Advances in Protein Chemistry: v. 10

ADVANCES IN PROTEIN CHEMISTRY

VOLUME X

This Page Intentionally Left Blank

ADVANCES IN PROTEIN CHEMISTRY EDITEDBY M. L. ANSON

KENNETH BAILEY

Cambridge, Massachusetts

University of Cainbridge Cambridge, England

JOHN T. EDSALL Biological Laboratories, Harvard Uni&ersity Cambridge, Massachusetts

VOLUME X

1955 ACADEMIC PRESS INC., PUBLISHERS NEW YORK, N. Y.

Copyright @ 1955, by ACADEMIC PRESS INC. 125 EAST 2 3 STREET ~ ~ NEW YORK 10, N . Y.

All Rights Reserved No part of this book may be reproduced in any form, by photostat, microfilm, or any other means, without written permission from the publishers.

Library of Congress Catalog Card Number, 44-8863

PRINTED I N T H E UNITED STATES OF AMERICA

CONTRIBUTORS TO VOLUME X

C. E. DALGLIESH, Postgraduate Medical School, Ducane Road, London, England G . H~MOIR,Laboratory of General Biology, University of LiBge, Belgium GERTRUDE E. PERLMANN, The Rockefeller Institute for illedical Research, New York, N . Y . JACINTOSTEINHARDT, Department of Chemistry, Massachusetts Institute o f Technology, Cambridge, Massachusetts BERTL. VALLEE,The Biophysics Research Laboratory of the Department o f Medicine, Harvard Medical School, and Peter Bent Brigham Hospital, Boston, Massachusetts LIONELA. WALFORD, Fish and Wildlife Service, United States Department of the Interior, Washington, D . C. CHARLES G. WILBER,Chemical Corps Medical Laboratories, Army Chemical Center, Maryland

ETHELM. ZAISER,Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts

This Page Intentionally Left Blank

CONTENTS CONTRIBUTORS TO VOLUME X . ..

...........................

v

The Nature of Phosphorus Linkages in Phosphoroproteins BY GERTRUDE E. PERLMANN, The Rockefeller Institute for Medical Research, New York, N . Y . I. Intxoductio .......................................... 1 from Phosphoprot,eins. . . . . . . . . . . . . . . . . . . . 2 111. Phosphoproteins and Phosphopeptones. . . . . . . . . . . . . . . . . . . . . . . . . . . 1V. Enzymatic Dephosphorylation of Phosphoproteins.. . . . . . . V. Possible Biological Function of Phosphoproteins. VI. Summary . . . . . . . . . . . . . . . . . . . . ....................................... 26 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolism of the Aromatic Amino Acids By C. E. DALGLIESH, Postgraduate Medical School, Ducane Road, London, England I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . ............................. 33 11. Biosynthesis of the Aromatic Amino ............................. 36 111. Degradation of Phenylalanine and Tyrosine to Acetoacetate ; the Principal Route Used by iVIammals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IIIA. Evidence Derived from Inborn Errors of Metabolism.. . . . . . . . . . . . . . . . . . 46 IIJB. Enzymic Experiments on the Normal Pathway in Mammals. . . . . . . . . . . . 55 IV. Tyrosine Degradation by the Catechol Pathway.. ...................... 65 V. Tyrosine Metabolism via Thyroid Hormones and Other Halogenated Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Pathways of Phenylalanine and Tyrosine Metabolism Utilized Princip by Microorganisms. . . . . . . . . . . . . . . . . . . . . . . . . . VII. Tryptophan Degradation by the Kynurenine-Ni VIII. Tryptophan Degradation by the Enteramine-Serontion Pathway.. . . . . . . 103 IX. Routcs for Tryptophan Degradation Used Principally by Microorganisms. 108 X. Tryptophan Metabolism in Plants. Heteroauxin. . . . . . . . . . . . . . . . . . . . . . . . 113 X I . Natural Products Probably Related to the Aromatic Amino Acids.. . . . . . 115 XII. Future Problems.. . . . . . . . . . . . . . . . . . . . . . . .......................... 121 XIII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................. Hydrogen Ion Equilibria in Native and Denatured Proteins BY JACINTO STEINHARDT A N D EwmL M. ZAISER,Department of Chemistry, Massachusetts fnslitute of Technology, Cambridge, Massachusetts I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Acid-Base Diesociations of Native Proteins.. . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Iinreactive Prototropic Groups in Native Prot.eins... . . . . . . . . . . . . . . . . . . . References.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

152 153 180

221

...

CONTENTS

Vlll

Fish Proteins BY G . HAMOIR.Laboratory of General Biology. University of Lihge. Belgium I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 I1 Proteins from Skeletal Muscle ........................... 228 I11. Fish Enzymes . . . . . . . . . . . . . . . . .............................. 269 IV Fish Blood Proteins ......................................... 273 V . Fish Protamines., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .274 . V I . Connective Tissue Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 VII . Conclusion: The Comparative Biochemistry of Fish Proteins . . . . . . . . . 279 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282

. .

The Sea as a Potential Source of Protein Food BY LIONELA . WALFORD A N D CHARLES G WILBER,Fish and Wildlife Service, United States Department of the Interior, Washington, D C and Chemical Corps Medical Laboratories, Army Chemical Center, Md . I World Protein Problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 . 303 11 Proteins in Marine Organisms., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I11. Variations in Protein Conten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312 IV . The Biological Value of Mari roteins . . . . . . . . . . . . . . . . . . . . . . . 313 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

.

. .

. .

Zinc and Metalloenzymes BY BERTL . VALLEE,The Biophysics Research Laboratory of the Department of Medicine, Harvard Medical School, and Peter Bent Brigham Hospital, Boston, Massachusetts A . General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318 I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318 I1. Metalloproteins and Metal-Protein Complexes . . . . . . . . . . . . . . . . . 380 I11. Characteristics of Metalloenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 IV . Characteristics of Metal-Enzyme Complexes . . . . . . . . . . 325 V . Empirical Formulas for Metalloenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . 327 VI . Instrumental Methods for the Detection of Metals . . . . . . . . . . VII . References to Metalloenzymes Containing Copper, Iron, an 332 denum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Zinc Metalloproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 V I I I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 TX . Carbonic Anhydrase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 X . Experimental Approach for Studies on the Leukocyte Zinc Protein, Carboxypeptidase, and Yeast Alcohol Dehydrogenase . . . . . . . . . . . . . 337 X I . The Zinc-Containing Protein from Human Leukocytes . . . . . . . . . . . . . 339 . . . . . . . . . . . . . . . . . . . 3-13 XI1. Pancreatic Carboxypeptidase . . . . . . . . . . . . . XI11. Yeast Alcohol Dehydrogenase . . . . . . . . . . . . . . . . . . . . 353 XIV . Coordination Chemistry of Zinc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .370 References.,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 AUTHOR INDEX . . . . . . . . . .

SUBJECTINDEX .........

. . . . . . . . . . . . . . . . . . . . . . . . . .

.....................................

:155

lo!)

The Nature of Phosphorus Linkages in Phosphoproteins BY GERTRUDE E. PERLMANN The Rockefeller Institute for Medical Research, New York, N . Y .

CONTENTS 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Phosphoamino Acids Derived from Phosphoproteins. . . . . . . . . . . . . . . . . . . . . . 1. 0-Phosphorylserine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 0-Phosphorylthreonine . . . . ...................................... 3. Phosphoarginine . . . . . . . . . . ...................................... 4. 0-Phosphorylserylglutamic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Phosphoproteins and Phosphopeptones. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Casein.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Phosphopeptones from Casein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Vitellin and Vitellenin.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Phosphopeptones from Vitellin and Vitellenin . . . . . . . . . . . . . . . . . . . . . . . . . a . Vitellinic Acid.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b . Ovotyrines . . . . . . . . . . . . . . . . ................................ 5. P h o s v i t i n , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Enzymatic Dephosphorylation of Phosphoproteins, . . . . . . . . . . . . .. 1. Ovalbumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................. 2. Casein.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................. 3. P e p s i n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................ V. Possible Biological Function of Phosph ........................ VI. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................ References . . . . ...............................................

1 2 2

3 3 3 4 4 5 6 6 6

6 7 9 11 16 22 25 26

27

I. INTRODUCTION The almost universal occurrence of phosphoproteins and their abundance in embryonic and rapidly growing tissue and in foodstuffs like milk and eggs initiated a study of this type of material more than fifty years ago. Our incomplete knowledge, however, is mainly due to the fact that none of the phosphoproteins thus far isolated satisfies the presently adopted criteria of purity of proteins-they are mixtures. They may have as few as one to two, or as many as thirty to fifty, phosphorus atoms per molecule. Their classification is based entirely on the fact that in contrast to the nucleoproteins, they lack purine and pyrimidine bases and contain the phosphoric acid esterified to an amino acid residue. Their main characteristic is that the phosphate groups are readily hydrolyzed in dilute alkali a t room temperature, e.g., in 0.25 N sodium hydroxide at 25"C., but that they are stable in acid under these conditions (74).

No attempt will be made here to review the extensive literature pertaining to the chemistry of phosphoproteins. 111 this article tlic subject will tie limited to the qucstion of the position of thc phosphorus in the nioleculr and to sonic of thc methods used in eliicidating thtl c*hemicbalnatiirr of thv bonds in whivh this clcment occurs. hicf survey The followiiig plan has hccii adoptcd for this discussioii. of: 1 . The rhemistry of three phosphoainino acids isolated from biological material. 2. The properties of the dipeptide 0-phosphorylserylglutamic acid. 3 . The phosphoproteiiis casein, vitellin, vitellenin, and phosvitin. 4. The phosphopeptones derived from milk and egg proteins. 5. The use of enzymes in the study of the nature of phosphorus bonds in phosphoproteins. 6. Remarks 011 the biological function of these materials.

11. PHOSPHO MINO ACIDSDERIVED FROM PHOSPHOPROTEINS Let us first turn to the question of how the phosphorus is bound to a protein. Both -0-Parid -N-Pesters present themselves as possibilities. Such linkages would involve either alcoholic or aromatic hydroxyls, on the one hand, or free amino groups or the guanido group of arginine, on the other. T o date, only three phosphoamino acids have been isolated from biological materials: 0-phosphorylserine and O-phosphorylthreonine, both representatives of an -0-Pester, and phosphoarginine, which has a -N-P-bond. In addition it has been possible to prepare phosphorylated derivatives of other amino acids, eg., tyrosine, oxyproline (37, 39), glycine, alanine, glutamic acid, leucine, and glycylglycine (98), and the methylester of N-phosphorylphenylalanine (40). 1. 0-Phosphorylserine

This amino acid C3HsOaNP (P = 16.7%) and with a n [a]:3of +7.2 (1) was first obtained by Lipmann and Levene from vitelliiiic acid (44). Lipmann subsequently succeeded in isolating this substance from an acid hydrolyzate of casein (45) and thus established that phosphoserine may occur as B constituent in phosphoproteins. More recently Agren, de Verdier, and Glomset crystallized 0-phosphorylserine (1). The following structure is usually assigned to this amino acid 0

\\

€1O-P-O-CH2

HO

I

/

CH

/ \

NH2

COOH

NATUHE OF PHOSPHORUS LINKAGES IN PHOSPHOPROTEINS

3

Here one has to keep in mind, however, the experiments of Bergmann and Miekeley, who showed that in the case of benzoylserine the acidity of the medium determines whether the benzoyl residue is linked to the hydroxyl or to the a-amino group of the amino acid (9). That a similar situation exists in the case of phosphoserine follows from the work of Plapinger and Wagner-Jaureggl (73). These investigators found tjhat on treatment of the N-diisopropylphosphoryl derivatives of the DL-serine methyl ester with boiiing aqueous hydrochloric acid O-phosphorylserine is formed from the Kcompound. Therefore, the existence of phosphoserine with a -N-P-bond in a native protein is feasible, e.g., as the N-terminal amino acid of L: peptide chain. 2. O-Phosphorylthreonine

The second phosphoaniino acid with mi -0-Plinkage, phosphothreonine C4Hlo06NP(P = 15.5%), [ag4 -7.37, was isolated from aii acid hydrolyzate of casein by de Verdier in 1953 (94). Plapinger and Wagner-Jauregg showed that, as in the case of phosphoserine, migration of the phosphate group from the N to the 0 position also occurs in the case of this compound (73). 3 . Phosphoarginine

CsH160&J4P (1' = 11.5 %) is the only amino acid with a -N-Pbond thus far encountered in biological material (57). Its presence in the muscles of invertebrates suggests that this substance fulfills a role similar to that of creatinephosphate in vertebrates. The function of these two compounds as reservoirs of readily available energy is coufirnied by experimental facts (46). However, it is still uiikiiown whether or not phosphoarginine occurs in phosphoproteins. One of the characteristic features of these three phosphoamino acids is that in contrast to the intact proteins, the phosphate group is stable in 0.25 N sodium hydroxide (72). The N-P bond of phosphoarginine, however, is acid-labile.

4. O-PhosphorylserylglutamicAcid I n 1933, shortly following the discovery of phosphoserine (44), Schmidt, and Levene and Hill isolated from a casein hydrolyzate a dipeptide consisting of serine, glutamic acid, and phosphoric acid (38, 84). I n 1941, Posternak and Pollaczek demonstrated the presence of a free a-amino group in the serine moiety of the molecule and assigned the following structure 1 The possibility of a phosphate migration in phosphoserine from the Nto the 0- position had already been suggested by LinderstrZm-Lang in 1933 (Linderstr$mLang, Ii. (1933). Ergeb. Physiol. u . Exptl. Pharmakol. 36,415.)

4

GERTRUDE E. PERLMANN

to the dipeptide (77) COOH

I I CBZ I CH / \ CH,

0

\\

HO-P-O-CHe

I

/

CH

€10

N 11,

/

\

CO-NII

COOH

Moreover, these investigators showed that the pept ide bond of phosphorylserylglutamic acid was resistant to the action of a dipeptidase from pig intestine. However, after removal of the phosphate group with the aid of kidney phosphatase the dipeptidase readily hydrolyzed the peptide bond. It is thus clear that the phosphate group has a protective action on the peptide linkage, and one can conclude that not only the type of linkage but also the surrounding molecular configuration determines whether an enzyme will act.

111. PHOSPHOPROTEINS AND PHOSPHOPEPTONES Following this discussion of some of the properties of three naturally occurring phosphoamino acids and of the dipeptide, O-phosphorylserylglutamic acid, a few examples of phosphoproteins, i.e., casein, vitellin, vitellenin, and phosvitin, and of the phosphopeptones derived from these materials, will be considered. 1. Casein

Casein, the most important and most thoroughly studied phosphoprotein, accounts for 80 % of the total nitrogen of cow’s milk (56) and about 30% of that of human milk (8). The interest of a great number of investigators in this protein may have been stimulated in part by the ease with which it may be separated from the other milk proteins by acidification to pH 4.6 (26). For a long time casein was considered to be a pure protein (15, 26). As will be discussed in a later section of this chapter, its inhomogeneity was, however, demonstrated as early as 1927 by LinderstrGm-Lang and Kodama (41). Since then it has become clear that the products obtained during fractionation are determined by the method of purification. The different procedures, as well as the most commonly used ones of Warner (95) and of Hipp and co-workers (30), have been reviewed recently by McMeekin (51, 52) and will not be discussed here. Electrophoretic analyses of the acid-precipitated casein (Hammarsten

NATURE OF PHOSPHORUS LINKAGES I N PHOSPHOPROTEINS

5

casein) indicate its heterogeneity (54, 95). I n addition, there is in the literature a wide divergence among the values for its molecular weight. Svedberg, Carpenter, and Carpenter (91) dctermined a molecular weight of 75,000 t o 100,000 and a sedirncntation constant of Szo = 5.6 X In contrast, Pedersen (63) reported a value of SZO = 12 X 10-13. The type of buffer used by these two groups of investigators differed, and this may explain the discrepancies in the sedimentation constant. Burk and Greenberg (12), with the aid of osmotic pressure measurements, found a molecular weight of 33,000 for casein in 6.66 M urea. It is apparent, therefore, that this protein undergoes association in the presence of salts. If 33,000 is assumed to be the minimum molecular weight, the acidprecipitated casein-complex with 0.8 % of phosphorus contains eight atoms of this element which are labile in 0.25 N sodium hydroxide a t 25°C. (15). The phosphorus resists the action of purified phosphomonoesterases (3, 79, 85). The fission of a few peptide linkages, however, with the aid of proteolytic enzymes renders some of the phosphate groups accessible to enzymatic hydrolysis (78, 79). In contrast to these findings are reports of Travia and Veronese (93), Lofgren (47), and others, who state that intact casein can be dephosphorylated b y phosphatases of mammalian origin. The findings of these investigators, however, may be due to the fact that crude extracts of spleen or kidney were used as enzyme source and that such preparations were contaminated with proteolytic enzymes. 2. Phosphopeptones from Casein

On treatment of casein with the aid of proteolytic enzymes, phosphopeptones are formed. However, there is a striking variation in the composition of the individual peptones. Posternak (75) isolated from a tryptic digest of cow’s casein a phosphopeptone with 5.9 % phosphorus and 11.9 % nitrogen. Moreover, he was able to demonstrate the presence of glutamic and aspartic acids, isoleucine and serine. This, as well as similar findings on a phosphopeptone from vitellin, led him t o suggest that the phosphorus in casein and vitellin was esterified in part a t least to serine (75, 76). Independently, Rimington and Kay (79) also prepared a phosphopeptone from a partial tryptic hydrolyzate of casein. Their product, however, contained 7.05 % phosphorus and 10.13 % nitrogen. Using the peptictryptic digestion method of Damodaran and Ramachandran (16), Mellander obtained the barium salt of a peptone with 5.35 % phosphorus and 7.05 % nitrogen which contained half of the original casein phosphorus (55). In view of these variations in the composition, i t is clear that there are striking differences in the chemical structures of these products. As will be discussed below, the following facts emerge from the work of two groups of investigators. Rimington and Kay demonstrated that

G

GEltTltUDE E. I’EltLMANN

casein resisted the action of phosphatases. 111 contrast,, hoivever, their phosphopcptonr preparations, which consisted of tcri to twelw amino acid residucs, ~vcrcreadily dcphosphorylattd. Thus, kidiicy phosphat:rw rcmoved all of the pcl~toiic-pbospliorus,mhercas only ti\-o-thirds was libcrated by t,he act ion of boiw phosphatasc (79). ‘l’hcsc investigators cxplain their results oil thc basis of cnzyinc specificity for crrtain types of bonds, e g . , that part of the phosphorus may be present in the configuration of nionoesters, whereas some of it is a different type of linkage (80). In their work, Posternak and Pollaczek draw attention to the fact that both the rrlative position of a phosphate group in the peptide chain arid tlic adjacent amino acids may be the factors determiiiiiig whether or not (*w zymatic hydrolysis occurs. These authors isolated two phosphopeptoncls, each of which contained three phosphate groups. Oiie of these, phosphopeptone I, consisted of ten to eleven amino acid residues, whereas phosphopeptone II had only seven. From phosphopeptorie IT all three phosphate groups are liberated with the aid of kidney phosphatase, whereas this enzyme removes only two of the phosphorus atoms of peptone I (78). Moreover, it is of interest that, as in the case of the dipeptide, O-phosphorylscrylglutamic acid, an intestinal aminopeptidase did not act on these peptones but that after removal of the phosphate groups with the aid of a phosphatase some of the peptide bonds were hydrolyzed by the actioii of the proteolytic enzyme. 3. Vitellin and Vitellenin

Whereas casein has been investigated rather extensively, little is known about the phosphoproteins from eggs, i.e., vitellin, vitellenin, and phosvitin, respectively. Two of these proteins are present in combination with phospholipids representing 25% of the egg yolk solids (2, 14, 62). After removal of the lipids with 80 % ethanol, vitellin and vitellenin are obtained with a phosphorus content of 1% and 0.29 %, respectively. Nothing is known about the homogeneity of these preparations. Since the literature pertaining to the properties of these compounds has been presented in the recent review of Fevold (2l), only a few observations which have a bearing on the present discussion will be recapitulated here.

4. Phosphopeptones from Vitellin and Vitellenin a. Vitellinic A c i d . Levene and Alsberg prepared from vitellin a peptide of high phosphorus content by extraction with 12 % ammonium hydroxide (36). After neutralization with acid and removal of the nitrogenous material with picric acid the copper salt, of vitellinic acid was obtained. This complex contained 9 % to 10% phosphorus and 0.6% iron. The phosphorus was labile in weak alkali at room temperature. b. Ovotyrines. I n coiitrast to the findiiigs of Levene and Alsberg (30)

NATURE O F PHOSPHORUS LINKAGES I N PHOSPHOPROTEINS

7

TABLE I CowLposition of Digestion Products 01 V i t e l l i ~ Composition in per cent

a

Compound

N

I’

Fe

Ovotyrine CY Ovotyrine p1 Ovotyrine 8 2 Ovotyrine y

10.87 11.33 10.92 10.70

13.76 12.55 12.09 7.90

None None 3.31 None

Taken from Posternak (761.

digestion of vitellin with the aid of proteolytic enzymes yielded three fragments which were designated as a-,p-, and y-ovotyrine (76). Ovotyrine p could be fractionated into p1 and pz . The composition of these substances is given in Table I. On the basis of the iron content these investigators identified their ovotyrine 02 with the vitellinic acid of Levene and Alsberg. Moreover, they emphasized that the high content of serine in ovotyrine pz (i.e., vitellinic acid) indicated that the phosphorus-containing unit of vitellin was phosphoserine. As discussed earlier this suggestion then led to the isolation of O-phosphorylserine from vitellinic acid (44). I n 1934, Blackwood and Wishart studied the action of pepsin and trypsin on vitellin (11). According to these workers pepsin splits the protein into a phosphorus-poor and a phosphorus-rich fraction. The acid-insoluble portion with 73% of the total phosphorus and a ratio of P/N of 3.65 was resistant t o further action of the enzyme. illthough treatment with trypsin also yielded two fragments, tryptic digestion differed in that the acid-soluble residue contained only 30% of the protein phosphorus and had a ratio of phosphorus to nitrogen of 1.5. I n the light of the work of Hergmann and Fruton a great deal of knowledge has been gained on the specificity of proteolytic enzymes for certain peptide bonds (10). Thereforc, the appareut contradiction of the results of Blackwood and Wishart is not surprising. I n addition it should be pointed out that the crude proteolytic enzyme preparations used by these investigators may well have contained small amounts of phosphatases. Fission of certain pc.ptide linkages, through the action of one enzyme but not by the other, thus may have rendcrcd some of the vitellin-phosphorus accessible to subsequent hydrolysis by phosphatases. 6. Phosvitin

As sho~vnby Mecham arid Olcott (53) 6 % of the total solids of egg yolk consists of the protein phosvitin with 10% of phosphorus, i.e., GO% of the

8

GERTRUDE E. PERLMANN

phosphoprotein-phosphorus of egg yolk is in this fraction. Although homogenous in the ultracentrifuge, phosvitin has at least two eleetrophoretic components. On the basis of osmotic pressure measurements, this protein has a molecular weight of 21,000. I n the presence of magnesium sulfate a value of 38,000 was obtained both with the aid of ultracentrifugation and by osmotic pressure measurements, indicating an aggregating effect of this salt on the protein. Amino acid analyses of phosvitin preparations also indicated inhomogeneity. It is striking, however, that an equal number of P-hydroxyamino acids and of phosphorus atoms were found to be present. The phosphorus is alkali-labile at room temperature. Phosvitin is readily dephosphorylated with the aid of a n acid phosphatase from citrus fruits (5). Bone phosphatase, on the other hand, does not liberate phosphorus. Moreover, the base binding capacity of this protein indicates that all of the phosphate groups are present in the form of monoesters with two dissociable hydroxyls. Although not reported here in detail, a striking feature emerges from the early work on phosphoproteins and peptones. Invariably, amino acid analyses revealed that the portions of the peptide chains which contain the phosphorus-probably esterified to serine-always are rich in leucine, isoleucine, aspartic and glutamic acids (78, 79). As will be discussed later in this article, these observations and some recent results indicate that certain types of amino acid sequences recur in all phosphorus-containing proteins. Another point of interest is the following : in the preceding paragraphs the lability of the phosphate groups of phosphoproteins in 0.25 N sodium hydroxide a t 25°C. has been stressed repeatedly. Although normally esters of phosphoric acid are resistant to the action of dilute alkali, Fond has shown that the monoesters of the glycerophosphates are readily hydrolyzed in this medium (23). As pointed out by Todd (92) the simplest explanation of this phenomerion is the formation of cyclic intermediates, i .e., triesters of phosphoric arid; such compounds are readily hydrolyzed in weak alkali. It, therefore, is feasible that the instability of the phosphoproteins in 0.25 N sodium hydroxide at rooin temperature may be due to the fact that also in these materials the phosphorus may undergo intxamolecular migration through cyclization involving adjacent groups, i.e., -NH2, -OH, or -COOH. The formation of such cyclic compounds which are known to be unstable is favored a t alkaline pH values and would explain the alkali-lability of the phosphate group in the protein in contrast to its stability in the phosphoamirro acids (72). Likewise, it is not improbable that a reverse situation cbxists in the case of an K-P bond. Moreover, it is not unlikely that the lability of an N-P linkage in dilute acid

NATURE O F PHOSPHORUS LINKAGES I N PHOSPHOPROTEINS

9

decreases considerably or is lost if the N-phosphorylamino acid residue is present in a peptide bond. From these observations it follows that (1) some of the chemical properties of a phosphoaniino acid may change considerably on incorporation into a peptide or protein, (2) that the adjacent molecular configuration may be responsible for the stability of the phosphate group, and (3) that the acidity of the medium determines whether migration of a phosphoric acid residue from the -0to the -Nposition occurs. As outlined in a previous section in this article N-phosphorylserine and N-phosphorylthreonine, respectively, would represent possible configurations of the N-terminal amino acid of a peptide chain in a native protein.

IV. ENZYMATIC DEPHOSPHORYLATION OF PHOSPHOPROTEINS Considerations of this kind led the author to undertake a n investigation using enzymes t o reveal the chemical nature of phosphorus bonds that may occur in phosphoproteins. This interest came through the accidental observation that a variety of phosphomonoesterases of mammalian origin and from plants will dephosphorylate ovalbumin, a protein with a low phosphorus content. Of course, a prerequisite in the selection of the enzymes for such work is that the dephosphorylation process should not be accompanied by any other enzymatic reactions that might result from the presence of small amounts of impurities in even highly purified phosphatase preparations; in particular, an extensive proteolysis has to be excluded. The phosphomonoesterases that proved most useful in this work, although free of proteolytic impurities, were found to be complex in their behavior toward phosphate esters. As indicated in Table 11, if tested with the aid of low molecular weight substrates, the intestinal (85) and the potato phosphatase (34) act on 0-P and N-P bonds, whereas the prostate enzyme (86) hydrolyzes only 0-P linkages.2 After the discovery of the specificity of two of these enzymes for low molecular weight N-P esters, it was noticed that the intestinal enzyme, although classified in the literature as “alkaline” phosphatase, hydrolyzes N-P bonds both a t p H 5.6 and 9.0, but not at pH 7.0. Since the pH range of 5 to 6 is that of maximum stability of almost all proteins, most experiments were carried out in this pH range. Thus the use of these three enzymes, either alone or in combination with each other, proved to be quite a powerful tool. A less desirable feature, however, also indicated in Table 11, is that these three phosphatases contain ah impurities siiiall amounts of phosphodicsterase and pyrophosphatasc. In contrast t o the monoesterases these eiizyiiic’s require the preaeiice of magnesium ions and act at pTI values 1 IR a recent article Max-hlgiller reported that acid phosphatase from seminal plasma hydrolyzes both 0-P and N-P esters (50).

10

GERTRUDE E. PERLMANN

TABLE I1 Dephosphorylation of Low Molecular U‘eight Phosphate Esters as Function o,f p H ~

Type of Phosphate Bond ~~

0

// -0-P4H

Enzyme

\OH

PfI

Prostate phosphatase Intestinal phosphatase

+

5.6 7.0 5.6 7 .o

-

+ +

5.6

2I

0

0

II

\OH

II

-0-PUP4-

dH

dH

dH

f

f f f

+f + +

0

II

-0-P-0-

-

=t

9 .o

Potato phosphatuse

0

// -N-P4H

+ active, f slightly active, - inactive. //

0

-0-P-OH

: 8-glycerophosphate,serine phosphate. oxyproline phosphate

\OH 0

// -N -P-OH

A

0 : N-(p-chlorophenyl) amidophosphate

\OH

II

-04-0-

A

: p-bis(nitrop1ienyl)phasphate

different from those a t which the moiioesterases are active. Hence their interference with the monoesterase activity could be avoided and their preseiice did not complicate the work. I n addition to phosphomonoesters, the occurrence, in these materials, of 0 0

I

II

linkages such as those of pyrophosphates, -0-P-0-P-0-, phospho0 0 I I I II OH or1 tlicstcrs, -0--1’-0- or -N-P-0-, aiid thc preseticc of thew holds

1

I

I

OH I3 013 i i i cyc*licarraiigelueiil , have to he anticipated. Thus it should be possihlc to deiiioiistrate such structural units with the aid of specific eiizymes which hring about their triLiisforniatioti into niorioesters that are then readily hydrolyzed I)y the wtioii of phosphonioiioc,sterascs. l‘hc purified phosphodiesterasc froiii rat1 lrsiiakc vetioiii, (‘rotulits udainuritms (ST),acting o i i thv -0-I’ 1)oiicl of ti tlic~htt~, slid the crystalliiw pyrophosphzltase f r o ~ n yeast (35) should Ix specific for thrh type of linkages ~nentioiic~l ahovc. Iiere one has to keep i i i n i i i i d , however, that not oiily thc spwifivity of :in c~iieyiiic~ for a. wrtaiii lmid hut also its adjucriit inolrci~larconfiguration may determitic slid modify the riizymatic action. This f:wt has recwitly bccii dcmotistratc>d experimciitally by Dekker (18). ~

NATURE O F PHOSPHOIIUS LINKAGES I N PHOSPHOPROTEINS

11

The author's work has been developed according to these general ideas mid will be presented as follows: 1 . A study of the dephosphorylation of ovalbuiniri, a protriii with the phosphorus in form of monocsttrs. 2 , An investigation of two typicd phosphoproteiiis, a- aiid fl-caseiii, which coiitaiii their phosphorus us moiioestcrs, diesters, and pyrophosphates. 3 . Work on the phosphorus of pepsin and pepsinogen. 1. Oualhumin

That ovalhmin is a phosphorus-containing protein was first demonstrated in 1900 by Osborne and Campbell ( 6 2 ) and was later substantiated

a.

t

Time in hours

,A1

Time in Electrophoretic composition hours

Atoms phosphorus per mole protein Computed Observed

85% AI 14% A9

trace AS 58% At 40%A2 trace A3 47% A1 49% A2 4% Aa 36% AI 58% Az 6%A3 94%Az 6% A3

1.8,

1.82

1.56

1.48

1.43

1.35

1.30

1.20

0.9,

0.9,

FIG.1. Dephosphorylation of ovalbumin with prostate phosphatase as a function of time (taken from PerImann (68)). Each reactmionmisture contained 4.6% ovalbumin and 0.01% enzyme. Electrophoresis was carried out in sodium phosphate buffer of p H 6.8 and 0.1 r/2 for 12,600 seconds a t 6.2 volts per centimeter.

12

GERTRUDE E. PERLMANN

by Sgreusen and collaborators (49). In 1949, Linderstrgm-Lang and Ottesen suggestml (43) that thc failurC2 of the phosphorus content of ovalhumin to correspond to an integral number of atoms per inolecule if the molecular weight were 24,000 could be correlated with the electrophoretic complexity of this protein (48). Thus the major, fast-moving component A1 should contain two phosphates a i d the slower moving protein A2 , one. The hypothesis advanced by the Danish workers mas coilfirmed by the experiments presented in Figs. 1 and 2. As shown in Fig. la, prostate phosphatase releases about 46% of the ovalbumin phosphorus. If the liberation of this element is followed with the aid of electrophoretic analyses (Fig. l b ) , it becomes apparent that the component A1 is transformed into

Time in Electrophoretic composition hours

A1

0 *2

d

85% Ai 14% A2 trace As

Atoms phosphorus per mole protein Computed Observed

1.84

1.82

0.97

12

50% Az 40% Aa

0.50

0.58

lo%*

0

FIG.2. Dephosphorylation of ovalbumin with intestinal phosphatase as a function of time (taken from Perlmann (68)). Each reaction mixture contained 4.6% ovalbumin and 0.006% enzyme. Electrophoresis was carried out in a sodium phosphate buffer of pH 6.8 and 0.1 r/2 for 12,600 seconds a t 6 volts per centimeter.

* Probably

due t o proteolysis.

13

NATURE O F PHOSPHORUS LINKAGES I N PHOSPHOPROTEINS

a protein with an electrophoretic mobility similar to that of Az , which has one phosphorus atom per molecule (65). If, on the other hand, either the intestinal phosphatase a t p H 9.0 or the potato enzyme a t p H 5.6 are added t o ovalbumin, the reaction is more complex. The protein is rapidly dephosphorylated until 46 % of the phosphorus is released (Fig. 2a). Here, A1 is again converted into a protein with the properties of A2 . Dephosphorylation, however, eoiitinues and a iicw component, AS, appears which moves more slowly than A2 and is a phosphorus-free ovalbumin (line 4, Fig. 2b). Thus: Proytnte intestinnl or potato) phosphata’se

Oval bumiii (85% A1:2P/mole) (15% At:lP/mole)

Intestinnl or yotnto

,

phosphatltse

2‘

AI loses 1 atom of phosphorus

-42 loses 1 atom of phosphorus

-

At

One point of interest resulting from these experiiiients is the difference in the electrophoretic behavior of A 1 , Az , and A3 . In general, as the p H of a protein solution is increased, various groups within the protein molecule

I

4.0

I

5.0

I

I

6.0 7.0 pH - 0°C.

I

8.0

I

9.0

FIG.3. Mobilities of the ovalbumin component A1 and the dephosphorylated ovalbumin At and A2 as function of pH.

14

GERTRUDE K . PKltLMAITK

lose their protons. Therefore, a coniparisoii of the electrophoretic mobilities of two proteins that are almost identical, except that one is formed from the other by removal of a few charged groups, gives a qualitative picture of the type of groups involved. Morcw-er, as show1 helow, at a given pH the evaluation of the number of groups lost i n the reactiori is possible. In Fig. 3, the electrophoretic niohilities of A, , h2, and A 3 are plotted as a function of pII. 'L'hescl curves diverge until a coilstarit mobility difference of Au = 0.6 X l W 5 is reacbhecl in the p1-I range of 7.0 to 9.0. If this mobility differelwe is corrclated with the haw hidiiig capacity of ovalbumiri (13), Au corresponds to a charigeiu the net charge of -2 (64, 6'3). I t , therefore, can be inferred from these measurements that the phosphate groups of A1 and A:! in the pH raiige of 7.0 to '3.0 are present as nionoesters with two ionized hydroxyls. In the pH range of 4.5 to 5.0, however, the mobilities differ by a value of 0.3 x lW5. Here the removal of each phosphorus is accoiiipaiiied by the loss of one itegzbtive charge only, RS indicated in Fig. 3. That the phosphorus is present as a monoester is further supported by the finding that on pretreatment of ovalbuinin with the phosphodiesterase from snake venom and subsequent iricubatiori a t pH 5.6 with prostate phosphatase, the same amount of phosphorus is released as with this enzyme alone, i.c., 4G %. Moreover, no change in the electrophoretic behavior occurs (72). The presence of a diestm in ovalbuinin haviitg thus been excluded, 011 Iht. basis of electrophoretic aiid enzymatic evidence, the failure of the prostate enzyme to remove the remaining phosphorus may be taken as a11 indicatioii either that the two phosphate groups of A 1 arc csterified to two different amino acid residues of the protein or that the adjacent molecular configuration is different in the two cases aiid renders one of the phosphate groups inaccessible to the action of the prostate enzyirie. In this coiiriection it is of interest that if ovalbumin is digested with pepsin at pH 1.5, followed by treatment with trypsin and chyniotrypsiii at pH 5.0, prostate phosphatasr still fails t o remove more than 46% of the phosphorus. Moreover, 0 1 1 hydrolysis of the phosphorus both of the intact protein and the proteolytic digest with 0.25 N sodium hydroxide at 37"C., liberation of 46% occurs rapidly, followed by a much slower release of the remaining phosphorus. This indicates that even on extensive degradation of the protein to polypeptides, no additional phosphorus becomes more readily accessible to the action of the enzyme, nor does the kinetics of hydrolysis in 0.25 N sodium hydroxide change ( 7 2 ) . On fractionation on starch columns of a partial proteolytic digest such as that described above a peptide fraction was isolated containing aspartic,

NATURE O F PHOSPHORUS LINKAGES I N PHOSPHOPROTEINS

15

acid, glutamic acid, alaniiie, leucine, and seriiie and having about 50 % of the phosphorus of thc digest (68, 7 2 ) . I t thus caould bc c~onc~luded that half of the ovalbumin phosphorus is present :is phosphoserinr. Recently t l i ~orcurreiicbo of phosphoserinc i n ovalhumin has hren confirmed by Iplavin (22). I n addition to phosphosrrinc Flavin s i u from it partial acid hydrolyzatc the follon-ing pcpl ides: hsp.SerP; Asp.(Glu, SerP) ; Asp.(Crlu,Ileu,SerP) ; SerP. (hla,Glu,Ileu); Asp.(Ala,Glu,Ileu,SerP) ; SerP.Ala; aiid possibly Glu. (Ala,SerP).3 After dephosphorylatioii of ovalbumiii with the prost ate phosphatase, SerP.Ala aiid Glu.(Ala,SerP) could no longer be detected in the acid hydrolyzate. He therefore coricludes that the one phosphoric acid residue hydrolyzed by the prostate phosphatase is csterified to seririe and is present in the sequence Glu.SerP.Ala. One point of interest emerges from these experiments, namely, that ovalbumin is the secoiid protein from which phosphoserine has been isolated. As in the case of the dipeptide, SerP.Glu, isolated from casein (38,77) ovalbumiii also contains an amino acid sequence with phosphoserine adjacent t o a dicarboxylic acid. Moreover, the close association of this amiiio acid with aspartic and glutamic acids, isoleuciiie and alariiiie in ovalbumin (22, 68) and casein (78-80) suggests the existence of a systematically recurring sequence in phosphoproteins. The nature of the second phosphorus bond in A 1 is still uncertain. The observations made in this laboratory that the intestinal enzyme a t pH 5.6 hydrolyzes low molecular w i g h t substrates with N-P bonds only would suggest that thc sccoiid phosphate of ovalbumin is ail N-P ester, e.g., that the phosphorus is linked to the guanido group of arginine, t o the e-amino group of lysine, or t o a terminal a-amino group. 111 contrast t o this view based on the onzymatic findings are, however, the results of Flavin, who, in addition to the amino acid sequence Glu.(Ala,SerP), found a small amount of a second phosphoserine-(.oiitainiiig peptide Asp. (Glu,SerP) in the partial acid hydrolyzate of ovalbumin. From this he concludes that the second phosphorus of A1 is also linked to seriiie but that the adjacent amino acids are diffrrcrit. It is of course feasible that such a specific molecular configuration, i.e., Asp.(Glu,SerP), may modify the enzymatic reaction. However, in view of the increasing evidence of amino acid migration and rearrailgenielit of amino acid sequenres during acid hydrolysis (82, 83), caution has to bc exercised in drawing final conclusions as to the 3 The abbreviations for amino acids and the conventions for indicating their sequence in a peptide are those of Sanger and Tuppy (81) ;in a known sequence of amino acid residues, the amino acid symbols are separated by periods; if t h e sequences of amino acids in a peptide are unknown, they are enclosed in brackets and are separated by commas. SerP = 0-phosphorylserine (22).

1G

GERTRUDE E. PEHLMANN

nature of this second phosphorus bond until additional experimental evidence has been obtained. Esterification of the phosphorus to the polysaccharide moiety of the protein, however, can be excluded, since it was possible to isolate from a partial proteolytic hydrolyzate of ovalbumin a peptide fraction that contained all of the carbohydrate but was phosphorus-free (72).

2. Casein Although it has been reported in the literature (3, 79, 85) that casein is resistant to the action of purified phosphomorioesterases from mammaliati tissues, this protein was nevertheless chosen as an example of a typical phosphoprotein for the reasons outlined below: That so-called acid-precipitated casein (Hammarsten casein) is a mixture of several distinct proteins was first demonstrated by the solubility studies of Linderstrgm-Lang and Kodama (41). This led to numerous attempts to fractionate this protein into its components, and in 1929 LinderstrgmLang achieved a separation into three fractions characterized by a phosphorus content of 0.96 %, 0.52 %, and 0.1 %, respectively (42). Mellander, in his electrophoretic stJudies,showed that casein has three electrophoretic components which he designated as a-, p-, and y-casein (54). It was, however, only in 1944 that Warner succeeded in preparing a- and p-casein, proteins with a phosphorus content of 0.99 % and 0.6 %, respectively, the a-protein being present in the original mixture in concentrations of 75% to 80% (95). As can be seen from the electrophoretic patterns shown in Fig. 4, these two proteins, although not homogenous over the entire p H range, were distinct fractions, neither of which was contaminated with the

a.

a

A

Unfractionated Casein 0 a a d

-a

&

Y

d

a-caaein b . 1

d

-a

d-

D 1

C.

-a

a

A

E

p-casein 6

&

-

0

L

d -

FIG.4. Electrophoretic patterns of unfractionated casein, a-casein, and @-casein (taken from Perlmann (69 )). Electrophoresis was carried out i n sodium phosphate buffer of p H 6.8 and 0.1 r/2 for 14,400 seconds a t a potential gradient of 4.95 volts per centimeter.

17

NATURE OF PHOSPHORUS LINKAGES IN PHOSPHOPROTEINS

other (95). Amino acid analyses of the caseins revealed that the rat,io of the basic to the acidic amino acid residues is the same in both cases, namely, TABLEI11 Composition of a-Casein and @-Casein ~~~

Total nitrogen, per cent5 Total phosphorus, per cent= Total cationic groups, equivalents/l06 g . proteina Total anionic groups, equivalents/l06 g. protein" Cationic groups/anionic groups u X lo6 cm.* set.-' volt-lb

a-Casein

@-Casein

15.53 0.99 115 112 1.03 -7.5

15.33 0.61 91 88 1.03 -3.4

Taken from Gordon, Semmet, Cable, and Morria (26).

* Electrophoresis in sodium phosphate buffer, pH 6.8, 0.1 r/2. 1.03 (25). From this it is apparent that a-casein, the protein with the higher phosphorus content and an electrophoret#icmobility which a t p H 6.8 in a sodium phosphate buffer of 0.1 ionic strength is considerably more negative than that of p-casein, might possess a number of phosphate groups as monoesters. Therefore, a phosphomonoesterase should act on this protein but not on &casein. As shown in Table IV, prostate phosphatase liberates 40 % of the a-casein phosphorus but has no effect on the p-protein. On prolonged exposure of unfractionated casein t o the enzyme about 12 % of the total phosphorus is liberated (66). These findings were recently confirmed by Sundararajan and Sarma (90). TABLE IV Action of Prostate Phosphatase on Casein Fractions Each reaction mixture contained 0.5% protein and 0.005% enzyme in sodium cacodylate buffer of p H 6.1 and 0.1 r/2. Phosphorus released by Time of enzyme (per cent Phosphorus Incubation at of total proteincontent, (%) 37°C. (hours) phosphorus) Unfrrtctionutrd Csseiii

0.8

U-Caseiii

1.o

6 24 6

21 $-Casein

0.6

6 24

0 12.5

24 42 0 0

18

GERTRUDE E. PERLMANN

I n experiments in which a- and p-casein are remixed in different proportions, it is noticed that if the relative concentration of the p-casein in tile mixture exceeds 20 % the presence of this protein inhibits the enzymatic action, the degree of inhibition being proportional to the concentration of the 0-protein. These results inay be taken as an explanation for the failure of previous investigators to dephosphorylate unfractionated casein without preceding transformation to phosphopeptones. As in the case of ovalbumin, the dephosphorylation of a-casein is accompanied by a change in the electrophoretic behavior. With the aid of Fig. 5, it is illustrated that the liberation of phosphorus is accompanied by thc appearance of several new componeiits with lower mobilities. The mobility decrements of these components at pH 6.8 are 0.5 X l W 5 or a multiple thereof. If compared with the base binding capacity of a-casein (31) this value corresponds to ;t change in the net charge of -2. This supports the initial assumption that some of the a-casein phosphorus is present in form of monoesters. a - casein

I\

6

s a-

I

6hOUP5 a -

Q

6

d.

c-----+

Fit,. 5 . Tracings ot electrophoretic patterliS of a-Cabeln Iwloic :LMI alter treat ment Kith prostate phosphatase (taken from Pel lmann (6!))) F:lectrophoreqis \\its carried out in sodium phosplintc hultcr o i p I I (j X : i n t i 0 1 1'/2 for 10,800 seconds at 4 75 voIts per centimeter.

10

NATUHE OF PHOSPHORUS LINKAGES IN PHOSPHOPliOTEINH

The observatioii that prostate phosphatasc liheratrs only 40 % of the a-casein phosphorus and doc5 not act on S-casein foreshadowed the exist ence of phosphodiesters and pyrophosphate bonds in these proteins. Thus the action on these materials of enzymes specific for such bonds should cstablish not only the nature of the linkages but also the proportions in which they occur. The results of such stepwise enzymatic analysis can be best followed with the aid of Table V. If a-casein is treated with either the crystalline pyrophosphatase of yeast a t pH 7.0 (35) or with the snake venom diesterase a t pH 8.2 (87), no inorganic phosphorus is released. However, if the diesterase reaction is carried out in weakly buffered solutions a small drop of p H takcs place (71), indicating the exposure of acidic groups. Subsequent incubation of the diesterase-treated a-casein with prostate phosphatase a t pH 6.0 liberates no more phosphorus than in the absence of the diesterase. If, however, prostate and intestinal phosphatase are added, 78% of the a-casein phosphorus is set free. Since the intestinal enzyme at pH 6.0 acts on low

Yo

molecular weight substrates of the type -JS-P-OH but not on 0 I \ // H OH -0-P-OH, it can be inferred from these results that a-casein contains

\

OH diester linkages of the -N-P-0type (71). If pretreatment of a-casein with pyrophosphatase is followed by incubation with the prostate enzyme, approximately 60% of the phosphorus is liberated instead of the 40% with the prostate phosphatase alone. Thus the difference of 20% presumably originates from monoesters of the type 0 0 0

//

II

II

I

I

derived from a pyrophosphate bond -0-P-0-P-0-

-0-P-OH,

\

.

OH OH OH Finally, if a-casein pretreated with the diesterase a t pH 8.0 and the pyrophosphatase a t pH 7.0 is exposed to the action of both the prostate and the intestinal enzynie, all of the phosphorus is liberated. These experiments thus demonstrate that a-casein contains 40% of its phosphorus as mono0 O

//

esters -0-P-OH,

\

OH

40 % as diestcr -0-P-N-,

II

I

and 20 % as pyrophos-

I

OH H

TABLE V Slcpwise Enzyttmtic Dephospho rylation o j cY-Cusein<8 Distribution of Phosphorus as pH of the reaction mixture

Reaction mixture

0

Maximal amoun of phosphorus released per cent of tota

U P I 4 H

Conclusions

b H

0

--N-b--OI

A

0

-@-P+P4I/

AH

AH

(per cent of total)

0

39.6

6.0

//

34 of phosphorus present ns 4 - P 4 H

40

\ OH i.5

+

a-Casein (diesterasepretreated) prcatate and intestinal phosphatsse

a-Casein

+ pyrophosphntase

+

7.8

0

0-P of dieater bond broken

6.0

41.5

Same as in line 1

6.0

78.0

% of phosphorus present as U P - O H and % derived from N-P-0 \ OH

7.2

0

40

4

0 40

40

0

6.0

59.6

4

H of phosphorus present as 4 - P - O H derived from O - P 4 - P 4

a-Casein (diesterase- f pyrophosphatase-pretreated) prostate and inteatinal yhosphake

+

0

Taken from Perlmann (71).

6.0

100

and 56

20

40

\

OH 40

40

20

0

1 I

I

OH

21

NATURE OF PHOSPHORUS LINKAGES IN PHOSPHOPROTEINS

0

0

I

phate -0-P-0-P-0-

I

II

(71). Complete removal of the phosphorus

I

OH OH is accompanied by a disintegration of the protein into smaller units which are soluble in 10% trichloroacetic acid. I t thus appears that the phosphorus in proteins caii be present in diester and pyrophosphate bonds which crosslink polypeptide chains. The occurrence in a-casein, the protein with 0.99% phosphorus (Y5), of 40% of this element as monoester, 40% as diester, and 20% as pyrophosphate requires a minimum of at least ten atoms of phosphorus per molecule. This would correspond to a minimum molecular weight of 31,000, a value ill agreement with that of 33,000 reported from osmotic pressure measurements (12) or any multiple thereof. As is shown in Table VI, most of the phosphorus of p-casein is liberated by the prostate enzyme at pH 6.0 after pretreatment with the snake venom diesterase. This indicates that the @-proteincontains diester bonds of the -0-P-0type, which is in contrast to the -N-P-0of the a-casein (71). In a recent communication Sundararajan and Sarma report that a. “phosphoprotein phosphatase” from rat spleen dephosphorylates a-,p-, aiid unfractionated casein (90). Since these authors state that their enzyme differs in its action from that of a phosphomonoesterase, their results are in accord with the occurreiice of a variety of phosphorus bonds in proteins. I n this coiinection it should be noted that intestinal phosphatase used in our work at pH 9.0 also liberates all of the a-casein phosphorus (72). As discussed earlier, although this enzyme at pH 6.0 hydrolyzes -N-PTABLEVI Action of Prostate Phosphatase on B-Caeein Pretreated with Snake Venom Diesterase

Reaction mixture

+

&Casein in 0.05 M NaHCOl phosphodiesterase @-Casein,phosphodiesterase-pretreated f prostate phosphatase @-Caseinin 0.05 M NaHC03 , control &Casein, control prostate phosphatase

+

Time of incubation at 37°C. (hours)

Phosphorus released by enzyme (per cent of total)

6

0

5.8 5.8

14 24

8.2 + 8.2 5.8

6

54 72 0

24

0

pH of reaction mixture 8.2

+

7.9

22

GERTRUDE E. PERLMANN

bonds only, in the pH range of 8.0 to 9.0 activity toward -N-P-OH, 0 I \ / H ‘OH -0-P-OH, diester, and pyrophosphate linkages, was noticeable. I n \ ‘011 view of these facts it is feasible that the so-called phosphoproteiri phosphatases described in the literature (20, 24, 27, 59, 88L90) are mixtures of several closely related enzymes which act on different bond types. 3 . Pepsin If the molecular weights of pepsin and pepsiiiogen are 35,000 and 38,000, respectively ( G l ) , each of these molecules contains one atoni of phosphorus (%8,60). Since it had been show~ithat ovalbumiii and a-casein are readily dephosphorylated by certain phosphatases from nianimalian tissue, and from potato, the action of these enzymes 011 pepsin arid its precursor was studied. It mas found that only the potato phosphatase a t pH 5.6 dephosphorylates pepsin and pepsinogen, whereas prostate phosphatase does not act on these proteins. The intestinal enzyme, although not active at pH 6.0, liberates phosphorus a t pH 8.9 (67). Having demonstrated that the phosphorus of these two proteins call be removed enzymatically, the influence of the dephosphorylation on the proteolytic activity of pepsin and on the pepsinogen pepsin transforination becomes of considerable interest. Since exposure of pepsin to p H values more alkaline than pH 6.0 results in spontaneous loss of the proteolytic activity of this protein, removal of the phosphorus had to be achieved at pH 5.6 with the aid of the potato enzyme. ---f

TABLE VII Action of Potato Phosphatase on Pepsin and Pepsinogen Each reaction misture contained 1% protein and 0.003% enzyme in sodium acetate buffer of p H 5.6 and 0.1 r/2.

Reaction mixture Pepsin Pepsin Pepsin potato phosphatave Pepsinogen Pepsinogen potato phosPepsinogen phatase

+

+

a

Time of incubation a t 37°C. (hours)

Phosphorus released (per cent of total)

0 24 24 0 24 24

0 0 99 0 0 96

Relative specific proteolytic activity‘ Hemoglobiu

Synthetic Substrate

100 96 96 100 98

100 96 95 Not tested Not tested Not tested

113

The relative specific activity of a freshly prepared enzyme solution is taken aE 100.

NATUltI.: OF I’HOSI’HOHUS LINKAGES IS PIIOSI’HOl’ILOTF~INS

‘23

If, as is showi i n Table VII, these phosphorus-free proteiiis are then assayed for proteolytic activity, 110 loss is found when tested rithcr with hemoglobin (4) or with the synthetic dipeptidcl, acetyl-L-phenylalaiiyl d iodotyrosine (7) :is a substrate. This indicates that the phosphorus i i t these proteins is unessential both for the ciizymatir artiVity of pepsin a i ~ i for the activation of thc precursor, pepsiiiogcn. The failurr of the prostate enzyme to dephosphorylate pepsin aiid pepsinogen made it appear likely that the phosphate group in these proteins is not a monoester of the 0-P type. Pretreatment of pepsin with a diesterme permits subsequent dephosphorylatioii with the prostatic enzyme, a i d it thus becomes clear that the phosphorus of this protein is present as a diester of the -0-P-0type. Further evidence for the occurrence of a diester linkage in this protein is reflected in the electrophoretic behavior. Pepsin still moves aiiodically in 0.1 N hydrochloric acid, pH 1.OfL4 On the other hand, the phosphorusfree pepsin is positively charged a t this pH. It has an isoelectric p H of 1.7. As shown in Fig. 6, the mobilities of these two proteins differ by a

0

pH - 0°C.

FIG.6. Mobilities of pepsin nnd the dephosphorylated pepsin as a function of pH. 4The anodic migration of pepsin in 0.1 N hydrochloric acid has also been observed by Tiselius et al. (Tiselius, A,, Hensehen, G. E., and Svensson, H. (1938). Biochem. J., 32,1814.

24

GERTHUDE E. PERLMANN

below pH 4.0. In contrast to the constant amount, Au = 0.4 X three ovalbuniins, A, , hz, and Aa , however, the pH mobility curves of the two pepsins do not diverge in the pH range of 6.0 to 8.0. These results, therefore, support the dicster nature of the pepsin-phosphorus, i.e., only one hydrosyl group with a dissociable proton is present. It is know1 from the work of Hcrriott (29) and of Williamson and I’assman (97) that! pepsin is a protein with a single peptide chain. Hence the phosphorus must serve to link two sites of the peptide chain in a loop. With the aid of Fig. 7 it, is illustrated that if during the action of phosphodiesterase this cyclic bond is broken, the diester is converted into a monoester with two dissociable hydroxyls. Consequently, in the pH range of 7 to 9, the diesterase-treated protein should move faster than pepsin and be in contrast t o the more slowly migrating phosphorus-free protein. That this is the case is dcnionstrated with the aid of the superimposed tracings of the electrophoretic patterns shown in Fig. 8. Here the full line represents pepsin, the dashed line being the pattern after treatment with the diesterase, 0

\\

0

P

0-

\ / P / \

/O-

/ \

Diesterase (pH 8.2) 0-P broken

ascending

c__1

Pepsin lmes 1 atom of phosphorus

7 descending

Pepsin -Phosphodiesterase treated pepsin -------Phosphorus-free pepsin ......... FIG.8. Superimposed tracings of the electrophoretic patterns of pepsin, phosphodiesterase-treated pepsin, and phosphorus-free pepsin. Electrophoresis was carried out a t 1% protein concentration in 0.1 N sodium I)icarbonate, pH 8.4 for 9000 seconds a t 6.5 volts per centimeter.

NATURE O F

I’HOSPHOltUS

LINKAGES IN PHOSI’HOPROTEINS

25

and the dotted one is that of the phosphorus-free protein. From the relative position of these three peaks it is clear that the mobility decrement is t,he same in each step, i.e., A u = 0.4 X lr5. I n a n attempt to determine the points of attachment of the pepsinphosphorus, Flaviri succeeded in the isolation of three peptides Thr.SerP; SerP.Glu; arid Thr.(Glu, SerP) (22). This evidence indicates that the single phosphate group of this protein is esterified in part to a serine residue. Moreover, the amino acid sequence Thr.SerP.Glu represents the third example in which the phosphoserine residue in a phosphoprotein is linked to a dicarboxylic acid. The second point of attachment of this phosphate diester, however, is still unknown. From the work carried out by the author it is apparent that a variety of eiizymes will dephosphorylate phosphoproteins if the phosphate groups are present as monoesters. As shown by the study of the three ovalbumins and a-casein, together with the results on phosvitin reported in the literature (53), these phosphate groups contribute to the net charge of the proteins. I n addition, the protein-phosphorus may be present in form of diester and pyrophosphate bonds.

V. POSSIBLE BIOLOGICAL FUNCTION OF PHOSPHOPROTEINS The biological function of phosphoproteins is still unknown. Owing to their abundance in embryonic tissue and in foodstuffs it has often been suggested that one of the roles of these materials is that of supplying phosphorus and the essential amino acids to the developing organism. T h a t phosphoproteiris are indeed highly active metabolites and may enter a great number of metabolic processes follows from recent work of several investigators (17, 19, 32,33, 96). During the past years it has been shown repeatedly that the phosphorus of the phosphoproteiri fraction of a variety of normal and malignant tissues undergoes a rapid turnover as measured by the incorporation of radioactive phosphorus, P32. I n addition, Kennedy and Smith succeeded in isolating radioactive phosphoserine of high specific activity from the phosphoprotein fraction of Ehrlich ascites tumor (33), thus offering further evidence that the phosphorus is renewed a t a very high rate. Moreover, these authors point out that phosphoproteins may vary in their reactivity and suggest that the metabolically active materials function as phosphotransferases by cyclic-regulated phosphorylation and dephosphorylation of their serine residues (33). An apparent contradiction to this hypothesis is the fact that no enzymatic activity can be attributed to proteins such as the caseins or phosvitin. The occurrence in these materials of such bonds as those described recently, e.g., pyrophosphates and phosphodiesters (70, 71) seems to indi-

cate that the role of Ihe phosphoproteins, however, is inorc l i k ~ l ythat of a “storage reservoir” for phosphorus. There is iiicreasiiig cviclrncc iii t htb literature that i n the presence of a phosphate donor like admosiiielriphosphate and creatinephosphate, respectively, the nonspecific nionochtmwh aiid diestrrases also fiinction as phosphotransferases (6, 58). The rupt iirc of a pyrophosphnte bond of a protein thus would supply the energy I ~ C C C S sary for such reactions (46) and establish oiie of the biological functions of these materials. I n this connectioii it is of interest to consider recent reports on enzymes specific for the hydrolysis of the phosphoprotein-phosphorus (20, 24, 27, 59, 88, 90). Although the evidence of the occurence of phosphoproteinphosphatases is riot quite unequivocal, it is striking that such enzymes are found in tissues which are abundant in phosphoproteins. Thus it is not unlikely that these phosphatases also may act as transferases. After discussing the possibility that the phosphorus of phosphoproteins may enter metabolic processes, it is tempting t o speculate on the function of the protein moiety. It is of course clear that one of the roles is to supply amino acids which are utilized in protein synthesis. However, one has to keep in mind that these reactioiis as well as those described above occur a t a neutral pH. I t , therefore, is feasible that one of the roles of the protein moiety is that of reducing the acidity of the phosphate group to a level where metabolic processes occur spontaneously. [cf. (91)]. VI. SUMMARY

As discussed in this article, our knowledge of the chemical structure of phosphoproteins is still incomplete. Recent work of several investigators, however, permits a few gencralizations and stresses the importance of thest. materials as distinct chemical entities. There is increasing evidence in the literature that amino acid sequencrs with phosphoserine adjacent to a dicarboxylic acid recur systematically i n phosphoproteins. I t , therefore, seems probable that certain principles must exist which determine the biosynthesis of such amino acid sequences and their subsequent incorporation into the protein molecule. A second property in common to all phosphoproteins is the great lability of the phosphate groups in dilute alkali, in contrast to the stability of phosphoamirio acids in this medium. Such observations show that the physictochemical properties of the phosphorus-containing amino acids change on their incorporation into a peptide or into a protein aiid hence are sensitive to the adjacent molecular configuration. Such considerations led the author to the development of enzymatic methods for investigating the nature of phosphorus linkages

NATURE O F PHOSPHORUS LINKAGES IN PHOSPHOPROTEINS

27

that occur in phosphoproteins. The advantage inherent in these procedures is readily apparent. Thus iC has been possible to show that a variety of phosphatases from mammaliaii tissues and from plants will dephosphorylate phosphoproteins without the occurrence of an extensive proteolysis, if the phosphate groups are present in the form of monoesters. With the aid of such enzymes the electrophoretic inhomogeneity of crystalline ovalbumin has been explained by the demonstrutiori that these preparations are always mixtures of a protein A1 , with two phosphorus atoms per molecule, and A ? , an ovalbuiniri with only one phosphorus. In additiou to monoesters, structural units such as those of diesters or pyrophosphate bonds occur in phosphoproteins. A denionstration of these linkages was achieved by enzymatic hydrolysis, employing enzymes specific for diester and pyrophosphate linkages. This brought about their transformation into monoesters, which could then be released in the form of iiiorgaiiic phosphate with the slid of phosphomonoesterases. As indicated in this article, such an experimental approach not only determiiics the types of linkages but also the proportions in which they are present ill a protein. Thus a-casein contains 40% of its phosphorus in the form of monoesters, 40 % as diesters, and 20 % as pyrophosphates. Moreover the pyrophosphate appears to be present not as a free terminal pyrophosphate group but as a cross link between two peptide chains. 111 @-caseinall the phosphorus is found t o be present in the form of diester linkages betweeii peptide chains. A breakage of the cross linkages leads to splitting of both a- and @-caseininto smaller subunits, even though no peptide linkages are split by the enzyme preparations employed. The results obtaiiied by the author and described in this review represent the first experimental demonstration of the existence of diester and pyrophosphate bonds in proteins. Moreover, they add a new type of crosslinks to that of the disulfide bonds. As in the case of the sulfur bridges, the phosphorus in proteins either can link differeut peptide chains, as in a- and @-casein,or may serve to connect two sites of a single chain in a cyclic loop as in pepsin. It is clear from the material presented here that some progress has been made in the study of phosphoproteins. However, only when more is known about their exact chemical structure mill it be possible t o understand their role in metabolic processes. 4

REFERENCES

1. Agren, G., de Verdier, C . I$., and Glomset, J. (1951). Acta Chern. Scand. 6,324. 2. Alderton, G., and Fevold, H. I,. (1945). Brch. Riochem. 8, 415.

28

GERTRUDE E. PERLMANN

3. Anagnostopoulos, C., Pacht, &I,, Hourland, E., and Grabar, P. (1951). Bull. sac. chim. biol. 33, 699. 4. Anson, bl. I,. (1948). I n “Cryst:tlline Enzymes” (J. H. Northrop, M. Kunite, and 11. X I . Herriott, eds.), 2nd ed., p. 305. Columbia liniv. Press, New Yorlc. 5. Axelrod, 13. (1947). .I. Biol. (‘hein. 167, 57. 6 . Axelrod, 1 % . (19483. .I. Rial. C h e t i i . 172, 1; 176, 295. 7 . Baker, 1,. 15. (1951). ./. Hiol. (Ihottr. 193, XO!). H. 13eacli, 11;. F.,13eriiuteiii, S. S.,HofTman, 0. I)., T e u g i ~ t I~) ,. A[., :iiicl Macy, I. C;. (1941). J . Hiol. ( ‘ h e m . 139, 57. 9. Berbm:inii, hI., a n c l Miekeley, A. (19241. %. plrysiol. Chein. 140, 128. 10. Bergmunil, >I., : i t i d Fruton, J . H. (1941). :ttlo~ttcesi i i Enzymol. 1, 63. 11. I3lack\vood, d . I t . , :ind Wishart, (;. A l . (1934). R i o c h t m . . J . 28, 550. 12. 13urk, S . E’., :iritl C;reetil)erg, D. &I. (1!130). J . Biol.(‘hetit. 87, 197. 13. Canniiii, I t . I<., Kit)rick, A , , :tiid Palmer, A . I f . (1041). .Inn. M. l r . :ioitl. S c i . 41, 243. 14. Chargaff, 1;. ( I W ) . .I. Biol. (:hciir. 142, 4!)1. 15. Cohn, E. J., : ~ n d13erggre11,R. E. 1,. (1924). J . Ceii. I ’ h ~ a i o l .7, 45. 16. Damodaritn, hl., and Ititmachandran, B. IJ. (1941). Biochem. J . 36, 122. 17. Davidson, .J. N.,Frazer, H. C., and Hutchison, W. C. (1951). Biocheni. J. 49. 311. 18. Dekker, C . .I. (1954). Federation I’roc. 13, 197. 19. Engelhardt, V. A . , and Lisovskaya, N. P. (1953). A h t . 19th Interti. Physiol. Congr., Moiifrenl, p. 335; Doklady ?ilezhdunar.od. Fiziol. K o n g r . , Montreal, pp. 10, 209. 20. Feinsteiri, It. X., :ind Yolk, XI. 1G. (1949). J. B i d . (‘heiii. 177, 339. 21. Fevolcl, IT. I,. (1051). Advataces iii Protein (,’hem. 6, 187. 22. Flavin,hl. (1954). .l‘ature173,21~;.1.Biol.(‘h.ew.210,771. 23. FonS, A. (1947). .irkiv Kemi Ninerul. Geol. 24A, No. 33, 14. 24. Foote, bl. W., and Kind, C. A. (1953). Arch. Biocheni. cind Biophgs. 46, 555. 25. Gordon, W. G., Semmett, ‘VV. F., Cable, R. S., :tiid Morris, M. (1949). J. -4m. Cheni. Sac. 71, 3293. 26. Httmmarsten, 0. (1883). 2. physiol. Cheiir. 7 , 227; (1885). Z. physiol. Chem. 9, 273. 27. Harris, 1). L. (1946). ./. BioZ. Chem. 166, 5-11. 28. Herriott,, Ti. M. (1938). J . C e n . P h y s i o l . 21,501, 29. Herriott, It. M. (l!j54). I n “-4Symposium on tho Mechanism of Enzyitie Action” (W. I). MciSlroy, and B. Class, cds.), 1). 24. Johns Hopkitis Press, 13:iltimore. 30. Hipp, X. ,J,, Urovcs, A l . I,., Crester, J. H., arid hlchfeekin, T. 1,. (195”). J . Dairy Sci.36, 272. 31. Hipp, N . J., Groves, M. I,., and McMeelcin, T. I,. (1952). J . Am. Cheni. S o c . 74, 4822. 32. Johnson, R. &I.,and Albert, S. (1953). J. Biol. Chein. 200, 335. 33. Kennedy, E. P., and Smith, S . W. (1954). J. Biol. Chem. 207,153. 34. Kornberg, A. Unpublished. 35. Kunitz, M. (1952). J. Gem Physiol. 36,423. 36. Levene, P. A,, and Alsberg, C. (1901). 2. physiol. Chenr.. 31, 543; (1906). J. Biol. Chem. 2 , 127. 37. Levene, P. A,, and Gchormuller, A. (1933). J . Biol. Chem. 100,583. 38. Levene, P. A , , and 1511, D. W. (1933). J . Biol. Chent. 101, 711.

NATURE O F PHOSPHORUS LINKAGES IK PHOSPHOPROTEINS

29

39. Levene, 1’. A., and Schormiiller, A . (1934). ./. R i d . (‘heu!. 106, 595. 40. L i , Si-Oh. (1952). . I . . , t i t i . ( ‘ h m r . Sor. 74, 5!)59. 41. J~inderstr$rn-I,3rig,K . , :ind Kodama, P. (1927). (’o?itpt. rend. fraa. lab. Curlsberg 16, N o . 1. 42. Ihderstr@ni-I,ang,K. (1929). f ’ o t t i p l . r e n d . frav. lab. Curlsbery. 17, No. 9. 43. I,inderst,rpmi-I‘aiig, K . , nntl Ottesen, M . (1949). (‘ompt. rend. t m v . lab. (.‘orZsberg 26, No. 16. 44. Lipmann, F. aiid Levene, 1’. A. (1932). .I. B i d . C‘hewr. 98, 107. 45. Lipmann, F. (1933). i~-utztr2crissenscha~~ftell21, 236; Biochsm. Z. 262, 3, 9. 46. Lipmann, F. (1941). Advunces in Enzymol. 1, 99. 47. I,ofgren, T. (1945). Svcnsk. Kern. T i d s k r . 67, 95. 48. Longsworth, I,. G . (1939). J . A m . Cheiti. SOC.61, 529. 49. Macheboeuf, M . , P@rensen,M., and Sorcnseri, S . P. I,. (1927). Compt. rend. trav. lab. Curlsbeiy 16, No. 12. 50. Max-Evloeller, I<. (1955). Biochim. et Biophys. A d a 16, 162. 51. McMeekin, T. I,., and Polis, B. D. (1949). Advances in Protein Chem. 6. 202. 52. McMeekin, T. I,. (1954). I n “The Proteins” (H. Neurath and K . Bailey, eds.), Vol. 11, P a r t A, p. 239. Academic Press, New York. 53. Mecham, D. K., and Olcott, H. S . (1949). J . Am. Chem. SOC.71,3670. 54. Mellander, 0. (1939). Riochsni. 2. 300, 240. 55. Mellander, 0. (1947). Cpsala Lakarcforcn. Porh. 52, 107. 56. Menefee, 8 . G . , Overman, 0. R., and Tracy, 1’. H . (1941). J . D a i r y Sci.24,953. 57. Meyerhof, O., and Lohmann,K. (1928). Biochcm. Z. 196,49. 58. Meyerhof, O., and Green, H. (1950). J. B i d . (’hem. 183,377. 59. Norberg, B. (1950). Acta C‘hsn?. Scand. 4. 1206. 60. Nort.hrop, J. H. (1030). J . Gen. Yhysiol. 13,739. 61. Northrop, J. € I . , (1948). I n “Crystalline Enzymes” (J. €I. Northrop, M. Kunitz, and R . M. Herriott, eds.), 2nd ed., pp. 74, 81. Columbia Univ. Press, New York. 62. Osborne, T . B., and Campbell, G. F. (1900). J . Am. Chem. SOC.22,422. 63. Pedersen, K. 0. (1936). Biochcni. J. 30, 948. 64. Perlmann, G. E. (1949). J . Am. Chem. Soc. 71, 1146. 65. Perlmann, G. 14:. (1952). J . Gen. Physiol. 36, 711. 66. Perlmann, G. E. (1952). J . A m . (‘hem. SOC.74.3191. 67. Perlmann, G. E. (1952). J. Am. Chcnz. SOC.74,6308. 68. Perlmann, G. 1.:. (1952). I n “Phosphorus Metabolism” (W. D. McElroy and H . Glass, eds.), Vol. 2, p. 167. Johns EIopkiiis Press, Baltimore. 6’3. Perlmann, G. E. (1953). Disciissioris Faraday Soc. 13, 67. 70. Perlmann, G. E. (1954). Biochim. et Biophys. A d a 13, 452. 71. Perlmann, G. E. (1954). h’ature 174,273. 72. Perlmann, G. E. Unpublished. 73. Plapinger, R. E., and Wagner-Jauregg, T. (1953). J . Am. (‘hem. SOC.75, 5757. 74. Plimmer, R. H . A , , and Bayliss, VV. h l . (1906). J. Phfjsiol. (London) 33,439. 75. Posternak, S. (1927). Riochem. -1. 27, 289. 76. Posternak, S., and Posternak, T. (1927). ( “ o m p t .rend. soc. biol. 184, 909; 186, 615. 77. Posternak, T., and Pollaczek, €1. (1941). H e l v . (’him. Acta24, 921. 78. Posternak, T., and Pollaczek, H. (1941). Helv. Chinr. A c f a 24, 1190. 79. Rimington, C., and Kay, H. D. (1926). Biochcm. J. 20,777. 80. Rimington, C. (1927). Riochenr. J. 21, 1179, 1187.

XI. Sanger, F., :~iidTupl)y, 11. (1951). Bioehem. d . 49,463. X2. Sanger, F., and Thompson, E. 0. 1’. (1%“). Hiochim. e l H i o p / q s . Acfa 9, 22s. 84. Schaffer, S . I<., Harshman, S.,and Engle, R . R . (1854). Fctlernfion, Proc. 13, 28!1. I - o r t r . f & h I n t c r u . f:orcgr. I’hyslol. IZotuc, cit,rtl in (1933). X i . Schmidt, G . (1932) , ~ a f t ~ , r u , i s , s c t c . , ~ c 21, ~ i n 502; ~ l c n (1934). %. ph?/siinl. f‘hcm. 223,86. X5. Schmidt., G . , :tnd Th:urrthauscr, S. J . (1943). . J . Biol. (‘hrui. 149, X!). 86. Schmidt, O., Cuhiles, R., antl Thannhauser, S. .J. (1947). (‘old Spring Harbor Symposia Quant. B i d . 12,161. 87. Sinsheimer, R. L . , snd Koerner, J. F. (1!152). J . B i d . C‘hert,. 198,298. XX. Sund:warajtin, T. 11..antl Sarmn, 1’. S. (1954). R i o c h m . .I. 66, 125. X!). Sundanrajan, T. A., i i n t l Smna, 1’. 8. (1054). . V a / ~ r e173, 6x5. 90. Sunciar:traj:in, T. A , , : ~ n dS:irmn, 1’. S. (1!)64). Biochini. e / 1 2 i o p h p . Acta 13, 588. 01. Svedberg, T., Carpenter, L . M., and Carpenter, D. C. (1930). J . Am. Cheni. SOC.62,211,701. 02. Todd, A. It. (1951). Harvey Lecluies Ser. 47, 1. 9.7. Travia, J,., and I‘eroncsc, A. (1940). Arch. sci. bid. 26, 438; Bull. soc. itat. s p e r . 16, 467. 94. de Verdier, C. 11. (1983). Acta Chem. Scand. 7, 196. 05. Warner, R. C. (l944j. J . Ana. Chem. SOC.66, 1725. 3G. Williams-Ashman, €I. G., and Kennedy, E. 1’. (1952). Cancer Research 12, 415. 97. Williamson, hl. B., ;inti P:isumann, J. 11. (1952). J . Biol. C h e m . 199, 131. !)X. Winnick, T . , arid Swtt,, R. M. (1847). :Irch. Riochcui. 12, 201.

Metabolism of the Aromatic Amino Acids BY C. E. DALGLIESH Postgraduate Medical School, Ducane Road, London, England

CONTENTS I. Introduction . . . . , . . . . . . . . . . . . . . . . . . . . . . . ....

.. ...

11.

Page 33 34 35 35 36 36 36

...

. , . , . . ... . . . . , . . , 1. Historical . . . . . . . . . . . . . . . . . . . . .. . . .. .. . .. .. .. . . . . . . . . ,. , . . . . . . . .. . . . . , . . , . . , , . . . . . 2. General . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . 3. “Essentittl” N:tture of Aromatic Amino Acids for AIammaIs. . , , , , . . Biosynthesis of t h e Aromatic Amino Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Experiments Using Mutants of Rlicroorgnnisms. . . . . . . . . . . . . . . . . . 2. The “Common-Precursor” Pathway of Aromatic Iliosynthesis.. . . . . . 3 . Final Stages in Phenylalanine Biosynthesis . . . . . . . . . . . . . . . . , . . , 4. Final Stages in Tyrosine Biosynthesis . . . .. . .. .. .... . 5. Final Stages in Tryptophan Biosynthesis . . , .. ... ........... 6 . The “Straight-Chain” Pathway of Aromatic Biosynthesis.. . . . . . . . . . . .

.

.

.

39 40 40 42

7 . Isotopic Evidence on the Pathwtys of Sromatic Amino Acid I3iosy1ithesis.. . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . 43 111. Degradation of Phenylalanine and Tyrosine t o Acetoacet,nte; the Principal .. ... . . . . . . . . . . 40 Route Used by Mammals. . , . . . 111.4. Evidence Derived from Inborn Errors of Metabolism. . . . . . . . 46 1. Allcaptonuria, and Related Work on Man and 1nt:iot Anirnals . . . . . 47 2 . Tyrosinosis and Other Cases of p-Hydrosyphenylpyruvic Acid 1Sxcretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................... 51 3. Phenylketonuria (Oligophrenia I’henylpyruvica; Falling’s Syndrome). , 52 11113. Enzymic Experiments on the Normal Pathway i n Mammals.. . . , . , , . . 55 1. General Outline of the l’at,hway.. . . .... ... .. . . .. . . 55 2. Conversion of Phcny1:ilanine t o Tyrosine. . . . . . . . . . . . . 5s 3 . Conversion of Tyrosine t.o p - I I ~ t l r o s . p h e n ~ l p y r ~ ~Acid v i c . . . . . . . . . . . 3!) 4 . Conversion of p-Hydrox!.phcnylpa,ruvic Acid to 2,5-l)ihytlros~;phcnylpyruvic arid Homogentisic acids. E’unction of Ascorllic Acid and of Hematopoietic Factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511 5 . Conversion of Homogeritisic Acid t o YIaleylacetoacet,ic, Fumarylacetoacetic, Fumaric, and Acetoncet,ic Acids.. . . . . . . . . . . . . . . . . . . . . . 64 IV. Tyrosine Degradation by t,he Catechol P a t h w i y . . . . , . . . . . 65 1. Adrenaline, Noriidrenaline, and Thcir Biogenesis . . . . . . , . 66 2 . Metabolic Degradation of Noradrenaline and Adrenaline. Adrenochrome. . . . . . 68 3 . Alelanogenesis and Alhiriism. . .... . . , . . . .. . . ., 0n -1. T l i ~ Ciiiecliol h t h w n y i n thcb I n n c ~ ~ t. . . . . . . . . . . . . . . 71 \‘. Tyrosiire ;\Iet:il~olismvia Thyroid Hormones :ind Ot,lic:r Halogeniited Derivatives, . , . , . , . . . . . . . . . . . . . , . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . 71 ,

,

,

..

,

,

31

,

32

C

. E. DALGLIESH

1’:tge 1 Thyroxine and It.s Biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 74 2 . Triiodothyroninc and Its Biogenesis . . . . . . . . . . . . . . . . . . . . . . . 75 3 . Metabolic Fate of the Thyroid Hormones . . . . . . . . . . . . . . . . . . . . . . 4 . Other Naturally Occurring Halogenated Tyrosines . . . . . . . . . . . . . . . . . . . 75 V I . Pathways of Phenylalanine and Tyrosine Metabolism Utilized Principally by Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 1 . Products Based on Decarboxylation and Amine Oxidation 2 . Products Based on Oxidative Deamination or Transamination. . . . . . . 77 3 . Phenol Formation from Tyrosine . p-Tyrosinase . . . . . . . . . . . . . . . . . . 78 4 . Degradations Involving Opening of the Aromatic Ring . . . . . . . . . . . . . . 78 VII . Tryptophan Degradation by the Kynurenine-Nicotinic Acid 1’at.hway . . . 70 1 . Establishment. of the Relation between Tryptophan and Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 80 2 . The General Outline of the Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . The Conversion of Tryptophan t o Formylkynurenine and Kynurenine . 83 4 . Tryptophan Pcroxidase-Oxidase Adaptation . . . . . . . . . . . . . . . . . . . . . . . 85 5 Conversion of Kynurenine t o Hydroxykynurenine . Role of Ribo86 flavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 . Iiydroxykynureninc and Insect Eye Pigments . . . . . . . . . . . . . . . . . . . . . 87 7 . Kynureninase, Kynurenine Transaminase, and the Formation of Anthranilic, Kynurenic, Hydroxyanthranilic., and Xanthurenic 88 Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Mechanism of Action of Pyridoxal Phosphate in Reactions Involving Aromatic Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . !)1 0 . Excretion of Hydroxykynurenine and Xanthurenic Acid by Man . . . . 04 10. Side Reactions of Kynurenine, Hydroxykynurenine, Anthrariilic Acid, and Hydrosyanthranilic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . !)5 11 . Conversion of Hydroxyanthranilic Acid t o Sicotiriic Acid . . . . . . . . 97 12 . Tryptophan, Nicotinic Acid, and the Pyridine Sucleotides . . . . . . . . . . 100 13 . Further Metabolism of Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . 101 V I I I . Tryptophan Degrttdrttion by the Enl.eramine-Serotonin Pathway . . . . . . . . 103 104 1 . Biosynthesis of 5-IIydroxytryptamine . . . . . . . . . . . . . . . . . . . . . 2 . Degradation of 5-Hydroxytryptamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 3 . N-Methylated Derivatives of 5-Hydroxytryptamine . . . . . . . . . . . . . . . . . . 107 I X . Routes for Tryptophan Degradation Used Principally by Microorganisms . 108 1 . Urinary Indolcscetic Acid and Indoleuceturic Acid . Urorosein . . . . . 700 2 . Bacterial Degrttdat.ion via Indole . The Tryptophnnrtsc Reaction . . . 110 3 . Fiirthcr Degradation of Indole by Hacteria . . . . . . . . . . . . . . . . . . .111 4 . Origin of IJrinitry Indican, Iudigo, Indirubin, Bkat.oxyl :tnd Skatole Red 111 X . Tryptoph:in Metabolism in Plants . Heteroauxin . . . . . . . . . . . . . . . . . . 113 1 . Riogenesis a r i d Ilegradntion of Indoleacetic -4rid i n Plant8 . . . . . . . . . . . 114 2 . Other Jnclolic €’l:int Growt Ii 1Iormont.s . . . . . . . . . . . 1 I4 XI. N:iiiiral Products I’rolxthly Related t o the Aromatic Amino k i d s . . . 115 1. Probably 1lel:itcd hIet:tt)olic Products in Microorganisms . . 115 2 . Probably Relat.et1 Metabolic Products in Plants and Fungi . . . llfi I l(j 3 . Flnvorioids a n d I, ignin . . . . . . . . . . . . . 4 . Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 X I 1 . Future Problems . . ............................................... 121 XI11. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

.

.

METABOLISM O F THE AROMATIC A M I N O ACIDS

33

I. INTRODUCTION I’henylalanine,1 tyrosine, and tryptophaii are unique among the amino acids for the wide variety of reactions which they undergo.

o7

Phenglalanine

Tyrosine

CH,.YH*COIH SHZ

H Try p top hail

Besides being fundamental constituents of proteins they are the parent substances from which powerful hormones are derived, for example, adrenaline (epinephrine), noradrenaline (norepinephrine), thyroxine and related substances, 5-hydroxytryptamine (enteramine, serotonin), and the plant hormone indoleacetic acid. Tryptophan is also the precursor of the B vitamin nicotinic acid and hence of part of the important pyridine nucleotides. All three aromatic amino acids are potential precursors of other substances having powerful physiological activity, for example, many of the alkaloids. Errors in the metabolism of the aromatic amino acids in man can give rise to sometimes serious, but fortunately comparatively rare, disorders such as alkaptonuria and phenylketonuria. The numerous metabolic pathways involved in aromatic amino acid metabolism therefore make a n important as well as an interesting study. Protein ingested by a living organism in general undergoes proteolysis, the liberated amino acids joining the respective amino acid “pools” of the organism. Simultaneously the organism’s own protein is being broken down and the resultant amino acids also join the amino acid “pools” together with newly formed amino acid molecules in those organisms in which biosynthesis occurs. Once amino acid molecules from these various sources have entered the “pools” they are indistinguishable (e.g., 614). From these pools material is withdrawn for ( I ) synthesis of new protein and (2)degradation or other metabolic transformations. I n the fully grown organism in the steady state as much amino acid must be metabolized as is ingested and/or biosynthesized (though not, of course, necessarily the identical molecules). This review covers these metabolic degradations and transformations and also biosynthesis. The incorporation of the aromatic amino acids into proteins will not be considered. Throughout this review i t is t o be assumed unless otherwise stated that amino acids are of the L-configuration.

34

C. E. DALGLIESH

Many thousaiids of papers have hcen written on the subject of this rc\.iew, of which only a sinall fraction tan Ite cited here. The aim has been to give some idea of the historical development of our knowledge, followed by a detailed statenient of the present state of that knowledge. The main emphasis has been laid on those pathways which appear to account for the greater part of normal aromatic amino acid metabolism in man; subsidiary pathways are dealt with more briefly. An attempt has been made to cover relevant papers received in England up to the end of 1954. 1. Historical

Tyrosine was the first of the aromatic amino acids to be discovered, iii 1846, by Liebig (558), who obtained it on acidifying and cooling a solution of casein which had been fused with potash. Better methods of hydrolysis than alkali fusion were soon discovered, and tyrosine was isolated from numerous other sources. The relative insolubility of tyrosine (0.048 g./100 ml. water a t 25°C. as compared with corresponding values of about 3 g./100 ml. for phenylalanine, 1.14 g./100 ml. for tryptophan, and 25.3 g./100 ml. for glycine) causes tyrosine, often mixed with cystine (solubility 0.01 1 g./ 100 ml. a t 25”C.), to separate particularly readily from protein hydrolyzates and also, in certain pathological states, from body fluids. The correct structure of tyrosine was first suggested by Barth (32), was strongly supported by Baumann (36), and was finally proved by synthesis by Erlenmeyer and co-workers (227, 229). Emil Fischer was the first to resolve synthetic m-tyrosine into its optical isomers (266). Phenylalanine was first obtained by Schultze and Barbieri in 1879 (779, 780) from etiolated lupine sprouts. Its structure was shown (781) by comparison with synthetic material prepared independently by Erlenmeyer and co-workers in 1882 (228). Synthetic DL-phenylalanine was first optically resolved in 1900 (267). The difficulty of isolating phenylalanine from protein hydrolyzates prevented recognition of its widespread occurrence in proteins until after Emil Fischer had developed his method for separation of the amino acids by fractional distillation of their esters. Tryptophan was first isolated only a t the beginning of this century (411). A number of color reactions of proteins were extensively studied in the latter half of last century and numerous attempts were made to isolate the chromogen responsible. The name “tryptophan” was given to this chromogen in 1890 by Neumeister (645). The chromogen was soon associated with the substance giving rise to indole on bacterial putrefaction of proteins. The failure of many early attempts to isolate tryptophan was probably due to the fact that it is destroyed on acid hydrolysis. The successful isolation by Hopkins and Cole (411) used enzymic hydrolysis of casein, but the chief reasons for their success were their discovery of mercury salts as

METABOLISM OF THE AROMATIC AMINO ACIDS

35

precipitating agents for tryptophan and their prior development (410) of a more sensitive version of the original Adamkiewicz reaction (2, 3) with which they could follow their isolation procedure. The structure of tryptophan was the subject of much discussion but mas finally settled in 1907 by the synthesis of Ellinger and Flainand (221). This discussion has surnniniized only the priticipal laiidinarks in the early history of the aromatic arniiio acids. For a complete account the excellent review by Trickery and Schmidt (894) should be consulted.

2. General The geiierd chemical and physicochemical properties of the aromatic amino acids are outside the scope of this review, but attention is called to the following prime sources of information: synthesis and criteria of purity (207) ; stereochemistry and optical properties (640) ; physicochemical behavior (152). 3. “Essential” Nature of Aromatic Amino Acids for Mammals

For higher organisms, e.g., maninials, phenylalanine and tryptophan are so-called “essential” amino acids, i.e., they cannot be synthesized by the animal and must be supplied in the diet (for man, see 728, 729). Tyrosine is derived, as we shall see later, from phenylalanine, and is not therefore itself an essential amino acid as long as the phenylalanine intake is adequate (726, 950) ; if the conversion of pheiiylalanine to tyrosine is inhibited, as in phenylketonuria, tyrosine can become essential (65). The “essential” nature of the aromatic amino acids is a reflection of the general inability of higher organisms to synthesize the benzene ring. For the requirements of phenylalanine and tryptophan by man and the rat, see 14, 15a, 153,630, 631, 653,728,729,732a1 732b, 750,757a1and the review by Albanese (6). &Amino acids vary in availability with the species. For example Dphenylalanine is used by rat, mouse, and man (15, 35, 727, 730, 962), whereas D-tryptophan is used by the rat (53, 54, 759, 895), is partially used by the mouse and pig (139,867), and is not used by man (7,29). The utilization of the D-amino acids is probably determined by the relative rates of absorption of the D-amino acid from the intestine, and of conversion of D- to L-amino acid in the liver (288). The conversion of D- to L-phenylalanine is reduced in vitamin-B6 deficiency (52),as is to be expected for a transformation involving transamination to phenylpyruvic acid. Phenylpyruvic and indolepyruvic acids, the a-keto acids corresponding to phenylalanine and tryptophan, may also, to an extent varying with the species, satisfy growth requirements (e.g., 55, 109,436, 725, 911).

36

C. E. DALGLIESH

11. BIOSYNTHESIS OF THE AROMATIC AMINOACIDS Biosynthesis of the carbon skeleton of the aromatic amino acids is coiifined to the lower organisms. Plant material is the main ultimate source of the aromatic amino acids for animals and man, though some may also derive from bacteria, cr.g., in the gut. Sumerous theories have been advanced in the past as to the origin of aromatic rings (e.g., 707), but oiily rcceritly has definite cviderirc beeii forthcoming, derived from work with microorganisma. There o nay well be a similar biosynthetic pathway iu plants, but there is its yet little direct evidence oil this point. Two main techniques have been uscd. Davis and his school have studied the requirements and excretion products of mutants of microorganisms, aiid several workers have studied biosynthesis from labeled precursors using isotope techniques. 1 . Experiments Using Mictants of JIIicrooryaniswu

Mutants of microorganisms can iiow be conveniently produced arid isolated from parent wild-type strains (for review, see 186). Such mutants are invaluable for the determination of metabolic sequences. If, for example, there occurs a sequence

A-+B+C-+D

...+ X

in which X is necessary for growth of the organism, then a mutant blocked in the coiiversiori of I3 to C will not in general grow unless C, or some suhstance subsequent to it iu the metabolic chain, is supplied to the organisin. Similarly 13, or a derivative, may accumulate in the culture medium, especially if A is supplied. Many complications occur owing to alteriiative pathways, ((leakage” due to incomplete blocks, and competitive inhibition of one metabolite by another close to it in the chain (e.g., 187-189). Severtheless simple relationships exist in a sufficient number of cases to make the method a most fruitful one. 2 . The “Common-Precursor” Pathway of Aromatic Biosynthesis

Davis (184) selected a large iiuinbcr of mutants of Escherichia coli requiring two or more aromatic amino acids for growth, and then tested a large number of substances to see if any could relieve the growth inhibition. Success was attained with shikimic acid (215,268), at that time a relatively obscure natural product, This indicated either that shikimic acid (structure diagram 1) was a true aromatic precursor or that it could readily be transformed into a true precursor. The likelihood that shikimic acid was a true precursor was increased when other mutants were found to accumulate shikimic acid in the medium, from which it could be isolated (184, 185). Not all the mutants responding to shikimic acid responded t o a mixture of

METABOLISM OF THE AROMATIC AMINO ACIDS

37

the aromatic amino acids. Some of these, however, responded when paminokmzoic acid was also supplied. The biosynthesis of p-aminohenzoic acid is therefore obviously related, a t least in E. coli, with biosynthesis of the aromatic amino acids. In further mutants a mixture of the aromatic amino acids and p-aminobenzoic acid still did not produce growth, unless supplemented with a trace of shikimic acid or with a culture filtrate of wildtype organisms. These mutants therefore required still another substance related to aromatic biosynthesis. Extracts of wild-type organisms were examined by paper chromatography, and it soon became clear that paper itself contained the unknown factor. A study of likely trace contaniinants of paper finally led to the identification of this bacterial growth factor as p-hydroxybenzoic acid (183). Evidence was also obtained that under certain conditions of pH, still another growth factor, called the sixth factor, could become necessary (e.g., 185, 188). This has not yet been identified. Davis concluded that shikimic acid was a common precursor of phenylalanine, tyrosine, tryptophan, p-aminobenzoic acid, p-hydroxybenzoic acid, and an unknown sixth factor, and he next set out to determine other substances lying on the biosynthetic pathway. The various mutants were therefore tested for syntrophism, i.e., for the ability of one mutant to produce a substance necessary for the growth of another mutant. There was thus found a thermolabile substance, X, which was a true precursor of shikimic acid (184). X was isolated from culture filtrates and identified as 5-dehydroshikimic acid (744). Similar experiments revealed a substance, W, which was a true precursor of substance X (187, 193). This also was isolated and shown to be 5-dehydroquinic acid (906). The enzyme, named 5-dehydroquinase1 converting dehydroquinic acid to dehydroshikimic acid has been partially purified (606). It is fairly stable, has a high specificity, appears to have no cofactors, and is of wide occurrence in bacteria, algae, yeasts, and plants but, as expected, could not be found in mammalian liver. The enzyme, 5-dehydroshikimic reductase, converting dehydroshikimic to shikimic acid, has also been studied (964a). It requires triphosphopyridinenucleotide as cofaetor . These results are summarized in diagram 1. Substance CP represents a common precursor which has not yet been identified but the existence of which is shown by the existence of mutants which require all six aromatic factors but excrete shikimic acid into the medium. The six aromatic factors are not all synthesized equally readily. Study of mutants with double, up to quintuple, requirements has shown (187) that the growth requirements for the various factors are always satisfied in the following order; p-hydroxybenzoic acid, p-aminobenzoic acid, tryptophan, and finally phenylalanine and tyrosine. This order parallels the quantita-

38

C. E. DALGLIESII

Glucose

110%COzH Quinic

0

0 dehydrogena3e

-Qo,, OH

o..L,Il

I10 COZH

IIO..’

OH Quinic acid

5-Dehydroquinic acid

i

5-Dehydroquinnse

c ( ):I

x z x z 7 €10J$

0JOH

OH 5-Dehydroshikimic acid

I

1

Side rcactiun nhich mag stnge blucked

OH Protocatcchuic acid

Dehydroaliikimic reductnse

YOzH Compound 21 I I

HO-’

I I

OH OH

I I

Shikimic acid

\

I

I? I

CP (common precursor)

1

I

* I

Phenylalanine

Phosphoshikimic acid

Tryptophan

p-Hydroxybenzoic acid

tive growth requirements, the amounts, on a molar basis, of p-hydroxybenzoic and p-aminobenzoic acids required for growth being about onethousandth of the requirement for tryptophan, which in turn is about one-quarter the requirement for pheriylalanine or tyrosine. The further metabolic changes undergone by shikimic acid are not yet clear. Mutants have been found that excrete two derivatives of shikimic acid (192). One of these, Z1, tentatively identified as a cyclic acetal of shikimic acid with pyruvic acid (192), may be a precursor of prephenic acid (191a). The other, 22, is a phosphoshikimic acid, and possibly, but not yet certainly, is a normal metabolite succeeding shikimic acid in the chain.

METABOLISM OF THE AROMATIC AMINO ACIDS

39

Similarly the immediate precursors of dehydroquinic acid, which must include open-chain compounds, are not yet known. Earlier work (301) with Neurospora suggested that quinic acid might be a precursor. However, quinic acid did not support growth of any of the E. coli mutants, though it did support growth of some Aerobacter mutants. It was established (193, 607) that quinic acid is not a true aromatic precursor, but that some organisms have the ability to convert it, as shown in the diagram, into dehydroquinic acid, which is a true precursor. The biosynthetic pathway in Neurospora appears to be the same as in E. coli (856). A Neurospora mutant blocked in the conversion of dehydroshikimic acid to shikimic acid converted a large part of the dehydroshikimic acid t o protocatechuic (3,4-dihydroxybenzoic) acid (856; cf. diagram 1). The ultimate derivation from glucose, shown in diagram 1, is considered in the section on isotopic evidence. A combination of mutant, enzymic, and isotopic techniques suggests that sedoheptulose-1 ,7-diphosphate is an intermediate in the conversion (456a,b, 823a). 3. Final Stages in Phenylalanine Biosynthesis Experiments with mutants have also revealed the final stages of phenylalanine biosynthesis. Both Davis (190) and Japanese workers (459) obtained mutants excreting a labile substance, subsequently called prephenic acid, which was very readily converted into a second substance, Y, which was in turn converted into phenylalanine. Y was identified as phenylpyruvic acid (190). Prephenic acid was isolated (907) and its structure (see diagram 2) demonstrated (cf. 288a). CP Common precursor

H02C CHz*CO*CO,H

CHz CO * COJI

Ciihiioao

stages I

OH Prepheiiic acid

l ’ l ~ e n y l ~ ~ y r u vacid ic

Phenylalanine Diagram 2. Final stages of phenylalanine biosynthesis in E. coli.

Hnddox (333), working with Neurosporn mutants, llns suggested that in this organism a-phciiylglycine is closely coiinected with phenylalanine biosynthesis. It was not possible to establish whether it mas a true precursor

40

C. E. DALGLIESH

or only a product of “shunt” metabolism formed from a true precursor, and more complete identification is in any case desirable.

4. Final Stages in Tyrosine Biosynthesis Some microorganisms may resemble the higher organisms in being able to convert phenylalanine directly to tyrosine; thus it can occur in Vibrio (167) and Pseudomonas (605) and has been claimed for E. coli (48; but cf. 807). However in Lacfobacillus arabinosus tyrosine is formed by a route not involving phenylalanine (20), as is apparently also the case in Aerobacter aerogenes (605). The direct conversion of phenylalanine to tyrosine is claimed by advocates of the “straight-chain” pathway of aromatic biosynthesis described later. Otherwise no immediate precursors of tyrosine appear t o have been reported. Transamination of p-hydroxyphenylpyruvic acid has been suggested to be the final stage in yeasts (474), and may occur in E. coli (809), and isotopic evidence, discussed later, suggests that even if tyrosine is not formed from phenylalanine, the method of introduction of the tyrosine side chain is very similar t,o that postulated above for phenylalanine. Formation of p-hydroxyphenylpyruvic acid from prephenic acid can be readily visualized. 6. Final Stages i n Tryptophan Biosynthesis

Anthranilic acid and indole are precursors of tryptophan in numerous microorganisms and fungi (e.g., 5, 263, 264, 602, 741, 783, 785, 816, 854, 855, 876), and i t is probable that anthranilic acid is derived, with intermediate steps, from the common precursor, CP of diagram 1. The conversion of anthranilic acid to indole and tryptophan has been shown unambiguously in Neurospora with the use of isotopic techniques (93, 663). There may, however, be other pathways for tryptophan biosynthesis (45, 702). Tryptophan can, for example, be formed by transamination of indolepyruvic acid (e.g., 470, 912), which might be formed other than via aiithranilic acid. Thus aromatic-requiring mutants have been found which accumulate unidentified indole compounds (307).

A i i t h ~ ~ i i lacid i~.

Ill~lolt-

Diagram 3. Final stages in tryptophan biosynthesis.

llaskins and Mitchell (365) foulid that a it’eiirosporu iiiutaiit blocked IJOtween anthranilic acid and tryptophan accumulated aiithranilic acid when

METABOLISM OF THE AROMATIC AMINO ACIDS

41

grown on a high-tryptophan medium. They therefore postulated a “tryptophan cycle”: Tryptophan

T

indole

+ kynurenine + hydroxykynurenine,

I

+ anthranilic

etc.

acid

Partridge and co-workers (663) showed that such a cycle plays no part under conditions of normal tryptophan synthesis, and Adelberg (4)has plausibly ascribed the original results to adaptive formation of the enzyme kynureninase (discussed in detail later) induced by the high tryptophan content of the medium. I n the conversion of anthranilic acid to indole it has been found that in Neurospora the carhoxyl group is lost (652), whereas in R. coli it has been claimed that the carboxyl group is retained and the additional carbon atom required is supplied by methionine (56). However, Yanofsky has prepared from E. coli a cell-free system carrying out almost quantitatively the conversion of anthranilic acid to indole. This system requires adenosine triphosphate (ATP) and ribose-5-phosphate or ribose or, in certain circumstances, hexose diphosphate or hydroxypyruvic acid in place of the ribose (967). Tracer experiments showed that the carboxyl group was lost in the coilversion (967) so that a two-carbon (or higher) unit must be added, and it has been shown that this unit becomes attached to the benzene ring a t the same position as the displaced carboxyl group, aniline not being an intermediate (969). Acetate may be the unit involved, becoming attached to the benzene ring through the methyl group (686a,b). Harley-Mason (361a) has realized a similar conversion chemically. The conversion of indole to tryptophan has been much more extensively studied. This is brought about by direct reaction of indole and serine under the influence of the enzyme tryptophan desmolase (better named tryptophan synthetase) (302, 853,854) which requires pyridoxal phosphate as a coenzyme (890). The enzyme has been obtained in the cell-free state (890) and partially purified (965) and its genetics in Neurospora studied in detail (966). Zinc appears to play some part in tryptophan desmolase formation or function (628). The mechanism of function of pyridosal phosphate in tryptophan biosynthesis is considered in more detail later in discussing other pyridoxinedcpeudeiit cnzyrnes. . t i i activated fornl of serinc is formed which reacts with indole. I>ehyclration call take place in two ways: intermolecularly, involving loss of water between the p-hydroxyl of activated serine and the 0-hydrogen of indole, or intramolecularly, involving loss of water from act ivated seriiie to give activated aminoacrylic acid, which theri adds on to the P-position of indole. Tatum and Shemin (858) in ingenious experiments

42

C. E. DALGLIESH

involving triply labeled (D, N”, p-C”) serine, concluded that the latter mechanism is more probable, but did not distinguish between the possible tautomeric intermediates. 6. The “Straight-Chain” Pathway of Aromatic Biosplhesis

Though the evidence for the “common-precursor” pathway is weighty, this pathway has not been universally accepted as the sole route for aromatic biosynthesis. An alternative “straight-chain” pathway has been proposed, largely based on evideiice obtained with competitive inhibitors of the aromatic amino acids such as phenylserine and fluorophenylalanine. Beerstecher and Shive (47) originally suggested that in 23. coli tryptophan could be directly converted to phenylalanine. Bergmann and co-workers (57, 58) studied the question in greater detail both by use of analogue inhibitors and by studying the “sparing effect” of one aromatic amino acid on the requirement for the others. They deduced the metabolic scheme shown in diagram 4, the dotted lines representing alternative pathways : precursor

1

~--------------+ phenylalanine

-

tyrosine t------- - - - - 3

tryptophan

-3

+

metabolic product of tyrosine

Diagram 4. “Straight-chain” pathway proposed for aromatic amino acid biosynthesis.

The possibility does not appear to have been sufficiently coilsidered that apparent conversion might be due to reversible transformations to a common precursor (cf. 187). Evidence obtained from inhibition studies is suggestive rather than conclusive, and other interpretations are possible; for example, Pimmonds ct al. (808), studying phenylserine inhibition in B. coli mutants, concluded that the inhibition produced by pheriylserine mas iiihibition of use of phenylalanine for growth, and not of phciiylalanint. tksyiithesis, aiid they wnsidered that their results supported tlic (‘coinnion-precursor” rather than the “straight-chain” pathway. S y c ri al. have suggested (650) that in Neurospora phenylalaniiie and tyrosine arc precursors of tryptophan, and 13eerstecher has suggested (45) that) in lactic acid bacteria phenylalanine can be converted to tryptophan. Until more evidence is forthcoming the direct transformation of t ryptophan to phenylalanine implied in the “straight-chain” pathway should not be considered as finally established. The observation (458) that the inhibition of l d . coli by azaserinr can be reversed by tryptophan, I,hciiylnlaiiiii~., or tyrosiiicl m i y Ic:~dto future useful rcsults.

METABOLISM O F THE AROMATIC AMINO ACIDS

43

7. Isotopic Bvidencc on thc Pathways of Aromatic A m i n o Acid Biosynthesis Ehrensviird and co-xvorkers (25) grew the yeast Torutopsis utilis on, as sole carbon source, acetate labeled with C14in the carboxyl carbon and C13 in the methyl carbon. They then isolated and degraded the tyrosine and studied the distribution of activity in various positions. Acetate carboxyl contributed to the tyrosine carboxyl and to C-4 of the aromatic ring, whereas the a- and &carbon atoms of the side chain and carbons 1, 2, and C, of the ring came predominantly from the acetate methyl carbon. This result immediately excluded cyclization of a straight chain of fatty acid type in which alternate carbon atoms should derive from the acetate methyl and carboxyl. It was suggested that the most plausible explanation of (cf. also 804) was that the aromatic ring was in some way connected with triosehexose systems. Gilvarg and Bloch (289, 290) grew the yeast Saccharomyces cerevesiae on a mixture of either unlabeled glucose and labeled ( l-Ci3, 2 G 4 ) acetate, or on labeled (l-Ci4) glucose and unlabeled acetate. In the presence of glucose no incorporatlon of labeled acetate occurred into phenylalanine or tyrosine, though there was extensive labeling of glutamic acid, nspartic acid, leucine, and lysine. Glucose was therefore the aromatic precursor, and not acetate or metabolically related substances such as ketoglutarate or oxalacetate. When (l-C14) glucose was substrate the label appeared in both ring and side chain of phenylalanine and tyrosine. On degradation none of the label appeared in carbons 1,3, or 5 of the ring, whereas the greater part occurred in carbons 2 and/or G (carbons 2 and 6 were not distinguishable by the degradative procedure). These results made it unlikely that direct cyclization of glucose had occurred, and ruled out condensation of two isotopically equilibrated three-carbon units which should give labeling in ortho or para positions thus:

c’

C*

cI

c* cI

C’ Head-to-tail union of &carbon unlts resulting in p a w labeling

Tail-to-tail union of 3-carbon units resulting In orlho labeling

More extensive studies (291) on biosynthesis from ( l-C14)glucose, using improved procedures for degradation and location of the activity, revealed high activity only in the /3-carbon of the side chain and in carbons 2 and/or G of the ring, confirming the previous results. The direct cyclization of glucose still remained a possibility; this should give rise to label in one position only, but it could not be determined whether activity appeared in both

C. E. DALGLIESH

44

carbons 2 and 6 or in only one of these. Direct cyclization was, howcvoi~, considered unlikely, and it was suggested (cf. also 211) that there might, o(*cur a condensation of a triose with a tetrose t o give sedoheptulose or sorw other seven-carbon sugar. Ehrcnsvard and lteio (213) grew both h'. coli and Neurospora crassa O I I (l-CY4, acetate. Both these organisms used the same mechanism for aromatic biosynthesis, which was similar to that in yeasts. Degradation of tyrosine showed that the ring could not have been formed by direct cyclization of glucose, and these authors favored a scheme involving co11densation of erythrose and triose in presence of aldolase (cj'., e.g., 41 5 , 861 ), or less probably, a C6-G union. This scheme can be represented as follow, M representing a carbon derived from acetate methyl and C, from awtate carboxyl :

C

/

&I-c

-If

C-C

Acetate

/

Glucose

+ c-c

- "":

FC '0 \

M \

c--c or

1

II

C, and C3fragments

lieptuloses etc.

