphatase by, 556 of alkaline phosphatase by, 538 of 5'-AMP deaminase by, 472 of aspartase by, 388 of D P N H cytochrome c reductase by, 692 competition with cytochrome c, 692 of ~-glutamyltransferase (brain) by, 272 of homogentisate oxidase by, 295 of iminodipeptidase by, 100 of leucine aminopeptidase by, 93 of luciferase (firefly) by, 856 of prolidase by, 105

protection of hydroxylamine reductase by, 419 of nitrate reductase by, 414 of renin by, 129, 133, 134 Pyruvate, activation of hydrogenase system by, 866 decarboxylation of as TPP assay, 636 determination of, 316, 730 formation of by cysteine desulfhydrase, 315-318 by cystathionine cleavage enzyme, 314 by exocystine desulfhydrase, 319 by pyruvate phosphokinase in test for adenosine kinase, 497 by L-serine dehydrase, 319-322 by tryptophanase in E. coli, 238 P: O ratio during oxidation of, 613, 616 protection of hydrogenase by, 218 reduction by molecular hydrogen, 729-730 *Pyruvate phosphokinase, role in adenosine kinase assay, 497 *Pyruvic decarboxylation system, CO2 versus tICO3- as product of, 841

Q Quinaerine (atabrine), inhibition of amine oxidase by, 393 of D P N H cytochrome c reductase by, 693, 698 of TPNH cytoehrome c reductase of yeast by, 703 as uncoupling agent, 615 Quinic acid, over-all equilibrium constant for conversion to shikimic acid, 311 in scheme for aromatic biosynthesis, 300, 301 Quinic dehydrogenase, from A. aerogenes, 307-311 Quinine, inhibition of n-amino acid oxidase by, 203 Quinoline, activation of thiaminase by, 625

SUBJECT INDEX

p-Quinone, see p-Benzoquinone Quinones, as electron acceptors in bacterial luciferase system, 857, 860 apparent inhibition by, 860 inhibition of homogentisate oxidase by, 295 R

Rapid-flow methods, for carbonic anhydrase, 840 Rattlesnake, diamond, 5'-nueleotidase in venom from, 561 phosphodiesterase in venom from, 564, 565 Reaction inactivation, as characteristic of ascorbic acid oxidase, 835 of plant tyrosinase, 826 Red blood cells, see Erythrocytes Reductic acid (1,2,3-triketocyclopentane), as substrate for dehydroascorbic reductase, 850 Reduetone, oxidation of by peroxidase complex II, 810 Reinheitszahl, see RZ Renin, from kidney, 124-135 as antigen, 129-130, 133 a-, f~- and w-forms of, 135 methods of stabilization of, 129, 133, 134 presser effect on dog, 124-125 purification procedures, 125-133 resistance to acid, 134 ultraviolet spectroscopy of, 135 Renin-protein complex, 135 Renin substrate (hypertensinogen), 124, 135-139 from hog serum, 135-139 electrophoretic analysis of, 138-139 identification in as fraction, 139 Rennet, commercial, as source of rennin, 73, 74 Rennin, from calf stomach, 69-77 amino acid composition of, 77

971

crystallization of, 74-76 formation from prorennin, 72, 73 peptone formation from a-casein by, 77 phosphatase activity of on phosphopeptone, thermostable activator for, 77 phosphoamidase activity of, 77 physical properties of, 77 proteolytic action on casein, 77 on hemoglobin, 77 in preparation of lactoperoxidase, 813 of medium for propionic acid bacteria, 388 Resolution of a-amino acids, use of aminoacylases for, 119 Resorcinol (1,3-benzenediol), detoxication of diphtheria toxin by myeloperoxidase and, 800 oxidation of by peroxidase complex II, 810 Respiratory enzymes, 681-870 Rhodanese, from beef liver, 334-337

Rhodopseudomonas fluorescens, cytochrome c in, 759

Rhodospirillum rubrum, hydrogenase in, 870 F t k f i a v i n (vitamin B2), distribution coefficient (benzyl alcohol: water) for, 642 inhibition of L-amino acid oxidase by riboflavin analogs and, 208 of bacterial luciferase by, 860 of DPNH cytochrome e reductase by, 693 kinase for in pyridoxal kinase preparations, 648-649, see also Flavokinase molecular extinction coefficient for, 641 phosphorylation of, 640, 644 relationship to old yellow enzyme, 712 spectrophotometric determination of in flavoproteins, 709 Riboflavin kinase, in FAD synthesizing system, 675 Riboflavin-5'-phosphate, see also Flavin mononucleotide (FMN), formation from FAD by nucleotide pyrophosphatase, 655

972

SUBJECT INDEX

Riboflavin, reduced, as electron donor in bacterial luciferase system, 861 Ribonuclease, amino acid composition of, 434 as antigen, 435 assay in tissues, unreliability of, 436 of beef spleen, 436 action of on product of pancreatic RNase action, 436 from bovine pancreas, 427-436 crystallization of, 430-432 as byproduct of DNase preparations, 439 chromatographic fractionation of into two components, 435-436 elementary composition of, 434 physical constants for, 435 preparation of radioactive, 429 in prostatic phosphatase preparations, 526, 529 in rye grass 3'-nucleotidase preparations, 553-554 various sources of, 427, 436 Ribonucleic acid, enzymatic hydrolysis of, 427-436 changes in solubility, diffusibility and ultraviolet absorption during, 427 extent of hydrolysis by prostatic phosphatase, 526 inhibition of streptococcal DNase by bacterial, 447 3'-nucleotidase action on, 553-554 preparation of solution of, 428 presence of in thromboplastin, 150 purification of commercial samples of, 428 resistance to DNase action, 441 ribonuclease-resistant "core" from, 566, 568-570 resistance to intestinal phosphodiesterase, 569, 570 as substrate for spleen phosphodiesterase, 566, 568 spontaneous hydrolysis of, 433 as substrate for intestinal phosphodiesterase, 569 for spleen phosphodiesterase, 565, 568

