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.

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[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.

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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

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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.

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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

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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.

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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.

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ENZYMES OF PROTEIN METABOLISM

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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).

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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].

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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

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oo

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~

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~. ~

•

~

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

~

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~

.

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

-

m ~.~

.=~

I

~,~

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.~

~I~,i 0 .~

m~ ~

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

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~

=

=

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.

.

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~~~

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.~ ~ . . ~~.; ~. ~. 0,~ •

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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).

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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).

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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).

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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).

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(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,

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ENZYMES OF PROTEIN METABOLISM

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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.

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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.

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ENZYMES OF PROTEIN METABOLISM

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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

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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).

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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

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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

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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.

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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).

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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).

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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

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ENZYMES OF P R O T E I N METABOLISM

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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.

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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).

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ENZYMES OF PROTEIN METABOLISM

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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).

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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

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ENZYMES OF PROTEIN METABOLISM

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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).

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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.

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ENZYMES OF PROTEIN METABOLISM

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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).

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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

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ENZYMES OF PROTEIN METABOLISM

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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.

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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).

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ENZYMES OF PROTEIN METABOLISM

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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

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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).

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ENZYMES OF PROTEIN METABOLISM

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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

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[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).

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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.

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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).

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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).

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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).

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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)

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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.

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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

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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

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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

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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.,

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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.

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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).

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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).

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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).

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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).

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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).

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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).

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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).

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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).

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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).

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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-~

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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).

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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).

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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

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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).

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ENZYMES OF PROTEIN METABOLISM

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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).

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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).

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ENZYMES OF P R O T E I N METABOLISM

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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).

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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

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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).

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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

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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).

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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).

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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).

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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

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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.

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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].

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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).

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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].

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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).

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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

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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.

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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).

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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

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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).

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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

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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.

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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).

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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).

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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.

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ENZYMES OF NUCLEIC ACID METABOLISM

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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

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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.

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[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]

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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

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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.

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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 META