Two possibilities were considered for the origin of the side chain, ( a ) direct addition of a two-carbon unit to a cyclized seven-carbon unit, or ( b ) addition of a three-carbon unit to the seven-carbon unit with expulsion of the one-carbon side chain:

\ + c-c-c pp--c Ehrensvard and Reio favored alternative ( a ) . Alternative ( b ) strikingly resembles the mechanism of phenylalanine biosynthesis revealed hy mutant techniques (see p. 39) ; that this mechanism can apply to tyrosine was rendered possible by work of Thomas and co-workers and was rendered extremely probable by work of Sprinson and co-workers. Thomas P t al. (866) degraded yeast grown either aerobically or anaerobically on carboxyl-labeled acetate or carbonyl-labeled pyruvate. I n aerobic pyruvate cultures the side chain of tyrosine was in all probability de-

METABOLISM OF THE AROMATIC AMINO ACIDS

45

rived from an intact pyruvate molecule (cf. also 166). The distribution patterns of activity supported conclusions of earlier workers ( 1 ) that direct cyclization of glucose formed from pyruvate did not occur, and (2)that none of the intermediates involved could have been in equilibrium with symmetrical products of glycolysis or the Krebs cycle. (Krebs cycle intermediates can, however, cause an increase in tyrosine biosynthesis; 473, 475.) Spriiisori i t al. (824) have settled thc origin of the side chain in. E. coli by growing the organism on a mixture of unlabeled glucose and labeled shikimate, and subsequently isolating and degrading the tyrosine. -4s required by alternative (b) above, the carhosyl of shikimate had been eliminated and, as carbons 2 and 6 of shikimate corresponded to carbons 2 and 6 of the ring of tyrosine, the side chain of tyrosine must have entered the same position in the ring that was occupied by the shikimate carboxyl. Furthermore it was shown that the side chain, in contrast to the ring, could have been in equilibrium with three-carbon glycolytic intermediates. The above results make it clear that glucose should be regarded as the precursor of the aromatic amino acids. In an attempt to elucidate the steps in this conversion Sprinson and co-workers have therefore combined isotope and mutant techniques. Using a mutant of E. coli accumulating shikimic acid in the medium, labeled bicarbonate, formate, and acetate did not, as expected, act as shikimic acid precursors, whereas ~ - ( l - C l glucose ~) gave shikimic acid of specific activity comparable to that of the precursor. With the use of glucose labeled in various positions, it was found (804, 805 as modified by 805a) that the shikimate carboxyl carbon arises from C-3 and C-4 of glucose; C-1 of shikimate arises from C-2 and C-5 of glucose; and C-2 of shikimate arises from C-1 and C-6 of glucose. Thus the carbon triad carboxyl . . . C-1 . . . C-2 of shikimate is in all probability derived from a three-carbon product of glycolysis. The remaining four-carbon portion of shikimate has a more complex origin, with C-3 t o C-6 of shikimate corresponding in some degree to C-3 to C-G of glucose, but with C-1 of glucose also contributing to shikimate C-6 to a significant extent (cf. diagram 5). The results are compatible with the postulated role of sedoheptulose diphosphate as an intermediate in the glucose-shikimate conversion (45Ga,b, 823a).

4. 6 CHAOH

Ho-. HO

5

0

'OH

B

CO,H

@

HO"

,

"OH

OH

OH

a-D-Glucose

Shikimic acid

a

CIIr' CH(XlIz) C02H

f$ <4

3

(OW Phen>lalauir,e and tyrusine

Diagram 5 . Numbering of carbon atoms in various substances discussed.

46

C. E . DALGLIESH

The isotope labeling of the benzene ring of tryptophan formed from carboxyl-labeled acetate and (3 ,4-CI4)glucose suggests that this has the same origin as the benzene ring of tyrosine and phenylalanine (686a,b), thus providing strong confirmatory evidence for the common-precursor pathway. The information at present available is thus sufficient to exclude certain possible routes for aromatic biosynthesis, but is not yet sufficient to reveal the actual mechanism or mechanisms used, or to define parts of the pnthway. But it seems probable that the techniques available are adequate t o deal with the problem and that perhaps in a short time the present obscurities will be made clear. Further information and pertinent speculations 011 aromatic biosynthesis can be found in reviews by Ehrensvard (212) and Davis (191).

111. DEGRADATION

O F PHENYLALANINE AND

THE PRINCIPAL

ROUTEUSED

TYROSINE TO ACETOACETATE ; BY nfAMM.4LS

The distinction between ketogenic and glycogenic substances was one of considerable importance to the earlier biochemists, and that phenylalanine and tyrosine are ketogenic was established a t an early date. Embden and co-workers in 1906 (226) showed that phenylalanine and tyrosine yield acetoacetic acid when perfused through a surviving liver. The ketogenicity was confirmed by numerous workers (e.g., 42, 131, 169, 208, 225, 706, 902) using classical methods and was put beyond all doubt by use of isotopically labeled precursors (905, 933). Phenylalanine and tyrosine were later shown also to be glycogenic (130, 131), and this, too, has been confirmed isotopically (552), the glycogen and keto-bodies being derived from different parts of the amino acid molecule. I n this review it is hoped to give some idea of the historical development of the subject as well as of its present status, and the elucidation of the pathway for the conversion of phenylalanine and tyrosine t o acetoacetate is therefore set out in a manner corresponding in some degree to the development of knowledge on the subject.

IIIA. EVIDENCE I ~ E R I V EFROM D IXBOF~N ERROWOF A~ETABOLISM I n discussing the use of mutants of microorganisms in the study of aromatic biosynthesis it was pointed out that valuable information could thus be obtained. An organism with a metabolic block rendering it unable t o convert a substance X into its metabolite Y is likely either to excrete X, or to metabolize X by an alternative pathway if such is available, or to excrete metabolites of X formed by the action of relatively unspecific “detoxicating” systems. Accumulation or excretion of abnormal substances may therefore indicate an erizyniic deficiency of this type. In the latter part of

47

METABOLISM O F T H E AROMATIC AMINO ACIDS

the last century there came to be recognized various rare humaii disorders in which excretion of abnormal substances occurred. The reinarkable insight of Garrod (ass),in particular, led to the realization that these “inborn errors of metabolism” were due to a congenital iiiability to perform a normal metabolic reaction; the patients were in fact natural mutants providing invaluable material for metabolic studies. Comparatively few metabolic disorders of this type are known; it so happens that an appreciable proportion of these are related to pheiiylalanine and tyrosine metabolism and gave the first clues as to metabolic pathways. The relationship of these defects is summarized in diagram 6, aiid the evidence for each will be discussed. Three of the defects, alltaptonuria, tyrosinosis, arid phenylketonuria will be considered immediately, and a fourth, albinism, will be considered (p. 69) under a n alternative pathway for tyrosine metabolism. Blocked iri plienylketoouria

Phenylalanine

It

&*

Phen ylpyruvic acid

lyrosine

I

It

-

p-Hydroxy’I)henylpyruvie acid I

Blocked in tyrosinosis

-t

+

3,4-DihydroxyphenyIalanine Blocked iu albinism

RIeIanin, etc.

2,j-Dil,ydroxyplieiiylpyruvicacid

I

+

Homogentisic acid Blocked in alkaptonuria

Acetoacetic acid, etc. Diagram 6. Relationship of metabolic defects concerned with phenylalanine and tyrosine metabolism. For structural formulas see diagrams 8, 11, and 12.

i . Alcaptonuria, and Related Work on M a n or Intact A n i m a l s For centuries rare cases have been noted (cf. 286) of persons passing urine which turned black. The condition was first accurately described in 1859 by Boedeker (86), who found that the urine contained a strongly reducing chromogen which he called “alkapton.” The chromogen was first isolated in 1891 by Wolkow and Baumanii (949) and identified as 2,5-dihydroxyphenylacetic (homogentisic) acid (949, cf. also 638, 657; structure, diagram 8). Wolkow and Baumann showed that homogentisic acid was derived

48

C. E. DALGLIESH

from tyrosine but thought that it was primarily a product of bacterial metabolism. Abderhalden ct al. (1) made a bacterial origin seem less likely by subcutaneously injecting glycyl-L-tyrosine into an alkaptoriuric aiid finding exccss honiogtwtisic acid excretion. Excess homogrntisic acid excretion also occurs on :Idministering phenylalanine (250). hfeycr (597) and Friedmaiiii (279) postulated that the side chain of homogentisic acid arises from migration of the side chain of phenylalanine and tyrosine. The metabolic defect involved in alkaptoiiuria was suggested by Bateson (34) as early as 1902 to be inherited as a recessive Mendelian character, and later evidence has supported this prediction (395,643). Gross (322)in 1914 concluded that it was due to lack of a specific enzyme. Alkaptonuria, unlike phenylketonuria, is not accompanied by mental symptoms arid is not an incapacitating disorder except insofar as it may lead to ochronosis and arthritis (cf. 598). Homogentisic acid is excreted by the alkaptonuric 011 giving tyrosine or p-hydroxyphenylpyruvic acid, but not on giving o- or m-hydroxyphenylalanines or their corresponding keto acids (84,636),and is also excreted on giving 2,5-dihydroxyphenylpyruvic acid (636). Homogentisic acid gives acetoacetic acid when perfused through a surviving liver (226)and so does p-hydroxyphenylpyruvic acid, but not phenylpyruvic acid (225). It soon became accepted that a probable pathway of phenylalanine metabolism was as follows: phenylalanine -+ tyrosine

-+

p-hydroxyphenylpyruvic acid

I

acetoacetic acid +- homogentisic acid

This picture (correct as later evidence has shown) was complicated by a number of other obscrvations. Phenyllactic acid was apparently ketogenic, whereas p-hydroxypheiiyllactic acid was not (226). Phenyllactic acid apparently iiicreased homogentisic acid excretion in the alkaptmuric, whereas p-hydroxyphenyllactic acid did not (636). Thesc ohservatioiis suggested that phenyllactic acid was also on the normal metabolic pathway, but it was not clear how it fitted into the chain. The difficulty was partly resolved in 1935 when Edsoii (208) showed that the ketogenicity of pheriyllactic acid mas due to the use of the ammonium salt, and that it was the ammonia aud riot the phenyllactate which was ketogenic; on the other hand, p-hydroxyphenylpyruvic acid and homogentisic acid were truly ketogenic. A complete and critical account of the large amount of early work on alkaptonuria (up to 1928)has been given by Keubauer (637). Additional interest arose when it was found possible to produce experimental alkaptonuria in animals. This can, for example, be brought about

METABOLISM OF THE AROMATIC AMINO ACIDS

49

by feedirtg the rat or mouse excess phenylalanirie (130,527,528, 662). Sealock and Silberstein (793) then found that in the guinea pig given excess tyrosine the excretion of homogen tisic acid was inversely proportional to the ascorbic acid intake, and that administration of excess ascorbic acid could prevent homogentisic acid excretion. Moreover homogentisic acid excretion occurred on giving tyrosine to the human being on an ascorbic acid-free diet and ceased when ascorbic acid was also administered. The homogentisic acid excretion differed from that found in true alkaptonuria in being accompanied by excretion of considerable amounts of p-hydroxyphenylpyruvic acid and p-hydroxyphenylacetic acid, it being possible in some cases t o account for the greater part of administered tyrosine as these three metabolites (794). A similar excretory picture was obtained on giving phenylalanine (792, 794). This work showed, as is discussed in more detail later, that ascorbic acid is connected with tyrosine and homogentisic acid metabolism. However, human alkaptonuria is not due to a simple ascorbic acid deficiency, as additional ascorbic acid does not prevent, homogentisic acid excretion (199, 610, 643,788). Experimental alkaptonuria has also been produced in rats on a diet deficent in sulfur-containing amino acids (295). Pimilar excretory patterns were produced after additional phenylalanine, tyrosine, or their corrcsponding keto acids, and thc condition was relieved on giving cysteine, but not ascorbic acid (644). Moreover the p-hydroxypheuylpyruvate excretion was much lower, relative to the homogentisic acid excretion, than in the type of ascorbate-dependent alkaptonuria studied by Pealock in the guirien pig (rats cannot in any case be made ascorbic acid-deficient). Seuberger and Webster (G44) also showed that this second type of experimental alkaptoriuria could be produced in many types of amino acid imbalance, or in protein deficiency, and that the threshold intake of phenylalanine or tyrosine required to produce the condition varied with the nutritional state and also with the acid-base balance, acid urines heing asmc-i;ttrdwith a decreased homogentisic acid excretion (cf. also 160, 273, 787). Seubauer (637) thought it possible that the initial formation of a 2 , 5 dihydroxyphciiyl compound took place at the amino acid stage to g iw OH

50

C.

E. DALOLIESH

various routes, e.g. : 2’,6-I)ihydroxyplieiiylpyruvic acid

or

homogentisic acid

2,5-Dihydi oxy~~lienyletliylurnlllc

2,5-Dihydroxypheriylalariinewas synthesized arid resolved by Keuberger (641) and its metabolism in an alkaptonuric examined as part of a detailed and extensive investigation (643). The conversion of pheriylalanine or tyrositie in an alkaptonuric was estimated to be 70 % t o 90 %. Homogrntisic acid was shown to be actively secreted by the kidney tubules, the concentration in blood being very low even after giving pheiiylalanine. 2 , s Dihydroxyphenylalanine gave rise to homogentisic acid excretion, but the evidence was insufficient to show whether i t was a normal intermediate. T,aiiyar (529) found that in the alkaptonuric phenylpyruvic acid was converted to homogeritisic acid with a high efficiency, whereas the conversion of p-hydroxyphenylpyruvic acid was much lower, arid that in experiment a1 alkaptonuria in rats neither of the keto acids could induce alkaptonuria or maintain an alkaptoiiuria previously induced by tyrosine. He therefor(. coiicluded that the keto acids are not normal intermediates in homogciitisic acid formation. I-auyar’s findings are opposed by earlier results (225) and by all the enzymic work on the subject, discussed later, and their explariation is not obvious. Experiments on, or related to, alkaptonuria led t o a provisiorial picture of phenylalaniue and tyrosine metabolism shown in diagram 7 . Phenylalanine

phenylpyruvic acid

I I

V

V

Tyrosinc

p-hydroxyphenylpyruvic acid

I

:1

2,SDihydroxyphenylalanine I

i?

V

2,ii-Diliydroxyplienylphenyletliylamine

phenyllactic acid

, I

I?

I?

v

.

I?

.

V

p- hydroxyplienyllactic acid

:?

<: :=>

V 2,5-dihydroxyphenylpyruvic acid I

V

- -- --* 2,5-dihydroxyplienylacetic (homogcntisic) acid

-

fui ther metabolism

Diagram 7 . Possible pathways for homogentisic acid formation suggested b y early work, particularly on alkaptonurics.

METABOLISM OF THE AROMATIC AMINO ACIDS

51

This picturc has been clarified t)y cnzyinic experimcnts which will be described later. Various other comments are, however, appropriate here. 2,5-Dihydroxyphenylalaninecan be converted to 2,5-dihydroxyphenylc%hylamine (83), which is a substrate for amine oxidase (80) and which if formed would therefor(. be expected to give lionlogentisic acid. It is uiilikely that either phenylacetic acid or p-hydroxypheiiylacetic acid could be intermediates in homogentisic acid formation, as these are known to be treated as foreign substances by the body and converted to glycine conjugates (cf. 930) ; moreover p-hydroxyphenylacetic acid does not give homogrntisic avid in the alkaptonuric (635). The formation of a 2,5-dihydroxyphenyl compound must therefore be brought about a t the amino acid or correbponding keto acid stage, or, much less likely, from the lactic acid derivative.

.

2 . Tyrosinosis and Other Cases of p-Hydroxgphenylpgruvic Acid Excretion

I n 1932 RiIedes (590) reported on a patient who excreted large quantities (c. 1.G g. per day on a normal diet) of p-hydroxyphenylpyruvic acid. This condition she named tyrosinosis. On adding increasing amounts of tyrosine (either pure or as protein) to the diet, the p-hydroxyphenylpyruvste excretion increased and further excretory products appeared in the order, tyrosine, p-hydroxyphenyl-L-lactic acid, and 3,4-dihydroxyphenyl-~-alanine. Additional phenylalanine gave rise to increased excretion of phydroxyphenylpyruvate and also of tyrosine and p-hydroxyphenyllactate ; additional p-hydroxyphenylpyruvate was excreted mostly unchanged and partly as p-hydroxyphenyllactate, but no tyrosine was excreted; additional p-hydroxyphenyllactate was excreted unchanged; homogentisic acid was metabolized completely. Medes concluded (1) that p-hydroxyphenylpyruvate formation was an early step in tyrosine metabolism, ( 2 ) that p hydroxypheiiyllactate was the product of a side reaction, formed enzymically as shown by the optical activity, (3) that in tyrosinosis the formation of 2,5-dihydroxyphenyl compounds is prevented, tyrosinosis involving a block one stage earlier in the metabolic chain than that involved in alkaptonuria. It is fortunate that Medes was available to make such a complete and able investigation, as no further cases of tyrosinosis have been reported and the metabolic defect must therefore be exceedingly rare. (There having been oiily one case recorded iiothing is, of course, known of the genetics of its inheritance). Recently, however, other cases of p-hydroxyphenylpyruvic acid excretion have been observed, and though these are probably of a different type it is convenient t o consider them here. Felix and co-workers developed a specific method for estimating urinary p-hydroxyphenylpyruvate (549) and then investigated the possible use of

32

C. E. DALGLIESH

this substancr for testing liver function (258,260). After giving p-by(IroxyDhciiylpyruvate to norma! persons arid patients with liwr dgsfunctioir the intensity of the. Millon reactioii of the urinc. w:ts roughly proportional io the dcgrce of liver d:image. Much of the Nilloii rcaction was chiti to phe.rtol arid not to unc~haiigetlp-hydroxyphcnylpyrii~ at c, a i d quite apart from i hr ibility of thc starting material thc method tloes itot, make ;t good liver function test ( c j . 313). During this work, however, two cases were eiicountered in which spontaneous p-hydroxyphenylpyruvate excretion occurred (259,546). Gros and Kirnberger (313)then made an extensive survey and found p-hydroxyphenylpyruvate excretion to be much less rare than was previously supposed. It was observed in 99 of 122 cases with liver disease (iiicluding all of 43 cases with liver cirrhosis and none of 4 cases with fatty liver) and in 35 out of 41 cases with miscellaneous blood disorders (including all cases with severe anemias). The amount excreted was far less than in tyrosinosis; in liver disorders excretion values went up. to 100 mg. per day with a mean of 40 mg. per day, and in blood disorders excretion was rather less. Moreover exogenous tyrosine seemed not to be involved, no increase in p-hydroxyphenylpyruvate excretion being observed even after considerable additions of tyrosine t o the diet. The excretion was attributed to a decrease hi the general oxidative capacity of the liver, due cither to cellular damage or an inadequate oxygen supply. p-Hydroxyphenylpyruvate excretion was also observed in various miscellaneous disorders such as endocarditis and Salmonella infections. p-Hydroxyphenylpyruvate excretion can also occur in aiiiinals, for example, in the rabbit fed large amounts of phenylalariirie (511)and, as already noted, in experimental alkaptonuria ( e g , 295, 644). Both p-hydroxyphenylpyruvate and 2,5-dihydroxyphenylpyruvateare excreted on giving sodium butyrate aiid tyrosine to the guiiiea pig (852). When given in large amounts to ftnimals p-hydoxypheriylpyruvate is in part converted to p-hydroxypheiiylacetate (471); the same conversion can occur in man. Levine and co-workers (555-557) made the important observation that p-hydroxyphenylpyruvate (and also p-hydroxyphenyllactate, but not in general homogentisic acid) is excreted by prematuie infants especially when the intake of phenylalanine and tyrosine is high. This excretion ceases on giving ascorbic acid, suggesting that asrorbate is concerned in the further metaholism of p-hydroxypheiiylpyruvate (cf. also 961). This coriclusioii was strengthened by the finding, already discussed, that, p-hytlroxyphenylpyruvate is excretrd i n artificial scurvy in animals aiid man (e.g., 792,794). 3. Ph~nyllictonrrria (Oligophrmia Phenylpynii~ica, Folling’s Syndrome)

Folling (272)in 1934 first described a syndrome characterized clinically by mental defect and hiochemically by the presence in the urine of phenyl-

METABOLISM OF THE AROMATIC AMINO ACIDS

53

pyruvic acid (cf. also 445, 44G). The disorder is a good deal more widespread than alkaptonuria, the number of cases in Great Britain being estimated a t 1600, or about 4 per 100,000population (615), but there are considerable geographical differences in its incidence, which may be due partly to inbreeding in isolated commuiiities and partly to the ultimate racial group. The condition is iiilierited as a recessive Meudeliaii character. It has been calculated (cf. 138) that about 1 in 200 people carry the recessive gene of phenylketonurin. There is some evidence that carriers are more liable than noncarriers to mental instability (663a). Fortunately a t present few, if any, pheiiylketonurics have offspring, but if treatment were sufficiently improved to allow phetiylketouurics to reproduce, thc incidence of the disorder might he expected to rise. The urine also contains considerable amounts of phenylalanine (180, 274), phenyllactie acid (150, 151), atid phenylacetylglutamine (832, 960), typical urinary excretioii values being (9GO), in milligrams per 100 ml., phenylalanine 38, phenylpyruvic acid 50, phenyllactic acid 54, and phenylacetylglutaminc 53. Pheriylacetylglutamiiie oc('urs in iiormal urine in appreciable quantities (0.25 t o 0.5 g. per day), but excretion by thc phenylketonuric is much higher (e.g., 2.4 g. per day, ref. 832). Folling suggested that the fundamental metabolic error was an abnormal racemization of phenylalaninc, thc D-isomer giving rise to the phenylpyruvic acid. 13ut although there are ahnormally high amouiits of phenyl:danine in the blood of the phenylketonuric (274, 450),iioiie of this is of the wconfiguration. Penrose and Quastel (664) suggested that the metabolic error was aii iiiability to break down pheriylpyruvic acid. But the blood of these patients contains negligible phenylpyruvic acid, and both phenylpyruvic aiid phenyllactic acids can be aminated by the phenylketonuric (450). (Subsequently Jervis (448) has found small amounts (c. 0.7 mg. per milliliter) of phenylpyruvic acid in the blood of phenylketonurics, this being increased on administering phenylalaninc, wpecially the D-form, or phenylpyruvate). The correct interpretation wits made in 1947 by .Jervis (447), who showed that in the normal fasting animal or man ingestion of phcnylalanine or phenylpyruvic acid is followed by a rise in Millon-reacting (i.e., phenolic) substances in the blood, whereas in pheny1ketonuric.s this docs not occur. He therefore deduced that the metabolic error in phenylketonuria is an inability to bring about hydroxylation. It was still uncertain whether the substance normally hydroxylated is phenylalanine (giving tyrosine directly) or phenylpyruvic acid (the conversion of phenylalanine to tyrosine therefore occurring via the keto acids), or whether both types of hydroxylation occur (cj. diagram 7). The direct conversion of phenylalanine to tyrosine was suggested by the carly work on alkaptonuria already described. Direct conversion was

54

C. E. DALGLIESH

rnade very probable by the work of Moss and Schoenheinier (614) iii 1940, who found that in the rat deuterium-labeled pheriylalariine was converted to tyrosine arid that the Conversion continues even iii preseircie of excess tyrosine, so that the process must be automatic and iiidepcirdent of the animal’s tyrosine rcqirenicint. Similarly pheiiyllactic :wid is uutomnt 1cally converted to pheiiylalaninc and tyrosine (613). Thcl cwzyiiie coilveriing phooylalariine to tyrosiiie was isolated from rat liver by lidc~iifricmcl a i d Coopcr (886) and partially purified (the dctails of this reaction arts (libcussed later). When Jervis (149) examined livers of pheiiy1ketoiiui.ic.ssouir after death using Utlcnfriend arid Cooper’s conditioiis hc could find no evidence for any formation of tyrosine froin phenylalaninc, whereas livers of controls showed a high conversion. The metabolic defect in phenylkctonuria was thus finally tied dowii :IS a failure to hydroxylate phenylnlanirrc to tyrosine. Afore recent isotopic investigations by Uiidenfricnd and Beseinaii (880) have shown that a small conversion of phenylnlanine to tyrosine can occur in phenylketonuria. Four possible explaiiations of the primary block werc advanced: ( 1 ) there may be a reduced amount of the appropriate enzyrne system, L-phenylalaiiitie oxidase, in the livcp, or (2) ti complete absence of the enzynic with conversion by alternative pathways (cf. 174), or ( 3 ) a iiorma1 amount of apoeiizynie with a coenzyme inissing or (4) a normal amount of enzyme inactivated by an inhibitor. It is known from work on microorganisms that metabolic blocks are by no means always complete (cf., e.g., 187, 188) and this may well apply to phenylketonuria, but which on(’ or more of the above four possibilities is correct must await further work. The relation cif tht. biochemical findings to the mental symptoms in phenylketoiiuria is still obscure. Toxicity of abnormal nietaholites actiiig over long periods is an obvious possibility. The finding (17b, 64, 65,961a) that the mental conditioii improves on a continuous low-phenylalaniiie diet, provided this is started before pernianent brain damage appears, gives some support to this cwiicept. It is to be expected that a deficiency of tyrosine, to which the phenylketonuric is liable, may also contribute t o the symptoms. The abnormal response of the phenylketonuric to adrenaline (138) may be due to a lowered endogenous adrenaline production due to a lack of tyrosine. The excretion of abnormal indolic metabolites (17) may indicate t hat tryptophan metabolism is also modified in phenylket onuria . The absence of pigmentation associated with phenylketonuria may be due either to lack of tyrosine as substrate for melanin formation, or t o inhibition of tyrosinttse by the abnormal amounts of phenylalanine (179a), or to both. Pigmentation is restored on giving tyrosine (816a). The cxisttwce in phrnylkrtonuria of a single metabolic block is in accord

METABOLISM OF T H E AROMATIC AMINO ACIDS

55

with the apparent determination of inheritance by a single recessive gene. Ho\vevcr, Boscott and Bickel (97, 98, and cf. 16) have found that the urine of phenylketonurics contains metabolites such as o- and p-hydroxyphenylacetic acids and p-hydroxyphenyl lactic acid (cf. 17a, 17c). They explained these by postulating that ortho-hydroxylation played a part in normal phenylalanine metabolism and that in phenylketonuria there were a whole series of metabolic blocks. Dalgliesh (174) has shown that the hydroxylated metabolites are probably due to action on the abnormally accumulated nonhydroxylated metabolites of the normal detoxicating mechanisms of the body, and that a single metabolic block adequately explains the known facts.

EXPERIMENTS ON THE IIIB. ENZYMIC

NORMAL

PATHWAY IN MAMMALS

The work already described on man and intact animals allowed a tentative scheme to be formulated for phenylalanine and tyrosine metabolism (diagram 7). In this section the enzymic and isotopic experiments will be described which have filled in many details of the picture. The subject will again be outlined in a semihistorical manner, but for convenience the complete pathway is summarized at this stage, in diagram 8. 1. General Outline of the Pathway

Early experiments by Bernheim, Felix, Sealock, and their co-workers on oxidation of tyrosine by liver breis showed an uptake of four atoms of oxygen per mole of tyrosine, with the production of one molecule each of carbon dioxide and acetoacetate, but no ammonia (60, 61, 261, 262, 789, 976). Felix and Zorn (261) found alanine to be formed and considered this to arise from a direct splitting of the tyrosine side chain. Although the experiments with man and intact animals already described made it seem very probable that p-hydroxyphenylpyruvic acid and homogentisic acid were normal intermediates in tyrosine metabolism, and although homogentisic acid was known to be readily metabolized by normal liver (e.g., 208, 695, 976) Felix and co-workers (262) considered p-hydroxyphenylpyruvic acid and homogentisic acid not to be intermediates in the breakdown of tyrosine by the liver system. Isotopic experiments shed more light on the contradictory evidence. Weinhouse and Millington (905) incubated liver slices with tyrosine labeled with C14in the 0-position of the side chain, and found that the activity appeared in the methylene carbon of acetoacetate. Schepartz and Gurin (772, 773) incubated with liver slices phenylalanine labeled with C14in the carboxyl group or a-position, or in positions 1, 3, and 5 of the aromatic ring. They found that the a-carbon of the side chain became the carboxyl-carbon of acetoacetate; either C-1 or C-3 of the ring became the terminal methyl

56

C. E. DALGLIESII transnminntion,

Id-phenylnlnninc

Phenylalaniric

e.g., xitli

(cofnctors: nscorbic wid, ? vitamin B,z.

HOOCH,.CO.COJI

6ce disgrnnis 9 and 10)

p-HgdroxyphenJ.lpyruvicacid

Tyrosine

$C&*CO

-CO,H

HO 2,5-Dihydroxyphenj Ipyruvic acid

1 direct conversion

/

lioningent isicnse (cofactors: ferrous iron,

&C"2*Co2H

HO'

1 ascorbic 1 iolie acid) acid,

*

CHzCO,H CO. CHt AIaleylacetoacetic acid

Homogentisic acid

+ CH,*CO*CHz*CO,H CO. CHz Furnarylscetoacetic acid

I

Fumaric acid

Acetoacetic acid

Diagram 8. Summary of the steps involved i n the degradation of phenylalanine and tyrosine t o fumaric and acetoacetic acids by t h e principal pathway used by m i mals and man.

of acetoacetate (thus providing a direct proof of the suggestion of Meyer (597) and Friedmann (279) that the phenylalanine side chain shifts to an adjacent ring carboiil ; there was no randomization between methyl and methylerie carbons of acetoacetate, so that the latter must have been derived from an intact four-carbon unit, and no participation of two-carboil units occurred in the degradation. Lerner (552) synthesized phenylalanine labeled uniformly in the ring with C1*and labeled in the a-position of the side chain with C13, and incubated this with liver slices. The dilutions of C14and C13in the acetoacetate mere the same, showing that breakdown and resynthesis could not have occurred. Lerner considered the fate of the four ring carbon atoms not accounted for, and concluded that it was unlikely that breakdown to two-carbon units had occurred) so that a four-carbon unit such as malate or fumarate (Neubauer (637) had earlier suggested

57

METABOLISM OF THE AROMATIC AMINO ACIDS

formation of the latter) should be formed. He diluted his preparations ivith inactive malic acid which he then reisolated and found t o be active. The activity was moreover equivalent in all carbons, consistent with its deriv:ttion from the original aromatic ring of phenylalanine, and the amounts of acetoacctate and malate formed appeared to he equivalent. Later isotopic work (eg., 201, 752,933) has supported various aspects of their results, which are all consistent with degradation in the following manner: (OH)

0B

'

- I

CH2-?O-H

CHz

I

CO,H

a

= ling

xCHOH

xCO

= CH1 I

xC02H

1

CH*XHz

CH,

I

. Q): -Q \*

ICO~H

I

+

I

CH1 I

*CO,H

carbon

= a-carbon of side chain

The apparent conflict between enzymic and isotopic evidence was resolved by the work of La Du and Greenberg (523),Sealock and Goodland (789,790), and, in particular, Knox and Le May-Knox (489,543). a-Ketoglutarate and ascorbate (cf. 661,700)were found to be cofactors for the tyrosine-oxidizing system of liver (543),and the dialyzed soluble fraction of liver had high tyrosine-oxidizing activity on addition of these substances. As the complete system took up four atoms of oxygen per mole of tyrosine and not the five atoms which would have been required had oxidative deamination occurred, it was concluded (489,523,769)that trarisamination is the first step in tyrosine metabolism; this step can be made rate-limiting, in which case i t shows a dependence on pyridoxal phosphate (489). Transamination was confirmed by demonstration of the formation of glutamate from a-ketoglutarate and by isolation of p-hydroxyphenylpyruvate as its 2,4-dinitrophenylhydrazone(489). The system could also use oxalacetate (523)or pyruvate in presence of a-ketoglurarate (489),in which case the corresponding amino acids were formed. 2,5-DihydroxyphenyIalanine was not attacked by the tyrosine-metabolizing system (489) and could therefore be excluded as a normal intermediate (cf. p. 50). The oxidation of p-hydroxyphenylpyruvate also involved four atoms of oxygen per mole, and ascorbic acid was involved in the first oxidative step (790). Its effect was catalytic and it could be replaced by iso-ascorbic acid but not by substances such as glutathione (489). The oxidizing system was highly specific, oxidizing only L-tyrosine, p-hydroxyphenylpyruvate, 2,5dihydroxyphenylpyruvate, and homogentisate. Ravdin and Crandall (695)had previously shown that the intermediate formed from homogentisic acid is fumarylacetoacetic acid and the combined results all fitted in with

the following scheme (for structural formulas see diagram 8) : 1. Tyrosine a-kctoglutarate --+ p-hydroxyphenylpyruvatc gluttimate 2 . p-Hydroxyphenylpyruvate "pt","o"p 2,5-dihydroxyphenylpyruvntr 3. 2,5-Dihydroxyphenylpyruvate 'lP:agk: F COZ 1iomogciitib:tttI of 4. Homogentisate uptake o2 F fuinarylacetoacetate 5 . Fumarylacetoacetate 3 fumarate acetoacetate This scheme has been confirmed in numerous experiments, especially those of Felix and co-workers (257) in which they re-examined and clarified their earlier results. The various steps, including some which have more recently become cvident, will now in turn be considered in detail. Before doing this, however, comment should be made on the site of the normal degradation of tyrosine. All the above work was carried out on liver slices, homogenates, or extracats derived from many different species, and the liver probably makes the predominant contribution to tyrosine degradation. Degradation can, however, occur in other organs. The kidney can also carry out tyrosine degradation by the same pathway as in liver but more slowly (160, 101), oxidation in kidney, like liver, homogenates depending on availability of keto acid (164). Tyrosine oxidation by kidney extracts can also lead to accumulation of p-hydroxyphenylacetic acid (257, 724), possibly owing to loss of ascorbate in preparation of the extracts. Brain, intestine, spleen, and blood appear to be unahle to oxidize tyrosine (160); indirect evidence has suggested that some oxidation may occur in muscle (314), though this is not supported by experiments with homogenates (160).

+

+

+

+

2 . Conversion o,f Phenylalanine to Tyrosine

This transformation, Considered likely on the basis of experimeuts with the intact animal, was proved by the isotopic experiments of Moss and Schoenheimer (614), which also showed it to be an automatic process. Udenfriend and Cooper (886) found a highly specific enzyme in liver mhichh carried out this reaction, possibly in two stages. The system required oxygen and DPN (diphosphopyridinenucleotide) and was considered responsible for the greater part of the normal metabolism of phenylalanine. Mitoma and Leeper (605) resolved the system into two components, separated from rat liver homogenate supernatant by differential precipitation with ammonium sulfate. Their combinedsystem of enzyme I, enzyme 11, DPN (or reduced DPN), and an aldehyde or alcohol, specifically hydroxylated L-phenylalanine but not other aromatic compounds. Enzyme I is labile and only a tenfold purification could he achieved. Enzyme I1 is relatively stable. Many, but not all, aldehydes and alcohols tested rould participate in the reaction.

METABOLISM OF T H E AROMATIC AMINO ACIDS

59

The conversion of phenylalanine to tyrosine occurs in muscle as well as liver extracts (560) and is decreased in the liver of ACTH-treated rats (428). The reverse transformation, of tyrosine to phenylalanine, does not occur even in phenylalanine-deficient animals (309). 3 . Conversion of Tyrosine to p-Hydroxyphenylpijruvic Acid

The experinicnts described earlier showed that in liver homogenates and extracts this reaction is brought about by transamination, which is an obligatory first step in the oxidation of tyrosine by such systems. The existence of such a transamiiiating system was already known (133, 134, 393), and the observed pyridoxal phosphat e-dependence when transamination mas was made rate-controlling (489) was in accordance with the known behavior of transamiriases (cf. 482). The absenre of oxidative deaniination was shown by the oxygen uptake and the abseuve of ammonia formation (257, 489, 523, 769). Such results with tissue extracts indicate only that no oxidative deaminase survived the isolation procedure. On the other hand, in rats with a severe pyridoxine deficiency no interference with over-all tyrosine metabolism was observed (141), and though these results are inconclusive because of uncertainty of the degree to which pyridoxine deficiency interferes with transaminase activity, they are supported by the finding (134) that liver of some species, including the rat, contains a powerful tyrosine deaminase (cf. 68, 69). It seems probable that in the intact animal both routes for conversion of tyrosine to p-hydroxyphenylpyruvic acid are used. Slight support derives from results of Schoenheimer and co-workers, who, in the first metabolic experiments with a Ws-labeled amino acid, found tyrosine nitrogen to appear in the amide- as well as amino-nitrogen of other amino acids with a distribution similar to that obtained after giving NI5-labeled ammonia (774).

4. Conversion of p-Hydroxyphenylpyruvic Acid to 2,5-Dihydroxypheny1pyruvic and Homogentisic Acids. Function of Ascorbic Acid and of Hematopoietic Factors The intermediate formation of 2,5-dihydroxyphenylpyruvicacid in this conversion has not been proved by isolation. But as this is readily metabolized by tyrosine-oxidizing systems (e.g., 489), unlike the possible alternative intermediate p-hydroxyphenylacetic acid, the pathway is not in doubt. On the other hand, the detailed mechanism of this conversion is probably the major unsolved problem in the study of tyrosine metabolism. The influence of ascorbic acid on tyrosine metabolism in man and intact animals has been discussed under alkaptonuria and tyrosinosis (q.v.). Tyrosyluria, also called hydroxyphenyluria, i.e., the excretion of p-hydroxyphenyl compounds in the urine, can be affected by factors other than ascor-

60

C. E. DALGLIESH

bic acid. It can occur, for example, in many types of congenital or acquired anemia in animals and man (e.g., 846) and in steatorrhea (99). A few such cases are relieved by hematopoietic factors, such as folic acid or vitamin Bu as well as by ascorbic acid (182, 648a, 847, 952, 953), though more usually such factors relieve the hematological symptoms without affertiiig the tyrosyluria (588, 589, G11, 747-749). Livers of folic acid-deficient rats have a redured capacity to oxidize tyrosine (722). Vitamin Blz has iio effect on the excretion of homogentisic acid by the alkaptonuric (271). It is uiicertain from all this evidence whether folic acid and vitamiii B12 arc directly concerned in tyrosine metabolism, for they could well be iiidircrtly concerned either because of the mutual influence of vitamins upoil cadi other (646,963) or by their effect on the general level of nietabolic activity, or by their influence on the availability of, for example, carriers such BY the cytochrome system. The adrenal, for example, possibly exerts an indirect effect on tyrosine metabolism (33,749). Positive evideiice on the function of the hematopoietic factors should he given by enzymic experiments, but these, as we shall see below, are still contradictory. Tyrosyluria can also occur in vitamin-A deficiency. This is probably due to the accompanying vitamin-C deficiency (596, 806). The demonstration by Knox that ascorbic acid is a cofactor in tyrosine metabolism has been discussed earlier. Though ascorbic acid is essential both for this purpose and for the prevention of scurvy, the latter is probably not due to any appreciable extent to inadequate tyrosine metabolism (e.g., 723). Further experiments with liver preparations have shown that a number of other substances can replace ascorbic acid, D-isoascorbic acid being as effective as ascorbic acid itself (160, 489). Many other ene-diols are effective in tissue homogenates (522,791) but not necessarily in the intact 5111imal, where poor absorption or retention reduces their efficacy (661,975). Substances such as 3-methylascorbic acid (791), which do not contain an ene-diol structure, cannot replace ascorbic acid. More detailed enzymic studies of the changes involved in the conversion of p-hydroxyphenylpyruvic acid to homogentisic acid have been made by two groups of workers, Williams and Sreenivasan (927-929) in rat liver preparations, and Uchida and co-workers (878) in rabbit liver preparations. Unfortunately the initial results of the two groups differ appreciably, but not all the conclusions are necessarily mutually exclusive. The American workers studied liver tyrosine oxidase with the aid of 2,6-dichlorophenolindophenol,which destroys reduced ascorbic acid. The inhibition was only partly reversed by ascorbic acid, and glutathione was found also t o be involved (928). Under certain conditions the dichlorophenolindophenol could be stimulatory and was thought to replace yet another factor (927). (The work of the Japanese authors discussed below

METABOLISM O F T H E AROI\IATIC AMINO ACIDS

ti1

suggests that this could be vitamin Bl2 .) They then studied aged tyrosine oxidase preparations which had lost their activity (920). Activity was restored by adding ascorbic acid, glutathione, and an extract of heated fresh enzyme which supplied, besides pyridoxal phosphate, another, anionic, factor. They found no stimulatory effect 011 adding DPN or flavinadeninedinucleotide, and a possible stimulation by folic acid which they considered an artefact. They suggested the mechanism shown in diagram 9, which is based on the presumed mode of action of glyceraldehyde-3-phosphate dehydrogenase (686). :Or

HO -C,H,-CHt*CO* CO,H

Ar *CHn*CO.CO,H

1

(glutathione)

OH

Ar*CHt*CO-SG+ C02

jtI'0

Ar'CHz'CozH

I

ton 4

(unknoun factor)

Ar * CHz. C: * COZH

I

SG Oa (ascorbic acid)

*

HOzC *CH CH.CO.CH2.CO CHz. COzH

+ GSH Ar = 2,5-dihydroxyphenyl GSH = glutathione Diagram 9. Pathway proposed by Williams and Sreenivasan (929) for formation and further degradation of homogentisic acid.

Uchida, Suzuki, and Ichihara (878) isolated a soluble enzyme system (thereby possibly excluding mitochondria1 participation) from rabbit liver, and partially purified it. Two enzymes were involved. The first of these converted p-hydroxyphenylpyruvic acid to 2,5-dihydroxyphenylpyruvic acid. If this enzyme was resolved, vitamin C alone did not restore the activity, but vitamin C and vitamin Blz did. The amount of BIZ required was very low, and they suggested that the true enzyme was a B12 derivative, possibly aquocobalamin hydroxide bound to enzyme protein, and that the function of the ascorbic acid was solely to stabilize the reactive form of the coenzyme. This agrees with the work of La Du and Greenberg (524), who considered the role of ascorbic acid to be quite unspecific. Ascorbate increased the rate of tyrosine oxidation in liver preparations but the net consumption was zero, and moreover numerous ene-diols were just as effective on a molar basis. La Du and Greenberg considered that ascorbic acid participates in a cyclic oxidation-reduction and happens to be a substance of the correct oxidation-reduction potential either t o participate directly or t o protect some other participant. Uchida et al. excluded the possible function of ascorbic acid as a peroxide source and considered their enzyme to be an oxidase, not a peroxidase. Their second enzyme converted dihydroxyphenylpyruvic acid to homo-

C . E. DALGLIESH

I

p-Hydroxgphenylpyruvic acid Cofactor vitamin BIZ, Enrymo I stabilized 111 ieactivc form by ascorbic acid

I

2,5-Dihydroxyphenylpyruvicacid Enzyme II Cofactors eocarboxylase and DPN

Homogentisic acid

1

Cofactnr folie acid

Further oxidation products Diagram 10. Summary of v i e w of Uchida, Suzuki, :ind Ichihara (878) on hoinogcritisic acid formation a n d degradation.

gentisic acid. This was apparently a straightforward oxidative decarboxylation, with cocarboxylase and DPN as cofactors. Folic acid was thought to play no part in the formation of homogentisic acid but to be concerned in its further metabolism. They considered that their stages I and I1 might be interconnected and t o some extent mutually interdependent. This recalls the coupled reactions of the tryptophan peroxidase-oxidase system (see p. 83), and La DU and Zannoni (525) consider that the p-hydroxyphenylpyruvate oxidase system resembles tryptophan peroxidaseoxidase in many respects. The views of the Japanese workers are summarized in diagram 10 (for structural formulas see diagram 8). Knox (483) has purified 100-fold an enzyme converting p-hydroxyphenylpyruvate to homogentisate. The system requires either ascorbic acid or dichlorophenolindophenol, and appears to be much more active than systems previously reported. The conversion occurs in one step. Details of the reaction are awaited with interest especially as 2,5-dihydroxyphenylpyruvate appears to be neither ail intermediate nor an inhibitor (209a). The mechanism of the changes under discussion is still obscure. Keubauer (637) suggested the following scheme : R

No hydroxydienone type of int.ermediate in tyrosiiie metabolism has ever been isolated, but analogous chemical changes can be realized (see below). This scheme was expanded by Neuberger (642), who suggested that the phenolic ion in its para-quinonoid resonance form was oxidized with loss of two electrons to give a positively charged carbonium ion; the latter on reaction with a negatively charged hydroxyl would give the hydroxydi-

63

METABOLISM OF T H E AROMATIC AMINO ACIDS

enone. OQ

0

0

0

R

R

R

HO R

Q-Q+Q’Q

Such a reaction would be expected only in alkaline solution. Moreover, analogous reactions with the resonance forms of the phenoxide ion carrying the negative charge in the ortho position should give 3,4-dihydroxy compounds. The function of an enzyme in this reaction could therefore be to activate the phenolic molecule at physiological p H and to direct subsequent reaction to give para hydroxylat,ion. The reaction has many analogies with that involved in the biogenesis of thyroxine ( q . ~ . ) . Witkop (946) has discussed chemical analogies to these changes. Reactions of the following type can be realized:

I

benzilic acid rcarrangemcnt

HO D

O

00

H R -

Mechanisms such as the above do not involve ortho-quinonoid or catecholtype intermediates. ,4n alternative suggestion (96) involves ortho-dihydroxylation (peroxidation) and a pinacol-pinacolone rearrangement :

6-:q

pinncolpina~ulonc reatrangenlent

\

OH

OH

*

Rd26 H \

\

OH I

OH

(or a tautomer)

There is 1 1 0 direct evidence for or against either of these schemes; nor is it obvious how (lither ascorbic acid or vitamin I312 would participate in them. The function of ascorbic acid iii tyrosine metabolism is complicated by the fact that not only does it participate in more than one stage of the normal pathway, but it can also participate in certain nonspecific reactions. It is not kiionw to what extent the mechanisms of these various functions are related. Nonspecific hydroxylatioii of aromatic (.ompounds (22, 23,

64

C. E. DALGLIESH

106, 881, 882), which can also under some circumstances play a part in phenylalanine and tyrosine metabolism (174), is carried out by ascorbic acid (or a number of other ene-diol or diketo compounds) and iron in the presence of oxygen. The active oxidizing agent is not ascorbic acid, dehydroascrobic acid, or peroxide and is considered (882) to be some unknown oxidation product of ascorbic acid. The results recall the enzymic oxidation of reduced pyridine nucleotides, which in all probability (465, 629) involves a one-electron transfer with a still unidentified oxidation product of ascorbic acid which may be a free-radical electron acceptor of the type of monodehydroascorbic acid. On the other hand, hydroxylation appears to be confined t o electronegative sites on the aromatic ring (106, 881) Mechanistic studies of the reactions involved are highly desirable. 5 . Conversion 0.f Homogentisic Acid to Maleylucetoacetic, Pumarylacetoacetic,

Fumaric, and Acetoacetic Acids Ravdin and Craiidall (695) isolated a protein fraction from rat liver which converted homogentisic acid to a P-keto acid decarboxylated slowly hy aniline citrate a t 38°C. A second enzyme fraction was obtained which converted this keto acid to acetoacetic acid. The P-keto acid was isolated as its silver salt and was found also to be a dicarboxylic acid and a P-diketone, and to give fumaric and acetoacetic acids on hydrolysis. The proposed formulation as fumarylacetoacetic acid (see diagram 8) has since been amply confirmed. Conversion of homogentisic acid to fumarylacetoaretic acid by a liver preparation involves uptake of the expected two atoms of oxygm (e.g., 489, 523). I n the previous section (cf. diagrams 9 and 10) conclusions were cited that ascorbic acid and folic acid may participate in the reactions involved in homogentisic acid degradation. Our principal knowledge of the enzymes involved is due t o Suda and co-workers. They first studied homogentisic acid degradation in a Pseudomonas species (840), from which they were able to obtain a cell-free enzyme preparation, and then studied the reactioii in rabbit liver (841). In the latter they found a requirement for a dialyzable cofactor arid for ferrous iron. Ferric iron was ineffective and thv function of ascorbate in the reaction was considered to he the reduction of ferric iron to ferrous. In livers of scorbutic guinea pigs they found (842) the level of homogentisicase to be much lower than in riornial animals. h r tivity was restored by ferrous iron or, less effcrtively, by ascorhic. avid. ad-Dipyridyl (which coordinates with iron) inhibited homogentisicasc :ivtion and administration of tyrosine to dipyridyl-treated rats was followcd 113’ homogentisic arid excretion. The influence of iron and ascorbic acid a11cl the irihibitioii by dipyridyl have been confirmed by Crandall (160) mid Schepartz (770, 771).

METABOLISM OF THE AROMATIC AMINO ACIDS

65

The quinonoid form of homogentisic acid, i.e., benzoquinone-acetic acid, is probably not a n intermediate in the reaction (160,770, 841), in which it acts as a n inhibitor, but may be slowly oxidized. The high redox potential of this quinone makes it unlikely to exist in the free state for any length of time in body fluids. Many other chelating substances, e.g., versene and oxine, inhibit homogentisicase (770,771), and the inhibition is reversed by ferrous iron. The extent of inhibition by versene depends on the time of incubation, indicating that the iron is not simply in ionic association with the apoenzyme. Iron cannot be replaced by other metals. Ascorbic acid can be replaced by many ene-diols (771); this supports a nonspecific action. Glutathione may also be concerned in the reaction, possibly protecting essential -SH groups. An iron-sulfur bond may be involved (163). Homogentisicase occurs in kidney as well as liver (160, 161, 164) but not in appreciable amount in other organs. Direct formation of a fumaric acid derivative (having a trans arrangement about the double bond) from a benzenoid compound (having a cis arrangement about the double bonds) would be surprising. Knox (486) has resolved this difficulty by showing that the more probable maleylacetoacetate is the first product of the reaction, and that this is then converted to fumarylacetoacetate by an isomerizing enzyme. The latter can be separated from homogentisicase by alcohol fractionation and the reaction thereby stopped a t the maleylacetoacetate stage. Maleylacetoacetate is formed from homogentisate with uptake of the expected two atoms of oxygen per molecule. The isomerase requires glutathione as a cofactor (cf. 209, 485). The enzyme hydrolyzing fumarylacetoacetic acid to fumaric and acetoacetic acids has as yet been little studied. It may be the same as acylpyruvase (592) or the triacetic acid hydrolyzing enzyme (154).

IV. TYROSINE DEGRADATION BY

THE

CATECHOL PATHWAY

3,4-Dihydroxyphenylalanine(for structure see diagram 11) is a n amino acid isolated (from the pods and sprouts of Viciu fubu) and first definitely identified in 1913 by Guggenheim (323),who showed it to be identical with synthetic material previously prepared (280). It is widely distributed in certain types of plants (beans, etc.) but is not a normal protein constituent. However, it plays an important part in mammalian metabolism of tyrosine, as it is the precursor of adrenaline (epinephrine), noradrenaline (arterenol, norepinephrine), and melanin. In this review these substances will be considered only insofar as they account for a portion of normal tyrosine metabolism.

60

C. E. DALGLIESH

1. Adrenaline, Noradrenaline, and Their Biogenesis

Adrenaline and noradrenaline (structures; diagram 1 1) are the chemical mediators of sympathetic nervous transmission and as such are indispensable for normal function of highcr organisms. Numerous reviews are available on their physiology and pharmacology and on the relation of structure to activity (e.g., 02, 74, 114, 244, 324, 735). Adrenaline was first isolated in the crystalline state from natural sources in 1901 by Takamine (851), and its structure soon proved by synthesis and optical resolution of the racemate to give the Zcwo isomer (168,270,834). Noradrenaline was known as a chemical substance before the remarkably recent realization (cf. 297, 850,875) that it, rather than adrenaline itself, is the chief sympathomimetic agent serving t o transmit adrenergic impulses under normal conditions (for reviews see 243, 849). The assumption of early workers that!adrenaline is derived from phenylalanine and tyrosine was conclusively proved by the demonstration (328, cf. also 888) that phenylalanine labeled in the a-position of the side chain with C14 gave adrenaline labeled in the corresponding position. It is reasonable to assume that tyrosine is an intermediate in this conversion. The further conversion of tyrosine to adrenaline involves four changes: (1) introduction of a further phenolic group, ( 2 ) decarboxylation, ( 3 ) introduction of the side chain hydroxyl, and ( 4 ) N-methylation. A great deal about the biosynthesis of adrenaline remains obscure, but it is nevertheless possible to advance a tentative hypothesis concerning the order in which these changes occur. The conversion of tyrosine to 3,4-dihydroxyphenylalanineoccurs both in vivo in man (590) and in vitro by the action of tissue tyrosinase (205,688). Mammals can decarboxylate both tyrosine (402,407)and dihydroxyphenylalanine (406), tyrosine decarboxylase and dihydroxyphenylalanine (dopa) decarboxylases being quite distinct and separable (405), though both are dependent on pyridoxal phosphate (73, 758, and review 72). I n mammals dihydroxyphenylalanine is the most readily decarboxylated of all amino acids, and it is therefore not unreasonable t o assume that this is the substrate normally decarboxylated in adrenaline biosynthesis (cf. 74, 75). Support for this concept derives from the fact that both the substrate and the product of the reaction (3 ,4-dihydroxyphenylethylamine; diagram 1 I ) can or do occur in the adrenal (298, 299, 802), and the amine is moreover, like adrenaline and noradrenaline, a normal urinary excretion product (245,404). If the above are accepted as the first two steps in adrenaline biogeriesis it becomes possible to predict that the remaining two steps occur in the order shown in diagram 11.

67

METABOLISM OF THE AROMATIC AMINO ACIDS

Tyrosine

3,4-Dihydroxyphenylalanine @+dopa; dopa)

HO

m -

c H 2 - m .CHI

OH Adrenaline (epinephrine)

3,4-Dihydroxyphenylethylamine

(hydrosytryamine; dopamine)

-

H

i

HO

o

b c H . cH2.NHz 1 OH

Noradrenaline (norepinephrine, arterenol)

Diagram 11. The pathway considered most probable for the biogenesis of adrenaline from tyrosine.

The widespread co-occurrence of noradrenaline arid adrenaline in itself suggests that noradrenaline is the immediate adrenaline precursor. This had been considered probable even before the natural occurrence of noradrenaline was known (70, 71), and the methylation of noradreiialine has since been shown both in vitro in adrenal preparations (110) and in vivo on perfusing the surviving adrenal (111). The methyl group can arise from methionine, probably formed from choline, in which the adrenal is extremely rich. A large proportion of the activity of administered (methy1-Cl4) methioniiie appears in the adrenal (460,569). As three of the four stages in adrenaline biogenesis are thus provisionally established, the remaining stage, introduction of the side-chain hydroxyl, should occur by conversion of dihydroxyphenylethylaniine to noradrenaline. There appears to be little information available on this reaction (cf. 195a). This picture of adrenaline biogenesis may well be an over simplification. Thus 3,4-dihydroxyphenylserine,but not its N-methyl derivative (177), can also be decarboxylated, to give noradrenaline (63,78); this mould imply that a sequence 3,4- dihydroxypheriylalanine + 3,4- dihydroxyphenylserine noradrenaline should be considered. 3,4-Dihydroxyphenylserine can be split to 3,4-dihydroxybenxaldehydeand glycine (594), and this might conceivably be a minor pathway of tyrosine metabolism. I n the octopus p-hydroxyphenylethanolamine (octopamine) occurs (373,762); this might imply the existence in this species of one of the sequences tyrosine tyramine + octopamine noradrenaline, or tyrosine -+ p-hydroxyphenylser---f

-

---f

68

C. E. DALGLIESH

HO

T H ~ HC-O ~ H OH NHZ 3,4-Dihydroxyphenylserine

H O ~ ~ H . C H ~ . N H ~ OH Octopamine

ine +octopamine --+noradreiialiiie. However, iii the mammal, though tyramine can be formed, especially in the pancreas (373), it seems to be cntirely metabolized by amine oxidase to give ultimately p-hydroxyphenylacetic acid (762), and such sequences as these would be unlikely to occur. Many reactions of possible significance in adrenaline biogenesis can also take place nonenaymically, e.g., tyrosine to dihydroxyphenylalanine (13, 18,174, 277,690) ; tyramine to dihydroxyphenylethylamine (106,403,690) ; octopamine to noradrenaline (801). Such reactions might in part be responsible for the contradictory evidence from early experiments with tissue slices and honiogenates (e.g., 198, 247, 777, 778, 896). The site of the biosynthesis of adrenaline is moreover not established, and there is good evidence (888) that the adrenal itself is by no means necessarily the principal tissue concerned. Different steps in the hiosynthesis may occur in different tissues. Different species, or even different organs of the same species, may use different routes for biosynthesis. It has even been suggested (803) that a still unsuspected biosynthetic route might owur.

2. Metabolic Llegradation of Noradrenaline and Adrenaline. Adrenochrome Adrenaline and noradrenaline can both give rise to melaniris (cf. below). An intermediate in such a transformation of adrenaline is adrenochrome, a molecule stabilized by resonance (359). Adrenochrome has a powerful effect on the maturation of reticulocytes (281) and might play a part in normal physiological processes. The extent to which adrenaline is converted to adrenochrome and melanin, and its significance, is still unknown.

I

CH, Resonance forms of adrenochrome

I CHs

The degradation of adrenaline to nonpolymeric materials has been studied especially by Schayer and co-workers, using various isotopically labeled adrenalines (760, 761, 764-766). At physiological concentrations no conjugation (e.g., with sulfate or glucuronic acid) occurs. The molecule is almost entirely degraded by t,he action of amine oxidase, which brings about

69

METABOLISM O F T H E AROMATIC AMINO ACIDS

demethylamination with subsequent excretion of a product still containing both a- and P-carbon atoms of the side chain. (For a review on amine oxidase, see 77.) 3. Melanogenesis and Albinism

Extensive reviews on melanogenesis are available (269, 553, 554, and especially 581), and the subject is dealt with only in outline here (diagram 12). Tyrosine

-""a""'; tyrosinase

+t02,

H

HO

o

~

c

*

COlH ~ ~

+$ 0H2

-GzGG+ noneiirymic

\

+:Or

internal

CH'C02H

reduction

/CH*Co2H

HO

internal oxidntionreduction, decarboxylation

\

nonenzymic-

KH

HO

*

HO

H Diagram 12. Pathway for the conversion of tyrosine t o 5,6-dihydroxyindole, the precursor of melanin.

The transformations involved in melanogenesis were largely worked out by Raper and his co-workers (205, 682, 688, 689, 691) and have been confirmed spectroscopically by Mason (579, 580). Tyrosine, like many other phenols, is attacked by the copper-containing enzyme tyrosinase (phenoloxidase; for review see 814) to give dihydroxyphenylalanine. This is further oxidized, enzymically or spontaneously, to the quinone, which by an internal oxidation-reduction cyclizes to 5,6-dihydroxy-2, S-dihydroindole2-carboxylic acid; this undergoes spontaneous oxidation to the corresponding quinone (dopachrome (113), formerly incorrectly identified with the natural pigment hallachrome) which by a further internal oxidation-reduction, possibly catalyzed by zinc (466), and subsequent loss of COz, gives 5,6-dihydroxyindole, the probable precursor of melanin. The melanogen in melanuria is a mixture of conjugated derivatives of 5,6-dihydroxyindole (547, 548, 548a, 563). Similar series of reactions can occur with other derivatives of 3,4-dihydroxyphenylethylamine,such as adrenaline, and these also give rise to melanins. Polymerization of 5,6-dihydroxyindole probably (112) occurs by oxidation t o the quinone, which then polymerizes through the 3-, 4-,7-, and occasionally 2-, positions to give a polymer as in diagram 13.

70

C. E. DALGLIESH

5,B-Dihydroxyindolc (cf. diagram 12) (*)

0 e.g.

8

-

0

etc.

b

<*I

*

Indole-5,G-quinone

*

0

Diagram 13. suggested type of polymerization in melanin formation (rj”. Du’T,ock and Harley-Mason; 112). Asterisks represent positions taking part i n polymerisation reactions, those in hrackets heing less likely.

Alternatively (108) a seven-membered ring might be formed before polymerization as in diagram 14. 2 Molecules of indole-5,6-quinone

i

1 HOn
polymers

I

I-I 0 (or isomers)

Diagram 14. An alternative suggested route (108) for melanin formation.

Melanin is an extremely inhomogeneous substance, bound to protein, (cf. 581a), and also binding metals such as iron (e.g., 531, 532). It is

probable tthat a “pure” melanin, in the sense of a product derived froni a single precursor, rarely occurs naturally. Melanin is the normal pigment of the skin arid mammalian hair. Carcinomatous growths in which abnormal melanin formation occurs are known as melanomas. A congenital metabolic defect in which skin pigmentation does not occur is known as albinism, and is inherited as a recessive Mendelian character (cf. 40). Albinos occur in many species besides man (e.g., the pink-eyed white rabbit). As adrenalhe formation is apparently unimpaired in albinos, the metabolic block presumably lies in the conversion of dihydroxyphenylalanine to melanin, as shown in diagram A, rather thaii in the conversion of tyrosine to dihydroxyphenylalanine. However, the exact nature of the block has not been established. It seems probable that melanin is not further metabolized, a t least in

METABOLISM O F THE AROMATIC AMINO ACIDS

71

the mammal, and can therefore be looked on as a true end product of tyrosine metabolism.

4. The Catechol Pathway in the Insect The insect cuticle is formed from a water-soluble protein and an orthodihydric phenol (677) shown to be 3,4-dihydroxybenzoic acid (679). This phenol probably ‘‘tans” the protein by oxidation to the quinone and reaction with protein side chains, cross-linking the chains and causing a hardening and darkening. Besides 3,4-dihydroxybenzoic acid the cuticle also contains 3,4-dihydroxyphenylacetic acid, 3,4dihydroxyphenyllactic acid, and 3,4-dihydroxyphenylalanine(332, 680, and cf. 331), obviously suggesting biogenesis from the latter. 3-Hydroxykynurenine (q.v.) may also be concerned in both hardening (678) and darkening (431) of insect cuticles.

V. TYROSINE METABOLISM VIA THYROID HORMONES AND OTHER HALOGENATED DERIVATIVES In higher organisms a portion of the tyrosine is metabolized via the thyroid hormones. Physiologically these are highly active substances, and the amount of tyrosine metabolized by this pathway is probably relatively small. However, no quantitative figures appear to be available. The exact nature of the thyroid hormone, whether protein or otherwise, and the physiological and endocrinological aspects, such as the mutual interaction of thyroid and pituitary, cannot be discussed here. Excellent reviews are available elsewhere (e.g., 8, 31, 358, 673, 703, 751). 1 . Thyroxine and Its Biogenesis

Baumann (37, 39) was first to show that the active principle of the thyroid gland contains iodine. It was only 20 years later, in 1915, that Kendall (461) reported his isolation, after a most laborious separation, of the pure substance, to which he gave the name thyroxine (462, 463). The analytical difficulties with a substance containing some 65 % of iodine are considerable, and the analyses of Kendall’s material misled him into formulating it as an iiidole derivative. Harington (349) considerably improved the isolation procedure and showed that Kendall’s formulation was untenable. Harington also found (350) that iodine c+ould be removed from thyroxine by catalytic hydrogenation to give an iodine-free phenolic amino acid, desiodothyroxine, which was latcr named thyronine. Degradative studies suggested the presence of two benzene rings, a phenolic oxygen, an ether oxygen, and an alanine side chain. Of the possible structures, that nou7 accepted for thyronine was considered most likely and was proved by synthesis (350). The position of the iodine

72

C. E. DALGLIESH

Thyroxine, R = I Thyronine, R = H

R

R

NH1

atoms in the previously known iodo-gorgoic acid (3 ,5-diiodotyrosine; described later) suggested that in thyroxine the iodine occupied the 3,5,3' ,5'-positions. As direct iodination of thyronine introduced only two iodine atoms, a new synthesis was devised by Harington and Barger (355)) and iodinated this to 3,5,3' ,5'who prepared 3 ,5-diiodo-~~-thyronine tetraiodothyronine, identical with the thyroxine from thyroid tissue (this had been obtained by a procedure involving alkaline hydrolysis and consequent racemization). The synthetic material was resolved by Harington (351) and the L-isomer shown to be identical with natural thyroxine isolated using nonracemizing enzymic procedures (137, 358). Inspection of the structure of thyroxine suggested tyrosine and diiodotyrosine as precursors (355)) and this supposition was strengthened by the isolation of 3,5-diiodotyrosine from thyroid tissue (276, 357). Between them thyroxine and diiodotyrosine account for the greater part of the organically bound iodine of thyroid. In addition, however, there also occur monoiodotyrosine (265, 860) and triiodothyronine, which is discussed in more detail below. Von Mutzenbecher (566) showed that iodination of casein under appropriate conditions gave a product from which thyroxine could be isolated; moreover, thyroxine was formed t o a small extent even on incubating diiodotyrosine in alkaline solution (625). These results were confirmed by Harington and Pitt-Rivers (356), and it was subsequently found (670, 671) that if simple peptide derivatives of diiodotyrosine were used, thyroxine derivatives could be obtained in quite high yields at pH values nearer neutrality; for example, incubation of N-acetyl-3 ,5-diiodo-~~-tyrosylglut&Illic acid a t pH 7.5 gave N-acetyl-DLthyroxylglutamic acid in a yield of 36 %. The study of the thyroid hormones was greatly facilitated when radioactive isotopes of iodine became available. Using II3l it was soon confirmed (576, 612, 665, 666) that activity administered as iodide was converted in the thyroid to diiodotyrosine and then thyroxine. The mechanism of thyroxine biogenesis was considered by Johnson arid Tewkesbury (451)) who recognized the similarity between the conversion of diiodotyrosine to thyroxine and the type of oxidation of ortho- and para-substituted phenols extensively studied by Pummerer (684). They suggested that oxidation gave rise to two types of free radical, one with the electron lost from the para position of the quinonoid form and one from the phenolic oxygen. Addition of these two, as in diagram 15, would give

73

METABOLISM O F THE AROMATIC AMINO ACIDS

I

Hu

Q-c y x a m NHZ

I

oxidation

1

1

o@Hza‘:“:’”.: -I- -0

I

D‘

CHZ.CH(SHZ)*COZH

I (free radical intermediates) o I

~

.1

~

~

*

C

O

~

H

CHz.CH(NHz) *COzH

I’

I

J.

Thyroxine -k amino-acrylic acid

+

pyruvic acid h’H, Diagram 15. Scheme of Johnson and Tewkesbury for thyroxin biogenesis.

an intermediate from which they postulated loss of aminoacrylic acid which would be expected to be spontaneously hydrolyzed to pyruvic acid and ammonia. They claimed to be able to detect the latter two substances in their reaction solution, but other workers have not been able to repeat this. Though the main outline of Johnson and Tewkesbury’s hypothesis is accepted, the mechanistic details and the fate of the ejected alanine side chain are not known. The hypothesis was considerably extended by Harington (353) and Neuberger (642), who pointed out that the chemical reaction is likely to occur in alkaline solution and is more probable with diiodotyrosine than with tyrosine itself, as the phenolic group of the former is more acidic. Moreover, the iodine ortho substituents inhibit various other reactions occuring with phenols not so substituted. The only probable product of the reaction is in fact thyroxine. The considerable similarity between thyroxine forinatiori and homogeritisic acid formatioil was pointed out by Neuberger (642), who also advanced an alternative to

74

C. E. DALGLlESH

the free-radical mechanism. Instead of loss of one electron each from two molecules, oxidation of a diiodophenoxide ion could give a positively charged carbonium ion which would then add to a second negatively charged unoxidized diiodophenoxide ion :

+ I’

* Hydroxydienone intermediate It is not known whether or not the conversion of diiodotyrosiiie to thyroxine is enzymic (cf. 354, 672). If enzymic, the function of the enzyme might be t o produce a t a physiological pH the type of reactive intermediate only obtainable in vitro a t a more alkaline pH. It may be significant that tyrosine, di-iodotyrosine and thyroxine all occur as N-terminal groups in pork thyroglobulin (7204. More detailed discussions of many aspects of the chemistry and biochemistry of thyroxine are available elsewhere (320, 352, 353, 048, 719). 2 . Yriiodothyronine and Its Riogensis

3,s,3’-Triiodo-~-thyroninewas found in plasma and thyroid by Gross and Pitt-Rivers (316, 318) and identified with synthetic material. It was identified almost simultaneously both free and in the circulating hormone thyroglobulin by Itoche and co-worker,. (714, 715, 717). It is several (probably three to five) times as active physiologically as is thyroxine in many different types of test (317, 319), and the question has been argued a t some length as to which is the true thyroid hormone. The question is not settled, and for the present it is convenient to regard both as hormones or hormone precursors. There are three plausible pathways for the biosyrithesis of triiodothyronine. It could be formed from one molecule each of mono- and diiodotyrosiiie in the same way as thyroxine can be formed from two molecules of diiodotyrosine; or it could be formed by incomplete iodinatiori of preformed thryoiiine or diiodothyronine; or it could be formed by deiodination of thyroxine. Formation by deiodination is favored by English work-

lIo&p,,. .CH I .C02H

NHz I Triiodotliyronine

METABOLISM OF T H E AROMATIC AMINO ACIDS

75

ers (e.g., 320, 570,571) and formation by one of the other routes, by French workers (e.g., 716, 718, 720). Only contradictory evidence is available on enzymes which might specifically bring about synthesis by any of the likely routw. The effect of various thyroxine antagonists has been attributed to inhibition of deiodinatioii (e.g., 923 for references). The biogenetic pathway is still an open question (321), and the coexistence of two pathways is not excluded. Significant recent papers are 624a, 860a, 863a, 963a. 3 . Metabolic Fate of the Thyroid Hormones

Both thyroxine and triiodothyronine are excreted unchanged and as their glucuronides in the bile (476, 721, 859). The greater part, however, probably undergoes deiodination, possibly in the salivary glands (252). An enzyme, tyrosine iodinase, occurs both in thyroid and, to a greater extent, in the salivary glands. This can either synthesize moiioiodotyrosine from tyrosine, or deiodinate monoiodotyrosine, the direction of reaction varying with the oxidation-reduction potential of the tissue (251, 253). The relation of this enzyme to thyroxine metabolism is still uncertain. It has been suggested that this or a similar enzyme brings about synthesis in the thyroid, where there is a high local concentration of iodine and the product is removed as thyroglobulin, whereas in the salivary gland conditions favor breakdown. It is not yet known whether deiodination of thyroxine and triiodothyronine is complete or partial, or what route the degradation of the deiodinated products takes. Mono- and diiodotyrosine are said not to leave the thyroid as such, but are deiodiriated and the iodine reutilized (320, 720). However, in liver and kidney diiodotyrosine can be converted to the corresponding pyruvic and lactic acids (873), and triiodothyronine and thyroxine give the corresponding pyruvic acids (721a).

4, Other Naturally Occurring Halogenated Tyrosines Drechsel (203) in 1896 reported isolation from the coral Gorgonia cauolinii of an iodine-containing amino arid, which he named iodogorgoic acid. Its formulation as 3,5-diiodotyrosine mas established by synthesis (388, 910). It has since been found that halogenated tyrosines are widely distributed in marine organisms, especially in the corneous skeleton of various Anthozoa and sponges. Besides diiodotyrosine there occur monoiodotyrosine, thyroxine, monobromotyrosine, and dibromotyrosine, and their distribution can be used for biological classification. Such organisms concentrate considerable amounts of halogens from sea water, the amount of halogen fixed as halogenated tyrosine depending on the organism’s tyrosine rontent. The field has been well reviewed by Rorhe (713).

76

C . E. DALGLIESH

VI. PATHWAYS OF PHENYLALANINE AND TYROSINE METABOLISM UTILIZED PRINCIPALLY BY MICROORGANISMS It is probable that the route for pheriylalanine and tyrosine degradation via homogentisic acid, the principal route obtaining in mammals, is also used by a t least some microorganisms (e.g., 453, 840). But not all microorganisms necessarily use it, and many can carry out a number of other types of degradative reaction. The chemical potentialities of microorganisms are so enormous that only some of the more important reactions can be considered here. These are summarized in diagram 16. Baumann (36, 38) suggested the following scheme for tyrosine degradation by the bacterial flora of the gut: tyrosine

-+

p-hydroxyphenylpropionic acid

I

p-hydroxyphenylacetic acid +- p-ethylphenol

I

p-cresol -+ p-hydroxybenzoic acid

+ phenol

These substances are all excreted in normal human urine (535) and are thought probably t o arise from bacterial action, but not all the transformations postulated by Baumann are probable. For example, as we shall see below, phenol is formed directly from tyrosine. Though benzoic and p-hydroxybenzoic acids can be formed from phenylalanine and tyrosine, for example, by Pseudomonas species (346, 833), a plant rather than bacterial origin has been suggested for the benzoic acid moiety of the hippuric acid excreted by man (59). Recent work suggests that benzoic acid can be formed in mammalian tissues (330a, 776a). 1 . Products Based on Decarboxylation and A m i n e Oxidation

Decarboxylation of amino acids is a typical feature of the bacterial decomposition of proteins. Both phenylethylamine and tyramine were isolated from putrid meat by Barger and Walpole (30), who considered it L‘extremely probable” that they were derived from phenylalanine and tyrosine, respectively. No cell-free preparation of phenylalanine decarboxylase appears to have been reported, but decarboxylation by a crude Streptococcus faecalis preparation provides a valuable method of phenylalaiiine assay (887). Bacterial tyrosine decarboxylase has been studied in detail (495), especially by Gale and co-workers (summarized in 284). It requires pyridoxal phosphate as coenzyme (26, 326, 327) and, unlike mammalian tyrosine decarboxylase, also attacks dihydroxyphenylalanine. Decarboxylation normally only occurs in acid media and is considered primarily to be a protective mechanism tending to restore the pH to neu-

METABOLISM OF T H E AROMATIC AMINO ACIDS

+

77

+

ArH NHa CHa.CO.CO2H (e.g., phenol from tyrosine)

T 8-tyrosinaue

I

(o.g.,

I)hetiJ’lethyl:irniiic.

I :tmirie oxidasr

I

Ar .CH 2 . CBO

dearninases or

I t ransaminases

(e.g., phenylacetaldehyde)

1 Ar.CH2.CO.COsH (e.g., phenylpyruvic acid)

>

-

1

1

Ar.CH2.C02H (e.g., phenylacetic acid)

A~.CI-IZ.CH,C>O?H

I

OH (e.g., phenylluctic acid)

?I

> Ar.CHz.CH?.COzH ---+Ar. CH: CH. C02H ? (e.g., cinnamic acid) (e.g., phenylpropi oriic acid) Diagram 16. Interrelationships of some side-chain metabolic reactions used b y microorganisms. Ar can be phenyl, p-hydroxyphenyl (as in tyrosine), indol-3-yl(as in tryptophan), 3,4-dihydroxyphenyl, etc. Names given above are in general for substances derived from phenylalanine.

trality. Under alkaline conditions splitting of tyrosine to phenol, described later, is favored (495). The amine resulting from decarboxylase action can be oxidatively deamiriated to give the aldehyde or, more usually, the acid by further oxidation : It.CHzNH2 -+ Ilt,CH:NH]

--t

RCHO

-, It.COrH

This reaction has been primarily studied in higher organisms (review 77) but probably occurs equally in microorganisms and also in plants, where it is probably intimately connected with biosynthesis of alkaloids ( q . ~ . ) . 2 . Products Based on Oxidative Deamination or Transamination Both reactions give the corresponding keto acid. Oxidative deamination has been studied by, e.g., Stumpf and Green (839). For cases of trans-

78

C . E. DliLGLIESH

amination see, e.g., references 739, 912. The keto acid can be further oxidized to the acetic acid derivative (the same as is formed by the decarboxylase-amine oxidase pathway). Thus the following pathways have been demonstrated in a Vibrio species (167): phenylalanirie -+ phenylpyruvic acid -+ phenylacetic acid

L

1

tyrosine

---f

p-hydroxypheriylpyruvic acid --+ hornogentisic acid

1

furthcr metabolism

Reduction of the pyruvic acid would give the lactic acid (214, 50’21, and dehydration of this would give the acrylic acid (p-coumaric acid from tyrosine (e.g., 391) and cinnamic acid from phenylalanine). However, the acrylic acid derivative might be formed directly from the amino acid (cf. the direct conversion of histidine to urocanic acid). Reduction of the acrylic acids might be the origin of the propionic acid derivatives sometimes encountered (e.g., 100). 3 . Phenol Formation from Tyrosine. p-l’yrosinase

Tyrosine is converted to phenol by an enzyme, p-tyrosinase, studied especially by Japanese workers (456, 654, 658, 879). The enzyme, which has been partially purified, is inhibited by carbonyl reagents and is dependent on pyridoxal phosphate. The reaction is mechanistically probably (593) very similar to the tryptophanase reaction and is discussed when considering the function of pyridoxal phosphate (p. 91).

4. Degradations Involving Opening

of the Aromatic Ring

The microbiological degradation of many substances allied to the aromatic amino acids has been studied (reviews 346, 825). Thus the ring of catechol is opened to give ultimately p-ketoadipic acid (469) via cis-cismuconic acid (246, 813, 826) :

various

OH

stapes

I

H&,

,CO,H C H?

Analogous reactions might occur with, for example, dihydroxyphenylalanine or its derivatives, (e.g., 568, 829).

METABOLISM O F T H E 4ROMATIC AMINO ACIDS

70

VII. TRYPTOPHAN DEGRADATION BY THE KYNURENINE-NICOTINIC ACIDPATHWAY Among the most remarkable features of tryptophan metabolism is the widespread use of a common pathway by a wide range of organisms. Thus many microorganisms growing on tryptophan as sole carbon source metabolize it by a route very similar to that used in man, animals, insects, and probably also to a considerable extent in plants. I n this discussion, therefore, evidence leading to the elucidation of the pathway will be drawn from many fields, and more detailed consideration of the individual steps will cover a range of species. The structures of the substances discussed can be found in diagrams 17, 18, 20, and 21. 1. Establishment of the Relation between Tryptophan and Nicotinic Acid

Nicotinic Acid It was only many years after the discovery of tryptophan that a plausible degradative pathway could first be outlined, but during this early period a few tryptophan metabolites were identified. The long-known (559) kynurenic acid (structure, diagram 20; cf. 408) was shown in 1904 to be derived from tryptophan (220), but the considerable amount of work on kynureiiic acid formation (reviewed by Neubauer, 637) gave few useful results. Neubauer (637), however, made the plausible (and correct) suggestion that i t was derived from o-aminobenzoylpyruvic acid (structure, diagram 20). It was not till 1925 that Japanese workers (587) reported a substance, kynurenine, subsequently shown (507) also to be derived from tryptophan. Unfortunately the position was confused by the incorrect structure originally assigned to kynurenine, and the correct structure (diagram 17) was only established in 1942 by Buteiiandt and co-workers (127, 129). Meanwhile Musajo (616, 617) isolated (from the urine of rats on a fibrin-based diet fed added tryptophan) a substance which he called xanthurenic acid and which he showed to be 4,8-dihydroxyquinoli1ie-2-carboxylicacid (diagram 20). Lepkovsky and co-workers (550) later found the urine of pyridoxine-deficient rats to contain a green pigment which was (551) an iron complex of xaiithureriic acid. A plausible hypothesis at this stage (about 1943) was that a sequence occurred : tryptophan -+ an intermediate -+ kynurenine + kynurenic acid. I t was thought that the intermediate between tryptophan and kynurenine might be the so-called a-hydroxytryptophan (for structure see p. 83), which had been obtained (917) on hydrolysis of phalloidine, a toxic peptide from the fungus Amanita phalloides (567) ; such a pathway received

80

C. E. DALGLIESH

further support from the study of insect eye-pigments. From genetic experiments (reviews: I 16, 467) with mutants of Drosophila melanogaster and Ephestia kuhniella the brown eye-pigment, ommochrome, was known to be derived from tryptophan by the following sequence: tryptophan 3 V+-substance -+ rn+-substance ommochrome. The V+-substance was identified as kynurenine (125, 857), and hydroxytryptophan was thought to act as a “prokynurenine” (126). Metabolic experiments with animals had meanwhile suggested that L-, rather than D-, tryptophan was precursor of the known metabolites and also suggested that an abnormal excretion of these metabolites occurred in pyridoxine deficiency (for further details see an earlier review by the author, 170). I n 1945 Elvehjem and co-workers (518) reported that nicotinic aciddeficient rats would grow if given tryptophan, suggesting conversion of tryptophan to nicotinic acid. Rosen and co-workers (731) showed that administration of tryptophan t o rats increased the urinary excretion of nicotinic acid derivatives, and numerous workers confirmed the conversion of tryptophan to nicotinic acid in man (399, 667, 755) and many other species (summary, 820). In the last ten years there has been intensive investigation of tryptophan metabolism. ---f

2 . The General Outline of the Pathway

It was quickly established by many techniques (381, 430, 782, 817, 957, and review 170) that the conversion of tryptophan to nicotinic acid occurred in body tissues and was not due (except perhaps in part in exceptional circumstances; cf. 170) t o intestinal bacteria. Moreover nutritional studies showed that kynurenine was probably also a precursor of nicotinic acid (457) and that kynurenine and xanthurenic acid excretion were increased in pyridoxine deficiency (21). The probable course of tryptophan-nicotinic acid interconversion first became clear from experiments with microorganisms, mainly due to Mitchell, Nyc, Bonner, and their co-workers. In 1947 it was found (41) that a Neurospora mutant requiring tryptophan or nicotinic acid could equally tryptophan peroxidnse

Formylkynurenine

Unknown intermediate

oxidsse ___*

K ynurenine

Diagram 17. Reactions involved in transformation of tryptophan t o kynurenine.

METABOLISM OF THE AROMATIC AMINO ACIDS

81

utilize L-kynurenine, and converted excess kynurenine to nicotinic acid. It was thought that nicotinic acid might be derived from the pyridine ring of kynurenic acid, but this was excluded (603) by tests with various potential oxidation products, and it was shown that the 8-hydroxy group in xanthurenic acid was in all probability introduced before cyclization. This immediately suggested 3-hydroxykynurenine (diagram 18) and the derived 3-hydroxyanthranilic acid as intermediates. Hydroxyanthranilic acid (651) was found to be rapidly converted t o nicotinic acid, and was also accumulated by another Neurospora mutant (90, 92). Hydroxykynurenine was isolated from the pupae of CaEliphora erythrocephala (115, 623) and was identified as the cn+-substance referred to above, and its constitution was proved by synthesis (128, 500, 623). It was also obtained from the larvae of the silkworm Bombyx mori (392) and was shown to give nicotinic acid in a Neurospora mutant blocked after the kynurenine stage (970), which accumulated Na-acetylkynurenine (diagram 20) in the medium. Interest then moved to animals. Both isotopic and nutritional experiments showed that the pathway established in microorganisms applied equally t o mammals. Thus hydroxyanthranilic acid was converted to nicotinic acid (9, 604), which it could replace as a growth factor (944), whereas there was no similar conversion of anthranilic acid (343). An outstanding series of isotopic experiments, especially by Heidelberger and co-workers, showed that the @-carbonatom of the tryptophan side chain became the ,&carbon atom of the kynurenine side chain and that the side chain was lost in conversion of kynurenine to nicotinic acid (369, 371, 427). Moreover the carbon in the 3-position of the indole nucleus became the carboxyl carbon of nicotinic acid (370; this experiment proved conclusively the reality of the tryptophan-nicotinic acid conversion) and the indole nitrogen appeared with only slight dilution in kynurenine, kynurenic acid, and xanthurenic acid (759). All these relations are those to be expected for the pathway tryptophan -+ kynurenine -+ hydroxykynurenine (or its phosphate) 3 hydroxyanthranilic acid (or its phosphate) + nicotinic acid, illustrated in diagrams 17 and 18. By 1950 the outline of the main pathway for tryptophan metabolism was therefore established, and it was becoming apparent that the over-all conversion of tryptophan to nicotinic acid was markedly reduced in many B-vitamin deficiencies. Thus tjhis occurred in pyridoxine deficiency (50, 387,732,784), riboflavin deficiency (387,455, G75), and thiamine deficieilcy (455, 675) but not in pantothcnate or folic acid deficiencies (455). I n the succeeding sectlions the various transformations involved will 1)e considered in detail.

M E3

Kynurenic acid, xanthurenic acid, and derivatives (diagram 19)

CO .CHz*CH*COzH

CO.CHz.CH.COzH

I

PiHz

Kynurenine

Anthranilic acid

I

h

NHz

NHz

OH Hydrosxkynurenine

Hydrosyanthranilic acid

(cf. intermediate Unknown diagram 21)

O'POaHs Hydroxykynurenine phosphate

Hydrosyanthranilic acid phosphate

*

Xicotinic acid

Diagram 18. Transformation of kynurenine to h\droxynnthmnilic acid or i t s phosphate.

?

METABOLISM OF THE AROAlATIC AMINO ACIDS

83

3 . The Converszon o j Trgptophan lo Formyllcynurenzne and Kynurenine

Details of the mechanism of tryptophan-kynurenine interconversion are largely due to Knox and his co-workers, in work originally arising from a wartime study of the quininc-oxidizing erizyine (477). I n 19-19 it was reported (492) that kynurenine was formed from tryptophan by liver homogenates under aerobic c*onditions,and detailed studies were published soon after (490, 591). L-Tryptophan was converted t o kynurenirie by a highly specific enzyme contained in liver homogenates of all species tested. The over-all reaction involved uptake of one molecule of oxygen, with liberation of one molecule of formic acid, but 110 carbon dioxide. The system was separable into two fractions. The first fraction converted tryptophan to a substance identified as formylkynureriine (diagram 17), and the second fraction contained another highly specific enzyme, formylase, which converted the formylkynureriine to kynurenine and formic acid. The enzyme system in the first fraction was itself divisible into two components. The first enzymic component required hydrogen peroxide generated in situ, added peroxide being iiieff ectivc, and converted tryptophan to an unknown intermediate, A. This was converted by the second enzymic component t o formylkynureiiine with simultaneous formation of peroxide, which was then utilized by the first component. The three stages are therefore as folloms: 1 Tiyptophan

A

oxidase + o2

+HD2

peroxidtrse ~

formyllipureiiine

3 Formylkyriurenine

+ A

+ I-1?01

formylase

kynurenine

+ H COIH

and the first two of these stages are coupled t o form the tryptophan pcroxidase-oxidase system. Differences exist between tryptophan peroxidase and other peroxidases, especially in their relation t o iron and copper (479, 481). Similar enzyme systems occur in ba ria (e.g., 366) and plants (e.g., 932). The nature of the intermediate A aroused widespread interest. The work already described suggested that it was a-hydroxytryptophan (more correctly described as oxindolylalanine, 157), which chemically is a plausible precursor of formylkynurenine (cf. review, 170).

a-Hydroxytryptophan

Oxindolylalanine

Rut when synthetic oxindolylalanine became available (156, 499) it8was

84

C . E. D.\LGLIESH

OH

Possible dial intermediate

OH

Witkop’s proposed intermediate (0-hydroxy-$-tryptophan)

quickly shown iiot to be a tryptophan metabolite (11 , 117, 176, 584, 743). Alternatively hydrogen peroxide might add to tryptophan to give a diol (176, 490). This has not yet been synthesized and so cannot be tested i n biological systems (nor has the intermediate, A, been isolated), From a consideration of numerous oxidations, using different types of reagent, of a variety of indoles and indole alkaloids Witkop arid co-workers concluded (216) that intermediate A was most likely to be of the reactive indolenine type (i.e., having a double bond between nitrogen and a-carbon of the pyrrole ring). They considered as the most likely substance P-hydroxy-$-tryptophan (structure above), which they proposed to synthesize, though no synthesis appears yet to have been reported. P-Hydroxy-+-tryptophan might be converted to formylkynurenine either directly or by addition of water to give the previously proposed diol, which might then undergo dehydrogenation and ring-opening. A third possible route was considered, by way of the more highly oxidized hydroperoxide, but was thought to be less likely for an in vivo reartion. Indirect support for this third route, however, derives from work of Weiss and co-workers (442), who studied the breakdown of tryptophan 011 X-irradiation of aerated solutions. Such conditions bring about reactions analogous t o biological degradations in a large number of cases, and in agreement with this, formation of formylkynurenine from tryptophan was observed. The reaction required molecular oxygen, providing strong eviderire for a hydroperoxide type of intermediate which, for mechanistic reasons, they eonsidered t o have the hydrated structure depicted ahove rather than the indolenine structure considered by Witkop.

Possible hydroperoxide intermediate

Weiss’s proposed interniediatc

The structure of the first intermediate in the normal biologiral degradation of tryptophan is therefore still uncertain. The difficulties in synthesizing any of the proposed intermediates are very considerable, and till such synthesis is accbomplished conclusive evidence will not be availablr. The problem is made somewhat less urgent by the fact that, whatever the

METABOLISM OF T H E AROMATIC AMINO ACIDS

85

intermediate may be, no evidence (except possibly 310) has apparently yet been reported for its separate existence in vivo for any appreciable length of time. No cofactors have been reported in the tryptophan peroxidase-oxidase reaction, but the marked reduction ill the conversion of tryptophan to nicotinic acid i n thiamine deficiency has been found in al! probability to be due to interference with the reaction at the tryptophan peroxidaseoxidase stage (173; cf. diagram 19). The evidence is still inadequate to show how enzyme function and vitamin are related. Biotin may also be concerned in the reaction (800; but see 175a). The enzyme called formylase by Knox and Mehler (490, 591) arid kynurenine formamidase by Jakoby (437) is present in liver in a considerable excess relative t o tryptophan peroxidase-oxidase (e.g., 491), and formylkynurenine is therefore not normally found in tissues or excreted in urine (e.g., 171). Partially purified tryptophan peroxidase-oxidase, from which formylase activity has been removed, accumulates formylkynurenine, shown (591) to be identical with synthetic (947 or better, 172) material. Formylase occurs widely in bacteria, and has been partially purified from Neurospora (437). I n both higher and lower organisms the enzyme shows considerable specificity.

4, Tryptophan Peroxidase-Oxidase Adaptation Although enzymic adaptation has been known for some time in microorganisms, tryptophan peroxidase-oxidase was probably the first animal enzyme t o be proved to be adaptive. Knox and Mehler showed that if tryptophan is given to an animal orally, subcutaneously, or intraperitoneally the liver tryptophan peroxidase-oxidase activity can rise up to ten-fold (491), and similar adaptation occurs in liver slices but not in homogenates. These results have been confirmed by many workers (e.g., 210, 536, 537), and investigation of adaptive formation of the enzyme, which probably occurs by de novo synthesis of enzyme protein rather than by rearrangement of existing protein, has been used as an approach to the problem of protein biosynthesis (314a, 535a, 536, 537). Adaptation also, as might be expected, occurs in bacteria (366). Tryptophan is, however, not the only agent which can bring about an increase in tryptophan peroxidase-oxidase. A smaller effect can be produced by substances which initiate the stress reaction of the adrenalpituitary system (478). High X-irradiation produces a similar effect in normal, but not in adrenalectomized, animals (869). Cortisone reverses the effect of adrenalectomy (868), and glucocorticoids (e.g., cortisone and hydrocortisone) can themselves cause an increase in the enzyme (484). How these changes are brought about is still obscure; their elucidation

80

C . E. DALGLIESH

should give valuable information on the normal mechanism of adjustment of physiological processes. 6 . Conversion o./ K!inureninc to tly~roxijkyrL~irciLine.IZole oJ IZibofEavivi

Experiments with mutants of microorganisms and insects, which made it likely that hydroxykynurenine is an intermediate in tryptophan nietabolism, have been described above. Further evidence for the occurrence of hydroxykynmenine in insect larvae has since heen reported (575, 84-1, 845), and its relation to eye-pigments is discussed below. It is also formed in plants (932). Strong support for participation in manimaliari metabolism was provided by its identification in mamrrialiati urine (171, 176, 292, and c.f. further discussion later). No enzyme system which will oxidize kyiiurenine to hydroxykynureriirie has yet been isolated in a cell-free state from any species, and there is some evidence that direct conversion may not normally occur, a t least in mammals. Riboflavin tvas suggested to be concerned in hydroxykynurenine formation a t a comparatively early stage (387), and this has been supported by nutritional experiments. Tryptophan metabolism can be briefly represented as in diagram 19. Tryptophan'

1

c__--- - - - - - c Formylkynurenine

Iiynurenic acid and conjugated derivatives of kynurenine

-

t

Kynurenine

A

I

Xanthurenic acid and conjugated derivatives of hydroxykynurenine

t

B

I

_f__c

Hydroxykynurenine

_ _ _ _ _ _ _ _ _ _ _ _ _ A_ _ _ _ _ _ - _ __ _ Anthranilic acid

4

________A

Hydroxyanthranilic acid

Diagram 19. Relation of vitamins t o tryptophan metabolism. Deficiencies of pyridoxine, riboflavin, and thiamine cause blocks a t A - - - A , I3 - - - H ,and (' - - - P , respectively.

It has been shown by the author that examination of the products excreted after administration of tryptophan to vitarnin-deficient animals can give valuable information on the function of that vitamin in tryptophan metabolism (142, 171, 173). When tryptophan is given to the riboflavindeficient rat there is a large excretion of those substances which lie t o the left of line BB in diagram 19 (142, 582). This clearly indicates that this is the step a t which riboflavin functions, and this is strongly supported by the fact that riboflavin deficiency can reduce up to ten-fold the conversion of t,ryptophan t,o quinolinic acid, whereas similar conversion of hydroxykynureiijrie is unaffected (385). On the other hand, the excretory pattern

iMRT.\BOLISM OF THE AltOMSTIC .\MINO hCIDS

87

obtained after feeding tryptophan to riboflavin-deficient rats is not clean cut as therc is also an increase in the excretion of xanthurenic acid, which lies to the right of line BB in diagram 19 (142). A possible reason for this emerged from work of Wiss and Hellniann (375, 942), who found that tryptophan and kynureninc were converted by liver homogenates not to 3-hydroxyanthranilic acid but to a derivative, identified as its O-phosphate, which also arts as a nicotinic acid precursor. Both kynurenine and hydroxykyriurenine are split by an enzyme, kynureninase (considered in more detail below), to give anthranilic acid and hydroxyanthranilic acid, respectively. As hydroxyanthranilic acid was not converted by liver to the phosphate, it is reasonable to suppose that the hydroxyanthranilic acid phosphate was formed by action of kynureiiinase on hydroxykynurenine phosphate (structure: diagram 18). This would indicate that the hydroxylation stage in tryptophan metabolism is an oxidative phosphorylation rather than a simple oxidation, energy from this phosphate bond possibly being concerned in subsequent conversion of hydroxyanthranilic acid t o nicotinic acid. Urinary excretion of hydroxykynurenine, rather than its phosphate, is not surprising in view of the widespread occurrence of phosphatases in many organs including the kidney. Riboflavin might therefore be converned with a phosphorylative rather than a purely oxidative function, and the increased xaiithureriic acid excretion in riboflavin deficiency can be plausibly explained on this basis (142). It is of interest that in Knox's early work on the yuinine-oxidizing system of liver (477) the partially purified enzyme which hydroxylated quinolines appeared to be a flavoprotein. I n view of the current interest in hydroxylation reactions in vivo it is to be hoped that this stage of tryptophan metabolism mill receive more detailed attention from enzymologists. At present it can be stated definitely only that kynurenine (or possibly a derivative such as N"-acetylkynureniiie; 170, 173) is converted either to hydroxykynureiiine or to some simple derivative a t the same level of oxidation. It is of interest that both kynurenine and hydroxykynurenine are formed on photooxidation of tryptophan, especially in the presence of ferrous iron (972). 6 . Hgdroxgkynurenine and Insect Eye Pigments

The identification of hydroxykynurenine as the cn+-substance which is the precursor of insect eye pigments (ommochromes) has already been described. Ommochromes had previously been considered to be pterin derivatives (140, 534), but there is no obvious route for the conversion of hydroxykyriurenine to the pterin type of structure. However, pterins also occur in insect eyes (275). More recent work has shown that the eye pig-

88

C. E. DALGLIESII

ments, which appear to be similar to the pigments of insect eggs, are fornicd directly from hydroxykynureiiine to give derivatives forming complrxcs with metals, especially copper arid iroii (468). In the eggs the pigmc3iit seems to serve a definite physiological function, as pigmented eggs arc more viable than noiipigmented (467). Hydroxykynurenine also seems to participate in the hardening of the insect cuticle (678). The chemical nature of the pigments is becoming evident from work in Butenandt's laboratory, which has eliminated the pterin type of structure. Ommochromes can be divided into ommins, of high molecular weight, and ommatins, of low molecular weight. Various ommatins were isolated (122) and studied chemically (120) and shown by comparison with model compounds (121) probably to be phenoxazine derivatives. Thus two molecules of hydroxykynurenine can react together to give a phenoxazine as follows (where R is the side chain .C0.CHz.CH(NH2)vCOnH): R

R __c

013 Hydroxykynurenine

R

R

f2H

OH phenoxazine derivative

J

transamination and cyclization of one side chain

The phenoxazine can then participate in reversible oxidation-reduction reactions, and moreover by transamination and cyclization of one side chain, in a manner analogous to xanthurenic acid formation discussed below, can give a pyridinophenoxazine derivative which equally undergoes reversible oxidation-reduction, and which, being a derivative of 8-hydroxyquinoline, would bind metals strongly (see also 122a).

7. Kynureninase, Kynurenine l'ransaminase, and the Formation of Anthranilic, Kynurenic, Hydroxyanthranilic, and Xanthurenic Acids Reference has already been made to the abnormally high excretion of kynurenine and xanthurenic acid in pyridoxine deficiency. This was confirmed by many workers (e.g., 372, 599, 620, 674, 698, 732), and the reason for it became evident from enzymic experiments. The first mention of enzymic conversion of kynurenine to kynurenic

METABOLISM OF THE AROMATIC AMINO ACIDS

89

acid and anthranilic acid was by Kotake (501, 512), the enzyme forming anthranilic acid being named kynureninase. Braunshtein and colleagues (102) showed that alanine was also formed, and that the enzyme occurred in the livers arid kidneys of all species tested. It was inhibited by carbonyl reagents and was dependent on pyridoxal phosphate. The amount of enzyme was markedly reduced in the liver of pyridoxine-deficient rats, but the pyridoxine deficiency affected only the degradation of kynurenine and not its formation from tryptophan. Kynureninase activity was restored to normal on adding pyridoxal phosphate. The formation of anthranilic acid and alanine was confirmed by Wiss (936, 941, 945) and the pyridoxal phosphate requirement by Knox (176). The enzyme attacks kynurenine and hydroxykynurenine at comparable rates (176, 480, 940) and is unspecific in the sense that it will attack (at varying rates) many substrates containing the .CO.CH2.CH(NH2).COzH side chain (123, 124, 376, 939, 940). Powerful support for these enzymic results was obtained from nutritional experiments (171, 176, 293) which showed that in pyridoxine deficiency tryptophan gave rise to urinary excretion of many metabolites besides the previously known kynurenine, kynureriic acid, and xanthurenic acid. The majority of these were identified (171), and all still carried the carbon skeleton of the original tryptophan side chain. It was therefore apparent that pyridoxine functioned in tryptophan metabolism at the stage a t which the original side chain was removed. Knox et al. (176) also found that after prolonged pyridoxine deficiency kynureninase activity of the liver is not restored by adding pyridoxal phosphate in vitro, and this, too, is in accord .with results of nutritional experiments (732, 768). As pyridoxine deficiency has no effect on conversion of tryptophan to kynurenine (102) or of hydroxyanthranilic acid to nicotinic acid (387), it must participate in tryptophan metabolism as indicated in diagrams 18 and 19. Knox et al. (176) found that formation of anthranilic acid from kynurenine was always accompanied by formation of kynurenic acid, and they considered that the keto acid (0-aminobenzoylpyruvic acid, diagram 20) might be a common intermediate in formation of both substances. This was soon disproved, and it is now clear that two independent reactions are involved, as illustrated in diagram 20. Wiss (937, 938) fractionated crude liver extracts to give a kynureninase fraction which would form anthranilic acid, but not kynurenic acid, and a transaminase fraction which mould not form anthranilic acid, but formed kynurenic acid provided an a-keto acid was present. o-Aminobenzoylpyruvic acid, the keto acid corresponding to kynurenine, is known to cyclize spontaneously to kynurenic acid (622), and the absence of ammonia production and requirement for an a-keto acid (cf. also 434) suggests that

00

1

[p

C . E. DALGLIESH

OH

CO.CH2. CO * COZH

spontsneoua _____)

N'

NHz

R

L

J

o-Aminobenzoylpyruvic acid derivatives

I

COzH

It Kynurenic acid (R- H) Xanthurenic acid (R- OH)

kynurenine transaminase

8:

CO *CHI*CH *COzH KH2

I

NH2

R

kynureninase (cofactor: pyridoxal phosphate)

-

Kynurenine (R * H) Hydroxykynurenine (R OH) ,

q

C

0 *CHz*CH.COzH

I

NH * CO * CHI NHz

R X@-Acetylkynurenine(RE H)

f CHJ*CH*CO~H

I

NHz

'

R

NHz

+

An thranilic acid (R H) alanine Hydroxyanthranilic acid (R = OH)

Conjugated derivatives of hydroxykynurenine

-

Glucuronide (R 0.C6HPOD) Sulfate ( R = O*SO,H) Diagram 20. Interrelationship of derivatives of kynurenine and hydroxykynarenine. Na-Acetylhydrosykyn(Irenine (R = OH)

it is formed by transamination alone and not by oxidative deaminatioii. 3-Hydroxykynureniiie undergoes similar splitting (kynureninase) and cayclization (transaminase) reactions. Pyridoxal phosphate is coenzyme for both reactions, mid comparison of activities of various fractions made it clear that the same enzymes attack both kynurenine aiid hydroxykynurcnine (938). These results have been confirmed by Knox (480),who showed that under normal circunistances the formation and degradation of kynurenine in the liver proceed at comparable rates. Iiynurenine and hydroxykyiiurenine are also metabolized by both kynureninase and kynurenine transaminase in the kidney. But whereas kynureninase activity of kidney is only about one-tenth that of liver (480),the transaminase activity of kidney is much higher (583), aiid kynurenine is therefore converted almost exclusively to kynurenic acid by kidney preparations (cf. 583a, 585). The detailed mechanism of pyridoxal phosphate participation in the kynureninase and kynurenine transaminase reactions is considered in detail later. Of interest in this connect,ion is the finding that other amino

METABOLISM O F THE AIEOMATIC AMINO ACIDS

91

acids inhibit mammalian kynureninase (306). This is probably due to the requirement for pyridoxal phosphate oE most reactions involving amino acids, and resultaut competition for the coenzyme. Such competition probably explains, a t least in part, the interference with tryptophail metabolism observed 011 giving excess of threonine, or in general amino acid imbalance (12, 315, 384, S l l ) , which has been attributed to interference with tryptophan absorption from the gut and to preferential use of tryptophan for protein biosynthesis. Microorganisms also utilize both the kynureninase and kynuren ine transaminase reactions. Bacterial kynureninase has been partially purified and, like the mammalian enzyme, requires pyridoxal phosphate (367) and givs anthranilic acid and alanine, but not pyruvic acid (600). Moreover bacterial kynureninase has no transaminating activity (GOO), and bacterial kynurenine transaminase has no kynureninase activity (601). The kynureninase of Neurospora has been studied in some detail (438, 433). This again resembles the mammalian enzyme in attacking both L-kynurenine and 3-hydroxy-~-kynurenine(it also attacks formyl-L-kynureniiw, but not D-kynurenine or N"-acetyl-L-kynurenine) and in requiring pyridclxal phosphate. Magnesium also activates the system. The Neurospora enzyme is also inhibited by amino acids and amines, probably by competitive removal of pyridoxal phosphate. Xanthureriic acid excretion can be caused in the rabbit by a vitamin-E as n.ell as vitamin-Be-deficiency (200). This is more likely to be due to vitamin interaction than to a direct effect of vitamin E on tryptaphan metabolism. 8. Mechanism of Action of Pyridoxal Phosphate in Reactions I7i volving Aromatic A m i n o Acids

Pyridoxal phosphate is the coenzyme in a large number of amino acid reactions. At this point it is convenient to consider together the mechanism of those pyridoxal-dependent reactions concerned with aromatic amino acids. The reactions concerned are (1) keto acid formation (e.g., from kynurenine, above), (2) decarboxylation (e.g., of 5-hydraxytryptophan t o 5-hydroxytryptamine, p. loo), ( 3 ) scission of the side chain (eg., p-tyrosinase, p. 78; tryptophanase, p. 110; and kynureninase, above), and (4) synthesis (e.g., of tryptophan from indole and serine, p. 40). Many workers have considered the mechanism of one or more of these reactions (e.g., 24, 216, 361, 595), but a unified theory is primarily due t o Snell and his colleagues (summarized in 593). Snell's experiments have been carried out largely in vitro, and it should be emphasized that in vivo it is the enzyme protein which probably directs the clectromeric changes.

92

C. E. DALGLIESH

The amino acid, coordinating metal, aud pyridoxal phosphate are considered to react to give a complex having a conjugated system of double bonds extending from the electron-attracting nitrogen of the pyridoxal to the site of reaction. The coordinating metal probably functions both by promoting Schiff’s hsse formation and by maintaining planarity in the conjugated system through chelate-ring formation. In the following diagram [PI represeiibs the phosphate group of pyridoxal phosphate which combines with the apoenzyme (cf. 300) :

Aromatic amino acid

Coordinating metal

Pyridoxal phosphate or its complex with the apoenzyme

Resonance forms of the coenzynie ainiiio acid complex, A; electromeric displacements indicated by the curly arrows.