Ribonucleic acid, deaminated, degradation of by RNase, 432 Ribose, inhibition of uridine nucleosidase by, 462 as product of nucleosidase action, 456, 460-461 determination of, 457 D-Ribose, paper chromatography of, 513 Ribose-l,5-diphosphate (R-1,5-P2), formation by phosphoglucomutase, 503 as intermediate in nueleotide synthesis, 501 Ribose-l-phosphate (R-l-P), conversion to R-1,5-P:, 503 elimination as intermediate in nucleosidase action, 460, 461 formation by nicotinamide riboside phosphorylase, 454 preparation of, 448-449 as substrate for nucleoside phosphorylase, 448 Ribose-5-phosphate (R-5-P), conversion to 5-phosphoribosyl pyrophosphate, 504 degradation in cells grown on xylose, 460-461 in de novo purine nucleotide synthesis, 506 elimination of as intermediate in nucleosidase action, 460, 461 in glycinamide ribotide synthesis, 510 " 5 " nucleotidase action on, 550 phosphorylation of, 502 heat-lability of enzyme for, 504 prostatic phosphatase action on, 525 synthesis of IMP from hypoxanthine and, 503 of nucleotides from, 501-504 Riboside (s), of 8-azaguanine, enzymatic synthesis of, 448 of hypoxanthine and guanine, action of phosphorylase on, 448 Rice bran, metaphosphatase from, 579 Robison ester, see Glueose-6-phosphate

SUBJECT INDEX

Rosindulin GG, as electron donor to FMN in bacterial luciferase system, 861 Rubidium ion, activation of tryptophanase by, 242 Rye grass, 3'-nucleotidase from, 551-555 RZ (Reinheitszahl) unit, purity of horseradish peroxidase measured by, 803 of myeloperoxidase measured by, 798, 801

Saccharomyces cerevisiae, see Yeast Safranin T, as electron donor to FMN in bacterial luciferase system, 861 Salicylaldoxime, inhibition of hydroxylamine reductase by, 419 of tyrosinase by, 826 Salicylic acid, inhibition of L-amino acid oxidase by, 2O8 Salmine sulfate, see also Protamine, precipitation of nucleic acids with, 499 Salmonella enteritidis, aspartase in, 388 Salts, inhibition of 7-glutamyltransferase (brain) by, 272 Sarcina spp., aspartase in, 388 Sarcina lutea, cytochrome b in, 745 Sarcosine (N-monomethylglycine), as substrate for glycine oxidase, 225~ 227 Sarcosylglyeine, as substrate for glycylglycine dipeptidase, 107 Sarcosyl-L-leucine, hydrolysis by glycyl-L-leucine dipeptidase, 107 Schmidt's deaminase, see 5'-Adenylie acid deaminase Selenite, catalysis of COs hydration by, 836 inhibition of DNase by, 442

973

Semen, acid phosphatase of prostatic origin in, 523, 540 Semicarbazide, inhibition of amine oxidase by, 393 of arginine desimidase by, 376 of aspartic acid decarboxylase by, 188 of kynureninase by, 253 Seminal vesicles, acid phosphatase in, 523 Seminal plasma, " 5 " nucleotidase from, 547-549 Sequential induction, in aromatic oxidations by Ps. fluorescens, 274 L-Serinamide, hydrolysis by leucine aminopeptidase, 92 Serine, conversion of/3-carbon of to formyl group, 518 in peptide A from fibrinogen, 160 >tryptophan synthesis from, 233 D-Serine (D-threonine) dehydrase, in E. coli, 322, 323 from Neurospora crassa, 322-324 L-Serine (L-threonine) dehydrase, distribution of, 320 from Neurospora crassa, 319-322 Serratia marcescens, aspartase in, 388 Serum, acid phosphatase of prostatic origin in, 524 L-tartrate inhibition as criterion for, 528 value in diagnosis of prostate carcinoma, 524 L-asparaginase from, 383-384 convertin from, 156 DNase in, 447 as-globulin fraction of, renin substrate in, 139 prolidase in, 105 prothrombin-free, Ac-globulin activity of, 141 renin substrate from, 135-139 stabilization of carbonic anhydrase by, 846

974

SUBJECT I N D E X

Serum At-globulin (accelerin), preparation of from plasma Acglobulin, 153 use of thrombin for, 152, 153 in prothrombin-free serum, 141 role in clotting mechanism, 139, 156 Serum prothrombin conversion accelerator (SPCA), see Convertin SH compounds, see Sulfhydryl Compounds Shellfish, thiaminase from, 622-626, 627-628 Shikimic acid, as intermediate in aromatic biosynthesis, 300, 301 over-all equilibrium constant for conversion to quinic acid, 311 as product of 5-dehydroshikimic reductase action, 301-304 Silica-celite, lactoperoxidase purification by chromatography on, 815 Silicone technique, for collection of blood, 149 Silver ion, inhibition of aspartase by, 388 of glutamic dehydrogenase by, 224 of hydrogenase exchange reaction by, 867 of metaphosphatase by, 579 of renin by, 134 of RNase by, 434 Skim milk powder, as substrate for rennin assay, 70 Skin, tyrosinase from, 829, 831 Snake venom(s), L-amino acid oxidase from, 205-209 DPNH cleavage by nucleotide pyrophosphatase from, 654 5'-nucleotidase from, 549-550, 561 phosphodiesterases from, 561-565 relative activities of, 565 pyrophosphatase accompanying phosphodiesterase activity in, 565 source of dried, 563 Sodium azide, see Azide Sodium chloride, effect on Cypridina luciferase, 853 on RNase, 433-434

inhibition of DNase (pancreatic) by, 442 of glutathione reductase (yeast) by, 725 of nitroethane oxidase by, 402 of xanthine oxidase by, 485 stabilization of renin by, 129 Sodium dehydroepiandrosterone sulfate, as substrate for steroid sulfatases, 330 Sodium dichlorophenoxyethylsulfate, as substrate for alkylsulfatases, 330 Sodium fluoride, in oxidative phosphorylation assay, 611 Sodium hydrosulfide, see Sulfide Sodium hydrosulfite, see Dithionite Sodium ion, effect on RNase, 433 inhibition of DPNH cytochrome c reductase by, 692 of pantothenate-synthesizing enzyme by, 621 Sodium nitrate, inhibition of nitroethane oxidase by, 402 Sodium sulfide, see Sulfide Sodium usnate, inhibition of DNase by, 442--443 dependence on cobaltous ion, 443 Soja hispidus, see Soybean Solanain, protease from horsenettle, 57, 63-64 Solanum elaeagnifolium, see Horsenettle Solubilization, of particulate enzymes, with n-butanol, 112 by cholate, 743 by cholate plus trypsin, 739 by deoxyeholate, 741 with digitonin, 686-687 with ethanol, 689, 708 Solubilized aminopeptidase, see Dehydropeptidase I Soluble acylase I, see Amino acid acylase I L-Sorbose-l,6-diphosphate, hydrolysis by fructose diphosphatase, 545-546 Soybean (Soja hispidus), protease (soyin) from, 56, 63 ribonuclease in, 427