The complex may then split in various ways. If splitting occurs a t aa, keto acid formation can occur by way of the following prototropic changes:

The keto acid and pyridoxamine phosphate result, and the latter can

METABOLISM O F THE AROMATIC A M I N O ACIDS

93

then transaminate by a reverse series of changes with another a-kc:to acid. The pyridoxal phosphate thus acts essentially as a nitrogen carrier between amino acid and keto acid, and the reactions may well be linked so that pyridoxamine phosphate as such has no independent existence. If, however, the complex breaks a t point bb in the first diagram above, decarboxylation results as follows :

+

Complex A, above

amine COz+ pyridoxal phosphs te 4

The above reactions are common to most amino acids. I n the case of aromatic amino acids the aryl group (except for the unactivltted phenyl group of phenylalanine) can itself act as an electron-attracting group. If this occurs the aryl group can be split off by the following series of rertctions:

Scission of the side chain leaves an Ar- ion which takes up a proton to give indole from tryptophan (as with tryptophanase) or phenol from tyrosine (as with @-tyrosinase). The side chain of the original molecule is left as the pyridoxal phosphate complex of aminoacrylic acid, and on hydrolysis the aminoacrylic acid tautomerizes to the imine of pyruvic acid which is hydrolyzed to pyruvic acid and ammonia: CHz=C*COzH --+ CHs.C.COzH + CHS.CO*COzH

I

NHz

II NH

+ NH3

Although the kyriureriinase reaction involves a scission, it is probably of

94

C . E. DALGLIESH

a differmt type. The Schiff’s base from kynurenine arid pyridoxal phosphate is an imine of a p-diketone, aiid it is to be supposed that the enzynw probably carries out hydrolysis analogous to the ready chemical hydrolysis of such cwmpoundtj. But the intermediacy of the Schiff’s base is not universally accepted (439), and it is significant that the side chain iii this rcwtion is liberated as alauine, aiid not as pyruvic acid and ammonia. It has also been suggested that the result can be explained by a scission of the tryptophanase type, together with an internal oxidation-reduction t o give the observed products (561.a). The pyridoxal phosphate complex of aniiiioacrylic acid can also be formed from seriiie by loss of an OH- radical in a manner aiialogous to loss of the Alir radical depicted above. This complex contains a reactive double bond to which the reactive @-hydrogenof indole can add, giving a complex which on hydrolysis yields tryptophan. Such a mechanism is in accord with the known facts on tryptophan hiosynthesis (cf. 858, and previous discussion, p. 41). 9. Excretion of Ilydroxykynurenine and Xanthurenic Acid by M a n

Excretion of hydroxykynurenine or xanthureiiic acid by man has been coniiected with pathological states. Excretion of xanthurenic acid by pyridoxiiie-defic.ient animals was observed a t an early stage, as already described. Xanthureiiic acid is also excreted, especially after a loading dose of tryptophan, during pyridoxine deficiency in man, whether this is produced by a true dietary deficiency (311) or by administration of the vitamin antagonist deoxypyridoxine (294). Pyridoxine deficiency in man caii to some extent be judgod by determining (599, 621, 732,733, 898) the amount of xanthurenic acid exrreted after a standard dose of tryptophan, the value so obtained being known as the xanthurenic index (145). il high xaiithurenic index is found in pregnancy (155a, 618, 891, 899), when the extra demand for the vitamin may cause a “physiogical” pyridoxine deficiency. This excretion caii be reduced by pyridoxine administration (e.g., 900). An especially high xanthurenic index may be found in certain disorders of pregnancy (823, 899). After giving a loading dose of tryptophan in pregnancy not only xanthurenic acid is excreted, but also (901) the whole range of tryptophaii metabolites observed in the pyridoxine deficient rat (cf. 171). Further interest was aroused when Kotake (503) claimed that xanthurenic acid would cause diabetes, that it occurred in diabetic urines (515), and that diabetes induced by xanthurenic acid was reduced by insulin administration (509). Further supporting work has since been published from the same laboratory (50-1-506, 508), but German workers (400) have been quite unahle t o demonstrate any diabetogenic action of xanthurenic acid,

METABOLISM OF T H E AROMATIC AMINO ACIDS

95

except for a transient hyperglycemia occurring when xanthurenic acid administration was superposed on a pyridoxine deficiency (908). Hydroxykynurenine excretion in pathological states was also first reported by Japanese workers (573))who identified it as the substance causing the diazo reaction and the Weiss urochromogen reaction in urines from cases of severe tuberculosis. This was confirmed in the author’s laboratory (178), where it was also shown that the excretion is unrelated to tuberculosis as such. Hydroxykynurenine excretion occurs in a large proportion of patients with fevers of varying etiology and is in all probability due to the increased rate of breakdown of body proteins in fever. Presumably the protein breakdown induces an adaptive increase in tryptophan peroxidase-oxidase, and the capacity of the available kynureninase, which comes laterinthe metabolic chain and is not an adaptive enzyme (480), is exceeded. Hydroxykynurenine excretion in fever is sometimes accompanied by smaller amounts of kynurenine, whereas if excess tryptophan is taken by mouth large amounts of kynurenine, but negligible hydroxykynurenine, are excreted. These differences in metabolism of exogenous and eiidogenous tryptophan are unlikely to be due to use of different metabolic pathways and suggest that tryptophan molecules from endogenous protein breakdown and from exogenous sources do not necessarily equilibrate in a common body tryptophan “pool.” Hydroxykynurenine and kynurenine excretion in leukemia (618, 619) and in diabetes (515) have also been reported. I n both cases fever is the probable cause. Diabetes as such does not result in hydroxykynurenine excretion (178), which probably occurs only when there is fever due to complications. Traces of kynurenine may also be encountered in normal urine (818a). For recent work on kynurenine and hydroxykynurenine excretion see (6194. 10. Side lieactions o j Kynureninc, Hydrozyliynurenine, Anthranilic A c i d ,

and Hydroxyanthranilic Acid Na-Acetylkynurenine (i.e., acetylated on the aliphatic amino group) was isolated from a Neurospora mutant culture (970) and was also found in the urine of pyridoxine-deficient rats together with the analogous Nu-acetylhydroxykynurenine (171). It is possible, though there is no direct evidence, that these N‘-acetyl derivativcs play some part in normal tryptophan mctabolism in the rat (ef. 170, 173)) or they may, as is probably the case in Neurospora, merely be products of side reactions occurring in the presence of abnormal amounts of kynurcnine and hydroxykynurenine. Hydroxykynurcniric is also cwreted by the pyridoxine-deficicnt rat as its O-sulfate

96

C. E. DALGLIESH

and 0-glucuronide (171). These are probably simple “detoxication” products without other metabolic significance. The formation of all these compounds is illustrated in diagram 20 above. Xanthurenic acid if present in unusual amount is also treated as a foreign phenol and excreted in conjugated form (171, 513), though the nature of the conjugating groups has not been established. Xanthurenic acid can probably also be degraded to some extent by an enzyme, xanthurenicase, in liver and kidney (510), but the products have not been identified. Kynurenic acid and xanthureriic acid are both metabolically speaking waste products in that they cannot be converted to nicotinic acid. The same applies t o anthranilic acid, which apparently cannot be directly converted to hydroxyanthranilic acid to any appreciable extent in mammals or even in microorganisms (9 1, 663). Under normal circumstances mammalian metabolism of tryptophan is so well regulated that kynureiiine is apparently converted to hydroxykynurenine (or its phosphate) as fast as it is formed. As a result, under normal circumstances only hydroxykynurenine (or its phosphate) is a kynureninase substrate and therefore no anthranilic acid is formed. Supporting evidence is that in man and other mammals anthranilic acid taken by mouth is excreted either unchanged, as the glucuronide, or as the glycine conjugate, o-aminohippuric acid (103, 142, 582), though a small amount of 3-hydroxyanthranilic acid and 5-hydroxyanthranilic acid (514) may be formed, possibly by unspecific hydroxylation (cf. 174). If anthranilic acid were formed in normal metabolism a t least part would therefore almost certainly be excreted. However, anthranilic acid excretion does not occur in man or mammals except after intake of excess tryptophan. It is of considerable interest that a rare human disorder, congenital hypoplastic anemia, is accompanied by anthranilic acid excretion (lo), indicating an associated anomaly in tryptophan metabolism. Both anthranilic acid and hydroxyanthranilic acid call he formed from t ryptophan in insect mutants, and both arc conjugated with glyvine to give substituted hippuric acids (706), whereas in plants or bacteria anthranilic acid tends t o be conjugated as the 6-glucoside (848). Another interesting variant of tryptophan metabolism has been fouiid in silkworm pupae ( I 18), which form kyriurine (4-hydroxyquinoline). This might arise by direct decarboxylation of kynurenic acid, but it is more likely (1 19) that it is formed by decarboxylation of kynureiiine to kynurcnaminc, which would be expected to give kynurine through the action of aminc oxidasc. T-Iydroxykynureiiiiie can also be converted by mouse liver, probably via the corresponding amine, to 4: 8-dihydroxyquinoline (571a).

METABOLISM OF T H E AROMATIC AMINO ACIDS

97

11. Conversion of Hydroxyanthranilic Acid to Nicotinic Acid

The discovery that 3-hydroxyanthranilic acid is a precursor of nicotinic acid in Neurospora (YO, 516, 603) and the rat (9, 516, 604, 943, 944) has already been mentioned. It had been known for some time (810) that rats fed tryptophan excreted in their urine a substance which, after hcing autoclaved in acid solution, had nicotinic acid activity. This was identified by Henderson (379, 383) as quinolinic acid (structure, diagram 21). Mutants of Neurospora were also found which accumulated quinolinic acid, and this could be used by some, but not all, nicotinic-requiring Neurospora strains (94). I n the rat quinolinic acid was mostly excreted unchanged, but part was converted to nicotinic acid, though much less efficiently than was hydroxyanthranilic acid (380). Hydroxyarithranilic acid was converted t o quinolinic acid by liver slices (386)) and it was considered possible that quinolinic acid was a normal intermediate in nicotinic acid formation. The lesser effectiveness as a nicotinic precursor of quinolinic acid relative t o hydroxyanthranilic acid was attributed to the conversion of quinolinic t o nicotinic acid being slou and rate-determining. At first the position was, moreover, complicated by isotopic experiments with NI6-ammonia and unlabeled hydroxyanthranilic acid in Neurospora (541). Isotope was incorporated in the nicotinic acid to a degree suggesting formation of a symmetrical intermediate. Sucl an intermediate was difficult to visualize, but the difficulty was later removed when the isotope incorporation was shown (971) t o be due t o simultaneous active growth of the organism. If no growth occurs the nitrogen of nicotinic acid is derived entirely from the nitrogen of hydroxyanthranilic acid. It is of considerable interest that recent evidence shows (968) that in 8.coli and B. subtilis, but not in Neurospora, nicotinic acid can be formed by a route not involving tryptophan. The results with Neurospora led Bonner and Yanofsky (94) to suggest that the conversion of hydroxyanthranilic acid t o nicotinic acid went by way of Intermediates A and B of diagram 21. Quinolinic acid formation was thought t o be a shunt or side reaction of intermediate A, slow conversion t o nicotinic acid possibly providing an alternative pathway. A similar conclusion was drawn from experiments in the rat (971), and it is now generally agreed that the conversion of quinolinic acid t o nicotinic acid is a t best of the order of a side reaction (e.g., 685,754, and in man, 397,696). The conversion of hydroxyanthranilic acid to nicotinic acid in the rat has been shown unambiguously in isotopic experiments (344). Conversion to either quinolinic or nicotinic acids must involve open-chain intermediates such as A and B of diagram 21. Japanese workers (572) suggested that these were formed by scission between the catechol hydroxyls of the ring

08

C . E. DALGLIESH

-QZ

3-Hydroxykynurenine (or its phosphate)

1

I

3,4-Dihydroxykynurenine

b +

c-g:

OH 3-Hydroanthranilic acid (or its phosphate)

HO

OH 3,4Dihydroxyanthranilic acid

\

I I I ?

1 2 stages

I

t

COiH

/ 9

Intermediate A

nCoiH

OHC Quinolinic acid V

Isocinchomeronic acid

... ...

NH,

Intermediate B

Nicotinic acid

Diagram 21. The probable pathway (via Intermediates A and B), and other suggested pathways, for conversion of hydroxyanthranilic acid t o nicotinic acid.

of 3,4-dihydroxyanthranilicacid (diagram 21), and it was claimed that synthetic dihydroxyanthranilic acid gave rise to nicotinic acid (433,572). However, many workers in enzymic and growth experiments on both animals and microorganisms could find no evidence that dihydroxyanthranilic acid was a nicotinic precursor (89,'376,382). Moreover, its presumed progenitor, 3,4-dihydroxykynurenine,has also been shown not to be B tryptophan metabolite (123,171,795). Isotopic experiments (763)with tryptophaii labeled with NI5 and deuterium in the indole ring have shown that quinolinic acid nitrogen is probably entirely derived from the indole nitrogen of tryptophan, and that scissioii of the benzene ring probably occurs between carbons 3 and 4. Presumably, therefore, the hydroxyanthranilic acid is converted to intermediate A without participation of a catechol-type intermediate, and it is possible that the phosphate-bond energy of hydroxyanthranilic acid phosphate (if this is in fact an intermediate) may contribute to the transformation. It is known

99

XETABOLISM OF THE AROMATIC AMINO ACIDS

that phenols can be converted to ortho-quinonoid compounds without the necessity of passing through a catechol stage (946), and it may well be that such a quinonoid compound participates in the conversion of hydroxyanthranilic acid to intermediate A. This is supported by spectroscopic evidence suggesting that two intermediates, the first possibly quinonoid, are involved in the conversion of hydroxyanthrariilic acid to quinolinic acid (89). A quirioneimiiie structure has been suggested (608) for the first intermediate, but no attention appears to have been paid to the necessity for a fumaric rnaleic isomerizatiori of intermediate A (but cf. 938a). An alternative route to nicotinic acid involves scission of hydroxyanthraiiilic acid between carbons 2 and 3, with intermediate formation of isocinchomeronic acid (diagram 21). But the latter does not act as a nicotinic precursor in Neurospora (374), and this route can probably be excluded. The enzyme (386, 971) converting hydroxyarithranilic acid to quirioliiiic acid has been widely studied. Rat liver homogenates or acetone powder can carry out the conversion in yields of 73 % to 100% (88), though an enzyme located in the heavy particles of the cell can also convert hydroxyanthranilic acid to a red-colored product (897). The quinolinic-forming system occurs widely in liver and kidney (676) but not in other organs (812), and the activity is associated with the nonparticulate fraction of an homogeriate (812). The spectroscopic changes occurring in the reaction are referred to above. The enzyme shows a requirement for ferrous iron under certain conditions (564, GOS), and to some extent resembles the enzymes oxidizing homogentisic acid, catechol, and protocatechuic acid (162). It is remarkable that the intermediates in the conversion of hydroxyanthranilic acid to nicotinic acid are still not known with certainty. Intermediates A and B of diagram 21 are plausible suggestions, but no synthesis of either has been reported. Both are extremely unlikely to be stable in the free state, but should be obtainable as simple derivatives. In the free state Intermediate A, for example, might be expected to tautomerixe to the iniino acid, and hence give keto acid and ammonia, or it could cyclixe to a piperazine or t,o quinolinic acid. Tautonierism through the iniino acid would eliminate the necessity for a fumaric maleic isomerization. It is quite possible that spontaneous cyclization explains the prominent part quinolinic acid plays in work 011 hydroxyanthranilic-nicotinic conversion. If the latter occurred in the following .way: ---$

---f

Hydroxyanthranilic acid

enzyme -4

Intermediate A (diagram 21)

spontaneous

+

I

!

I

-coz

-COa; enzyme B

Intermediate

B (diagram 21)

Quinolinic acid

I

?

.1 spontaneous +

Nicotinic acid

100

C. E. DALGLIESH

and if eiizyme B vere more labile than enzyme A, or if the two enzymes became spatially separated on disorganization of the cell as in a n homogenate, then hydroxyaiithranilic acid mould be expected to give quinolinic acid. There is good evidence that the conversion of intermediate A to quinolinic acid is spontaneous (564, 590a). The low activity of quinolinic acid as a nicotinic precursor might be due to some much slower and less specific8 decarboxylation process. If a large dose of hydroxyanthranilic acid is given to nu aiiinial n small proportion is excreted unchanged (104). Hydroxyanthranilic acid is also excreted by man after a large dose of tryptophan ((i96), and has been found in human urine in tuberculosis (624). The latter is probably due to high eridogeiious protein breakdown (cf. 178). It is possible that quiiioliuic acid might he decarboxylated to picolinic (pyridine-karboxylic) acid as well as to nicotinic (pyridine-3-carboxylic) acid. Such may be the origin of the hoinarine (picolinic acid betaine) widely occurring in marine organisms (e.g., 423). 12. Tryptophan, Nicotinic Acid, and the Pyridine Nucleotides

The pyridine nucleotides are the functional form of nicotinic acid, but still comparatively little work has been done on their relation to tryptophannicotinic acid metabolism. Two possibilities must be considered; the pyridine riucleotides may be formed from tryptophari without intermediacy of nicotinic acid and only give nicotinic acid on breakdown, or nicotinic acid first formed from tryptophan may be incorporated into pyridine nucleotides. The latter now seems the more likely possibility, though the former has not been excluded. pyridine nucleotides

nicotinic acid

?further

------+

breakdown

nicotinic metubolitcs

Injected tryptophan causes a rise in erythrocyte DPN in the rat (562). The problem was taken up by Elvehjem and his school (cf. review, 224), who a t first found tryptophan to be more active than nicotinamide in stimulating synthesis of rat-liver DPN and TPN (924, 925). Nicotinamide had, however, a sparing effect in young, but not in adult, rats (925). In pyridoxine deficiency conversion of tryptophan to pyridine nucleotides

METABOLISM O F THE AROMATIC AMINO ACIDS

101

appeared to be unaffected (926) unless deoxypyridoxine were also given (519). These results suggested that nicotinic acid and amide were not, iiccessary intermediates, hut later work (254, 255) showed that tryptophan aiid nicotinic acid were equally good pyridine nucleotide precursors on a molar h i s (previous results having been due to use of a relatively much larger amourit, of tryptophan), whereas nicotinamide was more efficient than either. But it was still felt that nicotinic acid was not a necessary intermediate. Conversion of nicotinic acid to pyridine nucleotides was, as is to he expected, unaffected by pyridoxine deficiency (519). Other workers (206,533) have studied the pyridine nucleotides of human red blood cells, which are known to be increased after nicotinic acid administration (342, 394). Sarett (206) found that both tryptophan and nicotinic acid given orally to man cause a rise in blood pyridine nucleotides, the peak of the increase occurring after 10 hours with nicotinic acid and after 18 t o 20 hours with tryptophan. Pyridine nucleotides can be synthesized by washed human red cells when incubated in Ringer phosphate with nicotinamide but not with tryptophan (159), and this is probably brought about by the enzyme, diphosphopyridinenucleotidase,studied by Colowick and his colleagues, which catalyzes exhange of the nicotinamide moiety of DPN with free nicotinamide (973) and also with related substances such as isonicotinic acid hydrazide (974). Improved methods of determining pyridine nucleotides and their precursors in blood (e.g. 520) should lead to further advances in this field (cf. 113b). Bacterial synthesis of pyridine nucleotides may proceed somewhat differently. Hughes (421) has produced evidence for the following pathway of cozymase synthesis: 1. nicotinic acid + nicotinamide 2, nicotinamide -+nicotinamide riboside 3. nicotinamide riboside -+ nicotinamide ribonucleotide 4. nicotinamide ribonucleotide -+ cozymase

However, isotopically labelednicotinic acidreadily gives rise to labeled DPK and TPN attached to the outside of certain bacterial cells (95), suggesting that exchange reactions may also occur. The biosynthesis, function, and degradation of the pyridine nucleotides have been reviewed by Singer and Kearney (812a). 13. Further Metabolism of Nicotinic Acid

I n the greater part of the preceding discussion nicotinic acid has, for brevity, been treated as the end product of tryptophan metabolism. In fact the end products consist of several metabolites of nicotinic acid, which can

102

C . E. DALGLIESH

flCO

*

NH * CHs*COnH

_____c

QcoaH

”

Nicotinic acid

Nicotinuric acid (nicotinyl glycine)

It c

1

-------

pyridine nucleotides

Nicotinarnide

1 ____c

QCO’”l” I Me

N-iJ4ethylnicotinamide

flco”H*

O1;J Me 6-Pyridone

Diagram 22. Further metabolism of nicotinic acid in animals.

only briefly be considered here. The particular metabolites formed vary with the species. Failure to realize this fact has in the past led to many erroneous conclusions owing to failure to estimate a metabolite appropriate to the species investigated. On the whole herbivorous species tend to excrete nicotinic acid free or as a conjugate, whereas carnivorous and omnivorous species tend to carry out N-methylation (668). But a herbivorous animal such as the guinea pig, which normally excretes nicotinic acid unmethylated, is said to carry out methylation during starvation, when i t is living on its body-protein stores and is temporarily “carnivorous” (144). Moreover ability of rats to methylate nicotinamide is known to vary with the straiii, such variations being genetically inherited (223). Birds, e.g., the chick (181), can conjugate nicotinic acid with ornithine. The compounds principally encountered in the animal kingdom are illustrated in diagram 22. For more extensive reviews see references 493 and 812a. Identification of N-methylnicotinamide as an important nicotinic metabolite is due to Huff and Perlzweig (418,429),and the 6-pyridone was identified by Knox and Grossmann (487,488). Care should be taken over nomenclature. The naturally occurring metabolite (diagram 22) may be referred to as N-methyl-6-pyridone-3-carbonamide or (more correctly from the chemical point of view) as N-methyl-2-pyridoned-carbonamide. Chemical oxidation of N-methylnicotinamide can give both possible a-pyridones (683), that illustrated in diagram 22 and its isomer with the keto group between nitrogen and carbonamide groups. The latter is systematically named N-methyl-2-pyridone-3-carbonamide. It is therefore probably

METABOLISM OF THE AROMATIC AMINO ACIDS

103

better, to avoid confusion, to refer to the natural metabolite as the 6-pyridone. Nicotinic acid can be amidated fairly readily in mammals, but hydrolysis of the amide occurs to a lesser extent. In microorganisms (e.g., 655) and insects (e.g., 843) deamidation appears to occur more readily. Nicotinic acid is also probably more easily broken down by microorganisms, this degradation occurring via 6-hydroxynicotinic acid (420). Nicotinic acid is also excreted as the glucuronide by the rat (249a). The conversion of N-methylnicotinamide to its 6-pyridone has been studied enzymically (429). The isomeric 2-pyridone may well be formed in small amount (542)) but most methods for determination do not distinguish between the isomers (e.g., 397). The 6-pyridone is not hydrolyzed to the corresponding acid (398; but see 1012). The enzyme of rat liver converting nicotinic acid to nicotinuric acid occurs in the mitochondria (454). Methylation of nicotinamide is carried out by a soluble enzyme, nicotinamide methylkinase, which has been studied by Cantoni (135, 136). The methyl group is derived from methionine. The fate of administered nicotinic acid and nicotinaniide has been studied in many species including man (696), the rabbit (143), rat (426, 542, 561, 696)) and mouse (542, 738) with the use of isotopic and other techniques. The results agree in showing that the amount of nicotinic acid or nicotinamide degraded completely to carbon dioxide is comparatively small ; the greater part is excreted as a mixture of the metabolites shown in diagram 22, the proportions of the mixture varying with the species and the dose level. These results imply that there is no readily available route in higher organisms from nicotinic acid and its simple derivatives to aliphatic compounds, and they therefore raise the question of how tryptophan, which is generally supposed normally to be degraded via nicotinic acid, is ultimately metabolized. A possible route for the latter is suggested by irradiation experiments on pyridine nucleotides, using both ultraviolet (797) and X-irradiation (831). Such experiments may give results analogous to those normally occurring in biological oxidations. It was found that splitting of the pyridine ring could occur, and also reduction to unidentified products. Such products if formed naturally might be degraded to aliphatic compounds. Is it therefore through the pyridine nucleotides, rather than through nicotinic acid, or even through an earlier precursor of nicotinic acid and the pyridine nucleotides, that the main stream of tryptophan metabolism flows?

1’111.

‘rRYPTOPHAN DEGH.\DATION B Y T H E E N T E R A M l N E -

SEROTONIN PATIIWAY As a result of extensive study of a system of cells, which he called the criterochromaffin system, occurring particularly in the skin and gastroin-

104

C. E. DALGLIESH

testinal mucosa of vertebrates, Erspamer deduced that it must contain a physiologically highly potent material to which in 1933 (892) he gave the name enteramine. Particularly rich sources of enteramine were found in the skin of amphibia and in the salivary glands of the octopus. Erspamer (review, 237) made detailed chemical (893) and pharmacological (230-232) studies which enabled him by 1948 (234) to characterize enteramine as an indole derivative also carrying one or more phenolic groups and a side chain having a terminal primary or secondary amino group. Meanwhile Rapport and his colleagues had been investigating the vasoconstrictor substance in serum which they named serotonin, and isolated in 1948 (693,694). The isolated material was shown (692) to be a complex of creatinine with what was thought to be 5-hydroxytryptamine (structure diagram 23) and identification of the latter was confirmed by synthesis (19, 341, 363, 821). It then became clear that enteramine and serotonin were identical (e.g., 238, 239), and an enormous amount of work on the physiology and pharmacology of 5-hydroxytryptamine has subsequently appeared (reviews 235a, 237, 660, 958). 5-Hydroxytryptamine is a substance of great physiological potency and is agreed t o be probably a hormone as important in normal physiological processes as are adrenaline, noradrenaline, acetylcholine, and histamine. Even so, its exact significance is not yet clear. It has a profound effect on smooth muscle (e.g., 699 and reviews above) and hence influences blood vessels and blood pressure; its vasoconstrictor properties were of course the basis of Rapport’s isolation. Gaddum (282, 283) has shown that specific receptors exist, called by him “tryptamine receptors,” which are insensitive t o adrenaline, acetylcholine, and histamine, and very sensitive to hydroxytryptamine. Hydroxytryptamine also occurs in the central nervous system, and Woolley and Shaw (958, 959) consider it to play a vital part in mental proceses. They attribute various mental disorders, such as certain forms of schizophrenia, to inadequate production of hydroxytryptamine in the brain. On the other hand, in various forms of cancer an excessive production of hydroxytryptamine appears to occur in the tumor (544, 545). This can be reflected in the symptoms (e.g., 870) or in an excessive urinary excretion of its degradation product, 5-hydroxyindoleacetic acid (149,389). Erspamer (236) considers the prime role of enteramine to be that of a hormone controlling renal function. 5-Hydroxytryptamine is also an active constituent of many venoms, e.g., wasp venoni (441) and toad venom (237,883). 1. Biospthesis of 6-Hydroxytryptamine

It seemed likely that enteramine was formed either froni tryptophan or possibly from tyrosine. If tryptophan were the precursor two biosynthetic

105

METABOLISM OF THE AROMATIC AMINO ACIDS

pathways were possible; tryptophan -+ tryptamine -+ 5-hydroxytryptamine, or tryptophan -+ 5-hydroxytryptophan -+ 5-hydroxytryptamine, If tyrosine were the precursor this might be converted to 2,5-dihydroxyphenylalanine and then to 5-hydroxyindole1a cyclization occurring readily in vitro (165, 360), followed by addition of the side chain:

-HoacHz ' 'CH.COzH

\

a-.

etc.

OH NHz

H

2,5-Dihydroxyphenylalanine

5-K ydroxy indole

Elucidation of the actual biosynthetic route in mammals as that shown in diagram 23 is due to Udenfriend and his colleagues. Biogenesis from tyrosine almost certainly does not occur in the animal organism (in which 2,5-dihydroxyphenylalanineis not a primary tyrosine metabolite) but may well occur in plants and may play a part in biogenesis of certain alkaloids. Udenfriend and his colleagues (885) found in animal kidney extracts an enzyme which specifically decarboxylated 5-hydroxytryptophan (217, 218) to 5-hydroxytryptamine, but was without action on tryptophan, 7-hydroxy-

Tryptophan

I

5-hydrosytryptophan 5-hydroxytryptophan decarboxylaae

-,

amine

~ ~ H~ C ~-IiydIoa)-indolcncetaldehyde

I

5-liydiosyindoleacet ic :xiti

O

]

H

o

~

-

-

~

~

2

-

H 5-hydroxytryptamine (enteramine, serotonin)

methylated conjugated derivat ivcs derivatives (Diagrani 24) Diagram 23. Pathway for mammalian synthesis and breakdown of 5-hydroxytryptamine.

~

~

2

106

C. E. DALGLIESH

tryptophan, or tyrosine. This obviously suggested that 5-hydroxytryptophan, rather than tryptamine, was the intermediate in hydroxytryptamine biosynthesis. Moreover 5-hydroxytryptophan, 5-hydroxytryptamine, and N-methylated derivatives (discussed later) occur together in the venom of the tropical toad (883). On giving (2-C'*)-~~-tryptophan to the toad the hydroxytryptamines became radioactive. Moreover radiocative 5-hydr~xy- C~~- t r y p to p (isolated h an after addition of carrier) was formed from the labled tryptophan by liver slices (884), conclusively demonstrating the the pathway for enteramine bi osynthesis . 5-Hydroxytryptophan was not metabolized by a tryptophan-adapted strain of Pseudomonas (217) and was not attacked by the tryptophan peroxidase-oxidase system (217, 884). The enteramine and kynurenine pathways are quite distinct, as is supported by the facts that synthetic 5-hydroxykynurenine (124, 574), the expected product of tryptophan peroxidase-oxidase action, does not act as an ommochrome precursor in insects or as a nicotinic acid precursor in Neurospora (124). No details have yet been reported of the system converting tryptophaii to 5-hydroxytryptophan. However 5-hydroxytryptophan decarboxylase has been studied in some detail (148). It occurs in the kidneys and livers of all animals tested; in the guinea pig the stomach contains more than the liver, and it also occurs in the lung. The enzyme is inhibited by carbonyl reagents (148)) and though this inhibition is not reversed by pyridoxal phosphate, the enzyme is considered probably to be Bs-dependent, as are other amino acid decarboxylases. It is also inhibited by chelating agents (49), and this inhibition is reversed especially by Mn* and Mg+. Dialysis does not remove the coenzyme. The enzyme is highly specific, attacking L-, but not D-, 5-hydroxytryptophan, and is quite distinct from dopa decarboxylase. The amounts of the enzyme and its wide distribution emphasize the importance of the pathway. The circulating hydroxytryptamine in the blood occurs solely it1 the platelets (424, 889,978), provided no platelet damage has occurred allowing its release into the serum. On the other hand, no 5-hydroxytryptophaii (977) or, in general, hydroxytryptophan decarboxylase occurs in platelets, and they are therefore unlikely to be in the normal site of hydroxytryptamine biosynthesis. Humphrey and Toh (425) have shown that 5-hydroxytryptamine is absorbed by blood platelets and they consider that it is formed in some other orgaii, such as the gastrointestinal tract (872),which is known to contain large quantities of enteramine (179,256))and is then absorbed by the platelets during circulation. They consider that platelets have the dual function of keeping the blood plasma normally free of hydroxytryptaminr, and also of allowing local release of the vasocoiistrictor from damaged platelets, bringing about hemostasis. On the other hand, Udciifriend and

METABOLISM OF T H E AROMATIC AMINO ACIDS

107

colleagues (889) have been able to introduce C1*-hydroxytryptaniine into the rabbit platelet. The half-life of the hydroxytryptamine in the platelet was equal to the half-life of the platelet, and they consider that the normal site of hydroxytryptamine biosynthesis is therefore the same as the normal site of platelet formation, which is generally considered to beeither the bone marrow or the lung (935). But the wide distribution of hydroxytryptophan decarboxylase suggests that such are not the only sites of synthesis. Erspamer (236) has advanced evidence against a common site for cnteramine biosynthesis and platelet formation. 2. Degradation of 5-Hydroxytryptumine

When 5-hydroxytryptamine is liberated into the plasma rapid degradation occurs. It has been known for a considerable time that tryptamine is rapidly converted to indoleacetic acid both in vivo and in perfused tissue (249, 325). This reaction is brought about by amine oxidase (82, 681, review 77), which equally attacks 5-hydroxytryptamine (76, 81, 278) and can conveniently be thus detected (79). 5-Kydroxytryptamine is readily converted to 5-hydroxyindoleacetic acid by liver and kidney homogenates (871) and the reaction is blocked by semicarbazide, suggesting that the expected 5-hydroxyindoleacetaldehyde (diagram 23) is an intermediate. 5-Hydroxyindoleacetic acid is a normal urinary excretion product, about 10 mg. per day being excreted by man (871). Experiments with isotopically labeled tryptamine in the mouse (767) suggest that amine oxidase is the sole degradative pathway used, and the same may well apply to 5-hydroxytryptamine. A large part of the urinary excretion of indoleacetic acid is in the form of the glycine conjugate, indoleaceturic acid (249, 767). The same may also apply to 5-hydroxyindoleacetic acid (but cf. 23513, 236). 5-Hydroxytryptamine may itself undergo conjugation. Substances closely related to it, e.g., Erspamer’s enteramine I, have been reported in several cases (e.g., 179, and review 237). N-Met,hylated derivatives may also be formed in mammals (113a). 3 . N-Meth ylated Derivatives of 5-H ydrox ytr yptamine

Wieland and co-workers (913) in 1931 isolated from the skin secretion of the toad two indole derivatives, bufotenin and bufotenidin, whose correct structure (diagram 24) was established soon after (914) and confirmed by synthesis (416, cf. 363). Dehydrobufotenin was isolated in 1935 (443) and the previously isolated bufothionin (915) shown t o be its sulfate ester (916, cf. diagram 24). These substances are fairly widely distributed amongst amphibia (197, 444, review 196) and have a powerful physiological action (e.g., 233,235). Their occurrence and pharmacology have been extensively studied by Er-

108

C. E. DALGLIESH

Tryptophan I

J 5-hydroxytryptophan

Hoa~ CHn.CI12* ;Me*

1tryptaniinc

OH-

H Bufot enidin

5-h ydroxy

(enteramine)

J

Hoa7 CH, C H ~N. M ~ ~

H o o 7 ~ ~ 2 s ~ ~ , v ~ ~ ~ e €1

N-Methylenteramine

.H Bufotenin

Bufothionin Dehydrobufotenin Diagram 24. Methylation products of enteramine (6-hydroxytryptamine) occurring especially in amphibia.

spamer (e.g., 237, 240, 242). The coexistence of 5-hydroxytryptophan, 5-hydroxytryptamine, and N-methylated derivatives (883) together with the frequent co-occurrence of related derivatives of tyrosine, suggests the biosynthetic sequences shown in diagram 24 (cf. 242). Bufotenin also occurs (919) in Amanita mappa, a poisonous fungus species related to Amanita phalloides, which produces phalloidin. Such organisms can obviously carry out varied types of tryptophan metabolism.

IX. ROUTESFOR TRYPTOPHAN DEGRADATION USED PRINCIPALLY BY MICROORGANISMS The work already described has shown that a large number of microorganisms degrade tryptophan partly or entirely by the rovte used in higher organisms. Stanier and his co-workers (826-828, 830), using the method of simultaneous adaptation, examined many strains of Pseudomonas and found that all degraded tryptophan to kynurenine. The majority of the strains then split kynurenine t o anthranilic acid, which was converted to catechol and the latter degraded as already described (p. 78). This they called the “aromatic pathway.” A few strains converted kynurenine to kynurenic acid, which was further degraded by a route not as yet established. This they called the “quinoline pathway.” One strain could use both routes (874).

109

METABOLISM OF T H E AROMATIC AMINO ACIDS

anthranilic acid aromatic ptlthww

/

/

--t

catechol --* etc.

P

Kynurenine

\\ ;l;ylog

I kynurenic acid

-

unidentified products

The bacterial enzymes involved in these changes, where known, have already been described. Besides using such specific pathways, bacteria can degrade tryptophan by many of the more general pathways applicable to other amino acids. Such pathways in the case of phenylalanine and tyrosine have been given in diagram 16 (p. 77), and similar pathways apply to tryptophan (putting Ar = indol-3-yl in this diagram). The existence of tryptophan decarboxylase has been claimed by many workers (e.g., 248). Gale and co-workers (cf. 284) failed to find it, but this might be due t o enzyme lability. It is probable that if tryptamirie were formed a t least some orgariistns could degrade it by amine oxidase to indoleacetic acid. Tryptophan can be converted to indolepyruvic acid either by oxidative deamination or by transamination (e.g., 739, 912) and the indolepyruvic acid can give rise to indoleacetic acid. The fate of indoleacetic acid formed by the bacterial flora of the mammalian gut is discussed below. Bacterial indolelactic acid (e.g., 757) is presumably derived from indolepyruvic acid, but indolelactic acid excreted by mammals (e.g. 17) may be of true mammalian rather than bacterial origin. Indolepropionic acid can also be formed by bacteria (e.g., 412, 633), but further metabolism in mammals of any indolepropionic acid formed in the gut is still obscure (904). Skatole (3methylindole) has long been known as a product of bacterial decomposition of protein and is formed from tryptophan not only by the bacterial flora of the gut but also in putrefying secretions, e.g., sputum (756). It may well arise by decarboxylation of indoleacetic acid. 1. Urinary Indoleacetic Acid and Indoleaceturic Acid.

Urorosein

Nencki and Sieber (634) in 1882 described a beautiful red color obtained on adding concentrated hydrochloric acid to certain pathological urines. Much confusion exists in the older literature between this Nencki-Sieber reaction, the Salkowski reaction (417, 745) (a red color with hydrochloric acid and dilute ferric chloride), and the urorosein reaction (a red color with hydrochloric acid and nitrite), but the same chromogen is probably mainly

110

C. E. DALGLIESH

responsible for all. Rosin (736,737) considered the chromogen to occur to some extent in all urines, but not all workers agreed with this (e.g., 287) The reaction was studied especially by Herter (390), who isolated the chromogen and identified it as indoleacetic acid. Later work (249,409) has show1 that in fresh urine the principal chromogen is iiidoleaceturic acid (indol-3ylacetylglycine). The colored compound formed has been isolated (“22) and shown to be a diindolylmetheiic (362). 111 the author’s experience indoleacetic acid and indoleaceturic acid are both normal human uriiiary constituents (cf. also 401, 659), the latter predominating. Indoleacetic acid is the sole plant auxin occurring in urine (918). It is probable that much of the indoleacetic acid is derived from bacterial degradation of tryptophan in the gut, and is then absorbed into the blood stream and to a large extent conjugated with glyciiie in the liver. Thus excretion can be increased in presence of an abnormal intestinal flora (390). On the other hand, at least part may arise from plant material in the diet (330, and cj. subsequent discussion of heteroauxin). Moreover the possible formation of indoleacetic acid from tryptophan in animal tissues deserves investigation. Indoleacetic acid has a marked effect on amino acid transport into cells (147, 704) and may conceivably have a normal physiological function. The suggestion (494) that indoleacetic acid affects a mammalian growth has not been substantiated (517, 733, 753). 2. Bacterial Degradation via Indole.

The Tryptophanase fieaction

The formation of indole during bacterial putrefaction of protein-containing materials has been known for a considerble time (e.g., 632, 746). Hopkins and Cole (412), soon after their discovery of tryptophan, investigated the action on it of Escherichia coli. They found that aerobic degradation of tryptophan gave indole and indoleacetic acid (cf. 746), whereas anaerobic degradation gave indolepropionic acid. The formation of indole was, and still is, widely used for differentiation of bacteria, but the biochemistry of indole formation was somewhat neglected. It was generally Considered that indole was formed by way of a number of derivatives wit,h successively shorter chains, eg., indoleacrylic acid, indolepropionic acid, ethylindole, indoleacetic acid, skatole, and indolecarboxylic acid. The direct formation of indole by cleavage of the tryptophan side chaiu, without participation of any indolic intermediate, became clear from the work of Woods (954) and Happold and Hoyle (347), and the enzyme bringing this about was named tryptophanase (347). Considerable work has been carried out on the structural requirements of the tryptophanase substrate (e.g., 28,46). The reaction is catalyzed by its products, indole and pyruvic acid (46). Besides pyridoxal phosphate, discussed below, potassium or ammonium ions (348) are required and possibly also iron (194).

METABOLISM OF THE AROMATIC AMINO ACIDS

111

Numerous hypotheses, subsequently proved incorrect, have been suggested for the mechanism of tryptophanase action (see review by Happold, 345, covering the subject up to 1950). With the demonstration, already discussed under biosynthesis, that tryptophan is formed from indole and serine, i t was thought possible that tryptophanase carried out the reverse of this reaction. However the synthesizing enzyme (890) was distinct from tryptophanase (951), though both require pyridoxal phosphate as coenzyme. Tryptophanase gives rise (951) to equimolar amounts of indole, pyruvic acid, and ammonia, but riot to serine, and no oxygen uptake is involved. Neither alanine nor serine is deaminated, and these cannot therefore be intermediates. The mechanism of function of pyridoxal phosphate in the reaction has already been discussed (p. 91). It has been concluded (300) that in the enzyme-coenzyme-substrate complex, L-tryptophan is bound t o the enzyme through the pyrrole nitrogen and the side chain carboxyl, the a-amino group is combined with the carbonyl group of pyridoxal phosphate, and the phosphate group binds coenzyme and apoenzyme. Experiments with atomic models are consistent with this interpretation (27). The adaptive formation of tryptophanase has been studied by Happold and his colleagues (202, 339, 340). 3. Further Degradation of Indole by Bacteria

Some bacteria can further degrade indole. This reaction has been studied by Uchida and colleagues. Experiments using successive adaptation (742) suggested the following pathway:

Indole

Isatin

etc.

Catechol c-Salicylic acid

Formylanthranilic acid

-

Anthranilic acid

Ring-opening to give formylanthranilic acid is analogous to the formation of formylkynurenine from tryptophan. Enzymic experiments on the ringopening step (877) led to the conclusion that rupture of the C-C bond in~ possibly concerned as coenzyme; volved a n oxidase in which vitamin B I is this is thought to be followed by a dehydrogenase connected with the pyridine nucleotides, and possibly linking up with the folic acid system.

4. Origin of Urinary Indican, Indigo, Indirubin, Skatoxyl, and Skatole Red Urinary indican is the 0-sulfate of indoxyl (usually isolated as the potassium salt, 414) and is excreted by mammals as a detoxication product of the

112

C. E. DALGLIESW

indole formed from tryptophan by the bacterial flora of the gut. The horse excretes particularly large amounts (364) The indole is absorbed through the gut wall into the blood stream. Part is detoxicated in the gut wall (647), the rest is detoxicated mostly in the liver, but to some extent in other organs (835). Some indoxyl may also be conjugated as the 0-glucuronide (639).

QQ

-

O - - O H

H Indole

H

Q

aC0

NH

,\ C=C

NH

c''

0

Indirubin

/

glucuronide

H Indican

ace,

,c=c < N H y J NH co \ Indigo

Indican was early considered to be a normal constituent of human urine (413), but this was disputed by later workers, probably owing to the inadequate sensitivity of the tests employed. Its normal occurrence is now established (87,175). Indole produces nausea, headache, and other unpieasant symptoms (649), whereas indican is pharmacologically almost inactive (435). Indican formation is therefore a true detoxication, but in man only about 30 % to 50 % of ingested indole is converted to indican (87,649). A good deal of the indole produced in the gut appears in the feces, indole and the related skatole (3-methylindole) being largely responsible for the typical fecal smell. Indican excretion tends to rise when there is an increase in protein intake, or an increase in endogenous protein breakdown as in fever, or in constipation or other intestinal blockage owing to the increased opportunity for bacterial action. Though there is little doubt that a bacterial origin is to be attributed to a great part of normal indican and indole excretion, there is evidence suggesting that there may be other sources. This point was early debated (e.g., 85, 219). Homer (409) suggested that indican could arise from indolealdehyde formed by a disturbed metabolism of tryptophan in the liver. But indolealdehyde is not indoxylogenic if the digestive tract is removed (835). On the other hand, removal of indole-formingorganisms

METABOLISM O F THE AROMATIC AMINO ACIDS

113

from the gut by intensive chemotherapy does not necessarily prevent, of even diminish, indican excretion (155, 956). It remains possible that indole could arise in some mammalian tissue. Chromogens related to indican may be precursors of the indigo and dibromoindigo (“Tyrian purple”) formed in some marine organisms (100a). Hydrolysis of indican gives indoxyl, which is readily oxidized to indigo and indirubin, as shown in the diagram above. “Indigo stones,” though rare, have been known for a considerable time (e.g., 146, 656), and these could be formed from indoxyl which has not been conjugated with sulfate, or from indican, which is readily hydrolyzed under acid conditions and may also be hydrolyzed by arylsulfatases. Indirubin is encountered more frequently in urine (cf. 618), and though its occurrence has received detailed study (705,736), it is not clear whether it is formed in the body and excreted as such or is merely derived from urinary indican after excretion Skatole also arises by action of the intestinal flora and was early claimed (105) t o give rise to a substance “skatoxyl,” supposedly analogous to indoxyl, and to “skatole red.” Homer (409) produced suggestive evidence that “skatole red” is probably a mixture of indigo and indirubin The formulation of skatoxyl is inherently improbable chemically, and unpublished preliminary experiments of the author suggest that its postulated existence will not withstand modern methods of investigation.

X. TRYPTOPHAN METABOLISM IN PLANTS.HETEROAUXIN Tryptophan can be metabolized in at least some plants by the kynurenine or closely analogous pathways. For example, tryptophan gives nicotinic acid in many green plants (329) ; both tryptophan and hydroxyanthranilic acid are converted to nicotinic acid in maize (626, 627) and kynurenine is also known in plants (e.g., 521). A mutant of maize obtained in the Bikini experiments accumulates large quantities of anthranilic acid (862). Tryptophan appears t o be converted to nicotinic acid in germinating green gram (PhaseoZus mungo) by a pathway similar to that in animals (798, 799). Wiltshire (932) in a detailed enzymic study of tryptophan oxidation in pea seedlings concluded that the greater part of the tryptophan was metabolized by a peroxidase system showing marked analogies t o the Knox-Mehler system in animals, and the metabolism probably proceeded through kynurenine and hydroxykynurenine. The rate of degradation of tryptophan by this pathway was more than 100 times as fast as the rate of conversion of tryptophan to indoleacetic acid reported in tobacco (922). A remarkable advance in plant physiology occurred when Kogl and coworkers (496) isolated a plant growth hormone, called heteroauxin, from human urine. This was identified as indole-3-acetic acid and was shown (497, 498) also to occur in plants, and in yeast from which it was isolated.

114

C. E. DALGLIESH

The work of many investigators soon showed it to be extremely widespread in plants, and it became clear that it was a fundamental plant hormone (e.g., reviews 530, 655a). Excessive production of indoleacetic acid by parasites is responsible for certain types of plant tumor (e.g., 948). The synthetic plant growth regulators, which are in general substituted phenoxyacetic. acids, probably function as indoleacetic acid analogues. 1. Biogenesis and Degradation of Indoleacetic Acid in Plants

Tryptophan has been clearly established as the precursor of indoleacetic acids in both plants (e.g., 303, 921, 922) and fungi (e.g., 864), and in plant tumor tissue (e.g., 378, 948). Two routes are possible for indoleacetic acid formation from tryptophan as follows: Tryptophan

decarboxylation

amine oxiduse P

tryptamine

1

I

1

dpamination or transamination

indolepyruvic acid

indoleacetaldehyde

-

-1 *

indoleacetic acid

I

indoleaoetic acid oxidaso

unknown products

There is evidence that both these routes can occur. The enzymes coilverting tryptophm to indoleacetic acid can be obtained in maize embryo juice; the tryptophaii is thought to arise from the endosperni (964). Indolepyruvic acid is also present in maize endosperm (837, 838), suggesting it to be an intermediate. On the other hand, tryptamine is converted to indoleacetic acid in plants (304, 815) and the amine oxidase responsible has been studied by Kenten and Mann (464). Consideration of the biogenesis of alkaloids, discussed later, suggests that both tryptainine and indoleacetaldehyde are likely to occur in plants. Indoleacetic acid is degraded in plants by a specific iiidoleacetic acid osidase. This is a light-activatable flavoprotein enzyme coupled through hydrogen peroxide to a peroxidase (285; but cf. 463a, 805b). It apparently uses phenols as cofactors (296) but can be inhibited by polyphenols (305). The product of the reaction is still unidentified (836). 2. Other Indolic Plant Growth Hormones

An auxin in apple endosperm (5G5) not identical with indoleacetic acid ivas identified as ethyl indoleacetate (863, cj. 697), but this may be an artifact due t o esterification during isolation (377).

115

METABOLISM O F T H E AROMATIC AMINO ACIDS

Other indolic auxins besides indoleacetic acid occur in plants (e.g., 396). Both indoleacetaldehyde (51, 107) and indoleacetonitrile (51, 377, 452) have been claimed to be plant growth hormones. Indoleacetonitrile has heen isolated from plant sources (377) and shown to be of wide distribution, but whether it is a hormone per se or only acts as a precursor of indoleacetic acid is not yet clear (cf. 838, 865). A11 these indolic plant hormones or hormone precursors are likely to arise from tryptophan. Other reactions of trytophan, for example, in alkaloid hiogenesis, are discussed in the next section.

XI.

RELATED TO AROMATIC AMINOACIDS

N A T U R A L PRODUCTS P R O B A B L Y

THE

I n higher organisms the number of products derived from the aromatic amino acids is comparatively small. In lower organisms, such as bacteria and plants, the biosynthetic possibilities are enormously greater. In this section very brief mention will be made of some types of compound probably metabolically related to the aromatic amino acids. A large proportion of the compounds mentioned will be of plant origin. Plant biochemistry has lagged considerably behind mammalian biochemistry, and relatively much less is known of the metabolic processes occurring, Though it is usual to regard many of the substances discussed as ultimately derived from the aromatic amino acids, it should be emphasized that there is still little direct evidence on this point. The changes postulated in theories of alkaloid biogenesis in general involve biochemically plausible reactions, but the result in some cases could be equally well explained on the assumption that both alkaloids and aromatic amino acids are derived from common precursors, e.g., aromatic amino acids

natural products

This applies especially to substances such as the essential oils or lignin. The comparatively small amount of work so far done on the biogenesis of alkaloids from isotopically labeled amino acids suggests that theories of hiogenesis of alkaloids from amino acids are probably justified.

I. Probably Related Metabolic Products in Microorganisms Of simpler substances can be mentioned phenylacetic acid as a constituent of benzylpenicillin; O-methyltyrosine in, e.g., the antibiotic puromy-

116

C. R. DALOLIESN

ciii (903) ; the antibiotic. chloramphenicol; the Pyo antibiotiw (2-nlkyl--lhydro?cyyuiiiolints, 368) and their N-oxides ( I 581, prohably derived froin kynurmic acid. 2. E'rohubly 11'clatcd dlctabolic Products in Plants and Fungi

Simple examples are: p-hydroxybenzylisothiocyanatc in white mustard .CH :CT3 .CsH6 i i i (472); the substance p-Me0.CBHI.CH2.CH2.NMe.C0 Southern Prickly Ash (526) ; circumin (cf. 822) ; chlorogeiiic and isochlorogenic acids, which are widely distributed glycosidic derivatives of 3,4-dihydroxycinnamic (caff eic) acid (cf. 432) ;vanillin (3-methoxy-4-hydroxybtllzaldehyde) and related substances such as many of the essential oils, e.g., anethole, eugeiiol, saff role; gallic acid (3,4,5-trihydroxybenzoicacid) and numerous other phenolic substances from, e.g., tannins; 3 ,4-dihydroxyphenylacetic acid, 2,5-dihydroxyphenyIglyoxylic acid, and many related substances in fungi (e.g., 609,687) ; some fungus pigments such as cortisalin, p-HO.CeH:- (CH:CH),~COIH(312), and violacein which contains both 5hydroxyindole and oxindole nuclei (43,44) ; sulfur derivatives of oxindolylalaiiine (a-hydroxytryptophan) as in phalloidin (1 57,920). 3 . Flavonoids and Liynin

A vast number of flavonoid conipounds ocrur in nature and these may well be derived from the aromatic amino acids. Birch arid co-workers (66) have demonstrated the following hiosyrithetic pathway in the green alga C'hlamydomonas agamctos: Phenylalanine

1 tyrosine

53,4-dihydroxyphcnglalanine

1 :<,4-dihydroxyphenylpropionicacid

1

iriositol

+

3,4-dihydroxycinnarnic acid

1 phloroglucinol

A --

L--T precursor X

1 quercetin, etc.

Lignin, which represents some 20 % to 30 % of wood, can be regarded a h a polymer formed from phenylpropane skeletons, such as 3-methoxy-4-hy-

METABOLISM OF T H E AROMATIC AMINO ACIDS

117

droxycinnaniic acid (e.g., 740). This suggests a close relation t o the phenylpropane skeletons of phenylalanine, tyrosine, and dopa (cf. 107a).

4. Alkaloids The relation of many of the simpler alkaloids to the aromatic amino acids is obvious. For example, germinating barley contains (241), besides tyrosine and tyramine, N-methyltyramine, NN-dimethyltyramine (hordenine), and the trimethylammonium derivative (candicine). In this simple case the N-methylated derivatives are known to be derivable from isotopically labeled tyraniine (538) and the methyl groups are known to arise from methionine by transmethylation (540, 586). Similarly N-methyl derivatives of phenylethylamine, 3 ,4-dihydroxyphenylethylainine, and 3 ,4,5-trihydroxyphenylethylamine are well known alkaloids (cf. review, 701). NMethylated derivatives of tryptamine and hydroxytryptamine equally occur; for example, eserine has an obvious relation to 5-hydroxytryptamine. Methylated derivatives of metabolites of the aromatic amino acids also occur, for example, trigonelline (67), which is the betaine of nicotinic acid, and damascenine is probably similarly related to hydroxyanthranilic acid.

Eserinc

Cirainine

The comparatively simple alkaloid gramiiie is particularly interesting, as the nitrogen is here separated from the indole nucleus by only one carbon atom. Almost all other indole alkaloids contain the aminoethyl side chain found in tryptamine. If some alkaloids were formed from precursors of the aromatic amino acids rather than from the amino acids themselves, one would expect gramine t o fall into this category. Yet isotopic experiments have made it clear that the indole nucleus and the side chain CH2 group of gramine arise from the indole nucleus and side chain &carbon, respectively, of tryptophan (101, 539). By analogy alkaloidal derivatives based on benzylamine are probably derived from phenylalanine. Theories of the biogenesis of more complex alkaloids are due to Pictet (Wig), Winterstein and Trier (934), and particularly Robinson (707, and later discussions, 708, 709, 711, 712), and a vast amount of work has been done on syntheses under “physiological conditions,” i.e., under conditions and with the use of substances likely to occur in the plant (for reviews and discussions see, e.g., 422,577,578,775 and for reviews on more plant physiological aspects of alkaloids see 195, 440). Winterstein and Trier pointed out that benaylisoquinoliiie alkaloids

118

C. E. DALGLIESH

Me0 Me0

M Me0e o q N H +

Tetrahydropapaverine Dimethoxyptienylethylamine and dimethoxyphenylacetaldehyde Diagram 25. T h e type of reaction possibly involved in benzylisoquiIioline alkaloid biosynthesis.

could be formed from two iiiolecules of dibydroxyplienylalaiiine. To tahr a specific iristance (819) 3,4-diinethoxyphenylethylamirie condenses with 3,4-diniethoxyphenylacetaldehyde under “physiological miditions” to give tetrahydropapaverine (diagram 25). Amine oxidase occurs in plants (e.g., 464, 909), and if it were to act on the above amine, both aldehyde stud amine would be present together, and conditions for tetrahydropapaverine formation would therefore be very favorable. Similarly acetaldehyde with, say, tyramine would give a much simpler isoquirioline derivative (776). It may not, however be necessary for decarboxylatiori of the amino acid to occur. A condensation with the amino acid similar to that above \vould give the same result if decarboxylation occurred after condensatiori. Similarly an aldehyde coinponerit need not be present as such; a potential aldehyde, such as a keto or imino acid, also condenses readily (c.g., 334,337). Again the same product is obtained by subsequent decarboxylation. Transaniination is known to occur in plants (e.g., 931) and the occurrence of keto acids is to be expected. Both amino slid keto acids could of course be formed from the amino acid by oxidative deamination. The comparatively complicated molecule of emetiiir (diagram 26) can be derived (710) from three molecules of dihydroxyphenylalaiiiiie by making use of the Woodward (955) hypothesis discussed below. In this, as in iiiany other cases, biogenetic considerations have played a large part in dttermilling the correct structure, and biogenetic theories thereby gain strong albeit indirect, support. The indole alkaloids provide an even richer source of biogenetic interrelationships. Thus, condensation of tryptamine and dihydroxyphenylacetadehyde (or equivalent precursors) under conditions similar to those already described gives a tetrahydro-harman derivative (diagram 26; cf. 336,338). Further condensation of this with formaldehyde (cf. 335) (which may be biogerietidly derived from, say, serine or glycine) gives the same basic skeleton as in the alkaloid yohimbine.

119

METABOLISM O F THE AROMATIC AMINO ACIDS

OMe

OMe

H Tryptaminc

+

Et Emetine

CHO

OH Dihy droxy phen y lacetaldeh yde

oxql+*

CHnO

/

/

'

'

OH OH OH OH Compound with yohimbine A benzyltetrahydroharman skeleton derivative Diagram 26. Emetine; and biosyrlthesis of yohimbine-type alkaloids.

I n yohimbine formation the initial Mannich-type condensation of the aldehyde with the tryptamine was assumed to occur in the a-position of the indole ring. Similar condensation in the ,&position (which already carries the ethylamine side chain) can give an intermediate which Woodward (955) pointed out can give rise to the very complicated molecule of strychnine. The stages, shown in diagram 27, involve a ring scission at the catechol grouping and a rearrangement. Similar ring scission is probably also involved in the biosynthesis of emetiiie (diagram 26) and of the quinine alkaloids. The derivation of two of the latter is show in diagram 28 (cf. 308). The starting product in diagram 28 can be derived from tryptophan, dihydroxyphenylalanine, and formaldehyde in ways obvious from diagram 26. Scission of the catechol grouping can give a dialdehyde compound, in which one aldehyde group can react with the secondary aliphatic amino group (dotted arrow 0) to give, ultimately, cinchonamine. If there is a further scission of the pyrrole ring (dotted line aa) and a rearrangement, cinchonine results. It will be seen from this very brief treatment that the essential step in

120

C. E. DALGLIESH

Tryptamine

&CH-I!JH

+

dihydroxyphenyl acetaldehyde (ct. diagram 26) OH

several stages

Strychnine

' OH Diagram 27. Woodward's hypothesis for biosynthesis of strychnine from, ultimately, tryptophan and dihydroxyphenylalanine. CHz * CHzOH

CHz. CHnOH

OH' in stages

CH2 * CHzOH

CHa CH * CH :CHZ

H Cinchonine Cinchonamine Diagram 28. Derivation of two typical quinine alkaloids from, utlimately, tryptophan and dihydroxyphenylalanine.

the biosynthesis of many alkaloids is a Mannich-type reaction between an active position on an aromatic ring, an aldehyde (which may also be derived from an aromatic amino acid), and the amino group of the original amino acid side chain. Other changes are fairly simple and plausible, such as the introduction of one-carbon and two-carbon units. Besides these reactions

METABOLISM OF THE AROMATIC AMINO ACIDS

121

the various members of the alkaloidal families may differ by the number of phenolic groups and the extent to which 0-and N-methylation has occurred, Isotopic experiments have shown that both 0-and N-methyl groups arise hy transmethylation reactions (132, 204, 540, 586, 786) analogous to those in the animal, and this is in accord with the virtual absence of 0- and N-alkyl substituents other than methyl (or the related methylenedioxy group). The biogenesis of alkaloids from the aromatic amino acids forms an enormously rich field for work with modern isotopic and chromatographic Cechniques, which as yet has hardly been touched. XII. FUTURE PROBLEMS Phenylalanine and tyrosine are degraded in large degree to aliphatic substances, acetoacetic and fumaric acids, which are metabolically highly active, and there is no difficulty in accounting for the amounts of these amino acids metabolized. Of the various transformations involved, the conversion of p-hydroxyphenylpyruvic acid to homogentisic acid, in particular, needs further investigation. Our inadequate knowledge of this reaction is a reflection of two major biochemical problems: the nature of aromatic hydroxylation reactions, of which the enzymes have been obtained in very few cases; and the biological functions of ascorbic acid, which appears to participate both specifically and unspecifically in a wide range of transformations. In addition, much is still to be learnt of the adrenaline and thyroxine pathways. No such tidy ending can a t present be given to our picture of tryptophan metabolism. In the adult animal in the steady state as much tryptophan must be degraded as is ingested (irrespective of the fraction used in protein biosynthesis, as old protein is being broken down as fast as new protein is being formed). To take man as an example, tryptophan can, so far as is at present known, be metabolized by three main routes: (1) by the nicotinic acid pathway, (2) by the 5-hydroxytryptamine pathway, and (3) by bacterial action in the gut. The 5-hydroxytryptamine pathway gives as principal end product 5-hydroxyindoleacetic acid. Bacterial action gives rise to a number of excretory products, including indican, indoleacetic, indoleaceturic, and indolelactic acids. In addition some tryptophan is excreted unchanged. All these indolic compounds taken together account for only a small part of ingested tryptophan. The nicotinic acid pathway potentially gives rise to a number of excretion products, largely due to side reactions, such as anthranilic acid, kynurenic acid, and xanthurenic acid, but normally these are excreted in, at most, small amounts. Presumably, therefore, the greater part of ingested tryptophan should give nicotinic acid or simple derivatives. But normal excretion of nicotinic acid and its derivatives is in any case low, and when

122

C . E. DALGLIESH

nicotinic acid or likely derivatives are given to an animal a large ftwtioii is excreted in forms with the ring unbroken. There would therefore seem to be no readily available pathway from nivotinic acid to simple aliphatic compounds (unless, as already suggested, this occurs via the pyridirie nucleotides), and hy iiiferencc no rcwlily available pathway for degradation of tryptophan to simple aliphatic, compouids. In short, in our present picture of tryptophan metabolism thcre seems to 1 ) ~ considerable discrepaiicy between our knowledge of what goes in aiid what comes out, and a major problem is to resolve this discrepancy. It may he that some a t present unsuspected pathway exists (the 5-hydroxytryptaminc pathway was only recently discovered). It may be that some intermediatc in one of the known pathways is metabolized to aliphatic compouiids by route or to a degree at present unsuspected. It may be that degradation of many intermediates occurs, so that there is a “leakage” to aliphatic compounds all along the pathway. It may even be that metabolism in sonw organ other than liver or kidney plays a prominent part. I n additioti many stages in known pathways of tryptophan metabolism require further investigation, in particular, the intermediate lying betweet 1 tryptophan and formylkynurenine, the hydroxylatiori reaction in conversion of kynureiiie to hydroxykyiiurenine, the intermediates in the wiiversion of hydroxyaiithranilic acid to nicotinic acid, aiid the site of synthesis and hormonal function of 5-hydroxytryptamine. And in the plant kingdom, especially, there lies a rich and barely touched field of investigation-the relation between aromatic amino acids and numerous natural products.

XIII. SUMMABY The reviex covers those aspects of the metabolism of phenylalanine, tyrosine, and tryptophan not concerned with peptide or protein synthesis or breakdown. Phenylalariiiie arid tryptophan are “essential” amino acids for higher organisms, i.e., they cannot be synthesized by the organism and must, he supplied in the diet. Tyrosiiie is formed from pheiiylalaniiie aiid is tiot essential if the pheiiylalaniiie intake is adequate. The first section of the review covers biosynthesis, which is confined t o lower orgariisnis (bacteria, fungi, plants, etc.). Glucose is the precursor of the aromatic ring and is converted to shikimic acid, such intermediates as are known bciiig showti in diagram 1 , Shikimic acid is a common precursor of the aromatic amino acids and of the bacterial growth factors, p-amino- and p-hydroxybenzoic acids. Such intermediates as are yet known in the final stages of aromatic amino acid biosynthesis are showii in diagrams 2 and 3. Much isotopic

METABOLISM OF T H E AROMATIC AMINO ACIDS

123

work has becii done 011 thc conversion of glucose t o shikiniic acid, but the evideiice is stJillinadequate to defiiie the pathway in detail. In higher organisms pheiiylalanine is normally transformed to tryosine, a i d then lirokeii down by the route, p-hydroxyphenylpyruvic acid, 2 , 5 dihydr'oxypheiiylpy~.uvicacid, hoxnogeritisic acid, rnaleylacetoacetic acid, fumarylacetoaretic acid, aiid finally fumaric and acetoacetic acids (diagram 8). The elucidation of this pathway is described scmihistorically; first are considered in vivo rsperimeiits aiid experiments i i i those rare metabolic disorders (c.g., phenylkctoiiuria and alkaptonuria; diagram 6) associated with inhibition of relevant normal degradativc reactions. Then in witro reactions arc coilsidered, followed by individual consideration of the various steps taken in turii. Phenylalaniiie and tyrosiiie are also metabolized in higher organisms by two routes which are quantitatively less important but physiologically of the highest importance. The first leads to the adrenal hormones adrenaline (epinephrine) arid rioradrerialirie (norepinephrine) ,which may be formed as in diagram 11; this pathway also leads to melanin (diagram 12). The second leads to the thyroid hormones thyroxine and triiodothyronine, the synthesis and breakdown of which are also discussed. Tryptophan is metabolized in a wide range of organisms by a pathway involving: an unkno~vnintermediate, forinylkyriureiiiiie, kynurenine, hydroxykynureiiinc (or its phosphate), hydroxyanthrariilic acid (or its phosphate), two unknown intermediates, and nicotinic acid (diagrams 17,18,21). The pathway is considered both in outline and in detail by individual steps. The relation of other vitamins to the pathway (diagram 19), side reactions giving such substances as aiithrariilic acid, kynureriic acid, and xanthurenic acid (diagram 20) and the further degradation of nicotinic acid (diagram 22) and its relation to the pyridine nueleotides are also considered. Some tryptophan is also degraded to 5-hydroxytryptamine (enteramine, serotonin; diagram 23) and some by iiitestiiial bacteria, using many pathways. These, a d pathways involved in bacterial degradation of phenylalaiiine and tyrosiiie are all considered (diagram l G ) , as are the chromogens excreted by man as a result of bacterial action, and such minor pathways as those concerned with hardening of the insect cuticle and formation of insect eye-pigments. Tryptophan also gives rise to the important plant hormone, indoleacetic acid, and microorganisms and especially plants metabolize the aromatic amino acids to a wide range of natural products, for example, certain antibiotics, alkaloids (e.g., diagrams 25-28), flavonoids, and possibly lignin. These are briefly considered. The final section mentions various problems likely to be the basis of future work.

124

C. E. DALGLIESH

ACKNOWLEDGMENTS

I thank Prof. A. Neuberger for his stimulating and valuable criticism, and Dr. B. D. Davis for his consideration of the section on biosynthesis. ItEPERENCES 1 . Abderlialdeii, 15., Block, U., and Rona, 1'. (1907). Z . physiol. (,'hem, 62, tX3. 2. Adamkiewicz, A. (1874). Pflugers Arrh. g r s . Physiol. 9, 156. 3. Adamkiewicz, A. (1875). Ber. 8, 161. 4. Adelberg, E. A. (1!)53). Bacteriol. Revs. 17, 253. 5. Akiba, T., and Irai, I. (1051). Japan J. Ezptl. Illed. 21, 7. 6. Albanese, A. A. (1947). Advances i n Protein C'hctn. 3, 227. 7. Albanese, A. A., Davis, V. I., and Lein, h9. (1948). J. B i d . C / w w . 172, 3!1. 8. Albert, A. (1952). A m . Rev. Physiol. 14, 481. 9. Albert, 1'. W., Scheer, U. T., and Deuel, H. J. (1048). J . Biol.('hetn. 176, 47s) 10. Altman, K. I., arid Miller, G. (1953). Nature 172,868. 11. Amano, T., Torii, ill., and Iritani, H. (1950). Med. J. Osaka Vriiu. 2, 46. 12. Anderson, J. O., Combs, G. F., Croschke, A . C., and Briggs, G. ill. (1951). J . Nutrition 46, 345. 13. Arman, C. G. van, and Jones, K. I<. (1949). J. Znvest. DermatoE, 12, 11. 14. Armstrong, M. D. (1953). Federation Proc. 12, 171. 15. Armstrong, M. D. (1953). J. Biol. Chem. 206, 839. 15a. Armstrong, M. D. (1955). J. Biol. Chern. 213, 409. 16. Armstrong, M. D., and Robinson, I<. S. (1954). Federation Proc. 13, 175. 17. Armstrong, M. I)., and Robinson, I<. S. (1954). Arch. Biochem. and Biophys. 62, 287. 17a. Armstrong, M. D., and Shaw, K. N. F. (1955). J. Biol. Chem. 213, 805. 17b. Armstrong, M. D., and Tyler, F. H. (1955). J. Clin. Znvest. 34, 565. 170. Armstrong, M. D., Shaw, K. N. F., and Robinson, K. S. (1955). J. BioE. Chem. 213, 797. 18. Arnow, L. E. (1937). J. B i d . Chewi. 120, 51. 19. Asero, B., Colo, V., Erspamer, V. and Vercellone, A. (1952). A n n . 676, 69. 20. Atkinson, D . E., and Fox, S. W. (1951). Arch. Biochem. and Biophys. 31, 205. 21. Axelrod, H. E., Morgan, A. F., and Lepkovsky, S. (1945). J . Biol.Cheni. 160, 155. 22. Axelrod, J., Brodie, B. U., and Udenfriend, S. (1952). I'ederation Proc. 11,3l!J, 23. Axelrod, J., Udenfriend, S , and Rrodie, R. I%. (1954). .I. Pharmacol. E z p f l . Therap. 111, 176. 24. Baddiley, J. (1952). h'alure 170, 711. 25. Baddiley, J., Ehrensviird, G., Klein, E., Iteio, L., und Saluste, E. (1950). J . Biol. Cherri. 183, 777. 26. Baddiley, J., and Gale, E. F. (1945). Nature, 166, 727. 27. Baker, J. W. (1953). Biochem. J . 66, proc. xxxii. 28. Baker, J. W., and Happold, F. C. (1940). Biochem. J. 34, 657. 29. Baldwin, H. R., and Berg, C. P. (1949). J. Nutrition 39, 203. 30. Barger, G., and Walpole, G. S. (1909). J. Physio2. (London) 38, 343. 31. Barker, S. B. (1951). Physiol. Revs. 31, 205. 32. Barth, I,. (1869). A n n . 162,96. 33. Basinski, D. H., Williams, J. D , and Freund, €1. A. (1952). J . B i d . ( ' h i / . 197,433.

METABOLISM OF THE AROMATIC AMINO ACIDS

34. I3ateson, W .

125

(1902). Report of the Evolution Commit,tee of the R o y 1 Socirt y (London). No. 1, p. 133. 35. Baucr, C. D., and Berg, C . P. (1943). J . iVutrition 26, 51. 36. Baumann, I<. (1879). Ber. 12, 1450; cf. also 2. physiol. Chein. 3, 250; (1880). 4, 304; (1882) 6, 183; (1886) 10, 123. 37. Baumann, E. A. G. (1895). 2.physiol. Chem. 21,319. 38. Baumann, E. A. G., and Herter, E. (1877). 2.physiol. Chem. 1, 244. E. (1895). 2. phvsiol. Chem. 21, 481. 39. Baumann, E. A. G., and ROOB, 40. Beadle, G. W. (1948). Fortschr. Chem. org. Naturstofle 6,300. 41. Beadle, G. W . , Mitchell, H. K., and Nyc, J. F. (1947). Proc. X d . Arad. S c i . (U.S.) 33, 155. 42. Bear, J., and Mum, 1,. (1907). Naunyn-Sch*niedebery’s Arch. c x p l l . l’athol. 11. Pharmakol. 66, 92. 43. Beer, R. J. S., Clarke, K., Khorana, H. G., and Robertson, A. (1949). .I. Chem . Sac., p. 885. 44. Beer, R. J. S., Jennings, B. E., and Robertson, A. (1954). J . Chem. SOC., p. 2679. 45. Beerstecher, E. (1954). J. Bacteriol. 68, 152. 46. Beerstecher, E., and Edmonds, E. J. (1951). J. B i d . Chem. 192, 497. 47. Beerstecher, E., and Shive, W. (1946). J. Biol. Chem. 164, 53. 48. Beerstecher, E., and Shive, W. (1947). J . Biol. Chem. 167, 49. 49. Beiler, J. M., and Martin, G. J. (1954). Federation Proc. 13, 180. 50. Bell, G. H., Scheer, B. T., and Deuel, H. J. (1948). J. Nutrition 36,239. 51. Bentley, J. A , , Honsley, S., and Bickel, A. S. (1952). J. E z p t l . Botany 3,303, 406. 52. Berezov, T. T. (1952). Doklady Akad. N a u k . S.S.S.R. 86,605. 53. Berg, C. 1’. (1934). J. Biol. Chem. 104, 373. 54. Berg, C. P., and Potgieter, M. (1931). J. Biol. Chern. 94, 661. 55. Berg, C. P., Rose, W. C., andMarvel, C. S. (1929-30). J. B i d . Chenz. 86,219. 56. Bergmann, E. D., and Sicher, S. (1952). Nature 170,931. 57. Bergmann, E. D., Sicher, S., and Volcani, B. €2. (1952). Biochim. et Biophya. Acta 9, 220. 58. Bergmann, E. D., Sicher, S., and Voleani, B. E. (1953). Biochem. J . 64, 1. 59. Bernhard, K., and Vuilleumier, J. (1952). Helv. Physiol. el Pharinacol. Actn 10, c47. 60. Bernheim, F. (1935). J. B i d . Chern. 111,217. 61. Bernheim, F., and Bernheim, M. L. C. (1934). J . Biol.Chenz. 107, 275. 62. Beyer, K. H. (1946). Physiol. Revs. 26,169. 63. Beyer, K. H. (1950). Nature 166,926. 64. Bickel, H., Gerrard, J., and Hickman, E. M. (1953). Lancet 266, 813. 65. Bickel, H., Gerrard, J., and Hickman, E. M. (1954). Acta Paediat. 43, 64. 66. Birch, A. J., Donovan, F. W., and Moewus, F. (1953). Nature 172, 902. 67. Blake, C. 0. (1954). Am. J. Botany 41, 231. 68. Blanchard, M. H., Green, D. E., Nocito, V., and Ratner, S. (1944). J. Biol. Chem. 166, 421. 69. Blanchard, M. H, Green, D. E., Nocito, V., and Ratner, S. (1945). .I. Biol. Chem. 161, 583. 70. Blaschko, H. (1939). J. Physiol. (London) 96, proc. 13. 71. Blaschko, €-I. (1942). J . Physiol. (London) 101, 337. 72. Blaschko, H. (1945). Advances in Enzymol. 6, 67. 73. Blaschko, H. (1948). J . Physiol. (London) 107, proc. 18.

12G

C. E. DALGLIESI-I

74. Hl:Lschko, 11. (1050). 172 “The Hormoties” (G. Pincus and I<. V. T h i m i ~ ~ l t ~ , eds.), Vol. 11, Chapter 9. Academic Press, h’ew York. 75. Blaschko, €1. (1950). Riochitii. ct Biophys. -4cta 4, 130. 76. Blaschko, H. (1952). Biochem. J. 62, proc. x. 77. Blaschko, €1. (1952). Pharmacol. Revs. 4, 415. 78. Blaschko, [I., 151irn, J. II., and I,:Lngemann, TI. (1950). Brit. J . P h a r n m o l . 6 , 431. 79. Blaschko, II., and Hellmann, K. (1953). J. Physiol. (London) 122, 419. 80. Blaschko, H., Ilolton, P., and Sloane-Stanley, G. 11. (1949). J . Physiol. (London) 108, 427. 81. Hlaschko, I I . , and Philpot, F. J. (1953). J . Physiol. (London) 122, 403. 82. I3luschko, II., Richter, D., and Schlossmnn, H. (1937). Biocheiti. J . 31, 2187. 83. 13laschk0, TI., and Sloane-Stanley, G. H. (1948). Biochetn. J . 42, proc. iii. 81. Blum, I,. (1908). ,Vaunyn-Schmiedcber~’s Arch. ezptl. Pathol. I ( . I’harnmkol. 69, 283. 85. Blumenthal, F., and Rosenfeld, F. (1903). CharitC-Ann. 27, 46. 86. Boedeker, H. (1859). 2. rat. Med. 7, 130. 87. Bohm, F. (1937). Biochem. 2.290,137. 88. Bokman, A. R.,and Schweigert, B. S. (1950). J. Biol. Chem. 186, 153. 89. Bokman, A. H., and Schweigert, B. S. (1951). Arch. Biochcm. and Biophys. 33, 270. 90. Honner, D. hl. (1948). Proc. Nail. Acad. S c i . ( U S . ) 34, 5. 91. Bonner, D. XI. (1951). (‘old Spring Harbor Sginposia Q u a n f . B i d . 16, 143. 92. Bonner, 1). M., atid Beadle, G. W. (1946). Arch. Biochem. 11, 319. 93. Bonner, D. XI.,and Partridge, C. H. W. (1950). Federation Proc. 9, 154, 94. I30ntlCr, D. M., and Yanofsky, C. (1949). Proc. R‘all. Acad. Sci. (U.S.)36,576. 95. Boone, I. U., Turney, D. F., and Woodward, K. T. (1954). Science 120, 312. 96. Boscott, It. J. (1953). 322nd meeting of the Biochemical Society, October 1953. 97. Boscott, R . J., and Bickel, H. (1953). Scand. J. Clin. &. Lab. Invest. 6, 380. 98. Boscott, R. J., and Bickel, H. (1954). Biochcm. J. 66, proc. i. 99. I3oscott, 13. J., and Cooke, W. T. (1954). Quart. J. Med. 23,207. 100. Boscott, R. J., and Greenberg, S. (1953). Bzochem. J . 66, proc. xviii. 100a. Bouchilloux, S., and Roche, J. (1955). Bull. soc. chim. b i d . 37, 37. 101. Bowden, K., and Marion, L. (1951). (’an. J . Chem. 29, 1043. 102. Braunshtein, -4.E., Goryachenkova, E. V., and Pashkina, T. S. (1949). Biokhitniya 14, 163. 103. Bray, H. G., Lake, H. J., Neale, F. C., Thorpe, W. V.,and Wood, P. 13. (1948). Biochem. J. 42,434. 104. Bray, 1%.G., Thorpe, W. V., and Wood, P. B. (1951). Biochctri. J. 48, 304. 105. Brieger, L. (1880). 2. physiol. Chem. 4, 414. 106. Brodie, B. B., Axelrod, J., Shore, P. A., and IJdenfriend, S. (1954). J . Biol. Chcm. 208, 741. 107. Brown, J . D . , Hcnbest, H. B., and Jones, K. It. €I. (1952). J . (‘hem. Soc., p. 3172. 107a. Brown, S. A,, and Neish, A. C. (1955). Nature 176, 688. 108. Bruce, J. M. (1954). J. A p p l . Chcni. (London),p. 469. 109. Bubl, 13. C., and Butts, J. S. (1949). J . Biol. Chem. 180, 839. 110. Bulhring, E. (1949). B r i t . J. Pharnmcol. 4, 234. 111. Biilbring, I$., and Burn, J . H. (1949). Brit. J . Pharnmcol. 4, 245. 112. Hu’T,ock, J . D., and Harley-Mason, J. (1951). . J . Cherti. Soc., p. 703.

METABOLISM O F T H E AROMATIC AMINO ACIDS

127

113. Bu’Lock, J. D., Harley-Mason, J., andMason, H. S. (1950). Riochem. J . 47, proc. xxxii. 113a. Bumpus, F. M., and Page, I. H. (1955). J . Biol. Chem. 212. 111. 113b. Burch, H. B., Storvick, C. A., Bicknell, R. L., Kung, H. C., Alejo, L. G., Everhart, W. A., Lowry, 0. H., King, C. G., and Bessey, 0. A. (1955). J. Biol. Chem. 212, 897. 114. Burn, J. €1. (1945). Ph@ol. Revs.26, 377. 115. Butenandt, A . (1949). Angew. chem. 61, 262. 116. Butenandt, A. (1052). Endeavour 11, 188, 117. Hutenandt, A , , Hellmann, H., and Hanser, G. (1952). 2. physiol. Chern. 269, 225. 115. Butenandt, A., Iiarlson, P., and Zillig, W. (1951). 2. physiol. Chew. 288, 125. 119. 13utenandt, A., and Itenner, U. (1953). Z. Kuturjorsch. 8b, 454. 120. Butenandt, A,, Schiedt, U., and Riekert, E. (1954). A n n . 686, 229. 121. Hutenandt, A , , Schiedt, U., and Hiekert, E. (1954). Ann. 688, 106. 122. Butenandt, A,, Schiedt, U., Bielrert, E., and Kornmann, P. (1954). Ann. 686, 217. 122a. Butenandt, A., Schiedt, U., Biekert, E., and Cromartie, R. I. T. (1954). Ann. 690, 75. 123. Hutenandt, A , , and Schlossberger, H. (1952). Chein. Ber. 86, 565. 124, I h t e n a n d t , A,, Schulz, G., and IInnser, G. (1952). Z. physiol. C l e m . 296,404. 125. Butenandt, A , , Weidel, W., and Becker, E. (1940). XalurwissenschaJten 28,63. 126. Butenandt, L4.,Weidel, W., and Becker, E. (1940). Il;atiirwissenschuf~en28, 447. 127. Uutenandt, A4., Weidel, W., ctnd Derjugin, W. von (1952). Autul.wisserischufien 30, 51. 128. Butenandt, A,, Weidel, W., and Schlossbergcr, H. (1949). Z. A-afurforsch. 4b. 242. 129. Hutenandt, A , , WeidcI, W., Weichert, It., and Dcrjugin, W. von (1943). 2. p l i p i o l . (‘hein. 279, 27. 130. Butt>s,J. S., Dunn, bl. S., and Hallman, I,. F. (1938). J . Biol. (’hem. 123, 711. 131. Butts, J. S., Sinnhuber, It. O., and Dunn, PI. S. (1941). Proc. SOC.h’xptl. Biol. &/led. 46, 671. 132. Byerrnm, It. IT.,Hitmill, It. L.,and Ball, C. D. (1954). J. Biol. (’hem. 210, 645. 133. Cammrtr:it:i, 1’. S., and Cohen, P. 1’. (1950). J . Biol. (‘hein. 187, 439. 134. Cammar:rt:i, 1’. S., and Cohen, 1’. 1’. (1051). . J . B i d . Cheni. 193, 45. 135. Cantoni, G. 1 , . (1951). J . B i d . (‘hem. 189, 203. 136. C:intoni, G. 1,. (1951). J . Biol.Chem. 189, 745. 137. Cmzanelli, A , , Harington, C. It., and Randall, S. S. (1934). Biochem. J. 28, 68.

138. Ca\rte, J . I<. (1054). ]fed. ./. Aztslralia 11, 15. 130. Celander, 1). It., and Berg, C. 1’. (1953). J . Biol. L‘heni. 202, 339. 140. C h m , F. I ., IIrym:inn, TI., :inti CI:incy, C. W. (1951). .I. < 4 t i t . Chetn. Soc., 73, 54.18. 1-11, C1i:irconiict llarcliiig, h‘., arid Lhlgliesh, C. 13;.

(1852). Abstt,. 2rcd IrL/c,rrL, ( ‘ o n g r . Riochetti., Paris, p. 208, 11’2. C1i:trconnrt-IIarding, F., I):ilgliesli, C. h:,, and Neubcrger, A. (1953). Biorhrtn. J . 63, 513. 1-13. Chuttop:idhyay, D , , Chosh, S . C . , Ch:it,topndhyay, 11., and U:krierjee, H. (1953). J . B i d . Chetn. 201, 529.

128

C. E. DALQLIESH

144. Cherkes, G. A. (1952). Biokhimiya 17, 706. 145. Chiancone, F. M. (1950). Acta Vitaminol. 4, 193. 146. Chiari, H. H. (1888). Prager med. Wochschr. 60, 541. 147. Christensen, H. N., Riggs, T . R., and Coyne, B. A. (1954). J. Biol. Chem. 209, 413. 148. Clark, C. T., Weissbach, H., and Udenfriend, S. (1954). J . Biol. Chew. 210. 139. 149. Clerc-Bory, M., Pacheco, H., and Mentaer, C. (1954). (‘ompt. rend. 238, 525. 150. Closs, K., and Braaten, K . (1941). 2. physiol. Che7n. 271,221. 151. Closs, K., and Folling, A. (1938). 2. physiol. Chem. 264, 250. 152. Cohn, E. J., and Edsall, J. T. (1943). “Proteins, Amino Acids and Peptides as Ions and Dipolar Ions.” Reinhold, New York 153. Cole, A. S., and Robson, W. (1951). Brit. J. Nutrition 6, 306. 154. Connors, W. M., and Stotz, E. (1949). J . Biol. Chem. 178, 881. 155. Conochie, J. (1953). Australian J. Exptl. Biol. Med. Sci. 31, 373. 155a. Coppini, D., and Camurri, M. (1954). Giorn. biochim. 3, 270. 156. Cornforth, J. W., Cornforth, R. H., Dalgliesh, C. E., andNeuherger, A. (19.51). Biochem. J . 48,591. 157. Cornforth, J . W., Dalgliesh, C. E., and Neuberger, A. (1951). Biochem. J . 48, 598. 158. Cornforth, J. W., and James, A. T. (1954). Biochewi. J . 68, proc. xlvii. 159. Costabile, L., Scala, E., Scardi, V., and Virgilio, A . (1952). Boll. S O C . ilal. biol. sper. 28, 1850. 160. Crandall, D. I. (1953). Federalion Proc. 12, 192. 161. Crandall, D. I. (1954). Federation Proc. 13, 195. 182. Crandall, D. I. (1955). Zn “Symposium on Amino Acid Metabolism’’ (N‘. McElroy and B. Glass, eds.), p. 867. Johns Hopkins Press, Baltimore. 183. Crandall, D . I. (1955). J . Biol. Chem. 212, 565. 164. Crandall, D . I., and Halikis, D. N . (1954). J . Biol. C‘hcm. 208, 629. 165. Cromartie, R. I. T., and Harley-Mason, J. (1952). J . Chem. SOC.,p. 2525. 166. Cutinelli, C., Ehrensvard, G., Reio, L., Saluste, E., and Stjernholm, R. (1951). Acta Chem. Scand. 6, 353. 167. Dagley, S., Fewster, M. E., and Happold, F. C. (1953). J . Gen. Microbiol. 8, 1. 168. Dakin, H. D. (1905). Proc. R o y . Soc. B78,491,498. 169. Dakin, €I. D. (1913). J . Biol. Chem. 14, 321. 170. DaIgliesh, C. E. (1951). Quart. Rev. Chem. SOC.6, 227. 171. Dalgliesh, C. E. (1952). Biochem. J . 62, 3. 172. Dalgliesh, C. E. (1952). J . Chem. SOC.,p. 137. 173. Dnlgliesh, C. E. (1954). Biochini. el Biophys. Acta 16, 205. 174. Dalgliesh, C. E. (1954). Biochem. J . 68, proc. xlv. 175: Dalgliesh, C. E. (1955). J. Clin. Pathol. 8, 73. 175a. Dalgliesh, C. E. (1955). Biochem. J . 80, xxiii. 176. Dalgliesh, C. E., Knox, W. E . , and Neuberger, A. (1951). Nature, 188. 20. 177. Dalgliesh, C. E., and Mann, F. G. (1947). J. Chem. SOC.,p. 658. 178. Dalgliesh, C. E., and Tekman, S. (1954). Biochern. J. 68, 458. 179. Dalgliesh, C. R., Toh, C. C., and Work, T. S. (1953). J . Physiol. (London) 120, 298. 179a. Dancis, J., and Balis, M. E. (1955). Paedialrics 16, 63. 150. Dann, M., Marples, E., and Levine, S. 2. (1943). J. Clin. Znvc,sf. 2 2 , 8 i . 181. Dann, W. J . , and Huff, J. W. (1947). J . Biol. Cheni. 188, 121.

METABOLISM OF THE AROMATIC AMINO ACIDS

129

182. Darby, W. J., McGanity, W. J., Stockell, A. K., and Woodruff, C. W. (1953). Brit. J. Nutrition 12, 329. 183. Davis, B. D. (1950). Nature 166, 1120. 184. Davis, B. D. (1951). J. Biol. Chem. 191,315. 185. Davis, B. D . (1952). 2nd Congr. Intern. Biochem., Paris. Symposium sur le metabolisme microbien, p. 32. 186. Davis, B. D. (1952). Experientia 6, 41. 187. Davis, B. D. (1952). J. Bacteriol. 64, 729. 188. Davis, B. D. (1952). J. Bacterial. 64, 749. 189. Davis, B. D. (1953). Proc. Natl. Acad. Sci. (U.S.) 39, 363. 190. Davis, B. D. (1953). Science 118, 251. 191. Davis, B. D. (1955). I n “Symposium on Amino Acid Metabolism” (W. McElroy and B. Glass, eds.), p. 799. Johns Hopkins Press, Baltimore. 19la.Davis, B. D. (1955). Private communication. 192. Davis, B. D., and Mingioli, E. S. (1953). J . Bacteriol. 66, 129. 193. Davis, B. D., and Weiss, U. (1953). Naunyn-Schmiedeberg’s Arch. exptl. Pathol. u. Pharmakol. 220, 1. 194. Dawes, E . A., and Happold, F. C. (1949). Biochem. J. 44, 349. 195. Dawson, R. F. (1948). Advances i n Enzymol. 8, 203. 195a. Dennis, D. J., Blaschko, H., and Welch, A. D. (1955). J. Pharmacol. Ezptl. f i e r a p . 113, 14. 196. Deulofeu, V. (1948). Fortschr. Chem. org. Naturstofe 6 , 241. 197. Deulofeu, V., andDuprat, E. (1944). J . Biol. Chem. 163,459. 198. Devine, J. (1940). Biochem. J. 34, 21. 199. Diae, C. J., Mendoea, H. C., and Rodriguez, J. S. (1939). Klin. Wochschr. 18, 965. 200. Dinning, J. S. (1953). Federation Proc. 12.412. 201. Dische, R., and Rittenberg, D. (1954). J. Biol. Chem. 211, 199. 202. Dolby, D. E., Hall, D. A., and Happold, F. C. (1952). Brit. J. Exptl. Pathol. 33. 304. 203. Drechsel, E. (1896). 2. biol. 33, 96. 204. Dubeck, M., and Kirkwood, S. (1952). J . Biol. Chem. 199, 307. 205. Dulibre, W. L., and Raper, H. S. (1930). Biochem. J. 24,239. 206. Duncan, M., and Sarett, H. P. (1951). J . Biol. Chem. 193,317. 207. Dunn, M. S., and Rockland, L. B. (1947). Advances i n Protein Chem. 3, 296. 208. Edson, N. L. (1935). Biochem. J. 29,2498. 209. Edwards, S. W., and Knox, W. E. (1955). In “Methods in Enzymology” (N. 0. Kaplan and S. Colowick, eds.), Vol. 11,p. 287. Academic Press, New York. 209a. Edwards, S. W., Hsia, D. Y-Y., and Knox, W. E. (1955). Federation Proc. 14, 206. 210. Efimochkina, E. F. (1954). Biokhimiya 19,68. 211. Ehrensvard, G. (1952). 2nd Congr. Intern. Biochem., Paris. Symposium sur le metabolisme microbien, p. 72. 212. Ehrensvard, G. (1954). Svensk kem. Tidskr. 66,249. 213. Ehrensvard, G., and Reio, L. (1953). Arkiv Kemi 6, 229. 214. Ehrlich, F., and Jackobson, K. A. (1911). Ber. 44,888. 215. Eij kmann, J. F. (1885). Rec. trav. chim. 4, 49 (1886); 6, 299. 216. Ek, A., Kissman, A., Patrick, J. B., and Witkop, B. (1952). Experientia 8,36. 217. Ek, A., and Witkop, B. (1953). J. Am. Chem. SOC.76, 500. 218. Ek, A., and Witkop, B. (1954). J. Am. Chem. SOC.76,5579.

130

C. E. DALGLIESH

Ellinger, A. (1903). Z . physiol. Chem. 39,44. Ellinger, A. (1904). Z . physiol. Chem. 43, 325. Ellinger, A., and Flitinand, C. (1907). Ber. 40, 3029. Ellinger, A,, and Flamand, C. (1909). Z. physiol. (‘heni. 62, 276 Elliriger, P., and Armitage, 1’. (1953). Biochem. J . 63, 588. JSlvehjem, C. A . (1952). Z n k r n . 2. Vitaniinforsch. 23, 20!). Embden, G., :ind lhldes, K. (1913). Biochem. Z . 66, 301. Embden, O . , 8ulomori, H., and Schmidt, F. (1!106). Beitr. chern. Physiol. u . Pathol. 8, 129. 227. Erlenmeyer, E., Jr., and Halsey, J. T. (1899). Ann. 307, 138. 228. Erlenmeyer, E., Jr., and Lipp, A. (1882). B e r . 16, 1006. 229. Erlenmeyer, E., Jr., :1nd Lipp, A. (1883). Ann. 219, 161. 230. Erspamer, V. (1940). Naunyn-Schmiedeberg’s Arch. exptl. Pathol. I L . Pharuiakol. 196,343, 366, 391. 231. Erspamer, V. (1942). Naunyn-schmiedeberg’s Arch. exptl. Pathol. u. Pharuiakol. 200, 43, 60. 232. Erspamer, V. (1943). Naunyn-Schmiedeberg’s Arch. ezptl. Pathol. u.Pharrriakol. 201, 377. 233. Erspamer, V. (1946). Arch. sci. b i d . ( I t a l y ) 31, 63. 234. Erspamer, V. (1948). Arch. intern. pharmacodynamie 76, 308. 235. Erspamer, V . (1952). Nature 170, 281. 235a. Erspamer, V. (1954). Pharmacol. IZevs. 6, 425. 235b. Erspamer, V. (1955). J. Physiol. (London) 127, 118. 236. Erspamer, V. (1954). Experientia 10, 471. 237. Erspamer, V. (1954). Rend. sci. Farniitalza 1. “I1 sistema cellulare enterocromaffine e 1’enter:imina.” 238. Erspamer, V., arid Asero, B. (1951). Ricerca sci. 21, 2132. 239. Erspamer, V., and Asero, B. (1953). J. B i d . (’hem. 200, 311. 240. Erspamer, V.,and Boretti, G. (1950). Experientia 6,348. 241. Erspamer, V., and Falconieri, G. (1952). Naturwissenschajtm 39, 431. 242. Erspamer, V., arid Vialli, M. (1952). Ricerca sci. 22, 1420. 243. Euler, U. S. von, (1950). Ergeb. Physiol. u.exptl. Pharmakol. 40, 261. 244. Euler, U. S. von, (1951). Pharnzacol. Revs. 3, 247. 245. Euler, U. S. von, Hamberg, U., and Hellner, S. (1951). Biochem. J. 49, 655. 246. Evans, W. C., Smith, B. S. W., Linstead, It. P., and Elvidge, J. A. (1951). h’ature 168, 772. 217. Ewiris, A. J., and I,aidlaw, P. 1’. (1910). J. Physiol. (London) 40, 275. 248. Ewins, A. J., and Laidlaw, P. P. (1910). Proc. Chem. Soc. (London) 26, 343. 249. Ewins, A. J., and Laidlaw, P. P. (1913). Biochem. J. 7, 18. 24%. Eys, J. van, Tonster, O., and Darby, W. J. (1955). Federation Proc. 14, 296. 250. Faltit, W., and Langstein, L . (1903). 2. physiol. Chem. 37, 513. 251. Fawcett, D. M., itnd Kirkwood, S. (1953). J. Biol. Chem. 206, 795. 252. Fawcett, I).M., snd Kirkwood, S. (1954). Science 120, 547. 253. Fawcett, D . M., and Kirkwood, S. (19%). J. Biol. Chem. 209, 249. 254. Feigelson, P., Williams, J. N., Jr., and Elvehjem, C. A. (1951). J . Biol. (‘hem. 193, 737. 255. Feigelson, P., Williams, J. N., Jr., itnd Elvehjem, C. A. (1951). Proc. SOC. E x p t l . Biol. hled. 78,34. 256. Feldberg, W., imd Toh, C. C. (1953). J. Physiol. (London) 119, 352. 257. Felix, K., Bock, E. G., Geratz, D., Glasenapp, I. von, Roka, I,., and Weisenberger, I<. (1953). Z . physiol. Chem. 292, 157. 219. 220. 221. 222. 223 224. 225. 226.

METABOLISM OF T H E AROMATIC AMINO ACIDS

131

258. Felix, K . , Leonhardi, G., and Glasenapp, I. von (1951). 2. physiol. Chem. 287, 133. 259. Felix, K., Leonhardi, G., and Glasenapp, I. von (1951). 2. physiol. Chem. 287, 141. 260. Felix, Ii.,and Teske, R . (1941). 2. physiol. Chem. 267, 173. 261. Felix, I<., :ind %om,W. (1941). 2. physiol. Chem. 268, 257. 262. Felix, Ti., Zorn, I<., arid Dirr-Kaltenbach, H . (1937). 2. physiol. Chem. 277, 141, 263. Fildes, 1’. (11141). Brit. .I. Exptl. Pathol. 22, 293. 264. Fildes, 1’. (1945). Brit. J . E z p t l . Pathol. 26, 418. 265. Fink, I<., :ind Fink, It. M. (1948). Science 108.358. 266. Fixcher, 15. (1899). Rer. 32,2451, 3638. 267. Fischer, TC., and Mouneyrat, A. (1900). B e r . 33, 2383. 268. Fischer, H. 0. I,., and Dangschat, G. (1937). Helv. Chim. ilcta 20, 705. 269. Fitxpztrick, T. 13., and Lerner, A . U. (1954). rirch. Dermatol. and Syphilol. 69, 133. 270. Fliicher, F. (1908). 2. physiol. C‘hem. 68, 189. 271. Flaschentriiger, B., Halawani, A,, and Nabeh, I. (1954). K l i n . Wochschr. 32, 131. 272. Folling, A . (1934). 2. physiol. C‘henz. 227, 169. 273. FoIling, A, and Closs, K. (1938). Z. physiol. Chem. 264, 256. 271. Folling, A , , Closs, I<.,and Gammes, T. (1938). 2. physiol. Chem. 266, 1. 275. Forrest, €1. S., and Mitchell, H. K. (1954). J. Am. Chem. Soc. 76, 5656, 5658. 276. Foster, G. I,. (1929). J. Biol. Chem. 89,345. 277. Foster, M. (1950). Proc. .Vatl. Acad. Sci. (U.S.) 36, 606. 278. Freyburger, W. A , , Graham, B. E., Rapport, hl. M., Seay, 1’. H., Govier, W. M., Swoap, 0. F., and Vander Brook, M. J. (1952). J. Pharmacol. 66,SO. 279. Friedmann, E. (1908). Beitr. chem. Physiol. u. Pathol. 11, 304. 280. Funk, C. (1911). J . Chem. Soc. 99,554. 281. Gad, I., Jacobsen, E., and Plum, C. M. (1944). A c t u Physiol. Scand. 7, 244. 282. Gaddum, J. H. (1953). J . Physiol. (London) 119, 363. 283. Gaddum, J. H., and Hameed, K. A. (1954). Brit. J. Pharmacol. 9, 240. 281. Gale, E. F. (1946). Advances in Enzymol. 6, 1. 285. Galston, A. W., Bonner, J., and Baker, R. S. (1953). Arch. Biochem. and Biophys. 42,456. 286. Garrod, A . Is. (1923). “Inborn Errors in Metabolism,” 2nd ed. Henry Froude and Holder & Houghton, London. 287. Garrod, A. E., and Hopkins, F. G. (1896). J . Physiol. (London) 20,112. 288. Gibson, Q. H., Newey, H., Smyth, D. H., and Whaler, B. C. (1954). J. Physid.(London) 126, 65P. 288a. Gilvarg, C. (1955). I n “Symposium on Amino Acid Metabolism” (W. D. McElroy and B. Glass, eds.), p. 812. Johns Hopkins Press, Baltimore. 289. Gilvarg, C., and Bloch, K . (1950). J. Am. Chem. SOC. 72, 5791. 290. Gilvarg, C., and Bloch, K. (1951). J . Biol. Chem. 193, 339. 291. Gilvarg, C., and Bloch, K. (1952). J . Biol. Chem. 199, 689. 292. Cinoulhiac, E., and Sensi, P . (1951). Actu Vitaminol. 6, 111. 293. Ciinoulhiac, E., and Tenconi, L. T . (1951). dctu Vitaminol. 6, 246. 294. Glazer, H. S., Mueller, J. F., Thompson, C., Hawkins, V. R., and Vilter, R. W. (1951). Arch. Biochem. and Biophgs. 33, 243. 295. Glynn, I,. E., Himsworth, H. P., and Neuberger, A. (1945). B r i t . J . Ezptl. Paihol. 26, 326.

132

C. E. DALGLIESH

296. Goldacre, P. L., Galston, A . W., and Weintraub, R. L. (1954). Arch. B i o chem. and Biophys. 43,358. 297. Goldenberg, M.,Faber, M., Alston, E. J., and Chargaff, E. (1949). Science 109, 534. 298, Goodall, McC. (1950). Acta Chem. Scarid. 4,550. 299. Goodall, McC. (1951). A c t a Physiol. Scand. 24, suppl. 85. 300. Gooder, H., andHappold, F. C. (1954). Biochem. J . 67, 369. 301. Gordon, M., Haskins, F. A., and Mitchell, H. K. (1950). I’roc. n’utl. A c a d . Sci. (U.S.) 36, 427. 302. Gordon, M.,and Mitchell, H. K. (1950). Genetics 36, 110. 303. Gordon, S.A., and Nieva, F. S. (1949). Arch. Biochem. 20, 856. . 367. 304. Gordon, S.A , , and Nieva, F. S. (1949). Arch. B ~ o c h c m 20, 305. Qortner, W.A,, and Kent, hl. (1953). J . Biol. Chem. 204, 593. 306. Goryachenkova, E. V. (1951). Doklady A k a d . h’auk S.S.S.R. 80,643. 307. Gots, J. S.,Koh, W. Y., and Hunt, G. R., Jr. (1954). J . Gen. Microbiol. 11, 7. 308. Goutarel, R., Janot, M-M., Prelog, V., and Taylor, W. I. (1950). Helv. Chitti. Acta 33,150. 309. Grau, C. R., and Steele, R. (1954). J . Nutrition 63,59. 310. Green, M.M. (1952). Proc. N a t l . Acad. Sci. (U.S.) 38,300. 311. Greenberg, L. D., Bohr, D. F.,McGrath, H., andRinehart, J. F. (1949). A r c h . Biochem. 21, 237. 312. Gripenberg, J. (1952). A c l a Chem. Scand. 6,580. 313. Gros, H., and Kirnberger, E. J. (1954). K l i n . Wochschr. 32, 115. 314. Gros, H., and Kirnberger, E. J. (1954). K l i n . Wochschr. 32, 645. 314s. Gros, P., Talwar, G. P., and Coursaget, J. (1954). Bull. SOC. chim. biol. 36, 1569. 315. Groschke, A. C.,and Uriggs, G . pvl. (1946). J . Biol.Chem. 166,739. 316. Gross, J., and Pitt-Rivers, R. V. (1952). Lancet 262, 439. 317. Gross, J., and Pitt-Rivers, R. V. (1952). Lancet 262, 593. 318. Gross, J., and Pitt-Rivers, R. V. (1953). Biochem. J . 63,645. 319. Gross, J., and Pitt-Rivers, R. V. (1953). Biochem. J . 63, 652. 320. Gross, J., and Pitt-Rivers, R. V. (1953). V i t a m i n s and Hormones 11, 159. 321. Gross, J., and Pitt-Rivers, R. V. (1954). Recent Progr. Hormone Research 10, 109. 322. Gross, 0 . (1914). Biochem. 2.61, 165. 323. Guggenheim, M. (1913). Z.physiol. Chem. 88,276. 324. Guggenheim, &I. (1951). “Die biogenen Amino.” S. Karger, Basel. 325. Guggenheim, M., and Loeffler, W. (1916). Biochern. Z.72, 325. 326. Gunsalus, I. C.,and Bellamy, W. D. (1944). J . Biol. Chem. 166, 357. 327. Gunsalus, I. C.,Bellamy, W. D., and Umbreit, W. W. (1944). J . Biol. (,’hem. 166, 685. 328. Gurin, S.,and Delluva, A . M . (1947). J . Biol. Chem. 170, 545. 329. Gustafson, F. G. (1949). Science 110, 279. 330. Haagen-Smit, A. J., Leech, W. D., andBergren, W. R . (1942). Am. J . Botany 29, 500. 330a. Haberland, G. L., Bruns, F., and Altman, K. I. (1954). Biochem. 2. 326, 107. 331. Hackman, R. H. (1953). Biochem. J . 64, 371. 332. Hackman, R. H., Pryor, M. G. M., and Todd, A. R. (1948). Biochear. J . 43, 474.

METABOLISM OF THE AROMATIC AMINO ACIDS

133

Haddox, C. H. (1953). Proc. S a f l . Acad. Sci. (U.S.) 38,482. Hahn, G., nlrwald, L., Schales, O., and Werner, H. (1935). A n n . 620, 107. Hahn,,G., and Hansel, A. (1938). Ber. 71, 2192. Hahn, G., and Ludewig, H. (1934). Ber. 67, 2031. Hahn, G., and Rumpf, F. (1938). Ber. 71, 2141. Hahn, G., and Werner, H. (1935). Ann. 620, 123. Hakim, A. A., and Happold, F. C. (1952). Biochem. J. 62, proo. xxv; (1953). 63, proc. xxxvi. 340. Hakim, A. A,, and Happold, F. C. (1954). h’afure 174,358. 341. Hamlin, K. E., and Fischer, F. E. (1951). J . Am. Chem. SOC.73, 5007. 342. Handler, P., and Kohn, H. I. (1943). J . B i d . Chem. 160, 447. 343. Hankes, L. V., Lyman, R. L., and Elvehjem, C. A. (1950). J . Biol. Chem. 187, 547. 344. Hankes, L. V., and Urivetsky, M. (1954). Arch. Biochem. and Biophys. 62, 484. 345. Happold, F. C. (1950). Advances i n Enzymol. 10.51. 346. Happold, F. C. (1950). Biochem. SOC. Symposia No. 6, 85. 347. Happold, F. C., and Hoyle, L. (1935). Biochem. J. 29, 1918. 348. Happold, F. C., and Struyvenberg, A. (1954). Biochem. J. 68,379. 349. Harington, C. R. (1926). Biochem. J. 20, 293. 350. Harington, C. R. (1926). Biochem. J. 20,300. 351. Harington, C. R. (1928). Biochem. J. 22, 1429. 352. Harington, C. R. (1939). Fortschr. Chem. org. Naturstoffe 2, 103. 353. Harington, C. R. (1944). J. Chem. SOC.,p. 193. 354. Harington, C. R. (1951). Endocrinology 49, 401. 355. Harington, C. R., andBarger, G. (1927). Biochem. J. 21, 169. 356. Harington, C. R., and Pitt-Rivers, R. V. (1945). Biochem. J . 39,157. 357. Harington, C. R., and Randall, S. S. (1929). Biochem. J . 23,373. 358. Harington, C. R., and Salter, W. T. (1930). Biochem. J. 24,456. 359. Harley-Mason, J. (1948). Experientia 4, 307. 360. Harley-Mason, J. (1952). Chemistry & Industry, p. 173. 361. Harley-Mason, J. (1954). Ezperientia 10, 134. 361a. Harley-Mason, J. (1955). Chemistry &Industry, p. 355. 362. Harley-Mason, J., and Bu’Lock, J. D. (1952). Biochem. J. 61,430. 363. Harley-Mason, J., and Jackson, A. H. (1954). J . Chem. SOC.,p. 1165. 364. Harrison, G. A., and Broomfield, R: J. (1928). Biochem. J . 22,43. 365. Haskins, F. A., and Mitchell, H. K. (1949). Proc. Natl Acad. Sci. (U.S.) 36, 500 366. Hayaishi, O., and Stanier, R. Y. (1951). J . BacterioE. 62,691. 367. Hayaishi, O., and Stanier, R. Y. (1952). J. Biol. Chem. 196, 735. 368. Hays, E. E., Wells, I. C., Katzmann, P. A,, Cain, C. K . , Jacobs, F. A., Thayer, S. A., Doisy, E. A , , Gaby, W. L., Roberts, E. C., Muir, R. D., Carroll, C. J., Jones, L. R., and Wade, N. J. (1945). J . BioZ. Chem. 169,725. 369. Heidelberger, C. (1949). J. BioZ. Chem. 179,139. 370. Heidelberger, C., Abraham, E. P., and Lepkovsky, S. (1949). J . Biol. Chem. 179,151. 371. Heidelberger, C., Gullberg, M. E., Morgan, A. F., and Lepkovsky, S. (1949). J. Biol. Chem. 179, 143. 372. Heimberg, M., Rosen, F., Leder, I. G., and Perlzweig, W. A. (1950). Arch. Bioc hem. 28, 225.

333. 334. 335. 336. 337. 338. 339.

134

C. E. DALGLIESH

373. Heinsen, 11. A . (1937). 2 . physiol. Chem. 246, 1. 37.2. Hellmann, €I., and Eggstein, M. (1954). Nnturwissenschaften 41, 335. 375. Hellmaiin, H., and Wiss, 0 . (1952). Helv. Physiol. et Pharniacol. Actn 10, C16, C35. 376. IIellmann, H., : ~ n dWiss, 0. (1952). 2. physiol. Cheni. 289, 301). 377. Henbest., 11. H.,Jones, E. R . I I . , and Smith, G . F. (1953). .I. (’hew. Soc., p. 3796. 378. Henderson, J . M., and Bonner, J. (1952). Am. J. Botany 39,444. 379. Henderson, 1,. M. (1949). J . Biol. Chem. 178, 1005. 380. Henderson, L . M. (1949). J . Biol. Chem. 181,677. 381. Henderson, I,. RI., and Hankes, I,. V. (1949). Proc. Soc. h’xptl. Rial. illetf. 70, 26. 382. Henderson, 1,. M., Hill, H . N., Koski, R. E., and Weinstock, I. XI. (l!M). Proc. SOC.E x p t l . Biol. Med. 78, 441. 383. Henderson, L. hf., and Hirsch, H. M. (1949). J. Biol.Chem. 181. 667. 384. Henderson, L. M., Koeppe, 0. J., and Zimmerman, H. 1%. (1953). J . Biol. Chem. 201, 697. 385. Henderson, I,. M.,and Koski, R. E. (1954). Federation Proc. 13, 228. 386. Henderson, I,. M., and Ramasarma, G. B. (1949). J. Biol. Chem. 181, 687. 387. Henderson, L . A t . , Weinstock, I . M., and Ramasarma, G. I3. (1951). J . Biol. (.‘hem. 189, 19. 388. Henze, M. (1907). Z. physiol. (’hem. 61, 64. 389. Hermanns, I,., and Sachs, 1’. (1921). 2. physiol. Che7n. 114, 79, 88. 390. Herter, C. A. (1908). 1 . Biol. Chem. 4, 238, 253. 391. Hirui, K. (1921). Biocheni. Z . 114, 71. 392. Hirata, \-., Kakanishi, K., and Kikkawa, H . (1950). Science 112, 307. 393, Hird, F. J. R . , and Ronsell, R. V. (1950). Nature 166, 517. 394. Hoagland, C.I,., Ward, S. M., and Shank, R. E. (1943). 1.Biol. (’hem. 161, 369. 395. Hogben, I,., Worra11, R. L., arid Zieve, I. (1932). Proc. Roy. Sac. Edinburgh 62, part 111, No. 13. 396. Holley, R. W., Boyle, F. P., Durfee, A. K., and Holley, A. D . (1051). Arch. Biocheni. and Biophys. 32, 192. 397. Holman, W. I. hf. (1954). Biochem. J. 66,513. 398. Holman, W. I. M., and de Lange, D. J. (1950). Biocheni. J . 46,47. 399. Holman, W. I. M., and de Lange, D. J.- (1950). Nature 166, 112; 166, 468. 100. Noh, C. von, €leinrich, W. D., and Holt, L. von (1954). 2. physiol. Chenz. 297, 241. 401. Holt, P. F., and Callow, H. J. (1943). Analyst 68, 351. 402. Holtz, 1’. (1938). Z. physiol. Chem. 261, 226. 403. Holtz, I’., and Credner, K. (1943). Naunyn-Schmiedeberg’s Arch. exptl. Pathol. u. Pharmakol. 202, 150. 404. Holta, P., Credner, K., and Koepp, W. (1942). n’aunyn-SchmiedebergJs Arch. exptl. Pathol. u. Pharmakol. 200,356. 405. Holtz, P., Credner, K., and Walter, H. (1939). 2. physiol. Chem. 262, 111. 406. Holta, P., Heise, R . , and Ludtke, K. (1938). Naunyn-Schniiedeberg’s Arch. expll. Pathol. I ( . Pharniakol. 191, 87. 407. Holta, P., and Janisch, H. (1937). Naunyn-Schmiedeberg’s Arch. exptl. Po.thol. u . Pharmakol. 186, 684. 408. Homer, A . (1914). J . Biol. Ckem. 17, 509.

METABOLISM OF THE AROMATIC AMINO ACIDS

135

Homer, A. (1915). J. Biol. ('hem. 22, 345. Hopkins, F. G., and Cole, S. W. (1901). Proc. Roy. Soc. 68, 21. Hopkins, F. G., and Cole, S. W. (1902). J. Physiol. (London) 27, 418. Hopkins, F. G., and Cole, S. W. (1903). J . Physiol. (London) 29, 451. Hoppe-Seyler, F. (1863). Virchow's Arch. Pathol. A n a t . u.Physiol. 27, 388. Hoppe-Seyler, G. (1883). Z. physiol. Chem. 7, 423. Horecker, 13. I,., and Smyrniotis, P. Z. (1952). J . A m . ('hem. Soc. 74, 2123. Hoshino, T., Kobayashi, T., and Shimodaira, K . (1935). Proc. Imp. Acad. Tokyo 11, 192; Ann. 620, 11, 19. 417. Houff, W. H., Hinsvark, 0. N., Weller, I,. E., Wittwer, S. H., and Sell, H . hZ. (1954). J . .4m. Chem. Soc. 76, 5654. 418. Huff, J. W., and Perlzweig, W. A. (1943). Science 97, 538. 419. Huff, J. W., and Perlzweig, W. A. (1947). J. Biol. Chem. 167, 157. 420. Hughes, D. E. (1952). Biochim. et Biophys. Acta 9, 226. 421. Hughes, I). E. (1954). Biochem. J. 67, 485. 422. Hughes, G. K., and Ritchie, 13. (1952). Rev. Pure A p p l . (,'hem. Australia 2. 125. 423. Hultin, T., Lindvall, S., and Gustafsson, K. (1954). Arkiv Kemi 6,477. 424. Humphrey, J . H., and Jaques, R. (1954). J. Physiol. (London) 124, 305. 425. Humphrey, J . H., and Toh, C. C. (1954). J . Physiol. (London) 124, 300. 426. Hundley, J . &I., and Bond, H. W. (1948). J. B i d . ('hem. 173, 513. 127. Hundley, J . hl., and Bond, H . W. (1949). Arch. Biochem. 21, 313. 428. Hunter, S. F. (1953). Proc. Soc. Ezptl. B i d . M e d . 82, 14. 429. Hunter, S. F., arid Handler, 1'. (1952). Arch. Biochem. and Biophys. 36,377. 430. Hurt, W. W., Scheer, B. T., and Deuel, H.J . (1949). d r c h . Biochem. 21. 87. 431. Inagami, K . (1954). A'ature 174, 1105. 432. Ingritham, I,. I,., and Corse, J. (1951). J . ,4m.('hem. Soc. 73, 5550. 433. Ito, F. (1951). V i t a m i n s ( J a p a n ) 4, 269. 431. Tto, T., Uchida, bZ., and Ichihara, I(. (1952). nletl. J. Osaka liniv. 3, 285. 435. Izquierdo, J. A , , and Stoppani, A. 0. M. (1953). Brit. J . Pharmacol. 8, 389. 436. Jackson, R . W. (1929). J. Biol. Chem. 84, 1. 437. Jakoby, W. B. (1954). J. Biol.Chem. 207,657. 438. Jakoby, W. H., and Bonner, D. M. (1953). J . B i d . Chern. 206, 699. 439. Jakoby, W. H., and Bonner, D. M. (1953). J . Biol. Chem. 206, 709. 110. James, W. 0 . (1953). Endeavour 12, 76. 111. Jaques, R., and Schachter, hl. (1954). Brit.J . Pharmacol. 9, 53. 442. Jayson, G. C., Scholes, G., and Weiss, J. (1954). Biocheni. J. 67,386. 143. Jensen, H. (1935). J. Am. Chem. Soc. 67, 1765. 444. Jensen, H., and Chen, K. K. (1932). Ber. 66, 1310. 445. Jervis, G. A. (1937). Arch. iVeuro1. Psychiat. 38,944. 446. Jervis, G. A. (1938). J. Biol. Chem. 126,305. 447. Jervis, G . A. (1947). J. B i d . Chern. 169, 651. 448. Jervis, G. A. (1952). Proc. Soc. E x p t l . B i d . filed. 81, 715. 449. Jervis, G . A . (1953). Proc. SOC.E x p t l . H i o l . M e d . 82. 514. 450. Jervis, C:. A , , Block, It. J . , Holling, D., s n d ICitnze, 15. (1940). J . U i o l . ( " h e m . 134, 105. 451. Johnson, 7'. H., :incl T e a k e s b u q ~ ,1,. H., J r . (1942). €'roc. .\at!. ,4cntl. Sci. ( U . S . )28, 73. 452. Jones, 15. R. FI., Henixst, €1. U.,Smith, G . I?., and Hentley, J . A. (1942). AVature 169, 485. 409. 410. 411. 412. 413. 414. 415. 416.

136

C. E. DALGLIESH

453. Jones, J. D., Smith, B. S. W., and Evans, W. C. (1952). Biochem. J. 61, proc. xi. 454. Jones, K. M., and Elliott, W. H. (1954). Biochim. et Biophys. Acta 14, 586. 455. Junqueira, P. B., and Schweigert, B. S. (1948). J. Biol. Chem. 176, 535. 456. Kakihara, Y., and Ichihara, K. (1953). Med. J. Osaka Univ. 3,497. 456a. Kalan, E., and Davis, B. D. Unpublished observations. 456b. Kalan, E., Srinavasan, P. R., Sprinson, D. B., and Davis, B. D. Unpublished observations. 457. Kallio, R. E., and Berg, C. P. (1949). J. Biol.Chem. 181,333. 458. Kaplan, L., and Stock, C. C. (1954). Federation Proc. 13,239, 459. Katagiri, M., and Sato, R. (1953). Science 118,250. 460. Keller, E. B., Boissonnas, R . A., and du Vigneaud, V. (1950). J. Biol. Chem. 183, 627. 461. Kendall, E. C. (1915). Trans. Assoc. Am. Physicians 30,420; Collected Papers Mayo Clin. and Mayo Foundation 7,393. 462. Kendall, E. C. (1919). J. Biol. Chem. 39, 125. 463. Kendall, E. C., and Osterberg, A. E. (1919). J. Biol. Chem. 40,265. 463a. Kenten, R. h. (1955). Biochem. J. 69, 110. 464. Kenten, R. H., andMann, P. J. G. (1952). Biochem. J. 60,360. 465. Kern, M., and Racker, E. (1954). Arch. Biochem. and Biophys. 48,235. 466. Kertesz, D. (1953). Bull. S O C . chim. biol. 36, 1157. 467. Kikkawa, H. (1953). Advances in Genet. 6, 107. 468. Kikkawa, H., Ogita, Z., and Fujito, S. (1954). PTOC. J a p a n Acad. SO, 30; (1955). Science 121, 43. 469. Kilby, B. A. (1951). Biochem. J . 49,671. 470. King, K. W., Wilson, D. G., and Burris, R. H. (1953). Federation Proc. 12,230. 471. Kirberger, E., and Bticher, T. (1952). Biochim. et. Biophys. Acta 8, 294. 472. Kjaer, A,, and Rubinstein, K . (1954). Acta Chem. Scand. 8, 598. 473. Kleinzeller, A., Burger, M., and Kubie, G. (1952). Chem. Listy 46, 171. 474. Kleinzeller, A., Burger, M., and Kubie, G. (1952). Chem. Listy 46, 474. 475. Kleinzeller, A., and Kubie, G. (1952). Chem. Listy 46, 106. 476. Klitgaard, H. M., Lipner, H. J., Barker, S. B., and Winnick, T. (1953). Endocrinology 62, 79. 477. Knox, W. E. (1946). J . Bioo. Chem. 163, 699. 478. Knox, W. E. (1951). Brit. J. Exptl. Pathol. 32,462. 479. Knox, W. E. (1952). Federation Proc. 11,240. 480. Knox, W. E. (1953). Biochem. J. 63, 379. 481. Knox, W. E. (1954). Biochim. et. Biophys. Acta 14, 117. 482. Knox, W. E. (1955). I n “Methods in Enzymology” ( N . 0. Kaplan arid H. Colowick, eds.), Vol. 11, p. 242. Academic Press, New York. 483. Knox, W. E. (1954). Private communication, and cf. ref. 482. 484. Knox, W. E., and Auerbach, V. H. (1955). J. B i d . Chem. 214,307. 485. Knox, W. E., and Edwards, S. W. (1954). Federation Proc. 13, 242; and c f . “Symposium on Glutathione” (S. Colowick et d., eds.), p. 183. Academic Press, New York. 486. Knox, W. E., and Edwards, S. W. (1954). Abstr. 126th meeting Am. Chem. Chem. SOC.,Kansas City, p. 21C; J . Biol.Chem. In press. 487. Knox, W. E., and Grossman, W. 1. (1946). J . Biol. Chem. 166,391 488. Knox, W. E., and Grossman, W. I. (1947). J . Biol.Chew. 168, 363. 489. Knox, W. E . , and Le May-Knox, M. (1951). Biochem. J . 49, 686.

METABOLISM O F THE AROMATIC AMINO ACIDS

137

490. Knox, W. E., andMehler, A. H. (1950). J. Biol. Chem. 187,419. 491. Knox, W. E., and Mehler, A . H. (1951). Science 113, 237. 492. Knox, W. E., Nero, K., Grossman, W. I., and Auerbach, V . H. (1949). Federation Proc. 8, 455. 493. Kodicek, E. (1951). Ann. Repts. Progr. Chem. Chem. SOC.London 48,276. 494. Kodicek, E., Carpenter, K . J., and Harris, L. J. (1946). Lancet ii, 491. 495. Koessler, K. K., and Hanke, M. T. (1922). J. Biol. Chem. 60, 131; (1924). 69, 835, 855. 496. Kogl, F., Haagen-Smit, A. J., and Erxleben, H. (1934). 2. physio2. Chenz. 228, 90. 497. Kogl, F., Haagen-Smit, A. J., and Erxleben, H. (1934). 2.physiol. Chem. 228, 104. 498. Kogl, F., and Kostermans, D. G. F. R. (1934). 2.physiol. Chem. 228, 113. 499. Kotake, M., Sakan, T., and Miwa, T. (1950). J. Am. Chem. SOC.72, 5085. 500. Kotake, M., Sakan, T., and Senoh, S. (1951). J . Am. Chem. SOC.73, 1832. 501. Kotake, Y. (1939). J. Chem. SOC.(Japan) 60, 632. 502. Kotake, Y., Chikano, M., and Ichihara, K. (1925). 2.physiol. Chem. 143,218. 503. Kotake, Y., Jr., and Inada, T. (1952). Proc. Japan Acad. 28, 68; (1953). J. Biochem . Japan 40, 291. 504. Kotake, Y., Jr., and Inada, T. (1954). J . Biochem. Japan 41, 263. 505. Kotake, Y., Jr., Inada, T., and Matsumura, Y. (1954). J . Biochem. Japan 41, 255. 506. Kotake, Y., Jr., Inada, T., and Matsumura, Y. (1954). Proc. Japan Acad. 30, 626. 507. Kotake, Y., and Iwao, J. (1931). 2.physiol. Chem. 196,139. 508. Kotake, Y., Jr., and Kamada, J. (1954). Proc. Japan Acad. 30, 122. 509. Kotake, Y., Kotake, Y., Jr., Hishikawa, M., Sakan, T., and Yamaguchi, M. (1953). Proc. Japan Acad. 29,143. 510. Kotake, Y., Kotake, Y., Jr., and Inouye, A. (1954). Proc. Japan. Acad. 30, 36. 511. Kotake, Y., Masai, Y., and Mori, Y. (1922). 2.physio2. Chem. 122,195. 512. Kotake, Y., and Nakayama, Y. (1941). 2.physiol. Chem. 270, 76. 513. Kotake, Y., Jr., and Nogami, K. (1954). Proc. Japan Acad. 30, 492. 514. Kotake, Y., and Shirai, Y. (1953). 2.physiol. Chem. 296, 160. 515. Kotake, Y., Jr., and Tani, S. (1952). Proc. Japan Acad. 28, 596; (1955) J . Biochem. Japan 40, 295. 516. Krehl, W. A , , Bonner, D., and Yanofsky, C. (1950). J . Nutrition 41, 159. 517. Krehl, W. A,, Henderson, L . M., de la Huerga, J., and Elvehjem, C. A. (1946). J. Biol. Chem. 166, 531. 518. Krehl, W. A., Teply, L. J., Sarma, P. S., and Elvehjem, C. A. (1945). Science 101, 489. 519. Kring, J. P., Ebisuzaki, K., Williams, J. N., Jr., and Elvehjem, C. A. (1952). J . Biol.('hem. 196, 591. 520. Kring, J. P., and Williams, J. N., Jr. (1954). J. B i d . Chem. 207, 851. 521. Kulescha, Z., and Gautheret, R. (1951). Compt. rend. S O C . biol. 146,245. 522. La Du, H . N. (1952). Federation Proc. 11, 244. 523. La Du, B. Y . , and Greenberg, D. M. (1951). J . Biol. Chem. 190, 245. 524. La n u , 13. N., and Greenberg, D. M. (1953). Science 117, 111. 525. La Du, 13. S . , and Zannoni, V. (1954). Federation Proc. 13, 246. 526. La Forge, F. B., and Barthel, W. F. (1944). J . Org. Chern. 9,250.

138

C. E. DALGLIESH

I,:myar, 11'. (1942). Z. physiol. C'hein. 276,225. Lanyar, F. (1943). Z. physiol. Chem. 278, 155. Lanyar, F. (1953). 2.physiol. Cheni. 293, 32. Larsen, 1'. (1951). Ann. Rev. Plant Physiol. 2, 176. Laxer, G . , Sikorski, J., Whewell, C. S., and Woods, H. J. (1954). Biochint. et Biophys. Acta 16, 174. 532. Laxer, G., and Whewell, C. S. (1954). ('heinisfry & Industry, p. 127. 533. I,eder, I. G., and Handler, P. (1951). J. Biol.Chem. 189, 889. 534. Lederer, 12. (1940). Biol. Revs. 16,273. 535. Lederer, I?. (1949). J. Chew/. Sac., p. 2115. 535a. Lee, N. D. (1955). Federation Proc. 14, 242. 536. Lee, N. D., and Williams, R. H. (1952). Biochivr. et Biophys. Acta 9.698. 537. Lee, X . D., and Williams, R. H. (1953). J . Biol.('hem. 204,477. 538. Leete, I<., Kirkwood, S.,and Marion, I,. (1952). Can. J. Chem. 30, 749. 539. Leete, I<., sntl Marion, 1,. (1953). Can. J. Chein. 31, 1195. 540. Leete, Ti;., :ind Marion, I,. (1954). Can. J . Chem. 32,646. 541. Leifcr, I<,,I,angham, W. H., Nyc, J. F., and Mitchell, H. K . , (1950). J. Biol. ('hem. 184, 589. 512. Leifer, E., ltoth, I,. J., Hogness, D. S., and Corson, M. 1-1. (1951). J . Biol. ('hein. 190, 595. 543. Le May-Iinox, M., and Rnox, W. E. (1951). Biochein. J . 48, proc. xxii. 514. Lembeok, F. (1953). .Vatwe 172, 910. 545. ternbeck, F. (1954). ~niinyn-Schnriedebe,.g'sArch. exptl. I'athol. 71 phar.~rakof. 221, 50. 546. I,eonhartli, G. (1952). Klin. Wochschr. 30, 168. 547. Leonhardi, C . (1953). Saturwissenschaften 40, 621. 548. Leonhardi, G. (1954). Naturwissenschaften 41, 141, 305. 548a. Leonhardi, G. (1955). Naturwissenschaften 42, 17. 549. Leonh:trtii, G . , Glasenapp, I. von, and Felix, K. (1950). Z. physiol. ('hem. 286, 19. 550. I,epkovsky, S., arid Nielsen, B. (1942). J . Biol.Chem. 144, 135. 551. Lepkovsky, S., Roboz, E., and Haagen-Smit, A. J. (1943). J . Rial. ('hem. 149, 195. 552. Lerner, A . 13. (1949). J. Biol. Cheni. 181. 281. 553. T,erner, A . 13. (1958). Advances in Enzymol. 14, 73. 554. I.erner, A . B,, and Fibpatrick, T. 13. (1950). Physiol. Revs. 30, 91. 555. J.evine, S.Z . , Mtirples, E., and Gordon, H. TI. (1939). Science 90, 620. 556. Levine, S.Z., Marples, E., and Gordon, H. H. (1941). J . C'lin. Invest. 20, 199. 557. I,evine, S. Z., Rlarples, E., and Gordon, H. €I. (1941). J. Clin. Invest. 20, 200. 558. Iiebig, J. (1846). , I n n . 67, 127. 559. Iietjig, J . (1853). Ann. 86, 125. 560. l i e n , 0.( i . , and Grcenberg, I). hl. (1952). .I. Niol. (:hem. 196, 637. 561. I h , P - l l . , and Johnson, 13. C . (1953). .I. Ant. C'hem. Soc. 76, 2974. 562. Ihi g, C. T . , IIegsted, D. &I.,and Stare, 17. J. (1948). J . H i o l . V h m . 174, 803. 563. I,innell, I,., and Rnper, 11. 8 . (1935). Biochevr. J . 29, 76. 564. Long, (!. I , . , Hill, l€.N., Weinstock, I. M., arid IIenderson, I,. hl. (1954). J . Hid.( ' h e u i . 211, 405. 5 6 4 ~Imigenecker, J. B., rind Snell, I<. 13. (1955). -1. Hiol. Chenr. 213, 229. 565. I,uck\rill, I,. C . (1952). 'Valure 169, 375. 566. r,ud\rig, W., a n d Mhlut.zenhecher,P. von, (1939). Z. physiol. Chem. 268, 195.

527. 528. 529. 530. 531.

METABOLISM O F THE AROMATIC AMINO ACIDS

139

567. I,yncn, F , :ind Wieland, U. (1937). Ann. 633, 93. 568. h‘laedonald, n. I,‘, Stanier, R.Y , and Ingraham, d. I,. (1954) J . Biol. Chetn. 210, 809. ,569 hlackenzie, C . G . , Chandler, J. P., Keller, E. B., Itachele, J. It., Cross, N . , and tlu Vigneaud, V . (1949). J. Biol. Chem. 160.99. 570. ~ I a c l a g a n S , . I<’.,and Sprott, W. 13. (1954). Lancet 267, 368. 571. blaclagan, N. F., and Wilkinson, J. H. (1954). J. Physiol. (London) 126, 405. 571a. Makino, K., and Arai, K. (1955). Science 121, 143. 572. hlakino, K., Itoh, F., and Nishi, K . (1951). Xature 167, 115. .573. Alakino, K., Satoh, K., Fujiki, T . , and Kawaguchi, I<. (1952). A-atzcre 170,977. 574. Makino, I<., and Takahashi, H. (1954). J. A m . Chem. SOC.76,4994. 575. Makino, K., Takahashi, H., Satoh, I<., and Inagami, I<. (1954). S a l w e 173, 586. 576. Mann, W., Leblond, C. P., and Warren, S. L. (1942). J. H i d . (‘hem. 142, 905. 577. Manske, R . H. (1954). J. Chem. Soc., p. 2987. 578. Marion, L. (1941). Rev. trimestr. can. 27, 342. 579. Mason, H. S. (1947). J. B i d . Chenz. 168,433. 580. Mason, €1. S. (1948). J . B i d . Chem. 172, 83. 581. Mason, H. S. (1953). I n “Pigment Cell Growth” (M. Gordon, ed.), p. 277. Academic Press, New York. 581a. Mason, H. S. (1955). Nature 176, 771. 582. blason, M. (1953). J . B i d . Chem. 201, 513. 583. Mason, M. (1954). Federation Proc. 13, 260. 583a. Mason, M. (1954). J. Bid. Chem. 211. 839. 584. Mason, M.,and Berg, C. 1’. (1951). J. B i d . Chem. 188, 783. 585. Illason, bl., and Berg, C. 1’. (1952). J. Biol. Cheni. 196, 515. 586. RIatchett, T. J., Marion, I,., and Kirkwood, 8. (1953). Can. J. (‘hem. 31, 488. 587. Matsuokn, Z., and Yoshimatsu, N . (1925). 2. physiol. Chem. 143, 206. 588. May, C . D., Hamilton, A,, and Stewart, C. T. (1952). Blood 7,978. 589. May, C. D., Nelson, E. N., and Salmon, R. J. (1949). J . Lab. C‘lin. Med. 34, 1724. 590. Medes, G . (1932). Biochem. J . 26, 917. 590a. Mehler, A. H. (1955). Federation Proc. 14, 254. 591. Mehler, A. H., and Knox, W. E. (1950). J . Biol. Cheni. 187.431. 592. bleister, A , , and Greenstein, J. 1.’ (1948). J . Biol. Chem. 176, 573. 593. hletzler, D. E., Ikawa, M., and Snell, E. E. (1954). J . Am. Chew. SOC.76,648. 594. hletzler, D. E., Longenecker, J. B., and Snell, E. $2. (1953). J . Am. Chcnr. Soc. 76, 2786. 595. Metzler, D. E., and Snell, E. E. (1952). J. Biol.Chenb. 198,363. 596. Meunier, P., Zwingelstein, G., and Jouanneteau, J. (1953). B u l l . S O C . chinr. b i d . 36, 1189. 597. Meyer, F. (1901). Deut. Arch. klin. M e d . 70,443. 598. Milch, H., and Milch, R. A. (1951). J . Intern. ColZ. Surgeons 16, 669. 599. Miller, E. C., and Baumann, C. A. (1945). J . B i d . Chem. 167, 551. 600. Millcr, I. L., and Adelberg, E. A. (1953). J . B i d . Chem. 206,691. 601. Miller, I . I,., Tsuchida, M., and Adelberg, E. A. (1953). d . B i d . Chem. 203, 205. 602. hlitchell, H. Ii.,arid Lein, J. (1948). J. B i d . Cheni. 176,481. 603. Mit,chell, If. K., and Nyc, J. F. (1948). Proc. Null. Acad. Sci. ( U . S.) 34, 1. 604. Mitchell, H. K., Nyc, J. F., and Owen, R . D. (1948). J . B i d . Chem. 176, 433.

140

C. E. DALGLIESH

Mitoma, C., and Leeper, I,. C. (1954). Federation Proc. 13, 266. Mitsuhashi, S., and Davis, 13. U. (1954). Biochim ct Biophys. Acla 16, 54. Mitsuhashi, S., arid Davis, B. D. (1954). Biochina et Biophys. Acta 16, 268. Miyake, A., Bokmsn, A. H., and Schweigert, B. S. (1954); J . Biol. Chcm. 211, 391. 609. Moir, G. F. J., and Ralph, B. J . (1954). Chemistry & Industry, p. 1143. 610. Monsonyi, 1,. (1939). Presse mkd. 47, 708. 611. Morris, J. E., Harpur, E. R., and Goldbloom, A . (1950). J . (‘Zin. Invest. 29, 325. 612. Morton, M. E., and Chaikoff, I. L. (1943). J . Biol. (‘hem.147, I . 613. Moss, A. 11. (1941). J . B i d . Chem. 137, 739. 614. Moss, A. R., and Schoenheimer, R. (1940). J. Biol.V h c n t . 136, 415. 615. Munro, T. A. (1947). Ann. Eugenics 14,60. 616. Musajo, L. (1935). Atti reale. accad. nazl. Lincei 21, 368. 617. Musajo, L. (1937). Gaz. chim. ital. 67, 165, 171, 182. 618. Musajo, L. (1953). Bull. soc. chim. biol. 36, 707. 619. Musajo, L., Benassi, C. A., and Parpajola, A. (1953). Ricerca Sci. 23, 1407. 619a. Musajo, L., Benassi, C.A., and Parpajola, A. (1955). Nature 176, 855. 620. Musajo, L., Chiancone, F. M., Coppini, D., and Ginoulhiac, E. (1951). Bull. S O C . chim. biol. 33, 1292. 621. Musajo, L., and Coppini, D. (1951). Experientia 7, 20. 622. Musajo, L., Spada, A,, and Bulgarelli, E. (1950). Gaz. chim. ital. 80, 161. 623. Musajo, L., Spada, A , , and Casini, E. (1950). Gaz. chinz ital. 60, 171. 624. Musajo, L., Spada, A., and Coppini, D. (1952). J . B i d . (‘hem. 106, 185. 624a. Mussett, M. V., and Pitt-Rivers, R. (1954). Lancet 267, 1212. 625. Mutzenbecher, P. von, (1939). 2. physiol. Cheni. 261, 253. 626. Nason, A. (1949). Science 109, 170. 627. Nason, A. (1950). A m . J. Botany 37,612. 628. Nason, A., Kaplan, N. O., and Colowick, S. P. (1951). J. Biol. Chem. 188,397. 629. Nason, A., Wosilait, W. D., and Terrell, A. J. (1954). Arch. Biochem. and Biophya. 48,233. 630. Nasset, E. S., and Ely, M. T. (1953). J. Nutrition 61,449. 631. Nasset, E. S., and Siliciano, A. M. (1952). J . Nutrition 47, 437. 632. Nencki, M. (1875). Ber. 8, 336. 633. Nencki, M. (1889). Monatsh. Chem. 10, 506. 634. Nencki, M., and Sieber, N. (1882). J . prakt. Chem. 26, 333. 636. Neubauer, 0. (1904). 2. physiol. Chem. 42,81. 636. Neubauer, 0. (1909). Deut. Arch. klin. Med. 96, 211. 631. Neubauer, 0. (1928). In “Handbuch der normalen und pathologischen Physiologie” (Bethe et aZ., eds.), Vol. 5, p. 851. Springer, Berlin. 638. Neubauer, O., and Falta, W. (1904). 2. physiol. Chem. 42,81. 639. Neuberg, C., and Schwenck, E. (1917). Biochenr. 2.79,387. 640. Neuberger, A. (1948). Advances i n Protein Cheni. 4, 297. 641. Neuberger, A. (1948). Biochem. J . 43, 599. 642. Neuberger, A. (1949). Ann. Rev. Biochem. 18, 243. 643. Neuberger, A., Rimington, C., and Wilson, J. M. G . (1947). Biochem. J . 41, 438. 644. Neuberger, A., and Webster, T. A. (1947). Biochem. J. 41, 449. 645. Neumeister, R. (1890). 2. B i d . 26, 324. 646. Nichol, C. A., and Welch, A. D. (1950). Proc. SOC.Exptl. Biol. Med. 74.52.

05. 606. 607. 608.

METABOLISM O F THE AROMATIC AMINO ACIDS

141

647. Nicolai, H. (1942). K l i n . Wochschr. 21, 538. 648. Niemann, C. (1950). Fortschr. Chem. org. Naturstojtfe 7, 167. 648a. Nitowsky, H. M., Govan, C. D., Jr., and Gordon, H. H. (1953). Am. J . Diseases Children 86, 462. 649. Novello, N. J., Wolf, W., and Sherwin, C. P. (1925). A m . J . M e d . Sci. 170,888. 650. Nyc. J. F., Haskins, F. A,, andMitchel1, H. K. (1949). Arch. Biochem. 23,162. 651. Nyc, J. F., andMitchel1, €1. K. (1948). J. Ani. Chevn. SOC.70, 1847. 652. Nyc, J. F., Mitchell, H. I<., Leifer, E., and Langham, W. H. (1949). J . Biol (‘herti. 179, 783. 653. Oesterling, M. J., and Rose, W. (2. (1952). J . B i d . C h e v i . 196.33. 654. Ohigashi, K., Tsunetoshi, .4., Uchida, RI., and Ichihara, I. (1952). J . Uioche9ri. J a p a n 39, 211. 655. Okn, Y . (1954). J . Biochem. J a p a n 41, 89, 101. 655a. Olson, R. A. (1955). Physiol. ZZevs. 36, 57. 656. Ord, W. If. (1878). B e d . klin. Il’ochsclrr. 16, 365. 657. Osborne, W. A. (1903). J . Physiol. (London) 29, 13. 658. Oshima, A. (1940). J . OsakaMed. Assoc. 39, 1523. 659. Pacheco, H. (1953). Compt. rend. 237, 110. 660. Page, I. If. (1954). Physiol. Revs. 34,563. 661. Painter, H. A., and Zilva, S. S. (1950). Biochem. J. 46,542. 662. Papageorge, E., and Lewis, H. B. (1938). J. Biol. Chem. 123,211. 663. Partridge, C. W. H., Bonner, D. M., and Yanofsky, C. (1952). J. Biol. Chein. 194, 269. 663a. Penrose, I,. S. (1935). Lancet 229, 192. 664. Penrose, 1,. S., and Quastel, J. H. (1937). Bioclievi. J . 31,266. 665. Perlman, I., Chaikoff, I. I,., and Morton, M. 15. (1941). J. Biol. Chenz. 139, 433. 666. Perlman, I., Morton, M. E., and Chaikoff, I. I,. (1941). J. Biol. Chem. 139, 449. 667. Perlzweig, W. A,, Rosen, F., Levitas, N., and Robinson, J. (1947). J . Biol. Chem. 167, 511. 668. Perlsweig, W. A., Rosen, F., and Pearson, P. B. (1950). J. Nutrition 40, 453. 669. Pictet, A. (1906). Arch. Pharm. 244, 389. 670. Pitt-Rivers, R. V. (1948). Biochent. J. 43, 223. 671. Pitt-Rivers, R. V. (1948). Nature 161, 308 672. Pitt-Rivers, R. V. (1950). Biochem. SOC.Syniposzu, No.6,55. 673. Pitt-Rivers, R. V. (1950). Physiol. Revs. 30, 194. 674. Porter, C. C., Clark, I., and Silber, R. H. (1947). ./. Biol. Chem. 167,573. 675. Porter, C. C., Clark, I., and Silber, R. €1. (1948). drch. Biochem. 18, 339. 675a. Price, J. M., and Lindenblad, G. E. (1955). Federation Proc. 14, 264. 676. Priest, R. E., Bokman, A. H., and Schweigert, B. S. (1951). Proc. Soc. Exptl. Biol. Med. 78, 477. 677. Pryor, M. G. M. (1940). Proc. Roy. SOC.Bl28,378. 678. Pryor, M. G. M. (1955). Nature 176, 600. 679. Pryor, M. G. M., Russell, P. B., andTodd, A. R. (1946). Biochem. J. 40,627. 680. Pryor, M. G. M., Russell, P. B., and Todd, A. R. (1947). Nature 169,399. 681. Pugh, C. E. M., and Quastel, J. H. (1937). Biochem. J. 31,2306. 682. Pugh, C. E. M., and Raper, H. S. (1927). Biochem. J. 21, 1370. 683. Pullmann, M. E., and Colowick, S. P. (1954). J . Biol. Chenz. 206, 121. 684. Pummerer, R., and co-workers (1914-1926). Numerous papers in B e r .

142

C . E. DALGLIESH

685. Quttgliarello, E . , and della l’ietra, G . (1954). Boll. sac. ital. biol. sper. 30, 374, 377. 686. Itacker, I<., cmd Krimsky, I. (1952). J . Biol.Chent. 198, 731. 686a. Itafelson, M. 1C. (1955). J. Biol. Chem. 212, 953; 213, 479. 686b. Itafelson, M . 15., EhrensvBrd, G., Bashford, M., Reio, L.,Saluste, E., arid IIedeti, C . G . (1954). 1.R,iol. (,’/Letti. 211, 725. 687. Italph, 1%.J., : ~ i i dItobertson, A. (1950). ./. (’hen,. Soc., p. 3380. 688. Raper, 11. S. (1926). Biochem. J . 20, 735. 689. Raper, 13. S . (1927). Biocheni. J. 21, 89. 690. Raper, H . S. (1932). Biocheni. J . 26, 2000. 6!31. Raper, 11. S. (1938). J. Chent. Soc., p. 125. 692. Itapport, R l . R f . (1949). .J. 13iol. Cheiti. 180, 061. 693. Rapport, 3’1. M., Green, A . A . , and Page, I . I-[. (1948). J . Biol. (‘hem. 174, 735; 176, 1237, 1243. 694. lt:tpport,, RI. RI., Green, A . A . , and Page, I. H. (1948). Sczcnce 108,320. 695. Ravdiri, It. G., and Cranddl, D. I . (1950). Federation Proc., 9, 218. 696. Reddi, K. K , , and Kodicek, E. (1953). Biocheni. J. 63, 286. G97. Itedemanii, C . T., Wittwer, S. H., and Sell, H. M. (1951). Arch. Biocheni. and Biophys. 32, 80. G98. Reid, D. F., Lepkovsky, S., Bonner, D. M., and Tatum, E. 1,. (1944). J . Biol. Chem 166, 209. 699. Reid, G., and Rand, M. (1951). Austrialian J . Exptl. B i d . M e d . Sci. 29, 401; (1952). Sattire 169, 801. 700. Reinits, I<. 14;. (1950). J. Biol.Cheni. 182, 11. 701. Reti, I,. (1950). Fortschr. Chern. org. Natiwstofle 6 , 242. 702. Ithul:md, I,. E., and Bard, R. C. (1952). J. Bacfeyiol. 63, 133. 703. Riggs, D. 6 . (1952). Pharniacol. Revs. 4, 284. 704. Riggs, T. It., Coyne, B. A., and Christensen, H. N . (1954). Federation f’roc. 13, 280. 705. Rimington, C . (1946). Biochetn. J . 40,669. 706. Ringer, A. J., and I m k , G. (1910). 2. physiol. Cheni. 66,106. 707. Robinson, It. (1917). J . Cheni. Sac. 111,876. 708. Robinson, It. (1934). 9th Congr. intern. Quinz. pura applicada. Madrid, 6 , 17. 709. Robinson, R . (1936). J. Chem. Sac., p. 1079. 710. Robinson, R . (1948). Nature 162, 524. 711. Robinson, R . (1949). f s t Intern. Congr. Biochein., Cambridge, Report of Lectures. p. 32. 712. Robinson, It. (1952). Bull. A’orld Health. Org. 6 , 207. 713. Roche, J. (1952). Experientia 8 , 45. 714. Roche, J., Lissitsky, S., and Michel, R. (1952). Conipt. rend. 234,997. 715. Roche, J., Iissitsky, S., and Michel, R . (1952). Compt. rend. 234, 1228. 716. Roche, J., Jhsitsky, S., and Michel, R . (1952). Compt. rend. sac. biol. 146, 1474. 717. Roche, J., Lissitsky, S., and Michel, R. (1953). Biochim. el Biophys. Acta 11, 220. 718. Roche, J. Michel, O., Michel, R., Gorbman, A., and Lissitzky, S. (1953). Biochini. et Biophys. Acta 12, 570. 719. Roche, J.,arid Michel, R . (1951). Advances i n Protein Chem. 6,253. 720. Roche, J., Michel, R . , Michel, O., and Iissitaky, S. (1952). Biochirn. e t Riopays. cia g, 161.

METABOLISM O F THE AROMATIC AMINO ACIDS

143

720a. Roche, J., Michel, R., Nunez, J., and Lacombe, It. (1955). Conipt. rend. 240, 464. 721. Roche, J., Michel, R.,and Tata, J. (1953). Bzochirn. ~1 Haophys ilctn 11, 543. i21a. Roche, J., RIichel, R , and Tata, .J. (1954) Riorhinr. el Bzophys. Acla 16, 500. -, 12,. ‘, Itotlney, G., Swendseid, M. E., and Sw:tiison, A . I,. (19l!)) .I. H ~ o l Chcnr. . 179, 18.

Kogeru, W.F., and Gardner, F. €1. (1949). J . hub. (‘lin. M e d . 34,1491. Roka, L. (1952). Abstr. 2nd Intern. Congr. Bioehenz., Paris, p. 302. Rose, W. C. (1937). Science 86, 298. Rose, W. C. (1938). Physiol. Revs. 18, 109. Rose, W. C. (1949). Federation Proc. 8, 546. Rose, W. C., Ilaines, W. J., and Warner, D. T. (1954). J. Biol. Chem. 206, 421. 729. Rose, W. C., Wartier, D. T., and Haiiieu, W. J. (1951). .1. Biol. Chetu. 193, 613. 730. Rose, W. C., and Womack, L. (1946). J. B i d . (’hem. 166, 103. 731. ltosen, F., Huff, J. W., and Perlzweig, W. A. (1946). J . Biol. Chem. 163, 343. 732. Itosen, F., Huff, J. W., and Perlzweig, W. A. (1947). J. Nutrition 33,561. 732a. Rose, W. C., Lambert, C. F., and Coon, M. J. (1954). J. Biol. Chem. 211,815. 73%. Rose, W. C., Leach, B. E., Coon, M. J., and Lambert, G. F. (1955). .I. Biol. Chem. 213, 913. 733. Rosen, F , I,owy, R. S., and Sprince, €1. (1951). Proc. SOC.E z p l l . Biol. Med. 77, 399. 734. Rosen, F., arid Perlaneig, W. A. (1947). Arch. Biochenr. 16, 111. 735. Rosenblueth, A. (1937). Physiol. Revs. 17, 514. 736. Rosin, H. (1891). Virchow’s Arch. pathol. Anat. u . Physiol. 123, 519. 737. Rosin, H. (1893). Deut. med. Il’ochschr. 19, 519. 738. Roth, L. J , Leifer, E., Hogness, J. R., and Langham, W. H. (1948). J. Biol. (’hem. 176, 249. 739. Rudman, D., and Meister, A. (1953). J . Biol. (‘hem. 200,501. 740. Russell, A. (1948) J.Am. Chem. SOC.70, 1060. 741. Rydon, H. N . (1948). Brit. J. E x p t . Pathol. 29, 48. 742. Sakamoto, Y., Uchida, M., and Ichihara, K. (1953). dled. J . Osaka U n i v . 3, 47.7 713. Sakan, T., and Hayaishi, 0. (1950). -1.Biol.C‘hem. 186, 177. 744. Salnmon, I. I., and Davis, B. D. (1953). J. Am. Chem. Soc. 76, 5567. 745. Salkonski, E., and Salkowski, H. (1884). 2.physiol. Chem. 9, 8. 746. Salko\rski, I<., and Salkowski, H. (1885). 2. physiol. Chem. 9,491. 747. Salmon, 11.J., andMay, C. D . (1950). J. Lab. Clin.Med. 36,591. 718 Salmon, It. J , and May, C. D. (1951). Arch. Biochenz. and Biophys 32, 220. 749. Salmon, R . J , andMay, C. D. (1953). J . Lab. Clin. M e d . 41,376. 750. Salmon, W. D. (1954). J. Nutrition 61,30. 751. Salter, W T. (1950). In “The Hormones” (G. Pincus and K. 1’. Thimann, eds.), Yol. II., Chapter 4. Academic Press, New York. 752. Sanadi, J. It., and Greenberg, D. hi. (1950). Arch. Biochem. 26, 257. 753. Sarrett, H . P. (1948). Pror. Soc. E x p t l . B ? d . Med. 69, 394. 751. S:irett, €1.1’. (1951). J . Biol Chcm. 193, 627. 755. Sarett, H. P., and Goldsmith, G. A. (1947). J. Biol. (”hem. 167, 293. 756. Sasaki, T., and Otsuka, J. (1913). Deut. w e d . B70chschr. 39, 159. i23. 724. 725. 72G. 727. 728.

144

C. E. DALGLIESH

757. Sasaki, T., and Otsuka, J. (1921). Biochem. 2.121, 167. 757a. Sauberlich, H. E., and Salmon, W. D. (1955). J. Biol. Chem. 214, 463. 758. Schales, O., and Schales, S. S. (1949). Arch. Biochem. 24, 83. 759. Schayer, It. W. (1950). J . Biol.Chem. 187, 777. 760. Schayer, R. W. (1951). J. Biol. Chem. 189,301. 761. Schayer, R. W. (1951). J. BioZ. Chem. 192, 875. 762. Schayer, R. W. (1953). Proc. SOC.Exptl. Biol. Med. 84,60. 763. Schayer, R. W., and Henderson, L. M. (1952). J. Biol.Chem. 196,657. 764. Schayer, R. W., Kennedy, J., and Smiley, R. L. (1953). J . Biol. Chrm. 202, 39. 765. Schayer, R. W., and Smiley, R. L. (1953). J. Biol. Chem. 202,425. 766. Schayer, R. W., Smiley, R . L., and Kaplan, E. H. (1952). J. Biol. Chent. 198, 545. 767. Schayer, R . W., Wu, K. Y. T., Smiley, R. L., and Kobayashi, Y. (1954). J. Biol. Chem. 210, 259. 768. Scheer, B. T., and Deuel, H. J. (1948). J. Nutrition 36,239. 769. Schepartz, B. (1951). J. Biol. Chem. 193,293. 770. Schepartz, B. (1953). Federation Proc. 12, 265. 771. Schepartz, B. (1953). J . Biol. Chem. 206, 185. 772. Schepartz, B., and Gurin, S. (1949). Federation Proc. 8,248. 773. Schepartz, B., and Gurin, S. (1949). J. Biol. Chem. 180,663. 774. Schoenheimer, R., Ratner, S., and Rittenberg, D. (1939). J. Biot. Chem. 127, 333. 775. Schopf, C. (1937). 2. angew. Chem. 60,787. 776. Schopf, C., and Bayerle, C. (1934). Ann. 613, 190. 776a. Schreier, K., Altman, K. I., and Hempelman, L. H. (1954). Proc. SOC. Exptl. Biol. Med. 87, 61. 777. Schuler, W., Bernhard, H., and Reindel, W. (1936). 2. physiol. Chem. 243,90. 778. Schuler, W., and Wiedemann, A. (1935). 2.physiol. Chem. 233, 235. 779. Schulze, E., and Barbieri, J. (1879). Ber. 12, 1924. 780. Schulze, E., and Barbieri, J. (1881). Ber. 14, 1785. 781. Schulze, E., and Barbieri, J. (1883). J. prakt. Chem. 136, 337. 782. Schweigert, B. S., German, H. L., and Gardner, M. J. (1948). J. Biol. Chem. 174. 383. 783. Schweigert, B. S., and Paneer, F. (1947). J. Biol. Chem. 168,283. 784. Schweigert, B. S., and Pearson, P. B. (1947). J. Biol. Chem. 168,555. 785. Schweigert, B. S., Sauberlich, H . E., Baumann, C. A., and Elvehjem, C. A. (1946). Arch. Biochem. 10, 1 786. Scribney, M., and Kirkwood, S. (1954). Can. J. Chem. 32,918. 787. Sealock, R. R. (1942). J. Biol. Chem. 146,503. 788. Sealock, R. R., Galdston, M., and Steele, J. M. (1940). Proc. SOC.Exptl. Biol. Med. 44, 580. 789. Sealock, R. R., and Goodland, R. L. (1949). J. Biol. Chem. 178, 939. 790. Sealock, R. R., and Goodland, R. 1,. (1951). Science 114,645. 791. Sealock, R. R., Goodland, R. L., Sumerwell, W. N., and Brierley, J. M. (1952). J . Biol. Chem. 196,261. 792. Sealock, R. R., Perkinson, J. D., and Basinski, D. H. (1941). J . B i d . Chem. 140, 153. 793. Sealook, R. R., and Silberstein, H. E. (1939). Science 90,517. 794. Sealock, R. R., and Silberstein, H. E. (1940). J. B i d . Chem. 136,261. 795. Senoh, S., and Sakan, T. (1952). J. Inst. Polytech. Osaka Citg Uniu. Ser. C 3, 53 (Nov.).

METABOLISM O F THE AROMATIC AMINO ACIDS

145

796. Senoh, S., Seki, T., and Kikkawa, I€. (1952). J. Inst. Polytech., Osaka City Univ. Ser. C 3, 59 (Nov.). 797. Seraydarian, M. W., Cohen, A. I., and Sable, H. Z. (1953). A m . J . Physiol. 177, 150. 798. Shanmugasundaram, E. R. B., and Sarma, P. S. (1954). J . Sci. I n d . Research ( I n d i a ) 13B, 21. 799. Shanmugasundaram, E. R. B., Tirunarayanan, M.O . , and Sarma, P. S. (1953). Current Sci. 22, 211. 800. Shanmugasundaram, E. R. B., Tirunarayunan, M. O . , and Sarma, P. S. (1954). Biochem. J. 68,469. 801. Shepherd, D. M., and West, G. B. (1952). J. Pharin. and Pharmacol. 4,671. 802. Shepherd, D. M., and West, G. B. (1953). J . Physiol. (London) 120, 15. 803. Shepherd, D. M., West, G . R., and Erspamer, V. (1953). Nature 172, 508. 804. Shiguera, H., and Sprinson, D . R. (1952). Federation Proc. 11,286. 805. Shiguera, H., Sprinson, D. B., and Davis, €3. D. (1953). Federation Proc. 12, 458. 805a. Shiguera, H. T., Srinivasan, P. R., Sprecher, M., Sprinson, D. B., and Davis, B. D. Unpublished observations. 805b. Siegel, S. M., and Galston, A. W. (1955). Arch. Biochenz. and B i o p h p . 64, 102. 806. Simic, B. S., Sinclair, H. M., and Lloyd, 13. B. (1953). Intern. Rev. T'ititamin Research 26, 7. 807. Simmonds, S. (1950). J. Biol. Chem. 186, 755. 808. Simmonds, S., Dowling, M. T., and Stone, D. (1954). J . Biol.Chem. 208, 701. 809. Simmonds, S., Tatum, E. L., and Fruton, J. S. (1947). J . Biol. Chem. 169,91. 810. Singal, S. A , , Briggs, A. P., Sydenstricker, V. P., and Littlejohn, J. M. (1946). J . Biol. Cheni. 166, 573. 811. Singal, S. A., Sydenstricker, V . P., and Littlejohn, J. RI. (1947). J . Biol. Chenz. 171, 203. 812. Singel, S. A,, Sydenstricker, V. P., and Littlejohn, J. M. (1953). Federation Proc. 12, 429. 812a. Singer, T. P., and Kearney, E. B. (1954). Advances zn Enzymol. 16,79. 813. Sistrom, W. R., and Stanier, R. Y. (1954). J . B i d . Chem. 210,821. 814. Sieer, I. W. (1953). Advances in Enzymol. 14, 129. 815. Skoog, F. (1937). J . Gen. Physiol. 20,311. 816. Snell, E. E. (1943). Arch. Biocheni. 2, 389. 816a. Snyderman, S. E., Norton, P., and Holt, L. E., ,Jr. (1955). Federation &ro'c. 14, 450. 817. Snyderman, S. I(;., Betrun, B.C., Carretero, It , :md Holt, L. I,. (1949). Proc. SOC.Exptl. B i d . Med. 70, 569. 818. Spacek, M. (1953). 1Yatitre 172, 204. 818a. Spacek, M. (1955). Can. J . Biochem. Physzol. 33,14. 819. Spiith, E., and Berger, F. (1930). Ber. 63, 2098. 820. Spector, H. (1938). J . Bid.Cheni. 173, 659. 821. Speeter, M. I<., Heinzelmann, R . V . , and Weisblntt, D. I. (1951). J . Am. Cheni. SOC.73, 5511. 822. Spicer, J. S., arid Strickland, J. D. H. (1952). J . ( ' / w n l . Soc., p. 4644. 823. Sprince, H., I,otvy, R. S.,Yolsome, c' E., :ind Behrmann, J. S. (1951). A m . J. Obstet. Gynecol. 62, 84. 823a. Srinivasan, P. R., Kalan, E., Sprinson, D. B., and Davis, B. D. Unpublished observations.

146

C. E. DALGLIESH

821. Srinivasan, 1’. It., Sprecher, hl., and Sprinson, D. B. (1954). Federation Proc. 13, 302. 825. Stanier, It. Y. (1952). 2nd Intern. Congr. Biochern., Paris. Symposium sur le hletabolisme Microbien, p. 64. 826. Stanier, R . Y., and Hayaishi, 0. (1951). Science 114,326. 827. Stanier, R. Y., and Hayaishi, 0. (1951). J . Bacterial. 62, 367. 828. Stanier, R. Y., Hayaishi, O., and Tsuchida, M. (1951). J . Bacteriol. 62, 355. 829. Stanier, It. Y., and Ingraham, J. L. (1954). J. B i d . Chem. 210,799. 830. Stanier, R . Y., andTsuchida, M. (1949). J. Bacterial. 68, 45. 831. Stein, G., and Swallow, A. J. (1954). Xature 173, 937. 832. Stein, W. H., Paladini, A. C., Hirs, C. €1. W . , and Moore, S. (1954). J . A m . Cheni. Soc., 76, 2848. 833. Stephenson, M. (1930). “Bacterial hletabolism.” Longmans, Green, New York. 834. Stolz, F. (1904). Ber. 37, 4140. 835. Stoppani, A. 0. M. (1945). J . Riol. C ‘ h e ~ i .167, 1. 836. Stowe, B. H . , Ray, P. M., and Thimann, I<. V. (1954). Cu7ntnitr~s.8th Z I L ~ C ~ I I . Congr. B o t a n y , P a r i s , 8, Section 11/12, 151. 837. Stowe, B. B., and Thimann, I<. V. (1953). AVature 172, 764. 838. Rtoae, B. B., and Thimann, K. 17. (1954). Arch. Biochenz. and B i o p h y s . 61, 499. 830. Stnmpf, 1’. K., and Green, D. E. (1944). J . Biol. Cheni. 163,387. 840. Suda, 31., and Takeda, Y. (1950). M e d . J . Osaka U n i v . 2, No. 1, 37. 841. Snda, M., and Takeda, Y. (1950). M e d . J. Osaka L’niv. 2, No. 1, 41. 8-12. Suda, M , , Takeda, T.,Sujishi, K., and Tanaka, T . (1951). M e d . .I. Osuha 1?niv. 2, No. 4, 79; J .Biochem. J a p a n 38,297. 843. Snndaram, T. I<., Radhakrishnamurty, It., and Sarma, P. 8. (1051). (’urrent S c i . 23, 92. 844. Sundaram, T. K., Rudhakrishnamurty, R . , Shanmmgnsundaram, €3. R. 13., :inti Sarma, P. S. (1953). Proc. Sac. E x p t . Rial. M e d . 84, 544. 845. Sundaram, T. K., and Sarma, P. S. (1953). X a t u r e 172, 627. 846. Swendseid, M. E., Burton, I. F., and Bethell, F. H. (1943). Proc. Sac. E x p t l . Biol. Msd. 62, 202. 817. Swendseid, M. R., Wandruff, B., and Bethell, F. €I. (1947). J . Lab. f ’ l i n . &fed. 32, 1212. 818. T:il)one, J., Tnhone, D., and Thomassey, S. (1951). Hicll. soc. chini. biol. 36, .565.

8-10, Teinter, 32. I,., and Ludueiia, F. 1’.

(1950). Recent Progt-. Hormone Reseurch 6 ,

3.

850. Taint,er, M . I,., Tullar, 13. F., and Luducfia, F. P. (1948). Science 107,39. 851. Takamine, J. (1901). J . Physiol. ( L o n d o n ) 27, proc. xxix; Am. J . Pharrn. 73, 523. 852. Tnkeda, Y., Sujishi, I<.,TI:tra, kl., andTanakz, T. (1952). ,!led. J. Osaka linizj. 3, 313. 853. Tatum, 11;. I,., arid Uonner, D. R.1. (1943). J . B i d . (‘he7n. 161,349. 854. Tatum, E. I,,, and Bonner, D. hI. (1944). Proc. Natl. A c a d . S c i . ( U S . ) 30.30. 855. Tatum, I<. I,., I\onner, D. h f . , and Beadle, G. W. (1943). -4rch. Biochem. 3, 177. 856. l‘:it.um, 13. I , . , Gross, S. I{., Iq:hrensviirtl, G., :ind Garnjobst, I,. (1954). I’roc.. S a i l . Acad S c i . 40, 271.

METABOLISM O F THE AXOMATIC AMINO ACIDS

147

857. Tatum, E. I,,, and IIaagen-Smit, A. J. (1941). J . Biol.Chem. 140, 575. 858. Tatuni, E. I,., and Shemin, L). (1954). J . B i d . C h e n ~209, 671. 859. Taurog, A Briggs, F.N., and Chaikoff, I. L. (1952). J. Biol. Chenz. 194, 655. 860. Taurog, A., Chaikoff, 1. L., andTong, W. (1949). J. Biol. Client. 178, 997. 860a. Taurog, A., Potter, G. D., and Chaikoff, I. L. (1955). J. Biol. Chem. 213, 119. 861. Taylor, I. F., Green, A. A., and Cori, G. T. (1948). J . Biol. Chem. 173, 591. 862. Teas, H. J., and Anderson, F,. G (1951). Proc. N a t l . Acad. S c i . ( U . 8.) 37, 645. 863. Teubner, F. G . (1953). Science 118, 418. 863a. Thibault, O., and Pitt-Rivers, It. (1955). Lancet 268,285. 864. Thimann, I<. V. (1935). J . Biol. Cheni. 109, 279. 865. Thimann, I<. V. (1953). Arch. Biocheni. and Biophys. 44, 242. 866. Thomas, R . C . , Cheldelin, V. I€., Christensen, B. E., and Sang, C. H. (1953). J . AWL.Chem. Sac., 76, 5554. 867. Thompson, C. M., Reber, E., Whitehair, C. I<.,and MacVicar, R. (1952). J . A n i m a l Sci. 11, 712. 868. Thomson, J. F., and Mikuta, E. T. (1954). Endocrinology 66, 232. 869. Thomson, J. F., and Mikuta, E. T. (1954). Proc. SOC.E x p t l . BioZ. Med. 86,29. 870. Thorson, A , Biorck, G., Bjorkman, G., and Walderistrom, J. (1954). A m . Heart J . 47, 795. 871. Titus, E., and Udenfriend, S. (1954). Federation Proc. 13, 411. 872. Toh, C. C. (1954). J. Physiol. (London) 126,248. 873. Tong, W., Taurog, A , , and Chaikoff, I. I,. (1951). J . Biol. Chem. 207,59. 874. Tsuchida, AT., Hayaishi, O., and Stanier, It. Y. (1952). .I. Bacterial. 64, 49. 875. Tullar, 13. I?. (1949). Science 109, 536. 876. Tyler, V. E., and Schwarting, A. F. (1953). Science 118, 132. 877. Uchida, M., Sakamoto, Y., and Ichihara, I(. (1953). Med. J . Osaka Univ. 3, 487. 878. Uchida, M., Suzuki, S., and Ichihara, K. (1954). J . Biocheni. J a p a n 41, 41. 879. Uchida, M., Takemoto, Y., Kakihara, Y., and Iehihara, K. (1953). Med. J . Osaka Unio. 3, 509. 880. Udenfriend, S., and Bessman, S. Y . (1953). J. B i d . C'hem. 203, 961. 881. Udenfriend, S., Clark, C. T . , Axelrod, J., and Brodie, B. B. (1952). Federation Proc. 11, 303. 882. Udenfriend, S., Clark, C. T., Axelrod, J., and Brodie, 13. B. (1954). J . B i d . Chcm. 208, 731. 883. Udenfriend, S., Clark, C . T., and Titus, E. (1952). Experientza 8, 379. 884. Udenfriend, S., Clark, C. T., and Titus, E. (1953). Federatian Proc., 12, 282. 885. Udenfriend, S., Clark, C. T., and Titus, E. (1953). J. Am. Chem. Sac. 76, 501. 886. Udenfriend, S., and Cooper, J. R. (1952). J. B i d . ('hem. 194, 503. 887. Udenfriend, S., and Cooper, J. R. (1953). J . Biol. Chem. 203,953. 888. Udenfriend, S., Cooper, J. R., Clark, C. T., and Raer, J. E. (1953). Science 117, 663. 889. Udenfriend, S., and Weissbach, H. (1954). Federation Proc. 13,412. 890. Umbreit, W. W., Wood, W. A , , and Gunsalus, I . C. (1946). J . B i d . Chem. 106, 731. 891. Vandelli, I. (1951). Acla Vitaminol. 6, 55. 892. Vialli, M., and Erspamer, V. (1933). 2. Zellforsch. u.niikroskop. A n a t . 19,743. 893. Vialli, M., and Erspamer, V. (1942). Arch. sci. b i d . ( I t a l y ) 28, 101,122. ~

148

C. E. DrlLGLIESH

891. Vickery, 11. II., :tnd Schmidt, C. I,. A. (1931). Chenr. Revs. 9, 169. 895. du Vigneaud, f r . , Senlock, R . R . , ant1 JSlten, C. van, (1932). J. Biol. Chcni. 98, 565. 896. Vinet, A. (1940j. Compt. rend. 210, 552. 897. Viollier, G., and Slillmann, H. (1950). Helv. Chini. Acta 33, 776. 898. Wachstein, M., and Gudaitis, 11. (1952). Am. J . Clin. Pathol. 22, 652. and I., Gudaitis, I€. (1952). J . Lab. Clin. Med. 40, 550. 899. Wachstein, & 900. Wachstein, M., and Gudaitis, H. (1953). J. Lab. Clin. Med. 42,98. 901. Wachstein, M., and T>obel,S. (1954). Proc. Sac. E s p t l . B i d . &fed. 86, 624. 802. Wakeman, A. J., andDakin, H. D. (1911). J . Rial. Cheni. 9, 139. 90.7.Waller, C. W . , Fryth, 1’. W., T-Iutchings, R. I,., and Willinnis, ,J. €1. (19.53). .I. :I )ti. Chew. SOC.76, 2025. 004. Ward, F. W. (1023). Biochem. J . 17,007. 905. Weinhouse, S., and Millington, R. H. (1948). J . Biol. (’hem. 176.995. 906. Weiss, U., Davis, B. D., and Mingioli, E. S. (1953). J. A m . Chem. Soe. 76, 5572. 907. Weiss, U., Gilvarg, C., Mingioli, E. S., and Davis, B. D. (1954). Science 119, 774. 008. Weitzel, G., Buddecke, E., Strecker, F. J., and Roester, U. (1951). 2. p h y i o l . Chem. 298, 169. 909. Werle, E., and Rower, F. (1950). Biochem. 2. 320, 298. 910. Wheeler, H. I,., and Jamieson, G. S. (1905). Anz. Chem. J . 33, 365. 911. White, J., St,ander, R. S., Dayton, A . , and David, P. (1953). Federntion I’roc. 12,289. 912. Wiame, J . M., and Storck, R. (1953). Biochinz. Biophys. Acta. 10, 268. 913. Wieland, H., Hesse, G., and Mithsch, H. (1931). Her. 64, 2090. 914. Wieland, H., Konz, W . , and Mittasch, H. (1934). Ann. 613, 1. 915. Wieland, H., and Vocke, F. (1930). Ann. 481, 215. !)16. Wieland, H., and Wieland, T. (1937). Ann. 628, 234. !)17. Wieland, H., and Witkop, B. (1940). Ann. 643, 171. 918. Wieland, 0. P., Hopp, R . S. de, and Avener, J. (1954). Natitre 173, 776. 919. Wieland, T., and Motzel, W. (1953). Ann. 681, 10. 920. Wieland, T . , and Schmidt, G. (1952). Ann. 677, 215. 921. Wildman, 8. G., Ferri, M. G . , and Ronner, J. (1947). Arch. Biochent. 13, 131. 922. Wildman, S. G., and Muir, It.M. (194!1). Plant Physiol. 24,84. 923. Wilkinson, ,J.€ I . , and Maclagan, N. F. (1954). Biochem. J. 68,87. 924. Williams, J . N., Jr., Feigelson, P., and Elvehjem, C. A . (1950). J . Biol.(‘hein. 187. 597. 925. Williams, J. N . , Jr., Feiyelson, I’., Shahinian, S. S., and Elvehjem, C. A . (1951). .I. Biol. Chem. 189. 659. 926. Williams, J. N., Jr., Feigelson, P., Shahinian, S. S., andElvehjem, C. A . (1951). Proe. SOC.E x p t l . Rial. Med. 76, 441. (327. Williams, J. N., Jr., and Sreenivasan, A. (1953). J . Biol. Chem. 203, 605. 928. Williams, J. X., J r . , and Sreenivasan, A. (1953). J. Biol. Chem. 203, 109. 029. Williams, J . N., Jr., and Sreenivasan, A. (1953). J . Biol. Chem. 203, 613. 930. Williams, R. T. (1949). “Detoxication Mechanisms.” Chapman and Hall,

London, 931. Wilson, D . G., and Burris, R. H. (1953). Federation Proc. 12, 230. 932. Wiltshire, C . €1. (1953). Biochem. J. 66,408. 9.73. Winnick, T . , Friedherg, F., and Greenberg, D. ill.(1948). J . Biol. (‘hem. 173, 189.

METABOLISM OF THE AROMATIC AMINO ACIDS

149

934. Winterstein, E., and Trier, G. (1910). “Die Alkaloide.” Borntraeger, Berlin. 935. Wintrobe, M. M. (1951). “Clinical Haematology,” 3rd ed., Chapter 5 . Kingston, London. 936. Wiss, 0. (1949). Helv. Chim. Acta 32, 1694. 937. wiss, 0 . (1952). 2 . hTatzirforsch. 7b, 133. 938. Wiss, 0. (1!)53). Z . physiol. Cheni. 293, 106. 93th. Wiss, 0. (1954). 2. Naturforsch. 8b, 740. 939. Wiss, O., niitl E’uchs, If. (1949). H e h . (’hirn. Acta. 32, 2553. 940. Wiss, O., uiid Fuchs, 11. (1050). h’xperientia 6, 472. 941. Wiss, O . , :md lIat,z, F. (1949). Hclv. (,’him. Acta 32, 532. 942. Wiss, O., and Ilellmanri, 11. (1953). %. Naturjorsch. 8b, 7 0 . 043. Wiss, O., Viollier, G., and Miiller, M. (1949). Helc. physiol. Pharmucol. A d a 7, C64. 944. Wiss, O., Violher, G . , arid hluller, M. (1950). Helv. (‘him. Acta 33, 771. 945. Wiss, O., Viollier, G., and Waldi, D. (1951). Helv. Physiol. Pharmacol. A d a . 9, C40. 946. Witkop, B., and Goodwin, S. (1952). Experientia 8 , 377. !)47. Witkop, B., and Gmser, G . (1944). Ann. 666, 103. 948. Wolf, F. T. (1952). Proc. Nat l . Acad. Sci . (U.S.)38, 106. 949. Wolkow, M. M., and Baumann, 13. A. G. (1891). Z. physiol. Chem. 16, 228. 950. Womack, M., and Rose, W. C. (1934). J . B i d . Chem. 107, 449. 951. Wood, W. A , , Gunsalus, I. C . , and Umbreit, W. W. (1947). .I. Biol. Chem. 170, 313. 952. Woodruff’, C . W., Cherrington, M. 15., Stjockell, A. K., arid Darby, W. J. (1949). J . B i d . Chem. 178, 861. 953. Woodruff, C . W., and Darby, W. J. (1948). J . Biol.(:hem. 172,851. 954. Woods, D. D. (1935). Biochem. J . 29, 640, 649. 955. Woodward, It. 13. (1948). Nature 162, 155. 956. Wooldridge, W. E., Mast, G. W., ttiid Hoffnxtn, RI. (1050). J . Lab. Ctin. Med. 36, 501. 957. Woolley, D. W. (1946). J. Biol. (’hem. 162, 179. 958. Woolley, D. W., and Shaw, E. (1954). Bri t . Med. J. ii, 122. 959. Woolley, D. W., and Shaw, E. (1954). Science 119,587. 960. Woolf, L. I. (1951). Biochem. J. 49, proc. ix. 961. Woolf, I,. I., and Edmunds, M. E. (1950). Biochem. J . 47, 630. 961a. Woolf, L. I., Griffiths, R., and Moncrieff, A. (1955). Brit.Med. J. i , 57. 962. Wretlind, I<. A. J. (1952). Acta Physiol. S c a d 26, 276. 963. Wu, $1. C . C . , and Sealock, R. It. (1953). Biochini ct Bzophys. A d a 44, 31%. 963a. Wyngaarden, J. B., and Stanbury, J. B. (1955). J. Biol. Chem. 212. 151. 064. Yamaki, T., and Makamura, K. (1952). Sci . Papers CoEE. Gen. Educ. L’jliiii. Tokyo, 2, No. 1, 81; Chem. Ahstr. 47, 2837h (1953). 964a. Yaniv, H., and Gilvarg, C. (1955). J . Biol. Chem. 213, 787. 965. Yanofsky, C . (1952). J. Biol. Chem. 194, 279. 966. Yanofsky, C . (1952). Proc. Nat l . Acad. Sci . (U.S.) 38,215. 967. Yanofsky, C. (1955). Biochim.et. Biophys. Acta 16,594; andprivatecommunication. 968. Yanofsky, C . (1954). J. Bacteriol. 68, 577. 969. Yanofsky, C. (1955). Science 121, 138. 970. Yanofsky, C . , and Bonner, D. M. (1950). Proc. N a t l . Acatl. Sci. ( U . S . ) 36, 167. 971. Yanofsky, C., and Bonner, D. M. (1951). J . B i d . Chenz. 190, 211.

150

C. E. DALGLIESH

972. Yoshida, Z-I,and Kato, M . (1954). J . A m . Chern. Soc. 76, 311. 973. Zatman, L. J., Kaplan, N. O., and Colowick, S.I-’. (1953). J.Biol. Chem. 200, 197. 974. Zatman, I,. J., Kaplan, N. O., Colowick, S.P., and Ciotti, M. ILI. (1954). J . Riot. Chem. 209, 453, 467. 975. Zilvu, 8 . S. (1935). Biochem. J . 29, 1612. 976. Zorn, I<. (1940). 2. physiol. f’hem. 266, 239. 977. Zucker, RI. 13., Friedmm, 13. K., and Rapport, RZ. RZ. (1954). I’roc. SOC.h’xptl. Biol. Aled. 86, 2x2. 978. Zucker, RI. U., ant1 Rapport, &I. b l . (1954). Federation Proc. 13, 170.

Hydrogen Ion Equilibria in Native and Denatured Proteins BY JACIETO STEISHARDT

.4 XI)

ISTHEI, > .! ZAISElt I

Department o j Chemistry, Massachusetts Institute Massachusetts

0j

Technology. Cambridge.

COXTENTS 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 I I . Acid-Rase Dissociations of Native Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 1 . Scope of This Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 2 . General Features of Protein Titration Curves. . . . . . . . . . . . . . . . . . . . . . . 153 3 . Theoretical Analysis of Titration Curves . . . . . . . . . . . . . . . . . . . . . . . . . 157 a . Outline of Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 6 . Comparison of t.lie Electrostatic Term w from Theory and 14xperi158 merit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c . Comparison of Salt Effect on Soluble and Insoluhle Proteins . . . . . . . 160 d . Intrinsic Dissociation Constants and Numbers of Groups . . . . . . . 164 4. Itelation of Stoichiometry t o Maxima in Binding and Dissociation of Protons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 5 . Stoichiometry itnd Differentiation of Prototropic Groups . . . . . . . . . . . . . . 166 a . Assignment of Segments of Titration Curve t o Specific Groups . . . . . . 166 b . Modification of Dissociation Constants of Groups . . . . . . . . . . . . . . . . . . . 168 c . Conversion t o Nonprototropic Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 (i. Heats of Ionization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 e . Spectrophotometric Titration of Tyrosine Phenoxyl Groups . . . . . . . . . 172 6 . Contribution of Ion-Protein Interactions t o Stoichiometry . . . . . . . . . . . . . 173 a . Stoichiometric Combinatiori . . . ................................ 173 b . Effect on Titration . . . . . . . . . . . . ............................. 174 7 . Effects of Less Specific Ion-Protein Combination on Titration and Electrophoresis of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 8 . Summary of Section I1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 111. Unrenctive Prototropic Groups in Native Proteins . . . . . . . . . . . . . . . . . . . . . . 180 180 1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Evidence for Unreactive e-Amino and Imidxzole Groups from EndGroup Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 3 . Titrimetric Criteria for Iiberat.ion of Unreactive Prototropic Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 4 . Early Titrimetric Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 5 . Unreactive Prototropic Groups in the Hemoglobins and Globins . . . . . . 186 a . Titrimet.ric Evidence for Unmasking of Groups . . . . . . . . . . . . . . . . . . . . . 186 h . Identity of Groups Liberated . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 c . Mechanism of Masking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194 rl . Trigger Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 e . R.elation of Unmasking t o Heme Splitting . . . . . . . . . . . . . . . . . . . 151

152

J. STEINHAHDT A N D E. M . ZAISER

6 . Titrimetric Evidence of Unmasking in Other Proteins. . . . . . . . . . . . . . . . . 198 7 . Relation of Kinetics of Denaturation and Liberation of Groups in Car-

bonylhemoglobin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

199

8. Kinetics and Thermodynamics of Denaturation and Liberation of Groups

in Ferriliemoglobiri . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 a. Rates and Kquilibria Ihsed on Spectroscopic and Solubilit,y Criteria for I)enat,uration.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 b. Rate of Increase of Proton-Uinding Groups.. . . . . . . . . . . . . . . . . .205 c. Separation of the Fornard and Reverse Rate Constants.. . . . . . . . . . . . 206 d . Effect of Temperature on Kinetics and Equilibria. . . . . . . . . . . . . . . . .206 e. Model of the Unmasking Reaction in Ferrihemoglobin. . . . . . . . . . . . 210 f . Thermodynamic Functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 g. Energy of Activation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 9. Kinetics and Thermodynamics of the Reversal of Denaturation of Ferrihemoglobin.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 10. Kinetic Data Suggesting Unmasking in Other Proteins. . . . . . . . . . . . . . . . 217 11. Summary of Section 111.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 ....... ............................. 221 References. ........

I. INTI~ODUCTION The acid-base dissociations of proteins have been the subject of continuing investigation ever since the development of suitable techniques for their study and the realization of their fundamental importance. All properties of proteins must be attributed ultimately to their structure (the unique arrangements of their constituent amino acid residues) arid the interaction of elements of this structure with the environment. A comparison of the acidic dissociations of proteins with those of their constituent amino acids or polypeptides may be expected to illuminate their manner of linkage and the nature of the modifications they undergo when they are incorporated into the higher structure. Conversely, investigations of the dissociations of proteins may often furnish indications as to their content of particular amino acids, or of the extent to which the carboxyl groups of the dicarboxylic acids are combined with ammonia in the unhydrolyzed protein. Furthermore, since a great many properties, such as physiological activity, enzymic function, and denaturation by acid or base, are markedly dependent on ~ € 1 studies , of acid-base dissociations of proteins often have special interest. Early attention was focused on the maximum amounts of acid and base with which proteins can combine, as indicative of the number of dissociable cationic and anionic groups which they possess. (Actually the terms “acidbinding” and “base-binding” as used in the older literature really refer to the binding slid retease of protons.) Later, analysis of the dependence of the extent of dissociation on pH was employed to give iiot only the numbers but the equilibrium constants of the various types of dissociating groups. Discrepancies between the dissociation constants found and those

HYDROGEN ION EQUILIBRIA

153

of the corresponding amino acids have been interpreted in terms of (1) changes in molecular polarity associated with the formation of the peptidc bond; ( 2 ) electrostatic interactions of dissociable groups which may alter their acidity; and ( 3 ) combination with ions other than hydrogen ion which may change the electrostatic condition of the protein and affect the hydrogen ion equilibrium, with resultant shifts in the titration curve. Such shifts thus afford insight into the structure of the protein, particularly where the site of a highly specific combinatioii with an ion can be inferred from them. Still other differences in titration curves brought about by various treatments may be attributed to the appearance or disappearance of dissociable groups (and thus profound rearrangements) as a result either of chemical modification or denaturation of a protein. This review deals first (Part 11) with recent work on the acid-base dissociations of native proteins. This is followed (Part 111) by a discussion of unreactive prototropic groups in certain native proteins, with emphasis on conditions for their detection, and their relation to protein denaturation and the structural changes which accompany it.

11. ACID-B.~SE DISSOCIATIONS OF NATIVEPROTEINS 1. Scope of This Review A detailed summary of the literature prior to 1942 on the acid-base dissociatioiis of proteins has been given by Cohn and Edsall (1943a). The present review relies chiefly on data on the titration of proteins with strong acids and bases published since that time1 to illustrate the present state of our knowledge of the hydrogen ion equilibria of native proteins. Since all the newer literature are not treated in detail, recent studies in this field are listed in Table I. Although the desired interpretation of titration data as equilibria requires demotistration that equilibrium data are actually obtained, the possibility that pH drifts or irreversible dissociations may occur within the region of pH studied is sometimes ignored. Table I indicates which papers contain explicit mention of observations bearing on this vital point. 2. General Features of Protein Titration Curves

The appearance of the titration curve of a protein depends primarily upon the relative numbers and dissociation constants of the dissociating groups of its constituent amino acids. The curves observed experimentally differ markedly, however, from a curve derived by merely adding the titra1 The titration of proteins with weak acids is not considered in this review, since in the only detailed study of the subject (Steinhardt, Fugitt, and Harris (1943) on wool keratin) the effect of selective solvation of the fiber by undissociated weak acid was added to the effect of combination with hydrogen ion.

I54

J. GTbXSHA1:l)T ANH E . M . ZAISICIl

TABLEI Titration Curves Published &er 1942 Caseins (a,@, -,) Collagen Fibrin (human) Fibrinogen e Fibrinogen (bovine) Fibrinogen and fibrin (bovine) Gelatin c Globin, ferrihemoglobin, itrid derivutives (Lovine) Ilemoglohin, cnrbonyl- (horse) Hemoglobin, ferri- (horse) 6 Horse-radish perosidase and derivs tives c Insulin and two fractions a * Insulin, zinc-free r lnsuliri, zinc compound p-I actoglobulin, dodecylsulfate complex a Lysozyme e Myoglobin (ferro- aiid carboriyl) Myosin and denatured myosin a Myosin a d Serum albumin (human) Serum albumin (bovine) e Silk fibroin Trypsin Wool keratin

(1

Wool kerat,iri e Wool keratin I

IIipp, Groves, and McMeekin (1952) Howes and Kenten (1948) Ferry and Morrison (1945) Chaudhuri (1948) Shulman and Ferry (1950) hlih6lyi (1954a, b) Rousselot (1941) Theorell (1943) Steinhardt and Zaiser (1951) Steinhardt and Zaiser (1953) Theorell (1943) Fredericq (1954) Tunford arid Epstein (1954a) Tanford and Epstein (1954k)) RlcMeekin et a l . (1949) Tanford and Wagner (1951) Theorell and Ehrenberg (1951) Dubuisson and IIamoir (1943) iVIih6lyi (1950) Tanford (1950) Shore (1953) Elod and Frohlich (1949a, b) Duke, Bier, and Nord (1952) Steinhardt, Fugitt, and Harris (1943, 1946) Mod and Frohlich (1949a, b) Steinhardt arid Zaiser (1950)

Key to ‘I’able I :

Titration stated t o be fully reversible. stated t o be free of pll drifts. c Drifts in p H observed. d Slight drift in pII in alkaline solution only. e I’artial titration only.

* Titration

tion curves of the constituent dissociating groups. Figure 1 shows the result of such a summation based on the known amino acid composition of ovalbunlit1 and reasonable values for the dissociation constants2 of the acidic and basic groups which do not form peptide bonds in the protein; it presents for comparison the titration curve of ovalhumin at low salt con-

* The values used are those ordinarily found for the groups in peptidrs arid pro teiiis, and differ of course from those for free amino acids.

HYDROGEN ION EQUILIBHIA

155

PH 1 . Xcid-Ixtse dissociation of ovalbumiii :it ionic strength 0.033 (solid line), data of Carinan, Iiibrick, and l’almer (1941). Broken line is summation of dissociations, without interaction, of individual groups: 52 carbosyls (pK 4.5),7 imidazoliurn (pK 6.8), 20 s-ammonium (pIi 10). &’I(;.

centration. Wherem the curves approach a common niaximum a t lonpH, the dissociation of any set of groups ill thc protein occupies a wider range of pH than that characteristic of a sum of nioiiobasic dissociations. This spreading of the titratioii curve is due, as in any polybasic acid, t o a cumulative clectrostatic repulsion (or attraction) between the iiicreasingly positively (or negatively) charged protein and hydrogen ion as the amount of the latter combined increases (or decreases). The maximum at low pH corresponds to the condition in which the protein holds its niaximuni complement of hydrogen ions and hence bears its maximum net positive charge. In that state all the dissociable groups of the protein exist in their acidic forms, which are either electrically neutral or positively charged as in the case of imidaaolium, ammonium, and guanidiiiium groups. As the pH iiicreases the prototropic groups dissociate in the order of decreasing acidity, the carboxyls from about pH 2 to about 5.5, imidazolium from about 5.5 to 8, followed by ammonium, phenoxyl, sulfhydryl, and guanidinium groups. The four groups last named have overlapping ranges of dissociation above pH 8. Although a maximum in the dissociation of hydrogen ion is theoretically reached a t high pH, it is never observed, because the weakly acidic guanidinium groups are not completely dissociated in the range accessible to ordinary p H measurements. If this point were reached all the acidic groups named above would exist in the form of their conjugate bases: carboxylate, imidazole, amino,

1st;

J. STEINHARDT AND E. M. ZAISER

pH FIG.2. Effect of added IiCl on dissociation of P-lactoglobulin. Data of Cannan, Palmer, and Kibrick (1942).

pheuoxylate, aid guanidino. The dissociatioii coilstarits of the groups differ in their dependelice 011 temperature, which is small for the carboxyl groups, larger for imidazolium, and still larger for the more basic groups. Electrical effects on the titration curve of a proteiii account not only for the wide spread of each of its constituent dissociation functions over the pH scale but also for the influence of the ionic strength and dielectric constant of the medium as well. The effect of added neutral salts is typified by the curves in Fig. 2 for p-lactoglobulin at various constant ionic strengths. The curves approach the same maximum at low pH, but their slope increases with the concentration of salt, until the effect approaches a limit. Salt lesscns the electrostatic repulsion already mentioned, and hence the curves are steeper and the p H range of each group of dissociations smaller. Ail additional effect of salt (not manifest with @-lactoglobulin)is due t o the combination with some proteins of anions or cations other than hydrogen or hydroxyl ions. The binding of anions, for example, increases the steepness of the carboxyl region of the curve by lowering the net positive charge, and may be reflected in a shift with salt concentration of the pH of zero reaction with acid or base (isoionic pH). This shift is notably absent in 8-lactoglobulin, since all the curves intersect at zero acid bound and p H 5.18. In most protein titration curves, including the two examples already given, only two discrete steps are readily discernible, and it is difficult to pick out even from these thc numbers and dissociation constants of sets of

HYDROGEN ION EQlJILIBltIA

157

groups. The discussions of the theoretical analysis of titration curves and stoichiometry to follow will be devoted largely to the various means, both theoretical and practical, which are used to deduce from titration data the numbers and dissociation constants of prototropic groups, as well as other information bearing on thc structure of individual proteins. 3. Theoretical Analysis of Titration Curves

a. Outline of Theory. LinderstrGm-Lang (1924) was the first to apply the theories of Bjerrum and Debyc-Huckel to the quantitative description of the dissociation of a highly polyvalent ampholyte, in terms of the numbers of dissociations, their characteristic equilibrium constants, and thcir electrostatic iiiteractioris with one another and with the environment. His treatment was employed by Cannan, Kibrick, and Palmer in their analyses of the titration curves of ovalbumin (1941) and 0-lactoglobulin (1942). The subject was reviewed by Cannan (1942) and the theoretical aspect of the problem has been further explored by Scatchard (1949). The reader is referred for details of the development to the original papers or to detailed presentations by Cohn and Edsall (1943a) and Klotz (1953). The consequences of the theory alone will be treated here, and the aspects dwelt on will be those emphasized in recent work-for example, the interpretation of the electrostatic term w (vide infra), riot only as it reflects the influence of ionic strength but as it bears on the structure of the individual protein. The theory requires two assumptions: that the dissociable groups may be divided into a small number of classes, each of which may be characterized by a single intrinsic dissociation constant (Kint), ; and that the ampholyte may be represented as a sphere over which the net charge is uniformly distributed. If n, denotes the total number of groups of class i, arid ri of these are dissociated a t a given pH where the average net charge on the molecule is 2, then

The number of groups r in a protein which are dissociated at a given pH is equal to Zri . The electrostatic factor w is defined as

where N is Avogadro's constant, c the elertronic charge, D the dielectric constant of the solvent, R the gas coiistant, T the absolute temperature, b the radius of thc spherical hydrated protein molecule, a its radius of

158

J. BTEINHARDT .4N1) X. M. ZAISEI'L

(LscIusio~i, aiid K the quantity defiiicd in the Ilebye-Hiickcl theory as d(8?rNe2/1000kT)p (frequently rrferred to as thc reciproral of the thickness of the ionic atmosphere). At coilstant T , D,a d ionic strength p, III depends only on the size and shape of thc protein molecule. Thus, whrrc values of a and b may be estimated, a theoretical value for w may be cal(ulated. The net charge 2 as determined from titratioii data alone ( i t . , the. number of hydrogeii ions combined) must be correctecl if the protein hinds other catioiis or anions. The adequacy of such a theoretical analysis of a titratioii curve clan be tested i n certain cases by using such values of w and Z to construct sets of curves from equation 1 atid the relationship r = xr,. i

For this purpose sets of values of ~(k',,,~); arid n, are chosen aiid the titratioii curve of the protein is constructed from an assembly of such sets of curves for the various kinds of dissociating groups. The theoretical analysis is held to be adequate if the experimental data are reproduced by a set of values of p(Kint), ctnd n, which are in reasonable agreenierit with those obtained from other sources (such as the corresponding dissociation constants in peptidcs, and the numbers of groups determined by amino acid assays or indicated by other methods to be described later-Section 11, 5 ) . Another test is afforded by the effect of added salt, since the electrostatic term w should d e p d on the iortic strength of the medium. Reasonable agreenierit with the experimental facts, both as to the appearance of the titration curve (or parts of it) and the effect of ionic strength, has beeii deriioustrated for some proteins, e.g., ovalbumiii aiid &lactoglobulin (Cantian, Kibrick, and Palmer, 1941, 1942). Where agreement fails the coinplications indicated in the following paragraphs may explain the failure. 0. Comparison of the Electrosiatic Term w from Theory and Experiment. Comparison with theoretical values of w calculated from equation 2 is facilitated by detcriniiiiiig w as an cmpirical curve-fitting constatit from thc titratioii data. Cariiiaii, Kibrick, aiid Palmer (1941, 1942) used a graphical method for firidiiig w as a function of ionic strength from theslopcs of titration curves in regions of p1-I near the middle of the ranges in which a single type of group dissociates (e.g., pH 3 to 4.5 for carboxyl, arid 10 to 11 for e-ammonium groups). In the case of ovalbumin, for which the isoionic point varies with pH, an empirical correction was applied by shifting the pH scale for each ionic strength so that all the t itratioii curves coincided a t a single pH. The experimeiital values obtained from ovalbumin data between ionic strength 0.008 and 0.7 were 20 % lower than those calculated from equation 2, but atJ higher ionic strength approached the theoretical ones. For 0-lactoglobulin the discrepancy was much smaller. It was coilcluded that the theoretiral derivation of equations 1 aiid 2 which define w is valid, aiid that the assumptions required as to molecular shape and

HYDROGEN ION EQUILIBRIA

159

charge distributioii are reasonably \yell suited to these ti\ o protein nioleC U ~ S . At the time of Cannan, Kibrick, arid Palmer’s publication, however, there was little quaiititative information as to the extent to which dissolved proteins combine with mions and cations other than hydrogen and hydrovyl ions. The effect of such combination on the net charge of the protein and the slope of the titratioii c’urves was thus not considered, although Cariiian pointed out that the isoionic point of ovalbumin varies with salt cloncentratioii, whereas that of p-lactoglobulin does not. This difference suggests that the two proteiiis differ in their ion-binding behavior; thus the values of w, especially for ovalbumin, should he re-examined and recalculated, if need be, with the use of corrected values for the net charge.3 I t is likely that the ovalbumin proved anieriable to the application of the theory only because an empirical correction for anion Iindiiig was applied, shifting t hr pH scale of ovalbumin titratioii curves obtained a t different ionic strengths so that they coincided at the isoionic point.* Cannaii, Kihrick, and Palmer also studied the effect of protein concentration on the titration curve and concluded that although the protein does contribute to the ionic strength, its effect is small and can be neglected in the presence of salt. Liiiderstrplm-Laiig (1952) has discussed the ealculation of the coefficieiit of activity of the protein. It is customary to neglect this activity coefficient, i.e., to assume it equals unity, on the ground that the solutions of protein which are generally titrated are very dilute (lW6 to lop4mole protein per liter). ItecentJy Tanford and his collaborators have made use of the eleetrostatic term w to analyze the titration (wrves of a number of proteins. By combining graphical methods and trial-and-error selection they obtained values of p(Kint),and w from experimental titration curves. In each case the value for the apparent average net charge was corrected for the binding of anions and cations as determined by iitdependent experimental methods. Diffcreaces between their experinieiital values of w and those calculated from thcorv W F ~ Pinterpreted by thwr isorkers in tcrnis of alterations iii molecular parameters or structure. For example, observed values which isere low, especially when they became low only at pH’s far removed from iieutrality, were attributed to alteration in the size or shape of the mo!ecule brought about by electrostatic repulsion. Thus, with human serum albumin Taiiford (1950) fourid that although the hasic branch of the titration curve could be computed from theory, the acid branch was much too steep The electrical inhomogeneity of both these protein prepariitions is not strongly relevant t o w,since the average net charge is used. Longsworth’s (1941) demonstration of constancy in the ratio of electrophoretic mobilities t o net charge attests the approximate adeqiiacy of this expedient of shifting the curves on the pH axis.

160

J . STEINHARDT A N D E . M. ZAISER

(i.e., w too small). Later work on bovine serum albumin by Tanford a i d Roberts (1952) aridshore (1953) suggests that this behavior of w may reflect a change in molecular shape due to the reversible rupture of carboxylphenolic hydroxyl hydrogen bonds. In the case of insulin Tanford and Epstein (1954a) found wide variations with pH in the values of w derived from experiment; these were attributed primarily to changes in molecular weight (the degree of polymerization of the protein monomer varies with pH). With lysozyrne Tanford and Wagner (1954) calculated w theoretically only for the isoelectric region; and although corresponding values of w could not be precisely determined from the experimental data they were at least as large as, and possibly larger than, the tjheoretical ones. Thcse authors believed that such values indicate that the lysozynie molecule is compact and sparingly hydrated near its isoelectric point. The unusual observation that the slope of the carboxyl portion of the titration curve of this protein is less than the theoretical awaits final explanation, although Tanford and Wagner suggest some possible causes for this aberration. A recent mathematical analysis by Laskowski and Scheraga (1954) demonstrates qualitatively that hydrogen bonding may so affect the reactivity of polar side chains of proteins as t o increase or decrease the pK's of such groups. Thus, depending on the participants in the hydrogen bond, the titration curve may be spread or steepened. (Electrostatic repulsions alone can cause only spreading.) I n one case, that of the phenoxylcarboxylate interactions in serum albumin, these authors have calculated thermodynamic quantities, on the basis of a hydrogen-bonded model, which agree well with those observed by Tanford and Roberts (1952). Direct quantitative application of their analysis to titration curves has so far been prevented by mathematical complexity. Their estimates of the magnitude of the role of hydrogen bonding between polar side chains must be regarded as an upper limit, since they ignore the possibility of hydrogen bonding between such groups and water. c. Comparison of Salt Effect on Soluble and Insoluble Proteins. The theory of Linderstr@m-Langexpressed in equation 1 adequately predicts the effect of salt on the titration curves of soluble proteins if consideration is given not only to the influence of ionic strength on the electrostatic factor w but also to the effect of bound ions (other than hydrogen ions) on the net charge 2. In the case of an insoluble fibrous protein, such as wool keratin, w cannot be calculated directly, partly because of the difficulty of assigning a molecular weight to the protein. Obviously other factors peculiar t o the presence of two phases also affect the results here. Thus the effect of added KC1 on the titration of wool with HC1, as shown in Fig. 3 (Steinhardt, Fugitt, and Harris, 1940a), is strikingly different from that for a soluble protein (e.g., p-lactoglobulin, Fig. 2). In the absence of salt the

HYDliOGEN ION EQUILIBRIA

ra

I

161

I

I

PH FIG.3. Effect of ionic strength on carbosyl titration of wool a t 0". From Steinhardt,, Fugitt , and Harris (1940a).

curve for wool is centered about a pH considerably below that of the usual pK for carboxyl groups. In the presence of different concentrations of salt a family of considerably less steep curves is obtained which for a considerable portion of their length are parallel to one another. The tendency to reach a limiting curve at high ionic strength is found here as in soluble proteins. Steinhardt, Fugitt, and Harris (1940s) observed that the displacement, of the mid-point of the curves was related stoichiometrically to the total roncentration of anion and could be ascribed to the binding of definite amounts of chloride ion by the wool The effect of added salt on the position of the titration curve on the pH scale was described in terms of a dissociation constant for the anion as well as for hydrogen ion. Their analysis also successfully predicted the gradual approach, at high salt concentrations, to titration curves entirely comparable with those of a soluble protein of similar composition. The slope of the mid-section of these curves is exactly half that which an ideal monobasic acid should give, and equal to that found for the titration of numerous polybasic electrolytes, e.g., polymethacrylic acid (Katchalsky and Spitnik, 1947). & T h echoice between this treatment and the consideration of a Donnan equilibrium between the water-containing fiber phase and the solution is not easy t o make. The existence of a real affinity of ions for wool, differing widely for different ions (Steinhardt, Fugitt, and Harris, 1941) lends support t o t h e simple stoichiometric explanation of these authors.

162

J. STEINHARDT AND E. M. ZAISER

Myosin is another protein to which the theory of Linderstr@m-Laiigin its present form is not applicable, since in niyosin the ratio of molecular length to width is 100/1-far from the sphericity on which the theory is based. Thus experimental values of the parameter w cannot be easily interpreted quantitatively. Myosin is soluble in the presence of salt on the alkaline side of its isoionic point only, and thus should behave as a soluble protein above p H 5.7 to 5.8 and as an insoluble one below this. Mihdlyi (1950) has studied the effect of salt on the titration of myosin and reports that its insolubility in acid in the presence of greater than 0.05 M KC1 does not affect the revcrsibility of the titration; nor are there any obvious discontinuities in his titration curves, shown in Fig. 4. The data for basic solutioiis appear to be affected by salt very much as those of other soluble proteins, ant1 reach an apparent limiting curve a t a fairly lorn ionic strength (0.15). In acid solution where the protein is insoluble, however, the effect of salt closely resembles that for wool, except that the displacements of the parallel central portions of the curves are somewhat less thaii for wool, consistent with a lower affinity of myosin for chloride ion. The slopes of these portions of the curves are within 10 % of those observed for

3

4

5

6

7

8

3 '

10

PH

FIG. 4 . Titration curves of myosin a t different ionic strengths. Temperature 25°C. Empty circles: 0. M KCI; vertical circles (right side black): 0.05 A4 KCI; full black circles 0 15 A4 KC1; horizontal half circles (black top) : 0.30 M KCI; vertical half circles (left black) : 0.60 M KCl; horizontal half circles (white top) : 1.20 M KCl Ordinate: equivalents of hydrogen ions bound or dissociated by lo6 g. myosin. Ab scissa: pH. The curve in the inset shows the difference in the number of equivalents of base bound by 106g , myosin in the presence of potassium chloride (r/2 > 0.15)and in the absence of salts. From Mihhlyi (1950).

163

HYDROGEN ION EQUILIBRIA

wool and long-chain polymeric acids. Similar observations have been made by Nanninga (1954) on L- and H- meromyosin (produced by the treatment of myosin with trypsin). Nanninga has concluded that the extremely low experimental values of w (1/45 those for ovalbumin) are associated with the high asymmetry of the meroniyosiri molecules. A satisfactory theoretical interpretation of the siopcs of titration curves of proteiiis to which Linderstr@m-I,ang;’sspherical model is inapplicable is still lackitrg, the chief difficulty being the calculatiorr of the clectrostatic free energy of a system of charged rods, probably the most suitable model for elongated proteiiis (e.g., myosin). Iiatrhalsky and Gillis (194!)), Katchalsky arid Lifsoii (1953), and Iiatchalsky, Shavit, and Eiseribcrg (1954) have made important contributioris to this problem for polymeric acids. Their model (an uncharged random coil which becomes elongated as charges are successively added) and the molecular parameters which figure in their formulation are not directly applicable to the protein case. TABLEI1 I n f r i n s i c Dissociation Constants from Anallisis of Titration Cicrves

Protein and reference

Carbosyl

I8,r

ff

~

Ovalbumin (Cannan et al., 1941) plactoglobulin (Cannan et al., 1942) Human serum albumin (Tanford 1950) Insulin (Frcdericq, 1954) Insulin (zinc-free (Tanford and Epstein, 1954a) Lysozyme (Tan ford and Wagner, 1954) Peptides (Cohn and Edsall, 1943c) Peptides (Ellenbogen, 1952)

_

Ammonium

I

__ I’henosyl ff

E

10.07

_

-

I Sulf-

Guanihydinium dry1 -

-

4.29

6.7-6.8

-

4.6

6.7-6.8

-

-

4.00

6.10

8.00

-

6.6

8.3

-

6.40

7.45

11.9

6.5-7 .O

7.5-

3.3 3.6 -

4.1, 5.0 4.73

-

3.0- 3.03.2 4.7 3.7

Imidaxolium

-

--

10.310.6

10.410.9

-

7.9

12.6513.3

5.6-7 .O

7.68.4

9.410.6

9.810.4

9.110.8

11.612.6

-

7.8

I-

-

164

J. STEINHARDT AND E . M. ZAISER

The only published solution for the electrostatic free energy of a system of charged rods is for the restricted case of parallel rods equidistant from one another (Lifson and Katchalsky, 1954). d. Intrinsic Dissociation Constants and Numhers of Groups. Table II presents a summary of values of intrinsic dissociation constants p(Kint), as determined for the ionizable groups of several proteins by the application of the theoretical treatment previously described. For comparison, values observed from the titration of peptides, as summarized by Cohn and Edsall (1943c), are included, as well as the revised values proposed for three sudi groups by Ellenbogen (1952). I n some cases the quantities p(K,nt)l mid ni yielded by analysis of titration curves may furnish special clues to the structure of particular proteins. For example, if ordinarily dissociable groups participate in formation of intramolecular bonds abnormal values for their intrinsic dissocintion constaiits may result, or their number may even appear to be smallrr than that found by amino acid assay. Examples will be furnished in thc later sections on stoichiometry and on unreactive prototropic groups.

4. Relation

of Stoichiometry to Maxima in Binding and Dissociation of

Protonse The ~iuniberof equivalents of hydrogen ioii which are bourid when a protein is brought from the isoionic condition to a state in which all dissociable groups have assumed their acidic forms is equal to the number of cationic acid groups in the molecule. Similarly, the equivalents of hydrogeii ion dissociated between the isoionic and fully basic states of the protein are equal to the number of anionic basic groups. Thus, inspection of titration curves for their limiting values a t low and high pH should reveal the number of cationic and anionic groups. These limits cannot be so interpreted, however, unless certain necessary precautions have been taken. At the high concentrations of acid or base required to reach the maxima only a small fraction of the total acid or base present it5 bound. Small errors in the estimation of the concentration of free acid or base produce large errors which can be minimized if high concentrations of protein are used. The concentrations of free acid or base calculated from the measured values of the activity of hydrogen ion in the solution are subject to two important sources of error: errors in activity coefficients in solutions containing protein; and uncertainties in the relation between pH and H+ ion concentration which arise from conventional standardization of the pH scale, and from the large and not exactly known liquid junction potentials which arise in measurements of strongly acid solutions. In addition, the combination 6 Throughout this review t h e amino acid assays referred t o are those tabulated by Tristram (1953),unless otherwise indicated.

HYDROGEN ION EQUILIBRIA

165

or dissociation of the maximum number of protons must be tested for reversibility by back titration before it can be safely ascribed to the native protein. If denaturation results in an increase in the number of prototropic groups, or if primary amide linkages are hydrolyzed, the ohserved maximum in protons bound or released will refer to a t least partially denatured protein (see Section 111 of this review) or to protein plus the ammonia liberated. Most titration curves approach a limiting value of hydrogen ion bound at pH between 1 and 2. In some cases there is evidence of additional comhiiiation of hydrogen ion at very low pH which may be attributed to titration of the weakly basic amide groups, but this ordinarily occurs in a region of pH where the data are subject to very large errors. It seldom interferes with the evaluation of the maximum hydrogen ion bound, since there is a reasonably well-defined plateau in the curve after all the carboxyl groups have been titrated-i.e., below pH 2. Steinhardt (1941) showed that in the case of wool this plateau is shifted to higher pH (along with the rest of the acid branch of the titration curve) in the presence of certain large anions which have high affinities for wool. The plateau of “maximum” hydrogen ion bound is unchanged, and the displacement brings the portion of t,he curve which indicates further combination of hydrogen ion with basic groups weaker than carboxylate into a region of pH where much of the uncertainty is removed. There is thus no doubt that the indicated titration of weakly basic groups is real.? I n the past the maximum hydrogen ion-binding capacity has furnished a more reliable figure for the total number of cationic groups in proteins than have amino acid analyses. The latter tended to give low results particularly when isolation procedures were employed. More recently, however, amino acid assays have been greatly improved. With most proteins the total number of arginine, lysine, and histidine residues is now in reasonable agreement with the maximum hydrogen ion-binding capacity (allowing for the fact that in some proteins part of the histidine may be in its basic form a t the isoionic point). The apparent discrepancies noted earlier for ovalbumin (Cannan, Kibrick, and Palmer, 1941) and silk fibroin (Gleysteen and Harris, 1941) have thus now disappeared. Where analyses still give lower results than titration curves (as for p-lactoglobulin (Cannan, Palmer, and Kibrick, 1942), myosin (Mihhlyi, 1950), and H- and L- meromyosin (Nanninga, 1954), it appears likely that further improvement in the analyses will remove the discrepancy. Thus, new analyses by Kominz et al. (1954) have decreased though not eliminated the discrepancy for myosin; and Perlmann’s (1941) measurements of cationic groups by combina7 Elod and Frohlich (1949a) suggested that such combination occurs with HC1 for silk and polyamides, as well as wool.

166

J. STEINHAI1DT AND 14. M . ZAISElt

t ion with nietaphosphorir acid corroboratc the hydrogen ioii-binding cqapacity found for this protein. Where the assays give higher results than the maximuni hydrogen ion-binding indicates, as with native hemoglobins (Steinhardt and Zaisw, 1951, 1953) and human serum albumin (Tanford, 1950), there is reason to seek unreactive prototropic groups (see Section I11 of this review). Because of the high pK of the guanidinium groups of arginine the uppcr limit for hydrogen ioti dissociation of proteins i.e., “maximum base-biiiditig capacity,” can be reached only in the pH range above 13. The alkalinc branches of titration curvw are seldom explored beyond pH 12, because it is difficult to obtain tneaningful pH data beyond that value. Thus, a t high p H the effect of side reactions oil the apparent dissociation of protons is more important than at low pH: for example, disulfide bonds may be broken: and tyrosine may be oxidized. In practice, therefore, the maximum proton dissociation, arid thus the number of anionic groups in the isoioriic protein, is not observed. It is possible that an apparent maximum for a particular protein may appear t o be reached if the pK of the guanidinium groups is very high and well separated from those of the phenoxyl and c-ammonium groups. Values which have in the past he11 assigned to maxima in the proton dissociatioiis of proteins (“maximum base-binding”) on the basis of titration data have proved to be illusory.

5 . Stoichionaefry and Diflerentiation of Prototropic Groups

Rather than resort to purely empirical selection of suitable values of for cyuation 1 it is more usual to begin by fitting experimental data with values of n, chosen to conform with the numbers of prototropic groups determined by several more direct and specific methods of examination of titration data. Even where the theoretical analysis of a titration curve is iiot attempted and exact values of p(Kint),for each type of group are therefore lacking, the numbers of groups so determined may furnish valuable clues to the internal structure of the protein, especially when they are compared with the results of amino acid analyses. a. Assignment of Segments of Titration Curve to Specijc Groups. In accordaiice with thr discussion ill Section 11, 2 the carboxyl group is the oiily otie which dissociates between pH 2 and 5 . 5 ; in most cases its dissociation is practically complete at pH 5.5. The number of equivalents of hydrogen ion which dissociate when the protein is brought from its state 2) to pH 5.5 arc of maximum combination with hydrogen ion (pH approximately equal to the number of free carboxyl groups, i.e., the suni

n, and p(#&

-

Steinhardt, Fugitt, and Harris (1940b) and Steinhardt and Zaiser (1950) corrected for the hydrolysis of disulfide bonds by alkali in their work on t h e titration of no01 H ith base.

HYDROGEN ION EQUILIBRIA

167

of the aspartic and glutamic acid residues plus the terminal a-carboxyl groups, minus the primary amide groups. If the amide nitrogen is determined and the number of terminal carboxyl groups is known from endgroup assays, the number of dicarboxylic amino acid residues can be calculated. Comparison with amino acid analyses for these residues has in some cases (notably the hemoglobins) indicated that not all the carboxyl groups in the native protein are available for combination with hydrogen ion. It is more common, however, to find that the number of carboxyl groups titrated equals or exceeds that indicated by analysis. Agreement with the most recent amino acid assays appears satisfactory in the case of human serum albumin (Tanford, 1SSO), insulin (Tanford and Epstein, 1954a), ovalbumin and p-lactoglobulin (Caiman, Kibrick, and Palmer, 1941, 1942). Titration data continue to indicate more carboxyl groups than assays show for bovine serum albumin (Shore, 1953), lysozyrne (Tanford and Wagner, 1954), trypsin (Duke, Bier, and Nord, 1952), silk fibroin (Gleysteen and Harris, 1941), collagen (Bowes and Kenten, 1948), myosin (Mihhlyi, 1950), and L- and H- meromyosin (Nanninga, 1954). However, such discrepancies may be due to low results in the assay of dicarboxylic amino acids or to high values for amide nitrogen. It is worth mentioning that stoichiometric binding of cationic dye or detergent ions supports the conclusions drawn from titration data in the cases of lysozyine (Glassman and Molnar, 1951) and trypsin (Fraenkel-Conrat, Bean, and Lineweaver, 1949). With the possible exception of histidine it is ordinarily impossible to learn from inspection of the titration curve the number of residues of an iiidividual basic amino acid. The maximum hydrogen ion-binding capacity equals the sum of the guanidinium groups of arginine, the e-ammonium groups of lysine, the terminal a-ammonium groups, and the imidazolium groups of histidine. Depending on the position of the isoionic point, this sum may include all or only part of the histidine residues. The amount of hydrogen ion dissociated between pH 5.5 and 8, however, is generally due to imidazolium groups (with perhaps a small contribution from a-aniinoniurn groups), and should be approximately equal to the amount indicated by the amino acid assay of histidine if all the imidazole groups are reactive. The agreement with amino acid assays has been satisfactory for the cases in which such a comparison has proved possible: human and bovine serum albumins (Tanford, 1950; Shore, 1953), lysozyme (Tanford and Wagner, 1954), trypsin (Duke, Bier, and Nord, 1952), P-lactoglobulin and ovalbumin (Cannan, Kibrick, and Palmer, 1941, 1942), myosin (Mihhlyi, 1950), 1,-and H- nieromyosin (Nanninga, 1954), and hemoglobin (Wyman, 1939). It is difficult t o make such a coniparison for the other basic aniino acid residues, however, since (1) in some proteins (hemoglobins), the maximum

168

J. STEINHARDT AND E. M. ZAISER

hydrogen ion-binding capacity usually includes the contribution of an uiicertain fraction of the histidine residues; (2) their dissociations are not easily distinguishable from those of the phenoxyl groups of tyrosine and the sulfhydryl groups of cystine, since they occur in overlapping ranges of p H ; and (3) the titration with base is usually incomplete. There are, however, some proteins having amino acid compositions such that the dissociation of one type of basic group dominates a particular segment of the titration curve, which can thus be identified almost as readily as the portion due to carboxyl dissociations generally can be. Obvious examples are the protamines, salmine and clupeiii, strongly basic proteins of low molecular weight. The titration of clupein (Rasmussen and Linderstrgim-Lang, 1935; T,inderstr@m-Lang,1935) is due entirely to arginine, which furnishes the only dissociable groups (except for one t,ermiiial carboxyl and one terminal amino group). Another rase is that of silk fibroin, which contains very little histidine and lysine, and approximately ten times as many tyrosine as arginine residues. Gleysteen and Harris (1941) were thus able to study the dissociation of the phenoxyl groups of tyrosine with little interference from other dissociable groups. Similar possibilities might appear to exist for zeiii and pepsin, which also contain relatively large numbers of tyrosine residues. Because xejn is insoluble in water, however, it has been titrated only in alcohol-water mixtures (Neuberger, 1934; Cohn, Edsall, aiid Blanchard, 1934), and pepsin is so unstable in neutral and alkaline solution that its dissociation curve above pH 6 (which must refer to the denatured protein) is know1 only approximately (Herriott and Northrop, 1934). With collagen (Bowes and Kenten, 1948) the number of groups dissociating in the region of p H attributed to r-ammonium groups is less than the lysine content from amino acid assay; heiice the possibility that some of these groups are unreactive must be considered, especially since lysine assays are more often low than high. I n most cases, however, recourse must be had to other methods to disentangle the dissociations of the several prototropic groups which are stronger bases than imidazole. b. Modijication of Dissociation Constants of Groups. French and Edsall (1945) have reviewed the reactions of formaldehyde with amino acids and proteins, including the effect of formaldehyde on their titration (wrves. Formaldehyde is of interest in this discussion chiefly hecause it reacts rapidly arid quantitatively with amino groups to give tddition products which are far weaker bases (Levy, 1935). Thus in the forniol titration of a protein the apparent dissociation equilibrium of the ammonium groups of lysiiie is displaced toward acid p H by as much as 3 units. Differeiitial titratioii in the presence and absence of formaldehyde thus gives the num-

HYDROGEN ION EQUILIBRIA

169

ber of e-ammonium groups. The ionization of imidaaolium groups is similarly affected, but if the differential formol titration is carried out at pH 8.5 neither imidazolium nor cr-ammonium groups should interfere. Carboxyl and phenoxyl groups are unaffected by this reagent. Cannan, Kibrick, and Palmer titrated ovalbumin (1941) and P-lactoglobulin (1942) in the presence of formaldehyde, and concluded that the numbers of t-ammonium groups were higher than the results of amino acid assays for lysine available at the time. Subsequent assays (see footnote 6) have increased the amount of lysine indicated, so that the discrepancy is now smaller for P-lactoglobulin and has vanished completely for ovalbumin. The titration by Dubuisson (1941) of myosin in the presence of formaldehyde indicated fewer lysine residues than the then current amino acid assay, but MihAlyi (1950), using a purer preparation, found more lysine by formaldehyde titration than in the earlier work, enough in fact to reverse the discrepancy. Although a recent analysis by Kominz et al. (1954) gives a higher result for lysine than the older one, the titration figure still remains slightly higher than the assay. However, Nanninga’s (1954) formol titrations of L- and H-meromyosin unexpectedly indicate amounts of lysine respectively equal to, and less than, the amino acid assays of these proteins (Komina et d.,1954). Steinhardt, Fugitt, and Harris (1946) found that the effect of formaldehyde on the titration curve of wool keratin was consistent with the combination of this reagent with the ammonium groups of lysine, but that the guanidinium groups of arginine appeared also to be affected, at least when salt was absent. This effect on guanidinium is not usually observed, in part because formaldehyde titrations have not generally been pursued to as high pH as in this work. In addition, the reaction of formaldehyde with the guanidinium groups may be very slow (Frieden, Dunn, and Coryell, 1943), possibly requiring times as long as those required for the equilibration of wool with base (over 24 hours at 25°C.). The conversion of e-ammonium groups to guanidinium by such reagents as 0-methylisourea results in a substantial elevation of their pK, and thus has been used for purposes similar t o those just described in connection with formaldehyde. Tanford (1950) found that the titration of guanidinated human serum albumin could be fitted by a curve computed on the assumption that all the e-ammonium groups had been changed to guanidinium, i.e., that below pH 12 only the readily separable dissociations of histidine and tyrosine were involved. The reaction of iodine with proteins is neither quantitative nor completely specific, and since it may be expected to result in chemical substitution or oxidation of a variety of groups the interpretation of the titration curve of an iodinated protein is subject to considerable uncertainty. At-

170

J. STEINHARDT AND E. M. ZAISER

tention is generally focused on the titration of the phenoxyl groups of tyrosine, which in the free amino acid have their pK lowered by about 3.5 units when two iodine atoms are introduced into the benzene nucleus. Zein, which contains a high proportion of tyrosine, little histidine, and no lysine, was compared with iodozein by Neuberger (1934). He found that the pK of the groups titrated between p H 8 and 13 was lowered by about 3.5 units, and this together with the known iodine content of this particular modified protein led him to conclude that the iodine was largely localized in the benzene ring of tyrosine. Similar conclusions were reached by Herriott (1936) from his titration of iodinated pepsin. Other iodinated proteins, however, have yielded less clear results. Cohn, Salter, and Ferry (1938) reported that the iodination of globin did not affect the ionization of tyrosine as expected. There was in fact a small effect in the opposite direction; after iodination less base was bound in strongly alkaline solution. Likewise Tanford (1950) found very little difference between the titration curves of human serum albumin before and after iodination, although this protein contains a large amount of tyrosine. The ionization constants of prototropic groups are substantially altered in alcohol-water mixtures, but the observed changes are not easy to interpret quantitatively and there is the additional disadvantage that alcohol may cause denaturation of the protein. Data of Lichtenstein (1940) on the titration of gelatin in water and alcohol-water mixtures show the effect of alcohol as a displacement of the acid branch of the curve toward higher p H and of the basic branch toward lower P H . ~ Zein, which has been titrated only in alcohol-water mixtures because of its insolubility in water, must be similarly affected, since the apparent pK’s are higher for the carboxyl groups and lower for the basic ones than is normally found for proteins in aqueous solution (Cohn, Edsall, and Blanchard, 1934; Neuberger, 1934). Fredericq (1954) titrated insulin in 40 % dioxane and interpreted his results in terms of a reasonable effect of the low dielectric constant of this solvent, i.e., an increase of I .I unit in the pK of the carboxyl groups and no effect on imidazolium and ammonium groups. The differential shift of the pK of dissociable groups in organic solvents has been adapted to determination of the acidic and basic groups of proteins by titration with acid or base t o an indicator end point in acetone-water or alcohol-water media (LinderstrGm-Lang, 1927; LinderstrGm-Lang and Jacobsen, 1941). Their use has been limited, however, since proteins tend to precipitate from organic solvents, and may even undergo denaturation. The total number of basic groups found by titration in acetone and the maximum hydrogen ionAlthough the problem of the definition of the pE-1 scale i n dcoliol-natw arises, the effect observed is riot due merely t o e shift in reference point, since components of the curve shift in opposite directions.