SUBJECT INDEX

trypsin inhibitor from, 36-37, 40-44, 50, 51, 52 Soybean leaves, cytochrome c peroxidase in, 764 nitrate reductase in, 414 Soyin, protease from soybean, 56, 63 Spermidine, amine oxidase action on, 393 Spermine, amine oxidase action on, 393 Spinach leaf (Spinacea oleracea, Tetragonia expansa ) , acetone powder preparation of, 789 carbonic anhydrase from, 842-843 catalase from, 789-791 5-dehydroshikimic reduetase and 5-dehydroquinase in, 304, 307 glutathione reductase in, 721 Spleen, adenylate kinase (myokinase) in, 602 carnosinase in, 94 cathepsin C from, 64-68 DNase from, 444-445 DPNase (pyridine transglycosidase) from, 660-663 L-glutaminase in, 382 kynurenine formamidase in, 248 nueleoside phosphorylase from, 448453 phosphodiesterase from, 565-569 RNase in, 436 Spores, Bacillus, alanine racemase in, 215 Squash, glutamie acid decarboxylase from, 190-194 keto acid decarboxylases in, 182, 192, 193 transaminases in, 182 Squash, yellow (Cucurbita pepo condensa), ascorbie acid oxidase from, 831-835 Stainless steel, adverse effect on renin, 130 Staphylococcus albus, cytochrome b in, 745 Staphylococcus aureus, arginine desimidase in, 376 glutamine synthetase in, 342

975

penicillin assay with, 121 Staphylokinase, activation of profibrinolysin by, 165 Steroid sulfatases, 324, 330, 332 in Patella vulgata (mollusk) , 332 Stomach, see also Gastric mucosa, rennin from calf, 69-77 Streptococcii group A hemolytic, DNase from, 446-447 Streptococcus faecalis, alanine raeemase from, 212-215 arginine desimidase in, 375, 376 in assay for "folic acid activity," 629 citrullinase in, 378 growth of, 188, 213, 647 transaminase in, 176 tyrosine decarboxylase from, 188, 646, 647-649 preparation of apoenzyme, 646, 647648 Streptococcus lactis, arginine desimidase in, 376 citrullinase in, 378 Streptokinase, activation of profibrinolysin by, 165 Streptomycin, effect of on RNase, 434 Succinate, activation of hydrogenase system by, 866 in assay of cytochrome c, 750 oxidation of, blocking by malonate of, 616 measurement of rate of, 748 P:O ratio during, 613, 616 role of cytochrome b in, 744, 745 of cytochrome bl in, 745-748 reduction of hematin peptide by, 167-169 stabilization of pyrimidine nucleoside by, 459 Suecinate-fumarate system, oxidation-reduction potential of, 740 *Sueeinic dehydrogenase, in assay of cytochrome b, 740, 741742 of cytochrome c, 750, 758 comparison of heart and Ustilago cytochrome c preparations, 758

976

SUBJECT I N D E X

cytochrome b preparations containing, 744 eytoehrome bl (diphtherial) and, 748 in heart particles, 737 manometric methods for, 748 Succinie dehydrogenase-cytochrome e linking activity (SC activity), of cytochrome b from heart, 742 *Succinic oxidase, cytochrome bl (diphtherial) and, 748 in heart particles, 738, 739, 750 assay methods for, 739 reduction of endogenous cytochrome c in preparations of, 75O from kidney cortex, 750 cytochrome c assay with, 750 Succinylacetoacetate, action of hydrolase on, 299 Succinyl phosphate, hydrolysis of, 556 Sucrose, in oxidative phosphorylation assay, 612 Sulfadiazine, see Sulfonamides Sulfa drugs, see also Sulfonamides, activation of bacterial thiaminase by, 628 Sulfanilamide, acetylation of in liver extract, 633 determination of, 634 inhibition of erythrocyte carbonic anhydrase by, 845 nitrite determination with, 403, 411 Sulfanilic acid, nitrite assay with, 400 Sulfatases, 324-332, see also Arylsulfatases, alkylsulfatases, 324, 330 arylsulfatases, 324, 327-332 assay of, 324-330 by determination of organic part, 327-330 by determination of sulfuric acid formed, 324-327 in aqueous solution, 325-326 in tissue suspension, 324, 326-327 chondrosulfatases, 324 glucosulfatases, 324, 330 myrosulfatases, 324

steroid sulfatases, 324, 330 Sulfate, activation of prothrombin by, 145 inhibition of acetyl phosphatase by, 556 of carbonic anhydrase (erythrocyte) by, 845 of D P N H cytochrome c reductase by, 692 of sulfatases by, 326 stabilization of pyrimidine nucleosidase by, 459 Sulfathiazole, see also Sulfonamides~ inhibition of tyrosinase by, 826 Sulfhydryl (SH) compounds (Thiol compounds), see also Cysteine, Glutathione, Mercapto compounds, activation of amino acid reductases by, 220 of homogentisate oxidase by, 295 of hydrogenase by, 869 of guanidinoacetate methylpherase by, 260, 263 of thiaminase by, 625, 627 of thio ether cleavage enzyme by, 314 of xanthine oxidase by, 485 cytochrome c reduction by, 754 effect of on 3'-nucleotidase, 554 inhibition of cysteine desulfhydrase by, 317 of hippuric acid formation by, 350 Sulfhydryl (SH) groups, presence in myosin, 587 requirement for methionine-activating enzyme, 256 for methylation of guanidinoacetic acid, 260 Sulfhydryl reagents, see also under names of individual reagents, e.g. p-Chloromereuribenzoate, Iodoacetate, etc., inhibition of D-amino acid oxidase by, 2O3 of ATPase (insect muscle) by, 598 of ATPase (myosin) by, 587 of luciferase (bacterial) by, 860 of rhodanese by, 337 Sulfide, activation of hydrogenase by, 868