HYDROGEN ION EQUILIBRIA

171

binding capacity have been found to agree in insulin and 8-lactoglobulin (Jacobsen, 1947), and in L- and H-meromyosin (Nanninga, 1954). Some of the more coniplex reactions which proteins undergo may result in an alteration in pK or number of dissociable groups, and are profitably studied by differential titrations. The impressive body of evidence relating to the structure and reactivity of the hemoglobins concerns, in part, their heme-linked acid functions. Differential titration studies have revealed that the degree of oxygenation of hemoglobin and the state of oxidation of the heme iron atom have marked effects on the pK’s of certain imidazolium groups through which presumably the heme prosthetic group is linked t o the protein moiety. This work has already been the subject of more than one review (see especially the review on heme proteins by Wyman, 1948). Recently differential titrations have contributed to the understanding of the clotting of fibrinogen. Chaudhuri (1948) observed a shift in pK pursuant t o clotting which suggested that €-amino groups participate in the process. Further clarification has come with MihB1yi’s (1954a,b) differential titration of fibrinogen, activated fibrinogen, and fibrin (clotted activated fibrinogen). He concluded that the activation of fibrinogen results in the appearance of about 1.1 group per lo5g. of fibrinogen with a p K of 7.50 and the loss of about 4.2 negative charges per lo5 g. (due t o splitting off of a peptide fragment by the activating enzyme, thrombin). Polymerization of the activated material to form a clot involves, on the basis of lo5g., 1 group of apparent pK 7.00 which gives up a proton and one of p K 8.22 which takes up a proton. These groups have been identified tentatively by their pK’s as an imidazolium and an amino group. c. Conversion to Nonprototropic Groups. Most chemical modifications which would suppress the ionization of amino acid side chains either yield insoluble products or can be effected only under drastic conditions which destroy the native protein structure.1° A few reactions, however, have been successfully employed to eliminate at least partially the contribution of certain groups to the titration of a protein, by blocking their prototropic function or removing them completely. Methylation or acetylation of ammonium and phenoxyl groups destroys their ability t o dissociate hydrogen ion (e.g., acetylation of pepsin by Herriott and Northrap, 1934). Nitrous acid has been used t o remove ammonium groups, and partial esterification of carboxyl groups has also been accomplished. The studies of Lichtenstein (1940) on gelatin include a titration curve of this protein after treatment with nitrous acid which shows clearly that the free 10 Herriott (1947) has reviewed the reactions of native proteins with chemical reagents.

172

J. STEINHARDT AND E . M. ZAISEK

ammonium groups have been removed." Gleysteen and Harris (1941 ) found that methylated silk fibroin gave u p very little hydrogen ion in the range of pH in which phenoxyl groups normally have their acid disso&tion. The titration with acid of proteins whose carboxyl groups have been partially esterified is of less interest for stoichionietry, since here there is 110 overlap with the dissociation range of other prototropic groups to be untangled by differential titration of native and modified proteins. d. Heats of Ionization. Since the temperature coefficients of the dissociation of acidic groups differ widely (being generally larger the weaker is their acidic function), temperature changes may be used to differentiate between the various acidic groups. The determination of the identity of dissociating groups from the effect of small temperature changes on their pK's involves far less risk t o the integrity of the native protein molecule than the use of even the mildest of chemical reagents. The heats of dissociation of prototropic groups are known from studies on amino acids and peptides and from the steadily increasing body of data on proteins. The usual range of heats of dissociation in kilocalories per mole observed near 25°C. for proteins is f1.5 for carboxyl, 6.5 to 7.5 for imidazolium, 10 to 12 for e-ammonium, about 6.0 for phenoxyl, and 12 to 13 for guariidinium groups. Examination of the effect of p H on the heat of dissociation of a protein, as calculated from titration data a t different temperatures, often reveals not only the identity of the groups but also the limits of p H within which they are titrated, and thus how many such groups there are. A classical example is Wyman's (1939) analysis of thermal effects on the dissociation of horse oxyhemoglobin. I n the p H range 4.5 to 10 he found three distinct values for the heat of dissociation, each one characteristic of a well-defined region of pH, clearly ascribable to carboxyl, imidazolium, and e-ammonium roups. Similar use of heats of ionization has been made by Theorell and kesson (1941) with cytochrome c, Steinhardt, Fugitt, and Harris (1940b) with wool keratin, and Cannan, Kibrick, and Palmer (1941, 1942) with ovalbumin and P-lactoglobulin. I n more recent studies thermal effects have been utilized by Tanford (1950) with human serum albumin, Mihalyi (1950) with myosin, and Tanford and Wagner (1954) with lysozyme. e. Spectrophotometric Titration of Tyrosine Phenoxyl Groups. The ionization of the phenoxyl group of tyrosine is accompanied by a n alteration in its ultraviolet absorption spectrum. Crammer and Neuberger (1943) first used the change in spectrum with p H t o measure the number and pK of these groups in proteins. With insulin they found that the spectrum ac-

1

11 Philpot and Small (1938) present evidence, though not from titration data, that nitrous acid attacks the tyrosine residues of pepsin to yield a diazo derivative of the protein.

HYDROGEN ION EQUILIBRIA

173

counts for all the tyrosine known t o be present, whereas with ovalbumin none of the phenoxyl groups dissociated until after the protein was denatured by heat, acid, or urea. The method has since been applied to other proteins: lysozyme (Fromageot and Schnek, 1950; Tanford and Wagner, 1954), human serum albumin (Tanford, 1950), and bovine serum albumin (Tanford and Roberts, 1952). With each of these three proteins the ionization of the phenoxyl groups is reversible (up to pH 12)) and the pK is abnormally high (10 t o 11). The view that this is an effect of hydrogen bonding t,o other groups in the molecule is strengthened by the high heat of ionization (11.5 kcal. per mole) observed for these groups in bovine serum albumin. 6. Contribution of Iun-Protein Interactions to Stoichiometry

Study of the purely electrostatic effect of unbound ions in the environment upon the titration curve of a protein (discussed earlier in Section II,3) yields no information on the kinds or numbers of dissociable groups. When ions are actually bound covalently to a protein, however, stoichiometric information may sometimes be obtained. For convenience the bearing of ion-protein interactions on titration curves will be considered in two parts: first, ion-protein interactions which are interpretable in terms of the stoichiometry of dissociable groups (this section) ; and second, ion-protein interactions that are not interpretable in this way but which nevertheless affect both the titration and electrophoresis of proteins (Section 11,7). a. Stoichiometric Combination. The number of dissociable groups of a given type may be determined directly from the composition of an ionprotein complex if they combine specifically and quantitatively with the ion. Since ion-protein combination is governed by the law of mass action, the degree of specificity of ions often depends upon their concentration. Highly dilute mercuric ion, for example, combines specifically with the sulfhydryl group of mercaptalbumin (Hughes, 1950)) whereas at higher concentrations combination with other groups (such as carboxylate) occurs. Under controlled conditions ions of either mercury or silver (Benesch and Benesch, 1948) can be used for the quantitative estimation of sulfhydryl groups. Zinc ions combine selectively with imidazole groups (Gurd and Goodman, 1952), but also have a smaller affinity for carboxylate groups. p H is also a determinative factor in the combination of ions with groups whose reactivity may depend on whether they are in their acidic or basic forms. The most common application of ion binding to stoichiometric investigations has been the use of certain acids, whose anions are tightly bound to protein, to indicate the number of cationic groups. This type of combination often results in precipitation of the complex, which permits direct

174

J. STEINHARDT AND E. M. ZAISER

determination of the amount of anion bound.I2 I n this way Perlmann (1941) has demonstrated that metaphosphoric acid combines quantitatively with the cationic groups of several proteins (ovalbumin, insulin, edestin, hemoglobin, serum albumin, rl-pseudoglobulin, myosin, and clupein), and has thus corroborated the values found for the maximum hydrogen ion-binding capacities. Among the numerous other anions which are effective precipitants for proteins and combine quantitatively with cationic groups, arc included such widely different substances as acidic dyes (Chapman, Greenberg, aiid Schmidt, 1927; Fraenkcl-Conrat aiid Cooper, 1944), AuC14- (Craig, Garrett, and Williams, 19541, aiid many anionic detergents (Putnam and Neurath, 1944a, 1945; Lundgren, 1945; Bull, 1946; Glassman arid Moliiar, 1951) . Similarly, quantitative combination with the anionic groups of proteins in basic solution has been reported for a number of cationic detergents (Polonovski and Macheboeuf, 1948; Glassmarl 1950; Glassman and Molnar, 1951; Timasheff and Nord, 1951). Some earlier values for maximum binding or dissociation of hydrogen ion based on titration with dyes are summarized by Schmidt (1944). Such methods of estimating total anionic and cationic groups must be used with discretion, as higl. results maj be obtained with detergents because of their tendency to combine with one another to form micelles, and low results may be obtained if the size and configuration of the dye or detergent ion causes its combination with the protein groups to be hindered sterically. When these agents denature the protein in the course of combining with it, they furnish information only about the dissociable groups in the denatured protein. 6. Eflect on Titration. Ions which are tightly bound to specific otherwise dissociable groups shed light on the number of these groups, even without resort to chemical analysis. Such binding may affect the titration curve by altering the pK’s of the groups involved, and differential titration may then establish the number and identity of the groups affected. This is not always possible, however; differential titration of P-lactoglobulin and its dodecylsulfate derivative by McMeekiri et al. (1949) did not reveal the identity of the groups involved, possibly because the two equivalents of dodecylsulfate bound did not occupy specific sites and hence gave only a small and nearly uniform shift of the titration curve to lower pH. Massey and Alberty (1954) showed that the pK’s of the groups concerned with the catalytic action of fumarase are affected by various anions, including the substrates, and considered this effect to play a role in the activating and inhibiting influence of these anions at different values of pH. By far the most numerous examples of the alteration of the acid strengths l2 This method hits also been applied in an opposite se~isc, e.g , the usc of trichloroacetic arid :is a precipitant in the assay of protein.

175

HYDROGEN ION EQUILIBlUA

2

3

4

5

6

PH

FIG.5. Titration of trypsin in the presence of CaCL , MgCln , and KCl. From Duke, Bier, and Nord (1952), with difference curve added (bottom).

of protein groups by combination with ions are furnished by bivalent metal ions. Some of these have marked influence on enzymic activity. Combination of metal ions with proteins frequently results in a shift of p H to a lower value because of displacement of bound hydrogen ion. Two interactions, those of calcium ion with trypsiii and zinc ion with insulin, are discussed here because of their bearing on titrations.13 Calcium ion has a very specific effect on trypsin (Gorini, 1951), and M n+ +and Cd* act similarly. However, other bivalent ions such as Mg*, Batt-, S F , Co++, Cu*, or Ni++are without effect (Nord and Bier, 1953). Duke, Bier, and Nord (1952) found that calcium increased the acidity of the carboxyl groups of trypsin between pH 3.5 and 5, and concluded that this was due t o chelation of Ca* with carboxyl groups.14 Their titration curves for trypsin in the presence of CaCh, MgCl, , and KC1 are shown in Fig. 5. A t pH below 3 the enhanced acidity due to Ca++ disappears, since chelation with undissociated carboxyl groups is impossible. Nanninga (1954) has made a similar observation in the titration of L-meromyosin in the presence of Mg*. Understanding of the metal-protein interaction between zinc and insulin has recently been enhanced by studies of titration curves. Crystalline insulin as normally prepared contains a small amount of zinc, and moreover is insoluble between pH 4 and 7, where p H drifts are also observed. Fredericq (1954) titrated two fractions of insulin which differed in their solubility a t pH 8, and reported that two imidazole groups which dissociate near pH 7 in the soluble form are not evident in the insoluble fraction. The combination of metal ions with proteins has been reviewed by Klotz (1953). Chelation involving alkaline earth cations and carboxylic acids has been studied in detail by Cannan and Kibrick (1938).Greenberg (1944)has reviewed t h e interaction between alkaline earth cations and proteins. l3

14

176

3. STEINHARDT AND E. M. ZAISER

PH FIG.6. Titration curves of insulin, zinc-free ( A ) and with 1 mole zinc per 11,500 g. ( B and C). C is the direct titration of zinc insulin, B the reverse titration with acid or base. A is completely reversible. Dat a of Tanford and Epstein (1954a, b).

Presumably his preparation of insulin contained zinc, and his two fractions may have differed in their zinc content. Tanford and Epstein (1954a,b) titrated zinc insulin (1/1) and zinc-free insulin (see Fig. 6), and discovered that in the absence of zinc the titration curve is fully reversible, even in the zone where precipitation occurs. In the presence of zinc, the titration is reversible except in the region where precipitation occurs, where back titration with acid and with base give identical curves which drift slowly back to the direct titration curve. These investigators ascribed the direct titration curve to crystalline zinc insulin, and the curve initially obtained on reversal to amorphous zinc insulin which slowly reverts to the more ordered form. Two imidazole groups which were titrated a t pH 6.5 to 7 in zinc-free insulin no longer dissociate in this region in the zinc compound (cf. the observation of Fredericq) but near pH 4 instead. Tanford and Epstein suggest that zinc ion is combined with these two groups, and that the degree of crystallinity of the insoluble zinc compound (pH 3.5 t o 7) is related to the degree of order in the chelation of the metal with pairs of the imidazole groups.16 7. Efects of Less Specijc Ion-Protein Combination on Titration and Electrophoresis of Proteins Ion-protein combinations which do not specifically involve dissociable groups shed no light on acid-base stoichiometry. The number of sites 1 6 Zinc ion, insulin molecules, and imidasole groups were present in the relative molar quantities 1/1/4, assuming the molecular weight of insulin t o be 11,500.

HYDROGEN ION EQUILIBRIA

177

available for this type of combination is not unique, but for a given protein at a fixed p H depends upon the identity of the ion which is bound. Both human and bovine serum albumins, for example, exhibit a wide range of affinities for different anions. Lack of demonstrable specificity for acidic or basic groups, however, is not necessarily associated with weakness of the ion-protein interaction. With serum albumins, although the heats of binding of anions (calculated from dialysis equilibrium data) are very small, favorable free energy changes ranging from -3700 cal. for chloride binding t o -8700 for dodecylsulfate have been calculated by Klotz (1953) from data of Scatchard, Scheinberg, and Armstrong (1950a) and Karush and Sonenberg (1949). This must be contrasted with the case of an insoluble protein, wool keratin, for which Steinhardt (1941) reported heats of dissociation of anions (determined from thermal effects on titration curves) ranging from around 2000 cal. for chloride to over 12,000 for the dye Orange 11. No survey of the voluminous literature on this type of ion binding will be attempted;16 attention will be confined to its effects on the amphoteric properties of proteins. Steinhardt, Fugitt, and Harris (1941, 1942) found that titration of wool keratin with a large number of different acids yielded a series of well-separated titration curves whose positions on the p H scale were related by these investigators to wide differences of affinity of the anions of those acids for the protein. A similar phenomenon was demonstrated for ovalbumin by the same authors (1941). The observed shifts can be described with fair approximation merely by taking into account the binding of anions and its effect of decreasing the net positive charge of the protein a t a given pH, an effect which results in an increase in the number of hydrogen ions which are bound at that pH. An analogous effect of the binding of cations was demonstrated by Steinhardt and Zaiser (1950) in their study of the titration of wool with a number of strong bases. In general anions are bound by proteins which have a net positive charge and cations by proteins which have a net negative charge.” Human serum albumiu, for example, is unusually prone to combine with anions-a property reflected in the specific effects of neutral salts on its isoionic point (Scatchard and Black, 1949). On the basis of dialysis equilibrium and electromotive force measurements Scatchard, Scheinberg, and Armstrong (1950a,b) established that thiocyanate ion combines with this protein a t more sites and more tightly than does chloride ion. The effect of the binding of ions on the net charge, and hence on the steepness of the titration curve, has already been referred to in the section on the theoretical analysis of titration data. The data of Scatchard and his co-workers on chloride The subject of ion-protein interactions is discussed fully by Klotz (1953). Proteins differ so very greatly in their tendencies to bind ions of either charge type, however, t h a t exceptions occur in t h e vicinity of t h e usual isoionic point I7

178

J. STEINHARDT AND E. M. ZAISER

binding provided Tanford (1950) with a partial explanation of the unusual steepness of the acid branch of the titration curve of human serum albumin. The titration of myosin (Mihitlyi, 1950) is undoubtedly affected by the binding of ions in the same way; a study of the effect of neutral salts on its isoionic point has been made by Ghosh and Mihitlyi (1952). Such nonspecific binding of anions is the cause of many of the discrepancies which exist between values of the average net charge estimated from titration curves and from electrophoretic mobilities. Disagreement does not exist when a protein has little or no affinity for neutral salt ions. Thus the ratio of net charge from titration data to electrophoretic mobility for 0-lactoglobulin is constant (Cannan, Palmer, and Kibrick, 1942). The agreement reported in the case of trypsin (Duke, Bier, and Nord, 1952) is more unexpected, since it indicates that this enzyme has little affinity for the usual buffer ions despite its strong and specific interactions with certain bivalent cations. These cases of agreement are exceptional, however; the great majority of electrophoretic studies yield mobilities which do not correspond to the apparent net charges indicated by titration curves. Two factors contribute to these discrepancies: (1) the titration curve gives net charge as a function of pH only if the binding of ions other than hydrogen ion is taken into account; and ( 2 ) the amount and kind of ions bound at a given p H may be different under the conditions of electrophoresis (buffer ionsusually present) and titration experiments (unbuffered solutions). Longsworth (1941) showed that a relatively constant value for the ratio of the electrophoretic mobility of ovalbumin to the net charge indicated by its titration curve was obtained only when the titration curves were shifted along the pH scale to coincidence a t the isoionic pH. Thus the titration data were, in effect, corrected for binding of chloride ion. This correspondence between electrophoretic mobilities and the “corrected” titration curve existed only when the mobilities were determined in buffered solutions of the same ionic strength and containing only monovalent ions. Evidently the diff erences in the affinities of ovalbumin for the particular small monovalent anions concerned must be small.1s In phosphate buffers mobilities differed ah much as 28% from those in buffers having monovalent anions. This type of effect on mobility is very commonly observed. One further instance is so extreme, however, as to be worthy of special mention. Velick (1949) found that the isoelectric point of aldolase in phosphate and ncetatcb buffers, as determined by electrophoresis, is much lower than would be be predicted from the amino acid composition, and varies strongly with the I @ This inference is not, how ever, iiiconsirtent n i th the demonstration by Stein himlt, Fugitt, ctnd Harris (1941) that certain other monovalent anions do infliienw the acid titration of ovttlbumin.

HYDROGEN ION EQUILIBRIA

170

ionic strength and with the particular buffer employed. Velick’s calculation that the binding of 7 phosphate or 10 acetate ions, per mole of aldolase, could cause the discrepancy was confirmed by the results of dialysis equilibrium studies. However, the net charge indicated by Velick’s titration data (pH G to 7 only) is higher than is compatible with the electrophoretic data, even after correction for the binding of anions; and the slope of the titration curve remains too steep, as in the case of human serum albumin. The electrophoretic data of Alberty and Marvin (1951) on bovine serum albumin, unlike those discussed above, were obtained in unbuffered solutions. They show that chloride ion binding increases as the solution becomes more acid; the indicated net charge on the protein is therefore less than that predicted from the titration curve. The extent of chloride binding found by these authors agrees well with that determined by Scatchard, Scheinberg, and Armstrong (1950a) for human serum albumin from dialysis equilibrium and electromotive force data.

8. Summary of Section I1 The study of acid-base dissociations of proteins makes important contributions t o the knowledge of their molecular structure and of their interaction with the environment. The method used at present for theoretical analysis of titration curves of soluble proteins (essentially a development of Linderstrgm-Lang’s early model) accounts adequately in most cases for the shape of the curves in terms of the numbers and equilibrium constants of different kinds of dissociable groups and the electrostatic interaction of the protein with hydrogen ion and other ions. This theory has proved widely valid, especially when account has been taken of the effects on net charge of combination with ions other than hydrogen ion. The values of the dissociation constants of particular sets of groups have sometimes contributed to understanding details of the internal structure of the protein, especially when the observed constants differ from those generally found in other proteins or in peptides. The electrostatic factor w which enters into the theoretical treatment depends upon ionic strength in a manner which in many cases adequately explains the effect of neutral salts on protein titration curves. Comparison of values of w from experiment and theory has, in addition, permitted inferences as t o the shape of the protein molecule or its state of aggregation. Although a comparable theoretical treatment has not been achieved for insoluble proteins, the effect of salts and the differences in the results obtained with different acids or bases are explained a t least in part as the result of combination of anions as well as hydrogen ion. The stoichiometry of the hydrogen ion eqyilibria of proteins has been of particular interest in the detection of interactions of elements of the protein structure which may render inactive some of the prototropic groups. Sev-

180

J. STEINHARUT AND X . M. ZAISEH

era1 methods have been applied to determine the number of titratahlc groups of a given class directly from the titration curve of a protein. Thc total number of catioiiic groups (imidaeolium, ammonium, and guanidinium) is usually determined as the maximum amount of hydrogen ion that can be bound. The maximum hydrogen ion dissociation, whic.li should equal the anionic groups (carboxylate) of the protein, cannot in practice be determined with any degree of reliability. Iri addition, certain segments of the titration curve may generally be attributed to the dissociation of particular groups, e.g., carboxyl from pII 2 to 5.5 and imidazolium from pH 5.5 to 8. Differential titration is a means for determinirig not only the numbers but the pK’s, aiid thus (often) the identities, of sets of groups from the difference between the titration curve of the normal protein and some modified form of it. This modification may consist in: ( I ) alteration of the pK of groups by chemical modification or change of solvent; (2) selective suppression of ionization of certain groups by chemical modification; or ( 3 ) shifting the dissociation equilibrium of the protein by changing the temperature. In addition] the combination of ions other than hydrogen ion can sometimes be related to the numbers of dissociable groups. Comparison of the numbers of dissociable groups from stoichiometric investigations with amino acid assays reveals whether or not all prototropic groups which would be free on the Fischer peptide model are artually free to react. In the few cases for which amino acid assays predict fewer prototropic groups than titration data reveal, it appears likely that the assays are too low. Thus improved assays for arginine, histidine, and lysine have removed, or at least greatly diminished, numerous former discrepancies of this type. Although the assays for aspartic aiid glutamic acids have been similarly improved, there are still a few proteins for which titration data continue to indicate more free carboxyls than assays predict, possibly because of the use of excessively high values for amide nitrogen. On the other hand, since amino acid assays are more often low than high, wherever they predict more prototropic groups than are titrated it is virtually certain that the internal structure characteristic of the protein is responsible for the failure of dissociable groups to participate in acid-base equilibria as expected. Such cases are considered in the following section of this paper.

111. UNREACTIVE PROTOTROPIC GROUPSIN NATIVEPROTEINS 1. Introduction

It has long been known that many proteius are extremely sensitive to even dilute acids (e.g., hemoglobin) and bases (e.g., pepsin). Preparative

HYDROGEN ION EQUILIBRIA

181

instructions have commonly cautioned against exposing material to strong acids for any length of time unless very dilute and a t very low temperatures. Nevertheless, the determination of their maximum combination or dissociation of hydrogen ion has necessarily involved working near the extremes of the pH scale. Whereas many investigators have acknowledged that this procedure introduced a strong possibility of denaturation, others ' have analyzed titration data with little regard to the consequences of this possibility. This disregard has undoubtedly been due to the widely prevalent impression that the titration curves of native arid denatured proteins differ only slightly, except possibly in the region of precipitation of denatured protein. After the electrophoretic iiiveatigations of Moyer (1938) on ovalbumin aiid Heidelberger and Pederseri (1935) on thyroglobulin it was appreciated that the isoelectric points of native arid denatured proteins might differ by as much as 0.5 p H unit. Shortly thereafter it was noted (Cohn and Edsall, 1943b) that the back titration of horse CO hemoglobin (hereafter COHb) exposed to solutions of pH below 2 gave very different results from those obtained by starting the titration in the vicinity of neutrality (Cohn, Green, and Blanchard, 1937). This difference is prima facie evidence ( I ) that denaturation occurred during the process of titration, and (2) that the acid-base properties of denatured COHb differ from those of the native protein. It follows that combination with some of the hydrogen ion must have occurred in the p H range in which the number or acid-strength of titratable groups was altered rather than in the p H range normally characteristic of those groups in the native protein. Nevertheless efforts were still made to treat the titration curve as if it represented a series of purely acid-base equilibria. It is now realized that the shape of the acid branch of the titration curve of this protein cannot be represented solely by means of such a series of equilibria, with or without interactions between the dissociating groups (Steinhardt and Zaiser, 1951). If all the prototropic groups of native proteins were normally reactive to acids and bases, this normal reactivity would constitute a significant exception t o the well-founded generalization that the functional groups (sulfhydryl, disulfide, phenoxyl) of native proteins are usually less reactive in native proteins than in proteins denatured b y any means (Neurath et al., 1944), just as the susceptibility of proteins to hydrolysis by proteolytic enzymes is usually increased by denaturation. 2. Evidence for Unreactive €-amino and Imidazole Groups from

End-Group Analyses The first direct indication that the prototropic groups conformed to the general rule resulted from end-group analyses by the 2,4-dinitrophenyI

182

J. STEINHAIZDT A N D E. M. ZAISER

(DNP) and 1,2,4-fluorodinitrobenzene (FDNB) method of Sanger (1945) and Porter and Sanger (1948). Porter (1948) found that the number of lysine e-amino groups in denatured 0-lactoglobulin, horse serum pseudoglobulin, and several cow and pig globulins which reacted with FDNR was in excellent accord with the best analytical data on the content of this amino acid in these proteins. However, when the corresponding native proteins were treated with this reagent under identical conditions, substantially fewer groups combined. In p-lactoglobulin, 40 % did not react; in horse serum pseudoglobulin, 33 %; and in cow and pig p- and yglobulins, 38 % to 45 %. 111 native bovine serum albumin as in insulin, on the other hand, all the lysine groups combined with the reagent. The fact that the e-amino groups in bovine serum albumin react almost quantitatively must, be interpreted with caution. Porter and Sanger (1948) reported that similar quantitative reaction was obtained with native as well as denatured hemoglobins and globins and concluded that in these proteins the end-groups possessed normal reactivity. It is shown below that a t least one-half of the lysine groups of horse, pig, and human henioglobin are not free to react with acid until after they have undergone, by exposure t o dilute acid (pH below 4), a time-dependent alteration which is indistinguishable from denaturation as usually defined (loss of solubility a t isoelectric point). It is therefore natural to inquire whether the treatment of Sanger when applied to some native proteins does not itself c a u e them t o become denatured. This is surely the case with hemoglobins and globins. Sanger’s method involves prolonged exposure (2 hours at room temperature) to over 60% alcohol in the presence of the reagent and sodium bicarbonate (Sanger, 1945). This concentration of alcohol in the presence of saturated bicarbonate denatures about two-thirds of the protein in less than 2 minutes.lg Even when i t can be shown that the environment in which reaction with FDNB occurs does not itself denature proteins, the combination with FDNB may itself affect an existing equilibrium between native and denatured protein, thus permitting denaturation to proceed to completion. The results of treatment with FDNB are qualitatively unequivocal when 19 Thus about two-thirds of a dilute solution of horse ferrihemoglobin exposed t o this solvent loses its solubility at the isoelectria point in the presence of 12.5% sodium sulfate. I t s abeorption in the region of the Soret band also changes toward t h a t of denatured ferrihemoglobin, i.e., the peak a t 404 mp diminishes sharply, and a shoulder forms on the ultraviolet side, consistent with the presence of a maximum at about 370 mp uhich characterizes denatured ferrihemoglobin. In the case of 8-lactoglobulin Porter has presented evidence t h a t ethanol denaturation does not occur in the presence of FDNB, even though denaturation is fairly rapid in the ah sence of this reagent. No such evidence has been obtained for the hemoglobins or for bovine Berum albumin.

HYDROGEN ION EQUILIBItIh

183

fewer than all the groups present combine. When all the groups react, the results must be interpreted in the light of other evidence. The results obtained with FDNB do not always agree with those obtained with the smaller molecules, ketene and acetic anhydride, which also react with free amino groups (although under somewhat different conditions). Treatment of p-lactoglobulin with ketene a t about p H 5 in the cold results in acetylation of all the eamino groups. With horse pseudoglobulin, however, the same number of lysine groups which fail t o combine with F D N B also fail to undergo acetylation with either ketene or acetic anhydride. Nothing is known as t o why certain e-amino groups of lysine in native protein are not normally reactive. However, if this lack of reactivity extends to their acid-base function (as in the case of hemoglobin described below), then it is difficult to believe that the “masking,” whether steric or otherwise, does not involve a corresponding number of carboxyl groups. Any substantial discrepancy between the number of “masked” amino and carboxyl groups would be reflected in a large shift in isoelectric point. While a moderate shift in isoelectric point (up t o about 0.5 p H unit) does, in fact, often occur as a result of denaturation, the isoelectric p H in native proteins would be substantially lower than it is found to be if lysine groups alone were unreactive. Evidence will be presented later that the “masked” lysine groups are in fact linked with, or matched by, approximately equal numbers of masked carboxyl groups in a t least one group of proteins. The method of end-group reaction with FDNB has also shown that in native P-lactoglobulin half of the more weakly basic prototropic groups of the histidine imidazole ring are not free to combine (Porter, 1950). A smaller proportion of imidazole groups in zinc insulin is also unreactive (the proportion changes on conversion to the fibrous form). The imidazole groups of ovalbumin and bovine serum albumin, however, are fully reactive. Porter’s finding that all of the histidine of hemoglobin is also fully reactive, tends to support the view expressed above that treatment with alcoholic FDNB denatured the proteins and destroyed the specific hemeglobin linkage. It has long been known that histidine is involved in this linkage, and it will be shown below that nearly one-quarter of the total hemoglobin histidine (two histidines per heme) are probably blocked when hemin is combined in the native protein.20 2 O Since there is reason t o believe that hemin also combines with denatured globin (as uell as v i t h other proteins), i t folloms that this nonspecific combination does not involve the same histidine residues. The Soret band of “acid hematin” in organic solvents and the Soret band of native ferrihemoglobin in water have similar maaima a t about 404 mp; but this maximum shifts from 404 mp t o 379 mp when other proteins are added t o the solution of acid hematin. Likewise, when ferrihemoglobin is denatured the absorption maximum of this band in water shifts from 404 to about 370 mp, near its position when hemstin is added t o other proteins.

184

J. STEINHARDT AND E. M. ZAISER

3. Titrimetric Criteria for Liberation of Unreactive Prototropic Groups

Two of the other prototropic groups of native proteins, the phenoxyl group of tyrosine and the sulfhydryl group of cysteine, are often partially unreactive to specific reagents. In only a few proteins, however, is anything known as to their availability in acid-base reactions, primarily hccause the titration curve of proteins with bases is difficult to analyze and the maximum number of dissociable hydrogen ions is commonly undetermined (see the discussion of stoichiometry in Section I1 of this review), and because titration with base is often demonstrably accompanied by irreversible chemical changes such as disulfide cleavage. Neither of these groups contributes to the titration curve with acid, to which much of the subsequent discussion will be confined. The other principal prototropic group is the guanidinium group of arginine. The position of the isoelectric point in most proteins demonstrates that this group must be free to accept protons from the carboxyl groups of aspartic and glutamic acid unless corresponding numbers of carboxyl groups are themselves unavailable in the native protein. Tanford (1954) has shown that there is no detectable tendency of guanidinium and acetate carboxyl t o associate. This evidence, however, is not fully conclusive for their reactivity in the native protein. The results of end-group analyses indicate that the maximum combination or dissociation of hydrogen ion by native proteins may be affected if groups are unreactive to hydrogen ion as well as to FDNB and are not “unmasked” by concentrations of acid or base encountered in the range of titration; if unmasking does occur the presence of such groups will affect the shapes of titration curves and the factors which must be considered in their analysis, even if the maxima are not affected. Unmasking, when it occurs, must be characterized ( 2 ) by differences between certain portions of the titration curves, or in the electrophoretic mobilities, of native and denatured proteins; (2) by noticeable pH drifts in the unmasking range (provided the unmasking reaction is neither too fast nor too slow to be observed with the electrode system employed) ; and (3) by differences between the titration curves as normally obtained and the corresponding back-titrations. In addition, (4) the shape of the titration curve should be anomalously steep in the pH region in which previously unavailable groups become available.21 When unmasking occurs during titration, the observation of maximal combining capacities in agreement with the analytical data on amino acid content, depends on whether or not residual stable cyclic configurations occur. When unmasking does not occur, one should look for marked discrepancies with analytical data in all cases where the end-group method has shown unreactive groups. However, as will be seen later, lack of reactivity t o FDNB need not necessarily imply lack of reactivity t o hydrogen ion.

HYDROGEN ION EQUILIBRIA

185

The signs of unmasking listed above can be manifested, of course, only if some of the prototropic groups are uiireactive in the native protein. It is well to bear in mind, however, that the observation of unmasking also depends 011 the susceptibility of the protein examined to denaturation by acid or base in the pH range of the titration curve at the temperature (usually 25" C.) at which it is determined, since the effects of pH and temperature on the velocity of denaturation are closely interrelated (Steinhardt, 1937). However, it will be shown that all proteins now known to have anomalously low hydrogen ion-binding capacities (see Section 11) show some or all of t,he four signs of unmasking listed when measured a t 25°C.

4. Early Titrimetric Evidence The early evidence bearing on differences in the acid-base equilibria of native and denatured proteins has been summarized in detail by Neurath Pt al. (1944). No differences were observed in some early work on hemoglobin (Lewis, 1927) or ovalbumin (Booth, 1930; Prideaux and Woods, 1932; Loughlin, 1933). Other investigators reported distinct effects with ovalbumin (Hendrix and Wilson, 1928; Wu and Chen, 1929; Wu, Liu, and Chou, 1931; Chou and Wu, 1934; Crammer and Neuberger, 1943) and with collagen denatured by salts (Neurath, 1940) or by heat (Theis and Jacoby, 1943). The data given for ovalbumin in several of these papers are inconsistent in detail with one another and must be considered to be inconclusive. However, the spectrophotometric titration data of Crammer and Neuberger (1943) establish that the tyrosine phenoxyl groups ionize only after the protein is denatured, and are thus fully consistent with the conclusion of Cannan, Kibrick, and Palmer (1941) that these groups do not dissociate in the native protein. Substantial differences in the electrophoretic behavior of native and denatured proteins in the region of the isoelectric point have been reported for ovalbumin (Moyer, 1938), serum albumin (Pace, 1931), and thyroglobulin (Heidelberger and Pedersen, 1935). When unbuffered ovalbumin is denatured in various ways (surface (Wu, Liu, and Chou, 1931; Bull and Neurath, 1937), heat (Bull, 1938), urea (Wu, Liu, and Chou, 1931), and ultraviolet radiation (Bernhart, 1939)), the pH of the solution rises. Thus, in all of these proteins a t least some of the signs of unmasking of unreactive groups listed in the preceding paragraph are actually observed. The electrophoretic evidence does not include a demonstration that liberation of masked groups may be brought about by acid or base in the range of the normal titration procedure at the temperature at which it i s carried out, and is thus incomplete.

186

J . STEINHAlt1)T AND E. M . ZhISEIZ

6. Unrcactivc Prototropic Groups in the Hemoglobins and Globins

a. I’itrimetric Evidence for Unmasking of Groups. All of the criteria of unmasking listed above were observed when both horse COHb and ferrihemoglobin were titrated with acid by Steinhardt and Zaiser (1051, 1953). By using a flow technique, however, which permitted making pH measurements within 3 seconds after mixing, these investigators succeeded i l l 01)taining for the first time a considerable portion of the acid titration curves of these proteins in the native state. Representative results with 0.4% horse COHb are shown in Fig. 7. No data are shown for values below pH 2, since a t this pH about 40% of the acid added remains uncombined with the very dilute protein, and the calculation of acid bound gives less exact results. The curve labeled 24 hours can be shown to represent attainment of an equilibrium; it may be obtained after any interval longer than about 12 hours. The back-titration curve, which much more closely resembles a “normal” protein titration curve, such as that of ovalbumin, drifts slowly back toward the “24-hour” (equilibrium) curve (Steinhardt and Zaiser, 1951). With ferrihemoglobin, which represents a much more completely reversible system (see below), the back-titration curve must be measured with a rapid-flow technique also, since the p H drifts rapidly until the hacaktitration is almost exactly superimposed on the equilibrium curvc. As the figure shows, there are great differences between the results ohtained when initially isoelectric native protein is exposed to various concentrations of dilute acid for 3 seconds, 10 minutes, and 12 hours or longer. On

FIG.7. The amounts of IlCl combined with COHb (0.4%) a t constant chloride concentration a s a function of pH and time. The two curves in the inset are obtained by difference: Curve 1, by subtracting the 3-second data from the 24-hoiir data; Curve 2, by subtracting the 3-second data from the back-titration data.

HYDROGEN ION EQUILIBRIA

187

the acid side of pH 4.2 there is a considerable range over which only half as much hydrogeii ion is bound instantaneously (3 seconds) as after some hours. The data shown for 10 minutes agree closely with the curves in the literature (Cohn, Green, and Blanchard, 1937), but both are wholly arbitrary sincc they represent neither instantaneous nor final values-neither wholly native uor wholly denatured protein, nor equilibrium mixtures of both. The data for 3 seconds, at pH values down to about 3, however, represent an instantaneous prototropic equilibrium involving unchanged COHb. Below this pH, appreciable denaturation occurs in less than 3 seconds and the color of the flowing material darkens. The data obtained in 10 minutes, 011 the other hand, represent darkened (denatured) material below about pH 4. When measured in 3 seconds, the data obtained down to p H 3 may be obtained by back-titration as well (if protein kept a t p H above 3 for less than 3 seconds is used), and thus represent a truly reversible equilibrium. Between pH 3 and G.6, the 3-second data represent titration of the native protein; the back-titration data represent the titration curve of protein in which the previously unreactive groups have been liberated. The difference between these curves (curve 2 in Fig. 7) thus represents the titration curve of the previously unreactive groups liberated by exposure to acid. It is clear from curve 2 that the pH range covered is so wide (over 3 units) that a t least two sets of groups having distinct dissociation constants must be invoked in any explanation. The maximum difference in hydrogen ion bound, 0.52 mmole per gram, is constant between about p H 3.1 and 3.5, and corresponds to at least 35 groups per molecule of COHb. It is assumed hereafter that the true figure is 36, since a number divisible by 2 is to be expected in a protein that dissociates into fragments which retain many of the significant properties of the aggregate (Steinhardt, 1938). The apparent decrease in the difference at pH below 3.1 is due to the fact that unmasking occurs so rapidly below this pH that the 3-second curve no longer represents unchanged native protein. Fortunately the increment, 0.52 mmole, is constant over a wide enough range of denaturation velocities, over tenfold (see below), to indicate that the maximum found is close to the true one, and corresponds to the number of groups initially unavailable for combination with hydrogen ion. This result with horse COHb has recently been confirmed for human oxyhemoglobin by Kistler, Buri, and Nitschmann (1953). An incremental binding of 0.50 mmole hydrogen ion per gram, corresponding to the unmasking of about 34 groups, was found at pH near 3.2. No details of the titration curve are reported. Almost exactly similar results have been obtained with horse ferrihemo-

188

J. STEINHARDT AND E. M. ZAISER I

II 1

I 2rrihsmbglobin Total chloride 0.02M-25” 3 9

3 seconds 2 rnmules

0

22 hours

id back titralm-3 seconds +

back lttral!On-

5

4

6

PH

FIG.8. Amount of HC1 combined with 0.4% ferrihemoglobin. Inset lower curvrs are obtained by subtracting titration curve a t 3 seconds from “equilibrium” curve (2 t o 22.5 hours) without correction (solid line) and with correction (see text) of 3second data (dotted line). Inset u p p er curves are obtained by subtracting 3-second titration from 3-second back-titration curves of COHb (solid line) and ferrihemoglobin A, with %second data for ferrihemoglobiri 0 included for comparison.

globiri (methemoglobin), which is even more sensitive to acids than COHb. The curves for native protein (3 seconds) are identical with those for COHb over a wide range. The chief difference (Fig. 8) is that the unmasking reaction is much more rapid in both directions (it is fully reversible). Thus, some new groups are liberated in 3 seconds a t p H values (final) as high as 4.0instead of 3.2.22 The position of the equilibrium curve is shifted t o higher pH values also, indicating that the reverse reaction is not as much faster as the unmasking reaction. Nevertheless, the reverse reaction is so fast that it is necessary to obtain the back-titration curve by a 3-second (flow) technique also. As the figure shows, the equilibrium curve is obtained within a few hours whether it is approached by titration with acid or by back-titration with base. The almost quantitative rapid reversibility of the unmasking reaction in this protein thus offered attractive possibilities for a complete kinetic and thermodynamic analysis of unmasking and its relation to denaturation. The inserted curves a t the bottom of Fig. 8 show the digerence between 22 Solutions with final pH values below this figure are exposed briefly t o much more acid reactions with consequently very rapid denaturation which cannot reverse fully in 3 seconds.

HYDROGEN ION EQUILIBRIA

189

the acid bound in 3 seconds, on titration with HCl, and on back-titration with KOH, for both COHb and ferrihemoglobin. The upper continuous curve represents the results for COHb. The curve traced by the open triangles a t equal pH intervals represents the digerences with ferrihemoglobin. The two sets of data agree down to p H values a t which the 3-second technique breaks down, as explained above. Thus, in this range, the groups liberated in both proteins have the same dissociation constant and are equal in number. Also shown on the upper curve of the insert are small circles representing the 3-second titration data from the main figure. It will be observed that the diference data and the 3-second data are practically superimposed a t pH values between 4.0 and 4.9 (the lower of these values is a t the limit of the range of validity, with ferrihemoglobin, of the 3-second technique). Thus, the number of groups liberated by acid is equal in this range to the number of initially titratable groups, i.e., j u s t one-half of the groups in native ferrihemoglobin (and COHb) which combine with hydrogen ion in this p H range are initially unavailable for such combination. Advantage has been taken of this simple relation (delayed acid uptake equal to initial acid uptake) to extrapolate the 3-second titration data (and therefore the digerence curve as well) to p H 3.5 (broken line through solid triangles). The extrapolation of the difference curve lies so close to the COHb difference curve that it has not been drawn; this similarity again shows that the maximum number of groups liberated must be about the same (36) in both proteins, corresponding to approximately 0.52 mmole of acid bound per gram of protein. b. Identity of Groups Liberated. Because the difference curves in Fig. 7 (curve 2) and Fig. 8 cover a wide range, they must be fitted by the titration curves of a t least two sets of groups. If, for example, each set corresponds t o just half of the total number of liberated groups, then the pK values characterizing these sets would be approximately 4.4 and 6.1. The first of these values (which would be slightly higher if the more acid set is present in larger amount than the less acid set) can only represent carboxylate (COO-) groups; the second set (which may be the smaller of the two) can only represent histidine. An independent method for demonstrating the involvement of carboxyl groups is based on the lower solid curve of the insert of Fig. 8 (a similar curve is shown in Fig. 7), which shows the difference between the instantaneous data (3-second) and the equilibrium (22-hour) data. Thus it should represent a product of two functions of pH: (1) the fraction of the total protein a t equilibrium in which 36 groups have been liberated, and ( 2 ) the fraction of the new groups which combine with acid at the same

190

J . STEINHAIZ1)T

Axil)

E. M. Z A I SE I

FIG.9. Dependence on pH of ferrihemoglobin denaturation equilibria in HC1-KCI solutions, = 0.02: A = fraction of maximum number of extra basic groups titrated in 0.4% ferrihemoglobin; B = fraction denatured by spectroscopic criterion in 0.06% ferrihemoglobin.

pH. Since the first function can be measured in other ways (as, e.g., spectrophotometrically), the second function can be ~ a l c u l a t e d . ~ ~ This has been done with the data for ferrihemoglobin (the spectrophotometric estimate of the fraction denatured is explained later in this paper); and it has been found that the liberated groups do not combine quantitatively with acid in the p H range of their liberation (Fig. 9). The fractioii combined with acid rises from about 25 % to 30 % a t pH 4.4 to over 80 CTO a t pH 3.8. The empirical “mid-point” pK of the liberated groups is therefore about 4.0 or 4.1, serving to identify them as predominantly carboxyl.74 Although there are 51 carboxyl and 36 histidine groups in hemoglobin, more than enough to account for these increments, it must be recalled that the only carboxylate groups which can take up protons are those which 2 3 As in the case of the upper solid curve, the loner solid curve falls a t low plI, because the 3-second technique does not give truly instantaneous values a t pH murh below 4. By making use of the same extrapolation device previously referred to, the broken curve is obtained, indicating that the same maximum of 0.52 mmole per gram is obtained here also. 24 As originally published, the equilibrium curve A (Fig. 9) was “corrected” for a difference in protein concentration between the spectrophotonietric and titrimetric experiments. When so adjusted the two curves were close enough t o encourage the conclusion that the liberated groups combined quantitatively with hydrogen ion. The correction made for concentration now appears t o be unjustified. Although a small correction for concentration difference must probably be made (since the regeneration reaction is not strictly first-order), the correction which can actually be demonstrated experimentally is quite small. Thus the conclusion in this review t h a t combination of liberated groups with hydrogen ion is incomplete in thin pH range cannot be avoided.

HYDROGEN ION EQUILIBRIA

191

have donated protons to guanidino groups in arginine residues, and to amino groups (predominantly t-amino groups of lysine). Thus, if the number of carboxylate groups which can combine with hydrogen ion increases, it must be postulated that the number of e-amino groups of lysine which accept protons from carboxyl groups increases, i.e., that a large number are initially unreactive, as Porter has established in a number of other proteins. Moreover, since the isoelectric point of native hemoglobin is near neutrality, and within the histidine titration range, the liberation by acid of 36 amino groups must be accompanied by the liberation of a corresponding number of carboxyl groups (or carboxyl groups plus a small number of imidazole groups). Thus, the analytical data (Tristram, 1949) indicate there are in addition to 51 carboxyl groups (uncombined as amides) and 36 imidazole groups, about 38 lysine residues and 14 arginine residues. The carboxyl groups are approximately equal to the sum of the strongly basic groups (t-amino and guanidino); thus the isoelectric point should be expected to fall squarely in the histidine range, as it does in the denatured protein. If the native protein contains only 2 reactive t-amino groups (38 minus 36), and only 15 reactive carboxyl groups (51 minus 36), the isoelectric point mill be slightly lower, but still well within the histidine range. On the other hand, if all the carboxyl groups of the native protein are free to react but half the e-amino groups are not, the isoelectric point of the native protein mould be a t least one p H unit lower, in a region where carboxyl and imidazoliuni dissociations overlap. In accordance with this reasoning it must be assumed that a little over half (about 2 ) , $ ~ ) of the total histidine buffering capacity in the native protein is exerted in the p H range below the isoelectric point. This assumption is i n fair agreement with Wyman’s (1939) careful study of oxyhemoglobin. It is clear from the titration curves (3-second curve) that about half of the histidine is demonstrably titrated in the unchanged proteins from pH 6.G to 5.4, a range in which little participation of carboxylate is to be expected. If a few imidazole groups are included along with carboxyl in the 36 liberated, the picture is not greatly changed, and remains consistent with Wyman’s data. The evidence therefore strongly favors liberation of an equal number of carboxylate (or carboxylate plus a few imidazole) and lysine t-amino groups, hut cannot exclude the possibility of participation of guanidino groups in the total basic set of 36. Confirmation of this model and definite exclusion of the guanidino could be arrived a t by making similar rapid titration measurements in the pH range 6.6 to 10. .Ilthough such measurements have not been made with hemoglobin itself, measurements made by Roche nearly 25 years ago on regenerated pig globiii furnish an opportunity to confirm these views.

192

5. STEINHARDT AND E. M. ZAISER

Roche (1930) prepared regenerated globin by exposing hemoglobin briefly in the cold to a minimal amount of acid (apparently pH 2.3), and then adsorbing the hematin liberated on ether-saturated kieselguhr before neutralizing the acid. He determined the amounts of acid and base required to bring solutions of definite amounts of the regenerated protein to a range of pH values between about 2.3 and 10.5. Roche’s data, interpolated and recalculated from his figures (no tables are given) to correspond to the units used in Figs. 7 and 8, are given in Fig. 10, together with the 3-second data obtained with COHb. It is apparent that Roche’s results would be in fairly good agreement with the COHb data at pH above 3.5, where the 3-second data represent unchanged protein, but for the fact that above pH 4.5 globin binds more hydrogen ion by an amount corresponding to about eight imidazole groups per molecule of weight 68,000 (these eight groups are not available in either native or denatured COHb in this pH range). Roche observed that when hemoglobin solutions were allowed to remain in acid (about pH 2.3) in the cold for 2 hours before neutralizing, a denatured form of globin, insoluble at the isoelectric point, was obtained (it had lost the power to regenerate on neutralizing). This material, paraglobin, bound much more base than regenerated globin. The titration data for paraglobin are included in Fig. 10 (the curves for globin and paraglobin

PH

FIG.10. Dissociation curves of globin (solid line) and paraglobin (broken line) from interpolated graphical data of Roche (1930). Open circles are 3-second titration data and solid circles are back-titration for COHb (Steinhardt and Zaiser, 1!151). IAower curve (inset) represents the difference between globin and paraglobin curvcs, and 0 the difference between 3-second and back-titration curves for COHh.

HYDROGEN ION EQUILIBRIA

193

have been made comparable by making them coincide a t p H 6.8).26 It is noteworthy that the data for paraglobin are practically identical with the back-titration curve of COHb shown in Fig. 10 except for the appearance of the effect of the same eight histidine residues in the heme-free protein. The insert a t the bottom of the figure shows the difference between the amounts of acid or base required to bring solutions of the same concentration of globin and paraglobin from pH 6.8 to the same pH. The maximum difference found a t pH values on the acid side is almost exactly 0.52 mmole per gram, the figure found by Steinhardt and Zaiser for COHb and ferrihemoglobin (the corresponding difference curve for COHb is also given in the inset, Fig. 10). Thus, as in horse hemoglobin, almost exactly double the amounts of acid are bound by the denatured protein. Of greatest interest, however, is the fact that a factor approaching 2 also appears to apply on the alkaline side of pH 6.8. Thus it is clear that approximately equal numbers of e-amino groups and acidic groups are unmasked when globin is denatured (converted to paraglobin). Roche obtained his titration data by the usual methods, i.e., without recourse t o a rapid-flow technique. Although his values, therefore, probably represent intervals of 10 minutes or more after mixing, rather than 3 seconds, his curve for globin is without the excessive steepness a t low p H that would signify that partial transformation to paraglobin had occurred. Clearly, therefore, “native” globin is much more stable t o acid than is native oxy-, carboxy-, or ferri-hemoglobin. Thus, combination with heme or hematin or changes in molecular configuration accompanying the combination render these proteins highly unstable.2‘j A recent report that the regeneration of globin is retarded or prevented, in part, by the presence of even small amounts of hematin (Tristram, 1949) may support this conclusion. However, since there is a pH-dependent equilibrium between native and denatured hemoglobins over a considerable range of pH, the observation cannot be generally applicable; it may apply only to the higher p H range in which denatured protein is precipitated. Such precipitation may be favored by the presence of hematin. 2 5 The d a t a for globin and paraglobin were obtained with solutions which differed slightly in salt concentration. This uncertainty is probably comparable in effect t o t h a t introduced by arbitrary selection of t h e p H a t which t h e curves were made t o superimpose. A shift of this pH t o 6.6 or t o 7.0 would have only a small effect on the difference curves described in the text. * e Hematin obviously combines with denatured globin, a s well as with other proteins (see footnotes 19 and 20), but i t does so i n a different way than when it combines with regenerated globin, Combination with denatured globin gives cathemoglobin, which on reduction in alkaline solutions yields a spectrum very similar t o t h a t given by heme with practically all nitrogenous bases. This nonspecific association with denatured protein occurs, a t least t o some extent, even a t pH values at which the native heme protein is split.

194

J. STElNHAltDT AND E. M . ZAISE11

c. Mechanism of Mashing. It will be shown later that not all proteins that have large numbers of unreactive groups are heme proteins. It is therefore expedient to discuss the nature of the masking first in general terms applicable to any protein before entering into the complicated field of heme-protein interactions. The nature of the binding (if any) between the acidic and basic groups remains obscure. Hydrogen bonding through a single loosely bound hydrogen ion cannot, be excluded and seems inherently much more likely than “salt-linkages.” However, as will be shown later, the energy change in unmasking is quite small, much less than would be expected to result from breaking 36 hydrogen bonds. The possibility that the impediment to combination before unfolding is entirely steric cannot be ruled out. In this case the forces holding the molecule together may come principally from a very small number of other bonds (trigger groups). The number of pairs (36) is so large as to suggest that two complementary molecular surfaces, or edges, may occur opposite t o one another in the native moleFurther fragmentary support for a steric interpretation will be mentioned later. Whatever the reaction that liberates the masked basic groups may be,2x it is indistinguishable experimentally from denaturation as usually defined. Equilibrium mixtures showing less than the full increment of acid-binding groups will, on neutralization in the presence of salt, precipitate an amount of denatured protein exactly equivalent to the proportion of “delayed” acid uptake characteristic of these mixtures. The implication that the liberation of all 36 pairs occurs in a single step (i.e., all molecules contain either the number of groups characteristic of native protein or the number of groups characteristic of denatured protein) has been thoroughly demonstrated by kinetic work on COHb (Zaiser and Steinhardt, 1951) and by kinetic and equilibrium studies on ferrihemoglobin (Steinhardt and Zaiser, 1953; Zaiser and Steinhardt, 1954 a,b) which will be described later. The denatured protein present in equilibrium mixtures (or by itself a t 27 Since native hemoglobin exists in acid solutions as a half-molecule (GralSn, 1939), the molecular-kinetic unit contains 18 pairs of masked groups, rather than 36. 28 Lumry and Eyring (1953) have recently stated that the appearance on acid denaturation of 36 new groups is “probably due t o the 36 acyl rearrangements possible when the secondary structure of the peptide is broken open.” Reference is made t o the rearrangement involving the peptide bond and serine or threonine studied by Elliott (1952), although Elliott’s work was done in much more strongly acid solutions and at higher temperatures. It does not appear t h a t such a n “acyl shift” need be invoked for this protein, not even (as appears later) for the reaction of the smaller Tanford (1955) has concluded number of trigger groups. NOTEADDEDI N PROOF: that there is no evidence for the occurrence of a n acyl shift during the titration t o pH 2 of ribonuclease, lysozyme, 8-lactoglobulin, ovalbumin, human serum albumin, or insulin.

HYDROGEN ION EQUILIBRIA

195

pH values below about 3.1 for COHb and 3.45 for ferrihemoglobin) is not, however, the paraglobin described by Roche (1930). Much longer exposure to acid is required before globin is quantitatively converted to p a r a g l ~ b i n . ~Moreover, ~ the denatured globiri (or hemoglobin) produced in these experiments regenerates rapidly and practically quantitatively a t pH values only slightly higher than those causing denaturation, and paraglobin does not. For these reasons it is referred to hereafter as Hemoglobin-D or Globin-D. d . Trigger Groups. Which groups in the protein interact with hydrogen ion to initiate the unmasking reaction? The equilibrium range of p H (3.1 to 4.2 with CO hemoglobin, 3.4 to 4.5 with ferrihemoglobiii) is characteristic of the dissociation of carboxyl groups. However, the groups liberated, half of which have already been tentatively identified as carboxyl, combine with quite large amounts of hydrogen ion a t pH values substantially above those at which even smaller amounts of unmasking occur; in fact they combine measurable amounts of hydrogen ion to well above pH 5.5. The dependence of rate of liberation on pH, to be discussed below, suggests that the number of trigger groups is small (possibly as small as two or three). The liberation of much larger numbers of groups than the number of trigger groups, would of itself act as an amplification factor of the very small amounts of acid combined by the trigger groups themselves at pH values as high as 5.5 or G. Such an amplification factor, however, would not be large enough to account fully for the discrepancy noted, and it must be concluded that the trigger groups are characterized by a somewhat lower pK value than the large number of groups liberated. Two possibilities present themselves: (1) the two propionate side chains on the heme; ( 2 ) two heme-linked imidazole groups per heme (as suggested by the difference between Roche’s globin and paraglobin data) which masquerade as stronger acids in the native protein because of an effect of the heme linkage on their acid strength. It is not possible to distinguish here between these two possibilities. Kinetic and thermodynamic data discussed later appear to show that the dissociation constant of the trigger groups differs i n the native and denatured protein. This requirement may favor groups involved in the specific heme linkage, i.e., the heme-linked imidazole groups of histidine. The acid strength of such groups might well differ in oxidized and reduced heme proteins such as ferrihemoglobin and COHb.30 2 9 Paraglobin formation may require lower p H than suffices t o produce the denaturedprotein in equilibrium mixtures. Thus, solutions of COHb exposed t o p H 1.9 absorb more strongly a t < 550 mfi than solutions exposed t o pH 2.5 t o 3.1 (absorption independent of pH in this range). 30 German and Wyman (1937) have shown t h a t certain imidazole groups in oxyhemoglobin change their dissociation constants on removal of oxygen. These groups, however, appear t o be weaker acids (pK about 5.7) than those hypothesized here, as

196

J. STEINHARDT AND E. M. ZAISER

e. Relation of Unmasking to Heme Splitting. The foregoing discussion makes it pertinent to enquire whether the liberation of 36 pairs of masked groups is initiated, accompanied, or followed by the separation of hemin from the protein, or by the destruction of the specific heme-protein linkage that is known to occur under approximately the same conditions as result in unmasking. A complete discussion of this question must be reserved until analysis of the kinetics and thermodynamics of the unmasking reaction is complete, but certain qualitative observations may be cited here. Spectrophotometric examination of ferrihemoglobiri in HC1 or formic acid a t pH 3.1 does not indicate that the heme is entirely present in the free form, but does suggest that the specific heme-globin linkage has been altered. Thus the intense Soret band found a t about 404 mp in the native protein becomes much less intense and its maximum is shifted to about 379 mp. This position is quite different from that of hematin alone, 400 mp, in organic solvents (Heilmeyer, 1943), but is very similar t o that which is found with hematin in glacial acetic acid or alcoholic HC1 in the presence of other proteins, i.e., gelatin (Hicks and Holden, 1929). Absorption in the red also shifts characteristically but differs appreciably from that of hematin itself in acid solutions (Lemberg and Legge, 1949; Heilmeyer, 1943). Thus, unless these differences from hemin or hematin are not attributed ad hoc to an effect of changes in state of aggregation, they seem to point to continued combination of hematin with the protein, although in a diflerent manner f r o m its combination with native protein. This conclusion is supported by the fact that all or nearly all the hematin present is precipitated with the denatured protein when ferrihemoglobin denatured by dilute acid is brought back rapidly to the isoelectric point. It appears very likely that only a small part, if any, of the hemin exists free in aqueous solutions a t pH 3. It is a common feature of the numerous met,hods of separating globin from heme or hematin that one component is removed from the mixture by adsorption, as on kieselguhr (hematin); or precipitation, as by acetone (globin). It is, in principle, impossible to determine the amounts of split products present in labile equilibrium by removal of one component, since removal of one product of the dissociation is bound t o result in a shift of the equilibrium point in the direction of increased dissociation. I n addition, when the medium is changed, as by addition of acetone, profound changes must occur in the acidic dissociations in the protein which affect the reaction (see Section I1 of this review). Both of these effects would ~~

~~

~~~

~

do the groups (pK 5.3) inferred by Coryell and Pauling (1940) on the basis of magnetic susceptibility measurements in ferrihemoglobin. If the heme carboxyls are involved in the protein linkage, as Holden (1945) believes, they appear t o be a more reasonable choice.

197

HYDROGEN ION EQUILIBRIA

1

'

I

'

I

'

Ferrihemoglobin in HCI + KC1

I

PH

FIG.11. Equilibria in solutions of ferrihemoglobin, HC1, and KCI. Open circles = fraction of protein denatured a t p = 0.02 from spectroscopic data of Zaiser and Steinhardt (1954a). Solid circles = fractional cleavage of heme a t p = 0.04 from acetone fractionation data of Lewis (1954).

probably operate in the same direction. It is not surprising, therefore, t,hat results recently published by Lewis (1954) on the amounts of hematin remaining in solution on addition of acetone to solutions of horse ferrihemoglobin initially brought to various p H values with HC1, do not agree a t all with the spectrophotometric results of Zaiser and Steinhardt obtained on mixtures (Fig. 11). However, the discrepancy is in the direction opposite to that which would be predicted from the considerations above, i.e., when allowance is made for a slight shift along the p H axis (Lewis' data to the left) which would be expected because of the small difference in ionic strength and because of the p H drifts which were to be expected and which Lewis reported. It appears that very little cleavage (less than one-third) occurs a t pH values a t which Zaiser and Steinhardt showed that about 90% of the protein had undergone unmasking and denaturation. Thus, one must conclude that hemoglobin-D still binds hematin very strongly, although its specific and characteristic hemoglobin-type linkage has been broken. This conclusion has an important bearing on the kinetics of the regeneration process in which denatured protein not only regenerates but must recombine with the prosthetic group; only a small amount of the latter is free in solution. A similar direct comparison cannot be made in the case of COHb, also studied by Lewis, since (1) Steinhardt and Zaiser (1951) made no spectroscopic observations on equilibrium mixtures of native and denatured protein; and (2) equilibrium occurs in a p H range in which the method used with COHb was interfered with by ferrihemoglobin formation. However, the equilibrium curve for unmasking itself (fraction of maximum delayed

198

J. STEINHARDT AND E. M . ZhISElt

acid uptake) may be compared with the Lewis data for heme cleavage. The comparison may be made with some assurance since denaturation of COHb occurs in more acid solutions than with ferrihemoglobin, arid nearly quantitative hinding of hydrogen ions by the liberated groups can be assumed. Unlike the case of ferrihemoglobin there is fair agreement with Lewis' data a t all pH values below about 4.2 (cleavage of more than 25 %). Above 4.2 Lewis finds more cleavage than would be expected from the results of Steinhardt and Zaiser. The theoretical objections to Lewis' method previously given may apply more strongly in the region of small cleavage and thus explain the discrepancy (as well as, possibly, some of the discontinuities in his data for this and the other proteins he studied). If the comparison is valid at all, however, the important conclusion results that heme (or hematin) is not bound to the protein moiety after denaturation of this protein (in a more acid range, and in the presence of air), although it appears to be bound to Clobin-D, formed 011 denaturation of ferrihemoglobin. The possibility exists, therefore, that the free protein moiety is altered more extensively when COHb (and OsHb) is exposed to dilute HCI than when native ferrihemoglobin is exposed to less acid solutions. 6.Titrirnetric Evidence of Unmasking in Other Proteins

No direct measurements have been made of time-dependent increases in maximum dissociation or combination of protons brought about by exposure to very dilute acid or base with other than heme proteins. However, a number of the symptoms of such lability listed on page 181 are shown unmistakably (at room temperature and in the same acid range of p H as in the hemoglobins) by at least two other proteins, edestin and conalbumin. With edestin large p H drifts occur when the protein is brought to p H values below 4.9 in 1.71 A l NaCl a t 20"C., and the protein is simultaneously transformed to edestan (Bailey, 1942), a three-component mixture of denatured proteins of lower molecular weight than the native protein (Lontie and Panier, 1951). Below p H 3.9 the reaction is too rapid to be measured and pH drifts therefore cannot be observed. As in the case of ferrihemoglobin (see below) the temperature effect on the rate of denaturation is very small (none was observed between 2" and 20°C). The kinetics are concentrationdependent and complex, only partly because of the drift in pH. As with COHb but not with ferrihemoglobin, the data do not clearly indicate the existence of a pH-dependent eq~ilibrium.~' Unfortunately, the only titration data on this protein, those of Hitchcock 31 Bailey did not test his d a t a for conformity with the kinetics of a fast reversible reaction having a pli-dependent equilibrium point, followed by a slower irreversible reaction. Such a slow irreversible reaction exists in the case of ferrihemoglobin.

HYDROGEN ION EQUILIBRIA

199

(1921), were obtained over 30 years ago, using the long equilibrium times required by a hydrogen electrode. The conditions used were such that the data obtained are surely those for the denatured protein, edestan, rather than for native edestin. Conalbumin (Warner, 1954) appears to duplicate very closely not only the unmasking effects reported for the hemoglobins and globin, but also the kinetics and equilibria involved in the reversal process of denaturation which accompanies unmasking and which are described later. The anomalously steep titration curve (pH 4 to 6) of collagen in 0.5 M NaCl (Bowes and Kenten, 1948) raises the possibility of the existence of masked groups in this protein. Drifts in pH could not be detected because attainment of equilibrium with the solid phase requires 3 days. The steepness is quite marked compared to that of t ~ o o l(Steinhardt, Fugitt, and Harris, 1940a) in the same p H range; thus effects due to the existence of separate phases are probably not responsible. The fact that only part of the lysine appears to be titrated lends strong support to the hypothesis of acid-labile masking in this protein (see Section TI, 5 of this paper). Smaller and less easily interpreted pH drifts have been reported by Kearney and Singer (1951) during inactivation of partially purified l-amino acid oxidases of snake venom. Reference mill be made later to other proteins that exhibit other less direct indications of the existence of labile masking of certain prototropic groups.

7. Relation of’ Kinetics of Denaturation and Liberation of Groups in Carbonylhemoglobin Further information as t o the nature of the states of the masked groups in native proteins, and as to the unmasking reaction, may be obtained by study of the kinetics and thermodynamics of the latter. By comparing the rates of unmasking, of spectroscopic absorption changes, and of changes in solubility, all as a function of pH, Zaiser and Steinhardt (1951) attempted to shorn in the case of COHb that, within the limits of experimental error and over a wide range of rates, all three processes occurred simultaneously. The first step in their demonstration is illustrated in Fig. 12, which shows that the first-order rate constants of spectroscopic change, and of solubility change, are the same over a very wide range of reaction velocities. The second step (Fig. 5 of Zaiser and Steinhardt, 1951) was an effort to shorn that the rates of liberation of acid groups (measured a t 10 minutes) and of spectroscopic changes (calculated from rate constants) are the same within the overlapping pH ranges in which the measurements of both are valid. On the basis of the foregoing it mas concluded that the liberation of 36 basic groups occurred as an all-or-nothing process coincident with denaturation. The evidence offered for the second step above, however, was

200

J. STEINHARDT AND E. M. ZAISER

PH FIG.12. Log first-order half-period as a function of p H and temperature for COHb in formate buffers, p = 0.02: circles, spectrophotometric method; crosses, precipitation method. From Zaiser and Steinhardt (1951). Broken line represents halfperiods for unmasking of groups, based on pH drifts over short times (see t e x t ) .

open to question for a number of reasons (see footnote 8 in Steinhardt and Zaiser, 1953). A more direct comparison of the rates of unmasking with the rates of denaturation has therefore been attempted by interpreting the pH drifts, observed when rapid flow is stopped, in terms of a rate of combination of hydrogen ion. The half-period of this process as a function of initial p H (rather than equilibrium pH) has been calculated from the initial rates (over a period of only 30 seconds, representing a very small change in pH). The results for 25°C. are shown by the broken line in Fig. 12. At first sight these results appear to show that denaturation is about four times as rapid as unmasking. Actually the comparison is inconclusive because of a peculiarity of the denaturation process (in the case of COHb) which must now be mentioned. The apparent first-order kinetics of the denaturation of this protein evidently concealed a more complex process. The rate of denaturation proved very sensitive to traces of oxygen-so sensitive, that reproducible kinetic data were obtained only if certain minimal amounts of air were deliberately added. Since ferrihemoglobin is formed rapidly when oxygen is present, and is denatured more rapidly than COHb itself, the transformation to denatured protein occurred a t least in

HYDROGEN ION EQUILIBRIA

20 1

part through the intermediate formation of ferrihemoglobin. Furthermore, at high pH where the reaction was slow, the spectrophotometric technique employed did not distinguish clearly between the rate of denaturation and the rate of ferrihemoglobin formation. The titration data were obtained under a good approximation to anaerobic conditions. The denaturation measurements were always deliberately made in the presence of air. The discrepancy in rates just noted is about as large as is t o be expected from this single difference in conditions. There is thus a strong presumption that the original conclusions of Zaiser and Steinhardt (1951) are valid. 8. Kinetics and Thermodynamics of Denaturation and Liberation of Groups in Ferrihemoglobin

a. Rates and ~ q ~ ~ l ~Based b r i aon Spectroscopic and Solubility Criteria for Denaturation. I n order to render these conclusions less equivocal and to study a much less complicated system, attention was turned to the oxidized form of hemoglobin. This choice minimizes the complication of successive or parallel reactions, and presents an additional attraction in that its denaturation by acid, unlike that of COHb (under the conditions of the early experiments), is completely rather than partially r e v e r ~ i b l e . ~Thus, ~ with COHb only the rates measured by the three methods could be compared. With ferrihemoglobin equilibria could be compared also. The balance of this discussion is therefore principally devoted to results obtained with horse ferrihemoglobin (Steinhardt and Zaiser, 1953; Zaiser and Steinhardt, 1954a,b). Analysis of the time course of the reaction requires constant p H and therefore buffered solutions. It is impossible to measure the rate of increase in the number of basic groups except by permitting the p H to change. Recourse was therefore had to following spectroscopic changes as in the work with COHb. The results of these, experiments (use was made of the Soret band a t 404 mp) strongly suggested the existence of an equilibrium involving hydrogen ion, since the reactions stopped short of completion a t endvalues which depended on pH. The time course of the reaction, over a t least three (usually four) half-periods was given by the isotherm of a reversible first-order process:

kl

+ Icz

1 In = -

t

xca x,-x

(3)

34 Qualitative demonstrations of the reversibility of the den:tturation of fcrrihemoglobin and evidencc bcaring on the identity of the regenerated material with native protein have been given by Anson arid Mirsky (1925, 1928, 1931a,h, 1934), Mirsky :md Anson (1929, 1930), arid Holden (1936).

202

J. STEINHARDT AND E. M. ZAISER

Formate buffers 25" lunic strength .02

time in minutes

FIG. 13. Iiinet,it*sof spectroscopic change at 4060 A. xith 0.0670 t'errihemogloth

:

upper curves, equilibrium optical density D, chosen by curve-fitting for best linear. relation of log (D - U,) t o time; lower curves, Guggenheim's method, where T = fixed time interval between density readings. From Steinhardt and Zaiser (1953).

where h , is the first-order constant for the denaturation reaction, 1c2 is the first-order constant for the reverse process, X , is the final concentration of denatured protein, and X is the concentration of denatured protein a t time 1. All values of X were determined by changes in absorption or solubility. Representative kinetic experiments by the spectrophotometric method are given in Fig. 13. The eqiilibrium points were obtained by Guggenheim's (1926) method in order to eliminate the effect of a very slow secondary reaction accompanied by irreversible loss of native protein ; values of X , so obtained when inserted in equation 3 give rate constants which agree with these obtained directly in the Guggenheim method.33 As would be expected with first-order processes the rates obtained arid the values of X , (obtained by Guggenheim's method) are independent of protein concentration over a very wide range (0.002%t o 0.1 %).34 It is 33 The agreement is excellent for pH values a t which most of the protein is denatured a t equilibrium; i t is least good a t high pII where very littlc protein is denatured, possibly because the regeneration reaction is complex. 34 This independence of concentration may not be true of the very mnch slo\\er reactions which follow the reaction measured.

HYDROGEN ION EQUILIBRIA

203

+

FIG.14. Variation with pH of log ( k l kp) and log half-period as measured rate of denaturation of ferrihemoglobin. From Steinhardt and Zaiser (1953).

unfortunate that the precision of these determinations is lowered a t the highest pH values where only a small fraction of the protein was transformed, since this is a region of great interest in making inferences as to the reverse reaction. The effect of pH on log (Ic, k t ) , determined spectrophotometrically, is shown by the circles in Fig. 14. The results already described for COHb are shown also. It will be observed that COHb is changed much more slowly than ferrihemoglobin a t each p H (the reaction is still slower if traces of air are rigorously excluded so that ferrihemoglobin can not be formed). With COHb the dependence on pH is greater (slope 3.2 instead of 2.5) and the reaction is slower throughout the pH interval. Because of the different functional dependence, the difference between the proteins is smallest a t the most acid pH, and may even reverse in sign a t still lower pH. With ferrihemoglobin, the proportionality of rate to the 2.5 power of the hydrogen-ion activity, while constant over a fifteen-fold variation in velocity, fails when pH exceeds about 3.75.' The leveling off of the rate constant a t high p H is not unexpected: the log of the sum of two rate constants, one or both of which depend on pH, cannot be linear over wide intervals of pH. It is shown below that kz is probably proportional t o [H+]-z,5. Thus

+

+

I c ~ kz

+

=

kl'[H+]"."

+ kz'[H+]-2.s

and log (kl k2) can be linear in p H only a t pH's well removed from the pH a t which the two terms on the right-hand side of the expression above are equal. The broken curve in Fig. 14 is calculated for kl' = 1.51 X I OR min.-'. min.-' and kz' = 5.62 x

204

J. STEINHARDT AND E. M. ZAISER

+

The practical upper limit of pH at which the rate constant (k1 h) can be measured is set by the small extent of reaction at p l I values much above 4, where the total change in density becomes only slightly larger than the experimental error. The fraction denatured a t equilibrium ( X , / A ) as a function of pH is shown in Fig. 15. Since the equilibrium constant K should equal k l / h while the observed "rate constant" is (Icl k z ) , it is of interest to determine the former quantity, in order to determine separately the dependence of lcl and ICZ on pH. I n Fig. 16 the data of Fig. 15 for formate buffer are replotted as log K against pH. Over a wide range K varies approximately inversely with the 5th power of [H+]. It should be noted that in Figs. 15 and 16 data are given for the approach to equilibrium from the denatured side as well as from the native side. The kinetics of the regeneration process will be discussed briefly later. Denaturation is commonly defined in terms of virtually complete loss of solubility in salt solutions a t the isoelectric point. Rate experiments and measurements of final values were therefore made by precipitating denatured protein and determining the soluble residue spectrophotometrically. I n Fig. 14 points are included which represent rate constants determined by the solubility method. It is clear that rates so determined are consistent with those found spectroscopically throughout the range of pH investigated. Figures 15 and 16 include data which show that equilibrium values determined by precipitation of denatured protein also agree with those determined by spectrophotometric determinations on the unfractionated reaction mixture. It is evident that both spectrophotometric

+

I

'

I

'

I

'

I

'

I

0.06% Ferrihemoglobin 25'

PH

FIG.15. Fraction of protein denatured a t equilibrium as a function of pH in buffers and HCl-KCl, ionic strength 0.02, from spectrophotometric studies of denaturation (A 4060 A.). Additional points in formate buffers were obtained by the precipitation method a, and by spectrophotometric @ and precipitation A studies of the reversal of denaturation. D a t a from Steinhardt and Zaiser (1953) and Zaiser and Steinhardt (1954s).

205

HYDROGEN ION EQUILIBRIA I

*

A

1

1

'

1

'

1

'

1

'

Ferrihemoglobin 25"

1

-

-

PH

FIG. 16. Equilibrium constant as a function of p H for denaturation of 0.06% t'errihemoglobin in buffers a t 25"C., ionic strength 0.02; spectrophotometry at 4060 A. Additional points in formate buffers obtained by precipitation (>,and by spectrophotometric @ and precipitation A studies of the reversal of denaturation. D a t a from Steinhardt and Zaiser (1953) and Zaiser and Steinhardt (1954a).

and solubility methods measure the transformation of native molecules to denatured protein. b. Rate of Increase of Proton-Binding Groups. The difficulty previously noted in making a complete kinetic analysis of the liberation of protonbinding groups (the fact that p H changes necessarily occur in the unbuffered solutions used for titration) can be partially circumvented by examining the pH drifts observed over small intervals of p H (0.1 unit) and correspondingly short intervals of time (12 to 60 seconds). I n this way approximate rate constants have been obtained from the initial rates of liberation of new groups. A more fundamental difficulty, however, is that the rates in HCI, used in titration, are demonstrably different from the rates in formate buffers, used in the kinetic measurements. The difference between the results obtained with formate and with other buffers (Zaiser and Steinhardt, 1954a) is therefore shown in Fig. 17,36 which also reproduces an estimate as determined above (shaded area) of log (kl kz) from the effect of pH on the rate of release of extra basic groups when ferrihemoglobin is titrated with HC1. Comparison of this rough estimate with the data for buffers other than formate shows that there is a t

+

36 Equilibrium data for the same buffers have already been presented in Figs. 15 and 16.

206

J. STEINHAHDT AND E. M. ZAISER

Ferrihemoglobin 25" o Formate A

Acetate Lactate Monochloroacetata

1.2

- .6

hading=delapd mid uplak (unbuffered)

3.4

3.6

50

4.0

4.2

0

PH

+

FIG. 1 7 . D e p e d e n c e of log ( k l k ? ) or log half-period 011 pfI in buffers, ionic strength 0.02 (from spectrophotometry a t 4060 A . ) . From Zaiser and Steinhartit (1954a).

most only a small discrepancy in rates of denaturation and increase in proton-binding groups. If this small discrepancy is significant, spectrophotometric changes precede unmasking rather than follow it. However, the fact that the titration measurements were made in more concentrated solutions than the spectroscopic measurements must not be lost sight of, for the small effect of concentration on the very slow reactions a t the equilibrium point is in the right direction to reduce even this small discrepancy. c. Separation of the Forward and Reverse Rate Constants. Since K and (lil X.2) are both known as functions of pH, it is possible to determine for the pH of each experiment separate values of lcl and kz . These values are shown in Fig. 18. The results show that the rate of the denaturation reaction (X.1) is proportional to the 2.5 power of [Hf] over the entire pH range investigated. They also indicate that the rate of the regeneration process (kz) is inverselv proportional to the 2.5 power of [H+].36 It must be emphasized that the values of kz calculated above, and their dependence on pH, depend critically on I<, especially a t high pH. Inspection of Fig. 16 will show that at pH above 4, values for K obtained in regeneration experiments do not agree as closely with those obtained in denaturation experiments as they do a t lower pH. Thus, confirmation of the exact dependence of kz on [H+] requires similar treatment of direct rate measurements on the regeneration reaction. d. Efect of Temperature on Kinetics and Equilibria. The effect of tem-

+

a R The specific effect of formste is a little greater on denaturation (kl) than on regeneration ( k ~ ) .

207

HYDROGEN ION EQUILIBRIA I

0-

'

l

-

Ferrhemoblobin 25"

cn

/

/

-3

!oe!oLka ,

B a r Acetate Lactate A CI-acetate a Formats

7

-

3

-

0

+

3.6

38

4.0 PH

I

42

4.4

FIG. 18. Dependence on pH of log kl (denaturation) and log ka (reversal) calculated from kinetics and equilibria of ferrihemoglobin in buffers, ionic strength 0.02 (slopes of lines f 2 . 5 ) . From Zaiser and Steinhardt (1954a).

perature on the kinetics and equilibria just described provides further information about the unmasking reaction, and therefore about acid denaturation. In the following, data obtained by the spectrophotometric method alone (Zaiser and Steinhardt, 1954b) are discussed, since it has been shown that this gives results identical with the precipitation method, and very closely similar to the titrimetric method. Figure 19 shows the dependence of log half-period and log (h kz)

+

DH

+

FIG.19. Variation with pH of log ( k l k,) and log half-period for denaturation of 0.06% ferrihemoglobin a t 15.5", 25.0°, and 34.9"C. From Zaiser and Steinhardt (195413).

208

J. STEINHARDT AND E. M. ZAISER

on pH a t 15.5", 25.0", and 34.9"C. The results are much the same at all three temperatures, except for the expected differences in velocity, which are much smaller than those usually found for protein denaturation. At the two lower temperatures, in fact, the resemblance is quite complete. Thus, the limiting slope a t low pH is 2.5 a t 15.5" and 25.0"C. (although only 2.1 a t 34.9"C.). It is apparent that temperature has little effect on the pH for minimum log (kl kz). Ordinarily, logarithmic plots of denaturation rate constants as a function of hydrogen-ion activity may be significantly compared a t different temperatures by translations parallel t o the two coordinate axes made in such a way that the data superimpose in a single unique fashion. I n the case of pepsin, for example (Steinhardt, 1937), where denaturation at p H 5 to 7 i s practically irreversible, such translations showed that, most of the temperature effect was manifested as a horizontal shift whose magnitude, in pH units, was related to the heat of dissociation of the groups which characterize the unstable ionic form, rather than to a large effect of temperature on the rate at which the reactive ionic species was transformed. I n the present reversible case, however, where the rates are determined by (kl k z ) , the curvature and the position of the minimum on the p H scale in Fig. 19 are determined solely by the relative rates of the forward and reverse reactions and their dependence on pH. Since kl depends directly, and kz appears to depend inversely, on pH, the occurrence of a minimum in log (Icl kz) arises from the mutual opposition of k1 and JGZ in a region of pH where their magnitudes are sensibly the same. I n general, if denaturation is proportional t o the zth power and regeneration to the inverse yth power of the hydrogen ion activity, (kl lcz) can be expressed (kl' [Hi]" kz' [H+]-u]. The p H of the minimum value of

+

+

+

+

(kl

+ k z ) is thus X-+-Y log

[;:;I. ~

+

It may be concluded, however, from

the fact that the position of this minimum is little altered by temperature that the effect of temperature on kl and kz cannot be widely different. Thus, very little, if any, effect of temperature on the equilibrium between native and denatured protein at each pH would be expected. No definite conclusion as t o the heat of dissociation of the trigger groups can be drawn. The effect of pH on the fraction ( X , / A ) of protein denatured a t equilibrium in the acid denaturation of ferrihemoglobin a t three temperatures is shown in Fig. 20. The points represent true equilibrium states, confirmed by approaching them by way of the regeneration of initially denatured protein. The equilibrium is the same a t 15.5" and 25.0°C., as predicted above, whereas a t 34.9"C. the curve is displaced slightly in the direction of increased denaturation a t a given pH. This shift cannot be a n anomaly caused by a temperature-dependent irreversible loss of pro-

209

HYDROGEN ION EQUILIBRIA

"

3.4

3.6

3.8

4.0

4.2

PH

FIG.20. Dependence on p H of fraction of protein denatured at equilibrium for 0.06% ferrihemoglobin a t 15.5', 25.0", and 34.9"C. From Zaiser and Steinhardt (1954h) ,

tein, since a reaction mixture equilibrated a t 34.9"C. can be partially regenerated by cooling to 15" to 25"C., with an extent of reversal which is compatible with the difference between the two equilibrium curves in Fig. 20. Figure 21 shows the dependence on p H of the equilibrium constant, defined as K = denatured/native = X,/(A - X,), and calculated from the equilibrium points in Fig. 20. It is evident that K is proportional t o

FIG.21. Log equilibrium constant as a function of pII for 0.06% ferrihemog1ol)itl a t 15.5', 25.0", and 34.9OC. From Zaiser and Steinhardt (195413).

210

3 . STEINHARDT AND E. M. ZAISER

the fifth power of the hydrogen-ion activity, as previously found a t the lower temperatures a t all pH values, but that its proportionality to the hydrogen-ion activity a t 34.9"C. decreases to the 4.6 power. (The equilibrium data a t high pH a t all temperatures are subject to the same reservations as have already been noted for the 25°C. data.) e. Model of the Unmaslcing Reaction in Ferrihemoglobin. Discussion of the energetics of unmasking will be facilitated by considering a simplified model of the reaction, intended to account for the Hfion equilibria which must be involved. The indication that both k1 aiid kz depend on pH, but in inverse ways, suggests that the role of [H+] is riot a simple catalytic one: i.e., [H+] must caornbine with protein (as previously suggested in the discussiori of "trigger groups") in the process of denaturation, and must he dissociated from protein in the process of regeneration. The validity of some details of the following model depends criticttlly on the demonstration (Figs. 18 arid 22) that k z is inversely proportional to [H+]. Because of the uncertainties described in considering Fig. 18, final acceptance of the model must await direct measurement of k.2 in regeneration experiments a t high pH. In the model, a distinction is made between native arid dcnatured protein, K and D (Globin-D or possibly Hemoglobin-D), arid thc ionic forms NH, and DH, , formed by the comhination of hydrogen ion with the trigger groups, r in number: N

+ xIi+-

KNH

I

NH,

4 11 i:k I>

+ rHf

c1

kO,

7 DH, KDH~

Besides actual deiiaturation arid regeneration steps (vertical arrows), tlixsociatiori equilibria (horizontal arrows) characterized by constants KNH, and KUH,are postulated, where KNW,and K D H , are each rompound constants for x steps, i t . , K I K r . . . K , . The equilibrium which is measured spectroscopically is hetiveeii total native protein (S SH,) ant1 total denatured protein (D DHz), so that the observed K,, = (D DH,)/ (N NH,) . At, equilibrium, d(D DH,)/dt = 1510(NHz)- L10 (D) = 0. In order to account for the effect of (DH,) k,O(N) -pH on the observed equilibrium it is assurned that denaturation of a molecule occurs only when a certain x groups (2.5 on the average) involved in the trigger react ion have combined with II+, atid that regeiieratioii call occur only when these groups dissociate again, i.e., that KNH,# KUIIzarid that kIo >> 1~~~ >> /;so >> k - 2°. The ftwmulation of the deiiaturation equilibrium will then contaiii iiot wily the ratio of forward :itrd revcrsc velority coilstants hit also a factor arising from the coritrolliiig influenre of pH

+

+

+

+

+

+

211

HYDROGEN ION EQUILIBRIA

on the dissociation of the trigger groups:

where 161 and k 2 are velocity constants equal to k I 0 [H+]'/KXH, and kz°KDH,/ [Hf]", respectively. f. Thermodynamic Functions. The heat of reaction which is observed to be zero, a t least between the two lower temperatures, is, according to the NH,) into above model, an over-all AH for the transformation of (N (D DH,), and is made up of the contributioiis of the heats of dissociation of NH, and DH, as well as the heats of reaction for the transformation of NH, into DH, and D into N. Accordingly, to state that AH = 0 in this temperature interval gives little information about the heat of transformation of K into D unless the dissociation constants K N H z and R D H ~ are identical. Since the equilibrium is affected by pH, KNHzand K D H ~ cannot be the same, in terms of the model above; the most that can be said is that AH for the conversion of N to D is probably small in this range of temperat~re.~' The standard free energy change AFO, defined formally as the change in available energy in going from a particular value of Keqwithout changing 1) DH, p H to an arbitrarily chosen standard state where 7 = 1, is equal 3 +NHZ to -RT In K,, . In the pH range studied, AFOfor 15.5' and 25.0"C. is small, ranging from -3.2 kcal. at pH 3.4 to +1.5 kcal. a t pH 4.1. This is to be expected, since observations were confined to the region of p H where a measurable equilibrium point is fouiid, and where K,, therefore does not depart grossly from unity. These small values of AFO do not, however, represent the free energy change in going from native to denatured protein, and the AF's for the processes D ---f N and NH, + IIH, and for the ionieation steps may coiiceivably be large. A similar difficulty is encountered i n the interpretation of the entropy of reaction. Since AH = 0 for the interval 15.5' to 2.5.0°C., A S varies with pH as AFO docs, and ranges from +10.9 E.U. a t pH 3.4 to -5.1 E.U. a t pH 4.1. Again this tells nothing about A s for the steps of the reaction that are of particular interest in N and NH, DH, . These limitations studyiiig denaturation, i.e., D in significance are applicable to thermodynamic data on the denaturation

+

+

+ ~

--$

--$

37 Forrest and Sturtevant (1954) report a value of 11.4 f 2.4 kcal. per mole for AH a t 25" C . , obtained by calorimetric studies of solutions of 0.6% t o 0.9% ferrihemo-

globin in formate buffers of pH 3.4 t o 3.8. Although they appear t o find slower rates than Steinhardt and Zaiser observed by other methods, their kinetics are also firstorder and show the same pH dependence.

212

J. STEINHAIlDT AND E. M. ZAISER

of riumerous other proteins, wherever the rate constaiits of the reactions of denaturation and regeneration are both functions of P H . ~ * Thermodynamic furictioiis have not been calculated for the temperature interval 25.0" to 34.O"C:. because the power of the dependence of K,.,, on hydrogen ion activity is different a t these two temperatures (Fig. 21) and the thermal functions thus depend on pH. The observed difference is not inconsistent with the model indicated above. Thus, beyond some particular temperature, the assumption that the reactions NH, + DH, and D ---f N go t o completion, subject only t o the control of the dissociation constants KNW,and KDH,, may no longer be valid. The maximum possible extent of denaturation or regeneration a t any pH would then be less than loo%, and the order of dependence of Kep on the hydrogen ion activity would be lessened. If this is the case a t 34.9"C., the calculated AH for the reaction between 25.0" and 34.9"C. a t each p H cannot have the same meaning as in the 15"-25"C. region discussed above. An alternative possibility is that the identity of the ionic species which most readily undergoes denaturation at 15.5" and 25.0"C. is not the same a t the highest temperature (i.e., that the average number of trigger groups changes). The second possibility is supported by the observed decrease in the dependence of log ( k , k2) on pH at 34.9"C. (Fig. 19).39 g. Energy of Ackivution. In order to obtain the energy of activation for the denaturation reaction, it is necessary to separate h-1 for denaturation from the measured velocity constant (kl k2) which contains the velocity constant of regeneration. Figure 22 shows the dependence on p H of log kl and log k z a t three temperatures as calculated from the measured rates and equilibria. (The values of lcz represented must be accepted with the same reservations as were stated above for those in Fig. 18.) At low pH, k 2 << kl , arid the calculated value of kl is practically the same as the observed rate constant, and exhibits the same order of dependence on the hydrogen ion activity (2.5 at 15.5" and 25.0"C. and 2.1 a t 34.9"C.). In view of the tentative identification of the trigger groups as earboxyl, however, it is likely that their heats of dissociation are almost negligible, and that the displacement of the curve for log kl against p H when the temperature is changed has no appreciable horizontal (ApK) component. Thus,

+

+

38 The authors know of only one well-studied case in which the velocity constants are demonstrably pH-dependent in one direction only. This is the reversible inactivation of snake venom Z-aminoacid oxidase studied by Kearney and Singer (1951). There is indirect evidence, however, that the reversible denaturation of soybean trypsin inhibitor reported by Kunitz (1948) is another such case. Thus Kunitz showed that AH = El - E Bfor this protein. The activated state must therefore be identical for the two reactions. 3 9 Unpublished kinetic measurements a t 1.7" C. show t h a t AH is not zero at temperatures below 15" C. also.

213

HYDROGEN ION EQUILIBRIA

Frc:. 22. Dependence on pll of log ki (denaturation) and log k z (regenerution), calculated from kinetics and equilibria for 0.06% ferrihemoglobin at 15.5", 25.0", and 34.9"C. From Zaiser and Steinhardt (195411) I

although the model for the reaction indicates that kl = lclo [H+Iz/KNH,, differences in log kl at two temperatures can be ascribed directly to the energy of activation, without the correction for a shift in p H which is sometimes required (Steinhardt, 1937). Figure 23 shows values of the Arrhenius energy of activation El for denaturation calculated from the effect of temperature on kl : l C 1 = 2.303 R [(T 'T )/ (T ' - T ) ] . Alog

h.1.

In the lower temperature interval ( 1 5 . 5 O to 25.0aC.), El = 1G.2 f 0.2 kcal. per mole over the entire range of pH. The difference in the pH dependence of the velocities a t 25.0"and 34.9"C. causes the energy of activation calculated in this interval to lose its usual meaning, and in fact appear to depend on pH. No attempt will be made here to interpret the rather ambiguous result of such a calculation. The value of the energy of activation, 16.2 kcal., falls well within the range commonly encountered in such chemical reactions as hydrolyses of ester or amide linkages. Reactions that depend solely on the rupture of a single hydrogen bond require much smaller energies. When the energy of activation is used in conjunction with the rates observed a t 25.0"C. to

214

J. STEINHARDT AND E. M. ZAISEIZ Ferrihemoglbbin Formale buffers-ionic strength 02

x

w

3.2

3.4

3.6

3.8

4.0

2

PH

FIG. 2 3 . iirrlieiiius energy of activation E , for dciiaturntiori From Zaiscr and Steirihardt (19541)).

:LS n

furirtioii o f pH.

calculate the apparent collision frequency factor 2 = /i1eh'1lRlV, the values of 2 of course vary with pH, reflecting the dependence on p H of the concentration of the ionic species which can undergo activation. The order of magnitude of 2 is loLD to 10" sec.-I, reasonably close to the value of lOI3 set.-' which collision theory would predict for the frequency of collision of a niolecule of the size and mass of the half-m~lecule~~ of ferrihemoglobin with water molecules. It i s important to note that the anomalously high values of E and 2, which are sometimes calculated (Neurath et al., 1944; Steinhardt, 1937) for protein denaturation from data uncorrected for the effects of ionic equilibria characterized by large AH values, are absent here (therefore the Eyring-La Mer entropy of activation is negligible). This further supports the suggestion that a few carboxyl groups, possibly in the prosthetic groups, are the ionizing groups responsible for the pH dependence of the reaction rates, since their heats of dissociation are small or zero, and would contribute very little to I3 or %. The application of absolute rate theory (Glasstone, Laidler, and Eyring, 1941) to values of kl a t 15.5' and 25.0'C. yields thermodynamic analogues as follows: AH* = 15.7 kcal. per mole, AF* varies from 17.2 kcal. per mole at pH 3.2 to 20.2 kcal. per mole at pH 4.1, and AS* varies from -5.1 E.U. a t pH 3.2 to -15.3 E.U. at pH 4.1. Such analogues are usually of meager value in model-building for such complicated molecules as proteins, and their precise meaning is subject to further uncertainty since the frequency factor k T / h , which is assumed in the theory of absolute rates, and on which the values just given depend critically, may not be appropriate for the collision of very large mole~ules(such as proteins) with solvent. According t o the cyclic model proposed above, the activated complexes

HYDROGEN ION EQUILIBILIA

215

for denaturation atid regeneration are not identical ; thus Id2 (the energy of activation for the reverse reaction) cannot be calculated from the known values of El and the over-all heat of the reaction, i.e., A11 # El - E 2 . The alternative possibility of calculating 132 from the variation in log k , with temperature (Fig. 22) is not attractive because the values of 1i2 giveti are very small a t low pH, while they arc subject to considerable error a t pH > 4,where the extent of denaturation is small and the precision necessarily limited. Furthermore, Equation 4 implies that the effect of temperature on k z will include an effect on KDH,of unknown magnitude. Preliminary examination of data on the regeneration reaction a t 15°C. and lower temperatures (Steinhardt, Zaiser, and Gibbs, 1954) suggests that the values of log k 2 shown in Fig. 22 are reasonable, aiid that the energy of activation for regeiieratiori E2 may be between 5 aiid 10 kcal. higher than 131 for denaturation, i.e., regeneration i s favored at high temperatures. 9. Kinetics and Thermodynamics of the Reversal oj

Denaturation of Ferrihemoglobin Equilibrium data on the regeneration reaction have already been presented (Figs. 15 and 16). No detailed description of the results of direct measurement of the rates of the regeneration reaction will be attempted here, although they add substantially t o an understanding of the unmasking reaction by revealing additional complications (Steinhardt and Zaiser, 1954). The rate of the regeneration reaction has been found to depend on the length of time that the denatured protein has been exposed to acid (pH 3.5); and the kinetics, a t p H values at which regeneration goes practically to completion, prove to be complex. Representative data obtained under such conditions are shown in Fig. 24 (data for 1.7"C.rather than 25°C. are shown because the regeneration reaction a t 25°C. a t this p H is so rapid that about half of the protein is transformed before the first reading can be made). The regenerated protein is identical spectroscopically with the native protein over the entire visible region. Because of the complex kinetics the rate constants cannot be compared directly with those predicted in the foregoing calculations based on denaturation rates and equilibria. However, in the p H range 3.8 to 4.2 where kinetic data may be obtained on both denaturation and renaturation, the half-period of the regeneration reaction ajter short exposure to acid is approximately equal t o the half-period of the denaturation reaction a t the same pH, in accordance with the requirements of even the simplest theoretical model.40 A 40 It is noteworthy that regenerated protein must be exposed t o regeneration p H for some time after the regeneration is complete by the spectroscopic criterion before it recovers t h e kinetic properties of the original native protein, i.e., denatures a t the same rate. Before such an interval, it denatures faster.

'210

I. STEINHARDT AND E. M. ZAISER

time (minutes)

FIG.24. Kinetics of regeneration of 0.06% ferrihemoglobin at 1.7"C. in formate buffer, p H 4.761. Protein previously denatured at p H 3.5, 25°C. for 2, 4, 8, and 16 times the half-period for denaturation under those conditions.

similar identity of denaturation and regeneration rates was demonstrated by Kunitz (1948) in the case of soybean trypsin in h ib it~ r.~ ' The dependence of rate on the duration of exposure t o acid and the complex kinetics of the regeneration process in the case of ferrihemoglobin have been attributed to the existence of successive reactions in denaturation and to competition a t higher p H between regeneration and a transitory equilibrium with another form of recombination (possibly with hematin, possibly molecular aggregation) which does not give native protein (Steinhardt , Zaiser, and Gibbs, 1954; Steinhardt and Zaiser, 1954). At very low temperatures (1.7"C.) the data a t high pH values yield to analysis in such terms. No complete kinetic analysis of the regeneration process a t higher temperatures has so far been successful. It appears that such an analysis may have t o take into account the combination of hematin with two forms of denatured protein in competition with native protein. Critical testing of this hypothesis will require experiments in which the total hematin present is systematically varied.42 4' Eisenberg and Schwert (1951) have given similar kinetic and thermodynamic data for chymotrypsinogen. They give a t two p H values, values of the apparent rate constant for denaturation (kl k 2 ) and the equilibrium constant ( k l / k * ) , and values of k l and l i e derived from these quantities, but i t does not appear t h a t the regeneration rate was actually determined experimentally. 4 1 It will be apparent that the kinetic and thermodynamic analyses just given differ in several important respects from that given earlier by Neurath et al. (1944), largely on the basis of Cubin's date on ox oxyhemoglobin. Some of the differences

+

HYDROGEN ION EQUILIBRIA

217

10. Kinetic Data Suggesting Unmasking in Other Proteins

The case of edestin has already been considered in detail (p. 1%) and need not be discussed here. Recently published data 011 the cleavage of horse-radish perozidaso (Maehly, 1052, 1953), although indicating other complexities, bear sufficient resemblance to the situation described above for ferrihemoglobin, to raise a strong presumption that cleavage is accompanied by the liberation of prototropic groups unavailable for combination with acid and base in the native enzyme. No direct titration data are available for comparison. Conalbumin in dilute acid (pH 3 to 4) is characterized by the same reversible kinetic and equilibrium behavior already described for hemoglobin (Warner, 195-1). As already stated (p. 199) theunmasking of a large number of groups can be demonstrated directly. Hog thyroglobulin exhibits great susceptibility to denaturation a t pH values below 4.8 (Heidelberger and Palmer, 1933) and the 0.42 unit shift of its isoelectric point on denaturation (Heidelberger and Pedersen, 1935) suggests that acid titration data should be obtained with a rapid-flow technique for comparison with the titration curve of the denatured protein. It is natural to seek further for other cases among the proteins which Porter found had t-amino groups unavailable for reaction with FDNB. Of those studied, critical titration data are available only for P-lactoglobulin (Cannan, Palmer, and Kibrick, 1942), for which the maximum amount of hydrogen ion bound corresponds very well with the amino acid content, assuming complete availability of the basic groups. Although the slope of the titration curve in the range p H 9 to 11 is somewhat steeper (ie., w smaller) than expected, the titration with base becomes irreversible only for p H 11 and above. The reaction that occurs there is probably the same as the pH-dependent denaturation of P-lactoglobulin studied by Groves, Hipp, and McMeekin (1951) between p H 8 and 10.5, where it is so slow that it does not affect the determination of the titration curve. It will be recalled that Porter found that all the a-amino groups of this protein reacted freely with ketene, although those of several serum globulins did may be due t o the difference in species and t o the fact t h a t OsHb was used by Cubin. Others, however, appear t o be due t o apparent combination by these authors of d a t a on different species and from different sources; and t o failure t o take into account different denaturation paths (as, for example, a t high temperatures a t the isoelectric point as distinguished from low temperatures in acid solutions). Their divergent conclusion as t o the pK of the trigger groups is due t o an oversimplified procedure for its determination. The equation given on page 203 of Neurath et al. (1944) is not exact and is only approximately true in the region in which log velocity is directly proportional t o pH (no curvature)-a condition which, when true, does not permit K t o be determined. Their conclusions as t o the bond energies involved cannot be accepted.

218

J. STEINHAHDT AND E. M. ZAISEll

not. A strictly steric interpretation suggests that groups inaccessible to FDKB may he easily reachcd by ketene, arid thus even more easily by hydrogen ion. If this is true, then the reaction of these groups, a t least with small molecules or ions, does not itself denature protein-rather, denaturation which is initiated by the ionization of “trigger groups” or by other cleavages results in rendering these groups more accessible. Electrophoretic experiments with dodecylsulfate ions arid serum albumin (Putnam and Neurath, l944b, 1945) are consistent with this interpretation. Titration data, when available, on the serum globulins studied by Porter which contain groups unreactive to ketene as well as to FDNR, may throw critical light on such steric interpretations. Bovine serum albumin. Levy and Warner (1954) have studied the kinetics of denaturation of this protein at temperatures above 46°C. and have shown that a very large decrease in the first-order denaturation rate occurs in the pH interval 2.5 to 3.5. This change, which occurred a t all the temperatures studied, is found in the same p H range in which Yang and Foster (1954) found changes in optical rotation and viscosity, and in which Gutfreund and Sturtevant ( I 953) found a fully reversible first-order evolutioii of heat of 3100 cal. The half-period of the effect was 2.5 minutes a t 25°C. a t pH 3.4, short enough to render it likely that titration data obtained a t this pH or lower characterize the reversibly altered protein. Since the reaction is fully and rapidly reversed on returning the pH t o 4.5, and since the protein is more stable a t the low pH, the titration curves, if affected, should also be completely reversible, as indeed they are for this protein and for human serum albumin as well (Shore, 1953; Tanford, 1950, 1952). Precipitation of denatured protein on neutralizing is not observed. Although the existence of pH-dependent reversible effects is not ipsofacto proof of the unmasking of prototropic groups, the fact that the published titration curves (Shore, 1953) deviate from those of “normal” proteins (ovalbumin, P-lactoglobulin) in the same direction as horse hemoglobin (Steinhardt and Zaiser, 1951) raises a question as to the existence of masked groups in this protein. The abnormal steepness of the acid branch of this tihation curve is only partly accounted for by the large anion-binding of this proteiri (Tanford, 1950; Shore, 1953). The residual effect has been attributed to reversible changes in shape, or swelling, due to reversible rupture of internal hydrogen bonds involving the phenoxyl groups of tyrosirie and (probably) carboxylate groups (Tanford and Roberts, 1952; Tanford, 1952; Shore, 1953). The unmasking of prototropic groups, however, might account for much more of the anomalous steepness of the curve than such changes in niolecular shape as may accompany the unmasking. However, efforts to detect a rapid p H drift in the titration of this protein have not been successful (Warner, 1954).

HYDROGEN ION EQUILIBRIA

219

Trypsin. Gutfreund and Sturtevant (1953) reported a reversible heat evolution with trypsin a t pH 2.5, similar to but larger (8000 cal.) than that found with bovine serum albumin. The titration data, however, are reversible and appear normal (Duke, Bier, and Nord, 1952) except for a strong influence of calcium ion and a larger hydrogen ion-binding capacity than can be accounted for from the analytical data. In the absence of denaturation experiments under comparable conditions, this observation of Gutfreund and Sturtevant can be considered only as an iiidicatioii that this protein should be investigated further from the point of view of this paper. Other proteins. All cases of “thermal” denaturation appear to be sensitive to pH. In some cases denaturation by dilute acid or base (pH 1 to 13) occurs a t the same temperatures (20” to 25°C.) a t which titration curves are normally obtained. The meaning of the interrelation between temperature and pH has beeii thoroughly elucidated for pepsiu (Steinhardt, 1937), ricin (Levy and Benaglia, 1950), ovalbumin (Gibbs, Bier, and Nord, 1952; Gibbs, 1952), and serum albuiniii (Levy and Warner, 1954; Gibbs, 1954).43 The existence of pH-sensitive reversible denaturations cannot be assumed in all cases to involve the liberation of prototropic groups. However, the fact that such denaturations in acid solutions have beeu shown in a number of cases, described above, to involve unmasking indicates that it should be looked for in every case in which denaturation occurs a t room temperature. The existence of p H drifts a t some pH in unbuffered solutions is easy to observe with the glass electrode, arid might serve initially to determine those cases which should be investigated further. When denaturation occurs at measurable rates in the p H range 2 to 12 only at elevated temperatures, there is 110 need to seek for evidence of unmasking, except in those cases in which discrepancies exist between maximum proton binding or dissociation, and amino acid analyses (where both sets of information are available). Abnormalities in the titration curve (steep slopes) may also be recognized in such cases. Kinetic experiments on denaturation are almost invariably conducted with buffers-it is quite possible, therefore, that drifts in pH, which mould otherwise have occurred, have not been observed. In the years since the publication of the comprehensive review of Neurath 43 Numerous other studies are, however, open t o serious criticism on grounds which have been stated most clearly by Neurath et a l . (1944): “Inasmuch a s . . . failure t o include the hydrogen-ion concentration properly in calculating the specific rate constants has been quite general in studies of the kinetics of protein denaturation, the calculation of the entropy of activation” (and one may add, the other “thermodynamic analogues” of absolute reaction rate theory) “must be formal insofar as the value of the activation energy is empirical and the standard states of the reactants remain unspecified.”

220

J. STEINHARDT AND E. M. ZAISER

et al. (1944) the number of protein denaturations which have been studied kinetically, some times in considerable detail, has been substantially enlarged. The best studied cases in which both temperature and pH have been varied include ricin (Levy and Benaglia, 1950), and both bovine and human serum albumin (Gibbs, Bier, and Nord, 1952; Gibbs, 1952; Levy and Warner, 1954) ; these studies have applied and extended the analytical procedure first made use of with pepsin (Steinhardt, 1937). Equally detailed are the careful studies of Kearney and Singer (1951) on partially purified snake venom Z-amino oxidase, the behavior of which with respect to chloride ions is unique in many ways. The denaturation of /3-lactoglobulin and a dodecylsulfate derivative by base in the pH range 8 to 10.5 has been studied by Groves, Hipp, and McMeekin (1951), and is probably related to the irreversible changes in acid-base behavior above pH 11 reported by Cannan, Palmer, and Kibrick (1942). The reversible acid inactivation at high temperatures of streptokinase (Christensen, 1947) and hyaluronidase (Mathews and Dorfman, 1953) occurs in the same pH range as the denaturation of hemoglobin, conalbumin, and bovine serum albumin, but the lack of highly purified preparations, or of work in unbuffered solutions, would prevent the observation of pH drifts if any occurred. Less detailed or less conclusive data on influenza A virus hemagglutinin (Lauffer and Scott, 1946), human plasma cholinesterase (Goldstein and Doherty, 1951), yeast raffinase (Wagreich, Abraham, and Epstein, 1948), and triosephosphate dehydrogenase (Rapkine, Shugar, and Simonovitch, 1950), do not furnish the information necessary for conclusions as t o concurrent unmasking of unreactive prototropic groups. 11. Summary of Section I I I

The prototropic groups of a number of native proteins, like their other functional groups, are less fully reactive than in the denatured protein, or than would be expected if all their side chains were freely available. Thus, in the hemoglobin of horse, pig, and human beings, large numbers of pairs of carboxyl and amino groups are hindered from dissociating or combining with hydrogen ion until after reaction of a smaller number of trigger groups initiates their liberation. The trigger groups in the heme proteins appear most likely to involve the propionate side chains of the prosthetic groups. Detailed similarities in either titration or denaturation kinetic or equilibrium behavior of a number of other proteins (at a minimum these include edestin, conalbumin, and horse-radish peroxidase) furnish strong indications that in these proteins also appreciable numbers of amino and carboxyl groups are not free to participate in acid-base equilibria. As with the hemoglobins, brief exposure to dilute acid (and presumably to dilute base) even a t room temperature, reversibly unmasks the unreactive groups.

HYDROGEN ION EQUILIBRIA

221

The possibility is shown to exist that this phenomenon is more general (involving possibly serum globulins, collagen, and hog thyroglobulin among others). Since the existence of unreactive groups and their reversible liberation have a profound effect on the titration curves of proteins in which they occur, the measurement of these curves by a rapid-flow technique is suggested in all doubtful cases. Because of the conditions under which Porter and Sanger’s end-group technique is applied to proteins, denaturation occurs with some, and in these all the masked groups may appear to be free. This is not always the case, however, and in globulins especially titration evidence for unmasking should be sought. The unmasking phenomenon is always accompanied by denaturation, as usually defined, although it is often fully reversible if the protein is not exposed for long periods to extremes of pH or temperature. More generally, denaturation (whether reversible or not) renders reactive such prototropic groups as were not free to react in the native state (as in the case of tyrosine phenoxyl of ovalbumin studied by Crammer and Neuberger, 1943). The conclusion is inescapable that the failure of large numbers of these prototropic side chains of certain proteins to react furnishes information as to the structural or configurational distinction between native and denatured proteins. It also explains a number of anomalies in titration or in electrophoretic behavior which have long been known. Although the evidence is inconclusive as to how unavailable acidic or basic groups are prevented from reacting in the native protein, the balance appears to favor a purely steric interpretation based on the existence of “folds” or “pockets” in the native molecule. Since only the small number of trigger groups, which initiate the process of unfolding or refolding, form bonds involving appreciable chemical energy, it is only these groups which are involved in the formation of internal cyclic structures in the native protein.

REFERENCES Alberty, R . A., andMarvin, H. H., Jr. (1951). J . Am. Chem. Soe. 73,3220. Anson, M . L., and Mirsky, A. E. (1925). J. Gen. Physiol. 9, 169. Anson, M. L., and Mirsky, A. E. (1929). J . Gen. Physiol. 13, 121. Anson, M.L . , and Mirsky, A. E. (1931a). J. Gen. Physiol. 14, 597. Anson, M . L., and Mirsky, A. E. (1931b). J . Phys. Chew. 36, 185. Anson, pvl. L., and Mirsky, A. E. (1934). .T. Gen. Physiol. 17, 399. Bailey, K. (1942). Biochem. J . 36, 140. Benesch, R.,and Benesch, R . E. (1948). Arch. Biochem. 19, 35. Bernhart, F. W. (1939). J . Am. Chem. SOC.61, 1953. Booth, N . (1930). Biochem. J. 24, 158. Bowes, J. H., and Kenten, R . H . (1948). Biochem. J . 43, 358. Bull, H. B . (1938). Cold Spring Harbor Symposia Quant. Biol. 6, 140. Bull, H.B . (1946). J. Am. Chem. SOC.68, 747.

222

J. STEINHAKDT AND E. M. ZAISElX

13~11,11. I<., ant1 Xeurath, H. (1937). J. Biol. Chem. 118, 163. Cannan, It. K. (1942). Chcm. Revs. 30, 395. Cannan, R. K., and Kibrick, A. (1938). J. Am. Chem. SOC.60, 2314. Cannan, R . K., Kibrick, A . , and Palmer, A. H . (1941). Ann. N . Y. Acad. Scz. 41, 243. Cannan, It. K., Palmer, A. H., and Kibrick, A. (1942). J. Biol. Chem. 142, 803. Chapman, 1,.&I., Greenberg, D. M., and Schmidt, C. L. A. (1927). J. BioZ. Cheni. 72, 707. Chaudhuri, I). It. (1948). H u n g . Acta Physiol. 1, 238; per Chew. Abstr. 43, 9097d (194!)). Chou, C., and Wu, 11. (1934). Chinese J . Physiol. 8, 145. Cliristen~c~i, 1,. It. (1947). J. Gen. Physiol. 30, 465. Cohn, E. J . , and Edsall, J. T. (1943%). “Proteins, Amino Acids and Peptides as Ions and Dipolar Ions,” Chapter 20. Reinhold, New York. Cohn, E. J., and Edsall, J. T . (1943b). “Proteins, Amino Acids and Peptides as Ions and Dipolar Ions,” p. 481. Reinhold, New York. Cohn, E. J., and Edsall, J. T., (1943~). “Proteins, Amino Acids and Peptides as Ions and Dipolar Ions,” Chapter 4. Reinhold, New York. Cohn, E. J., Edsall, J . T., and Blanchard, M. H. (1934). J . Biol. C h o n . 106, 319. Cohn, E. J., Green, A. A . , and Blanchard, M. H. (1937). J. Am. Chem. SOC.69,509. Cohn, E. J., Salter, W. T., and Ferry, R. Rf. (1938). J. Biol. Chem. 123, Proc. xxiv. Coryeil, C. D., and Pauling, L. (1940). J. Biol. Chem. 132,769. Craig, J. P., Jr., Garrett, A. G., and Williams, H. B. (1954). J . Am. Chem. SOC.76, 1570. Crammer, J. I,,, :ind h’euberger, A. (1943). Biochenz. J . 37, 302. Dubuisson, 31. (1941). A x h . intern. Physiol. 61, 133, 154. Dubuisson, M., and Hamoir, G . (1943). Arch. intern. Physiol. 63, 308. Duke, J A , , Bier, M., and Nord, F. F. (1952). Arch. Biochem. and Biophys. 40,424. Eisenherg, M. A , , and Schwert, G . W. (1951). J. Gen. Physiol. 34,583. 13llenbogeri, E. (1952). J. Am. Chem. SOC.74, 5198. Elliott, D. F. (1952). Biocheni. J . 60, 542. Nod, E., and Frohlich, H. G. (1949a). Melliand Teztilber. 1949, 239. Elod, 1 C , and Frdhlich, H. G. (1949b). Melliand Tezlilber. 1949, 405. Ferry, J. D., and Morrison, 1’. R . (1945). “The Physical Properties of Fibrin Films Prepared from Human Fibrinogen and Thrombin.” 11. Interim report under Contract OEMcmr-139 with the Committee on Medical Research and Development. Forrest, W. W., and Sturtevant, J. M. (1954). Paper read at the 126th National Meeting of the American Chemical Society at New York City in September. E’ruenkel-Conrat, H., Bean, R . S., and Lineweaver, H. (1949). J. B i d . Chew. 177, 385. Fraenkel-Conrat, II., and Cooper, M. (1944). J . Biol. Chem. 164, 239. Fredericq, 13. (1954). J. Polymer S c i . 12, 287. French, D., and Edsall, J. T. (1945). Advances in Protein Chem. 2, 277. Frieden, E. H., Dunn, M. S., and Coryell, C. D. (1943). J. P h y s . Chem. 47, 118. Fromageot, C., and Schnek, G. (1950). Biochim. et Biophys. Acta 6, 113. German, I<., and Wyman, J., Jr. (1937). J . B i d . Chem. 117, 533. Ghosh, B. N., and Mihdlyi, E . (1952). Arch. Biochem. and Biophys. 41, 107. Gibbs, R . J. (1952). Arch. Biocheni. and Biophys. 36, 229. Gibbs, R . ,J. (1954). Arch. Biochem. and Biophys. 61, 277; 62, 340.

HYDROGEN ION EQUILIBRIA

223

Gibbs, R. J., Bier,M., andNord, F. F. (1952). Arch. Biochem. and Biophys. 36,216. Glassman, H. N. (1950). Ann. N. Y. Acad. Sci. 63,91. Glassman, H. F., and Molnar, D. M. (1951). Arch. Biochem. and Biophys. 32,170. Glasstone, S., Laidler, K. J., and Eyring, H. (1941). “The Theory of Rate Processes,” Chapter VIII. McGraw-Hill, New York. Gleysteen, 1,. F., and Hahis, M. (1941). J . Research Natl. Bur. Standards 26, 71. Goldstein, A., and Doherty, M. E. (1951). Arch. Biochem. und Biophys. 33,225 Gorini, 1,. (1951). Biochim. et Biophys. Acta 7, 318. G r a l h , N. (1939). Biochem. J. 33, 1907. Greenberg, D. M. (1944). Advances i n Protein Chern. 1, 121. Groves, M. I,., Hipp, N. J., and McMeekin, T. L. (1951). J . Am. Chem. SOC.73, 2790. Guggenheim, E. A. (1926). Phil. Mag. [2] 7, 538. Curd, F. R. N., and Goodman, D. S. (1952). J . Am. Chem. Soc. 74,670. Gutfreund, H., and Sturtevant, J. M. (1953). J . Anz. Chem. SOC.76,5447. Heidelberger, M., and Palmer, W. W. (1933). J . Biol. Chem. 101,433. Heidelberger, M., and Pedersen, K . 0. (1935). J . Gen. Physiol. 19,95. Heilmeyer, I,. (1943). “Spectrophotometry in Medicine.” Hilger, London. Hendrix, 13. M., and Wilson, V. (1928). J. Biol. Chem. 79, 389. Herriott, R. M. (1936). J . Gen. Physiol. 20, 335. Herriott, R. M. (1947). Advances in Protein Chem. 3,169. Herriott, R . M., and Northrop, J. H. (1934). J. Gen. Physiol. 18,35. Hicks, C. S., and Holden, H. F. (1929). Australian J . Exptl. Biol. Med. Sci. 6, 175. Hipp, N. J., Groves, M. I,., and McMeekin, T. L. (1952). J . Am. Chem. SOC.74, 4822 Hitchcock, D. G. (1921). J . Gen. Physiol. 4,597. Holden, H. F. (1936). Australian J. Exptl. Biol. M e d . Sci. 14, 291. Holden, H . F. (1945). Australian Rev. Biochem. 14, 599. Hughes, W. L.,Jr. (1950). Cold Spring Harbor Symposia Quant. Biol. 14, 79. Jacobsen, C. F. (1947). Compt. rend. trav. lab. Carlsberg, SBr. chim. 26,326. Karush, F., and Sonenberg, M. (1949). J . Am. Chem. SOC.71,1369. Katchalsky, A., and Gillis, J. (1949). Rec. trav. chim. 68.879. Katchalsky, A., and Lifson, S. (1953). J . Polymer Sci. 11, 409. Ilatchalsky, A . , Shavit, N., and Eisenberg, H . (1954). J . Polymer Sci. 13, 69. Katchnlsky, A . , and Spitnik, P. (1947). J . Polymer Sci. 2, 432. Kearney, E. B., and Singer, T. P. (1951). Arch. Biochem. and Biophys. 33,377,397, 414. liistler, l’., Buri, A., and Nitschmann, € I . (1953). Helv. Chim. ’4cta 36, 1058. Tilotz, I. R I . (1953). I n “The Proteins” ( € I . Neurath and K. Bailey, eds.), 1‘01. I, Part B, Chapter 8. Academic Press, Nev York. Jlominz, D. R . , Hongh, A . , Sgmonds, P., and Laki, K. (1954). Amh. Biochem. and Biophys. 60, 148. Kunitz, 31. (1948). J . Gen. Physiol. 32, 241. Laskowski, N . , J r . , and Scheraga, H . A. (1951). .J. Am. Chem. SOC.76,6305. Lauffer, M. A . , and Scott, E. M. (1946). Arch. Biochern. 9, 75. Lemberg, R., and Legge, J. W. (1949). “Hematin Compounds and Bile Pigments,” pp. 373, 223-4. Interscience, New York. Levy, ill.(1935). J. Biol. Chem. 109, 365. l,evy, 31 , and Benaglia, A . E. (1950). J. Bzd. C‘henr. 186,829. I,evy, M . , and Warner, R. C. (1954). 1.Phys. Chein. 68, 106

224

J. STEINHARDT AND E. M. ZAISER

Lewis, P. S. (1927). Biochenz. J. 21, 47. Lewis, U. J. (1954). J. B i d . Chem. 206,109. Lichtenstein, I . (1940). Biochem. 2. 303, 22. Lifson, S., and Katchalsky, A. (1954). J. Polymer S c i . 13, 43. LinderstMm-Lang, K (1924). Compt. rend. trav. lab. ('arlsberg 16, No. 7. Linderstlybm-Lang, K. (1927). Conipt. rend. trav. lab. Carlsberg 17, No. 4 LinderstMm-Lang, K. (1935). Trans. Paraday Soc. 31, 324. Linderstrplm-Lang, K. (1952). ('ompt. rend. trav. lab. Carlsberg, SBr. chim. 28,281. Linderstlybm-Lang, K., and Jacobsen, C. F. (1941). ("ompt. rend. trav. lab. C a d s berg, S6r. chim. 23, 179. Longsworth, L. G. (1941). A n n . 'V. Y. Acad. S c i . 41, 267. Lontie, R., and Panier, A. (1951). Bull. soc. chim. biol. 33, 532. Loughlin, W. J. (1933). Biochem. J. 27,99. Lumry, R., and Eyring, H. (1953). J . Phys. Cheni. 68,110. Lundgren, H. P. (1915). Textile ResearchJ. 16,335. McMeekin, T . I,., Polis, B. D., DellaMonica, 12. S., and Custer, J. €1. (1949). J . Am. Chem. Sac. 71, 3606. Maehly, A. C. (1952). Biochinr. et Biophys. Acta 8, 1. Maehly, A. C. (1953). Arch. Biochem. and Biophys. 44,430. . Acta 13,354. Massey, V., and Alberty, R. A. (1954). Biochim. ~ t Biophys. Mathews, M. B., and Dorfman, A. (1953). J . B i d . Chem. 208, 143. MihAlyi, E. (1950). Enzymologia 14, 224. MihBlyi, E. (1954a). J . Biol. Chem. 209, 723. MihBlyi, E. (1954b). J. Riol. Chem. 209, 733. Mirsky, A. E., and Anson, M. 1,. (1929). J . Gen. Physiol. 13, 133. Mirsky, A. E., and Anson, M. 1,. (1930). J. Gen. Physiol. 13,477 Moyer, L. S. (1938). J. P h y s . ('hen,. 42, 71. Nanninga, 1,. B. (1954). Personal communicatioii. Neuberger, A. (1934). Biochetn. J. 28, 1982. Neurath, H. (1940). J . P h y s . Chem. 44, 296. Neurath, H., Greenstein, J. P., Putnam, F. W , and Erickson, J. 0. (1944). ('hem. Revs. 34, 157. Nord, F. F., and Bier, hl. (1953). Biochim. et Biophys. Acta 12.56. Pace, J. (1931). Biochem. J . 26, 422. Perlmann, G. E. (1941). J . B i o l . ('hem. 137, 707. Philpot, J. S t . L . , and Small, P. A. (1938). Biochetn. J . 32,542. Polonovski, J., and Macheboeuf, hI. (1918). A n n . i n s t . Pasteur 74, 196. Porter, R. R. (1948). Biochim. et Biophys. Acta 2, 105. Porter, R. R. (1950). Biochem. J. 46, 304. Porter, R. R., and Sanger, F. (1948). Riochern. J . 42, 287. Prideaux, E. B. R., and Woods, D. 15. (1932). Proc. Roy. SOC.B110, 353. Putnam, F. W . , and Seurath, H. (104la). J. A t t i . ('herti. SOC.66, 692. Putnam, F. W., and Seurath, 11. (1944b). J . A m . ('hettr. Soc. 66. 1902 Putnam, F. W., and Nrnmth, €1. (1945). .I H i d . ( ' h f , r t i . 169, 305. Rapkinc, I,., Shrigni, I)., and Simonovitch, I, (1O50). .4 / c h . N i o c h c t / t . 26, X I Rasmussen, I<. E., a n d I,intlrrstlybni-I,anff,K (1935) ( ' o t f t p t . ronrl. trav. I d ) . Carl\, berg 20, No. 10 Roche, J. (1930). ( ' o w p t . i v / i d YIP. lob. ('arlsbpry 18, N o 4. Ronsselot, A . (194-4). ( " o v i p t . rend 218, 716.

HYDROGEN ION EQUILIBRIA

225

Sanger, F. (1945). Biocheni. J. 39, 507. Scatchard, G. (1949). Ann. A-. Y. Acarl. S c i . 61, 660. Scatchard, G., and Black, E. S. (1949). J. Phys. 82 Pollozd (‘hem. 63.88. Scatchard, G., Scheinberg, I . H., and Armstrong, S. II., Jr. (1950a). .I. A m . V h m . Sac. 72, 535. Scat,chard, G., Scheinberg, I . H., and Armstrong, S. H., J r . (l950b). J . Am. (‘hettr. Sac. 72, 540. Schmidt, C. 1,. A. (1944). “The Chemistry of the Amino Acids and Proteins,” 2nd ed., p. 744. C. C. Thomas, Springfield. Shore, W. 8. (1953). Dissertation Abstr. 13, 318. Shulman, S., and Ferry, J. D. (1950). J. P h y s . & Colloid ChenL. 64, 66. Steinhardt, J . (1937). Kgl. Danske Videnskab. Selskab M a t . f y s . Medd. 14, No. 11. Steinhardt, J. (1938). J . Biol. Chem. 123, 543. Steinhardt, J. (1941). Ann. N . Y. Acad. S c i . 41, 287. Steinhardt, J., and Zaiser, E. M. (1950). J . Biol. Chenz. 183, 789. Steinhardt, J., and Zaiser, E. M . (1951). J. Biol. Chem. 190, 197. Steinhardt, J., and Zaiser, E. M. (1953). J. Am. Chem. Sac. 76, 1599. Steinhardt, J., and Zaiser, E. M. (1954). Abstract in Federation Proc. 13. Steinhardt, J., Fugitt, C. H., and Harris, M. (1940a). J . ResearchNatl. B u r . Standards 24, 335. Steinhardt, J.,Fugitt, C. H., andHarris,M. (1940b). J . Research-Vatl. Bur. Standards 26, 519. Steinhardt, J., Fugitt, C. H., and Harris, M. (1941). J . Research N a t l . Bur. Standards 26, 293. Steinhardt, J., Fugitt, C . €I., and Harris, M. (1942). J . Research N a t l . Bur. Standards 28, 201. Steinhardt, J., Fugitt, C. H., and IIarris, M. (1943). J. Research X a t l . Bur. Standards 30, 123. Steiuhardt, J., Fugitt, C. H., and Harris, h3. (1946). J . Biol. Chem. 166, 285. Steinhardt, J , Zaiser, E. M., and Gibbs, R. J . (1954). Unpublished observations. Tanford, C . (1950). J . Am. Cheni. Sac. 72,441. Tanford, C . (1952). Proc. Iowa Acad. Sci. 69, 206. Tanford, C. (1954). J. A m . Chem. Sac. 76, 945. Tanford, C., (1955). J . Am. Chem. Sac. 77, 1912. Tanford, C . , and Epstein, J. (1954a). J . Am. (’hem. Soc. 76, 2163. Tanford, C., and Epstein, J. (195413). J. Am. Chein. Sac. 76,2170. Tanford, C., and Roberts, C . I,., Jr. (1952). J . A m . Chem. Sac. 74,2509. Tanford, C . , and Wagner, M. 1,. (1954). J. Ant. C h e m . Sac. 76, 3331. Theis, 13. R., and Jacoby, T. F. (1943). J . Biol. Phenz. 148, 105. Theorell, H . (194!). i2rkivoKemi Mineral. Geol. MA, No. 14. Theorell, H., and Alreseon, A. (1941). J. Am. Chem. Sac. 63, 1818. Theorell, H., and Ehrenherg, A. (1951). Acta Chew). Scand. 6, 823. Timasheff, S. N ,and Nord, F. F. (1951). Arch. Biochem. and Biophys. 31,309. Tristram, G . It. (1949). In “Hemoglobin” (F. J . W. Roughton and J. C. Kendrew, eds.), pp. 109-111. Butterworth’s, London; Interscience, New York. Tristram, C i . R. (1053). In “The Proteins” (H. Neurath and K . Bailey, eds.), Yo]. I, Part A, Chapter 3. Academic Press, New York. Yelick, S. F. (1949). J . P h y s . & Colloid Chem. 63, 135. Wagreich, H., Abraham, S., and Epstein, M . E. (1948). Arch. Biochem. 18, 147.

226

J. STEINHAHDT AND E. M. ZAISER

Warner, H. <’. (1954) Personal communication. Wu, H , and C‘hrn, T. T. (1929). f’hinese J . I’hysiol. 3, 7. W u , €I., Liu, 8 . C . , :tnd Chori, C. (1931). (‘hinese J . Physzol. 6 , 30!J. Wyman, J., J r . (1939). J . Bzol. (‘herti. 127,1. Wyman, J., Jr. (1948). Advances z n Pr o t ei n Chctn. 4, 407. Ysng, J. T., and Foster, J . F. (1954). J .4m. (’heni. SOC.76, 1588. Zaiser, E. M., and Steinhardt, J. (1951). .I. .4m. C‘heni. Sac. 73,5568. Zaiser, Ii:. M., and Steinhardt, J. (1954a). .I. Am. Chevr. Sac. 76, 1788. Zaiser. E. hl.. and Steinhardt, J. (19541-3). J. Am. Chein. Sac. 76, 2866.

Fish Proteins BY G. HAMOIR* Laboratory of General Biology, University of Lihge, Belgium

CONTENTS I . Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Proteins from Skeletal Muscle.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. IIistology of Fish Muscle., . . . . . . . . . . . . . . . . . . . . . . ............ 2. Amount and General Composition of Fish Muscle 3. Classification and Nomenclature of the Muscle Proteins.. . . . . . . . . . . . . . 4. Earlier Biochemical Work on Fish Muscle Proteins.. . . . . . . . . . . . . . . . . . . a. Quantitative Fractionation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................... b. Some Abnormal Propcrties of Fish Proteins. 5 . Composition of Extract.s of Low Ionic Strength 0.15) . . . . . . . . . . . . . . . a . White Muscles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . h. Red Muscles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Izxtractability of the Structural Muscle Proteins. . . . . . . . . . . . . . . . . . . . . 7. Composition of Extracts of High Ionic Strength ( 2 0 . 5 ) . . . . . . . . . . . . . . . a. Extracts Obtained at p 0.5 and pH 5.9 and 6.2.. .................... b. Extracts Obtained a t p 1.0 and p H 5.0, 5.2 and 5.5.. . . . . . . . . . . . . . . . 8. Isolation and Properties of Some Protein Components of Fish Muscle. a. Henrotte’s Myogen.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Carp and Tuna Myoglobins.. . . . . . . . . . . . . . . . . . . . . . c. Actomyosin.. ........................................ d. Myosin ....... ..................................... e. Actin.. . . . . . ............................ .......... ......................................... 111. Fish Enzymes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Fish Blood Proteins.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Fish Protamines.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Connective Tissue Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Conclusion: The Comparative Biochemistry of Fish Proteins.. . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

227

231 235 235 235 237 238 239 241 243 245 215 246 251

255 262 265

274 277 279 282

I. INTRODUCTION The study of fish proteins were initiated by the Swiss physiologist Miescher (1897), who first isolated the protamiiies from the sperm cells of various fishes as early as 1868. The relatively simple amino acid composition of these proteins and their pharmacological action have since promoted extensive researches, first of all by Kossel and co-workers, whose important

* AssociO du Fonds National

do la Recherche Scientifiquc.

227

228

G. HAMOIR

contribution was summarized in a monograph (Kossel, 1928). Numerous papers have since been published on this group of proteins by various authors (see Geiger, 1948; Felix, 1953). Thiaminase, the enzyme cleaving thiamine, has also received much attention during recent years (see Harris, 1951). The interest in fish proteins has not, however, been limited to components endowed with some specific property. The first investigations on fish muscle proteins carried out by Reay and Kuchel (1936) showed that a loss of solubility was brought about by storing in the frozen state, owing to a specific denaturation of the actomyosin fraction, as demonstrated by Subba Rao (1948) and by Dyer, French, and Snow (1950). The impairment of quality occurring in frozen fish on storage is due both to fat spoilage and to protein denaturation (Dyer, 1951). Basic research on the proteins involved is necessary in order to elucidate the origin of these alterations, since our present knowledge of muscle proteins is gathered almost exclusively from investigations carried out on the rabbit. A similar development has now been undertaken as regards fish.

11. PROTEINS FROM SKELETAL MUSCLE 1. Histology of Fish Muscle The muscle biochemists have taken little account in the past of the various histological aspects of this tissue, but recent efforts have been made to attempt a correlation of cytological and biochemical aspects (Perry, 1952, 1953; Huxley, 1953; Lawrie, 1953a,b; Huxley and Hanson, 1954). The common development initiated by these researches makes it desirable to pay more attention to the histology of the material investigated. The occurrence of two kinds of muscular fibers different in color and iu sarcoplasmic content was established a long time ago (Ranvier, 1874; Knoll, 1891). In the case of fishes several histologists (Lavocat and Arloing, 1875; Stirling, 1886; Knoll, 1889, 1891; Greene, 1913; Kishinouye, 1921-23) have observed the presence of a red superficial muscular region underneath the skin along the lateral line (Musculus lateralis superficialis Trunci). This red muscle has been observed in many species (Stirling, 1886; Bolk et al., 1938; Maser, 1950) and is described in anatomical treatises (Vialleton, 1902; Ihle el al., 1927; Bolk et al., 1938) and in several papers (Stirling, 1886; Maser, 1950). Its width (in cross section) is greatest along the lateral line and decreases progressively dorsally and ventrally (Fig. 1). In the case of the Scombridae this “superficial” red muscle is prolonged along the horizontal myoseptum by another “deep-seated” one (Kischinouye, 1921-23). A red coloration in fin muscles has also been noticed by Lankester (1872) and Rollett (1888). Another general subdivision of skeletal muscles based on the appearance of transverse sections of the fibers has been proposed by Kruger (1952);

229

FISH PROTEINS

he distinguishes a “Fibrillenstruktur,” in which the myofibrils are uniformly distributed, and a “Felderstmktur,” in which they are associated in small bundles, in discrete fields through the whole section of the fiber. The various criteria used, color, amount of sarcoplasm, or appearance of transverse sections, cannot be correlated absolutely, but, as a first approximation, it call be assumed from the study of various vertebrates that the “Fibrillenstruktur” corresponds to the white muscle poor in sarcoplasm and the “Felderstruktur” to the red muscles rich in sarcoplasm (Barer, 1948; Kruger, 1950). The two aspects described by Kruger can be observed in muscles of the Selachii, whose histology is therefore similar to that of the other vertebrates

uperttrunci

a

b

FIQ.1. Transverse sections through the tail (a) and the abdomen ( b ) of the carp showing the lateral red muscular region (after NISHI, 1938).

(Kruger, 1950). Couteaux (1950) has furthermore noticed differences in the innervation of the white and red fibers of these fishes. I n the case of the teleosts, however, the appearance is too complicated to allow a classification based on direct microscopic observation. Their muscles contain not only the cylindrical myofibrils of all other vertebrates but also ribbonshaped ones (Emery, 1882; Lansimaki, 1910) (Figs. 2 and 3), and these two types of myofibrils occur in very variable arrangement and proportions. The study of the embryological development of these muscles, however, has improved the earlier classification based on their color, since a division into “primary” and “secondary” fibers has been established (Buhn, 1940). The primary fibers are fully developed at hatching, and their peripheral myofibrils are situated just beneath the sarcolemma from the beginning of the development (Fig. 2) ; they contain a great number of cylindrical or band-shaped myofibrils and correspond approximately to the white muscles. The secondary fibers develop only in adult animals. I n this case, sarco-

230

G. HAMOIIL

FIG.2. Transverse section of fish white muscle showing BUHN’Y (1940) prim:try fibers. Adductor superficialis of Leuciscus rutilus L. Magnification 700 X (after B U H N , 1940).

FIG.3. Transverse sections of fish red muscle showing BUHN’S (1940) secondary fibers. LqM: Musculus lateralis superficialis Trunci of catfish (Ameiurus nebulosus I,.). bhgnification XI400 (after BARETS, 1952). Right: pectoral fin muscle of stickleback (Gaslerosteus acideahs L.). Magnification X666 (after MASER, 1960).

plasm accumulates beneath the sarcolemma forming a wide circular zone, a sarcoplasmic mantle, and is also abundant among the myofibrils (Fig. 3). Moreover, the number of mitochondria is much higher (Uematsu, 1954). Different descriptions of the secondary fibers have been given (Rollett, 1888; Buhn, 1940; Maser, 1950; Barets, 1952; Uematsu, 1951, 1954) and zt subdivision into several types based upon the arrangement of the myofibrils has been proposed (Uematsu, 1951, 1954). This variability is illus-

FISH PROTEINS

23 1

trated in the two pictures of Fig. 3. The secondary fibers are red and usually rich in sarcoplasm, and a further difference between primary and secondary fibers is found in their innervation (Barets, 1952); it is likely therefore that they also differ in physiological behavior. On the other hand, we do not know whether the variation in cross section of the myofibrils themselves is of significance. I n the course of embryological development, the peripheral ribbon-shaped myofibrils split, giving rise t o the more axial cylindrical ones (Buhn, 1940). I n view of these interrelations, it is possible that these morphological aspects have little significance from the biochemical point of view.

2 . Amount and General Composition of Fish Muscle The amount of flesh in fresh fish has been evaluated in many species by Reay, Cutting, and Shewan (1943) and by Van Wyk (1944). The first authors took the edible portion as equivalent to the commercial fillet and did not attempt to correlate their results with size, age, sexual state, or season of capture. The yields obtained for fillets without skin usually varied between 40% and 50% according to species. I n the evaluations made by Van Wyk (1944), the flesh was removed as quantitatively as possible and the amounts obtained corresponded to 50 % to 60 % of the total weight of the animal. Such results compare quite favorably with the values given for domestic animals. The yield of meat from the ox amounts to approximately 42 % of the live weight, according to Reay, Cutting, and Shewan (1943), and Jean-Blain (1948); in the case of man figures of 43 % (Bjorck, 1949), 40% (Walker, Boyd, and Asimov, 1952; Forbes, Cooper, and Mitchell, 1953), and 32 % (Mitchell et al., 1945) have been given. The relative amount of muscular tissue is thus higher in fish than in domestic animals or in man. The composition of fish striated muscle with respect to water, proteins, and lipids differs widely from one species to another, and within a species seasonal variations may occur. The influence of this factor has been investigated by Van Wyk (1944). In fatty fishes, an important increase in fat content is observed in Spring or in Summer, with a corresponding decrease in water content, while the nitrogen values remain relatively constant. Determinations made on many species by various authors have been compiled by Jacquot and Creac’h (1950). Other evaluations have been done more recently on numerous Spanish fishes (De Las Heras and Mendez Isla, 1952) on a few Kolhapur fresh-water fishes (Airan and Joshi, 1952), and on Skipper (Cololabis saira) (Tsuchiya et al., 1953). Some of the figures given by Jacquot and Creac’h (1950) are reproduced in Table I, which contains for each species a mean value or sometimes two values corresponding to the limits of variation. Water accounts for three-

TABLEI Chemical Composition of the Flesh of Several Fishes (as Per Cent of Fresh Weight)

Scientific name Raja sp. Clupea harengus L. Clupea pilchardus Wslb. Gadus aeglefinus L. Gadus merlangus L. Gadus morrhua Day Hypoglossus vulgaris Flem. Lophius piscatorius L. Merluccius capensis Caste1 Merluccius vulgaris Cloquet Petroinyzon marinus L. Pleuronectes microcephalus (Donovan) Scomber sconibrus L. Thunnus lhynnirs Acipenser sturio 1,. Salmo salar L. Cyprinus carpio I, (breedingj Cyprinus carpio L. Salmo jario L. Tinca vulgaris Costa A N eontent of 16% is agsumed in all species.

Common name

Water

Lipids

Ray Herring Sardine Haddock Whiting Cod Halibut, Angler Stockfish Hake Sea lamprey Lemon sole Mackerel Tuna Sturgeon Salmon Mirror carp Carp Trout Tench

76.8-82.2 59.1-78.2 78.3 79.0-84.0

0.1-1.6 2. 0-22.0 2.0-12.0 0.14.6 0.2-0.6 0.14.9 0.5-9.60 7.51 0-1.77 0.4-1.94 13.0 0.5-3.8 5.14-8.36 13.0

80.0 80.3-82.6 75.4-79.0 68.4 78.1-84.0

79.580.9 78.9 68.8-71.3 58.5 79.0 66.6 73.0 i7.9-79.7 74.8-79.5 79.5

1.50 0.35-14.0 9.0 2.0-2.2 2.24.3 0.1

* Olhtined by fiubtmctionof the nonurotein N from the 6g11resof the adjacent column according to Shewan (1951).

Protein and nitrogenous extractives" (total N X 6.25) 18.2-24.2 10.1-19.2 16.3-21 .0 16.4-20.3 16.4-19.0 15.0-19.0 18.0-18.8 23.2 15.2-18.5 16.3-18.8 15.0 16.4-18.4 17.623.1 27.0 18.0 21.5 17.0 17.5-18.9 16.G20.1 18.0

Proteinb 11.8-15.6 8.7-16.6 14.0-18.0 14.3-17.7 14.6-16.9 13.0-16.4

-

P

13 .&16.4

E

14 .&16.6

0

-

-

14.6-16.3 15.2-M.0

El

FISH PROTEINS

233

quarters of the muscle mass (75% to 80%). The solid matter consists mainly of nitrogenous compounds (17 % to 24 %) and lipids (0 to 22 %). The carbohydrate content represents only a very small fraction. The only component of this type present in noticeable amount in muscle is the glycogen. Its content in fish muscle has been reviewed by Reay and Shewan (1949) and by Partmann (1954a). The recorded values vary between 0 and 0.85% of the fresh weight according to the state of fatigue of the muscles. The recent determinations of Amano, Bito, and Kawabata (1953) on the frigate mackerel ( A m i s tapeinosoma Bleeker) are also within this range: the glycogen content amounts t o 0.6% when the motor nerve is punctured immediately after catch, to 0.34 % after beheading, and to 0.11 % when the fish dies after struggling. The lipid fraction shows extremely wide variations; it corresponds to less than 1 % in lean fishes such as the gadoids and to 13 % in red tuna fish. The values are very variable in fatty fishes such as the clupeids. In these latter species, the flesh becomes infiltrated seasonally with fat, and there is no clearly marked and easily separable fatty tissue as in meat. A certain localization is, however, observed. The oil content in different parts of the snoeck (Thyrsites atun Euphr.) fillet in June is as follows: muscular walls forming body cavity, 37.5 %; reddish streak along the sides, 33 %; white flesh dorsal and ventral to the reddish streak, 7 % (Van Wyk, 1944). The nitrogen fraction of Table I has been calculated by assuming a nitrogen content of 16 %. As this percentage may vary from species to species and is somewhat higher in the case of rabbit myosins (Bailey, 1937, 1942), these figures represent only rough evaluations. Furthermore, total nitrogen is subdivided into nitrogenous extractives and proteins. Its partition between these two fractions in fishes has been reviewed by Shewan (1951). Nonprotein nitrogen amounts, according to this author, to 9 % to 14% of the total nitrogen in flatfishes and gadoids, to 14 % to 18 % in the herring group, and to 34% to 38% in the elasmobranchs, whose muscles contain approximately 1% trimethylamine oxide and 1 % to 2 % urea. More recent researches on the nitrogenous extractives of fish muscle (Shewan et al., 1952; Anderson and Fellers, 1952; Jones, 1954a, b) do not modify this general picture. Although a great mass of data on chemical composition of fish has accumulated, the accuracy of the determinations is usually poor. The variations occurring from one individual to another of t,he same species, and such factors as season or geographic area, have frequently been neglected; slid the sampling procedures used are often open tjo criticism. A project for the collection of more representative data has been recently set up by the Fish and Wildlife Service (Stansby, 1954). It is interesting to compare the composition of fish muscle with that of

234

G. HAMOIR

TABLEI1 Chemical Composition of Frog and Mammalian Striated Muscle (as Per Cent Fresh Weight) ~

~

~

_

_

_

_

_

~~~~

o.f

~

Water content

Protein content

Nitrogenous extractives

Glycogen

References

Frog Mammala

80 75

18 20

0.42 1-1.5

1 0.5-1

Dubuisson (1942) Zoethout and Tuttle

Mammal“

” C

la

-

-

0.5-1

MammalQ Manb

72-80 79.5

16.5-21 16.5

Walker, Boyd and Asimov (1952) I’olonovski (1952) Mitchell, Hnmilton, Steggerda, and Bean

Manb

70.1

21.9

(1943)

0.63-2.0

-

-

-

._

-

(1945)

Forbes, Cooper, Mitchell (1953)

and

Species not specifically stated.

* Each set of figures corresponds to only

one subject.

frog and mammalian muscle (Table 11). The figures given here pertain mainly t o lean muscle. The composition of frog muscle has been investigated by many authors (see Dubuisson, 1942). The relatively large range in the glycogen values reported (see also Partmann, 1954a) is due to the influence of factors such as proper resting and sometimes feeding before killing (Bate Smith, 1948). The amount in “normal” mammalian muscle may be considered approximately as about 1% (Bate Smith, 1948). A comparison of the figures of Tables I and I1 does not reveal any majordifference, except in the case of the glycogen, which is slightly but definitely lower in fish muscle. It is generally assumed that the content of nonprotein nitrogen is somewhat higher in teleosts than in mammals (Reay, Cutting, and Shewan, 1943; Jacquot and Creac’h, 1950). Some components of the noncoagulable nitrogen of beef muscle have been evaluated recently. The creatine amounts to 0.64 % of the fresh weight (Bigwood, Crokaert, and Bilinski, 1953); tho sum carnosine anserine, to 0.6% (Crokaert, 1953) (see also Conway and Hingerty, 1946; Bricas and Fromageot, 1953). These results suggest that a complete evaluation of this fraction should give values similar t o those obtained on flatfishes or gadoids. The great difference between fishes and frog or mammals from t8hispoint of view consists in the nature of the nitrogenous compounds. Creatine is the only component which can be found in fishes as well as in frog or in mammals. Carnosine and anserine are usually absent or present only i n small amount in fish muscle (Shewin, 195l), whereas trimethylamiric oxide, betainc, and ammonia, which occur in fish, do not exist in mammalian muscle. Urea

+

FISH PROTEINS

235

is found in not)able quantities only in the elasmobranchs. It may therefore be concluded that the nietabolism of these compounds is quite different in fish and in the other vertebrates. 3. ClassiJication and Nomenclature of the Muscle Proteins

One of the earlier attempts made by the biochemists in the study of the muscle proteins was to identify several fractions and to try to estimate them quantitatively (Weber and Meyer, 1933; Smith, 1934, 1937). These researches carried out on rabbit muscles led to the subdivision of the proteins into four fractions : an albumin fraction, a globulin “globulin X” extractable a t low ionic strength ( 6 0.15) and insoluble in water, myosiii extractable at high ionic strength ( 3 0.5), and the stroma, a residue insoluble in dilute solutions of hydrochloric acid or sodium hydroxide. Weber (1934) gave the name myogen to the albumin fraction of the muscle juice, Smith (1934, 1937) suggesting a further subdivision of this fraction, which he found t o contain a protein of low isoelectric point (3.0 to 3.5) called myoalbumin. Further research has notably altered this first picture. Fractionation methods (Raranowski, 1939a,b) and electrophoresis (Jacob, 1947, 1948) have shown that myogeii and globulin X are actually whole systems of proteins. Myogeii consists of a complex mixture of enzymes (see Mommaerts, 1950). Myoalbumiri needs to be defined again according t o electrophoretic data and corresponds only to a very small amount of the muscle albumiiis (.Jwob, 1947, 1948; Hamoir, 1951b; Connell, 1953a,b). The myosin of Edsall (1930) and Weber (1934) is a mixture of actomyosin and “crystalline” myosin (Banga and Szent-Gyorgyi, 1941-42). The term myosin” corresponds now to the protein defined by Szent-Gyorgyi (1943). In this review, the plural term “myosins” has been used for the mixtures of muscle globulins extracted a t high ionic strength and isolated b y dilution. Another muscle protein, tropomyosin, has also been characterized (Bailey, 1948). Actin, myosiii, and tropomyosin are all found in the myofibril (Perry, 1953) and are considered together under the more general name of structure proteins. They are characterized by the wide difference in the salt concentration necessary for their extraction and their salting in (Dubuisson, 1950). i(

4. Earlier Biochemical Work on Fish Muscle Proteins a. Quantitative Fractionation. The scheme of fractionation elaborated by Smith (1934, 1937) has been applied to fish muscle by several authors (Reay and Kuchel, 1936; Bailey, 1939; Subba Rao, 1948; Dyer, French, and Snow, 1950). The most reliable figures are given in Table I11 together with data relative to rabbit muscles. Wide differences are observed on the

TABLE I11 Content and Partition of Protein Nitrogen i n Rabbit and Fish Muscle

Scientific name Lepus euniculus L. White muscles

Protein N as per cent of the fresh AlbuCommon name weight mins Rabbit 2.54 22

Red muscles White muscles Gadus aeglefinus L.

Haddock

2.25

17

-

10

Torpedo marmorata Risso Ray 2.352.83 and Torpedo ocellata Rafinesque Gadus aeglefinus L. Haddock 2.54 Gadus morrhua Day Cod 2.50 Pleuronectes microcepha- Lemon sole 2.75 lus Day Raja batis L. Common skate 2.73~ Scyllium canicula Day Lesser spotted 2.71 dogfish Gadus morrhua Day Cod 2.50 Lepus cuniculus L. Rabbit White and red muscles

Myofibrils

-

Globulins soluble a t p 5 0.15

E3 W

Q,

Structural proteins

Stroma

References Weber and Meyer (1933) Weber and Meyer (1933) Smith (1937) Reay and Kuchel (1936) Bailey (1939)

22

39

17

17

39

27

9

63 67

18

30 26

64

10

3

?

15 15 16

-

17

-

13

8

3 3

-

28

76

}

Myosin 38 52 Actin 13-15 rropomyosin 4 Unidentified corn0 ponent Actomyosin 70-78

~

8 10

Subba Rao (1948) Subba Rao (1948)

3

Dyer, French, and Snow (1950)

19-21 Hasselbach and 15-17b Schneider (1951) 9-17c Perrj. (1953'1

2 4

cfr. also the probably more correct values of 2.63 (Rajaclamta L.)and of 2.41 (Raia r a d i a ~ D) determined by Bailey (1939). After extraction of the residue with 30% ." urea. C After exhuative extraction with 1.25 M KC1 or 0.9 M LiCl. dAfterexbaustiveextractionwith0.6MKCl,0.04MNaHCO:,and0.01 JINa2CO~orwith0.4 3f KCl, 0.6 .1-I KI, and 0.067 .If KzIIPO4. a

4

Subba Rao (1948) Subba Rao (1948) Subba Rao (1948)

E

0

FISH PROTEINS

237

same material because of the lack of accuracy of the earlier fractionation methods. A clear result is, however, obtained in the case of the stroma content, which shows very remarkable variations between teleosts, elasmobranchs, and the rabbit. It seems also that the amount of structure proteins is higher in fish than in rabbit. The earlier evaluations do not reveal this difference, and it is evident only when a better account is taken of the comminution of the tissue and of the conditions of stability of these proteins as in the determinations of Dyer, French, and Snow (1950) and of Hasselbach and Schneider (1951). It seems well established that in rabbit muscle, the structure proteins correspond to at least half of the total protein content. The estimation made in the case of fish muscle is not as reliable. Dyer, French, and Snow (1950) have corrected their experimental results following Smith (1934) for loss of myosin during precipitation (12 %). Such a procedure is questionable in view of the insolubility of the myosins a t low ionic strength (Jacob, 1947; Hamoir, 1951b; Nikkilg and Linko, 1954a) (see also Hasselbach and Schneider, 1951). If this correction is avoided, a lower value of 67% is obtained which agrees with another recent determination (Nikkila and Linko, 1954a). The discrepancy existing between this last value and that of Hasselbach and Schneider (1951) is still considerable and cannot therefore be due to an experimental error. It does not seem possible to draw other conclusions from the data of Table 111. The actual data on the quantitative analysis of the muscle proteins could be notably improved if new determinations would take into account the recent progress made in our knowledge of these compounds. b. Some Abnormal Properties of Fish Proteins. In the study of fish muscle proteins it was first thought that they did not differ from those of rabbit muscle. Some preliminary results, however, showed that definite discrepancies exist. Bailey (1937) has determined the content in cystine, methionine, tyrosine, and tryptophan of myosins of different origins (rabbit, dog, ox, chicken, fish, and lobster). The order of magnitude of the figures obtained is similar, but some variations occur: the highest values were found in fish, representing an average deviation of about 15%, the next in lobster, with an average deviation of 5 %. Bailey’s results on fish have been confirmed on lingcod muscle actomyosin by Deas and Tarr (1949). Guba (1943) has extracted muscles of several animals (rabbit, pigeon, frog, pike, catfish (Ameiurus nebulosus Les.), cattle, hen) under the same conditions and has compared the rate of formation of actomyosin with the time of extraction. The speed is the same for the different species investigated except for the fish, in which the reaction is about three times quicker. This result has been confirmed and extended by Roth (1947) in a more detailed study of carp muscles. She was the first to point out that myosin, which can so easily be isolated from rabbit muscle, seems absent in fish

238

G. HAMOIR

extracts. She has also observed a slight difference between carp and rabbit myogens, finding for these two fractions isoelectric points of 6 and 6.3, respectively. It follows from these preliminary investigations that differences do exist between fish arid rabbit muscle proteins. It was towards a better definition of the fish muscle proteins and the elucidation of these differences that rcsearch had first to be oriented. The important progress made in this direction will be described in the following sections. 5 . Composition of Extracts oj Low Ionic Strength ( 6 0.15 )

When the cell membranes are destroyed mechanically and the pulp obtained dispersed in water or saline solution of low concentration and approximately neutral pH, the muscle albumins and the globulins soluble at lorn ionic strength pass into solution. The ease of preparation of such extracts, their low viscosity, and the low ionic strength necessary to keep these proteins in solution have prompted their study. Some very preliminary results have been obtained with several fishes by paper electrophoresis (Ranke and Bramstedt, 1954) and a detailed study of 20 species of fish has been made with the Tiselius method by Connell (1953a,b). This last author did not take into account the differences existing between white and red muscles, though many scattered observations do suggest that differences exist. I n fatty fishes like the herring, the content of fat and of the enzymes catalyzing the oxidation of fat is higher in the red lateral band (Ranks, 1938; Reay, Cutting, and Shewan, 1943; Van Wyk, 1944; Reay and Shewan, 1949; Khan, 1952) (see p. 271). A preliminary note of Figikawa and Nagaiiuma (1936-37) suggests that sardine white and red muscles differ widely in composition. Shewan (1951) has reported that the dark lateral band of herring and that of tuna have only half the trimethylamine oxide content of that of the rest of the skeletal muscle. I n some fishes, the methionine content of red muscle is higher than that of white muscle (Matuura, Kogure, and Fukui, 1952). According to Kawabata (1953), the dark muscle of pelagic fish such as albacore (Germ0 alahmcnga G.) or frigate mackerel (Auxis tapeinosoma Bleeker) contains relatively large amounts of trimethylamine and a trimethylamine oxide-rcductase as compared with the white muscles of these fishes. Fish arginase occurs only or at least in higher amount in the superficial red muscle (Matsuura, Raba, and Mori, 1953; Connell, 1955). Some differences in carbohydrate metabolism itself are manifest, since immediately after death the pH of the white and dark muscles of the frigate mackerel are, respectively, 6.8 and 6.5 in fishes killed by decapitation and 5.8 and 6.4 in those killed after struggling (Amano, Bito, and Kawabata, 1953) (see also p. 269). We have investigated separately these two kinds of muscle in the case

FISH PROTEINS

239

of the carp (Hanioir, 1951b, 1953b, 1954, 1955). The red muscles amount to less than 10% in fishes, even in tuna (Kishinouye, 1921-23); the study of a mixture of both corresponds as a first approximation to that of white muscles and the results obtained by authors who have not distinguished the two will thus be discussed under this heading. a. White Muscles. (1) Content of albumins and globulins. New figures on cod (Dyer, 1954) and carp (Hamoir, 1955) have recently been added to the earlier ones given in Table 111. The mean values for carp white muscle correspond to 21.2% (whole extract) and 15.0% (albumins) of the total protein, assuming the latter is 18% of the fresh weight (Table I). The agreement with the data of the literature (Table 111) is satisfactory, but the scatter of the results is very wide particularly in the case of the globulin fraction. Storage is, however, apparently without eff ect on these proteins in carp (Hamoir, 1955) as well as cod (Dyer, 1954). These variations are due to the low stability of the niuscle globulins under the conditions of the experiments; better extraction media are needed to advance their study. (2) Electrophoretic analysis. This comparative work deserves special attention ;fish are the only class of vertebrates in which electrophoresis has been applied to many species. Carp (Hamoir, 1951b, 1954, 1955), freshly killed codling (Connell, 1953a), and post-rigor cod (Dingle, Eagles, and Neelin, 1955) have been investigated extensively, and a survey of many other species including several gadoids, flatfishes, elasmobranchs, and freshwater fishes has been made (Connell, 195313; Nikkila and Linko, 1955). The researches carried out on cod and codling agree fairly well; the diagrams obtained under the same conditions do not differ notably. According to Connell (1953a), they can be subdivided into three mobility groups as in the case of rabbit and frog diagrams (Jacob, 1945,1947):a fast-moving group containing only a small and diffuse peak, a second one somewhat arger with three peaks, and a third one subdividing also into three peaks

FIG.4. Ascending electrophoretic patterns of extracts of low ionic strength of carp (left) and codling (right) muscles. U p p e r part: whole extracts. Lower purl: albumin fraction (after HAMOIR, 1955 and CONNELL, 1953a).

240

G. HAMOIR

and contributing about 65 % of the total area (Fig. 4). The extracts of the other species investigated contain approximately the same number of components (7 to lo), which can also be distributed into these three groups. But within this over-all similarity, extreme differences are observed from species to species. A great diversity in the number and dispositiori of components occurs particularly in the case of the third main group; no similarities are found between related species except for the elasmobranchs, whose extracts contain important components of unusually high isoelectric point (about pH 7). With some slight modifications, the work carried out on the carp and the cod fits in with this general picture. The electrophoretic pattern of carp whole extract (Fig. 4) shows that the rapidly moving group may not be represented by a peak but by heterogenous material and that the fastest component of the second group can become prominent. The reproducibility of the patterns appears rather less than stated by Connell (1953a,b) and the resolution obtained is greatly influenced by the pH of electrophoresis (Dingle, Eagles, and Neelin, 1955). The comparative work carried out so far needs some confirmation. The over-all similarity of the electrophoretic patterns is not the only common property which can be noted. The major part of the turbidity migrates always with the fastest component of the intermediate group, which seems therefore to correspond to a constant constituent of these extracts. Furthermore, fish proteins generally have low isoelectric points. The mean mobilities of codling and carp extracts are at least 1.5 times greater than those corresponding to the rabbit calculated from Jacob’s (1947), Bosch’s (1951), or Haan’s (1953) data. The values of the other species are situated between these two extremes, except for the dogfish, whose mean mobility is approximately equal to that of the rabbit. A general trend towards higher mobilities therefore exists in the case of fishes. In the study of the carp and the codling, the albumins have also been examined by electrophoresis. The removal of the globulins by dialysis against water great,ly alters the pattern in the case of carp but does not notably affect that of the codling (Fig. 4). In the carp, the gradients of the whole extracts have a definitely albumin or globulin character, whereas in the codling the absolute amounts of all components, except the turbid one, decrease in approximately the same proportion-a behavior already observed with blood serum (Raffel, Pait, and Terry, 1940; Svensson, 1941) and with several muscle extracts (Jacob, 1947; Renard, 1952). The variability in protein composition which characterizes the muscle extracts of low ionic strength obtained from different species has already been established by several authors (Jacob, 1945; Hamoir, 1951b; Renard, 1952; Crepax, 1952; Dubuisson-Brouha, 1953; Haan, 1953). It is stressed further by Connell’s comparative study, which is the first one carried out

FISH PROTEINS

241

on related species of similar physiological activity and conditions of life. In spite of this similarity and although these proteins, consisting mainly of glycolytic enzymes (Mommaerts, 1950), have corresponding functions in the different species, wide differences are nevertheless observed. The specificity of these mixtures is thus extremely pronounced. (3) Ultracentrifugal analysis. The ultracentrifugal data relative to muscle extracts are very scarce. The only published work is that of Deuticke (1934) on whole extracts of frog and rabbit muscles. In the case of fishes, investigations have been limited so far to carp albumins. The electrophoretic pattern of this fraction is given in Fig. 4. Three components are also observed by ultracentrifugation, but the general appearance of the diagram is very different (Fig. 5). The main fraction, comprising about 50% of the mixture, sediments with a corrected rate (SZ~,,) of about 1.5 S; and the median one, which represents about 40%, with a corrected rate of about 4.8 S. A correlation of the electrophoretic and ultracentrifugal diagram has been made possible by the isolation from this mixture of a protein migrating at the rate of the main electrophoretic gradient and of a corrected rate of sedimentation of about 5 S (Henrotte, 1952, 1954). The median ultracentrifugal gradient corresponds to a part of the median electrophoretic one. The slowly sedimenting proteins correspond to a mixture of components of different mobilities.

-

FIG. 5. Ultraceiitrifugal pattern of the albumins of carp white muscles (after 1955).

HAMOIR,

Deuticke (1934) has described two main components in frog and rabbit extracts sedimenting with corrected rates of 1.1 and 7.6 S and of 5.4 and 7.7 S, respectively. These few results show that the ultracentrifugal behavior of these mixtures is very variable and that fish muscle differs qualitatively from that of the other vertebrates in this respect. No carp component seems to correspond to Baranowski’s myogen A (aldolase), isolated from rabbit muscles, which sediments at a rate of approximately 7.8 S. The occurrence in frog and fish extracts of a slow component suggests the possibility that components of low molecular weight could generally occur in cold-blooded vertebrates. The considerable amount of this fraction in (harp muscle is in any case very remarkable. b. Red Aluscles. (1) Content of albumins and globulins. Evaluations made a long time ago by Weber and Meyer (1933) on rabbit red muscles

242

G . HAMOIR

suggest that the amounts of albumins and globulin X in these muscles are lower than in white muscles (see Table 111). A few determinations have been made on muscles of the lateral line of the carp (Hamoir, 1955). The reproducibility of the results does not seem better than in the case of carp white muscles, although in this case each determination was made on muscles taken from several animals. The amount of protein extractable a t p 0.15 and pH 7.7 is approximately the same in white and red muscles, while the latter seem somewhat richer in albumins and poorer in globulins. These figures do not therefore reveal significant differences between these two muscles. ( 2 ) Electrophoretic analysis. The preparation of the extracts of carp red muscle and their electrophoretic analysis were carried out under the conditions used for white muscles. The variations observed are illustrated in

FIG.6. Ascending electrophoretic patterns of two l-hour extracts a t p 0.15 nnd p H 7 of carp red muscles. Upper part: whole extracts. Lower part: corresponding albumin fractions (after HAMOIR, 1955).

Fig. 6, in which an extract of low turbidity (left side) is compared with another of high turbidity (right side), the upper diagrams corresponding to the whole extracts and the lower ones t o the albumins of the same preparations. The migration of a red color (shaded line) and of the corresponding gradient is clearly visible in the left-hand diagrams, but it cannot he observed on the right-hand ones. The amount of rapidly-moving material is also very different in the two extracts. Although the patterns vary widely, their contrast with those of the white muscles (Fig. 4) is obvious. The resolution is much lower and the gradients seem to differ except in the case of the main one, which can be identified by its mobility to the major component of the white muscles. Furthermore, the appearance of the pattern is not altered by dialysis against water. These differences are much more pronounced than those observed between white and red rabbit muscles (Amherson et al., 1949; Bosch, 1951; Crepax, 1952). The identification of the red color observed in the left-hand diagram of Fig. G as rnyoglobin (see p. 263) also sheds a new light on the nature of this tissue. The

FISH PROTEINS

213

regularities found by Lawrie (1950, 1953a,b) in mammalian and avian red muscles may very likely be extended to this case. The cytochrome system must be more developed and a correspondingly higher capacity for energyrich phosphate resynthesis under aerobic conditions must also exist, These muscles are probably capable of enduring more prolonged muscular exertion than white ones, but this difference does not seem sufficient to explain their very abnormal protein composition. The metabolism of fish red muscle must be characterized by other pecularities, since its capacity to accumulate fat could induce changes which might well explain the big differences observed. 6. Extractability of the Structure Muscle Proteins

The solubilization of the structure proteins of muscle is influenced by many factors: the nature of the tissue, its physiological state, its degree of subdivision, the ionic strength, pH, and duration of the extraction. The minimal ionic strength necessary to bring these proteins into solution a t neutral pH varies between 0.3 and 0.45 according t o the species considered (seeHamoir, 1955). I n the case of the rabbit, Crepax (1952) finds that actomyosin is more easily extracted from the red muscles than from the white, and explains this behavior by the different adenosine triphosphate [ATP] content of these muscles (Crepax, 1952; Lawrie, 1953a,b). The influence of the physiological state has been investigated in rabbit and tortoise muscles (Dubuisson, 1950, 1953). The extractibility is maximal in normal, resting, and relaxed muscle and decreases in stimulated, contracted, or coiitractured muscle. Such an extensive study seems impossible with fish muscle in view of their quick inexcitabiiity after exeresis (Abbott, private communication). The changes occurring in rigor mortis are the only ones which have been investigated. Nikkila and Linko (1954a) have shown that the amount of actomyosin extractable decreases during the onset of rigor mortis and increases again after its completion. Connell’s work has been carried out on fish muscle preceding rigor, and fish muscle after rigor has set in, has been investigated by Dyer and co-workeis. A comparison of these two states would be relatively easy if due attention were given to the slight p H change occurring in rigor mortis (see p. 269), but it has not, so far as we know, been carried out. Extractibility is also influenced by the degree of subdivision of the tissue. When cod muscles are comminuted for 3 t o 5 minutes with a Waring Blendor, a quantitative extraction of the structure proteins is readily obtained and the nature of the salt used now has no influence (Dyer, French, and Snow, 1950). The last authors have also investigated the influence of pH on cod muscles blended for 3 to 5 minutes with a large volume of 5 % NaCl ( p 0.85) adjusted to various pH values with several buffers. The solubilization increases

244

G . HAMOIR

100

80 60

40 20 0

3

4

5

6

7

8

9

PH FIQ.7. Influence of pII on the extractability of the muscle proteins of cod a t ionic strength 0.85. Ordinate: per cent of the total protein N extracted as soluble protein N (X), as myosin N (0),and as non-myosin (A) (after DYER,FRENCH, and SNOW,

1950).

sharply between p H 5 and 6 (Fig. 7). Similar results have been obtained on carp muscles cut with a freezing microtome in slices 40 p thick and extracted with phosphate-NaCl solutions of p 0.5 or 1 and varying p H (Hamoir, 1955). A quick increase in extractibility is observed from p H G at p 0.5 and from p H 5.5 a t p 1. The curve of Fig. 7 does not depend on the species of fish or the method of subdivision of the tissue, but its position is slightly influenced by the ionic strength. The minimal salt concentration necessary to extract these proteins has been determined on carp muscles a t p H 7.5. This threshold value corresponds t o an ionic strength of 0.45. It is much higher than that of 0.3 given by Connell (1953a) for codling. The occurrence of variations due to the age or t o the species is not impossible in this last case. It is interesting to compare these results with those obtained on other animals and especially on the rabbit. Hasselbach and Schneider (1951) have shown that myosin can be quantitatively extracted at p 0.6 and p H G to 6.4 from rabbit muscles coarsely minced; actin can be brought into solution by a further mincing with a Waring Blendor. Rabbit muscles cut with a freezing microtome and extracted a t ionic strength 0.5 and 1.O and varying pH behave in the same way: myosin already dissolves a t p H 6, while actomyosin does not go into the solution below p H 7 (Van de Bergh, unpublished results). On the other hand, the fish extracts obtained at pH 6 have a strong flow birefringence and therefore contain actomyosin. The long F-actin filaments of the muscle fibers are apparently already cut into shorter fragments a t slightly acid p H and the selective extraction of the myosin according to Hasselbach and Schneider (1951), does not seem possible (see also Connell, 1954). The stability of fish and rabbit muscles a t

FISH PROTEINS

245

low pH is thus very different. A similar discrepancy occurs also at pH 7.5. In this case, the minimal ionic strength necessary to solubilize these proteins is not lower in fish than in other animals (Hamoir, 1955), but the speed of solubilization of actomyosin is much quicker (Guba, 1943; Roth, 1947). On the other hand, fish muscle is also less stable in the frozen state; it loses its tenderness and becomes tough whilst a simultaneous denaturation of actomyosin occurs (see Dyer, 1951). This general instability seems not to be attributable to the lower content of stroma in fish muscle. We shall show later on that it seems to be due to an abnormal organization of these proteins in the fish myofibril. 7. Composition of Extracts of High Ionic Strength ( 3 0.6) The analyses carried out up to now on such extracts have been based upon differences of solubility (see p. 235). The discussion of the results obtained has made clear that a reinvestigation of these mixtures is necessary in order to define more accurately the structure proteins of fish muscle. Electrophoresis, which permits the analysis of such mixtures with a minimum of alteration, appears a particularly suitable method. It has been applied as yet only to carp muscle extracts of high ionic strength (Hamoir, 1951b, 1954, 1955). In view of the very constant electrochemical behavior of the muscle structure proteins (see Hamoir, 1953a), it seems safe to assume that similar results will be obtained with other fishes. The results already obtained will therefore be more extensively described. The slight difference in extractibility previously mentioned between white and red rabbit muscles (Crepax, 1952) suggests that a separate study of both fish muscles would also be desirable in this case, but it has not yet been undertaken. a. Extracts Obtained at p 0.6 and p H 5.9 and 6.2. The electrophoretic patterns obtained after extraction for 10 minutes with phosphate-NaCl solutions at p 0.5 and pH 5.9 or pH 6.2 are reproduced in Figs. 8 and 9. Their comparison with the carp whole extract of Fig. 4 reveals the presence on the ascending side of one (Fig. 8) or two (Fig. 9) new peaks migrating in front of the main myogen component. The quicker gradient 2 which migrates with the turbidity remains very sharp, whereas the slower one 3 has a more symmetrical shape. On the descending side, the resolution is less sharp. When the extraction is carried out at neutral pH, gradient 2 is still observed but 3 is absent. The peaks 2 and 3 of Fig. 9 are identical in appearance and mobilities (see Table IV), respectively, to the (Y and /3 peaks of rabbit extracts (Dubuisson, 1946). The faster one seems therefore to correspond to actomyosin and the slower one to myosin. This last component is absent in extracts of neutral pH, in agreement with previous results (Roth, 1947) and would occur at lower pH. The solubility of the proteins found in gradients 2 and 3 confirms this identification. Both are

246

G . HAMOIR

precipitated quantitatively by dialysis against a solution of p 0.15 and p H 7.3. This result recently confirmed by Nikkila and Linko (1954a) invalidates the correction of Smith (1937) allowing for an incomplete precipitation of these components a t low ionic strength. On the other hand, it suggested the possibility of isolating myosin from extracts made a t low pH. The globulins of these extracts have been separated by dilution, purified by reprecipitation, and examined by viscosinictry, electrophoresis, and ultracentrifugation in order to test this possibility (Hamoir, 1955). It was

oc

la l b

5

I

6

FIG. 8 FIG. 0 FIG.8. Electrophoretic pattern of a 10-minute extract of carp muscles a t p 0.5 and pH 5.9 (after HAMOIR, 1955). FIG.9. Electrophoretic pattern of a 10-minute extract of carp muscles a t p 0.5 and pH 6.2 (after HAMOIR, 1955).

found that the ,8 peak of carp extracts corresponds to components having in common with myosin a low viscosity uninfluenced by ATP, a low turbidity, and no flon~birefringence but differing in ultracentrifugal behavior, The3!, peak of muscle extracts, which has always hitherto been identified with myosin, may thus correspond to mixtures of slightly different proteins. b. Extracts Obtained at p 1.0 and p H 6.0, 5.6, and 6.5. The incipient solubilization of the structure proteins of muscle has also been investigated with phosphate solutions of ionic strength 1.0 and low pH (Hamoir, 1955). The electrophoretic patterns of 10-minute extracts at p H 5.0, 5.2, and 5.5 are reproduced in Figs. 10, 11, and 12, respectively. They very sharply depend on the pH. Nevertheless the high ionic strength used for the ex-

TABLEIV Electrophoretic Mobilities of the Chief Components of Fish Muscle Mobility in Nature of the preparation

Animnl

Conditions of electrophoresis

Ascending values 1

Whole extracts at p 0.5 and p 1.0

Carp

Myosins extracted at p 0.5 and varioua p H Tropomy osin

Carp

Myosin

F-actin

Carp

Cod

Cod Carp

G-actin

Cod

Sodium phosphate -4.19 of p 0.1 NaCl 0.25 M - pN 7.1 Sodium phosphate of p 0.1 4- NaCl 0.25 ~ -lpH i 7.1 Sodium phosphate of p 0.1 NaCl 0.25 M - p B 7.1 Phosphate bufier of p 0.1 KCI 0.2 or 0.3 M - pH 7.5 Phosphate buffer of p 0.1 KCl 0.3 ill - pH 7.4 Sodium phosphate of p 0.1 NaCl 0.25 M - pH 7.1 Phosphate buffer of p 0.1 KCl 0.3 M - pH 7.4

+

+

3@)

4

5

6

1

2

3

-2.93

-2.81

-

-2.28

-1.36

-3.05

-2.24

-1.16

Refer enceS

Hamoir (1955)

-2.88

-2.82

-2.60

Hamoir (1955)

-4.30

-3.90

Hamoir (1951a)

-2.9'"

Connell

8 5

8 6

I2 Z

(1954)

-6.5"

+

+

Descending values

2(a)

+

+

cm.2 volt-' see.-'

Connell (1951)

-6.5

-5.3

-3.9"

Connell (1951)

*

f.3 a

Average values.

-I

248

G. HAMOIR

traction permits a good reproducibility. When carp muscles are extracted at pH 5 (M-KH~POI),the structure components do not go into solutioi1 (Fig. 10). At pH 5.2 corresponding to extractarit fluids of pH 4.5 t o 4.8, two new peaks are visible; the slower one migrates with the speed of th(’ myosins and the faster one represents a structure component, not ohseryable in extracts made at p 0.5 (Fig. 11). When the pH of extraction reaches 5.4 to 5.5 (Fig. 12), the solubilization of the myosins increases and the cr and /3 peaks already observed at p 0.5 (Fig. 9) become prominent. Further-

\ L

.

FIG. 11 FIG. 10 FIG. 10. I3lectrophoretic pattern of a 10-minute extract of carp muscles at 1 and pH 5.0 (after HAMOIR, 1955). FIG. 11. Electrophoretic pattern of a 10-minute extrart of rnrp muscles x t 1 and pH 5.2 (after IIAMOIR, 1955).

/.I

/.I

oc

P

FIQ. 12. Electrophoretic pattern of a 10-minute extract of carp muscles at 1 and pH 5.5 (after HAMOIR, 1955).

p

FISH PROTEINS

249

more, a fast ascending component, usually absent at p 0.5, is visible on the ascending side. It migrates with the speed of the quick ascending peak of Fig. 11; but i t does not diffuse during the electrophoresis and its concentration is much lower. It seems to correspond to a gel. The increase of the ionic strength from 0.5 to 1.0 gives rise in the case of the fish muscle to the extraction of new structure components migrating in front of the actomyosin. The mobilities of the gradients observed a t p 0.35 and p H 7.1 are given in Table IV together with figures relative to the different fish muscle structure proteins. The values of -2.7 X 1 k 6and -2.5 X 1 k 6 cm.2volt-' set.-' found under similar conditions for the (Y and peaks of the rabbit (Dubuisson, 1946) do not differ significantly. This constant electrochemical behavior first noticed in the case of the carp (Hamoir, 1949) has been confirmed since with striated muscles of very different properties and origins (see Hamoir, 1953a). The figures corresponding to the fast ascending components are also in agreement with earlier mobility determinations of carp tropomyosin (Hamoir, 1951a) and suggest th a t they correspond t o this structure protein. The fractionation of the mixtures obtained a t p H 5.2 and 5.5 allows the isolation of these different components and facilitates their characterization. The quick ascending gradient of Fig. 11 precipitates a t 1.1 0.05 and pH 4.6 and can be purified by ammonium sulfate fractionation at neutral pH between 50 % and 66 % saturation. The electrophoretic pattern of this fraction is given in Fig. 13. It contains only two peaks of very close mobilities, both of them corresponding to the quick ascending gradient of Fig. 11. The yield of this preparation indicates that all the gradient of Fig. 11 is isolated. It therefore entirely corresponds to tropomyosin. This protein can be extracted from fish muscle without previous dehydration of the muscle pulp with organic solvents. The slight electrophoretic heterogeneity of the preparations will be considered later (see p. 207). A detailed analysis of the mixture obtained a t p H 5.5 (Fig. 12) is also possible. The (Y peak is first removed by dialysis against a phosphate-NaC1

FIG. 13. Electrophoretic pattern of tropomyosin isolated from a n extract obtained a t p 1 and pH 5.2 (see Fig. 11) (after HAMOIR, 1955).

250

G. HAMOIR

solution of p 0.25 and pH 7.2 for 25 to 30 hours. The precipitate formed, washed with the dialysis fluid and redissolved, is pure by electrophoresis (Fig. 14). Its solution has a strong flow birefringence and high viscosity and obviously consists of actomyosin. The electrophoretic pattern of the supernatant is given in Fig. 15. The a! peak has completely disappeared. This method, which is a simplification of the procedure described by Portzehl, Schramm, and Weber (1950), allows the actomyosin to be separated quantitatively. Its elimination causes a n important change of

FIG. 14 FIG. 15 FIG. 14. Electrophoretic pattern of carp actomyosin isolated from an extract at p 1 and pH 5.5 (see Fig. 12) (after IfAMOIR, 1955). FIG.15. Electrophoretic pattern of the fraction soluble at p 0.25 and pH 7.2 of un extract obtained a t 1 and pH 5.5 (see Fig. 12) (after HAMOIR, 1955).

the fast ascending gradient (compare Figs. 12 and 15). As the precipitation of actomyosin does not involve any change of volume of the solution, the increased surface of gradient 1 of Fig. 15 can beexplained only by the occurrence of some interaction between actomyosin and tropomyosin : a fraction of tropomyosin with a gel structure (Fig. 12) migrates freely in presence of actomyosin while the other moves with the a! peak and is set free after elimination of this gradient. The mixture of Fig. 15 has been further fractionated by diluting to ionic strength 0.025. The precipitate obtained, after washing with KC1 0.025 M, redissolves in KC1 0.5 M. Electrophoresis usually reveals the presence of two gradients: on the ascending side, the fast one is identical with that of whole extracts and the second to the 0 peak; on the descending side the

251

FISH PIZOTEINS

faster one migrates much morc slowly and is much morc important (Fig. 16). A marked asymmetry therefore exists reminiscent of’ that described by Longsworth and Mac h i e s (1942) in the case of mixtures of ovalbumin and yeast nucleic acid. The similarity of t,he phenomena suggests that the formation of a dissociable complex probably occurs in this case too. The /3 gradient has also been found as a separate pure component in some preparations. When two gradients occur (Fig. 16), they can he separated by ammonium sulfate fractionation a t neutral pH. The fraction precipitating up to 43 % saturation gives rise oiily to the p component, while the one isolated between 50 % and 70 % saturation corresponds t o pure tropomyosin. The various structural proteins of Fig. I 1 and 12 may thus be isolated. Their behavior is very similar to the corresponding components of rabbit muscle, but their extractibility is quite different. Fish actomyosin goes easily into solution and rabbit actomyosin dissolves with difficulty. Fish tropomyosin can be selectively extracted a t p 1 and pH 5 . 2 , whereas similar experiments on rabbit muscle reveal only a very small solubilization of this protein (Van de Bergh, unpublished results). Fish muscle is fundamentally similar to other striated muscles but seems t o be characterized by a looser association of its structure components. 8. Isolation and Properties of Some Protein Components

OJ

Fish Muscle

The isolation of the proteins of fish muscle is still in its beginning, but is a necessary preliminary step to gain a deeper insight into the metabolism and structure of this tissue, thus providing a basis t o attack the problems of storage. For the present, our description will be limitcd to some pure

Fro. 16. 1’:lectrophoretic pattern of the glohuliris isolated t)et\zeen and 0.025 a t pH 7 from the mixture of Fig. 15 (after ii,ixo1it, 1055).

p

0.25

252

G. HAMOIR

FIG.17. Crystals of Henrotte’s myogen (after HENROTTE, 1952).

crystalline components recently isolated and to the different structure components of fish muscle. a. Henrotle’s Myogm. This protein has been isolated by Henrotte (1 952) from the mixture of carp albumins whose electrophoretic arid ultracentrifugal patterns are reproduced in Figs. 4 and 5. The precipitate obtained by animoniuni sulfate fractionation a t neutral pH between 40 % and 55% saturation is redissolved in the minimum of watcr, and solid amrnoiiiuni sulfate is added to slight turbidity a t pH 6.4 to 6.6. Very thin needle-like crystals separate within 1 hour (Fig. 17). Their solution examined by electrophoresis and ultracentrifugation appears homogeneous (Henrotte, 1952). The position of the corresponding gradient in the diagrams of the albumin fraction of the carp has been previously described (see p. 221). It sediments at approximately the same rate as a gradient of rabbit extrarts observed by Deuticke (1934), but its electrophoretic inobility is two t o three times higher than that of the fastest moving main component of these preparations (Jacob, 1947 ; Haan, 1953). It therefore differs widely from the albumins of this animal, as also from those of frog muscles, which do not sediment at this rate (Deuticke, 1934). Connell’s (1953b) comparative work suggests that it could be very generally distributed among marine and fresh-water fishes: an apparently well-defiiied component migrating with approximately the same speed is generally observed amounting to 20% to 30% of thc hole extract.

FISH PROTEINS

253

Some properties of this crystalline material have been investigated (Henrotte, 1954). Its molecular weight is 67,000, thus differing very much from the values of 150,000 of rabbit myogen A and B (Gralen, 1939; Bailey, 1940); i t has no aldolase or phosphoglyceraldehyde dehydrogenase activities, and its function in the metabolism of the cell is still unknown. b. Carp and T u n a Myoglobins. Although the wide distribution of this respiratory pigment in the animal kingdom is well known (Lemberg and Legge, 1949; Ladd Prosser, 1950; Hamoir, 1955), its occurrence in the lower vertebrates has not been ascertained until recently. Since Lankester's old observation (1872) describing its presence in the dorsal fin muscle of Hippocampus, no further research seems to have been carried out. Lemberg and Legge (1949) express the view that it is probably present in the cardiac muscles of fishes, amphibia, and reptiles, and Ladd Prosser (1950) believes that it also occurs in scattered skeletal muscles of these animals. On the other hand, other authors (Kishinouye, 1921-23; Roule, 1927) suggests that the coloration of fish red muscles is due to a larger number of capillaries. The isolation of carp and tuna myoglobins (Hamoir, 195313, 1954, 1955; Huys, 1954) has disproved this last interpretation and shown that the pigment is present in notable amounts in fish red skeletal muscle. As it has also recently been isolated from tortoise skeletal muscles (Renard, 1953), there is no doubt that it generally occurs in the red muscles of cold-blooded vertebrates. Fish myoglobin was first isolated from the red lateral bands of the carp (Hamoir, 195313, 1954, 1955), and some preliminary work on tuna myoglobin ( T h u n n u s thynnus) was carried out a little later in Bergen (Jebsen, unpublished results). The complete purification of this pigment has recently been reported (Huys, 1954). Both proteins have been prepared only from frozen muscles, essentially by the method of Theorell (1932) for horse heart niyoglobin. The pulp is extracted with water, the major part of the proteins present is removed by precipitation with 0.5 M basic lead acetate a t 30" C., and a final purification is carried out by ammonium sulfate fractionation a t neutral pH. Tuna myoglobin, separating between 80 % and 90 % saturation, is practically pure by electrophoresis and ultracentrifugation and crystallizes in sheaves of elongated plates (Fig. 18). Carp myoglobin precipitates bctwcen 75 % and 05 % saturation, but its solution still contains about 70 % of impurities and does riot crystallize. It is preferable in this case to substitute phosphate fractioiiatiori for ammonium sulfate. The myoglobin which precipitates in the phosphate range 2.35 to 3.0 M contains about 30 % of impurities. It crystallizes easily from concentrated phosphate buffer of p H 7 to 8 (Fig. 19). The corrected rates of sedimentation of carp and tuna myoglobins are, respectively, 1.7 to 1.8 S and 1.65 S; they are similar to those of other

254

G. HAMOIIL

myoglobins (Theorell, 1934a; Svedberg and Pedersen, 1940; Helwig and Greenberg, 1952; Renard, 1953). In the elwtrical ficld, rarp inyoglobin migrates more rapidly than tuna myoglobiii; their ascendiiig mobilities a t p 0.15 and pH 7.3 are, respectively, of -2.4 X lop5 and -0.3 X cm.2 volt-' set.-'. The well-known differences in :mino acid make-up of these pigments (Rossi, 1940; Rossi and Travia, 1941; Ilossi and Vesvia, 1941; Roche, Derrien, arid Vieil, 1942; Schmid, 1949) must therefore exist also in the case of fish inyoglobins. 130th oxymyoglobins have spectra in the visible region with three maxima of absorption, a very pronounced one a t about 415 mp arid two much smaller ones a t about 540 and 575 mp, in good agreement with previous determinations (Theorell, 1934b; Theorell and Ehrenberg, 1951; Renard, 1953; see Ladd Prosser, 1950). The variations of a few millimicrons which do occur, are of the order of magnitude observed for myoglobins of different origins (Ladd Prosser, 1950). This

FIG.19. Crystttls of carp oxymyoglobin. Magnification: left X120; riglit X620 (aftcr H A M O I R , 1954).

FISH PROTEINS

255

work has recently been extended by Japanese workers. Cytochrome C has been isolated from the red muscles of T h u n n u s orientalis (Matsuura, Yamada, and Hashimoto, 1955). Myoglobins of several species of tuna have been crystallized and examined by spectrophotometry' (Matsuura and Hashimoto, 1955). The amounts of hemoglobin, myoglobin, and cytochrome C in the red and the white muscles of several fishes have been determined (Matsuura and Hashimoto, 1954). The deep-seated red muscle of tuna contains from 1 to 2 % myoglobin on a wet basis. It is obvious from these results that the close parallelism between myoglobin content and physiological activity observed in mammalian and avian muscle (see p. 243) also occurs in fish muscle. c. Actomyosin. It is clear from the previously described electrophoretic study that all the fish myosins examined so far in the literature are in fact more or less pure actomyosins. The 0 gradient appears only when the extraction is carried out at low p H (see p. 245 and 246). The "myosin" isolated by Dyer, French, and Snow (1950) is actomyosin; and it amounts in fish muscle to approximately 67% of the total protein (see p. 237). If account is taken of the possible presence of tropomyosin in this fraction, this figure should be reduced to 64 % to 65 % (see p. 265). It is thus by far the most important component, and its study is of paramount importance for the understanding of the properties of this tissue. The investigations relative to fish actomyosin have first been oriented towards the study of its denaturation. Fish actomyosin becomes easily insoluble in salt solutions and its instability is increased by successive precipitations (Bailey, 1944; Atlantic Fisheries Experimental Station, 1953). This change of solubility has been used to study its denaturation. A comparison of the denaturation by freezing of actomyosin gels in vitro and of muscle in situ was made by Subba Rao (1948). He found that the influence of the speed of freezing and the duration of cold storage is the same in both cases but that denaturation occurs more slowly in muscle. Some of the factors occurring in freezing have been investigated on pure haddock actomyosin by Snow (1950). His main conclusions are as follows. Precipitated protein denatures much more quickly by freezing than when dissolved and the extent of denaturation parallels the freezing time, more rapid freezing causing less denaturation than slow freezing. Denaturation from solution is increased a t pH values lower than 6.5 or higher than 7.8. The nature of the salt used as dispersing agent has also been examined. Preparations of actomyosin dissolved in normal solutions of various salts were kept for the same time at -4", -8", -24", and -33" C. Denaturation occurs when the temperature of storage is below the eutectic point of the salt solution used (Table V).

256

G. HAMOIR

TABLE V Denaturation of Actomyosin Dissolved in Normal Solutions of Various Salts and Kept for the Same Time at Different Temperatures (Snow, 1950)

Salt

Eutectic point of the salt solution in "C.

-4°C.

-8°C.

-24°C.

-33°C.

Sodium sulfate Disodium phosphate Potassium sulfate Potassium nitrate Magnesium sulfate Barium chloride Potassium chloride Ammonium chloride Sodium nitrate Sodium chloride Magnesium nitrate Magnesium chloride Calcium chloride

-1.0 -1.2 -2.9 -3.9 -7.8 -11.1 -15.8 -18.5 -21.1 -29 -33.6 -55

36 45 83 0 0 0 0 0 0 0 0 0 0

95 95 73 78 70 0 0 0

86 80 84 79 59 60 78 88 50 40 55 0 2s

80 67 71 73 55 45 14 50

Percentage denaturation

51

4" 38 0 0

35 18 16 0 0

Denaturation considered negligible.

Actomyosin denatures also in situ under the influence of hypertonic salt solutions. When cod muscle is immersed in various concentrations of sodium chloride, there is a critical salt content in the fillet (8% to 10% NaC1) a t which denaturation occurs together with a rapid loss of water and uptake of salt (Duerr and Dyer, 1952; Fougere, 1952). In herring, however, this critical salt concentration is much lower (3 % NaC1) (Nikkila and Linko, 195413). The stability of the native configuration appears very variable, but we are unable to explain such differences. The need for a better knowledge of the protein itself is clearly stressed by these researches. Let us now consider the recent progress made in this direction. (1) Solubility. The application to fish extracts of fractionation methods elaborated for the separation of rabbit actomyosin and myosin has allowed the comparison of the two actomyosins (Hamoir, 1955). The precipitation of actomyosin at neutral pH in presence of a phosphate-NaC1 buffer is complete a t p 0.25 in the case of the carp (see p. 249) and a t p 0.30 in the case of the rabbit. A similar difference is observed with the phosphate-acetate method of fractionation (Hamoir, 1947). Carp actomyosin is therefore slightly, but definitely, more soluble than rabbit actomyosin. (2) Electrophoretic behavior. The very constant mobility of carp actomyosin whatever may be its conditions of preparation and its identity with

257

FISH PROTEINS

that of rabbit actomyosin have been previously described (see p. 249). The asymmetry of its electrophoretic pattern is shown in Fig. 14. It is due to the high viscosity of its solutions, which causes a slight blurring of the descending boundary, and to their thixotropy (Bailey and Perry, 1947; Jaisle, 1951). (3) Viscosity. The first viscosimetric determinations made by Guba (1943) and Roth (1947) have not shown clearly the very high viscosity of fish actomyosin. It has only been observed (Hamoir, 1949) and investigated (Hamoir, 1955) recently. Our determinations have been carried out with a slightly modified Ostwald's viscosimeter (MARTIN, 1925) a t 20" C. (f0.05"). The capillary was 18 cm. long and had a diameter of 0.63 mm. The kinetic energy correction amounts to less than 0.7% and was neglected. The average rate of shear B calculated with Kroepelin's (1929) formula varies between 800 (solvent) and 90 (0.42% actomyosin). The results obtained were evaluated as viscosity number 2, according t o PORTZEHI~,SCHRAMM, and WEBER (1950). This unit is calculated as grams per liter and is thus ten times smaller than the intrinsic viscosity 171. The carp muscles were extracted at /A 0.53 and approximately neutral p H (0.5 M KC1 and 0.03 M NaHC08) for 10 or 60 minutes. The actomyosin was isolated by ailution and purified by washing of the precipitate and by reprecipitation.

The viscosity of all actomyosin preparations investigated varies with concentration according to the Arrhenius formula log qrel = K.C. Their viscosity numbers 2, are therefore equal to 2.303 K . The results obtained with 1-hour actomyosins are reproduced in Fig. 20, but those which relate to 10-minute actomyosins are in no way different, in contradiction with 1.5

-E.

1.0

r

8 2 0.5

0

0.1

0.3

0.2

0.4

0.5

Per cent

FIU.20. Viscosity of several 1-hour actomyosins of the carp. The triangles correspond t o the same preparation without and with ATP (after HAMOIR, 1955).

258

G. HAMOIR

previous work (Guba, 1943 ; Roth, 1947 ; Hamoir, 1949) ; the duration of the extraction is therefore without influence on the viscosity of the globulins extracted. The conservation of the muscles used for the extractions in a frozen state a t -20' C. from two days to more than two months also does not change the viscosity. 2, varies only between 0.54 and 0.50. In presence of ATP, a considerable drop is observed, 2, falling from 0.54 to 0.25 (Fig. 20). These figures have been compared with those obtained for rabbit actomyosin and myosin (Hamoir, 1955). These last values differ notably from one author t o another especially in the case of actomyosin, but they always remain lower than our determinations. The viscosity numbers determined by Kerekjarto (1952) for rabbit actomyosin and myosin a t 15" C., which seem t o be the most reliable, are, respectively, 0.32 and 0.17 ( f O . O 1 ) . Both figures are definitely lower than our values carried out a t higher temperature. In the case of actomyosin, this difference appears to ke due to the structure of the solutions. Recent determinations (Hamoir, 1955) suggest that fish and rabbit actomyosins have approximately the same intrinsic viscosity at = 0. The axial ratio of these particles seems therefore not to differ notably. In view of this relationship, more accurate determinations are necessary in order to determine if the different viscosities observed in the presence of ATP are really due to a different shape of the particles. (4) Ultracentrifugation. The 10-minute and l-hour actomyosins whose viscosimetric behavior has just been described, have been ultracentrifuged a t different concentrations. All these preparations sediment as a single peak which remains very sharp during the experiment (Fig. 21). Some experiments have also been done on 10-minute actomyosins extracted a t acid pH and isolated a t p 0.25 and p H 7.2 (see p.249 and Fig. 14). A single peak is also observed which gives rise in this case to a slight spreading; some im-

a

FIG.21. Ultracentrifugal pattern of a 10-minute actomyosin of the carp extracted at p 0.53 and neutral pH (after H A M O I R , 1955).

FISH PROTEINS

259

60

50 40

30

20 10

Per Cent with concentration of thesolution for carp actoFIG.22. Variation of S P ~ X. ~ myosins prepared by ( a ) ten-minute or ( b ) l-hour extraction a t neutral p H of muscles kept frozen for different periods of time ( a ) 2 days 0 , 21 days A ; ( b ) 5 days X , 24 days 0, 38 days 0 . The lower figures (0 and +) correspond t o 10-minute actomyosins extracted at acid pH (after HAMOIR, 1955).

purities are present, however, amounting to less than 10 %. The slow peak of myosin which always occurs in preparations of rabbit actomyosin (Snellman and Tenow, 1948; Portzehl, Schramm, and Weber, 1950; Johnson arid Landolt, 1950, 1951) is absent here. The sedimentation-concentration curves of several preparations are reproduced in Fig. 22. The rate of sedimentation is not influenced by the duration of extraction and of storage frozen except apparently in the case of a 10-minute actomyosin extracted from muscles kept frozen for three weeks. The general aspect of the curves is similar to that given by rabbit actomyosin (Portzehl, Schramm, and Weber, 1950). But a more accurate analysis reveals a wide discrepancy. If the experimental data are extrapolated to C = 0 by plotting l/s as ordinate instead of s, linear relations are obtained corresponding to the equation l/s = l/so ICC as for rabbit actomyosin, but now they usually intersect the ordinate a t zero or a t slightly negative values (Fig. 23). This method of extrapolation is thus applicable to rabbit actomyosins and to the fish actomyosins extracted a t acid pH but not to those extracted a t neutral pH. It was desirable therefore to reconsider the method of extrapolation (Hamoir, 1955). Measurements have been recently extended a t low concentration (up to 0.01 %) making use of the new cell of longer optical path devised by the Spinco company. The curve obtained with carp actomyosin (Fig. 24) bend upwards a t low concentration and can be easily extrapolated a t about 90 8. At very low concentration, fish actomyosin does not therefore dissociate and sediments at about the same rate as rabbit actomyosin. In conclusion, fish and rabbit actomyosins are very similar from the

+

260

G . HAMOIR

0.12 0.10

0.08

r r(

0.06

0.04 0.02

0.5

'-0.7

0.9

1.1

1.3

Per cent

I

FIG.23. Variation of ~ / S ? OX,10~'3 with concentration of the carp actomyosins of Fig. 22. Samesymbols as in Fig. 22. The abscissae have been shifted for clarity by 0.2 (X), 0.4 ( 0 ) ,and 0.6 (A, 0 ) unit to the right (after HAMOIR, 1955).

electrochemical point of view :their maximum acid and base binding capacities (Hamoir, 1951b), their solubilities, and their electrophoretic mobilities are very close. But the properties related to the size and shape of the particles differ widely. The extrapolations of the viscosity at gradient 0 0.06 -

0.04

0.02

0.2

0.1

Per cent

FIG.24. Variation of l/SZQ,toX lol* with concentration for two 1 hour-actomyosins .of the carp ( 0 , X ) and a rabbit actomyosin (0)(after HAMOIR, 1955).

FISH PROTEINS

26 1

and of the sedimentation constant a t concentration 0 show however that these differences are due to the structure of the solution (cf. Tsao, 1953) which seems to be much more pronounced in the case of fish actomyosin. From the point of view of its extractibility, the peculiar behavior of fish actomyosin is not due t o a difference in the properties of solubility of this protein. Its origin must be related to secondary properties and correspond t o a higher level of organization; it must lie in the association of these particles within the myofibril. d. Myosin. The data with respect to this protein are still very scarce, although many attempts a t isolation have been made on carp (Hamoir, 1949, 1951b, 1955) and on cod muscles (Connell, 1954). It is always absent in carp extracts made at neutral p H but when the extraction is carried out a t p 0.5 or 1.0 and slightly acid pH, a 0 peak appears in the electrophoretic pattern (Figs. 9 and 12). Ultracentrifugation has shown, however, t ha t the peak occurring in extracts of p 0.5 does not correspond to myosin (see p. 246). The one observed in extracts of p 1.0 has been isolated as a pure electrophoretic component or as a mixture of two components (Fig. 16), and both solutions have been examined by ultracentrifugation (Hamoir, 1955). The /3 component gives rise to two peaks with nearly the same rates of sedimentation, usually varying between 4.0 and 5.0 S a t the total concentration of 1.1 % (Fig. 25). They therefore sediment a t the speed of rabbit myosin (Snellman and Erdos, 1949; Portzehl, Schramm and Weber, 1950; Johnson and Landolt, 1950, 1951). The relation to rabbit myosin is much closer than in the case of preparations extracted at p 0.5 but a discrepancy still occurs. On the other hand, a cod “myosin” was recently described by Connell (1954). Fresh coarse minced muscle was extracted for 5 minutes with a

FIG.25. Ultracentrifugal pattern of the j3 gradient isolated from a ten-minute extract at p 1 and pH 5.5 (see Fig. 12). Conc. 1.07%. (after HAMOIR, 1955).

262

G . HAMOIR

phosphate-KC1 solution of p 0.35 and p H 6.5 containing 0.01 M pyrophosphate. The myosin was separated by dialysis against water and the precipitate washed at p 0.05 and p H 7.5; i t was purified by a second precipitation under the same conditions. The clear solution obtained was pure by electrophoresis and had practically the same intrinsic viscosity and isoelectric point as rabbit myosin (Table VI). It combines with cod F-actin to give artificial actomyosins having intrinsic viscosities of about the half of the “natural” ones. But once more the ultracentrifugal study showed that the preparation differs widely in size and shape from rabbit myosin so far as can be judged from the examination of one sample. It sedimented with rates of 9.65 and 17.1 S at 0.9 % and 0.45 % concentration, respectively. Neither cod nor carp myosin has thus been isolated so far in homogeneous form. When the conditions are not favorable for the extraction of actomyosin, a fraction can be obtained from fish muscle which has many properties in common with rabbit myosin but differs from it by its heterogeneity and the size and shape of the particles. The fish “myosin” extracted so far seems to be an association of the pure protein with other components which confer upon the preparations a lower reproducibility and a more or less marked abnormal ultracentrifugal behavior. The existence of a true myosin in fish muscle has, however, been unequivocally established by the ultracentrifugation of carp actomyosin in presence of ATP (Hamoir, 1951b, 1955). The actomyosin peak disappears completely under these conditions and a new one sedimenting much more slowly becomes visible. Its sedimentation-concentration curve extrapolated to zero concentration gives a sedimentation constant of 6.55 (Fig. 26). The figure of 6.9 previously published (Hamoir, 1951b) has been corrected according to a new calibration of the rotor temperature. I n the

a2

0.4

0.6

0.8

Fro. 26. Variation of per cent 8 2 0 , ~X IOl3 for carp myosin with concentration. The crosses correspond to the 6ame preparation a t different dilutions (after HAMOIR, 1955).

TABLE VI Con-pnrison of Soiirc Propcrtics of r i s h and Rabbit Siruclure Proici/:s Actom yosin Cod S?O

-

Intrinsic viscosity Isoelectric point

-

Myosin

G - and F-actin

Carp

Rabbit

Cod

Carp

Rabbit

110-1555 5.0-5.4= 5.4Q

70-l1Oa 3-5' 5.6h

-

6.55a

2.0d

-

5.3d

-

7.1' 2.2c 5.4'

Cod

0.075 ( G ) d 4.74.8 (G &

Max. binding capacity in 10-6 eq./g. prot.: acid range 162' alkaline range 165' 4.30 Tyrosine content in per cent protein Tryptophan content in 0.950 per cent protein _ _ ~

-

155d 182d

156"-150" 180"-165" 3.4k 0.8k

~~

Hamoir (1955)--" Portzehl, Schrarnm, and Weber (1950)-c Weher and Portzehl (1952kdConnell (1954)'T s a ~(1953)J Tsao, Bailey, and Adair (1951)-@ Roth (1947)-' Hamoir (1947)--i Snellrnan and Erdk (1948)Debain (unpublished results)-k Bailey (1948)-' Suhba Rao (1948)-m Duhuisson (1941)-n Mihalyi (1950)'Bailey ( 1 9 3 7 1 3 Tsao and Bailey (1953)

a

F)d

1 .Od

Rabbit

Tropomyosin Carp

Rabbit

65 (F)c 2.70a 0.20 (G)' 1.40-0.39f 5.05 (G)- 5.0a 5.lk 4.85(F)j

4.7p

3.1k

1.2p

0k

2 ;

264

G. HAMOIR

case of rabbit myosin, figures of 7.2 (Snellman and Erdos, 1948; Johnson and Landolt, 1951), 7.1 (Portzehl, Schramm, and Weber, 1950), and 6.7 (Mommaerts and Parrish, 1951) have been found. Lower values of about 5.5 have, however, been recently obtained by Laki and Carroll (1955). The slight difference observed in the case of the fish myosin is therefore perhaps not significant. These two proteins appear so far very similar in all their properties if not identical, but the peculiar properties of fish actomyosin cannot be explained by our present knowledge of fish myosin. e. Actin. When carp muscles are soaked in an organic solvent for half a day and extracted, after removal of the solvent, a t p 1 and neutral pH as for the preparation of tropomyosin (Bailey, 1948), a new gradient appears in front of the electrophoretic pattern of such whole extracts (Hamoir, unpublished results) (Fig. 27). It migrates with the turbidity and does not diffuse during the experiment; the component involved shows flow birefringence, this property being more or less pronounced in the extracts according to the amount present. A few determinations of its mobilities have given values of -6.5 X 10-5 and -5.3 X volt' set.-' at p 0.35 (0.1 phosphate and 0.25 NaC1) and pH 7.1. for the ascending and descending limbs, respectively; it moves much more rapidly than the adjacent boundary which corresponds to tropomyosin. It is obviously F-actin. This protein was recently prepared from cod muscles and investigated by Connell (1954). Acetone-dried muscle fiber was extracted with water by the method of Feuer, et al. (1948) omitting the final treatment with Na2COS (Tsao and Bailey, 1953). The G-actin obtained polymerizes on addition of salts to a viscous solution of F-actin showing pronounced double refrac-

--

FIG.27. Electrophoretic pattern of an ll-hour extract at p 1 and pH 7 of carp muscles previously treated with ethanol. Ionic strength: 0.35; pH: 7.1. Electrical field: 1.75 volt/cm. Duration of the electrophoresis: 40.800 sec. Upper part: ascending limb. Lower purl: descending limb.

FISH PROTEINS

265

tion of flow. It loses its polymerizability by dialysis against water for several days and it may also be depolymerized with 0.6 M KI. The Gand F-forms could thus be examined by electrophoresis: their average and about - 6.5 X 1 0 - 6 mobilities at p 0.4 and pH 7.4 are of -3.9 X cm.2volt.--' set.-', respectively. The corresponding figures for rabbit actin and -6.5 X 1 0 - 6 in the same conditions are, respectively, -4.65 X cm.z volt.-' set.--' (Dubuisson, 1950). Rabbit, cod, and carp F-actin therefore migrate at the same speed while cod G-actin apparently moves more slowly than depolymerized rabbit F-actin. The ultracentrifugal analysis of a partially depolymerized sample revealed the presence of a light component (about 1.0 S) and a very heavy polydisperse one. It should be noted that such a slow component has been observed in carp myogen (see p. 241) and is absent in rabbit G- and F-actin, whose sedimentation constants are 3.2 and 5 0 4 5 S, respectively (Weber and Portzehl, 1952). In general, however, the similarity to rabbit actin is clear (Table VI). A better agreement should not be expected in view of the limited purity and reproducibility attained in the isolation of this protein. Further researches will show whether the slight differences observed are significant and whether some properties of fish actin may explain the peculiar behavior of fish actomyosin. f. Tropomyosin. This protein was first isolated from muscles of different origins (including whiting) previously dehydrated with organic solvents (Bailey, 1948). The amount in carp muscle determined under such conditions is approximately 0.4% of the fresh muscle weight (Hamoir, 1951c), and a figure of 0.47% was found by Bailey (1948) for rabbit. The preliminary treatment with organic solvents is unnecessary in the case of fish muscle, from which tropomyosin is particularly easily extracted (see p. 249), and the possibility of using very gentle conditions of extraction has prompted its study (Hamoir, 1951a,c; 1953a, 1955). These researches have shown that tropomyosins differing in solubility and state of aggregation from the protein described by Bailey (1948) may be isolated from such extracts. A detailed account of these investigations is given elsewhere (Hamoir, 1955). The occurrence of two fractions of different electrophoretic behavior and solubility in carp whole extracts of p 1 and pH 5.5 has been previously described (see p. 250). These two fractions are already present in extracts of p 0.6 prepared in presence of ATP from which they were first isolated (Hamoir, 1951a,c). We shall consider in turn the preparations obtained a t p 0.6 and 1.0. ( 1 ) Tropomyosin extracted at p 0.6, soluble at low ionic strength. Tropomyosin is already extracted in small amounts in 20-minute extracts a t p 0.20 and neutral pH or at p 0.25 and pH 5.5 and in 10-minute extracts at p 0.6 and pH 5.5 in presence of 0.1 % ATP. The major part of the globu-

<

266

G . HAMOIR

lins present are discarded by dilution a t p 0.05 and tropomyosin is isolated from the supernatant by precipitation at p H 4.6 and ammonium sulfate fractionation at neutral pH between 50 % and 66 % saturation (see p. 249). The yield is only 0.03 % of the fresh weight. The crystallization, electrophoresis, and ultracentrifugation of these preparations do not reveal any difference from Bailey’s rabbit tropomyosin (Hamoir, 1951a,c, 1955). A slight contamination with pentose nucleic acid (2% to 3%) is, however, shown by ribose and phosphorus determinations as well as by the ultraviolet spectrum. (2) Tropomyosin extracted at p 0.6, insoluble at low ionic strength (nucleotropomyosin). When the globulins extracted a t p 0.6 and p H 5.5 in presence of 0.1 % ATP which were discarded in the previous paragraph, are purified by a further precipitation and fractionated with neutral ammonium sulfate, another tropomyosin separates between 50 % and 66 % saturation (Hamoir, 1951a,c). The yield amounts t o 0.07% of the fresh weight. These preparations do not differ electrophoretically from the preceding one and crystallize under the conditions described by Bailey (1948) but in another form (Fig. 28). Their rate of sedimentation, however, is intermediate between that of the preceding tropomyosin and that of myosin (Fig. 29). Phosphorus and ribose determinations as well as ultraviolet absorption reveal the presence of 10% to 20% ribonucleic acid. As the ultracentrifugation and the electrophoresis diagrams do not show a corresponding boundary, it therefore seems that we are dealing with a ribonucleoprotein. We do not know whether it arises from the association of tropomyosin with nucleic acid during the extraction or whether it is a

FIG.28. Crystals of carp nucleotropomyosin. Magnification X54 (after 1951a).

HAMOIR,

FISH PROTEINS

267

Per cent

FIG.29. Variation of ~ 2 0 X , ~ loL3 of carp tropomyosin and nucleotropomyosin with concentration. Lower curve: tropomyosin extracted a t I.( 5 0.6 soluble a t low ionic strength. Median curve: nucleotropomyosin. U p p e r curve: carp myosin (sec Fig. 26). The two circles correspond t o rabbit tropomyosin (BAILEY, GUTFREUND, and OGSTON, 1948). (after HAMOIR, 1951a)

natural component of the muscle. Although this question has been considered by several authors (Perry, 1952; Tsao, 1953; Elson and Chargaff, 1954; Hamoir, 1955), it still remains open. The conflicting data could be reconciled by assuming that nucleotropomyosin occurs as a natural component but that its content in rabbit muscle is much lower than in carp muscle. (3) Tropomyosin extracted at p 1, soluble at low ionic strength. When the mixture of Fig. 15 is diluted to p 0.05, tropomyosin can be isolated from the supernatant by the usual procedure. In this case, however, the acidification has been carried out only as far as pH 5.5 instead of 4.6. The fraction examined thus corresponds to a part of the tropomyosin soluble a t low ionic strength present in the extracts. Its electrophoretic picture is similar to that of Fig. 13. When the preparation is precipitated a t p H 4.6 and a t relatively high salt concentration ( p l ) , it is profoundly transformed: the fast electrophoretic component which corresponds to Bailey’s tropomyosin increases notably a t the expense of the slow one. The same transformation is observed by ultracentrifugation : components sedimenting a t rates varying between those of tropomyosin and nucleotropomyosin become less important while the slow tropomyosin peak increases (Fig. 30). As the nucleic acid present in the preparations does not precipitate with tropomyosin at p 1 and pH 4.6, the question arises if this transformation is not due to its removal. As the amounts present are,

268

G . HAMOIR

however, usually lower than 1%, it seems unlikely that they could have such a marked influence on the stability of the heavy components. (4) Tropomyosin extracted at p 1 , insoluble at low ionic strength. The isolation of this fraction from extracts carried out a t p 1 and p H 5.5 has been previously described (see p. 251). It precipitates together with the globulins of the /3 peak between p 0.25 and 0.025. The electrophoresis of this mixture reveals a strong interaction: the ascending and descending fast peaks differ strongly in mobility and area (Fig. 16). This behavior does not occur with the tropomyosin fraction described in the previous paragraph. When it is added to the mixture of Fig. 16, the area of the fast ascending peak increases and on the descending side, a new one migrating

-

U

FIG.30. Ultracentrifugal patterns of the carp tropomyosin extracted a t p 1 soluble a t low ionic strength before (full line) and after (broken line) precipitation at p 1 and p H 4.6 (after HAMOIR, 1955).

in front of those of Fig. 16 is observed. The retarding influence of the interaction on the fast descending boundary of Fig. 16-an effect explained by Longsworth and Mac Innes (1942)-is not observed with the soluble fraction. A qualitative difference thus exists between these two fractions. The tropomyosin extracted a t p 1, insoluble a t low ionic strength, also differs from nucleotropomyosin; it contains only 5 % t o 6 % nucleic acid, and this nucleic acid very likely migrates independently in the U-tube. The high ionic strength of the extraction seems to have induced a splitting of the nucleoprotein. A stepwise degradation of the tropornyosin complex thus occurs in the course of the extraction, leading to a progressive increase of solubility and allowing the identification of more or less different fractions. The particle described by Bailey (1948) corresponds t o the final step of this transformation. Tropomyosin may actually exist in fish muscle as a ribonucleoprotein but as the ultracentrifugal behavior typical of nucleotropomyosin is ob-

FISH PROTEINS

269

served with tropomyosin preparations containing as little as 0.5 % nucleic acid, a reinvestigation of this problem is desirable. The final step in the degradation of the tropomyosin complcx seems in any case identical in fish and rabbit muscle.