SUBJECT INDEX compound of with horseradish peroxidase, 812 determination of, 315-316 inhibition of ascorbic acid oxidase by, 835 of carbonic anhydrase by, 845 of carnosinase by, 96 of DNase by, 442 of tryptophan peroxidase by, 246 of tyrosinase by, 826 ~-Sulfinyl pyruvic acid, desulfination of, 333 formation from cysteinesulfinic acid, 333 Sulfite, activation of amorphous form of asclepain (protease) by, 62 inhibition of rhodanese by, 337 Sulfonamides, accumulation of aminoimidazolecarboxamide riboside in E. coli cultures containing, 512-514 inhibition of L-amino acid oxidase by, 208 of carbonic anhydrase (erythrocyte) by, 845 p-Sulfonamidobenzoic acid, inhibition of erythrocyte carbonic anhydrase by, 845 Sulfonic acids, aromatic, inhibition of L-amino acid oxidase by, 2O8 Surface denaturation, serum albumin as protecting agent against, 724 T

Tabernamontana grandiflora, protease (tabernamontanain) from fruit of, 56, 63 Takadiastase, adenosine deaminase (nonspecific) from, 475-478 arylsulfatase from, 328 folic acid conjugase in, 630-631 metaphosphatase in, 579 source of, 476 triphosphatase ill, 580, 582

977

L-Tartrate, inhibition of prostatic phosphatase by, 528 clinical use of, 528 Tea tannins, oxidation by tyrosinase, 826 Tenbroeck homogenizer, extraction of liver acetone powder with, 653 L-Tertiary leueinamide, action of amidase on, 399 Testosterone, increased acid phosphatase in prostate gland after injections of, 523 Tetradecyl aldehyde, as component of bacterial luciferase system, 860 Tetraethyl ammonium chloride, in angiotonin assay, 136 Tetragonia expansa, see Spinach Tetraguaiacol, formation from guaiacol in peroxidase assay, 772 Tetrahydrofolic acid (FAH4), as cofactor in formylation of 5-IRMP, 519 of glycinamide ribotide, 510 formylation of, 516-518 by reaction with formate, 517-518 with serine, 518 Tetrazolium salts, see also Triphenyltetrazolium and Neotetrazolium, as electron acceptors for DPNHcytochrome c reductase of bacteria, 697 Thermal conductivity cell, for analysis of ortho-parahydrogen mixtures, 864 Thermobacterium acidophilus, growth medium for, 467 nucleoside transdeoxyribosidase in, 467 Thiaminase, 622-628 from Bacillus thiaminolyticus culture medium, 626-628 activation by sulfa drugs, 628 in Carassius carassius, 625 in carp, 625 in clam viscera (Meretrix meretrix), 622-626

978

SUBJECT I N D E X

kinetics of base exchange versus hydrolytic actions of, 626 sources of, 625 substrate specificity of shellfish and bacterial enzymes, 628 Thiamine, exchange of thiazole moiety of, for other bases by thiaminase, 622 inhibition of yeast phosphatase by, 638 phosphorylation of, 636 Thiamine derivatives, thiaminase action on, 624, 628 table for, 628 Thiamine pyrophosphate (TPP), cleavage by nucleotide pyrophosphatase, 659 as coenzyme for benzoylformic carboxylase, 279-280 determination of, 636-638 formation by thiaminokinase action, 636 thiaminase action on, 624 Thiaminokinase, 636-640 from baker's yeast, 639-640 from brewer's yeast, 638 Thiazole, as product of thiaminase action, 622 Thiobenzoic acid, inhibition of heparin cofactor by, 163 Thiochrome method, for thiamine determination, 622-623 Thiocyanate, colorimetric determination in rhodanese reaction, 334 Thio ethers, cleavage of, 313 Thioglycolate, activation of histidase by, 231 of hydrogenase by, 868 inhibition of DNase by, 442 of heparin cofactor by, 163 reduction of dehydroaseorbio acid by, 85O role of in blood clotting mechanism, 160 Thiolactic acid, reduction of dehydroascorbic acid by, 850

Thiol compounds, see Sulfhydryl compounds Thiopental (Pentothal), as uncoupling agent, 615 Thiophene-2-sulfonamide, inhibition of erythrocyte carbonic anhydrase by, 845 Thiophosphates, as probable substrates for alkaline phosphatase, 538 Thiosulfate, as sulfur donor for rhodanese, 334-337 Thiosulfonates, as sulfur donors in rhodanese reaction, 336 Thiouracils, inhibition of tyrosinase by, 826 Thiourea(s), activation of uricase by, 489 inhibition of nitrate reductase by, 415 of tyrosinase by, 826 Thrombin, 156-158 adsorption of during clotting, 158 preparation from prothrombin, 156, 157 role in clotting mechanism, 139, 140, 152, 156-158, 159 use in two-stage prothrombin assay, 141 Thromboplastin (thrombokinase), 139, 140, 146-151, 154 activation of proconvertin by, 154 of prothrombin by, 145 adsorption on calcium oxalate, 147 assay by one-stage method, 146 by two-stage method, 147 from brain, 140, 149 use in prothrombin assay, 140, 141 complexes with convertin and plasma Ac-globulin, 151 components of, 139 concentration in oxalated horse plasma, 147 heat-labile factor of (antihemophilic factor, AHF), 139, 147-148 occurrence in plasma, 139 from lung, 148-149 role in clotting mechanism, 139, 152, 154

SUBJECT INDEX

Thymic acid, degradation by RNase, 433 Thymidine (thyminedeoxyriboside), bioassay of, 464 R! values for, 466 in transdeoxyribosidase reaction, 464-468 Thymidine phosphorylase, from horse liver, 453 Thymine, as acceptor of deoxyriboside group, 468 oxidation to 5-methylbarbituric acid, 490 increase in optical density during t 490 Rs values for, 466 Thymine deoxyriboside, see Thymidine Thymine oxidase, identity with uracil oxidase, 491 Thymus, aminotripeptidase from, 84, 85-86 DNase from, 443-444 RNase in, 436 Tissue slices, assay of oxidative phosphorylation in, 614 d-biotin oxidase in, 631-632 a-p-Toluenesulfonyl derivatives of arginine and lysine, as substrates for trypsin, 36 Toluidine (o or p), detoxication of diphtheria toxin by myeloperoxidase and, 800 Toluquinone, reduction of by DPNH, 728 Top yeast, see Yeast, baker's Toxins, bacterial, detoxication by myeloperoxidase plus donor substances, 801 TPNH cytochrome c reductase, from liver, 704-706 inactivity with bacterial cytochrome c, 760 from yeast, 697, 699-703 rate constants for reduction and oxidation of, 697, 703 comparison with related enzymes~ 697

979

T P N H cytochrome reductases, TPNH-diaphorase activity of, 711

Tradescantia fluminensis, cyanide sensitivity of carbonic anhydrase from, 845 Transamidation, by cathepsin C, 65, 68 Transaminase (s), from bacteria, 170-177, 182, 184 for aliphatic amino acids (transaminase B), 176 for D-amino acids, 171-172 for aromatic amino acids (transaminase A), 176 for pyridoxamine-alanine, ]73 for valine-alanine and valine-aaminobutyrate, 176 distribution of (glutamic-oxalacetic enzyme), 184 estimation of, 172-176, 178-184 manometric, 182-184 quantitative filter paper chromatography, 178 speetrophotometric, 179-182 from liver, 289 role of in dcsulfinase system, 333 in oxidation of L-amino acids, 211 in squash, 182 Transhydrogenase, see Pyridine nucleetide transhydrogenase Transpeptidation, a-chymotrypsin and, 21 Transphosphorylation, by phosphatases, 556-561 efficiency of, 560 Treburon (synthetic sulfated polygalacturonic acid), inhibition of RNase by, 434 Tri-acetic acid-hydrolyzing enzyme, probable identity with fumarylacetoacetate hydrolase, 298 Triaminodiphenyl sulfones~ effects on prothrombin activation, 145 digestion by plant proteinases, 55 Trichloroacetic acid, use in purification of trypsin, 30 2,3',6-Trichloroindophenol, in nitrate reductase system, 415 Trichuris, digestion of, 55 Triethanolamine buffer, 676

980

SUBJECT INDEX

Trifluoroacetyl amino acids~ action of acylase I on, 118 q'rimetaphosphate, es substrate for metaphosphatase, 577-578 1,3,5-Trinitrobenzene, reduction of by Neurospora, 410

2,4,6-Trinitrophenol, inhibition of quinone reductase by, 729 2,4, 6-Trinitrotoluene (TNT), enzymatic reduction of, 406, 410 Tripeptidase, see Aminotripeptidase Tripeptides, resistance of to iminodipeptidase, 100 to prolidase, 105 as substrates for leucine aminopeptidase, 91 2, 3, 5-Triphenyltetrazolium, in assay of flavoproteins, 695 Triphosphatase, 580-582 orthophosphate as sole product of in yeast, 580 pyrophosphate as product of cleavage by A. oryzae enzyme, 580, 582 sources of, 580 Triphosphate, preparation of, 580-581 protection of nitrate reductase by, 414 Triphosphopyridine nucleotide (TPN), assay of, 652 of ATP and ADP by reduction of, 497 of glucose-6-phosphate by reduction of, 675, 676, 677 cleavage by DPNase (animal), 662 by DPNase (Neurospora), 666 by nucleotide pyrophosphatase, 655, 659 by plant enzymes, 720 as eoenzyme for benzaldehyde dehydrogenase, 280-281 for 5-dehydroshikimic reductase, 301 for L-glutamic dehydrogenase, 224 complex of with protein moiety of old yellow enzyme, 715 formation of by DPN kinase, 652-655 large-scale preparation, 655 liver (horse) as source of, 700

in oxidative phosphorylation assay, 612, 615, 616 reduction of by molecular hydrogen, 732 role of in formylation of FAH4 by serine, 518 in transhydrogenase reaction, 681-687 Triphosphopyridine nucleotide, reduced (TPNH), see also under TPNH, extinction coefficient for, 497 generation of by glucose-6-phosphate dehydrogenase, 699 as hydrogen donor for GSSG reduetase, 301, 719, 722, 724 for hydroxylamine reductase, 418 for luciferase (bacterial), 861 for new yellow enzyme, 715 rate constant for, 716 for nitrate reductase, 411, 414, 415 for nitroaryl reductase, 408, 410 for nitrogen gas formation from nitrite, 422 for old yellow enzyme, 712 rate constant for reaction, 716 for quinone reductase, 728 for transhydrogenase, 681-687 P: O ratio during oxidation, 616 preparation of, 411 Triphosphopyridine nucleotide, 3'isomer, action of nonspecific deaminase on, 477, 478 Triphosphopyridine nucleotide-linked dehydrogenases, determination with glutathione reductase, 721 Tris (hydroxymethyl)aminomethane (Tris buffer, THAIV[ buffer), L-glutamic dehydrogenase and, 224 low affinity of for zinc, 79 protection of hydroxylamine reductase by, 419 Tropaeolum majus, see Nasturtium Trypsin, activation of a-chymotrypsinogen to a-chymotrypsin by, 8, 12 of a-chymotrypsinogen to ~r- and ~chymotrypsins by, 8, 15-16 of procarboxypeptidase by, 80