111. FISHENZYMES Fishes are the only vertebrates able to split thiamine. Thiaminase occurs in muscles and viscera of many fishes and in some invertebrates; the advances made in its study have been reviewed by Harris (1951), though new contributions have recently appeared. The activation of carp preparations by nitrogenous compounds has been investigated by Tatarskaja (1952). Its distribution among invertebrates and vertebrates has been examined by Tatarskaja, Kudrjashov, and Fajn (1954). The occurrence of a t least two thiaminases differing in p H optimum and in activity in presence of heavy metals has been established by comparing fresh-water, brackish-water, and salt-water fishes (Deolalkar and Sohonie, 1954). The other enzymatic reactions found in fish seem to be common to all vertebrates. Study of these reactions has been mainly directed towards the biochemical changes occurring post-mortem in fish muscle, the so-called “internal” factors of fish spoilage which have involved investigations in glycolysis, lipolysis, and proteolysis. Let us first consider the breakdown of the carbohydrates, which has been extensively investigated on warm-blooded animals a t the Low Temperature Research Station in Cambridge (Bate Smith, 1948; Bate Smith and Bendall, 1949; Bendall, 1951; Marsh, 1952). The fragmentary data available on fish have been summarized by Reay and Shewan (1949) and recently reconsidered by Partmann (1952-53, 1954a) in view of modern theories linking glycolysis, muscle contraction, and rigor mortis. The post-mortem changes occurring in the muscle tissue of frigate mackerels killed by various methods have been examined by Amano, Bito, and Kawabata (1953) and by Fujimaki and Kojo (1953). Their simultaneous determinations of glycogen (see p. 233), lactic acid, ammonia, amide, and p H (Amano, Bito, and Kawabata, 1953) as well of the various acid-soluble phosphorus compounds (Fujimaki and Kojo, 1953) confirm earlier researches of Tarr (1950). The differences compared with domesticated land animals are small and mainly due to the lower glycogen content of fish muscle (see p. 234) and to the pronounced state of fatigue in which it is usually obtained. Fish muscle thus normally contains more lactic acid than mammalian muscle, but after rigor mortis the ultimate value reached is actually less (0.1% to 0.5% lactic acid instead of 1.1 % in mammals) and the p H decreases only to about 6.3 as compared with 5.6 found in beef (see Partmann, 1954a). The only exceptions recorded are the halibut (see Part-

270

0. HAMOIlL

maiiii, 1954a) and the frigate mackerel (Amano, Bito, and Kawabata, 1953) (see p. 238), whose muscles attain an ultimate pH of 5.6 to 5.8. Several other researches have shown that a third factor distinguishes fish and mammalian muscle: the total muscle ATPase activity. According to Steinbach (1949), KIT is split, a t about the same rate I)y the total muscli. ATPase of fishes, frog, mouse, bird, or turtle at 30" C., whereas wide differences occur a t about 0" C.; a t this temperature, the rate of ATP splitting remains fairly large in fishes (Ql0 = 1.4 to 1.7) and is much smaller in other vertebrates (Ql0 = 1.9 to 2.2). A short note of Davison and Richards (1954) has recently confirmed these results. Reuter (unpublished results) has found that the splitting of the labile phosphorus present in muscle extracts of high ionic strength and p H 8 a t 2" C. is extremely rapid in the case of the carp and much slower in the case of several vertebrates (Fig. 31). A comparison of ATP breakdown by muscle pulp of tench and cattle (gastrocnemius) has also been made by Partmarin (1954a,b, 1955). In presence of vrronal a t pH 7, half of the added ATP is split 2.8 times more 500

40C

300 3 VI

E

. bil

c ._

E 20c

a"

1oc

FIG.31. Variation of the labile phosphorus of muscle extractasmade under the same conditions G( = 0.5; pH 7.5; T:2" C.) from carp (O),frog ( O ) ,tortoise (X), hen: leg (A) and pectoral muscle (V)and rabbit (0). Ordinate: y of phosphorus hydrolyzed after 7 minutes in boiling HCl N pro gr of muscle. Abscissa: time in minutes (after REUTER, unpublished results).

FISH PROTEINS

27 1

quickly by fish white muscle than by cattle muscle at O", 20", and 30" C. In the case of fish red muscle, at 20" C., the rate is only 1.35 times faster than that of cattle muscle. The temperature coefficients found here arc thus the same for both animals (2.3 between 0" and 20" C.; 1.3 to 1.4 between 20" and 30" (3.). Furthermore, these enzymic activities both in tench and in beef are unimpaired by freezing and frozen storage, whereas the amount of actomyosin extractable is greatly reduced (Partmann, 1954b, 1955). It is obvious from these various researches that the ATPase activity of fish muscle is larger than that of the other vertebrates, but the value of its temperature coefficient remains uncertain. On the other hand, the differeiices observed between fish white and red muscles (see also p. 238) entirely agree with the results obtained with mammalian and avian white arid red muscles (Lawrie, 1952, 1953a,b). Glycolysis is very similar iii fishes, frog, or mammals, but fish white muscle seems to be characterized from this point of view by a higher glycolytic activity than that of all other white muscles. Another process of degradation in muscle which appears also more rapid in fish than in warm-blooded animals have been described by Tarr (1952, 1953, 1954) and by Tarr and Rissett (1954). An important liberation of ribose occurs post-mortem in fish from ribonucleic acid, ribotides, ribosides, ATP, and ribose-5-phosphate under the influence of riboside hydrolases. A partial purification of these enzymes isolated from lingcod and rock cod muscle has been carried out, recentJy (Tarr, 1955). As ribose plays a predominant role in the browning of heat-processed fish products, more work will certainly be done in this direction in the near future. Some other recent contributions to the carbohydrate metabolism in fish may also be mentioned: the description of the occurrence in fish muscle of the K-activated pyruvic phosphopherase already described in mammalian inuscle (Boyer, 1953),the observation of the glycolytic activity of the swim bladder gland (Strittmatter, Ball, and Cooper, 1952), the study of the respiration and glycolysis of the retinal tissues of fishes (De Vincentiis, 1952). Concerning lipolysis in fish muscle, an important advance has been made by Khan (1952). It was shown a long time ago (Banks, 1938) that a "lipoxidase" system contributing notably to the development of rancidity is localized in the lateral brown band of fatty fishes. This enzyme is very detrimental for the conservation of fish (Reay and Shewan, 1949; Dyer, 1951). Preparations over two thousand times as active as the raw material have been obtained (Khan, 1952), which require for activity some iron-containing, heat-stable activator. No way has yet been found of overcoming the detrimental action of this enzyme. Several enzymatic reactions involving nitrogenous compounds also occur post-mortem in fish muscle. They have been overlooked until recently

272

G. HAMOIR

because they are normally concealed by the predominant influence of bacteria on the alterations observed. The presence of urease (Ferguson-Wood, 1950), trimethylamine oxide-reductase (Nickerson, Goldblith, and Proctor, 1950; Nikkilii, 1951 ; Kawabata, 1953), arginase (Matsuura, Baba, and Mori, 1953), and anserinase (Jones, 1954b) has been reported. In view of the high proteolytic activity found by Snoke and Neurath (1950) in rabbit muscle extracts contrary to the generally admittcd opinion (Fearon and Foster, 1922; Smorodinzew, Schirokow, and Zyganowa, 1933) it, was zllso desirable to assess the importauce of proteolysis in fish muscle. This s u b ject has been reviewed by Partmann (1952-53, 1954a) and recently reiiivestigated a t the Massachusetts Institute of Technology (Proctor, Sic,kwson, and Goldblith, 1950; Nickerson, Goldblith, and Proctor, 1950) mid i i i Germany (Partmann, 1952-53, 1954a). The American authors observed a distinctly different behavior in haddock and mackerel muscle. Iu the first case, no changes could be detected after t~ two weeks ice storage, whereas in the second proteolysis and lipolysis were definitely measurable. It seems probable that this quicker autolysis is due to the greater development of the red muscles in mackerel, as suggested a long time ago by Oya (1928). I n muscle blocks of tench, the increase of amino nitrogen after a two months storage a t 20" C. corresponds t o the splitting of 1.8% of the whole nitrogenous substances (Partmann, 1954a). Although specific variations occur, the proteolytic activity of fish muscle is low arid does iiot materially contribute to fish spoilage. If the conservation of fish meat has promoted a much better knowledge of fish enzymes, these components have also been investigated in some cases for their own sakc. The pyloric caeca of fish, which have a function similar to that of niaiiimalian pancreas, are a potent source of tryptic enzymes. The crude enzyme preparations made by acetone drying of the comminuted caeca are as effective as commercial proteolytic preparations (Tarr, 1948). According to Kashiwada (1952), this proteolytic enzyme activity rises in spring and decreases gradually in summer in the case of skipjack (Katsz~wonus vagans). Crude preparations from red fish (Sebastes marinus) have been studied by Stern and Lockaert (1953), who have determined their optimal conditions of activity and have shown that this activity also compares favorably with that of commercial preparations. Some researches mainly interesting from the point of view of comparative enzymology have been recently carried out on fish. The important contribution of Augustinsson (1948) to the study of cholinesterase in various tissues of invertebrates and vertebrates including fishes is well known, Some further data on this enzyme have been published; Laurerit (1952a,b) has examined its activity in the case of the catfish (Ameiurus nebulosus

FISH PROTEINS

273

Les.), and Couceiro, de Almeida, and Freire (1953) have localized it histochemically in the electrical tissue of Ekctrophorus electricus I,. The distribution of carbonic anhydrase in several tissues of two teleosts and its inhibition in vivo by the sulfonamides have been investigated by Maetz (1953a,b). The presence of cathepsin in the stomachs of various animals including pike and trout has been established by Buchs (1954). A new advance has also been made in the comparative study of pepsin. This enzyme, previously crystallized from salmon (Norris and Elam, 1940), halibut (Eriksen, 1943), and shark (Sprissler, 1942), has now been crystallized from three species of tuna (Norris and Mathies, 1953). These interesting researches have shown that fish pepsinsdiffer in crystal structure, amino acid composition, and specificity from swine or bovine pepsins and show a closer relationship to one another. As pointed out by Velick and Udenfriend (1953), specificity requirements toward substrates are less exacting with extracellular enzymes.

IV. FISHBLOOD PROTEINS It has been stated that osmotic pressure measurements of animal plasmas have shown that ‘(anincontestable correlation exists between the protein concentration of the plasma and the position occupied by the animal in the scale of zoological classification” (Florkin, 1949). This rule, a t least to some extent, may also be applicable to certain classes of vertebrates. Numerous determinations by Deutsch and McShan (1949) on lower vertebrates indicate that fish plasmas are usually poorer in protein than those of higher vertebrates, although wide variations exist, e.g., 3.2 % for Cyprinus carpio and 5.8 % for Coregonus clupeaformis. An independent determination on Lophius piscatorius plasma by Brull and Niaet (1953) is well within the above range, but a larger variation may occur, judging by the figure of 2.1 % for Macrones gulio given by Menon (1952). On the other hand, the protein concentration given by Deutsch and Mc Shan (1949) and Menon (1952) for frog plasmas is only 2.5% (&O.l%). The trend, therefore, towards a higher plasma protein concentration as vertebrates evolve, exists, but it is not so marked as suggested by Menon (1952), and there are many exceptions. The comparative electrophoretic study of plasmas, which was a t first confined t o mammals and birds (Deutsch and Goodloe, 1945; Moore, 1945), was later extended to fish (Deutsch and Mc Shan, 1949). Other preliminary electrophoretic analyses of fish plasmas may also be mentioned (Connell, 1953b; nrull and Nizet, 1953; Drilhon, 1953; Irisawa and Irisawa, 1954) as well as the study of the distribution of lipids among the components of a few fish sera separated by means of paper electrophoresis (Ilrilhon, 1954). The protein components so observed are sometimes

274

G . HAMOIIt

similar to those of higher vertebrates (i.e., by having a large, faster nioviiig peak follomed by several smaller ones), whereas a t other times lhcy arc entirely different. Furthermore, the comparison of four species of catfish and three of trout (lkutsch and Mc Shan, 1949) shows that thr variations hetween closely related species are as great as those between unrelated species. The only general characteristic observed is the relative paucity or even absence of a component comparable to the y-globulin of mammals. Fish plasmas are thus as variable as fish muscle extracts of low ionic strength (see p. 240). The isolation of the different protein components of fish blood has not so far received much attention. The fractionation of the components has been limited to the determination of the albumin/globulin ratios, which are relatively low (Lepkovsky, 1929-30), a recent value being 0.9 for Macrones gulio by Meiiori (1952), which agrees with earlier results. Some further work has been carried out on fish hemoglobins, which were among the first well-defined proteins (see Wyman, 1948; Pedersen, 1950; Ladd Prosser, 1950). Brull and Cuypers (1954) have found that the hemoglobin of the blood of Lophiins piscatoriiis amounts to only approximately 2 %. Carp hemoglobin has been crystallized from a concentrated phosphate buffer of pH 6.8 by Huys (unpublished). The comparative study of hemoglobin has been reviewed by Ladd Prosser (1950) and further investigated by Korzuev (1952). The progressive iiirrease in amount of blood per kilo body weight and in capacity of absorption of oxygen in vols % as we go up the evolutionary ladder from fishes to mammals has been stressed again by the last author. The respiratory function is thus poorly developed in fishes. A good illustration of this fact has been recently given by liuud (1954). He has for the first time pointed out that some fishes living in the Antarctic are devoid of erythrocytes and also of respiratory pigment, the oxygen requiremeiits of these fishes being so low that they are adequately satisfied by the dissolved oxygen in the plasma. ltuud’s findings thercfow suggest that the function of hemoglobin in many species of fish is that of an emergency oxygen reserve (Munro Fox, 1954).

v. FISHPROTAMINES From the time of Miescher and Kossel, few proteins have attracted as much interest as fish protamines. This is due to several factors: their low molecular weight and simple chemical composition, their importance in the study of heredity, their use as precipitants in preparative protein (ahemistry, and their pharmacological action. These proteins are prepared from the cell nuclei of spermatozoa which are easily isolated from the milt (Felix et al., 1951). Herring testes have also been used recently (Felix and Krekels, 1953a). This material, however, is less favorable, for the isola-

FISH PROTEINS

275

tion of the nuclei is more laborious; moreover, when this preliminary isolation is omitted and the comminuted tissue directly extracted, the protamine is apparently split during the extraction by an enzyme present in the tissue. According to Felix and co-workers, the cell nuclei of fish sperm are essentially made of nucleoprotamines. They dissolve in 10% NaCl without leaving any residue, the dissolved material precipitating completely by dilution and the nucleoprotamine fibers obtained by repeated reprecipitations having the same nitrogen and phosphorus contents as those of cell nuclei (Felix, 1953; Felix and Krekels, 1953a; Fisher, 1954). Moreover the reactions for tryptophan, cystine, methionine, and phosphatase are negative on cell nuclei as well as on nucleoprotamines (Felix, 1953). Cell nuclei of fish spermatozoa therefore appear to be entirely built up from nucleoprotamines. The recent isolation of protamine (gallin) from the sperm nucleus of fowl (Fisher, 1954) furthermore suggests th a t these proteins are of wide biological importance. The main methods of preparation have been briefly reviewed by Sorm and Sormova (1951). As pointed out by these authors, little attention has hitherto been paid to possible alterations in the course of isolation, although these procedures usually involve excessively drastic conditions. The control of the homogeneity of the preparations by physical methods has also not been much considered. The study of their behavior in the ultracentrifuge, in the electrophoretic apparatus, and by countercurrent distribution is quite recent: a salmine (Genus Oncorhynchus) examined by the two first methods by Velick and Udenfriend (1951) has been found relatively homogenous; but various preparations investigated by Felix and co-workers were slightly heterogenous by ultracentrifugation (Daimler, 1952 ; see also Colvin, 1952) and electrophoresis (Rauen, Stamm, and Felix, 1953) and behaved as complex mixtures by countercurrent distribution (Felix, 1953; Zahn and Stamm, 1954). These proteins have low sedimentation constants of about 1 S (Velick and Udenfriend, 1951), which makes their study by centrifugation difficult ; some modifications of the technique have been introduced in order to improve results (Daimler, 1952). Although the two other methods are more easily applicable, the investigations carried on so far are still of a preliminary character. The use of chromatography is also of interest since dinitrophenyl-clupein has been split into two fractions by this method (Waldschmidt-Leitz and Pflanz, 1953). These results show that the evidence for the homogeneity of the preparations investigated is usually insufficient and the occurrence of several more or less closely related components is possible. While the physical characterization of these proteins is still very poor, their chemical composition is well kno\vn. Analyses carried out on herriiig protamine (clupein), on sturgeon protamine (sturine), and on protamines

276

G . HAMOIR

of various Salmonidae (salmines) have shown that they contain only from 6 to 10 types of amino acid (see Felix, 1953). This simple composition first prompted the well-known attempts to determine amino acid sequences in proteins (Waldschmidt-Leitz and Kofranyi, 1935; Felix and Mager, 1937). This early work has culminated in the formula proposed by the last authors for clupein: Pro (Arg. .4rg. Arg. Arg. N. M.),, Arg. Arg

It appeared particularly promising to resume these researches with the aid of the new techniques now available (Sanger, 1952), and their first applications t o protamines have been reviewed by Tristam (1949) and Sanger (1952). Many further investigations have been carried out more recently. The nature of the N-terminal residue has been investigated on a salmiiw (Genus Oncorhynchus) (Velick and Udenfriend, 1951), on clupein (Sorm and Sormova, 1951; Waldschmidt-Leitz and Pflanz, 1953; Felix and Krekels, 1953b) and on several protamines (Felix and Krekels, 1953b). Proline exists as N-terminal amino acid in salmine (Genus Oncorhynchus) as indeed in the other protamines examined except sturine, which apparently has two terminal residues: alanine and glutamic acid (Felix and Krekels, 19531s). DNP-clupein has been partially hydrolyzed and the terminal peptides have been isolated (Sorm and Sormova, 1951). According to these authors, the terminal amino acid sequence is Pro. Ala. Ser. and thus differs from that given in Felix and Mager's formula. On the other hand, the analysis of the basic peptides of clupein is in agreement: 35 % of clupein nitrogen has heen isolated as Arg. Arg. Arg. Arg., 2.5 % as Al. Arg. Arg., 6.7 % as Ser. Arg. Arg., 24.9% as Arg. Arg. and as arginine, while the whole neutral amino acid fraction represents only 6.4% (Felix, Rauen, and Zimmer, 1953). Salmine has also been extensively investigated. Its amino acid composition has been determined by Velick and Udenfriend (1951) and by Corfield and Robson (1953), who have discussed the discrepancies occiirring between various authors. These discrepancies have led to differelit minimum molecular weights: 7000 according to Velick and Udenfriend (1951), 10,000 according to Corfield and Robson (1953). It is probable that these variations may be partly accounted for by differences in origiri of the salmine samples. The arrangement of the amino acids in salmine of Oncorhynchus analyzed by Velick and Udenfriend (1951) has been extensively studied by Monier and Jutisx (1954a,b). Their main resulth are as follows: the N-terminal amino acid sequence is Pro]. hrg. Arg. T l i r ncutral pept,ides fraction isolated after acid hydrolysis is made of five> dipeptides, all of which have been ideutified. Trypsin hydrolysis gives pel)tides containing one OY sevrral nrgiriirie residues; the study of those with

FISH PROTEINS

277

one arginine residue has permitted the assignment of two neutral amino acids whose peptides are hydrolyzed by the acid treatment. The agreement of these results with Felix and Mager’s formula is remarkable. Furthermore the comparison with the analytical data of Velick and Udenfriend (1951) shows that nearly all neutral amino acids present in themoleculehave been isolated as peptides. Two different peptides have, however, been obtained for the single residue of isoleucine existing in the molecule. It is possible that the salmine preparations investigated contain two types of polypeptide chains. Several conflicting retults have however been obtained on salmine from keta salmon (Oncorhynchus keta) by Phillips (1955) who Euggests an average molecular weight of only 3.800. Nevertheless the clearing up of the whole polypeptide structure of the protamine seems near at hand. The agreement found in the numerous analytical determinations made so far suggests that the homogeneity of these proteins is probably higher than might be expected from physical methods. Should heterogeneity exist, i t seems to be limited to very small variations of the polypeptide chains. As regards the properties of protamines, some recent researches are also worthy of attention. The precipitation of several common proteins with salmine has been investigated and appears to be due to a neutralization of net charges of opposite sign (Ross, 1954a,b). Such complexes may be very insoluble; Madsen and Cori (1954) have shown that the inhibition of the phosphorylases a and b by salmine described by Krebs (1954) is in fact due to such a reaction. The growth of bacteria is also inhibited by protamines and can be restored by acidic compounds; a new contribution t o this problem has been published by Wolff and Brignon (1954). Other studies include the binding of organic anions by protamine sulfate (Colvin, 1952) and a determination of the ultraviolet spectrum of clupein sulfate (Mc Laren and Waldt, 1952). The pharmacological action of these proteins was examined some years ago by Geiger (1948) and will not be considered here. VI. CONNECTIVE TISSUEPROTEINS Our knowledge of the proteins of fish connective tissue is limited so far to collagen. This major fibrous component of fish skin and swim bladders is fairly well known (see Geiger, 1948; Hamoir, 1951b; Gustavson, 1953). The isolation of a collagen-like protein from herring scales has also been reported by Block, Horwith, and Bolling (1949). Fish and mammalian collagens transform into gelatin by the action of hot water; that of elasmobranch fishes is the only one which does not (Faure-Fremiet, 1937). Furthermore, fish wastes containing large amounts of this protein can be

27 8

G. HAMOIlt

manufactured into glue (see Geiger, 1948; Tarr, 1948). The preparatioii of these two degradation products and the study of their properties arc mainly of practical interest and will not he considered here. Fish collagen itself has been investigated i n order to determine the common features of the collagens in gerieral and the origin of the variations observed, a topic recently reviewed by 13ear (1952). The X-ray study of fish and mammalian collagens shows a remarkable constancy in the large and small structural spacings and points to the existence of a basic structural pattern. The variations observed in the case of the teleost collagen, ichthyocol, involve only slight alterations in small-angle intensities, while those corresponding to the elasmobranch collagen, elastoidin, are more pronounced, showing the greatest departure from other vertebrates. A similar relationship occurs between the amino acid compositions of teleost and mammalian collagens. Ichthyocol is very similar to mammalian collagen. It differs only in a relatively lower amount of hydroxyproline (9 % instead of 14 %) and in minor divergencies of some amino acids present in small amounts, such as serine, threonine, and methionine (Beveridge and Lucas, 1944; Neuman, 1949; Neuman and Logan, 1950). The comparison, however, cannot be extended to elastoidin because of the scarce analytical data available. But the chemical studies carried out on fish and mammalian collagen are only partially sketched in Bear's review, although these researches have also largely contributed to our present knowledge of collagen fibril. Interest in fish collagen was aroused by the observation that its shrinkage temperature is much lower than that of mammalian collagen (Gustavson, 1942a). Gustavson (1953) has found that skins freely suspended in water shrink in the temperature range GO" to 68" C. for mammals, 54" to 57" C. for pelagic fishes (warm- and surface-water fishes), and 37" to 42°C. for bathybic fishes (cold- and deep-water fishes). Takahashi and Takana (1953) have examined a greater number of species and have also observed the occurrence of intermediate values. It appears that the shrinkage temperature is characteristic of the species and varies continuously from 35 to 59" C. among fishes. The differences in chemical behavior occurring between bovine skin and codfish skin have also been extensively investigated, and the main results have recently been summarized (Gustavson, 1953, 1955). Fish skin collagen is more easily degraded by many agents (hot water, acids, alkalis, trypsin, polyacids, anionic detergents). After thermal denaturation its reactivity towards large molecules mainly interacting by means of hydrogen bonding (e.g., vegetable tannins), is unaltered, whereas a drastic increase o(:curs in the case of bovine collagen. Both types of collagen show, on the other hand, a close similarity in electrochemical behavior: the reactivit.y of agents principally reacting with proteins by elec-

FISH PROTEINS

279

trovalency is practically identical (Gustavson, 1942a,b); the acid and alkali binding capacities are identical and the titration curves do not differ signifirantly (Gustavson, 1950). The very clear-cut results obtained in this comparative work have suggested interesting working hypotheses of the structural organization of collageii. Its hydrothermal stability appears to be governed by the strength of the cross links of the ultimate protein units and mechanical strength of forces between larger units (Gustavsoti, 1949, 1950). Fish and mammalian collagens are stabilized by the same salt links but the hydrogen bridges are apparently very differently developed (Gustavson, 1950, 1953). More direct evidence in favor of this last interpretation has been given recently. New determinations of the hydroxyproline contents of various collagens (Gustavson, l954a, 1955; Takahashi, unpublished results) support the idea that this imino acid stabilizes the structure. The nature of the interchain link itself appears to have been established by the study of the acetylatiori of bovine collagen; arid would seem to occur between hydroxy- and keto-imide groups (Gustavson, 1954b, 1955). The comparative work (wried out on fish has contributed in no slight measure to the great advances made in the study of this protein.

VII. COSCLUSIOS : T HIC COMPARATIVE BIOCHEMISTRY OF FISHI’ROTEIXS The comparative tiorhemistry of proteins is still in its beginning. Research was first, directed towards the study of the protein components of mammals, but is now extending to other classes of vertebrates. In the case of fish, this study, of great economic importance, has been contributed to both by protein chemistry and technology. The main characteristics of fish protein are now satisfactorily defined, and a comparison of the highest and lowest classes of vertebrates is possible from the point of view of protein composition, This comparative work was initiated twenty years ago in Upsala by the study of the respiratory proteins of the animal kingdom. It revealed the occurrence of a close relationship between the hemoglobins belonging to all (*lassesof vertebrates (see Pedersen, 1950). The comparative study of plasma or serum proteins by electrophoresis shows a very diff ererit picture. The patterns obtained in the case of mammals and birds are generally similar in showing a large faster moving albumin component followed by several smaller, slower globuliri components (Deutsch and Goodloe, 1945). But those corresponding to the lower vertebrates are very variable and do not apparently have common characters inter se or with the mammalian ones; furthermore, when members of the same family such as various Salmonidae, Ameiuridae, or Reptilia are examined, the variations observed are not smaller (Deutsch and Mc Shan, 1939). Among lower vertebrates, the fast

280

G. HAMOIR

serum albumin peak of mammals and birds occurs only irregularly and a component comparable to the y-globulin of mammals does not seem t o exist. The muscle extracts of low ionic strength of many species have also been analyzed by electrophoresis. This comparative work began in Liege in 1945; frog, rabbit, carp, hen, pigeon, tortoise, and lobster extracts have been successively investigated by several workers (see Hamoir, 1955). Twenty species of fish have also been recently examined by this method hy Connell (1953b). The occurrence of differences was first established by compariiig the electrophoretic patterns of frog and rabbit extracts (Dubuisson and Jacob, 1945; Jacob, 1945). The further study of carp extracts made it clear that very wide qualitative variations may sometimes occur (Hamoir, 1951b). This variability has now been very nicely illustrated by Connell’s extensive work. The differences observed are so characteristic as to “fingerprint” any one species. The diagrams of genetically related species like those of the Gadidae or Pleuronectidae show as much variation amongst themselves as do the diagrams of unrelated species, except in the case of the elasmobranchs, which all contain proteins with unusually high isoelectric points. A similar discrepancy has been observed between hen and pigeon (Renard, 1952). The synthesis of the blood as well as of the muscle proteins may thus result in very different patterns even in closely related species. While the most striking fact emerging from this comparative work is the variability of protein composition, some regularities are, however, observed in the case of muscle extracts. The components of all electrophoretic patterns examined so far can be divided into three mobility groups according to Jacob (1947) and Conneli (1953a,b) (see p. 239); as the two faster of these three groups frequently comprise less than 35% of the total area of the diagram and as the third one is very variable, this similarity must not be overemphasized. All these patterns contain a small turbid component always situated in the same relative position (see p. 240), and this is the only directly observable common constituent. The consideration of the mean electrophoretic mobility appears also of great interest in the comparison of these patterns. It has been shown that this value is definitely higher in fish extracts than in rabbit extracts (see p. 240). Similar high figures are obtained in the case of tortoise and frog (Renard, 1952). Furthermore, the highest value obtained so far occurs in lobster (DubuissonBrouha, 1953) and the lowest in pigeon, hen, and rabbit (Renard, 1952). The mean electrophoretic mobility appears therefore to decrease progressively as we go up the evolutionary ladder. In the case of ultracentrifugal analysis, no comparative work seems to have been carried out 011 fish plasma proteins and the data relative to fish

FISH PROTEINS

28 1

muscle extracts of low ionic strength are limited to the carp. The comparison of these last results with the few data in the literature (see p. 241) suggests that the muscles of lower vertebrates contain low molecular weight proteins not present in higher vertebrates. Further research will establish whether this surmise is correct. Some recent ultracentrifugations of various myoglobin preparations (see p. 253) are also worth mention here, since they have allowed us t o extend the well-known size relationship of mammalian myoglobins to the myoglobins of the lower vertebrates. The regularities found so far are thus very limited. A much closer analogy could be expected, in view of the biological functions of these proteins, of their frequently very similar conditions of activity, and of the close relationship of some species investigated. I n fact, the properties examined appear so far unrelated to the biological activities of these proteins. The great similarity observed by Velick and Udenfriend (1953) between yeast and rabbit glyceraldehyde-3-phosphate dehydrogenases seems exceptional. Such extensive structural requirements do not apparently occur in the case of the other glycolytic enzymes. The contrast shown between the results just mentioned and those relative to the structure proteins is extreme. The great similarity existing between the structure proteins of carp and rabbit muscle has been shown in this review. Myosins and actomyosins prepared from mouse, carp, or lobster skeletal muscle, from white or red rabbit muscle, and from rabbit or horse heart muscle are indistinguishable electrochemically (Dubuisson-Brouha, 1953; see Hamoir, 1953a). A change in electrophoretic mobility is detectable only in embryonic muscle (Crepax, 1952). Tropomyosins of various origins seem so far to be identical (Bailey, 1948; see p. 265), and cod actin is very similar t o rabbit actin (Connell, 1954). Rat, mouse, and rabbit myosins examined by various met,hods (electrophoresis, ultracentrifugation, viscosimetry, refractometry) do not show any difference (Miller et al., 1952). Variations do, however, occur. The content in the muscle of stroma proteins amounts t o approximately 16% in rabbit, 10% in elasmobranchs, and 3 % in teleosts (Table 11). Carp myosin sediments a little more slowly than rabbit myosin (see p. 262). Carp and rabbit actomyosins differ slightly in solubility and strongly in ultracentrifugal and viscosimetric behaviors (see p. 256). As, however, fish and rabbit myosins and actins singly behave similarly from these points of view, these properties seem to be due t o a difference in aggregation state. The muscle fiber is thus made of a most remarkable association of extremely constant and very variable components. The main structural component of the skin, collagen, shows also a great constancy of structure throughout the animal kingdom. The differences occurring between mammalian and teleost collagen have been carefully

282

G. HAMOIlt

investigated (see p, 277). Those relative to the elasmobranch cdlageii are iiot h o \ v d l kiiowii; their main interest so far is t o draw our attcvitioii oiic’e more to the definite difference i n protrin composition existing twtivcwi lmny atid cartilaginous fishes. The different variations observed in the case of the tnuscle structure proteins and collagen occur only within iiarrom limits. The chemical strucbture of these proteins appears nearly as invariable as t h a t of the protein hormones.

ACKNOWLEDGMENTS The author ivishes to express his deep gratitude t o Dr. I(.Bailey, who gave valii: h l e advice and criticism and corrected the manuscript. He is grateful t o Dr. W. J. Dyer for several remarks, and t o Mr. A. Uarets and D r . K . H. Gustavson, who commented upon parts of the text. The author’s thanks are also due t o Professor pvl. Duhuisson for facilities put a t his disposal and t o Drs. J. J. Connell, W. Partmann, and J. It. Dingle, who kindly permitted the use of unpublished manuscripts.

REFERENCES Airan, J. W., and Joshi, J. V. (1952). J . U n i v . Boniba.y A21, 69. Amano, K., Bito, M., and Kawabata, T. (1953). R i d . J a p a n . Soc. Sci. Fisheries 19,487. . Amherson, W. R., ErdGs, T., Chinn, B., and Ludes, H. (1949.) .I. Biol. P h e ~ u 181, 405. Anderson, D. N., and E’ellers, C. R. (1952). Food Research 17,472. At.lantic Fisheries Experimental Station, Halifax (1953). Annual Report,. Augustinsson, K. I3. (1948). Acfa Physiol. Scand. 16, 182. Bailey, K. (1937). Biochem. J . 31, 1406. Bailey, K. (1939). Biochem. J . 33, 255. Bailey, K . (1940). Kature 146, 934. Ihiley, K. (1942). Biochena. J . 36, 121. h i l c y , K. (1944). Advances in Protein Chent. 1 , 289. Bailey, K. (1948). Biocheni. .I. 43, 271. Bailey, K., Gutfreund, € I . , and Ogston, A. G. (1948). Biochem. J . 43, 279. Ihiley, K., ant1 Perry, S. V. (1947). BioChiB2. et BiopAys. Acta 1 , 5 0 6 . Ihnga, I., and Szent-Gyorgyi, A. (194142). Studies Inst. M e d . Cherri. Uniti. Szeged 1, 5. I h n k s , A . (1938). J . Sac. Chem. I n d . 67, 124. lhrunowski, T. (1939a). Coinpt. rend. sac. bioE. 130, 1182. H:iranowski, T. (19391.)). 2.physiol. Cheni. 260, 43. I h r e r , R. (1948). B i d . Revs. 23, 159. l h r e t s , A. (1952). Arch. Anat. microscop. Alorphol. e q t l . 41, 305. Bate Smith, E. C. (1!148). Advances in Food Research 1 , 1 . Rate Smith, E. C., and Bendall, J. It. (1949). I . Physiol. (London) 110, 17. Bear, R. S. (1952). Advances in Protein Cheni. 7, 69. Bendall, J . It. (1951). J . Physiol. (London) 114, 71. Beveridge, J . M. R., and Lucas, C. C. (1944). J . B i d . Chew. 166, 547. Hignood, I<. J . , Croka.ert, R., and Hilinski, 15. (1953). H i ( / / .mad. roy. urCd. H c l g . 18, 82.

FISH PROTEINS

283

Bi6rck, G. (1949). A c t a Med. Scand. 133, Suppl. 226. Block, R. J., Horwith, M. I<.,and Rolling, D. (1949). Proc. Soc. E x p t l . Biol. Med. 70, 494. Holk, I,., Goppert, E., Kallius, I<;., arid Liiboseh, W. (1938). “Handbuch der vergleichende Anatomie der Wirbeltiere.” Urban and Schwarzenberg, Berlin. Ijosch, h4. W. (1951). Biochirn. et Biophys. A c t a 7, 61. 13oyer, P. D. (1953). J . Cellular Comp. Physiol. 42, 71. 13rieas, E., and Fromageot, C. (1953). Advances in Pyotein Cheni. 8, 4 . Rrull, I,., and Cuypers, Y. (1954). Arch. intern. physiol. 62, 70. Brull, I,., and Xizet, E. (1953). J . Marine Biol. Assoc. U . K . 32, 321. Buchs, S. (1954). Z . oeryleich. Physiol. 36, 165. Biihn, W. (1940). Z . Zellforsch. zi. mikroskop. A n a t . 30, 323. Colvin, J. R. (1952). C a n . J . Chem. 30, 320. Connell, J. J. (1953a). Biochem. J . 64, 119. Connell, J. J. (1953b). Biochem. J . 66, 378. Connell, J. J. (1954). Biochem. J. 68, 360. Connell, J. J. (1955). Nature 176, 562. Conway, IC. J., and Hingerty, D. (1946). Biochenz. J . 40, 561. Corfield, M. C . , and Robson, A. (1953). Biochem. J . 66, 517. Couceiro, A , , de Almeida, D. F., and Freire, J. R . C. (1953). A n a i s m a d . brasil. cienc. 26, 205. Cout,eaux, R . (1950). Compt. rend. Assoc. A n a t . 37, 80. Crepax, 1’. (1952). Biochim. et Biophys. Bcta. 9, 385. Croltaert’, It. (1953). ‘Contribution B 1’8tude de la B alanine e t de ses compos8s dans les milieux biologiques.” Act,a Medica Belgica, Brussels. Daimler, B. H. (1952). Koiloid-2. 127, 8, 97. Davisoii, J. A., and Richards, A . G. (1954). Arch. Biocheni. and Biophys. 48, 484. Deas, C. P., and Tam, H. I,. A. (1949). J . Fisheries Research Board (‘an. 7, 513. De 1,as Heras, A . R., and Mendez Isla, M . C. (1952). Anales bromutol. ( M a d r i d ) 4, 403. Deolalkar, S. T., and Sohonie, K. (1954). Nature 173, 489. Deuticke, I?. J. (1934). Z. physiol. Chem. 224, 216. Deutsch, H. F., aiid McShan, W. 1%. (1949). J . Biol. Chem. 180, 219. Deutsch, H. F., arid Goodloe, M. B. (1945). J . Biol. C h e w 161, 1. De Vincentiis, M. (1952). Publs. Stat. Zool. iVapoli 23, 214. Dingle, J. It., Eagles, D. I<., and Neelin, J . R4. (1955). J . Fisheries Research Board (‘an. 12, 75. llrilhon, A. (1953). Compt. w n d . 237, 1779. Drilhon, A . (1954). Compt. rend. 238, 940. Dubuisson, M. (1941). Arch. intern. physiol. 61, 133. Ihbuissori, M. (1942). Arch. intern. physiol. 62, 439. I)ubuisson, 31. (1946). Erperientia 2, 258. Iliibuisson, ill. (1950). R i o l . Reos. 26, 46. Ihihuissoii, M. (1953). Bull. ucad. roy. sci. Belg. 39, 35. Dubuisson, M., and Jacob, J. (1945). B u l l . S O C . ray. sci. Lihye No. 3, 145. I)ul,uisso~i-Rrouha,A. (1953). Bull. m a d . roy. sci. Bclg. 39, 121. Duerr, J. I]., and Dyer, W. J. (1952j. 1.Fisheries Research Board Can. 8, 325. Dyer, W . J. (1951). Food Research 16, 522. Dyer, W. J . (1954). “Symposium on Cured and E’rozcn Fish Technology.” Svenskn Institutet for Konserveringsforskning, Gotehorg.

284

0. HAMOIR

Dyer, W. J., French, H. V., and Snow, J. M. (1950). J . Fisheries Research Board Can. 10, 585. Edsall, J. T. (1930). J . B i d . Chem. 89, 289. Elson, D., and Chargaff, E. (1954). Nature 173, 1037. Emery, C. (1882). M e m . accad. sci. zst. Bologna 3, 613. Eriksen, N. (1943). Thesis, Univ. of Washington. FaurB-Fremiet, E. (1937). J . chim. phys. 34, 125. Fearon, W. R., and Foilter, D. I,. (1922). Biochem. J . 16, 564. Felix, K. (1953). I n “The Chemical Structure of Proteins” (G. E. W. Wolstenholme and M. P. Cameron, eds.) p. 151. Churchill, London. Felix, K., Fisher, H., Krekels, A., and Mohr, R. (1951). Z. physiol. chem. 287, 224. Felix, K., and Krekelu, A. (1953a). Z. physiol. chem. 293,284. Felix, K., and Krekels, A. (195313). 2. physiol. chem. 296, 107. Felix, K., and Mager, A. (1937). 2. physiol. chem. 249, 111. Felix, K., Rauen, H. M., and Zimmer, G. H. (1953). 2. physiol. chem. 291, 228. Ferguson-Wood, E. J. (1950). Australian J . Marine and Freshwater Research 1, 129. Feuer, G., Molnar, F., Pettko, E., and Straub, F. B. (1948). H u n g . Acia Physiol. 1, 150. Figikawa, K., and Naganuma, H . (1936-37). Bull. J a p a n . SOC.Sci. Fisheries 6,95. Fisher, H . (1954). Trans. Faraday SOC.60, 295. Florkin, M. (1949). “Biochemical Evolution.” Academic Press, New York. Forbes, R. M., Cooper, A. R., and Mitchell, H. H. (1953). J. B i d . Chem. 203, 359. Fougere, H . (1952). J . Fisheries Research Board Can. 9, 388. Fujimaki, M., and Kojo, K . (1953). Bull. J a p a n . Soc. Sci. Fisheries 19, 499. Geiger, E . (1948). Fortschr. Chemie org. Naturstofe 6, 267. Gralen, N. (1939). Riochem. J. 33, 1342. Greene, C. W. (1913). A n a t . Record 7, 99. Guba, F. (1943). Studies Znst. Med. Chem. Uniu. Szeyed 3, 40. Gustavson, K. H. (1942a). Soensk K e m . T i d s k r . 64, 74. Gustavson, K.H. (1!342b). Biochem. Z . 311, 347. Gustavson, K.H. (1!>49). Advances in Protein Chem. 6, 353. Gustavson, K. H. (1950). A c t a Chem. Scand. 4, 1171. Gustavson, K. H. (1953). Svensk K e m . Tidskr. 66,70. Gustavson, K. H. (1054a). A c t a Chem. Scand. 8, 1298. Gustavson, K. H. (1954b). A c t a Chem. Scand. 8, 1299. Gustavson, K.H. (1955). Nature 176, 70. Haan, A. M. F. H. (1953). Biochim. et Biophys. Acta 11, 258. Hamoir, G. (1947). Ezperientia 3, 498. Hamoir, G . (1949). BUZZ.SOC. chim. biol. 31, 118. Hamoir, G . (1951a). Riochem. J . 48, 146. Hamoir, G. (1951b). Biochem. SOC.Symposia, Cambridge, EnvI. No. 6, 8. Hamoir, G. (1951~). Biochem. J . 60, 140. Hamoir, G. (1953a). Discusszons Faraday Soc. No. 13, 116. Hamoir, G. (195313). Nature 171, 345. Hamoir, G. (1054). “Symposia on Cured and Frozen Fish Technology. Svenska Institutet for Konserverinbsforskning, Goteborg. Hamoir, G. (1955). Arch. intern. physiol. et biockim. 63, suppl. Harris, R. S. (1951). I n “The Enzymes.” (a. 13. Sumncr and K. hlyrlmck, r d s ), Vol. I, Part 2, Academic Press, New York. IIasselbnch, W., and Schneider, C. (1951). Riocheni. %. 321, 161.

FISH PROTEINS

285

Helwig, H . L., and Greenberg, D. M. (1952). J . Biol. Chem. 198, 695. Henrotte, J. G. (1952). Nature 169, 968. Henrotte, J. G. (1954). Arch. intern. physiol. 62, 14. Huxley, H.E. (1953). Biochim. et Biophys. Acta 12, 387. Huxley, H.E., and Hanson, J. (1954). Nature 173,973. Huys, J. V. (1954). Arch. intern. physiol. 62, 16. Ihle, J. E. W., Van Kempen, P. N., Nicrstrasz, H. F., and Versluys, J. (1927). “Vergleichende Anatomie der Wirbeltiere.” Springer, Berlin. Irisawa, H., and Irisawa, A. F. (1954). Science 120, 849. Jacob, J. (1945). Bull. S O C . ray. sci. LiBge No. 4, 242. Jacob, J. (1947). Bzochem. J . 41, 83. Jacob, J. (1948). Biochem. J . 42, 71. Jacquot, R.,and Creac’h, P. V. (1950). Les protides dupoissonet leur valeur alimentaire. Congr. intern. d’etude sur le r61e du Poisson dans l’alimentation. Inst. Oceanogr., Paris. Jaisle, F. (1951). Biochem. 2. 321, 451. Jean-Blain, M. (1948). “Ies Aliments d’origine animale destines B l’homme.” Vigot, Paris. Johnson, P., and Landolt, H. R. (1950). Nature 166, 430. Johnson, P., and Landolt, H. R. (1951). Discussions Faraday SOC.No.11, 179. Jones, N. R. (1954a). Biochem. J . 66, xxii. Jones, N. R. (1954b). Biochem. J. 67, xxiv. Kashiwada, K. (1952). Bull. Japan. Sac. Sci. Fisheries 18, 13. Kawnbata, T. (1953). Bull. Japan. SOC.Sci. Fisheries 19, 505. Kerekjarto, 13. V. (1952). 2. Naturjorsch. 7b. 94. Khan, M.R . (1952). J . Fisheries Research Board Can. 9, 393. Kishinouye, K . (1921-23). J . Coll. Agr. Imp. Univ. Tokyo 8 , 293. Knoll, P. (1889). Sittber. Akad. Wiss. Wien. Math. naturw. Kl. 98, 456. Knoll, P. (1891). Denkschr. K . Akad. Wiss. Wien Math. naturw. K1. 68, 633. Korzuev, P. A. (1952). Uspekhi Sovremennot Biol. 33, 391. Kossel, A . (1928). “The Protamins and Histones.” Longmans, Green, New York. Krebs, E . G. (1954). Biochim. et Biophys. Actu 16, 508. Kroepelin, H. (1929). Kolloid-2. 47, 294. Kruger, P. (1950). 2. Naturjorsch. 6b, 218. Kruger, P. (1952). “Tetanus und Tonus der quergestreiften Skelettmuskeln der Wirbeltiere und des Menschen.” Akademische Verlagsges., Leipzig. Ladd Prosser, C. (1950). “Comparative Animal Physiology.” Saunders, Philadelphia. I d i , K., and Carroll, W . It. (1955). .Vature 176, 380. Lankester, E:. R. (1872). Proc. Roy. Sac. B21, 70. 1-ansimaki, T.A. (1910). Anat. Hefte 42, 251. Laurent, P. (1952a). Con~pt.rend. sac. bid. 146, 1521. Laurent, P. (1952b). Conipt. rend. S O C . biol. 146, 1523. Lavocat, A , , and Arloing, S. (1875). MCm. acad. sci. Toulouse 7, 177. l,awrie, It. A. (1950). J . .Igr. Sci. 40, 356. Lawrie, R. A . (1952). N a t u r e 170, 122. 1 aurie, R. A. (1953:~). Riochem. J . 66, 298. Imvrie, 11. A . (195311). Riochem. J . 66, 305. Imntierg, TI., itrid Legge, J. W. (1919). “IIemntiri Compountls :ind Bile pigments.” Interscience, New York.

286

G . HAMOIH

Lepkovsky, S. (1929-30). J . Biol. Chein. 86, 667. Longsnorth, I,. G., and MacInnes, D. A. (1942). J . Gen. Physiol. 26, 507. Madsen, N. B., and Cori, C. F. (1954). Riochinr. et Biophys. A c f a 16, 516. Maetz, J. (1953a). C‘onipf. rend. soc. biol. 147, 204. Maet.2, J. (1953b). Conipt. rend. sac. biol. 147, 207. Marsh, B.B. (1052). Biochitn. et Biophys. Acla 9, 127. Wlartin, F. (1925). Bull. soc. chini. belg. 34, 8 . Maser, J. (1950). Zool. Jahrb. 70, 60. Matsuura, F.,and Hashimoto, K. (1954). Bull. J a p a n . Soc. S c i . Fisheries 20, 308. Matsuura, F., and Hashimoto, K . (1955). Bull. J a p a n . Sac. Sci. Fisheries 20, 946. hlatsuura, F., Baba, H., and Mori, T. (1953). Rirll. J a p a n . Soc. Sci. Fisheries 19, 893.

Matsuura, F., Yamada, S., and Hashimoto, I<. (1955). Bull. Japan. Sac. S c i . Fisheries 20, 951. Matuura, F., Kogure, T., and Fukui, G. (1952). Bull. J a p a n . Sac. Sci. Fisheries 17, 359.

McLaren, A. D., and Waldt,, I,. (1952). Biochint. el Biophys. A c f a 9, 240. Menon, K . It. (1952). J. L’niv. Bonibay B20, 19. Miescher, F. (1897). “Statistische und biologische Beitrage ziir Kenntniss von Lehen des Rheinlachses im Siisszwasser.” Leipzig. Mihalyi, E. (1950). Enzymologia 14, 224. Miller, G.I,., Golder, R. H., Eitelman, 15. S., and Miller, E. F:. (1952) Arch. B i o cheni. and Biophys. 41, 125. Mitchell, H. H., Hamilton, T. S., Steggerdn, F. R . , and Bean, H . W. (1945). J . Biol. Cheni. 168, 625. hlommaerts, W. F . H . M . (1950). “Muscular Contraction.’’ Intcrscience, New York. Momrnaelts, W. 17. €1. bl., and Parrish, R . G. (1951). J . BioZ. (‘heni. 188, 545. Monier, It., and Jutisz, M. ( 1 9 5 4 ~ ) . Biochin,. et Biophys. Acta 14, 551. Monier, It., and Jutisz, M. (1954b). Biochini. et Biophgs. Acta 16, 62. Moore, I). H. (1945). -1. Biol. Chevi. 181, 21. Munro Fox, H. (1954). I\iat7tre 173, 850. Neuman, R. 12. (1949). Arch. Biocheni. 24, 289. Neuman, R.E., and Logan, & A. ‘I. (1950). J . Rial. ( ‘ h e m . 184, 299. Nickerson, J. T. R . , Goldblith, S. A , , and Proctor, €3.12. (1950). Food. Y’cchnol. 4, 8.1, Xikkila, 0. E. (1951). Suornen keniistilehti 24, 54. Nikkila, 0.I<., arid Iinko, It. It. (19548). Food Rrsearch 19, 200. NikkilB, 0. E., and Iinko, 11. 11. (19541)). Buonien Keinistilchti B27, 37. Nikkila, 0. E., and Linko, R. It. (1955). Biocheni. J . 80, 242. Nishi, S. (1938). I n “Handhuch der vergleichende Anatomie dcr Wirbeltierc.” Vol. 5 , p. 351. Urban and Schnarzcnberg, Berlin. Norris, E. R . , :ind ISlnm, D. W. (1940). J . Biol. (*hem. 134, 443. Norris, I<. It., :~ntlhlathies, .J. C. (1953). J . Hid. ( “ h e t i / .204, 673. Oya, J. (1928). J . S c i . F i s h c i ~ i f ~IS’OC. s YO.8 . Partmnnn, W. (1952-53). A r c h . Fischereiwiss. 4, 40. Partmarin, W. (1954a’l. “Symposium on Curod a n d Frozen I M i Technology." Svenska Institutet fiir Iiortservc~iri~sforsl~~iing, Giitelmry. l’;wtm:inn, W. ( 1 9 5 4 ) ) . 2 . Lebensr/i. I;nlersitch. I ( . l ~ ’ o / w h99, . 3.41. I’nrtmann, W. (1055). Rioehetn. 2. 326, 260.

FISH PROTEINS

287

I’cdersen, K. 0. (1!)50). (’old Spring Harbor S y m p o s i a Qirant. R i d . 14, 1-10, I’crry, S.V. (1952). Biochirn. ct Bioph?la. Acta 8 , 483. Perry, S. V. (1953). Biochcni. .I. 66, 114. Phillips, D. AI. P . (1955). Riochcm. J . 60, 403. I’olonovski, M. (1952). “Biochimie M6dic:tle.” Masson, Paris. I’ortzchl, ]-I., Schrnnim, G . , itnd Weber, II. H. (1950). 2. iVaturJorsch. 6b. 61. Proctor, 13. l<;.,Kickerson, J. T. It., and Goldblit,h, S. A . (1950). Refrig. Eng. 69,

375. Itaffel, S., I’ait, C. F., and Terry, M . C. (1940). J . Zmniiinol. 39, 317. Iianke, I<.,and Bramstedt, F. (1954). Arch. Fischereiwiss. 6 , 43. Itanvier, 1,. (1874). Arch. physiol. norm. pathol. 2, 1. Itnuen, 1%.If.,Stnmm, W . , and Felix, I<. (1953). 2. physiol. cheni. 291, 275. I
262, 425. Snellmnn, O., and Erdos, T . (1948). Biochim. et B i o p h y s . A c t a 2, 650. Snellmnn, O., and Tenon, M. (1948). Biochim. el Biophys. A c t a 2, 384. Srioke, J. I<., and Seurath, H. (1950). J . B i d . Chenz. 187,127. Snow, J. M. (1950). J . Fisheries Research Board (’an. 7 (lo), 599. Sorm, F., and Sormova, Z. (1951). Collection czech. Chem. Conimuns. 16,207. Sprissler, G. P. (1942). Dissertation, Catholic Umiv. of America. Stanshy, M .E. (1954). Food Research 19, 231. Steinbach, H.R. (1949). J . Cellular Conzp. Physiol. 33, 123. Stern, J. A , , and Lockaert, E. E. (1953). J. Fisheries Research Board C a n . 10, 590. St,irling, W. (1886). (‘On Pale and Red Muscles in Fishes,” 4th Annual Report. Fisheries Board of Scotland, Appendix F, p. 9. Strittm:ttter, C. F., Ilall, E. G., and Cooper, 0. (1952). Biol.B u l l . 103, 317.

288

G . HAMOIR

S u h h ltao, Ci. S . (l!MS). l’hysico-chemical investigations on the muscle protehs of some commerc:i:il species of fish caught in North Enst, Scot t)ish {voters. Thesis, Univ. of hherdrcn. Svedberg, T., n r i d l’edersen, I<. 0. (1940). “Thc Ult,mcentrifuge.” Clnrendon, Oxfortl.

Svensson, 1-1. (1941). J . Biol. Cheni. 139, 805. Szent-Gyorgyi, A. (1943). Studies Inst. Med. Chem. Uniu. Szeged S. 76. Takahashi, T., and Takana, T. (1953). BuZE. J a p a n . SOC.Sci. Fisheries 19, 603. Tarr, H. I,. A . (1948). Food Tech.nol. 2, 268. Tarr, H. I,. A . (1950). J . Fisheries Research Board (’an. 7,608. Tarr, H. L. A. (1952). Fisheries Research Board Can. Progr. Repts. Pacific Coast Stas. No. 92,23. Tarr, H. L.A. (1953). Nature 171, 344. Tarr, H.1,. A. (1954). Food l’echnol. 8, 15. Tam, H. L. A. (1955). Biochem. J . 69, 386. Tarr, H.L. A., and Bisset, H. M. (1954). Fisheries Research Board Can. Progr. Repts. Pacijic Coast Stas. No. 98, 3. Tatarskaja, R. I. (1952). Biokhimiya 17, 598. Tatarskaja, R.I., Kudrjashov, J . B., and Fajn, F. S. (1954). Biokhimiya 19, 229. Theorell, H. (1932). Biochem. 2. 262, 1. Theorell, H. (1934a). Biochem. 2. 268, 46. Theorell, H . (193413). Biochem. 2. 268, 55. Theorell, H . , and Ehrenberg, A. (1951). Acta (‘hem. Scand. 6, 823. Tristram, G . R. (1949). Advances in Protein Chem. 6, 83. Tsao, T. C . (1953). Biochim. et Biophys. Acta 11, 236. Tsao, T.C., and Bailey, K . (1953). Discussions Faraday Sac. No. 13. 145. Tsao, T. C., Bailey, K . , and Adair, G. S. (1951). Biochem. J . 49, 27. Tsuchiyu, Y., Hata, M., Asano, M., Takahashi, I., Nomurn, T . and Suzuki, Y. (1953). B d l . J a p a n . Sac. S c i . Fisheries 19, 513. Uematsu, H. (1951). Arch. histvl. Japan. 3, 1. Uematsu, 11. (1954). Folia A n a t . Japon. 26, 51. Van Wyk, G . F. (1944). J . Soc. Chem. I n d . 63, 367. Velick, S. F., iirid ‘IJdenfriend, S. (1951). J . Biol. Chem. 191, 233. Velick, S.F., and Udenfriend, S. (1953). J . Riol. Cheni. 203, 575. Vialleton, 1,. (1902). Compt. rend. Assoc. Anaiom., p. 47. Waldschmidt-Leitz, E., and Kofranyi, E. (1035). Z . pkysiol. Cheni. 236, 181. Waldschmidt-Leit>z,E., and Pflanz, I,. (1953). 2. physiol. Chem. 292, 150. Walker, 13. S., Boyd, W. C., and Asimov, I. (1952). “Biochemistry and Human Metabolism.” Williams & WilkinA, Baltimore. Weber, H. H. (1934). Ergeb. Physiol. 36, 109. Weber, H.H., and Meyer, K. (1933). Biochenz. 2. 266, 137. Weber, H. H., and Portzehl, H. (1952). Aduances in Protein Chent. 7, 162. Wolff, R., and Rrignon, J. (1954). Bull. sac. chin].biol. 36, 1125. Wyman, ,J., Jr. (1948). Advances i n Protein Chem. 4, 407. Zahn, It. K., and Stamm, W. (1954). Naturwissenschaften 41, 95. Zoethout, W. D., and Tuttle, W. W. (1943). “Texthook of Physiology.” Moshy, St. Louis.

The Sea as a Potential Source of Protein Food BY LIONEL A. WALFORD AND CHARLES G. WILBE11 Fish und Wildlife Service, United Slates Depurtment of the Interior, Washington, D.C . and Chemical Corps Medical Laboratories, Arrriy Chewiical Center, M d . CONTEKTS

I. World Protein Problem.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Proteins in Marine Organisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Algae.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Invertebrates.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Fish.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Marine Mammals. .............................................. 111. Variations in Protein Content. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Geographical.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Anatomical.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Seasonal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The Biological Value of Marine Proteins., . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

289

303 303 304 308 312 312 312 312 312 313 315

I. WOI~LD PROTEIN PROBLEM Among the inany solutions that have been suggested for supplying the growing population of the world adequat.ely and cheaply with required protein food, the one advanced more frequently than most in the popular press and by many thinking scientists is that the sea is a great virgin area remaining t o be exploited for man’s needs. It contains all the soluble fertilizer and all the trace chemicals required b y living organisms. It occupies 2-56 times as much of the surface of the earth as does the land. It averages 2.38 miles in depth, whereas the soil averages only a few inches. Yet only a small fraction of the total produce man eats comes from the sea. Fishermen take only about 26 million metric tons of fish, crustacea, and mollusks-an amount which is less than half the annual world production of meat and eggs and about one-eighth the production of milk. The harvest of foodstuffs from all the oceans is only about X Z Oas much per acre as that from all the land and the edible portion of the yield of the seas is about 0.9 % of man’s total food supply. If the sea is vast and fertile why does i t yield so little? T o begin with, it is not evenly fertile any more than is the land. Productivity varies widely from one area to another, ranging from teeming luxuriance t o almost barren desert. In any given place it varies seasonally in a pattern which is subject to sporadic, great fluctuations. Owing to 289

290

L. A . W’ALFORD A S D C‘. G. WILBEH

sampling problems, the total productivity of all the oceaiis is exceedingly difficult t o measure precisely. Yevertheless, inariiie biologists geiierally agree that it is about the same as that of all the land; i.e., 011 the order of 1 . j x lo1‘’tons of organic carbon are produced yearly. They also gcnerally agree that sea water is not a rich medium for the production of plaiits and tltiirnals. On this question, l<edficld (1033) says, “The cycle of life is such that the greater part of the production and destruction of orgaiiic matter is dotie by relatively siiiall populations of floating aiiinials mid plants, highly dispersed in the water atid he.rtc.e difficult to harvest. Succgessful Iiariwt is limited for the most part to fishes whose habits of aggregatiolt :ttld niigratioii and whose size facilitate their capturc. These owur late it1 the food chain a t i d accumulate but a small fraction of the over-all prothic4ivity.” I t is an cweccli~iglyperplesiiig :irid elusive problem t o get a quaittitatively sigiiificaiit picture of the many populations of diverse species that inhabit any given area in the sea. Speries, and eveii races within species, differ in habits, in geographic distribution, in life cycle, in rate of production, in physicd characteristics. Their population numbers oscillate in differing patterits. Consequently sampling errors are high, and it is difficult to integrate thr samples knowledgeably for estimating total produrtiori in an area per unit of time. Nevertheless, this has been attempted for various areas. For example, Krey (1953) has compared sonie estimates with agricultural produc%ioiias follon-s: Yearly Production of Organic Su\xd:iiice it. .1qt iciclttcre 7t1 Gertrta,iy, 1937

Usable (g./m.2)

Oats Wheat rot:kt OCb

170 180 180 680

I3ect sugar

Total (g./m.21 380

160 520 920

13. Tropical proditciion

Caiie sugar Uananas AI:tnicli

:3:3GO 5000 7250 (

’. Ocrari prod cicl I 011

Sr:i gi:iss i i i 1):Liiisti \raters f’l:lnktoI1: West Atlantic (23” to 11’) Tropical Atl:tiitic Earents Sea Gulf of hlnirie Oresund Isefjord (I3enmitrk)

S10 :350-‘.)00 700

130-810 100

120 GOO

THE SEA -48 h POTENTIAL SOURCE O F PROTEIN FOOD

29 1

Sielsen (1951), working from the Danish research vessel Galathea 011 its voyage in the eastern part of the Atlantic Ocean from Lisbon to Capetoivn, estimated annual rates of production of 18 g. per square meter in the vicinity of thc Canary Islands, 90 to 180 g. per square meter in parts of the tropical Atlantic, and around 1500 g. per square meter in the Uenguela current. As for various types of organisms, Harvey (1950-51), gives the following estimates for the mean quantities occurring throughout the year in the English Channel: Type of Organism

Wet Weight of Tissue (Tons per square mile)

Phytoplankton Zooplankton Pelagic fish Demersal fish Ifpi- and in-fauna

57.6 22.4 25.6 16 256

Fish production varies even 011 dif'ferent parts of a single bank. Clyde Taylor, of the U. S. Fish and Wildlife Service (1955, personal communication), estimates roughly the total population of bottom fishes in different areas of Georges Bank, Gulf of Maine, in tons per square mile, as follow: 18.0 (area G), 10.5 (area H), 27.9 (area J); 9.9 (area M); and 4.8 (area S ) . From a far distant and evidently richer part of the world, Wheeler (1953) surveyed the commercially important bottom fishes of a number of banks in the Mauritius-Seychelles area. He denionstrated that measures of abundance of these fishery stocks depend on the kind of gear fished. Thus fishing with a series of drifting lines arranged in a row 40 feet wide yielded catches ranging from 2.1 to 48.4 tons of bony fish per square mile plus 5 to 81.3 tons of sharks. Fishing with nets on an offshore bank (the Soudaii Hank) indicated that the annual yield would become stabilized a t around 83 tons per square mile after a year of intense fishing. Thus the production of usable animal protein in thc sea varies widely from one area t o another and covers about the same range of variation as pasture land, which supports from 5 or less tons per square mile up to around 130 (Finn, 1954). It would seem that the most reasonable way to increase the exploitation of the sea mould be to open up virgin areas of unusual productivity. However, most of those which are knowii, for example, the Bering Sea and the Southern Ocean, are not easily accessible to port facilities and markets. The accessible productive parts of the sea are scarcely virgin territory. For wherever people live by the sea, they have exploited its resources, not with uniform intensity to he sure, but they have exploited them, even in the most primitive countries; and they have done this for a long time. In many areas they have reached the point where the best hope of increasing

292

Id. A. WALFORD AND C. G . WILBER

harvests is not so much in expanding into the unknown as in developing and applying a science of husbandry to the use of the known. So it is that people who live by the sea have come to face the same kind of problem as have those who live by the land. But whereas agriculturists have gone very far in developing a science of husbandry, fishery people have only just begun, and theirs is an enormously more difficult task, because they are more severely limited in their choice of possible techniques than are agricwlturists. For example, it is unlikely that we could make muchprogress i n selective breeding of marine organisms to improve stocks. Sessile animals like oysters might be bred t o produce strains that are disease-resistant or rapid-growing, but probably nothing of the sort could be done for mild, motile creatures like herring or cod or lobsters or shrimp. We cannot expect to control any of the diseases or pests that plague all marine animals and plants-the useful ones as well as the others-or reduce the abundance of predators. We don’t foresee furnishing veterinary service to sick whales; or modifying the spawning season of fishes to fit in with abnormal marine weather conditions. Nor is there any hope of inducing the desert areas of the ocean to flourish by fertilizing them. We can cultivate a few kinds of organisms that live very close to shore, like oysters and other shellfish among animals, or red algae among plants; and we can cultivate fishes and shrimps under certain special conditions in salt-water enclosures-in some oriental countries this is a highly developed ar t ; but these are the limits of any similarity between husbandry which farmers can practice and that which fishermen must learn to practice. The farmer’s principal resource is the environment, that is, the land, and scientific farming consists of controlling the ecology of the environment by planting appropriately profitable flora and fauna which will later be harvested, and in the planting, spacing them for maximal growth and survival. There is scarcely anything quite comparable that is possible in the sea. The fisherman’s principal resource is not the environment but the wild populations of creatures that happen to inhabit it. I n the sea, for the most part, man cannot control the environment, but only his harvest, that is t o say, his choice of wild resources to harvest, and his rates of harvesting them. It would seem that there is a very wide range of choices; for there are about 24,000 species of fishes in all the seas of the world, and many more kinds of invertebrates than that number. However, not all of these species are usable for human purposes. I n order to be usable, a species must have certain qualities, which are determined largely by economics and by various powerful human sentiments, such as aesthetics, which vary widely among different cultures. To be significantly useful, a stock must be : Abundant, i.e., voluminous, in the sense of being capable of yielding large volume.

THE SEA AS A POTENTIAL SOURCE OF PROTEIN FOOD

293

Aggregated in dense concentrations, or capable of being easily concentrated. Regular enough in habits that fisherrneii can depend on finding them year after year a t about the same places and times. Accessible to fishermen; that is to say, not so deep as to be beyond range of fishing vessels and their gear, not inhabiting unworkable ground, not so elusive as t o make the cost of catching prohibitive. Palatable or nutritious, preferably both. Firm-fleshed, so that they keep in good condition during the time of transportation from fishing grounds t o port. Amenable to preservation by such methods as salting, pickling, smoking, freezing, canning, or able to be kept alive in tanks until needed. Standards of these qualities vary widely among peoples of different cultures and different economic condition; and by any standards, qualities vary widely among different species of organisms. A very small part of all the species of vertebrates or of invertebrates is put to human use. The great majority of commercially useful fishes occur in less than 3 % of the 300 or 400 families which systematists recognize. About a third of all sea fishes caught, as reported in available statistics of the world, are herrings and their relatives (sardines, menhaden, and anchovies); about a quarter are cod fishes and their relatives (hakes, haddock, and pollack) ; about 4 % are flounders; about 6 % are tunas and their relatives (bonitos and mackerels). The remainder, a little less than a third of the catch of sea fish, is made up of rockfishes, bass, snappers, weakfishes, and a miscellaneous assortment of other groups. About 10% of the world’s harvest of sea food, that is to say about 2.7 million tons, consists of invertebrate animals. Of this quantity, about half are shrimps, crabs, and lobsters; about 36 % are mussels, oysters, clams, and scallops; about 5 % are squid. The rest are miscellaneous varieties like sea cucumbers, snails, abalone, sea urchin, and so on. Why is the selection of species restricted to so few groups? Because it is these which fishermen, from long experience, know how to catch; it is these with which people are familiar and which they are willing to buy; and as far as fishermen can determine their catches, they select these in preference t o others because they know they can sell them. But is this handful of families of fishes, these few invertebrates, all that is usable in the sea? .ire there not some unknown massive stocks of marine organisms which Iishermen could harvest profitably if they knew about them, which could contribute significantly to the human food supply? Perhaps fishermen are not adventurous enough. Perhaps they don’t go far enough out to sea. I t is true that most fishermen in most places w e more or less tied t o the land, being limited by small vessels and simple equipment t o a n operating radius of relatively few miles. The vessels of

294

L. A . WALFORD AND C . G . WILBER

highly advanced mechanized fisheries can go much greater distances; nevertheless, they too are tied to land. Such factors as the willingness to spend long periods a t sea and the high cost of far-flung operations limit the range of oceanic fisheries and selection of their fish t o a few species like tuna and cod 'i\ hich have special qualities, therefore relatively precious value and a high-paying market. But more than two-thirds of all the known species of marine fishes, aiid most of the larger usable invertebrates, are also bound to land in one way or another, a t least during part of their lives. Thus although cod and flounders may be caught hundreds of miles from port, they are limited to the plateaus and slopes of continental shelves, and in summer they tend to migrate towards shore and polewards. The continental shelves of northern seas are very large; elsewhere they are much narrower. Consequently, most species that are actually or potentially of commercial value spend their lives well within 20 miles or so of land. This is true not only of sedentary bottom-dwellers but of pelagic, surface-swimmers as well, including those whose habitat is usually referred to as oceanic. In general, these shore-fishes also tend to move landward in summer. Moreover, the young of many species, including shrimps, spend the first months of life close to the beaches, in estuaries, and sloughs, even moving far up into fresh water. As they grow larger they move out to sea, but not often, if ever, do they stray beyond the continental shelf, for the spawning grounds must be located in such a particular relation t o caurrents as to provide for transport of the young to their shoreward nursery grounds. In fact, nonc of the known commercially valuable food fishes or iiivertebrates is exclusively oceanic ; all make a t least occasional visits close inshore. Consequently, even the most primitive fishermen have a t least some acquaintance with the food organisms of a much larger area than is encompassed by the limited radius of their small local fisheries. They may see only an occasional specimen belonging to a very large stock; they may not know where to seek the main body of the stock or be equipped to reach it. They may even have no name for these occasional visiting species; but from long-continued, day-to-day fishing experience, they do know of their existence. And of course scientists know about most of the species from the collections which have been preserved over more than two centuries in museums and described by figures in literature. Therefore, it is quite clear that ignorance of what is in the sea plays :L rather minor part in limiting exploitation of the sea resources. Aesthetic standards are a major factor in determining the acceptability of food from the sea. Being often, if not usually, irrational, they are often exceedingly difficult for people of different cultures to understand ; and they are so variable from one culture to another that no universal formula

THIS SIC.\ .\S .1 POTENTI.AL SOUHCE O F PltO’l’lCIK FOOD

293

can be given for dealing with them. Such food prejudices arr not peculiar to primitive peoples. Americans think of themselves as 011c of the “advaimd” peoples. They live on thc edge of a region containing one of the largest fishery resources of the world, the Atlantic herring. This is one species that biologists consistently agree is far underesploitcd. Small, immature herring are used for canning. Large herring are as good and nutritious as the small ones. They wnuld make an excellent canned product; hut Americans have such a prejudice against the word “herring” that there is little market for these useful fish under that name. They are likewise prejudiced against eels, although these fish are superbly rich-fleshed, arid they are plentiful. 1-nfortunatrly they are repellent t o i I m c r i ( ~ aberause i~ of some fancied resemblarice to snakes. Similarly, Aniericmis nrglect periwinkles, squid, octopus, sea urchins, aud, of course, sharks. English people consider periwinkles a delicacy. Americans would not dream of eating one. Englishmen, on the other hand, eschew clams. One of our most precious fish is the albacore, a mild-flavored, whitemeated tuna. We like albacore. Japanese people consider it a most inferior fish, fit only for the Anierican trade. Americans like frozen fish fillets; Japanese people don’t. They prefer to buy their fish fresh and whole. T o Asiatic and Oceanian peoples, seaweeds are essential for flavoring food. Americans use seaweed extensively in manufacturing processes but are quite inimical to the idea of using the fronds themselv.es in their cuisine. Such food discriminations are found in all cultures; and it would be foolish to plan on tapping any new, exotic marine food resources, no matter how abundant they may be, without taking seriously into account the problem of changing human tastes, prejudices, taboos. Another group of influences which tend to limit the exploitation of the sea might be appropriately called the amateur conservationist complex. Much as people may talk about the sea frontier as our great resource for solving human food problems, they almost universally recoil from the idea of utilizing the sea resources fully, for fear of exhausting the supply; and almost universally are opinionated about the conditions of the fishery stocks. The widespread idea is that fish are never as plentiful as they once were, that they are getting scarcer every year-and alrnost universally people are sure that they know exactly what iieeds to be done to restore abundance. Many of these preconceptions (*oiicerii the techniques of caatching fish. For example, an idea commonly accepted, usually without supporting facts, is that nets are always harmful compared with hook-and-line; another is that purse seines are more destructive than other nets. There are many such notioiis: notions concerning times and places to fish; notions prejudicial to particular groups of people-to commercial fishermen, for example; prejudices against certain uses of fish-manufacture into meal is the favorite

296

1,. A. WALFORD AND C. G . WILBER

among thew!. Siwh preconceptions relating to conservation often bear no relation to sc~ientilic~ husbandry, and in many situations they do more to obstruvt than to forthcr the ideal of full utili7,at’ion. Thew are strong human sentiments that limit the harvest of the seathat determine the kinds of creatures that will be harvested and the times and the plac.es and the manner of harvesting. There is another, even more important, complex of influences; this is in the cold, hard realm of Economics. It is a costly, risky business to exploit the sea. Vessels and fishing equipment are expensive and vulnerable. Fishing is a hard life. Men are fishermen in order to earn a living, and they remain fishermen only so long as it pays better than anything else they might do. Fishery products must compete in the market with other protein foods, with meat, eggs, cheese, pulses. People buy fish of their choice only so long as the price is acceptable; when it becomes too expensive, they buy other foods. Even though the price is acceptable, they buy only as much as they want. Further reduction in price does not necessarily induce them to buy. One of the many problems that trouble Americans engaged in fishery industries is that it is hard to get risk capital to finance new vessels and equipment. Ranks are extremely reluctant to lend money on even the most conservative kinds of fishery ventures. This is one of the reasons why our fishing industries are characteristically backward and unenterprising. In other parts of the world, particularly in some Oriental countries, people with capital do invest in fishing vessels and they do lend money to fishermen to tide them over from one season to another, but they minimize their risk by imposing extremely onerous terms. Consequently, their fishermen never get out of debt and spend their lives in bondage hardly better off than slaves, Wherever this pattern obtains, fishery industries remain a t primitive levels. I n many areas, important increase in the harvest of the sea is possible only by employing more fishermen. This is easier to say than to do, for the fact is that in several parts of the world the number of fishermen is not keeping pace with the growth of population. I n the IJnited States, fishermen have lately been abandoning their occupation a t the rate of about 5000 a year t o go into other, more comfortable, or better paying occupations. A similar trcnd seems to be in progress in Sewfoundland, in Ireland, in the Setherlands, in India. In India, fishermen are close t o the bottom of the caste hierarchy. They are wretchedly poor, illiterate, conservative. Those among them who are ambitious try to elevate their status by finding more respected employment as cooks, water carriers, porters, domestic servants, and so on. How can men be induced to choose to be fishermen? How ran fishing be

THE SEA AS A POTENTIAL SOURCE O F PROTEIN FOOD

297

made into an attractive occupation without pricing fish out of the market? These are difficult problems, but they had better be attacked while the search goes on for new sources of sea food. The Food and Agriculture Organization has just published a (*hartshowing the geography of fishery resources. r‘ndcrexploited fishery stocks aw grouiid fish on the large coiitinental shelf areas of the I3ering Sea and Gulf of Alaska, and the mast of Patagoriia; also reef fishes throughout the ItidoI’acific region from hlladagascar to the farthest eastern reaches of Polynesia; tuna fishes in a xone rich in plankton arid nutrients near the Equator i n the Pacific and probably also in the Atlantic; and herring-like fishes around all voasts all over the world. If all these fishery stocks were fully utilized, they would yield about five million tons, u7hic.h is about 20% of the present world production. Howeyer, not many of these stocks arc located coiiveriiently close to fishing ports, market facilities, population centers; and that is one of the main reasons why they are underexploited. We have been talking about coiiventional kinds of fishery organisms, which are familiar to all maritime people, such as mackerels, flounders, basses, shrimps, oysters, arid so on. What about the dreams of feeding the world from the sea? What are the possibilities of increasing by many fold the producation of food by using some kind of sea creatures quite beyond present vommercial experience? Beyond the continental shelf are about 7000 species of fishes which live far below the surface of the deep sea outside the present range of fishermen. Among these are the bristlemouths arid lantern fishes, which are evidently more iiumerous than any other kind of fish, even more numerous than the herrings. They usually live beyond continental shelf influences, never take baits, are never caught in conventional nets or traps, and yet are widely distributed in the deep parts of all the oceans. They live in the community of plankton which concentrates in layers that move towards the surface by night aiid toward the deep by day. Abundant and ubiquitous though these fishes are, nutritious though they may be, they seem never to conrentrate in dense aggregations; arid what with the technical difficulties of reaching them and catching them in large quantities, the chances of developing any commercial fishery for them are not promising. It does not appear likely therefore that the supply of animal protein could be vastly increased by increasing the harvest of sea fishes. On the other hand, there has been much speculation in recent years about the possibilities of harvesting the plankton, i.e., the assemblage of plants and animals which drift more or less passively in the water aiid comprise the largest bulk of the organic material in the sea. Harvey gives the daily production in grams of dry organic matter under a square meter of sea surface in the

298 L. A . W A I 9 0 R D AND C . G . WILBElt

FIG.1. Marine fisheries of t h e world showing areas of established fisheries, those those intensively exploited, and those underexploited or entirely neglected. A: Tuna; B: Clripeoid; C : Percomorph-as reef stocks; D: Gadoid; E: Crustacea; F. Redfish (From Improving the Fisherics Contribution t o World Food Supplies, F.40 Fisheries Bulletin. Yo1 6. S o 5.)

THE S I S I AS .2 POTENTIAL SOURCE O F PROTEIN FOOD

299

English Channel as follows: 1’11 yt oplari k t on Zooplankton Pelagic fish Bottom fish

0.4-0.5 0.15 0.0016 0.001

Thus the volume of the zooplankton is vcry niuch greater than that of the fishes. Moreover, it is rich in the same nutrieiits that orrur in fishes and might make satisfactory raw material for such products as protein flour, animal feeds, and fertilizer. However, Jackson (1!%4), in reviewing the engineering and economic problems, estimated that the cost of producing a ton of dry plankton meal by each of four methods that have been proposed, would range from $5000 to $10,000. Careful designing of machinery and fishing techniques could no doubt reduce these rosts somewhat, but probably not enough to make plankton fishing a paying proposition. There are a number of reasons for this. There ran be :lo assurance of a steady supply of plankton in cconomically useful quantities. Even in the richest areas, plankton is not concentrated enough to supply a fishery; 0.5 g. (dry weight) per cubic meter is a frequent quantity occurring iu a rich area. I n such a c.oncentratioii, according to Jackson, it would be necessary to filter about one million gallons of water to recover a pound of plankton in such an area. Xot the least detriment to a profitable plankton fishery is the heterogeneity in quality. About 90% of the catch would be expected t o consist of phytoplankton, which would tend to plug the filtering device and to complicate manufacturing processes. What is more serious is that not all forms of plankton are equally iiutritious; indeed, some are severely tosic, such, for example, as dinoflagellates that cause “red tides.” The silicious frustules of diatoms would have the quality in meal of ground glass. The presence of such forms would probably preclude the bulk harvesting of plankton without regard to its composition. I n spite of all the talk about fishing for plankton 110 one has yet devised any technique of catching plankton nearly as efficiently and cheaply as a herring does it; nor of transforming plankton into any product as dependably uniform in quality as herring flesh. Taking all the technical difficulties into consideration, it seems wisest to leave this job to the creatures best fitted to work at it. In our opinion the most promising way to enlarge the harvest of the sea is through husbandry, husbandry not in the sense of being abstemious in making use of the fishery stocks, but rather by aggressively utilizing them to the fullest extent of their maximum sustained productivity, in the light of the best scientific principles. Although husbandry alone will not produce the spectacular quantities of food from the sea which some writers have

300

L. . i . W A L F O R D .iND C‘, G . N’ILBER

glihly promised i n popiilar literature, it should volitriht e sigtiifi(*aiit1y ellough to reptty manyfold the investment for thc coritiniious rcswwc.h which \vould l w rcquired to inakr it truly scientific. ‘l’hs the harvest of protein from the sea van be increased about, 20% by expanding fisheries for stocks that arc’ now virgin or oiily partially cxploited ; and it (mi he increased by some additional presently uriestimuble per cent by knowledgeable management of fishing rates. a n idea of how much this might be is given for the S e w England haddock fishery, on which extensive biostatistical data are available. The I’nited States Fish atid \I7ildlife Service has estimated that recently adopted regulation of fishing gear, designed to increase the age a t first capture, will effect an ultimate increase in the total catch by 35% to 50%. Apart from such measures, cbhanging the use of the catch might add still further t o the supply of protein available to people. As it is now, 2.7 million metric tons, i.e., about 13 per rent, of the world catch of sea fish goes into the manufacture of meal and oil. RIost of the protein, of (’oiirse, is contained in the meal, which is almost eiitirely used in tiiiimal feeds. Wheii the protein of the fish meal is transformed into chicken flesh, meat, or eggs, its aesthetic value is greatly improved, hut it derreases in volume by about 90%. It would seem desirable therefore to incorporate fish meal directly into the diets of people to whom protein is a clritical need (Taylor, 1953). The quality of fish meal varies with species, with location of catch, with season. It is also much influenced by the manufacturing process, being damaged by heat where the drying temperature goes over 100°C. (Carpenter ct ul., 1952; Render aiid Miller, 1953). It is not surprising, therefore, that there should be much confusion in literature as to “the” nutritive value of fish meal. There is 1x0 doubt, however, that cihickens fed on a diet of which 2 % to 5 % consists of fish meal or fish solubles, grow better than do chicks fed on a diet composed wholly of vegetable feeds. This is owing to the presence of all the essential amino acids in the right proportions and to an unidentified growth factor (S). The factor S is present also in grains such as corn and wheat and in soybean oil meal, but in niuch smaller yuantities than in fish meal (Berg, 1952; Biely et al., 1952). An animal fed fish meal requires less other feed. Thus, the addition of about, 5 % of fish meal to a diet increases the growth rate of chicks in the first year by about 7 %; aiid it decreasw the feed requiremcnt by about 20 %. It is therefore profitable to agriculturists to use fish meal in animal feeds. It is used extensively in Europe, Korth America, and Japan, and very little in Afrira arid South America. Most of what is produced in Africa is export,ed. Excellent though fish meal is for domestic animals, much as it may improve their growth and redure production costs, it cannot bring the pricc of meat and poultry anywhere near the reach of people who live in extreme

THE SEA AS A POTEKTIAL SOURCE O F PROTEIN FOOD

30 1

destitution, who are familiar with animal protein only from the juices which lhey use as seasoning for some carhohydrate staple such as rice (van Veen, 1953). What these people need is animal protein food a t a cost of a penny or two a pound, and in a form which keeps well without ice. One or the other of these requirements rules out fresh, canned, or pickled fish. On the other hand, fish meal can be produced from herring-like fishes for about 7 cents a pound i n the Vnited States, where labor costs are high. I t could probably be produced for much less in other countries. Why not use fish meal directly in humau diets? A pound of fish meal is equivalent in food value t o about A pounds of fresh fish. Herring-like fishes, which are cheaply caught, are exceedingly abundant around all coasts, including Africa and South America, where protein deficiency is serious in human diets. Fish meal keeps rather well and can be easily transported to inland areas. The manufacture of fish meal for animal feeds is a crude process. The product has a strong fishy flavor which may be acceptable in cbountries where people are acclustomed to pungeut taste, but not elsewhere. The Food and Agriculture Organization in experimenting with fish flours found that in Indonesia one sample which was nearly flavorless was not acceptable, while a different sample having a flavor resembling vertain salted fish products was well liked. I n Chile the F A 0 (1952) sponsorcd a series of tests in which fish flour was added to a number of recipes which were served to a few people. The results of the experiment with these recipes are shown in Tables I arid 11. All those sampling the dishes found no fault with the appearance. Among 89 persons, 14 noticed an abnormal odor in four of the dishes (vegetable soup, potato soup, cochayuyo, beef stew); 14 noticed an abnormal flavor in four of the dishes (potato soup, cochayuyo, beef stem, boiled potatoes); 20 considered the texture abnormal in four of the dishes (vegetable soup, tagliarini, beans, boiled potatoes). Everyone found the following dishes acceptable: vegetable soup, potato soup, fried potatoes, beet leaf pie, beans, cocktail crackers, and coffee cake. Only four dishes were considered unacceptable by 21 of the 89 people making the test. The acceptability test of bread with South African fish flour was carried out during 50 days among 140 school children belonging to poor families in Santiago. The children were not aware of the experiment. Every child received with the lunch on each of 5 days each week, a piece of bread weighing 90 g. which was made with nine parts of white flour to one part of fish flour. During the 50 days that the bread with fish flour was distributed the children ate all of it without comment. Moreover, the children had no complaints regarding digestive troubles that could be attributed to the fish flour. The results of these preliminary tests appear t o give promise that fish

TABLEI Fish Flour Experiiueni: Dishes Prepared with South African Fish Flour (Every person ate one serving) ~

Dish

~

~

Fish flour Sumber ( G . per of serving) persons

1. Vegetable Soup

2. Potato soup 3. Tagliarini Cochayuyo (edible alga) Fried potatoes with lettuce Beet leaf pie Beans Beef stew Boiled potatoes 10. Cocktail crackers 11. Coffee cake N

normal: A = ahnonnsl.

10

14 5

30

10 5 5

125 75

As felt in the mouth when eating the dish. =

3 5

10 10

4. 5. 6. 7. 8. 9.

a

10 10

1 14 6 3 4 20 14

Appearance

N

A

3 5 14 5 1 14 6 3 4 20 14

-

__ -

-

-

Odor N 2 14 1

14 6 -

4 20 14

Flavor

A

1 5

5 -

N

A

S

A

3 14 1 I4

-

5

-

-

3 -

-

Teuture"

6

3

20 14

5 5 3 1 -

-

-

5 1 14 4 3 3 20 14

3

14

-

Yet Accept- hcceptable able

303

TIIE Y E A AS A POTENTIAL SOURCE OF PROTEIS FOOD

TABLEI1 Fish Flour Experiment: Test of Receipes Prepared with and without South African Fish Flour (Percentage of 15 persons who found the samples normal for the listed qiiitlities.) Sample la _ I _

100

Odor Color Flavor Texture a

26.7 66.6 03.3

__

Sample 2”

S:tmple 3=

~ _ _ ~ ~ _ _ _ _

86.4 26.7 60 10

100

26.7 60 73.3

Ordinary bread.

’ Ordinary bread plus 10% fish flour. Ordinary bread lard and fish flour. plus

10%

meal, which is a cheap product, can be made acceptable in the diets of people who are suffering from protein starvation, who are not accustomed to eating meat, poultry, or fish in adequate quantities, and who could never afford to buy such foods.

11. PROTEINS IN MARINEORGANISMS 1. Algae The use of seaweed as a source of chemicals needed by man is not recent. The practice originated as far back as the beginning of the eighteenth century. In more recent years, The Scottish Seaweed Association has sponsored extensive studies t o provide basic information upon which an industry using seaweed as the raw material could be based (Woodward, 1951). The fronds of the alga, Laminaria, contain as high as 14% (dry weight basis) of crude protein in Scottish waters at certain times of the year (Black and Dewar, 1949). Channing and Young (1952) analyzed the following species of algae: Laminaria saccharina, L. cloustoni, Ascophyllum nodosum, and Pelvelia eundiculata. The major amino acids found were: aspartic acid, glutamic acid, glycine, alanine, valine, leucine, isoleucine. They also found some serine, threonine, proline, phenylalanine, lysine, and a trace of arginine. On the basis of dry weight there is an average of 6.3 % of peptide and protein in these species. In the Skagerrak, Fucits vesiculosus has been found to contain the following amino acids : glycine, a-alanine, tyrosine, serine, threonine, cystine, methionine, arginine, lysine, proline, histidine, aspartic acid, glutamic acid, valine, leucine, isoleucine, and phenylalanine. Hydroxyproline could be identified with ninhydrin but no tryptophan was ever found. Protein varied with the season of the year from 4 % to 14 % (Ericson and Sjostrom, 1952).

304

L. A. WALFORD AND C. G. WILBER

TABLEI11 Per Cent Diawiino Acids in Marine Plants (16% N ) Arginine

S

Histidine 3 ,8 0.7

4.6

:1.7 8.0 4.0

0'0 0 .0 0.0 4.5 1.s 6.1 3.5 3 5 0 .4

0.9 1.I) O.!) 0.6

1.1 0.0 0. !) 0 .n 0.0

" Only

1,ysine

5.0 2 ,3 2.4

knnwn tissue proteins which lack arginine.

TABLEIV Aromatic Amino Acids in Some Marine Plants __

Tyrosine(%)

Source _

-

Lu~tririutza I'a,yassuttl

3 4 0 0 9.0 2.9

Diatoms

0.4

I'(lLC1LS

1.7 0.6

I'hUrrklLdt II ttl ('11u

Jl acrocystes

Tryptophan(%) Phenylalanine(%)

___________~ 0.2 0 .6 1 3 1.8 7.3 0 6 0 6

2.1 4.3 1 !) 0 6 ..

__

The amino acid content of several species of marine algae is shown in Tables I11 and I V taken from Block and Bolling (1945). 2 . Invertebrates