S ~ J E C T INDEX cross reactivity with chymotrypsins on a synthetic substrate, 21 determination of activity of, 32-36 casein digestion method fo~, 33-34 hemoglobin digestion method for, 34 with synthetic arginine derivatives, amidase activity, 34-36 esterase activity, 23, 36 isoelectric point of, 35 effect of calcium on, 35 isolation of from beef pancreas, 26-36 crystallization of, 26, 29-31 improved method for, 26, 29 preparation as byproduct of DNase isolation, 439 triehloroacetic acid for purification of, 30 molecular weight of, 35 nitrogen content of, 32 optical factor for, 32 proteolytie action of, use in purification of cytochrome oxidase, 739 of myeloperoxidase, 796 of TPNH eytochrome c reductase, 7O6 of xanthine oxidase, 706 stability of, 32 effect of calcium and other cations on, 32 standard activity curve for, 33 Trypsin inhibitor(s), antifibrinolysin and, 165 determination of, 37-38 standard preparation of trypsin for, 38 general properties of, 37 isoelectric points of, 51 molecular weights of, 52 optical factors for, 50 trypsin complexes with, general properties of, 37 isoelectric points of, 51 molecular weights of, 52-53 optical factors for, 50 from various sources, Ascaris, 37, 54 blood plasma, 37, 49-54 multiplicity of inhibitors in, 49 colostrum, 37, 46-48, 50, 51, 52 crystallization of, 48

981

trypsin complex with, 47-48, 50, 51, 52 crystallization of, 47-48 lima bean, 37, 48-49, 51, 53 potency of amorphous and crystalline forms, 48-49 ovomucoid, 37, 49, 51, 53 pancreas~ as byproduct of insulin preparation, 40 removal from trypsin by trichloroacetic acid, 30 use in recrystallization of trypsinogen, 28 pancreas (Kazal preparation), 36, 40, 50, 51, 52 pancreas (Kunitz and Northrop preparation), 36, 38-40, 50, 51, 52 crystallization of, 39-40 trypsin complex with, 38-39, 50, 51, 52 crystallization of, 38-39 preparation as byproduct of DNase isolation, 439 soybean, 36-37, 40-44, 50, 51, 52 crystallization of, 42-44 trypsin complex with, 44-46, 50, 51, 52 crystallization of, 44-46 Trypsinogen, from beef pancreas, 26-36 as byproduct of DNase preparation, 439 crystallization of, 26, 27-29 molecular weight of, 35 recrystallization of, 28 use of diisopropyl fluorophosphate (DFP) during, 28 of pancreatic trypsin inhibitor during, 28 transformation into trypsin, 26, 29, 32 by autocatalysis, 26, 29 by enterokinase, 26, 29, 32 "inert protein" formation during, 26, 29 suppression by calcium ions, 26, 29 by kinase from Penicillium, 26

982

SUBJECT INDEX

mechanism of, 26 peptide formed during, 26-27 aspartic acid content of, 27 Tryptazan, inhibition of tryptophanase by, 241 Tryptophan, colorimetric determination of, 233 decarboxylation in mammalian tissues, 199 inhibition of tryptophanase by D-isomer of, 241 of tryptophan synthetase by, 237 of tryptophan synthetase formation by, 237, 238 oxidation of, enzymes for, 242-253 kynureninases, 249-253 kynurenine formamidase (formylase), 246-249 L-tryptophan peroxidase, 242-246 as precursor of authranilic acid and catechol, 273 as product of aromatic biosynthesis, 3OO L-serine (L-threonine) dehydrase action on, 322 as source of indole in E. coli, 238 as substrate for L-amino acid oxidases, 208, 211 for transaminase, 176 L-Tryptophanamide, hydrolysis by leucine aminopeptidase, 92 Tryptophanase, from E. coli, 238-242 kynureninase and, comparison of mechanisms, 253 Tryptophan desmase (Tryptophan desmolase), see Tryptophan synthetase L-Tryptophan peroxidase, adaptive increase by tryptophan administration, 244, 246, 253 differentiation from other peroxidases, 245 distribution of, 245 from liver, 242-246 in tryptophan-adapted Pseudomonas, 245 Tryptophan synthetase (tryptophan desmase, tryptophan desmolase),

in g. coli, 234 formation of in microorganisms, 237238 effect of nutrients on, 237 genetic control of~ 238 in Glomerella cingulata, 234 from Neurospora crassa, 233-238 Tsuchihashi procedure, for denaturation of hemoglobin, 102 use in prolidase purification, 102 Tumors, absence of cysteine desulfhydrase from, 318 Tungstate, use in purification of renin, 127-128 Turnover numbers, for carbonic anhydrase and catalase compared, 844 Tyramine [p-(~-aminoethyl) phenoll, amine oxidase action on, 393 detoxication of diphtheria toxin by myeloperoxidase and, 800 Tyramine phosphate, as standard for angiotonin assay, 136 L-Tyrosinamide, hydrolysis by leucine aminopeptidase, 92 Tyrosinase, insect, from grasshopper eggs, 831 activating agents for protyrosinase in, 831 Tyrosinase, mammalian, 827-831 assay methods for, 827-830 choice of method, 830 histochemical, 828-829 isotopic tyrosine method for pigmented tissue, 829-830 manometric, 827-828 from ciliary bodies, 829, 830 cytoplasmic particles as site of, 830 in melanomas, 828-831 existence in active state, 831 from pigmented eye tissue, 828 from skin, 829, 831 activation by ultraviolet irradiation, 831 distinction from tyrosinase of melanomas, 831 Tyrosinase, plant (polyphenol oxidase), 817-827, 830

S~ECT

comparison with mammalian tyrosinase, 830 distribution of, 817 from mushroom (Psalliota campestris), 822-827 chronometrie assay of catecholase activity, of, 819-821 diagram of apparatus for, 820 manometric assay of cresolase activity of, 818-819 properties of, 825-827 purification procedure for, 822-825 high catecholase preparation, 822-824 high cresolase preparation, 824825 reaction inactivation as characteristic of, 826 distinction from mammalian enzyme, 830-831 Tyrosine, standard solution of for proteinase assay, 55 Tyrosine, Cl 4 labeled, tyrosinase assay in melanoeytes with, 829 L-Tyrosine• conversion to acetoacetate, 287-300 decarboxylation in mammalian tissues, 199 in fibrin and fibrinogen, 160 oxidation by mammalian tyrosinase, 827-831 induction period preceding, 827 oxidation by plant tyrosinase, 826, 830 in peptide B from fibrinogen, 160 preparation of from DL mixture, 829 as product of aromatic biosynthesis, 3OO as substrate for ~amino acid oxidases, 208, 211 for transaminases, 176, 289 Tyrosine apodecarboxylase, from Strep. faecalis, 646, 647-648 activation of as measure of pyridoxal kinase action, 646 Tyrosine decarboxylase, in measurement of transaminase reactions, 171 from Strep. faecalis, 188

INDr.X

983

resolution of, 189 L-Tyrosine ethyl ester, as substrate for trypsin and chymotrypsin, 23 Tyrosine-glutamic acid transaminase, from liver, 289 L-Tyrosine-oxidizingsystem, distribution of, 289 from liver, 287-300 fumarylacetoaeetate hydrolase, 298-300 homogentisate oxidase, 292-295 p-hydroxyphenylpyruvate enol-keto tautomerase, 289-292 maleylacetoacetate isomerase, 295298 over-all system, 287-289 tyrosine-glutamic acid transaminase, 289 U

Ultracentrifuges, comparison of molecular weight values obtained with different, 808 Uncoupling phenomenon, agents for in oxidative phosphorylation, 615 Uracil, as acceptor of deoxyriboside group, 468 inhibition of uridine nucleosidase by, 462 oxidation of to barbituric acid, 490 • increase in optical density during, 490 Rt values for, 466 Uracil deoxyriboside (UDR), formation from cytosine deoxyriboside, 478 Rt values for, 466 in transdeoxyribosidase reaction, 464-468 bioassay of, 464 Uracil nucleosides, identification of, 479, 480 interference of pyrimidine nucleoside phosphorylase with, 480 Uracil oxidase, manometric determination of uracil with, 458

984

SUBJECT I N D E X

from Mycobacterium, 490-491 identity with thymine oxidase, 491 Uranium acetate, use in RNase assay, 427 "Uranium-soluble" phosphate, as measure of phosphodiesterase, 561 Uranyl acetate-perchloric acid reagent, in assay of phosphodiesterase, 565-566 Uranyl aeetate-trichloroacetic acid reagent, in assay of phosphodiesterase, 562 Urea, determination of~ 356, 364-365, 370371, 379 in arginine-synthesizing system, 356, 364-365 in blood with urease paper, 379 formation by arginase, 368 by barbiturase, 492 Urease, arginoly~ic activity of, 370 CO2 versus HC03- as product of, 841 from jack bean meal, 378-379 Ureidosuccinase, irreversibility of, 497 Ureidosuccinie acid, conversion to aspartic acid, NH3 and C02, 497 to 5-(acetic acid)-hydantoin, 496497 formation from dihydroSrotic acid, 496 Urethan(s), inhibition of enzymes by, 527, 867 hydrogenase, dependence on assay method, 867 prostatic phosphatase, 527 Uric acid, assay of, colorimetric, 486 spectrophotornetric, 458 cytochrome c reduction by, 754 formation by xanthine oxidase, 480, 481, 482 increased absorption during, 480 oxidation of, 485, 794, 810 in myeloperoxidase assay, 794 by peroxidase complex II, 810 peroxidase assay with, 774 preparation of lithium salt of, 485 RS values for, 466

Uric acid riboside (UAR), in beef erythrocytes, 459 nucleosidase action on, 459 Uricase, evidence for metal component in, 489 interference with guanase assay, 481 from kidney, 485-489 from liver mitochondria, 489 new procedure for purification of, 489 spectrophotometric method for, 458, 486 Uridine (UR), formation from cytidine, 478 molecular extinction coefficient, 478 nucleosidase action on, 459 Uridine nucleosidase, absorption changes during action of, 461 from baker's yeast, 461-462 Uridine triphosphate (UTP), hydrolysis by myosin, 586 as phosphate donor to AMP, 603 UDPG synthesis from glucose-l-P and, 675 Uridin ediphosphoglucose (UDPG), cleavage of by nueleotide pyrophosphatase, 659 determination of by galactowaldenase, 677 by a specific pyrophosphorylase, 676 by UDPG dehydrogenase, 677 synthesis from UTP and glucose-l-P, 675 Uridinediphosphoglucose dehydrogenase, UDPG determination with, 677 Uridinediphosphogluce se pyrophosphorylase, purification procedure for, 677 UDPG determination with, 676-677 from yeast (brewer's), 675-677 3'-Uridylic acid (3'-UMP), 3'-nucleotidase action on, 553 5'-Uridylic acid (5'-UMP), as acceptor in nucleoside monophosphate kinase reaction, 603 "5" nucleotidase action on, 549 Uridylic acids, prostatic phosphatase action on 2' and 3'-, 524

SUBJECT INDEX Urine, acid phosphatase of prostatic origin in, 523 fibrinolytic activator from, 165 Urocanase, in liver, 232 from Ps. fluoreseens, 231-233 Urocanic acid, degradation by urocanase, 231 formation from L-histidine by histidase, 228 molar extinction coefficient of, 228 Usnic acid, as uncoupling agent, 615

Ustilago sphaerogena, cytochrome c from, 755-758 content in dried cells, 757 growth of, 755-756 Uterine tissue, glycylglycine dipeptidase from, 108109 glycyl-L-leucine dipeptidase from, 105-107 V L-Valinamide, hydrolysis by leucine aminopeptidase, 92 L-Valine, as substrate for L-amino acid oxidase, 211 transaminase for alanine or a-amino butyric acid with, 176 transaminatiou with isoleucine in microorganisms and animal tissues, 171 Vanadate, inhibition of DPNH cytochrome c reductase by, 692 Vasoconstrictor substance, see Angiotonin Venom, see Snake venom Venus mercuriana (Quahog clam), thiaminase in, 625 Verdohemachrome peptide, formation from cytochrome c, 167-169 Verdoperoxidase (myeloperoxidase), see Peroxidase from leucocytes

985

Veronal (Barbital), catalysis of C02 hydration by, 836 in colorimetric assay of carbonic anhydrase, 839 inhibition of glycine oxidase by, 227 low affinity for zinc, 79 Versene, see Ethylenediamine tetraacetate

Vibrio, aromatic oxidations in, 273 Viper, Russell's, 5'-nucleotidase in venom of, 561 phosphodiesterase from venom of, 564, 565 Vitamin B~, see Riboflavin Vitamins, metabolism of coenzymes and, 619-677 W

Wheat, as source of protease, 63 Wheat germ, glutathione reductase in, 721 White blood cells, see Leucocytes X

XR, see Xanthosine Xanthine, as aceeptor of deoxyriboside group, 468 formation by guanase, 480 oxidation to uric acid, 484 as source of peroxide, 244 spectrophotometric assay of, 458, 480, 481 Xanthine oxidase, diaphorase activity of, 711 from milk, 482-485 reduction of trinitrotoluene by, 406 of dinitrobenzene by, 409 removal of product of nucleoside phosphorylase by, 448, 449 spectrophotometrie determination of adenine, hypoxanthine, and xanthine with, 458 Xanthosine (XR), nucleosidase action on, 459, 463 phosphorolysis of, 448

986

SUBJECT INDEX

Xanthydrol reagent, preparation for urea determination, 368-369 X-ray analysis, molecular weight of RNase by, 435 X-rays, inactivation of RNase by, 434 p-Xyloquinone, reduction of by DPNH, 728 Xylose, degradation of R-5-P in cells grown on, 460-461 Y Yeast, adenylate kinase (myokinase) in, 602 arginine-synthesizing system in, 357, 360, 362-364 asparaginase in, 384 cytochrome as in, 732, 734 cytochrome b~ (lactic dehydrogenase) in, 745 cytochrome e in, 745 5-dehydroshikimic reductase and 5-dehydroquinase in, 304, 307 DPN pyrophosphorylase from, 671 lactic dehydrogenase (cytochrome b2) in, 746 metaphosphatase in, 577, 578 phosphatase in, 638 inhibition by "pyrimidyl" and thiamine, 638 purina~cleeside phosphorylase from, 453 TPNH cytochrome c reductase from, 697 transhydrogenase in, 682 Yeast, baker's (top yeast), adenosine phosphokinase in, 498 alcohol dehydrogenase from, 660 I)PN assay with, 660 arginine desimidase from, 375-376 component of arginine condensing system from, 362-364 cytochrome b in, 744, 745 DNase from, 445-446 glutathione reductase from, 723-724 inhibitors of thaminokinase in, 640 inorganic pyrophosphatase from, 364, 570-576

plasmolysis of, 375, 376, 581,639 purine nucleosidase from, 462-464 thiaminokinase from, 639-640 triphosphatase in, 580, 581, 582 uridine nucleosidase from, 461-462 Yeast, brewer's (bottom yeast), adenosine phosphokinase from, 4975OO alkaline washed, preparation of, 637 thiamine pyrophosphate assay by carboxylase from, 636-637 aspartase in, 388 conversion of 5-amino-4-imidazolecarboxamide riboside to ribotide by enzyme from, 505, 514-516 FAD pyrophosphorylase from. 673675 flavokinase from, 640-645 glutathione synthetase from, 345-346 glycerol for extraction of, 581 new yellow enzyme from, 715-719 old yellow enzyme from, 712-715 plasmolysis by sodium chloride, 638 pyridoxal kinase from, 646-649 thaminokinase from, 638 triphosphatase in, 580, 581, 582 UDPG pyrophosphorylase from, 675677 Yeast, top ale, phosphorylating enzymes for glucose6-P preparation in, 699 T P N H cytochrome c reductase from, 699-703 "Yeast adenylic acid" (mixture of 2'and 3'-adenylic acids), prostatic phosphatase action on, 527, 528 Yeast extract (s), folic acid and CF content of, 630 effect of folic acid conjugase on, 630 as hydrogen donor for nitrogen gas formation, 422 Yeast protein, inhibition of yeast DNase by, 447 Yeast sodium nucleate, see Ribonucleic acid "Yeast uridylic acid" (mixture of 2'and 3'-uridylic acids), K,~ value for prostatic phosphatase action on, 527

SUBJECT INDEX

Yellow enzyme, new, from bottom yeast, 715-719 Yellow enzyme, old, from bottom yeast, 712-715 comparison with new yellow enzyme, 716 crystallization of, 714 non-identity of protein moieties of new yellow enzyme and, 718 rate constant for reaction with cytochrome e, 697 with oxygen, 697, 715 with TPNH, 715 Z

Zinc, as component of earboxypeptidase, 78 content in carbonic anhydrase from erythrocytes, 842, 843 from spinach, 843 content in uricase preparations, 489 deficiency, effect on Neurospora DPNase content, 666 purification of aminotripeptidase of thymus with, 85-86 requirement for formation of tryptophan synthetase, 237 Zinc ion, activation of alkaline phosphatase by, 538 of carnosinase by, 94, 96 effect of on pH optimum, 96 of citrullinase by, 378 of flavokinase by, 645 of glycyl-L-leucine dipeptidase by, 105, 106, 107 requirement of phosphate for, 107 time required for, 106 of metaphosphatase by, 579

987

effect on metaphosphatase content of A. niger, 579 inhibition of alkaline phosphatase by, 538 of amino acid amidase by, 399 of arginine desimidase by, 376 of aspartase by, 388 of ATP-creatine transphosphorylase by, 610 of 5-dehydroquinase by, 307 of DNase by, 442 of DPNH cytochrome c reductase by, 692 of fructose diphosphatase by, 546 of glutamic dehydrogenase by; 224 of glycylglycine dipeptidase by, 109 of leucine aminopeptidase by, 93 of pantothenate-synthesizing enzyme by, 621 of prolidase by, 105 of RNase by, 434 of D-serine (D-threonine) dehydrase by, 324 of splitting enzyme for arginine synthesis by, 367 of tryptophan synthetase by, 237 of uricase by, 489 low affinity of for Veronal and Tris buffers, 79 Zince~ ions, nonexchangeability with zinc of carbonic anhydrase, 843-844 *Zwischenferment, see Glueose-6-phosphate dehydrogenase

Zymobacterium oroticum, dihydroSrotic dchydrogenase from, 493-496 growth of, 494 Zymogen, 79, see also under names of individual proenzymes