Invertebrates from the sea are a rich source of protein of relatively high iiutritioiial quality. Table V shows that certain shellfish rimy have a s much as 2.5% protein (Jacobs, 1951). TABLE V Shelljish

~~~

Oil(%)

Protein (%)

1 2 1

9 17 7

1

25

-

Clams Crabs Oysters Shrimp

305

T H E SEA A S A POTENTIAL SOURCE O F PROTEIN FOOD

TABLE V1 Broinatic A m i n o Acids in Invertebrate Jfuscle 9 (yo)

Species ~~

.

Clam Crab Lobster myosin Shrimp Shrimp Sr;lllol,

16.0 16 0 16.1 16.0 -

17 1

Indian oysters are reported to contain from 5.7 % to 13.3% protein as compared with 6 % t o 12 % in clams from the same area (Venkataraman and Chari, 1951a). The clams are said to be inferior to oysters as a source of food. Squid, Loligo vulgaris, which is considered a delicacy among many Mediterranean folk, contains an average of 17% protein (de Couveis and de Gouveia, 1951). The amino acid content of invertebrate muscle has been studied and compared with that of other protein sources by Block and Bolling (1945). Table VI summarizes the per cent of various aromatic amino acids from selected invertebrates. Table \‘I1 shows the content of basic amino acids in a number of edible invertebrates. The content of aromatic amino acids from a wide variety of marine species and terrestrial animals shows little variation. Furthermore, the yield of cystine and of methionirie is essentially the same whether terrestrial animals, fish, or marine invertebrates are the source of protein. Crab and shrimp meal are very rich in protein. Crab meal coiitairis a t least 25 % protein, shrimp meal over 40 % (Yearbook of Agriculture, 1939). TABLEVII Basic A m i n o Acids in Shellfish Muscles Species

S

Histidine

Lysine

1.5 1.5 1.8 1.9 1.8 1.8

5.4 6.4 5.2 5.4 5.2

-

5.3 7.6 5.7 6.9 5.7 6.6

16.0

6.5

1.8

8 .O

6.2 f 0.9

1.7 f 0.2

5 . 5 f 0.5

Clam Crab Oyster Scallop Shrimp Shrimp

16.0 16.0 16.0 17.1 16

“Rest values” Mean W. 2s

Arginirie

8.3

TABLEV I I I

w

8

Total .Iriirogen, Protein lYitrogen, and Nonprotein i\;itrogen i n Fish

Sonprotein No. of saniples

Species

ELASYOBRANCHS Dogfish (Aca)zt h ias u u Zgar is)

1

1

Blue Skate (Raja batis) Cuckoo Ray (Raja circularis) Thornback Skate (Raja clavata) Rays (sp. I)

1

1

2

Total Protein nitrogen (%) nitrogm (%)

3.29-3.96 (3.48) 3.62 3.98 3.58 3.803.03 (3.92)

1.95-2.49 (2.16) 2.17 2.57 2.18

2.86 2.83 2.5G3.02 (2.80)

3.03

2.43 2.47 2.llr2.83 (2.48) 2.66 2.45-2.48 (2.47)" 2.74

2.70-2.78 (2.73) 2.763.14 (2.93)

2.23-2.34 (2.88) 2.27-2.59 (2.47)

(2.52)"

nitrogen as % of total

Sonprotein nitrogen (yo) nitrogen 1 .05-1.34

S3.0

(1.18) 1.20 1.34 1.38 1.38-1.42 (1.10)

33.7 38.6 35.6

0.41 0.36*

14.3 12.i

(0.37) 0.34 0.344.38

13.0 11.3

33 .0

TELEOSTS Gadoids

Cod (Gadus niorrhua) Haddock (Gadus aeglejinus) Whiting (Gadus nzerlangus) Ling (Molua niolva) Flatjishes Plaice (Pleuronectes platessa)

Lemon Sole (Pleuronectes microcephalus)

1

i

15

1

i

1 2 1

2

3

3 .O

2.81-2.86

@.@I

(0.36)

12.7

0.34

11.2

0.3M.31

(0.31)

11.:3

0.30-0.34 (0 33)

11.3

1

Dab iP1eiironecte.s linrando)

Megrini (drnoglosszts megastonla)

i

1

1 2 1

2.85 2.SS-2.93 (2.90) 3.07

2.38

0.38

13.3

(2.71)O

0.244.29 (0.265) 0.30

9.i

2.06

9.2

4

Herring (('lupea harengus) Sardine (Clupea pilchardus)

Combined snmple 6 fish

3

Herring (Finnish harengus wenibras)

2.70-3.01 (2.90) 2.98

2.36-2.63 (2.53)" 2.67

0.34-4.40

3.27-3.78 (3.46) 2.41-2.73 (2.60) 2.42-2.74 (2.57)

2.83-3.19 (2.97)"

0.444.59 (0.49) 0.344.51 (0.45) 0.314.60

(2.10)"

(0.47)

15.3

3.33

2.76

0.42

12.5

(2.15)"

(0.38)

0.41

13.1 13.7 14.1

17.3

Aliscellancous Species Xlackerel (Scowber sconibrus)

Combined sample 3 fish 7

Grey Gurnard (Trigla gurnardus) Horse hlackerel (Caranz lrachurus Monk fish (Lophius piscatorivs) Hake (ilferluccizts ~~ierlttccius)

1 1 1 2

Catfish (Anarrhicas lupus) Golden Perch (Sparus auratus)

1 1

2.CG3.10 (3.05) 3.20 3.12 2.70 2.76-2.90 (2.84) 2.56 3.22

2.3S2.76 (2.60)B 2.87 2.68 2.40 2.4S2.58 (2.54) 2.29 2.S5a

0.384.51 (0.45) 0.30 0.35 0.29 0.324.34 (0.33) 0.26 0.37

14.8 9.4 11.2 10.7

11.6 10.0 11.5

THE SEA AS A POTENTIAL SOURCE OF PROTEIN FOOD

('1 u peids

1

B y difference.

w

307

a

0 -.I

308

L. A. WALFORD .4ND C. G . WILBER

A few invertebrates which are not usually eaten have been analyzed. One of these, a starfish (Stichaster aurantiacus) from Peru, has an average protein content of 27% (Espinoza, 1950). Among species which do have commercial value, the abalone, Hatiotis gigantea, contains 23 % protein, the wreath-shell, Turbo cornutus, 19% (Simidu et at., 1953). Both these species were from Japanese waters. 3. Fish

Kumerous chemical analyses have been made of fish, both fresh and processed. Table IX after Shewaii (1951), shows one set of analytical results for a number of species. Protein nitrogen values can be converted to protein by niultiplying S by 6.03. The common marine fish available in India contain 9 % to 24 % protcin. The protein is 93% digestible t)y human beings (Qureshi, 1951). Table TI11 shows the average values for amino acids in fish proteins. Hock and Bolliiig (1945) emphasize the fact that “muscle proteins from a wide variety of speries show little if any significant differences in their content of aromatic amino acids . . . the relative constancy in the basic amino acids in all types of muscle, animal, fish, or crustacean, is noteworthy.” The authors use crustacean as synonymous with shellfish. Hence, their generalizations are meant to include oysters and rlams. High-grade fish meal vontains as much as 60% protein and relatively TABLEIX Average Percentage of Amino Acids i n F i s h Proteins (Calculated t o 16% ru’) Amino w i d

Muscle

Stic Water

Mcal

Argininc His t idi iic

5.6 f 1.0 1.9 f 0.6

5.9

1,ysitir~ ‘ryrosi ti(’

6.8 4 1.3

5.4 2.G 1.1 0.8 0.8

4 1.2 f 0 . 1

1.!)

1.8

3.4

1.5 2.3 2

3

Tryptophriti Phenylalanitie Cystine Met hionirie Threoninc Idcuriiie Isolericine l’alinr. Scrinr ( h t a m i c Acid .\l:triinc)

I’rolinc.

f

0.4

I!=

0.1

4.4

4.6 14 7 3

3.4 5.7 2.X 1.2

1

1 3

5 10 4 4

309

THE SEA AS A POTENTIAL SOURCB O F PROTEIN FOOD

little moisture (10%). (Yearbook of Agriculture, 1939; Anonyrnou~,1953). Fish roe, which is an expensive delicacy when packaged as caviar, is characterized by a high protein content: 23 ?' & to 29 % (Jones, Carrigail, arid I)assow, 1948). The average coiitent of essential amino acids ill salmoii r o ~ expressed , as per (wit of total protein, has been reported as follows: arginine, 7.3; histidine, 2.6; isoleucine, 7.4; leucine, 9.9; lysine, 8.8; methionine, 3.0; phenylalanine, 4.8; threonine, 5.7; tyrptophan, 0.9; valine, 7.2 (Seagran, 1953) (See Tables X and XI). Kumerous other recent anlyses of various kinds of fish nieals indicate the rich protein content of these products (Moen, 1951; Helmel, 1051; Balken, 1952; Buffa, 1952). Yalues up to 60% of nutritionally high-grade protein are reported. TABLE X Oil and Protein Content of the Edible Portion of F i s h

Species

Average oil content (%)

Protein content

5

19 17 18 16

(%I

Fish Alewives Cod Croaker Flounder Haddock Halidut Herring, sea Mackere , common Mullet Pollock Salmon, kinga Salmon, sockeye" Salmon, cohoa Salmon, pinka Salmon, chum" Sardine (pilchard) Squeteague (sea trout) Tuna, skipjacka Whiting

0.4 3 0.6 0.3 5 11 13 5 0.8 16 11 8 6

18

19 19 18 19

0.4

20 18 21 21 21 21 23 19 22 17

1 2 1 1

9 17 7 25

5

13 2 4

She1ljis h

Clams Crabs Oysters Shrimp a

Canned.

TABLEX I Percentage of Certain Essential Amino Acids i n Fish and Shellfisha Species Fish: Catfish Cod Croaker Haddock Halibut Herring : Lake Sea Lake trout Mackerel: Boston Spanish Mullet Pilchard Red snapper Salmon : Chum King Pink Silver Sockeye Shad Squeteague (sea trout)

Scientific name

Date sample was prepared

Ameiurus catus Gadus callarias Micropogon undulatus Melanogrammus aeglejinus Hippoglossus hippoglossus

Aug., 1937 June, 1936 May, 1936 Sept., 1935 Dec., 1936

5.58 5.81 5.70 6.00

Leucichlhys arledi Clupea harengus Cristivomer namaycush

Nov., 1937 March, 1937 June, 1937

5.09 5.73

Scomber scombrus Scomberomorus maculatus Mugil species Sardinops caerulea Lutianus blackfordii

Aug., 1936 Jan., 1936 Feb., 1937 March, 1937 Nov., 1936

Oncorhynchus keta Oncorhynchus tschawytscha Oncorhynchus gorbuscha Oncorhynchus kisuteh Oncorhynchus nerka Alosa sapidissima Cynoscion regalis

May, 1938 April, 1937 April, 1937 Jan., 1937 Sept., 1937 May, 1936 June, 1937

Arginine Histidine Lysine Tryptophan Cystine

-

1.24 0.85 1.64

1.41 1.15 1.16 1.45

1.25 1.23 1.17

-

1.72 1.37 1.17 1.66

6.83 6.10 6.41 6.16

-

-

1.56 1.40

7.03 7.15

5.78 5.27 5.78 5.60 6.18

1.93 1.48 1.61 1.23 1.57

7.13 6.53 6.74 6.78 6.72

1.36 1.37 1.36 1.30 1.22

1.18 1.25 1.29 1.29

5.55 5.02

1.30 1.41

5.68 4.54 5.90

1.87

5.69 6.27 6.57 6.45 6.78

1.33 1.20 1.09 1.44 1.25 1.22 1.01

1.27 1.15 1.39 1.17

-

-

-

1.09 1.42

0.97 1.06

F

?

3 9

m a

*z U

0

-

.D c

8m

Tuna: Albacore Bluefin Bonito Skipjack Shellfish and crustacea: Clam, hard Crab, blue Oyster Shrimp Proteins from other foods: Casein Beef round Egg albumin a

Pottinger, 6.

Gernio alalunga S'hunnus thynnus Sarda chiliensis h-atsuwonus pelainis

Sov., 1937 Oct., 1937 Sept., 1937 Dec., 1937

J-enus mereenaria t'allinectes sapidus Crassostrea virginica Peneits brasiliensis

Sept ., 1936 Nov., 1936 Oct., 1936 June, 1936

R. and Baldwin, W. H.

(1930). Proc. 6fA

2M -

5.27 7.61 5.71 7.50

1.45 1.51 1.79 1.61

5.40 6.38 5.24 7.35

1 .19 1.11 1.67 0.96

1.25

5.2 7.5 6.0

2.6 1.8 2.3

7.6 7.6 3 .8

2.2 0.9 1.3

0.3 1.3 0.9

ui

*m

Pm'fic Sci. Conpr., CalZX, P. 453.

d

?IMI

312

L. A. WALFORD b ND C . G. WILBER

4. Marine Mammals Seal liver is reported to contain essentially the same percentage of protein as does beef liver (Dugal and Riou, 1947). The fresh meat of four species of whales caught by Japanese whale fishers contained from 17 7% to over 24% protein. The epidermis of the head and back of these whales contained over 30 % protein; the pancreas and stomach were very low in protein-3 % and 6 %, respectively (Arai and Sakai, 1952). In view of the fact that Japanese whalers catch around 3500 whales per year, it is evident that these mammals are a source of appreciable protein.

111. VARIATIONS IN PROTEIN CONTENT 1. Ceographdcul

The location in which algae are collected apparently influences the protein content. Moss (1948) collected Fucus vesiculosus in protected loch areas, in the open ocean off Scotland, aiid a t a point intermediate between the two. He found that algae from the open ocean contained appreciably greater amounts of organic nitrogen than those from sheltered or intermediate areas. Seaweed meal produced from algae around Denmark has a raw protein content of about 13%, as compared with that from Scottish waters (11 %) or from Norway (7 %) (Chapman, 1950). I n Japan, Laminuria from Horotzumi contains about 6.4 % protein (dry basis); that from Mitsuishi, 8 %. Similar variations in content of algae along the California coast are also known (Chapman, 1950). 2. Anatomical

In Loch Melfort, Scotland, the distal port>ionsof the thalli of Pucus vesiculoszis contain 0.7 % of nitrogen on a dry weight basis; the middle portion contains 0.82 %; the proximal portion 1.07 (Moss, 1948). Like variations are found in the same species in different geographical locations. On the other hand, salmon contains comparable amounts of protein a t the head and tail ends: red chinook near head, 17.6%, near tail, 17.9%; white chinook near head, 19.0 %, near tail, 19.9 %. Yellowtail shows slight variation from 19.8% protein in the middle portion of the fish to 21 % near the tail. Skipjack belly muscle contains about 18 % protein, as does the dark meat alone; the white meat alone contains almost 22% protein (Jacobs, 1951).

3. Seasonal The content of crude protein in algae on a dry weight basis shows clearcut variations during different seasons of the year (Black, 1950). Figure 2, based on data reported by Black (1950), shows that the protein content of Laminaria off England is greatest during early spring and declines t o a

T HE SEA AS A POTENTIAL SOURCE O F PROTEIN FOOD

313

15 14

-

13

2

12

5 Im n n

p 11 U

g

10

al

Y

0

&

9

U al

2

0

8 7

6 5 I

XI

k-

1st

v

VII

tx

XI

I

111

v

VII

IX

1947 -1948-d

Month of year

FIG.2. Seasonal variation in crude proteins in L. saccharina. A : Open-sea stipes; B : open-sea whole plants; C : open-sea fronds; D : loch whole plants; F : loch stipes.

minimum in late summer. Puch seasonal variations have keen reported also by Scandinavian workers, who found that 1,. saccharina contains 4 % protein in late summer arid 14% in winter (Ericson and Sjostrom, 1952). Oysters and dams, on the other hand, are said not to fluctuate appreciably in protein content a t different times of the year (Venkataraman and Chari, 1951b). Herring taken off Sakalin Islarid show seasonal proteiii variations : spring herring are reported to contain 16.9% protein; summer herring, 18.1 % (Levariidov, 1950). The mackerel of India, Rastrelliger kangurta, shows little seasonal variation of protein content (Venkataraman and Chari, 1951a).

IT. THEBIOLOGICAL VALUE OF MARINEPROTEINS

It was pointed out before that studies on marine fish from India indicate that the proteins from them are about 93% digestible by man (Qureshi, 1951). The United States Fish and Wildlife Service has studied the nutritive value of the proteins extracted from the flesh of 17 species of fish. It mas found that these marine proteins were equally effective in promoting growth in rats. As compared with proteins from beef round they were comparable in growth promoting effectiveness (Xilson, Martinek, and Jacobs, 1947). Earlier studies had indicated that the growth-promoting

314

L. A . WALFORD AND C . G . WILBER

16 Dried fish

14

12 Soy.bean

10

Skimmilk powder Meat

E"

I M

6 5

8

I

e

a Beans and peas

6 Whole milk Ground.nuts

4 Wheat flour Millet: sorghum Maize Yams Rice (half.milled) White rice; taros

2

Plantains; sweet potatoes Fresh cassava Cassava flour

0

5

10

15

-

20

)

Age, years

FIG.3. Protein requirements per 100 calories, according t,o age.

value of fishery products was 1.3 to 1.G times greater than that of beef round (Lanham and Lcmoii, 1938). It is not easy to explaill the differences but one fact is clear: the growth-promoting value of proteins from fishery products is at least equal to that of proteins from beef. Reef round is about 89% digestible as tested with rats. Salmon, tuna, bonito, and herring are all close to 90% digestible (Nilson, Martinek, and Jacobs, 1947). Studies by Canadian workers on the biological value of fish proteiiis indicate that weight gains in rats fed on crude protein from lingcod, halibut, lemon sole, white spring salmon, red snapper, or herrririg are significantly greater than in rats fed on beef proteins or egg albumin (Reveridge, 1947).

THE SEA AS A POTENTIAL SOURCE OF PROTEIN FOOD

315

REFERENCES Anonymous.

(1951). Meldinger j r a SitdoFe-og Sildemetsindustrieus Forskningsin-

stitutt No. 3, 48. April 1951.

Anonymous. (1953). Food 22, No. 9. Arai, Y., and Sakai, S. (1952). Sci. Repts. Whales Research Inst. No. 7, 51. Balken, K. (1952). Tech. Research Rept. Norway Fish. I n d . 6, 61. Bender, A. E., and Miller, D . S. (1953). Biochem. J . 63, vii. Berg, L. R. (1952). Feedstufls 24, No. 22, 12. Beveridge, J. M. R. (1947). J . Fisheries Research Board Can. 7 , 3 5 . Biely, J., March, B. E., and Tarr, H. L. A. (1952). Fisheries Research Board Can. Progr. Repts. Pacific Coast Stas. No. 92,lO. Black, W. A . P. (1950). The seasonal variation in weight and chemical composition of the common British Laminariaceae. J . Marine Biol. Assoc. United Kingdom 29, 45.

Black, W. A. P., and Dewar, E . T. (1949). J . Marine Biol. Assoc. United Kingdom 28, 673. Block, R . J., and Bolling, D . (1945). “The Amino Acid Composition of Proteins and Foods.” C. C Thomas, Springfield. Brock, J. F., and Autret, M. (1952). Food and Agr. Organization Xutritional Studies, No. 8. Buffa, A. (1952). Conserve e deriv. agrumari (Palernzo) 1, 13. Cannon, P . R . (1050). “Recent Advances in Nutrition.” Univ. of Icansas Press, Lawrence. Carpenter, I<. J., Duckworth, J., Ellinger, G. M., and Shrimpton, D. H. (1952). J . Sci. Food Agr. 3, No. 6 , 278. Channing, D . M., and Young, G. P. (1952). Chemistry & Industry, p. 519. Chapman, V. J. (1950). “Seaweeds and Their Uses.” Methuen, London. de Gouveia, A. J. A . , and de Gouveia, A . P. (1951). Conservas Peixe 6 , No. 18. Dugal, L. C., and Riou, L. (1947). Fisheries Research Board Can. Progr. Rept. Atlantic Coast Stas. No. 37, 9 . Ericson, L. E., and Sjostrom, A. G. M. (1952). -4cfa Chew. Scand. 6, 805. Espinoaa, A. V. (1950). Rev. jac. f a r m . bioquini. y U n i v . nacl. mayor S u n Marcos L i m a , Peru 12, 76. Finn, D . B. (1954). Nutrition Abstr. & Revs. 24, 487. FAO. (1952). Food and Agr. Organization 11. N . , Rome, 2nd World Food Survey. Harvey, H. W. (1950-51). .I. Marine Biol. Assoc. United Kingdom 29, 97. ,Jackson, Philip. (1954). .I. d u Conseil 20, No. 2, 167. Jacobs, hf. 13. (1951). “Tho Chemistrv and Technology of Food and Food Protluct s.” Interscience, Xew York. Jones, G. I., Carrigan, E. J., and Dassow, J. A. (1918). Dept. Commerce, IRRD Rept. No. 2 , I). Krey, J . (1953). Veroflentl. Inst. Meeresforsch. in Bremerhaven 2, Part I, 1 . Lanham, W. B., and Lemon, J. M. (1938). Food Research No. 3 , 5 4 9 . I,evanidov, I. 1’. (1950). Rybnoe Khozyatstvo 26, No. 2 , 3 7 . Moen, A. (1951). Meldinger N o r g . Landbruksh$gskole 31, 23. Moss, B. I,. (1948). A n n . Botany (London) 12, 2 6 i . Sicken, 12, (1851). iYakrrt: 167, 684. Silson, 1%.W . , hI:irtinek,W. A , , ~ i n dJacobs, €3. (194i). ( ‘ o n ! .Fisheries Rcv.9, S o . 7 . Qureshi, 31. R. (1951). Pakistan Aletl. J . July 1951 3, 3.

316

L. A. WALFORD AND C. GI. WILBER

R.edfield, A. C. (1953). Symposium on the sea frontier. The sea as a prodiictive system. Address presented a t AAAS, Bost,on. Seagran, H. I,. (1953). Corn. Fisheries Rev. 16, No.3, 31. Shewan, J. M. (1951). Biochem. S O CSymposia, . Cambridge, Enyl. No. 6, 28. Simidu, W., Hihiki, S., Sibata, S., and Takedn, IC. (1953). Bull. Japan. 8 o c . Sci. Fisheries 19. N o . 8 . Stamp, L.D. (1952). “Land for Tomorrow: The Underdeveloped World.” Indiana Univ. Press, Bloomington. Taylor, 11. F. (1953). The determinnnts of food production from the sea. Paper presented a t t h e AAAS, Boston, 1953. van Veen, A. G. (1953). Advances i n Food Research 4.209. Venkataraman, It., and Chari, S. T . (1951a.). Indian .J. M e d . Research 39,533. Venkataraman, It., and Chari, S. T . (1951b). Proc. Indian Acad. Sci. B33, 126. Vergara, A. (1954). IJNESCO (UN Children’s Fund), E/ICEF/IJ.543/Add.l. 1954. Wheeler, J. F. G. (1953). Colonial Office (Brit,ish) Fishery Publications, Vol. I , X o , 3, Part’ 1 . Woodward, F . N. (1951). $1.Maritbe B i d . Assoc. I-nited Kingdom 29, 719. Yearbook of Agriculturt!. (1939). U . 8. Dept. Agr., Washington, D. C .

Zinc and Metalloenzymes

BY BERT L . VALLEE The Biophysics Research Laboratory o j the Department oj Medicine. Harvard Medical School. and Peter Bent Brighani Hospital. Boston. Massachusetts

CONTENTS A . General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Metalloproteins and Metal-Protein Complexes . . . . . . . . . . . . . . . . . . . . . . . 111. Characteristics of Met.alloenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

318 318 320 321 IV . Characteristics of Metal-Enzyme Complexes . . . . . . . . . . . . . . . . . . . . . 325 V . Empirical Formulas for Metnlloenzymes . . . . . . . . . . . . . . . . . . . 327 V I . Instrumental Methods for the Detection Metals . . . . . . . . . . . . . . . . 328 FrII. References t o Metalloenzymes Containing Copper, Iron and Molybd e n u m . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332 H . Zinc Metalloproteins . . . . ................................... 333 V I I I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 I X . Carbonic Anhydrase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 1. Physical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 2 . Chemical Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 3 . Inhibition by Metals and Zinc Complexing Agents . . . . . . . . . . . . . 336 X . Experimental Approach for Studies on the Leukocyte Zinc Protein, Carboxypeptidase, and Yeast Alcohol Dehydrogenase . . . . . . . . . . . . . 337 X I . The Zinc-Containing Prot.ein from Human Leukocytes . . . . . . . . . . . . . 339 1 . History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 2 . Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 3 . Physical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 4 . Chemical and Ihzymatic Properties . . . . . . . . . . . . . . . . . . . . . . . 343 XI1 . 1’ancre:ttic Carboxypeptidnsc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 1. Physical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 2 . Chemical Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 a . Basic Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 b . Magnesium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 r . Zinc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 3 . Inhibition Due to Metal-Binding Agents .................... 349 4 . Physiological Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353 ST11. Yeast Alcohol Dehydrogrnase . . . . . . . . . . . . . . . . . . . . . 353 1 . Physical Properties . . . . . . . . . . . . . . . . . . . . . . . 353 2 . Chemical Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 a . Basic Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 b. Zinc., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356 3 . Inhibition of Enzyme Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359 . 4 . Yeitst ADH as a Zinc Metalloenzyme.,. . . . . . . . . . . . . . . . . . . . . . 367 31 7

3 18

BERT L. VALLEE

XIV. Coordination Chemistry of Zinc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Successive Stabi1ity.Constants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. The Entropy Effect.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. The Coordinate Bond.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

370 370 371 374 376 378

A. GENERAL1 I. INTRODUCTION The importance of zinc in biological systems has been recognized for some time, and interest in many aspects of the problem dates back about a century. The requirement of zinc for the growth of Aspergillus niger was first recognized by Raulin in 1869, and the presence of zinc in tissucs of plants and animals was described by Lechartier and Bellamy (1877). The investigation of the functional roles of zinc and most of the metals was delayed by the fact that they occur in most tissues in very small amounts, making their quantitative identification, and therefore their functional assessment, difficult. Zinc and these other metals have been grouped together quite arbitrarily as “trace metals” because of their low concentration and their resistance to definitive appraisal (Vallee, 1952). The terminology employed for their description has implied conjecture concerning their biological role. In other instances, it has been based on the problems associated with their concentration, detection, and occurrence in biological material, thus adding to the confusion. The designations trace element, oligoelement, micronutrient, microelement, rare element, and minor element have been used interchangeably. None of them is a happy choice. Such nomenclature implies that the function of all these elements is similar -solely on the basis of the fact that all are present in small concentrations. Such abstraction may or may not prove to be correct, but little if any justification for it existed at the time it was made, nor does it indeed now exist. As information on each of t,hese elements has accumulated, some have successively been removed from the general group and arrorded slaturc i l l their own right; iron and copper are examples. The search for an explanation of the physiological and biochemical role of all these elements has emphasized their association with enzymes in cells 1 The following abbreviations will be used : ADII- alcohol dehydrogenase; D P N diphosphopyridine nucleotide; DPNH-reduced diphosphopyridine nucleotide; OP- 1,lO-phenanthroline: 8-OHQ-8-hydroxyquinoline; 01 ,a’-D-a ,a’dipyridyl; 8-OHQ5SA-8-hydroxyquinoline-5-sulfonic acid; TU-thiourea; EDTA-ethylenediamine tetraacetic acid; zincon-2-carboxy-2‘-hydroxy-5-sulf~rmazyl~ei~zene; diamox - sodium 2 - scetylamino - 1 , 3 , 4 - thiodiazole - 5 -sulfonamide; NaDDC - so dium-diethyldithiocarbsmate; CGP-carbobenzoxyglycyl-L-phenylalanine.

ZINC AND METALLOENZYMES

319

(Oppciiheimrr and S t t m , 1949; Green, 1941; Warburg, 1949; Lehninger, 1950; Vallee, 1951; Williams, 1953). Many of these studies have been conrerried purely with the fi~nctionulcharacteristics of a given system, using the rate of reaction catalyzed by an enzyme, and the effect of different metal ions upon it, as criteria of the interaction. Studies on alkaline phosphatase of intestinal mucosa (Cloetens, 1941a,b, 1942; Neumann, 1949), arginase (Edlbacher and Zeller, 1936; Hellerman and Stock, 1938; Richards and Ilellerman, 1940; Boyer, Sham, and Phillips, 1842; Mohamed aud Greenberg, 1945), phosphoglucomutase (Cori, Colowick, and Cori, 1938; Stickland, 1949), oxaloacetic decarboxylase (Speck, 1947, 1948), oxalosuccinic decarboxylase (Iiornberg, Ochoa, and Mehler, 1948), and enolase (Lohmann and Meyerhof, 1934; Warburg and Christian, 1941) may serve as examples of the experimental modes of approach chosen and the interpretations which have been made. It has been pointed out (Lehninger, 1950; Vallee, 1951; Williams, 1953) that the activating effect of a metal upon an enzyme in vitro defines neither its structural association with the enzyme nor the physiological function of the metal. It is virtually impossible to assign any functional meaning to many of these observations, which may be of passing interest only. The approaches and logic used by Warburg, as exemplified by his monograph (1949), have markedly influenced this area of study (James, 1953). The use of inhibitors of enzymatic action, assumed to modify the rate of a reaction by specific interaction with a given metal, constitutes a n important facet of this mode of investigation. Enzyme activity is the primary measurement, and the participation of the metal is inferred from the altered rate of reaction induced by the inhibitor, which is thought to combine with or remove a particular metal. Ideal metal inhibitors should act in very low concentrations and be completely specific for a given element, acting a t the pH optimum of the reaction without affecting any other component of the system. They should diffuse rapidly without being destroyed and be soluble in biological material. Since it is difficult to meet all of these conditions with any one reagent, it has been necessary to employ different agents having one or more of the desired properties, and not infrequently the prerequisite characteristics of inhibitors cannot be found at all. At best, conclusions are arrived a t by a scheme of successive, multiple, inferential approximations. Inhibitors are therefore only of limited usefulness in identifying the presence and presumable significance of metals in enzymes when direct analytical data are not on hand. On the other hand, the continuing trend toward explanation of function in terms of the chemistry of proteins and metals has been reflected in a different experimental approach to the study of metals in enzymatic catalysis.

320

BERT L. VALLEE

11. METALLOPROTEINS AND METAL-PROTEIN COMPLEXES

‘l’mo groups of proteins which associate with metals can be differc~ntiatetl. Members of each group may or may riot possess known enzymatic activities. In one group, the mctalloproteins, a given metal is combined with the protein in a unique manlier so that the two can be thought of as an “entity” iil nature. Isolation of highly purified proteins, homogeneous by physicalchemical criteria, has permitted the investigation of their metal content per unit of protein (Table I). The iron proteins (Granick, 1946; Lemberg and Legge, 1947;Hauronitz and Hardin, 1954),the copper proteins (Dawson arid Mallette, 1945; Singer and Kearney, 1954a,b), and the zinc proteins (Keiliii and Mann, 1940a; Vallee and Altschule, 1949; HochandVallee, 1953; Vallce, Hoch, and Hughes, 1954; Vallee and Neurath, 1954; Vallee and Hoch, 1955a,b) are examples. Vitamin Blz,containing 4 % cobalt, should be mentioned as a compound containing a metal and potentially related to this group of substances possibly as a prmthetic group (Sebrell and Harris, 1954). A second group, in which a protein combines reversibly with one of several different cations, the ?netal-protein complexes, has been much more resistant t o definitive appraisal. Such systems have been the subject of extensive studies concerning the site of binding between metal and protein (Tanford, 1952; Gurd and Goodman, 1952; Koechlin, 1952; Warner and Weber, 1953; Gurd, 1954; Klotz, 1954; Tanford and Epstein, 1954). The appraisal of the physiological significance and biochemical implications of the association of metals with this latter group has frequently of necessity been held in abeyance, since the studies with these substances involved the assessment of either physical or biological properties, while correlations of the two have been difficult, if not impossible. The assessment of functional significance of associations between metals and proteins is greatly facilitated when the pure proteins under study exhibit specific enzymatic function. These enzymes may fall in either group of proteins described above. Highly purified proteins which contain a metal firmly and apparently uniquely bound-metalloenxymes-represent particularly useful model systems for the study both of interactions of proteins with metals and of the biological effects of metals. Since the members of this group are structural and functional units, at least three primary pnrameters can be measured independently to ascertain the interdependence between structure and function: ( I ) The protein, (2) the metal, and ( 3 ) the activity. The opportunity for correlation of these objective parameters with biological specificity makes the metalloenzymes a particularly suitable group for extrapolations of information obtained on simple systems toward the understanding of metabolic mechanisms. The unique chemical specificity of metals provides a powerful tool for such studies.

ZINC AND METALLOENZYMES

32 1

Metal-enzyme complexes, a subgroup of metal-protein complexes, exhibit enzymatic activity consequent to readily dissociable combination with a variety of metal ions. Many of these studies have been performed with unpurified enzymes, a i d , even when pure enzymes were used, the stoichiometry of the interaction of the metal arid enzyme has riot been measured. Enhancement of enzymatic activity as a result, of the addition of metal ions and its partial loss on their removal has been the chief criterion of assessment of physiological significance. Only in a few instances, e.g., enolase, has the stability and stoichiometry been studied in relation to function (Malmstrom, 1953, 1954). The study of metal complexes and particularly metal chelates (Bjerrum, 1941 ; Martell arid Calvin, 1952; Calvin, 1954) has provided both new experimental and new theoretical backgrounds for the study of metals in relation to the specificity of enzyme action, metal-enzyme (Calvin, 1954), metal-substrate (Najjar, 1951), and metalloenzyme interaction, as well as nietal-enzyme inhibition (James, 1953). Although the precise mode of binding of metals by proteins is not understood in most cases, the vharacteristic stability, specificity, and versatility of chelate complexes, as a function of a variety of physical and chemical factors, offers particularly interesting possibilities for speculation and experimentation to explain the properties of metal-protein systems. Differences between metalloenzymes and metal-enzyme complexes can be viewed in thelight of the type of bond between metal and protein. The two types of metal-enzyme interaction perhaps correspond to the metal complexes and metal chelates encountered in simpler systems. In metal complexes, the number of donor groups is identical with the number of ligands. I n metal chelates, two or more donor groups are available to form a ring structure with one molecule of ligaiid which provides additional stability (Martell and Calvin, 1952). Nitrogen, sulfur, and oxygen atoms participate most readily in the format>ionof chelate structures with metals. The metalloenzymes have several distinct characteristics which it may be well to inspect and analyze.

I1 I. CHAI~ACTERISTICS OF METALLOENZYMES 1. ‘The metal is firmly associated with the protein moiety of the enzyme. Consequently, the metalloenzyme can be isolated from its matrix, retaining all of its metal complement in the “natural” state (Edsall, 1951). During purification, the ratio of metal to enzyme-protein increases. With complete purification, a protein homogeneous by physical-chemical criteria is obtained, and the ratio of metal to protein becomes constant (Keilin and Mann, 1940a). 2. In the highly purified state, the ratio of moles of metal/moles of enzyme becomes an integral number, attesting to the specificity of the inter-

TABLE I Metalloproteins (Including Metalloenzyntes) Previously Described

Protein

Source

Metal

pg

Metal/gm. protein

References

I

cu

Hepatocuprein Cu Protein of liver Ascorbic acid oxidase Butyryl CoA Dehydrogenase Ceruloplasmin Laccase T yrosinase Uricase

Bovine milk Bovine erythrocytes Helix pomatia Busycon canaliculatuni Octopus vulgaris Loligo peali Limulus polyphemus Homarus americanus Bovine liver Equine liver Cucumis sativus Bovine liver mitochondria Human serum Rhus vernicifera Psalliota campestris Pig liver

c 1 1

Chlorocruorin Ferritin Hemergthrin Hemoglobin hZyoglobin Catalase Cytochrome c DPPu’H-Cgtochrome Reduc-

Spirographis spallanzanii Equine liver Sipunculus nidus Equine erythrocytes Equine myocardium Equine liver Bovine myocardium Pig myocardium

Fe Fe Fe Fe Fe Fe Fe Fe

“Cu-bearing” Protein Hemocuprein Hemocyanin

tase

cu

cu

1,900 3,400 2,450

Dills and Nelson (1942) Manu and Keilin (1938) Hernlet and Philippi (1933)

2,450

2,500

cu cu CU cu

cu CU

cu

2,600 1,730 1,870 3,400 3,060-4,100 2,500 3,450 3,400 2,400 2,500 550

12.000 170, &230,000 9,900 3,350 3,450 900 4,300 2,700

W

n

Jlann and Keilin (1938) Mohamed and Greenberg (1954) Dunn and Dawson (1951) Mahler (1954) Holmberg and Laurel1 (1948) Kcilin and Mann (19100h) Danson and blallette (1945) Mahler, h u m , and Huebscher (1955) Roche and Fox (19333 Laufberger (1937) Roche (1935) Zinoffsky (1886) Theorell (1932) Agner (1938) Theorell and h e s o n (1939) Ilahler and Elowe (1951)

a e

r 4

+

P F

m crl

Lactoperoxidase Peroxidase Verdoperoxidase Liver aldehyde oxidase

Bovine milk Armarocia rustieana Human leukocytes Pig liver

Xanthine oxidase

Bovine milk

Arginine transphosphorylase

Crayfish muscle

Leukocyte Zn protein Carbonic anhydrase Carboxypeptidase Alcohol dehydrogenase

‘Human leukocytes Bovine erythrocytes Bovine pancreas Yeast

Fe Fe Fe Mo IFe(P0R)I Mo Fe Mn

700 1,200 1 ,ooo 160 95 300 11400 2,670

Zn Zn Zn Zn

3,000 3,300 1,820 1,sOp

Theorell and Pedersen (1944j Theorell (1942) Agner (1941) Mahler, et al. (1954) Richert and Westerfeld (1954) Szorenyi, Dvornikova, and Degtyar (1949) Vallee, Hoch, and Hughes (1954) Keilin and Mann (1940a) Vallee and Neurath (1954, 1955) Vallee and Hoch (1955a, b)

5

*z

w p.3 w

324

HEI1T L. VALLEE

action and implying stoichiometry with specific reactive groups of the apoenzyme (Dawson, 1950). 3. When a metalloapoenzyrne interacts tvith a coenzyme (vide infra) or :I prosthetic group, correspondence between the numbers of moles of coenzyme arid the riumher of moles of metal contained in thc apornzyme lrrids further significance to the presence of the metal (T’allee and Hoch, 1955b). 4. The metal, in all instances known thus far, is a reactive group of the enzyme molecule. During purification, the metal/protein ratio rises concomitantly with the specific activity of the enzyme, implying correlation between these two parameters. With complete purification, the ratio of metal/specific activity becomes constant, as does the ratio of metal/proteiii and activity/protein (Keilin and Mann, 1940a; Vallee and Neurath, 1955). The addition of agents known to have high affinity for the metal and steric properties compatible with the configurational properties of the site of location of the metal, results in complete inhibition of enzymatic activity when the metal-inhibitor complex is formed at appropriate pH, concentration of the inhibitor, time, and temperature. Inhibition may be reversible when a dissociable complex is formed between inhibitor and metal, and the latter remains bound to the protein molecule. It is apparently irreversible when the metal is removed from the protein (Keilin and Mann, 1940a). 5. The metal is bound very firmly to the protein molecule. The very fact that the metal-to-protein bond is maintained through purification under widely varying conditions of the physical-chemical environment attests t o the strength of the bond. The metal is removed with extreme difficulty and usually only by drastic measures. When this is accomplished, activity is lost proportionately to the metal removed implying the severance of a specific linkage.2 The addition of ions of the bound metal or In the case of the copper metalloenzymes, there have been several attempts t o reconstitute the enzyme by replacing the metal previously removed from the protein (apoenzyme) (Kubowitz, 1937,1938; Meiklejohn and Stewart, 1941 ; Allen and Bodine, 1941; Tenenbaum and Jensen, 1943; TissiBres, 1948; Lerncr el al., 1950; Mahler and Green, 1954). I n general, this has been accomplished by :mmonium sulfate precipitation and dialysis follouing treatment by cyanide. The addition of a two- t o teIifold excess of copper has been required for the restoration of full activity, although 1,erner el al. (1950) have been able to regain 90% activity in a preparation of mouse melanoma tyrosinase by the addition of exactly the amount of copper removed. Little to no activity is restored by the addition of iron, zinc, cobalt, magnesium, manganese, or nickel t o the apoenzyme. The interpretation of these results is somewhat complicated by the catalytic properties of copper ions and copper-protein complexes, but there seems little doubt a t present that the enzymatic activity can be a t least partially restored by the addition of copper t o the metal-freed enzyme Similarly, neither iron nor copper can substitute for molybdenum once removed from metal-free xanthine or aldehyde oxidase, and up t o 5000 times the number of molecules of molybdenum removed are necessary for reactivation of one molecule of apoenzyme (Mahler and Green, 1954).

ZINC AND METALLOENZYMES

325

any other riielal to the highly purified metalloenzyme may result in lowered, raised, or identical enzyme activities, but this is not necessarily a guide t o the presence or function of the “intrinsic” metal. Extraneous ionic species may be differentiated from the “intrinsic” metal by simultaneous measurement of total metal content, activity, and protein. While the “intrinsic metal”/protein ratio increases during purification, all other “extrinsic metals”/protein ratios decrease concomitantly, without affecting the activity. The ratio of “extrinsic metals”/protein may be lowered t o absolutely and stoichiometrically insignificant values by dialysis or recrystallization (Vallee and Hoch, 1955). 6 . The metal constitutes a “reactive” group of the enzyme. It represents a “tag” which has the chemical features typical of the metal in question and similarly entails to the enzyme attributes which allow of precise measurement of the apoenzyme, coenzyme, and substrate interaction by techniques characteristic of the study of metal complexes in simpler systems (Calvin, 1954). Though some of the features of the inorganic chemical behavior of the metal may be retained, the bonding to the prot,ein ligand usually alters many of them drastically (Williams, 1953). 7. This type of specific association permits the assignment t o the metal of specific biochemical and physiological roles in metabolism (Keilin and Mann, 1940a; Williams, 1953). 8. The metals in metalloenzymes belong to the first and second transition group (molybdenum) of the periodic system (Vallee, 1954). Element 30, zinc, takes a special place among the first transition group, encompassing numbers 22-30 of the periodic system, since in contrast to all others, it can assume only one oxidation state, having a complete 3 d shell in the ground state as the doubly charged ion.

IV. CHARACTERISTICS OF METAL-ENZYME COMPLEXES I . The metal is bound loosely to the protein and dissociates readily, and therefore the two cannot be isolated jointly in the “natural” state. Since the association is subject to variations in the physical-chemical environment, metal analyses of different preparations frequently yield varied and inconsistent results. The apoenzyme can be readily obtained metal-free, and the binding is therefore much less specific than that in metalloenzymes (Williams, 1953; Gurti, 1954). 3. Chemical stoichimnetry between nietal uiid eiizyiiie has not been studied exhaustively but appears less rigid than in the previous group. The degree of binding, in large part, seems to be t: function of the concentration of the metal, without reaching a specific and limitiiig integral ratio of moles of metal to mole of protein. The ligands involved seen1 to have relatively low affinity constants for the metals involved (Williams, 1953; Malmstrom,

326

BERT L. VALLEE

1953, 1954; Gurd, 1954), though the interaction of enolase with Zn++, for instance, is stronger than that found in its non-specific binding to imidasole groups, e.g., in serum albumin (Malmstrom, 1953 ; personal communication, 1955). 3. The lack of stoichiometry between cofactor and apoenzyme interaction is analogous to the variable metal content. 4. Strict proportionality of activity to metal content is absent. Complete removal of the metal may not result in the complete abolition of activity in manyinstances. The percentage increase in activity on addition of metal ions to the purified protein is generally used as the criterion of the “esseiitiality” of a metal in the catalytic process (Lehninger, 1950; Lardy, 1051; Najjar, 1951; Vallee, 1952; Williams, 1953; MrElroy, 1953). Since the ratios of metal/protein or metal/activity have not been ascertained prior to and after the addition of the metal, it is difficult to form judgments as to the function of the metal on the basis of available evidence. 5 . The association constant of the metal with the reactive group of the protein molecule is low, and the metal is therefore readily removed by dialysis; removal is accompanied by a partial loss of activity often restored by readdition of the metal. Diflerent metals may substitute for one another in bringing about “activation” of enzymatic activity. This substitution has not lent itself readily to systematization on the basis of known properties of the metals or the possible reactive groups involved. The elements substituting for one another in bringing about activity are frequently dissimilar chemically, and the physical parameters likely to provide a universally acceptable common denominator have not been descerned thus far (Lehninger, 1950; Martell arid Calvin, 1952; Williams, 1953). Some of these problems have recently been discussed in connection \I ith experimental data on the interaction of enolase with Mg++, Zn++, Mn++, and Fe++, which activate the enzyme, and of many other ions forming inactive complexes. These data give indication that methods for their analysis are beginning to be worked out, however (Malmstrom, 1955). 6. The difficulties encountered in the characterization of these enzymes are at!tributahle directly to the experimental and technical difficulties c i i couiitered in cstablishing the t’xistenoe of specific binding sites of metall protein responsihle for catalytic activity (Gurd, 1954). 7 . The physiological and biorhcniicd significnncc of‘ iiietul “ w ti~ n tio ii” in cnzynio catalysis is dificult to iiiterprcbt. Sinw tho ussoriutioti is t e i t u ous, specific physiological roles caniiot be assigned to one cleirient iri preference to another, SLIVCby teleology. Where this has been attempted, this cmweutrat ioii of zt nictal fouiitl i l l :t pnrtirular (*ellor tissuc oltrii Ii:ts beeit tukcii ns n guidv to tlic assigtinient of a specific role in the function of a11(’11zymc. Thus, while manganese, iron, cobalt, and nickel are all known to

ZINC AND METALLOENZYMES

327

activate arginase, niaiigaiiese is regarded as the “biological” activator (Richards and Hellerman, 19-20) because of its supposed coilcentrat iorial preponderanc’e--an assumption not borne out by available and singificant nnalyticd data. Similarly, studies of rneymatic activities in tishucs of plants and animals deprived of mctals havc. yielded iiic.onc~lusiveanswers (McElroy and Sason, 1954). 8. Elements of virtually all groups uiid periods of the periodic table have been found to “activate” various enzymes. Keodymium, lanthanum, and samarium, e.g., which have iiot been found in biological tissues, activate the succinoxidase system (Horecker ct al., 1939), further increasing the clomplex task of finding common denominators for the behavior of metals. The above characterization of the metal-enzyme complexes is obviously not as satisfactory as that of the preceding group, heing beset] with many ambiguities arid qualifications. Such limitations in our knowledge do not detract from the interest in these systems, but it is apparent that the establishment of more precisely defined chemical criteria is needed. T’. F!MPIHICALFOI~MULAS FOR METALLOENZYMES

The above categorization attempts to delineate metal-enzyme interactions in terms of structural and functional biochemistry and aims a t the establishment of a working hypothesis and a subsequelit operational approach. The characteristics of the metal-protein bond serve as the primary parameter for the differentiation of metalloenzymes from metal-enzyme complexes. The spectrum of bond strengths is continuous, of course, and the present discussion focuses attention 011 its extremes and not on its center, where overlapping behavior must be expected-a situation teleologically related t o the behavior of acids and bases. Consideration of only the high bond-strength end of the metal-enzyme spectrum-the metalloenzymes-suggests a different investigative approach than is presently feasible with the metal-enzyme complexes. For the metalloenzymes, an empirical formula based on the molecular weight of the protein, the ratio of moles of metal/moles of protein, and that of moles of nietal/moles of coenzyme when present, expresses the stoichiometric relationships of the components of the active holoenzyme to one another. Such a system of notation views the active holoenzyme as a chemical complex in which the ligands are groups of the enzyme (designated by a common abbreviation) and of the coenzyme or prosthetic group. Brackets denote a high degree of relative strength of the bond between protein, metal, or coenzyme or prosthetic group, when present. The simplest case of this type may be exemplified b y carboxypeptidase, a metalloenzyme which does iiot require a coenzyme. 1. [(CPD)Zn]

328

BERT L. VALLEI.:

The iiiteractioiL of yeast, alcohol dehydrogenase, zinr, and T)PN t o form the active holoerizynie is therefore indicated as follows: 2 [(AI)H)Znl](I)I’S) n

Zinc is bound firmly to the ADII protrin. DPS is rwersihly h u n t 1 t o zinc, 1 mole of DPK per mole of zinc. The porphyrin-containing enzymes may be represented by catalase, 3. (CAT)[Fe(POR)]r

designating the stability of the assoriation of iron with the porphyrin ring. The FAT1 metal enzymes might be represented by I1PiYH-rytoc.hrornc~ reduct asc, 4. [(CCR)(E’AI))]Fe4

indicating the intrinsic association constants between FAD arid the proteiii to be high relative to those of iron. No effort is made to decide the site of binding. Table I1 lists the metalloenzymes for which molecular weights, metal contents, and the molar ratios of metal/protein have been published. The empirical formula, most likely at present on the basis of the above discussion, is presented. A metalloenzyme may therefore be considered t o have the following characteristirs: 1. The ratio of moles of protein to metal is an integral number. 2. The ratio of metal to coenzyme, when this latter is part of the active complex, is an integral number. 3. The highly purified protjein can be isolated with its full metal complement and full activity. 4. The ratio of moles of metal/protein or coenzyme is a small number, in conformity with the law of multiple proportions. These concepts have proved themselves pragmatically useful and led to the identification of three metalloproteins (Vallee, Hoch, and Hughes, 1954; Vallee and Xeurath, 1954; Vallee and Hoch, 1955a,b). In all instances, the firm association of the metal made it possible t o follow the course of purification of the protein by metal analyses. By contrast, the loose binding of metals in metalloenzyme complexes makes a precise presentation of empiric structural formulas difficult, since the tenuous nature of the association of metal and protein obstructs a definitive characterization of the system and the role of the metal in it.

VI. TNSTRUMEKTAL METHODSFOR THE DETECTIONOF METALS In view of these considerations, the analytical detection of metals becomes a significant aspect of work with metalloproteins. Many of the sen-

ZINC A N D METALLOENZYMES

329

sitive and highly precise procedures common to analytical chemistry and instrumental analysis are applicable. Sensitivity of the techniques is of great importance, since the total material available for analysis is usually severely limited. Spectrophotometry, spectrography, polarography, and other electrovhemical techniques, X-ray and electroil optics, ion-exchangc and chromatographic separations, isotope methods including neutron activation analysis (Tobias arid Durin, 1949) are examples of methods conimonly cmployed. A few general references are given for the interested reader. ('ombinations of these methods often become useful and are sometimes indispensable (Harrison, Lord, and Iioofbourow, 1948; Tobias and 1)imn, 194'3;Feigl, 1949; Snell and Siiell, 1949; Sandell, 1950; Ahrens, 1950; Sachtrieb, 1950; Mellon, 1950; Muller, 1951 ; Kolthoff and Lingane, 1952; Boltz, 1952; Hall, 1953; Lederer and Lederer, 1953; Samuelson, 1953; 0. C. Smith, 1953; Lingane, 1953; Delahay, 1954; Weissberger, 1949 and 1954). KOattempt will be made to detail in any way any of these techniques here, many of which are of common usage and have been developed to a high degree of perf ectiori. Emission spectrography takes a special place in our minds, since our approach has rested heavily on its use, and since its principles, applications, and promise have received little attentioil as a tool in protein chemistry. Elements excited by sufficieiit energy cmit light of characteristic wavelengths. The accepted mechanism of this effect is the excitation of atoms to higher electronic energy states, followed by emission of radiation when the excited atoms return to lower energy states, or when ionization takes place by complete ejection of an electron from the atom. The various transitions from higher to lower states are accompanied by the emission of radiation of specific wavelengths, characteristic of a particular element. The appearance of different lines of the same element and of lines of different elements is a function of the particular source employed for the ex(+ tation of the sample (Nachtrieb, 1950). Two essentially independent, processes condition this phenomenon : (1) volatilization of thc inorganic salts which constitute the sample; ( 2 ) excitation of the atom to a higher energy state. When the d.-c. arc is employed as a source, its essentially thermal characteristics first cause volatilization of the elements into the arc column. The rate of diffusion into the arc column is a function of the anode temperature, which in turn is a function of the applied wattage, the mass of the atoms, and-for ions-the field of force. Since the d.-c. arc operates at temperatures varying from 3000" to 8000" K. (Seminova, 1946), elements contained in the sample as compounds of low boiling points may volatilize very rapidly and may be lost from the arc stream prior to excitation (Vallee and Peattie, 1952).

TABLE11 Metalloenzymes of Known Molecular 1Veight ard Metal

Enzyme

Metal/ CoenMoleMolezyme/ Abbre- cular Per cent cular AKoleculur Metal/ viation weight metal Weight Weight Coenzymv

('otiteiit

l h p i r i c u l i'orniith

References

Cz1:

W

8

Ascorbic acid oxidase (summer squash) Butyryl CoA dehpdrogenase Ceruloplasmiri

AAO

146,000

0.26

6

Diinn and Dawson (1951)

AAD

120,000 220,000

0.345

10-1;

CPXI

150,OOO

0.34

8

-

Phenol oxidase (cultivated mushrooms) Fe: Cytochrome c

TTS

100,000

0.25

4

-

Green ~t n l . (1954) Mahler (1954) Ilolmherg and LaureII (1048) Mallette and Dawson (1941))

CTC

13,200

0.46

1

l(PO€t)

lFe/POIt

DPiVH-cjTtochrome reductase

CCIt

80,000

0.27

4

1(FADj

3Fe/FAD

44, loo

0.12

1

1(I'OR)

IFe/POI1

93,000

0.07

1

1 (POItj

lFe/POR

I

(CYC)[Fc(POI%)]

Pederson quoted by Theorell (1951) Neilands (1952) Mahler et al. (1952)

Theorell (1940) Theorell andMaehly (1050) Theorell and Pedersen (193.1)

-~

~

I

I.iver catalase

CAT

4 (1’0R)

lFe/POH.

(CAT) ,Fe (FOR 1 1,

XTO

230,000 Mo:O.03/ l(?2; 2 (Fall) Fe: 0.14’

j<-lMo/ FAD 4Fe/FAD

[(XTO)(FAD)2]?rIol-tFe8 PhiIpot (1939) Richert and Westerfeld

1Zn/D PN

I (ADH)Zni]( D P S ) i

Theorell (1947) Suinner and Gralen (1938) Agner (1938)

Alo and Pe:

Xarithine osidase

Zn: Alcohol dehgdrogenase (yeast) Carbonic anhydrase Carboxypept.idase

(1954)

I

ADH

150,Ooo

1

0.i8

1

4

CAD

30,000 0.2-0.33 1(?Z:

CPD

34,300

0 18

1

4(DPK)

-

-

[ (CAD)Znl-?]

-

-

[(CPD)ZnI

Hayes and Velick (1954) Vallee and Hoch (1955) Scott and Fisher (1942) Keilin and Mann (1940) Green and Neurath (1954) Vallee and N w r a t h (1951)

332

T3lCltT L. V.4LLElC

The excitation of atoms and ions, once present in the d.-c. arc, is a fuiiction of the electrode material, shape and separation of the elrvtrode, the voltage drop across the system, and the amperage applied. When the radiation emitted from one or several elements is dispersed by a prism or a diffraction grating, its component wavelengths may be recorded on a photographic film or plate, where the light at each wavelength will bring about blackening in the form of a line. The light-gathering power of the spectrograph, the range of the spectrum recorded, and the characteristics of the spectrum plates employed add additional variables to be considered in the evaluation of both the qualitative and quantitative data obtained. The technique has the unique advantage of allowing the simultaneous qualitative and quantitative analysis of most of the biologically important elements quickly and accurately. This is true for concentrations as low in t o 1 X l0-O g. per gram of sample. Under apsome instances as 1 X propriately (+ontrolledconditions, the light emitted and hence the blackening of the photographic plates is proportional to the amount of an element present in the sample. The density of lines is therefore an index of the amount of the element present, and the blackening can bc measured accurately with a microdensitometer. We have recently developed emission spectrographic techniques (Vallee, unpublished data), allowing the simultaneous measurement of some 20 elements with high sensitivity, accuracy, and precision, and with samples no larger than a few milligrams. Whereas quantitative emission spectrographic analysis yields accurate identification and quantificoation of elements present, an attempt at semiquantitative interpretation frequently leads to erroneous conclusions. This unfortunate circumstance and its frequent abuse have brought the technique into disrepute, and, as,a result, its obvious and desirable features have been largely ignored. Although the technique is simple in principle, the achievement of accurate, quantitative data is a time-caonsumiiig arid tedious process, mostly owing t o photometric problems. With the advent of photoelectric devices for measurement of line intensities, this should be simplified greatly. VII. REFERENCES TO METALLOENZYMES CONTAINING COPPER,IRON, AND

MOLYBDENUM

The chemical characteristics of the metal ciit ail sperific properties to the metalloenzymes which are riot encountered in enzymes lacking such groups. Fe, Cu, and Mo generally contribute their typical absorptions of visible light to the proteins to which they are attached. Their oxida-

ZINC AND METALLOENZYMES

333

tioii/reduction potential can be measured. Their physical properties are guides to their sites of binding by the protein, and the characteristics of the bond and its strength can be examined. Similarly, the presence of a metal and its possible participation in the formation of a metal/substrate complex delineates the structural possihilit ies for binding arid indicates specific mechanisms for catalyses. This discussion does not attempt a review of all metalloeiizyrnes listed in Table 11. With the exception of the zinc enzymes, all of them have recently been discussed, arid an additional review would serve 110 purpose (Nelson and Damon, 1944; Granick, 1947; Leniherg and Legge, 1947, 1950; Warburg, 1949; McElroy and Glass, 1950; Dawsori and Tarpley, 1951; Chance, 1951; P a d , 1951; Theorell, 1951; Drabkin, 1951; Singer and Kearney, l954a; Haurowitz and Hardiii, 1954). The rapidly accumulating iiiformation on the nietalloflavoproteins (De Itenzo et al., 1953, 1954a, b; Richert and Westerfeld, 1954; Mackler, Mahler, and Greeii, 1954; Mahler and Elowe, 1954; Nason and Evans, 1953; Nicholas and Nason, 1954; Kicholas, Nason, and McElroy, 1954; Mahler, Fairhurst, and Mackler, 1955b) has been reviewed and brought up t o date (Mahler and Green, 1954; McElroy and Nason, 1954). The information on their structure, available at this time, has been incorporated in Table 11. 13. ZINC METALLOPROTEINS

VIII. INTRODUCTION Four zinc metalloproteins have been characterized thus far: the carbonic anhydrase of ox erythrocytes (Keilin and Mann, 1940a), the zinc-containing protein of human leukocytes (Hoch and Vallee, 1952; Vallee, Hoch, and Hughes, 1954), the carboxypeptidase of bovine pancreas (Vallee and Neurath, 1954,1955), and the alcohol dehydrogeiiase of yeast (Vallee and Hoch, 1955a, b). Until very recently, the presence of zinc in carbonic anhydrase was the only conclusively proved role of this metal in metabolism.

IX. CARBONIC ANHYDRASE 1. Physical Properties The nieasurement of activity, isolation and purification, activation, inhibition, and distribution of carbonic anhydrase have been reviewed (Van Goor, 1948; Vallee and Altschule, 1949; Roughton arid Clark, 1951; Weier and Stocking, 1952). A suniinary of its physical and chemical properties will be given here. The existence of carbonic anhydrase was first described by Brinkman, Margaria, Meldrum, and Roughton in 1932. Meldrum and Roughton (1932a, b, 1933) purified the enzyme by means of an ammonium sulfate pre-

334

BERT L. VALLEE

cipitation similar to one Tsuchihashi (1923) described for catalase. Erythrocytes are the most commonly used sources of the enzyme, which is most frequently prepared from bovine red cells (Keilin and Mann, 1940a). Carbonic anhydrase has not been observed in ultracentrifugally or electrophoretically pure form. The best preparations seemed to contain impurities amounting to about 15 % (Petermann arid Hakala, 1942). While Scott and Fisher (1942) have claimed to have crystallized the enzyme, there is doubt in this regard (Keilin and Mann, 1944). The molecular weight has been estimated to be approximately 30,000 (Eirich and Rideal, 1940; E. C. 13. Smith, 1940; Petermann and Hakala, 1942). The isoelectric point is in the neighborhood of 5.6 (Roughton, 1943). The enzyme is stable as a dry powder but has been found to lose activity in dilute solutioii as a function of its purity, dilution, and the temperature of storage. Davis (1955), however, has found no such change with carbonic anhydrase from human erythrocytes. Activity is destroyed below pH 3 and above p H 13. 2. Chemical Composition The amino acid composition of the enzyme has not been determined. The purest preparations obtained from ox erythrocytes were found to contain 14.95% N (Keilin and Mann, 1940a), 14.9% to 15% N (Leiner and Leiner, 1940), and 15.9% PI: (Scott and Mendive, 1941h). The presericc of tryptophan, arginine, and phenolic groups has been reported (Meldrum and Itoughton, 1933; Scott and Mendive, 1941b). The cystine content was found to be 1.3 % and the tryptophan content 4.1 % (Scott and Mendive, 1941b). Free-SH groups could not be detected (Haugaard, 1946). Keilin and Mann (1939, 1940a) first noted that zinc is a constituent of carbonic anhydrase. Leiner and Leiner (1940) and Hove, Elvehjem, and Hart (1940) corroborated these findings. Day and Franklin (1946) found zinc in carbonic anhydrase extracted from plants. The figures 011 the percentage zinc content vary. Keilin and RIann (1940a) found 0.33%, Hove, Elvehjem, and Hart, (1940) 0.318 % for enzyme preparations of coniparable purity, as judged by their activity. Scott and Mendive (1941b) reported 0.15% of zinc for their purified and 0.2% to 0.23% for their crystalline preparations (Scott and Fisher, 1942). Assuming a molecular weight of 30,000, the zinc contents reported by Keilin and Mann (1940a) and Hove, Elvehjem, and Hart (1940) correspond t o 1.52 and 1.46 moles of zinc per mole of ox carbonic anhydrase. The data of Scott and Mendive (1941b) are equivalent to 0.92 and 1.04 moles of zinc per mole of protein. Since the molecular weight is not known with certainty and the enzyme has never been analyzed for zinc when found homogeneous by gravitational or electrophoretic criteria, the e x a d stoichiometry of zinc to protein is not settled with finality. Though human carbonic an-

ZINC AND METALLOENZYMES

335

hydrase has never been purified adequately, the data of Keilin and Mann (1940a) may indicate a difference in zinc content for the ox, sheep, and human enzyme. Carbonic anhydrase activity and zinc content of human erythrocytes are closely correlated, indicating that all zinc in human red cells is accounted for by the enzyme (Vallee, Lewis, Altschule, and Gibson, 1949). It is thus feasible to estimate the enzyme conttmt of red blood cells (Keilin and Mann, 1940.). Based on these considerations, 100 cc. of erythrocytes are calculated t o contain 0.21 g. of carbonic anhydrase or 1% of the hemoglobin content (Vallee and Altschule, 1949). One liter of mammalian blood contains approximately I g. of carbonic anhydrase. Less sat,isfactory correlation between activity and zinc content in other orgaiis led to suggestions (Mann and Keilin, 1940a) that the existence of other zinc proteins accounted for the discrepancy. Keilin and Mann (1040a) noted (1) direct proportionality between enzyme, activity and zinc content; ( 2 ) similar zinc contents, proportionate to their activity, in preparations obtained by completely different procedures and from different sources; ( 3 ) the absence of iron, copper, manganese, lead, and magnesium from the highly purified enzyme. These facts, together with the high zinc content of purified preparations (vide supra) and inhibition data (vide infra), established carbonic anhydrase as a zinc protein. This view is enhanced by the fact that splitting off the zinc leads to irreversible inactivation. They also found that other zinc rompounds, such as inorganic salts, organic complexes, zinc porphyrins, zinc insulin, and zinc complexes of serum and tissues are devoid of carbonic anhydrase activity. The linkage between the metal arid protein is unknown (Roughton and Clark, 1951). Electrodialysis (Scott and Mendive, 1941b) and exchange experiments with purified carbonic anhydrase from ox blood and Znes (Tupper, Watts, and Wormall, 1951) have shown that zinc in carbonic anhydrase is very firmly bound. No exchange of the isotope against the nonradioactive zinc in the enzyme was observed over a period of 32 days. Van Goor (1945) reported that the enzyme could be split into thermostable and thermolabile fractions. This work was extended by Keller (1955), \\rho verified Van Goor’s observations. He reported the thermostable fraction to contain zinc and a tripeptide consisting of two glutamic acid and one glycine residues. This tripeptide does not have carbonic anhydrase activity itself, but augments the activity of dilute solutions of the enzyme. Elementary analysis gave the following values for the tripeptide, the amino acid composition of which was determined by paper chromatography : C : 36.41%; H: 4.25%; 0 : 32.8%; N : 10.6%; ZII: 16.4%

Significant details of the experimental procedures are omitted, and the conclusioiis are incompletely documented. These data are in need of sub-

536

BERT L. VALLEE

stantiation, and the supposition that this fragment is a coenzyme of carbonic anhydrase is open to question. 3 . Inhibition by Mctals a i d Z i n c C'ompl~ciugA gents

The ctizymc~is inhibited by several metals: Ag, Au, Cu, Hg, % I ) , cxrid V I)ring about 50 % inhibition in concentrations varying from to dT. Similar cwirentrations of RI, He, Ca, Cd, Cc, Co, Cr, Fe, li, Mg, Mil, Ka, Xi, Pb, Pt, and Ti have no effect (Meldrum a i d Itoughton, 1933; Van 6 o o r , 1934, 1918; Itoughton a i d Booth, 1946). It has been suggested that metals irihibiting the eiizynie arc protein precaipitants and exert, their inhibitory effect by virtue of this property (Roughton and Clark, 1951). Among the elements not found to inhibit, many are known to be protein precipitants also (Cd, Fe, Pb), leaving this explanation open to question. The enzyme is inactivated by a variety of agents thought to exert their effect by combining with zinc. Cyanide (Meldrum and Roughton, 1933; Stadie and O'Brien, 1933; Van Goor, 1934; Keilin and Mann, 1910a; Kiese and Hastings, 1940), sulfide (Meldrum and Roughton, 1933; Stadie arid O'Brien, 1933; Keilin and Marin, 1940a; Kiese and Hastings, 1940), azide (Meldrum and Roughton, 1933; Keilin and Mann, 1940a), and HAT, (Webb and Van Heyingen, 1946) produce marked inhibition of thc enzyme. The findings of Van Goor (1934) aiid Leiiier and Leiner (1040) disagree with regard to sulfide. Keilin and Mann (1 940a) reported that vyanide inhibits the enzyme 85 '3, i n concentrations as low as 4 X lop6M . They found inhibition completely reversible. The inhibition with carbon monoxide, found by Meldrum and Roughton (1933) and Van Goor ( ~ 3 4 was ) ~ not observed by Kiese and Hastings (1940). Keilin and Mann (1940a) adduce the instantaneous and reversible inhibition brought about by cyanide, sulfide, and azide as important evidence that a metal, in this instance zinc, must be the active group of carbonic anhydrase. Thiocyanate ioiis also strongly inhibit carbonic arihydrase (Davenport, 1939; Feldberg, Keilin, and Mann, 1940; Hove, Elvehjem and Hart, 1940). The latter authors thought that this action could not be attributed to complex formation between zinc and thiocyanate, since equimolar zinc ions preiricubated with this agent not only did not prevent the action but inhibited the enzyme further. This conclusion is operi t o question. No data on the inhibitory action of many of the chelating agents known to combine with zinc (Martell and Calvin, 1952; James, 1953) have been published. Similarly, there is little information on the kinetics of inhibition with the agents referred to above. Pending such data, therefore, their action on zinc can only be inferred, justifiable though this interpretation may bc.

ZINC AND METALLOENZYMES

337

Davis (1955), in unpublished kinetic studies of human erythrocyte carhonic anhydrase, has inyestigated the niechanism of inhibition of that enzyme a t pH 7 by sulfanilaniidc, various chrlating agents, and sodium sulfide. The chelatiiig agents, 1 ,l0-phenanthroliiicl tctrasodinm cthylcircdianiiiie tetraacetate, ethylenediamine, and methyl amine, produced no inhibition a t concentrations variously lo4 to lo6 times the enzyme colicentration, indicating to Davis that the zinc atom of carbonic anhydrase is very firmly chelated i n the protein molecular structure. Sodium sulfide (actually HS- or H2S at the pH of the experiment), which probably binds at the zinc atom, was found to be a veiy efficient iioiicompetitive inhibitor. Davis points out that inasmuch as the association constant for the enzyme and inhibitor was the same as that for the enzyme-substrate complex and inhibitor, the carbon dioxide cannot bind at the zinc atom. Davis suggests that the zinc niust participate in the catalysis either by imparting some special ccnfigurational property to the enzyme molecule or by contributing a n hydroxyl group or some related species to the hydration. Sulfanilamide was also found to be a noncoinpetitive inhibitor, possibly, like sulfide, binding to the zinc (Davis, 1955, personal communication). There is, however, no conclusive evidence that the marked inhibition of carbonic anhydrase by the sulfonamides, first observed by Keiliii and Mann (1940b) and ably reviewed by Van Goor (1948), results from the interaction of these compounds with zinc.

X. EXPERIMENTAL APPROACH FOR STUDIES ON PROTEIN,

THE LEUKOCYTE Zrsc CARBOXYPEPTIDASE, AND YEAST ALCOHOL DEHYDROGENASE

The studies on carbonic anhydrase just described were approached, in the main, from the enzymological and biochemical points of view, with chief emphasis on activity measurements as a criterion for purity. The following work, by contrast, was based on considerations emphasizing the metal chemistry of these proteins and involving metalloproteins as a group. The identification of zinc as a component of the leukocyte protein, carboxypeptidase, and alcohol dehydrogenase had certain common experimeiital features whichmight be summarized prior to a discussion of the details: 1 . Attempts a t establishment of protein purity. 2. Metal analysis of fractions. 3. Identification and measurement of enzymatic activity. 4. Inhibition of activity by zinc complexing agents. 5 . Examination of stoichiometry between zinc arid protein. 6. Correlation of zinc content, of fractions with activity. 7 . Demonstration of the absence of other metals. Since this approach is based on the contention that complexes containing

338

BERT L. VALLEE

molar metallprotein ratios exist in the natural state, the exact measuremelit of the metal content of the original material and knowledge of the molecular weight of the isolated protein is essential. During purification of the zinc protein from leukocytes, the catalytic activity of which, if any, has not been identified, rigorous avoidance of contamination with zinc was thought to be an absolute prerequisite. The zinc/protein ratio was considered a valid criterion of succesive purification for zinc-containing protein in thc natural state, provided that ( I ) the bond between metal and protein was sufficiently strong so that no significant amounts of metal dissociated from the protein during any of the operations performed; arid ( 2 ) all zinc found in the system was combined with the protein in the natural state, and none was introduced from extraneous sources. Only the ratio of moles zinc/moles ultracentrifugally homogeneous protein could serve as an ultimate index of metalloprotein purity. Therefore, elaborate precautions were taken to avoid “contamination” in the collection of material, its storage, and handling. Water was purified by repeated distillations or by ion exchange; chemicals employed in purification were recrystallized or extracted to be freed of metals. High-speed centrifugation could not be performed successfully, since the only containers withstanding the high centrifugal force were rich in zinc and readily released the metal to the sample. The reproducible isolation of similar fractions with comparable zinc content in successive fractionations seemed to justify these cumbersome precautions. The development, of emission spectrographic procedures and the choice of catalytically active proteins for purification have somewhat altered present evaluation and procedure. The simultaneous detection of all metals including zinc serves as a guide to contamination. The presence of extraneous ions and their decrease in concentration with increasing purity of the enzyme has become an index of the state of purity of the preparation, of the introduction of contaminants, and of the relationship of metals t o enzyme action. An increase of the zinclprotein ratio to a constant limiting value is found with increasing protein purity and increasing specific activity, while simultaneously all other metals decrease to absolutely and relatively insignificant cancent,rations. Contamination with both zinc and other metals is readily detected, and remedial action can be taken when indicated. The removal of extraneous metals often further increases specific activity. The procedures for the prevention of contamination during protein fractionation have therefore been modified. In particular, purifications of chemicals-employed during purification-with chelating agents are held t o a minimum to preclude their potentially deleterious effects on enzyme activity. High-speed centrifugations are carried out in metal-free plastic

ZINC AND METALLOENZYMES

339

containers now obtainable. Strict precautions are observed for all analytical work, aiming a t the differentiation of all “loosely bound” metals from “firmly bound” zinc.

XI. THE ZINC-CONTAINING PROTEIN FROM HUMAN LEUKOCYTES 1. History Emission spectrography and microchemical studies (230) showed that the zinc content of normal leukocytes is high (Vallee and Gibson, 1948a, b). Normal human white blood cells contain 3.2 f 1.3 X pg. Zn per niillion cells, a concentration higher by a factor of 25 than that found in the same number of human red blood cells (Vallee, Fluharty, and Gibson, 1949). Physiological studies with Zn66and enzymological investigations failed t o reveal any clue to its function (Vallee, Fluharty and Gibson, 1949; Gibson et al., 1950; unpublished data). The zinc content of white blood cells from the blood of human patients with leukemia is reduced to 10% of the normal value. Temporary therapeutic amelioration of the leukemic process, as manifested by a decreased number of circulating leukocytes, is accompanied by a rise in the concentration of zinc in these cells to normal values (Gibson et al., 1950). Since at the time a t which this work was undertaken carbonic anhydrase was the only known zinc metalloenzyme (Keilin and Mann, 1940a), and no carbonic anhydrase activity could be detected in leukocytes (Van Goor, 1948), the work on the purification of the zinc-containing protein of leukocytes (Hoch and Vallee, 1952, 1953) was undertaken to elucidate the physiological and biochemical significance of zinc in human leukocytes through a study of its association with other components of these cells. 2. Procedure

Preliminary experiments suggested that appreciable amounts of zinc in leukocytes are firmly bound to proteins, since only a very small fraction of the metal was found dialyzable. White cells from human subjects were frozen, thawed, diluted with distilled water, and then extracted with ether to remove lipids. The total white cells contained 13,000 pg., the materials extracted by ether contained 39 p g . of zinc, or 0.13% of the total zinc present. Investigations, therefore, centered on the zinc presumably bound t o lipid-free protein. Leukocytes were obtained from 450-ml. aliquots of the blood of human donors. The leukocyte “buffy coats” were poured off from the surface of erythrocytes, packed by centrifugation. To obtain adequate quantities of leukocytes, 400 to 500 bleedings had to be handled in this manner, and, consequently, relatively large quantities of erythrocytes were included, even though further centrifugations and decantations resulted in their progres-

340

BERT L. VALLEE

TABLEI11 Determination of Z i n c , Protein, and Oxyheinoglobin on Mi.cticres of H u m a n Leukoc!/lc.s and Erythrocytes after Thawing”

WBC sample no.

y

I1

I11

+ Buffer

IV

v

‘I

~-

(e)

(d)

(S)

Total protein (b)

!1.4 17. I* 8.2 8.2 8.4 10.8 3.0

mg. per mg. per ml. ml. 86.2 81.8 82.9 10.3 80.1 126.8 47.2 123.4 44.7 18.4 42.9 101.0 13.7

8.0

(h) =

Erythrocyte zinc

Total zinc (a)

per nil.

1

Oxy- Total 1,eukohemo- zinc/ cyte globin protein protein (c)

per mg. per gm. ml. 109 99 72.6 102 69.8 79.6 66 76.2 88 26.3 67 79

(9)

(h)

per ml.

y prr

-

-

0.7

7.8 7.8 6.5 8.9 2.3

107 112 82 117 88

0.6

7.4

85

(4)

y

87.3

TJcuko1,euko- cyt,c cyte zinc/ zinc protein

per ml.

y

0.4 1.9

y

gm.

(g)

x

10-3

From Hoch and Vallee (1952). Contamination.

sive elimination. Table 111 shows data on five representative mixtures of human leukocytes, identified by roman numerals, which were employed as the starting material of fractionations and in which zinc protein and oxyhemoglobin were measured. Column c shows the variation in amounts of red cells present with the leukocytes employed for fractionation. The weight of protein (Column 0 ) is composed of that contained in leukocytes and erythrocytes; an estimate of the protein content of leukocytes can be made by subtraction of the hemoglobin from the total weight of protein as in column c. The zinc to protein ratio of leukocytes is obtained by use of correcbted values for zinc and protein; such ratios are shown in column h and are irk good agreement with one another. Since all zinc of human erythrocytes is presumably structurally associated with carbonic anhydrase (Keilin and Mann, 1940a; Vallee et al., 1949), the identification of this enzyme in subsequent fractionations provided an indirect check on these calculations. The only fraction in the process of purification exhibiting significant carbonic anhydrasr activity was precipitated between 0.85 and 0.90 of full saturation with ammonium sulfate (Table IV).

341

ZINC AND METALLOENZYMES

TABLEIV Pilot Fractionation to Denionstrafe the Separalzon of (‘arbonic Anhydrase f r o m Other Fractions Containing Zinc Precipitates between ranges of saturation with ammonium sulfate 0 -0.20 0.20-0.30 0 30-0.40 0.40-0.50 0.50-0.60 0.60-0.70 0.70-0.80 0.8CkO. 85 0,854.90 ~

a

Zinc (M.1 16 40 38 148 41 21 24 70 63

Protein

(w.) 153 195 186 266 83 111 155 1,035 298

Zinc/l’rotein (pg./g.)

Carbonic arihydrase activit ya -

107 205 204 556 492 206 172 68 210

0 0 0 0 0 0 0 0 11.5

Units as defined by Vallee, Hoch, and Hughes (1951).

The total zinc content of this fraction corresponded closely to that calculated to come from erythrocytes on the basis of the data given in Table 111. Previous work (Meldrum and Roughton, 1932a; Tupper, Watts, and Wormall, 1951; Vallee, Hoch, and Hughes, 1954) has demonstrated that carbonic anhydrase is precipitated at high saturation of ammonium sulfate. These data and calculations indicate, therefore, by correlation of measurements of enzyme activity, zinc content, and the known characteristics of the protein, that two distinct and different zinc-containing proteins exist in leukocytes and erythrocytes and can be differentiated. A zinc protein containing 82 to 117 pg. of zinc per gram of protein was obtained reproducibly from leukocytes by extraction with phosphate buffer for a total period of 12 to 24 hours, p H 7.2 a t 4” C., and p = 0.01. In this manner, a soluble fraction comprising 81.1% of zinc of leukocytes and 79.6 % of protein of leukocytes could be obtained. The remaining 18.9% of leukocyte protein were insoluble under tlhe conditions of extraction. This insoluble zinc fraction has not been investigated beyond establishing it as distinctly different from the soluble zinc protein. Zinc was not associated with fractions of nucleic acids (Hoch and Vallee, 1952). The soluble proteins were precipitated between 0.4 % and 0.6 % of full saturation of ammonium sulfate, where a high zinc/protein ratio was obtained. This fraction xvas solubilized with phosphate buffer p H 7.2, p = 0.01, and the residue was removed by centrifugation a t 17,000 r.p.m. At pH 6.0, p = 0.1, and in the presence of 10%ethanol, a precipitate could be obtained. Extraction of this precipitate with carbonate-bicarbonate buffer, pH 9.4, p = 0.1, resulted in material which had a zinc content of 3000

Leukocytr

I’rotcjin

TABLEV Precipitated between 0.4 and 0.6% of Full Saturation with Ammonium Sulfatea Zinc/protein, Gcg./g .I

Experiment

a

Dialysis against :

Fractions

Dialysis against:

Fractions

I

I1

1.

HzO

FSupernatant fluid \Precipitate

841b 1,430b

96 1,070

2.

HzO

PSupernatant fluid \Precipitate

236 1,250 1,540

261 2,000 1,570

7 Supernatant

Buffer pH 9.0, pO.lLResidue

fluid

3.

Buffer pH 6.0 p-0.01 \

4.

Buffer pH 6.0 p-0.2

5.

Buffer pH 6.0 fi-0.1 7 Supernatant fluid LPrecipitate Ethanol 10%

6.

Buffer pH 6.0 p-0.1 f Supernatant fluid L Gxipitate Ethanol 10%

From Vallee, Horh, and Hughes, 1954. Contamination.

YSupernatant fluid

Precipitate

310 3,000 1,440

r r

n

m 689 308 895

PSupernatant fluid Buffer pII 9.4, fi-0.1\Residue

@

3

2r

229 788 423

td

444 2,930 760

ZINC AND METALLOENZYMES

343

pg. per gram of protein (Table V). Other metals were shown absent or present in insignificant concentrations by emission spectrography (unpublished experiments). 3. Physical Properties

Ultracentrifugal studies as t30t,he homogeneity of this fraction were unsatisfactory because of the limitations of materials. No final statement can, therefore, be made concerning the molar proportions of zinc to protein, pending establishment of the molecular weight. However, the minimum molecular weight based on zinc content would be 21,700. The magnitude of the zinc content is comparable to that encountered in other metalloproteins (Tables I , 11). The isoelectric point was found close to p H 6.5. 4. Chemical and Enzymatic Properties Sothing is known about the amino acid composition of the protein which absorbs light a t 280 my indicating the presence of aromatic amino acids. There were no characteristic absorption maxima in the visible part of the spectrum. Attempts a t identification of the activity of this protein have not been successful thus far. It does not have any activity akin to that of carbonic anhydrase, alcohol dehydrogenase, carboxypeptidase, or dehydropeptidase. In this sense, it can be compared to hepatocuprein, hemocuprein, or the copper protein of Mohamed and Greenberg (1954) (possibly identical with hepatocuprein), all metalloproteins without identified enzymatic act,ivity. Further studies along these lines may reveal the biochemical significance of the presence of this protein.

XII. PANCREATIC CARBOXYPEPTIDASE 1. Physical Properties The carboxypeptidase of pancreatic juice was first isolated in crystalline form by Anson in 1937a. The physical, chemical, and enzymatic properties of this enzyme have been the subject of several recent comprehensive rcviews (Keurath and Schwert, 1950; E. L. Smith, 1951a, b, e ; Green and New rath, 1954) to which the reader is referred. The known physical and chemical properties of the enzyme will be described here. The enzyme is prepared from the exudate of frozen sliced beef pancreas. The precursor, procarboxypeptidase, is activated at 37" C. pH 7.8 for I hour; a crystalline enzyme is obtained by fractionation (Green and Neurat,h, 1954). Sevenfold recrystallization yields an enzyme which has been found essentially monodispersed by electrophoresis a t pH 8.5, but a minor, faster inoving component appears at pH 9.3 (Putnam and Keurath, 1946). Thc molecular weight has been calculated to be 34,300 (Putnam and Neurath,

344

BERT L. VALLEIC

1946; E. 1,. Smith, Brown, and Hanson, 1949; Smith, 1951b, c ; Green arid Neurath, 1954). The enzyme is unstable below pH 6 (Anson, 1937a, b) and above pH 10.4 (Green arid Neurath, 1954). The p H optimum is 7.5, and the isoelectric point corresponds to p H 6 at ionic strength of 0.2. 2 . Chemical Composition

a. Basic Composition. Analysis of the amino acid composition of the enzyme reveals no characteristk features differentiating this peptidase from other proteolytic enzymes (E. I,.Smith and Stockell, 1954). The relatively high content of serine and threonirie and the presence of only one residue of inethionine were noted. The molecular weight, arrived a t on the basis of amino acid analysis, is in good agreemcnt with that calculated on the basis of ultracentrifugal data. b. il4agnesium. I t has been reported (E. L. Smith and Haiison, 1948) that qualitative emission spectrographic analysis of the ash of five times recrystallized carhoxypeptidase showed it t o contain significant amounts of M g and traces of Fe and Cu. Zii, Mn, Co, Ra, and I,i could not be detected. On the basis of these and other data (vide infra), carboxypeptidase was thought to be a magnesium metalloeiizyme with the metal firmly bound t o the protein and essential to its activity. E. L. Smith (1954) has since retracted this claim and stated that carboxypeptidase is not a metalloenzyme. lTntilrecently, no further metal analyses have been reported. Since quantitative data mere not given, and the methods employed for spectrographic analysis were not detailed, it is not possible to examine the validity of these observations. It should be pointed out that the usefulness of qualitative emission analysis is severely limited and does not readily permit accurate inferences concerning the distjribution of inorganic ions in biological material (vide supra). c. Zinc. Recent quantitative emission spectrographic examinations of carboxypeptidase prepared according to published methods (Neurath and Schwert, 1050; Green and Neurath, 1054; Neurath, 1955) do not confirni these earlier studies. Analyses of several crystalliiie preparations with the high voltage spark uniformly indicated the presence of large quantities of zirw. All other rnetals were either completely abscrit or present iri minute quantities (Vallee arid Seurath, 1954, 1955). Samples were dry-ashed a t 450’ C. in a thermostatically controlled iinilfie furnacc. Internal standards \\ere addrhd to the ash. Alicluots \v(’rc sparked iu porous c ~ i pelectrodes (Feldman, 1949; Vallee, i i i preparation for publication). Jalm4l-Ash “Varisources” were cmployed to geiirratc and caitrol the spark. The two spectrographs were 21 ’ Wadsworth niouritirigs hut difl’&ig irr the ch:wacteristics of‘ their grutiiigs. On(. instrument was provided with a 15,000 line per inch grating reflecting maximally in the first-

345

Z I N C .4ND METALLOENZYMES

order violet. The reciprocal linear dispersions were 5.18 A. per millimeter and 5.25 A. per inillimctrr, respectively. Eastniaii Kodak 103-0 photographic plates wrre cniploycd throughout, recording radiation between 2200 to 4800 A. in oiie txposure. I h e s and backgrounds were measured tlcnsit ornet rically. Working ciirvcs wrrc prcparcd with spwtrosropically purc chemicals. Zinc8 was determined both c‘hcinic*ally(Vallce and Gibsoii, 19 18; Hoch and Vallee, 19-1-9)and spectroscopically to ensure accuracy. All analytical procedures were carried out with precautions against melal contamination. Water was obtained by slow passage through mixed I R 120 and IRA 420 ion-exc.hange resins (Rohm and Hans). The effluent had a specific resistance of a t least 1.5 x 106 ohms. Reagents were freed of metals when necessary and stored in acid-cleaned polyethylene bottles throughout. Table VI shows spectrographic analyses for all metals, as well as microchemical analyses for zinr and copper performed on four different preparations of the crystalline enzyme, and Fig. 1 shows a spectrogram of a fractionation. Ten t,o twenty milligrams of the enzyme were employed for analysis. Preparations nos. 1 and 2 were prepared by Dr. Hans Neurath and recrystallized five and six times, respectively. Preparations 3 and 4 were obtained from two dif‘ferent commercial sources and had been recrystallized five and three times, respectively. All four preparations had high enzymatic activities and showed comparable and consistently high zinc rontents, both by spevtrographir and chemical analysis. The observed differTABLEVI Metal Analyses of Crystalline (‘arhox~peptidasea Prepnrat ion number c

Element ‘I

Method

1

2

3

4

Dithizone Spectrograph). Spectrography Spectrography Spectrography Spectrography Spectrography Sliectrogrnphy Spect rography Spectrography

1,820 1,820 33 40 6 6 3 18 S o t found S o t found

1,770 Not done Not done 54

1,800 1,700 Not done 36 11 17 16 48 1 S o t found

1,980 1,665 61

~ ~ _ _ _ _

~

Zinc Zinc Copper Iron Aluminum Magnesium Calcium Barium Strontium Mnnganese a

Data expressed as microgram of metal per gram

4 6 38 0 S o t fourid X o t found

carboxypeptidase.

75 73 16 6 114 1 1

* Not fonnd: beryllium, boron, cadmium, chromium, cobalt, lead, lithium. molybdenum, nickel, ptins-

phorus, potassium, silver, tin. For descriptinn ( t f preparations, see the text. \From Vallee and Neurath, 19551

34G

BERT L. VALLEE

FIG.1. Pertinent sections of spectrograms representing analyses on a carboxypeptidase fractionation are shown. The lines of manganese at 2576 A , magncsiurn a t 2779 and 2802 A, zinc a t 3345 A, and calcium a t 4318 A are identified by marks. The fractions, the protein weight taken for analyses-allowing for the difference between samples in total quantities taken for analyses-and the zinc/protein ratios are indicated on the right-hand side. The spectra show t h e aggregation of zinc with purification, and the simultaneous disappearance of magnesium, manganese, and calcium in duplicate analyses. The bottom spectrum represents a reagent control, the top spectrum an iron reference, and the second spectrum from the top a 3 fig. per gram standard.

ences were entirely within the error of the technique; since zinc is easily lost on dry-ashing, the microchemical data obtained by means of the TC-4 precipitation technique were considered more reliable. Based on a molecular weight of rarboxypeptidase of 34,300, the ratio of moles of zinc per mole of carboxypeptidase for these prcparations lvas 0.96, 0.93, 1.04, and 0.95, respectively. All other elements were present in stoichiometrically and absolutely insignificant amounts. Preparations nos. 1 and 2 contained 5.7 and 6.0 p g . of Mg per gram of protein, and the two commercial preparations, nos. 3 and 4, 16 and 17 pg. of Mg per gram, respectively. Lithium, cniployed in recrystallization, was not detected; barium, added in large quantities during purification, was apparently removed during recrystal-

ZINC AND METALLOENZYMES

347

lization. The three times recrystallized material, no. 4, contained larger amounts of extraneous metals than did those recrystallized five or six times. In order to follow the metal content and the specific enzymatic activity through the process of isolation and crystallization of carboxypeptidase, aliquots of several fractions were analyzed for zinc and other metals, nitrogen, and enzymatic activity. The fractions were: ( I ) the crude pancreatic juice; (2) the euglobulin precipitate obtained after tenfold dilution with water of the activated juice, at pH 4.6; ( 3 ) the supernatant solution of the euglobulin precipitate; (4) the enzymatically inactive solution obtained by extraction of the euglobulin precipitate with barium hydroxide a t pH 6.0 (referred to in Table VII as Ba(OH), extract); and (5-9) the enzyme obtained after the first through the fifth crystallizations. For metal analysis these protein fractions were dried to constant weight first in a vacuum desiccator and then in an oven at 108” C. Protein concentration of fractions containing nonprotein nitrogen was determined by the micro-Kj eldahl method on the precipitate obtained by addition of 10% trichloroacetic acid to aliquots of the solubilized fraction. Enzymatic activities were determined on the freshly prepared fractions. Table VII shows metal analyses and activity measurements (Vallee and Neurath, 1955) obtained on pancreatic juice, the fraction attending the isolation of carboxypeptidase, and the five recrystallizations of the enzyme. The zinc content per gram of protein of pancreatic juice mas 310 pg. per gram and rose to 1870 pg. per gram in the first crystals. There was virtually no change in zinc content with recrystallization. The ratio of moles of zinc per mole of crystalline carboxypeptidase remained close to 1 throughout five recrystallizations. The specific activity in active fractions increased and was parallel to the zinc content. The ratio of specific activity/zinc rose as “extraneous” protein and “extraneous” zinc were removed. The individual concentrations of all other elements and their sum decreased with purification as zinc and specific activity increased (Table VII). Magnesium concentrations fell from more than 1000 pg. per gram of protein in the pancreatic juice to 29 pg. per gram of protein in the euglobulin precipitate, and to even lower levels in the crystals. Strontium, molybdenum, and iron were apparently introduced with barium hydroxide and were removed during crystallization. Chromium and lead appeared as spurious contaminants. A slight rise in calcium, magnesium, aluminum, and iron after the third crystallization was attributed to impurities in LiCl and/or water employed for recrystallization. Data in Table VI and VII indicated that recrystallization accomplished the removal of extraneous metals. There was apparently little change in protein composition of the crystals, as evidenced by the constant Zn/protein ratio.

TABLEVII dletal Content0 of Fractions Attending the Isolation of Carboxypeptidase f r o m Pancreatic Juice Olhcr Metalsd I’raction

(x,

Pancreatic juice Euglobulin precipitate Supernatant solutiori Ba(0H)p Extract 1st Crystals 2nd Crystals 3rd Crystals 4th Crystals 5th Crystals

bp.,‘gm.

protein)

310

590 450 410 1,870 1,860 1,850 1,880 2,000

Zinc* Activity‘ Activity Protein C - - Zinc x 103 0.16 0.31 0.24 0.21 0.98 0.98 0.97 0.99 1.05

0.18 1.43 0.05 0.05 13.2

0.58 2.43 0.11 0.12 7.05

*

*

14.5 14.0 18.6

7.85 7.45 9.30

-~ Sr

Ba

Ca

Mp

0.3 1.6 520 1,160 2.4 6.9 71.0 29.0 5.7 300 0.7 1,250 212 1,050 135 >1,OOO 3.9 >1,OOO 32.0 13.0 1.1 219 23.0 20.0 98.0 33.0 1.5 52.0 30.0 43.0 28.0 50.0 29.0

A1

Fe

Mn

150

Cu

Cr

Pb

1.9 65.0 4.7 - 19.0 19.0 160 * 1.7 33.0 20.0 66.0 5.2 - 43.0 11.0 760 3.7 41.0 51.0 32.0 130 - - 82.0 12.0 36.0 - * 4.2 - * 12.0 51.0 - * - 12.0 50.0 - * 10.0 -

Unless otherwise indicated, metal content is given in microgram per gram of protein. All zinc determinations by the dithieone method in duplicate, and in quadruplicate for the crystalline enzyme preparations. Copper determined by the sodium dithiocarbamate method. Spectrographic analyses were done in duplicate, and separately on two different instruments. The zinc/protein ratio has been calculated as the moles of einc/mleerdar weight of carboxypeptidase. Expressed W I fimtdrder rate constant (in decimal logarithm) per mg. of enzyme N per cubic centimeter. The symbol - denotes not found; *, not determined. [From VaUee and Neurath, 19551

’

ZINC .4ND METALLOENZYMES

348

Dialysis of carboxypeptidase for 18 hours against water or ammonia did not remove zinc from the protein. A preparation contaiiiirig 2000 pg. of Zn per gram carboxypeptidascA licfore tlialysis coritairied I900 pg. of zint* per gram after dialysis against water. It, appears that one atom of zinc*is firmly bound to the protein of carboxypeptidase. The zinc content increases throughout the course of purification in the very fractions in which carboxypeptidase activity is increased. The zinc content becomes constant as purification is achieved. At the same time, other metals decrease to absolutely arid stoichiometrically insignificant amounts. Zinc is firmly bound to the protein. This is implied by the fact that the zinc to protein bond is maintained through the changes in pH, ionic strength, and temperature, arid against competition of other ions to which the enzyme is exposed in the course of fractionation. The metal is not removed by dialysis against water or by prolonged standing in water a t pH values considered suitable for the integrity of the protein. Zinc, not associated with the carboxypeptidase of pancreatic juice-as indicated by the absence of enzymatic activity-is removed during fractionation, as is ionic zinc, which may be introduced from reagents, water, or glassware (Table VII, lines 3 and 4). N o special and elaborate precautions against contamination with zinc or other metals were taken during fractionation of the enzyme. The levels of contamination introduced were probably different, yet the final ratio of moles of zinc per mole of protein is so close in all instances as to be virtually identical, indicating that the firm Zn to carboxypeptidase bond exists in the “natural” state. Zinc content and the activity of the enzyme are directly related. The metal is aggregated in the fractions of highest activity (Table VII). Throughout recrystallization, both the enzymatic activity and the zinc content remain a t a constant level. The presence of all other metals is apparently unrelated to activity through fractionation. Thus, constant zinc/ protein, zinc/activity, activity/protein ratios are achieved with purification of carboxypeptidase. It is unlikely that the metal is a chance contaminant, since the molar zinc/protein ratio of the crystalline enzyme is an integral number, i.e., 1. Zinc is an integral, structural part of the carboxypeptidase molecule; carboxypeptidase of pancreas thus should be classified as a zinc metalloenxymP. At this time, the sites of binding are not known, nor has its firmness been measured quantitatively. 3. Inhibition Due to Mefal-Binding Agents

E. L. Smith and Hanson (1948, 1949) reported that carboxypeptidase is inhibited by sodium sulfide, sodium cyanide, sodium citrate, sodium ox-

350

BElZT L. VALLEE

alate, sodium pyrophosphate, phosphate, cysteine, iodoacetate, cuprous chloride, arid lead acetate. Carbobenzoxyglycy~-~-leucine at a concentration of 0.05 M was employed as the substrate for the data presented, a t pII values between the limits of 7.4 and 7.8. The inhibitors and enzyme were preincubated for a few hours a t 25°C. or overnight a t 2°C. The inhibition by cyanide, sulfide, or phosphate was found reversible. However, 110 inhibition was observed with 0.1 M sodium fluoride or in the presence of 0.1 M sodium fluoride plus 0.01 ICI phosphate. These data were interpreted to be compatible with and to confirm the presence of magnesium as an active, firmly bound moiety of the carboxypeptidase molecule. The inhibitory actions of cyanide arid sulfide were not explained (E. L. Smith, 1949b). Apparent inconsistencies in the data were attributed to the firmness of the bond between magnesium and the protein. These inhibition data, together with the analytical information discussed (vide supra), served as the basis for kinetic studies, for a model for cnzyme-substrate interaction, and for a possible mechanism of action of this enzyme (E. I,. Smith and Hanson, 1949; E. L. Smith, 1949a, 195la, b, r ; E. L. Smith and Lumry, 1949). Further studies on this system with carbobenzoxyglycyl-L-phenylalanine (CGP) and carbobenzoxyglycyl-L-leucine (CGL) as the substrates led to the conclusion that orthophosphate, pyrophosphate, oxalate, citrate, and cyanide did not affect the initial rate of hydrolysis significantly (Neurath and de Maria, 1950). In the presence of these anions, competitive inhibition by the liberated amino acid was found t o occur. These authors coricluded that the anions had no effect on the enzymatic activation process and that the hypothesis that carboxypeptidase is a magnesium enzyme was devoid of experirnental evidence. The presence of zinc in carboxypeptidase would appear to elucidate the problem. It could be shown, moreover, (Vallee and Neurath, 1955) that five times recrystallized carboxypeptidase was completely inhibited by metal chelatiiig agents, such as 8-OHQ-5SA and 1-10 phenthroline at coneeritrations of 10-3M,CY ,a'D at conceritratioiis of lo-* M , and some 30 % by EDTA a t 10-3 M . These are all known to form complexes with zinc in simple systems. In these experiments, the buffered enzyme solutions mere incubated with the chelating agent at pH 7.5, 4"C., for 1 hour prior to the addition of the substrate. Inhibition did not occur when these chelating agents were first incubated with an equimolar amouiit of zinc, cupric, or ferrous ions. Sodium diethyldithiocarbamate, zincon, sulfanilamide, and diamox, the latter two employed because of their effect on carbonic anhydrase, had little, if any, effect on carboxypeptidase activity. DPN, nicotinamide, and N-methylnicotinamide, examined because of their effect on the ADH sys-

ZINC AND METALLOENZYMES

351

INHIBITORS: * 1-10 PHENANTHROLINE 8 OH OWINOLINE'S~SULFONIC ACID 1 w..' DlPYRlOYL

CONCENTRATION OF INHIBITOR

FIG.2. Effect of concentration of inhibitors on carboxypeptidase activity, following preincubation. Preincubation conditions : Carboxypeptidase f inhibitor, pH 7.5, O'C., 60 minutes. Activity measurements: 0.02 M CGP in veronal buffer containing 0.1 M iYaC1, pH 7.5,25"C. Activities were expressed as apparent proteolytic coefficients calculated from the strictly linear portions of first-order reaction plots. V. The percentage activity, X 100, is plotted as a function of the logarithm of the

vo

molar inhibitor concentration (log I ) . Lines were fitted by a modification of the "least squares" method. [From: Vallee and Neurath, 19551

tern, did not inhibit, but, if anything, increased activity slightly (unpublished). Iodoacetic acid, salicylaldoxime, sodium azide, and nitrilotriacetic acid did not inhibit in concentrations of 1 X 10-3 M when incubated with the enzyme for 1 hour a t 0" C (Vallee and Coombs, unpublished). The effects of increasing concentrations of 8-OHQ-5SA, OP, and aa'D on the activity of carboxypeptidase at a constant substrate concentration of 0.02 M CGP are shown in Fig. 2. (Vallee and Neurath, 1955.) Activity of the inhibited reaction was expressed as per cent of the proteolytic coefficient observed at zero inhibitor concentration. The conditions of preincubation are indicated. Recent and unpublished data indicate the time course of the inhibitory effects of these agents; OP in concentrations M causes 90 % inhibition of the reaction in 60 minutes. 80 % of 1 X of the inhibition occurs in the first 15 minutes (Fig. 3). Addition of 1 x M zinc ions to the enzyme thus inhibited restores enzymatic activity, demoristrat)ingthe reversibility of inhibition (unpublished results). Since inhibition did not occur when chelating agents were first incubated with zinc, cupric, or ferrous ions to form the respective metal chelate, it appeared that the sites of chelation of these compounds are responsible for the observed inhibition. Inhibition is therefore not caused by any structural similarity between the inhibitors and the substrate.

3.32

BEET L. VALLEE

100

1,lO.phenanthroline

0

0

5b

100 Time of incubation i n minutes FIG.3. Time course of 1,lO-phenanthrolirie inhibition, 1 X W 4ilf 01’ resulting

in 90% inhibition of carboxypeptidase in 60 minutes cent activity mere calculated as in Fig. 2.

Conditions of ass:ty ant1 per

The agents employed are known to form stable complexes with Zn++ i i i solution, and their physical chemistry has been studied (Kolthoff, Leussing, aiid Lee, 1951; Albert, 1953; Lauger, Fallab, and Erlenmeyer, 1953). These agents apparently inhibit carboxypeptidase through their effects on its zinc atom, possibly by formation of a complex. Calculations of the stoichiometry for similar reversible enzyme-inhibitor complexes have becii proposed (E. L. Smith aiid Hanson, 1949; E. L. Smith, 1951a, b, c). Such calculations are based on several tacitly assumed conditions. Inhibition is presumed to be fully and freely reversible, under the test coiiditions. The substrate is presumed not to compete with or interact with the inhibitor. Furthermore, the stale of knowledge of mixed complexes and their lwhavior niakes such calculations of dubious value (Coryell, 1955). The inhibitory effects of chelatiiig agents, although consistent with thr stability of their zinc complexes in solution, are no direct function of this parameter, and their physical-chemical interpretations preseiit the difficulties commonly encountered with data on mixed vomplexes (Klotz arid Loh-Ming, 1954; de Witt and Watters, 1954). The geometric arrangcments of the zinc atoms with respect t o protein and ligand molecules, thc steric and chemical factors contributed by the reactive polar groups of protein arid ligand and their respective charges would significantly alter the constants arrived a t on the basis of simpler systems (KolthofT, Leussing, and Lee, 1951; Albert, 1953; Lauger, Fallab, and Erlenmeyer, 1955). Table VIII gives the association constants of several chelating agents with Zll++.

A full elucidation of the role of zinc in the functions of carboxypeptidase has to await further experiments now in progress on the reversal of enzy-

353

ZINC AND METALLOENZYMES

TABLEVIII .Lssouinlior~ (‘onstants oJ’ Some

Synibol used in text

Iteagen t X-Hyc~rox3.quinoline

8-OHQ

X Hydroayquiiio

line-5 sulfonic acid Ilithisone 1- 10 Phenan t,hrol ine

a,a’

Dipyriciyl

Zinc (‘tielates Reference 3Z:trtell and Calvin (1952) Albert (1953)

10.9

20.8

8-OHQ5SA

8.4

15.1

DZ

-

20.1

01’

6.4

12.2

a,a’ 1)

5.0

1 1 . P t>o 16.95u t o 17.1 12.1

17.0

a

Hablio and l’hilipenko (1947) Kolthoff, Leussing, and Lee (1951) I.Luger, Fallsh, and Erlenmeyer (1955)

The constants fo ol,ol-‘dipyridyl were concentration-dependent.

matic inhibition, on the ease of exchange between bound zinc and free zinc or other metal ions, and on the contribution of zinc to the stability of the protein. It may be of interest that zinc chloride alone and zinc cysteinatc do not have any activity toward CGP (Vallee and Coombs, unpublished data). These studies of inhibition of carboxypeptidase support the conclusion that zinc is both a structural and functional component of the enzyme and that i t participates in its catalytic action.

4. Physiological Implications It has been reported that a large fraction of Zn66administered to dogs is excreted in pancreatic juice (Montgomery, Sheline, and Chaikoff, 1943). KO explanation for this finding has been offered. These data make it appear likely that a t least part of this zinc is associated with carboxypcptidase in pancreatic juice. XTII.

ALCOHOLDEHY1)ROGENASE I . Physical Properties

YEAST

Yeast alcohol dchydrogeiiaac was crystallized from brewer's yeast by Segelein and Wulff (1937) and found to be dependent upon DPN for its activity by Anderson (1934). A distinctly different alcohol dehydrogenase was crystallized from horse liver by Bonnichsen and Wassen (1948) and Bonnichsen (1950). The present discussion will be concert led primarily with those structural and functional aspects of the yeast enzyme which

354

BERT L. VALLEE

relate to its characteristics as a zinc metalloenzyme (Vallee and Hoch, 1955a, b). Its physical, chemical, and enzymatic properties have been reviewed and discussed extensively (Schlenk, 1951; Singer and Kearney, 1954; Velick, 1954; Racker, 1955). Data obtained with the mammalian enzyme will be referred to where deemed pertinent, since a completely separate discussion of these two systems is difficult a t best. The enzyme is prepared from dried baker's yeast by extraction, differential heat denaturation, and fractionation with acetone and ammonium sulfate (Racker, 1950; Hayes and Velick, 1954). Thin platelike crystals result. The enzyme is stable in the dry state and in solution a t pH 7 and 0" C. but is destroyed rapidly below pH 4.5 and above pH 8.5. Electrophoresis of a crystalline preparation revealed one major enzymatically active component and a minor inactive one (Negelein and Wulff, 1937). The smaller, inactive constituent varied from 5 % t o 20% of the total protein. Re-examination of the electrophoretic properties of yeast ADH crystals confirmed the presence of one slow, presumably active, and one fast-moving, presumably inactive component. Their relative amounts did not change systematically with recrystallization as shown by area analysis. The percentage of inactive component was a function of age of the solution and duration of preliminary dialysis. Preparations dialyzed for 20 hours a t pH 5 contained as much as 14 % to 25 % of the inactive coinponent, while dialysis for 4 hours showed as little as 6 %. The second component was assumed to be an inactive transformation product of the active enzyme (Hayes and Velick, 1954). The molar absorption coefficient, E , at 280 mp is EN, = 1.89 X mole liter-1/cm.-2. Measurement of the optical density a t 280 and 280 mp measures the degree of removal of DPN and DP N H from the apoenzyme. The O.D.zso/O.D.zeo of a highly purifiedpreparationwas 1.82. The sedimentation constant, S20,w = 6.72 X I&l3 sec-l. Diffusion measurements gave a diffusion coefficient D z o ,=~ (4.70 f 0.03) X lO-' cm.2sec-'. The partial specific volume D is 0.769 ml. per gram. The molecular weight, was cnlculatpd t o t e 150,000 (Hayes and Velick, 1954; Velick, 1954). Pedersen, quoted by Theorell and Bonnichsen (1951a), found a sedimentation constant S20,w = 7.61 X l&13. The molecular weight calculated 011 this basis is about 140,000 (Bonnichsen, 1953). The molecular weight of horse liver ADH was shown t o be 73,000, about half that of the yeast enzyme (Theorell and Bonnichsen, 1951a). Neither of the ADH enzymes is isolated as DPN complexes, but each has a strong and characteristic affinity for the reduced and oxidized forms of the coenzyme (Velick, 1954). The O.D. 280/260 ratio measures protein and nud~ot i dec*omponentsof a particular ADH preparation, and a high ratio of a pure protein denotes removal of DPN or DPNH from the apocnzyme.

ZINC AND METALLOENZYMES

355

Horse liver ADH binds two molecules of DPNH per molecule of ADH between pH 7 and 9; a t pH 10, about one DPNH per one ADH molecule is bound. These data were obtained by taking advantage of the lowering of the absorption maximum of DPNH and its shift from 340 to 325 mp on the addition of mammalian ADH to DPNH (Theorell and Bonnichsen, 1951a). While the velocity of association with the liver enzyme is apparently equal for DPN and DPNH, the ADH-DPNH complex has a dissociation constant, a t equilibrium, of lW7M at pH 7.0, and the calculated dissociation constant for ADH-DPN is 2 X lo+’ M . Thus D P N is bound about 200 times less “tightly” to the mammalian enzyme than is DPNH (Theorell and Bonnichsen, 1951a). It was concluded that both are bound by ADH a t the same locus. The difference in the turnover number of liver ADH for two substrates was explained in part on the basis of the difference in strength of binding between ADH-DPN and ADH-DPNH (Theorell and Chance, 1951). No change in wavelength or absorbance of DPNH a t 340 mp occurs on its combination with yeast ADH. However, Hayes and Velick (1954) demonstrated that a total of 4 moles of DPNH and/or DPN are bound to 1 mole of the yeast apodehydrogenase a t pH 7.8, utilizing an ultracentrifugal separation method (Velick, Hayes, and Harting, 1953; Velick, 1953). The four binding sites appeared to be equivalent. The dissociation constant was 2.6 x 10-4 M for yeast ADH-DPN and 1.3 X 1 e 6 M for yeast ADH-DPNH. The respective Michaelis constants were of the same order of magnitude. DPNH replaces D P N competitively, while the total amount bound remains constant. Evidence that DPN and DPNH compete for the same catalytic site on ADH was also obtained kinetically. These authors assumed the formation of a yeast ADH-acetaldehyde complex with a dissociation constant of 1.8 X lW4 M , a t pH 7.9, 26” C., to explain an observed equilibrium shift (Hayes and Velick, 1954).* It has been suggested that this apparent agreement between kinetic data and theory may be fortuitous, however (Racker, 1955). 2. Chemical Composition a. Basic Composition. The dry protein contains 52.8% C, 6.96% H, 16.54% N, 1.21% S, 0.015% P, 0.0027% Fe, 0 % Cu. The presence of aromatic amino acids has been inferred from the 280 mp absorption band (Negelein and Wulff, 1937). Though the amino acid composition is unknown, Hayes and Velick (1954) concluded that the yeast enzyme has a higher content of aromatic amino acids than the mammalian enzyme, based on the absorption coefficients a t 280 mp. The partial specific volume 3 They also obtained good correspondence between equilibrium and kinetic data by assuming the formation of a ternary complex, ethanol-ADH-DPN (Hayes and Velick, 1954).

BERT L. VALLEE

356

Tmm

1s

Mefal (‘onterbf vJ’ Different (‘rystulline I’eust A D H I-’reparulzvns (All values in pg. of metal per g. of protein) 4 5 2 3 1 Metal

Zinc Magnesium Calcium Aluminum Barium Strontium Lead Cadmium Chromium Iron Copper Moles zinc/molc protein Rloles maguesium/moles protein

Expt . tcxpt. 1Sxpt. Expt . Expt . Expt. Expt. 8-225 !6-156 8-200 3-178 23-91 13-135 23-148

___

___-

1 ,440

,600 130 0 500 28 0 0 0 0 0

296 105 48 20 2 0 0 0 81

1,660 1,180 39 79 11

4

3.3

3.7

45 13 8 80 165 3.8

1.8

0.80

7.3

*

*

,910 95 <1 53 27 0 0 0 0 0

1,800 630 679 34 0 0 0

0 0 0

’,050 28 <1 0 0 0 0 0 0 0

*

*

4.4

4.1

4.7

0.5‘

3. 9

0.1;

*

,900 25 <1 0

*

0 0 0 0 0 0

4.4

I

Expt. 26-165

1,800 2.4 0 0 9.1 0 0 0 0 0 4.1

Not done. 0 = Not detectable [From Vallee and Hoch, 1955bl

also implies that the yeast enzyme has a lower content of amino acids with polar side chains than its mammalian counterpart. The presence of some 22 free SH groups/mole of the yeast apodehydrogenase were reported (Barron and Levine, 1952). b. Zinc. Recent spectrographic and inicrochemical studies have shown that the alcohol dehydrogeriase of yeast is a zinc metalloenzyme (Vallee and Hoch, 1955aJ)). The investigation was carried out by techniques and procedures similar to those described for carboxypeptidase (vide supra) and elsewhere (Hoch and Vallee, 195513). Table IX shows analyses on eight different crystalline preparations of highly purified yeast ADH, and Fig. 4 shows a spectrum obtained with oiie of them. Preparations noK. 0 a i d 7 were didyzed against large volumes of metal-free distilled water for 24 hours at 0” C.; 110. 8 was recrystallized four times. This preparation, identical with crystals no. 4 in Table X, was subjccted t o ~iltraceiitrifugatioii a i d electrophoresis (courtesy Dr. Hans Xeurath) arid found to contain two components, the slower moving of which comprised more than 85 % of the total.

357

ZINC .4NI) METALLOENZYMES

5.0 rg of %I1

ADH No. 23-288

S:rmple

t Zn 3345.02 A

FIG.1.Showing the zinc line at 3345.02 in an emission spectrum taken \ \ i t h 2 mg. of highljr purified yc:tstc ADII. .4reference standard containing 5pg. of zinc is shown for comparison.

Zinc, the major metallic coristituent, varies in concentration from 1440 to 2050 pg. of zinc per gram of protein. The molar ratios of zinc/protein shown in line 12, Table IX, range from 3.3 to 4.7, averaging 4.1 moles of zinc per mole of ADH based on a molecular weight of 150,000 (Hayes and Velick, 1954). The zinc content of one crystalline preparation was lowered from 2210 to 1720 pg. of zinc per gram of protein by dialysis against OP, corresponding to 3.9 moles per mole of protein. Magnesium, the only other element present consistently, varied widely in different preparations and decreased to absolutely and stoicahiometrically insignificant cwiceiitrations in highly purified samples. It appears that four atoms of zinc are present in one molecule of AD13 apoenzyme. The zinc in these crystalline preparations is firmly bound ; the xinc/protein bond is maintained against competitive physical-chemical factors involved in fractionation, potentially capable of dissociating it. Recrystallization or dialysis against water fails to remove zinc. In some preparations more than 0.2 % of zinc was found. I n these instances, 'small amounts of zinc could be removed by relatively mild procedures such as dialysis against OP. Such dialyses have not, thus far, lowered the zinc content below 4 moles of zinc per mole of protein. This complex has been assigned the empirical formula [(ADH)Zn4]ill accord with the considerations and nomenclature discussed. It was shown that the zinc atoms are involved in enzymatic activity. Zinc, present in excess of four atoms/mole, represents contamination extraneous to

358

BERT L. VALLEE

TABLE Y Activity and Metal Content of Fractions i n the Course of Purification of ADH from Yeast

--

A

Fractions

ipecic Act ivitg 1'lg.P x 106)

C

B Activity: Zinc

E

1)

+

Calcium

Barium g.1'

~

1. Estraction 55" Precipitation ; Supernatant 2. Acetone Precipitation; Supernatant 1 3. Acetone Precipitation; Supernatant 2 4. Dialysis Supernatant 5 . b (NH4)$04 Precipitation, Supernatant 1 6. (NH4)?S04Precipitation; Precipitate 1 7. (NH4)pSOaPrecipitation; Supernatant 2 8. Crystals 1 ; Supernatant 9. Crystals 1 10. Crystals 2 11. Crystals 4 c

G

Aluminum *g./g.P g./g.F /a,/ M . /

MagZinc nesium

VIP% rg./ x 103) g.1'

F

~

17.2

47.5

362 D ,000

14.0

24.1

582

2,200

0

0

,695

24.8

91.5

1.5

6.6

g.1' _ _

H

Iron fig./

g.1' -.

10

63

530

0"

39

17

200

2,100

0

0

0

0

271

4,100

0

10

12

227

0

150

2.2

5.3

130

199

554

161

15

0.9

1.1

24

74.0

128

580

620

33

1.7

1.0

32

44.1

126

351

370

0

3.5

3.0

0

266 290 305

910 ,910 ,800

270 95 2:

0
27 9.1

110

242 555 550

--

,800

0

0 53 0

86

0 0 0

-

Present: K, Na, P. Absent; Ag, B. Cd, Co, Cr, Li, Mn, Mo, Ni, Pb, Sr, Ti. a 0 = Not detectable. Discard. The fourth crystals were more than 85 per cent pure withreapect toprotein. Dr. Hans Neurath, Department of Biochemistry, University of Washington, did the ultracentrifuge and electrophoreticanalyses. [From Vallee and Hoch, 1955131

[(ADH)Zn4]and does not increa.se enzymatic activity; nor does the removal of such zinc decrease it. Table X shows data on metal content and activity obtained on consecutive fractions during a purification of ADH from yeast according t o the method of Racker (1950) as modified by Hayes and Velick (1954). Enzyme activity was measured as described (Vallee and Hoch, 1955b). Specific activity (Column A , Table X) rises from 17.2 X los in the materials initially extracted from yeast (line 1)to 555 X lo6V per gram of protein in

ZINC AND METALLOENZYMES

359

the fourth rrystals (liiie 11). Concomitantly, zinc content (Column C) rises from 3G2 (line 1) to 1800 (line 11) pg. per gram of protein. The activity/zinc ratio (Column B ) reflects the relationship of the activity to the metal, rising from 47.3 X lo3in the extract (line 1) t o 305 X lo3 1’ per microgram of zinc in the fourth crystals (line 11). The magncsium concentration in the initial extract from yeast (line 1) is about 100 times as high as the corresponding zinc concentration and falls to 2.4 pg. per gram of protein in the fourth crystals (line 11), a 10,000-fold decrease. Activity and magnesium content vary inversely in the course of purification (Columns A and D). Magnesium in the initial yeast extract varied from 10 to 30 mg. per gram of protein, and its inconsistent removal during fractionation provides an adequate explanation for the variability of this element in the crystal analyses shown in Table X. During purification, magnesium concentrations decrease lO,OOO-fold, whereas specific activity increases about 30-fold. All other elements-xcept zinc-are also either reduced in concentration or remain a t their initial, insignificant levels (Columns E to H ) , and none of them are structurally or functionally associated with ADH. The second crystals are contaminated with aluminum and barium (line 10). The data in Table X extend the conclusions reached on the basis of the analyses of crystals. The concomitant increases of specific activity, activity/protein, and zinc content, zinc/protein, imply an interdependence typical of a metalloenzyme. The discard fractions during purification contain zinc. The presence of substantial concentrations of zinc in the absence of activity (Table X, fractions 3, 5) indicates that zinc extraneous to [(ADH)Zn4]is removed. The activity and zinc of fraction 8 probably reflect incomplete removal of crystals from this fraction by centrifugation. 3. Inhibition of Enzyme Activity

The yeast ADH is very sensitive to various metal ions: 1.14 X lW6 M Cu”, 1.52 X M Agl, and 2.28 X 10-6 Hgl result in 50 % inhibition of yeast ADH. Ferric iron and zinc ions were also found to inhibit, but much less so. Ferrous iron and manganous ions did not inhibit a t all (von Euler and Adler, 1935). The copper inhibition could be reversed by tenfold excess concentrations of glutathione and ryanide (Wagner-Jaueregg and Moller, 1935). The copper inhibition was confirmed by Negelein and Wulff (1937). It must be kept in mind, however, that the assay system of vou Euler and Adler measured oxidation rates of both the flavoproteins and ADH. Using measurement of DPNH formation, it has been found, however, that IW3 M zinc and 5 X lCF4cadmium inhibited the reaction when pre-

incubated with the enzyme for I hour at 0" C. Ferrous ions inhibited ahoiit 80% at conccntrations of 10P ill under the same conditions. Sickcl a w l robalt also inhibited the oxidation of ethanol (TIoch and T'nllec, utipiit)lished data). Contact with nibher stoppers inhihits the wizynw (Kac.ht-\r, 1955). Von Euler and Adler (1935) reported that 5 X lop3M rnonobrornoac.ettltc. inhibited 50%. 1 x M iodoacetate reduced thc activity by 92%; the degree of inhibition was a function of time of contact between inhibitor and enzyme (Dixon, 1937). Lutwak-Mann (1938) found that the yeast ADH was inhibited by cyanide, whereas the mammalian and bacterial enzymes were not. Urethane, oxalate, maleate, and pyrophosphate similarly inhibit yeast ADH; quinine, morphine, and nicotine did not. Voii Euler and Adler (1935) found no effect with sodium fluoride. Wald and Hubbard (as quoted by Theorell and Bonnichsen, 1951a,h) rioted that p-chloromercuribenzoic acid inhibits the mammalian enzyme. The inhibition was reversible with glutathione. Barron and Singer (1915) found that p-carboxy phenylarsine oxide reversibly inhibited yeast ADH. Barron and Levine (1952), using the yeast ADH, observed strong inhibition with p-chloromercuribenzoate, and weaker inhibitions with phenylarsine oxide, iodosobenzoate, iodoacetamide, and iodoacetate. Dixon (1937), 0 1 1 the other hand, observed strong inhibition with iodoacetate. Much of this work was designed to establish the participation of -SH groups in the mechanism of action of yeast ADH, though IIO final conclusions have been reached in this regard. I n view of its zinc content, the activity of yeast ADH was studied in the presence of a large variety of agents known t o combine with this metal ioii in simple systems as salts, chelates, or complex ions. It was expected that the formation of such compounds would reduce the activity of the enzyme if zinc were involved in its mechanism of action. I n addition to OP, aa'D, 8-OHQ-5SA, DZ, and TU (Vallee and Hoch, 1955), the following have since been found to inhibit yeast ADH activity: NaDDC, BAL, Cupferron, thiosemicarbazide, sodium sulfide, potassium cyanide, and sodium azide (Vallee and Hoch, in preparation for publication). Inhibition was found to be strongly dependent upon the time of contact between the enzyme and the inhibitor prior to the measurement of activity, the p H of the preincubation mixture, and the temperature a t which the preincubation was allowed to take place. Nicotinamide, nitrilotriacetic acid, EDTA, quinaldinic acid, imidazole, sulfanilamide, diamox, and zincon did not inhibit the oxidation of alcohol significantly in concentrations of 5 X M , 0" C., 1 hour preincubatiori, pH 6.0 t o 9.5. Figure 5 shows the effects of preincubation of one of these agents,

361

ZINC AND METALLOENZYMES 100,

0.150

I

0.140

0.130 LD

N

m

0.120

N W

0.1 I 0

0.100

PH

PIG.5 . A , left ordinate: Effect of pH preincukxttion with 01' on inhibition of ADH :Lctivity. Preincubation conditions: ADH f 5 X M OP, O'C., 60 minutes. Activity measurements: the rate of D P N + DPNH, pH 8.8,23"C.is measured. Each point represents an activity measurement after preincuhation with the inhibitors (I;;) as a percentage of the uninhibited control (V0). H , right ordinate: Effect of pH on the formation of [Zn (OP),l++ complexes. The absorption at 292.5 mF measures the formation of [Zn (OP),]++complexes; points are calculated from the d a t a of hfcClure and Banks (1951). [From Vallee and Hoch, 1955bl

5 x lW3 41 UP, with ADH for 1 hour at 0" C. a t the indicated p H levels. OP completely inhibits the enzymatic oxidation of ethanol a t pH values below 7.5, and inhibition diminishes with increasing alkalinity of the incubation niediuni. At pH levels below 6.0 and above 9.5, the activity of the uninhibited control reaction became too low and erratic to measure. Spectrophotoinetric data on the effect of pH on the formation of [Zn(OP).]++ complexes from the work of McClure and Banks (1951) are shown for comparison. With rising pH, the formation of these complexes is progressively suppressed (Fig. 5 ) . The enzymatic data plotted in Fig. 5 imply that, the inhibition observed is a function of the formation of a zincOP complex. The dependence of the effectiveness of other inhibitors (vide supra) on pH of the preincubation mixture has been studied (Vallee and Hoch, in preparation for publication). The effects of various concentrations of OP, 8-OH&,aa'-D,DZ and T U on the oxidation of ethanol are shown in Fig. 6 ; the conditions of preincubation are indicated.* The concentration of agents required to produce 50 % inhibition is in the order DZ < OP < %OH& < m'-D < 8-OH&-5SA < TU 4

The data are not corrected for ionization of these agents.

362

BERT L. VALLEE

-4

-2

-3

I

log I ( M I

FIG.6. Effect of concentration of inhibitors on ADIi activity, following preincubatiori. I'reincubation conditions: ADA zt inhibitor, pH 7.5, O'C., 60 minutes. Activity measurements: the rate of D P S DPNH, pI-K 8.8, 23°C. is measured. Vi The partial activity after inhibition - x 100 is plotted as a function of the loga--$

vo

rithm of the molar inhibitor concentration (log I ) . [From: Vallee and Hoch, 1955bl

-3 lop

I (M)

-2

FIG.6a. Effect of concentration of OP on ADH activity. Preincubation coilditions: ADH f OP, pH 6.5, 60 minutes. Activity measurements: the rate of DPNH -+ DPN, pH 6.5,23OC. is measured. [From: Vallee and Hoch, 1955bJ

(Vallee and Hoch, 1955). RAL, not reported on previously, is intermediate between DZ and OP, and thiosemicarbazide is between 8-OH&-5SA and T U (Vallee and Hoch, unpublished data). The slopes of the inhibition curves arc very much steeper than those observed for carboxypeptidase

ZINC AND METALLOENZYMES

363

(vide supra) (Vallee and Neurath, 1955), and the concentrations of inhibitor required to bring about 50% inactivation of yeast ADH are considerably higher than those required for carboxypeptidase. are required to bring about 50 % inhibiHigher concentrations of CYCY’-D tion than are uecessary for OP, as is the case for carboxypeptidase. This is presently explained on the basis of an additional degree of freedom in the aa’-D molecule as compared with OP, owing to rotation about the C-C bond, resulting in a lower statistical probability for chelation with aa’-D. The degree of inhibition may be related to the stability of the zinc complexes formed, but while consistent with this parameter, other factors enter which have been discussed above for carboxypeptidase (see p. 352, also Table VIII) and which apply for yeast ADH. Hayes and Velick (1954) have shown the four sites of D P N attachment to yeast ADH t o be equivalent. The steep slopes of the present inhibition curves are probably not solely due t o the concentration and chemical configuration of the inhibitors. Presuming chelation of the agents to zinc atoms which remain in situ on the enzyme, these curves suggest that a greater number of moles of inhibitor are bound to zinc than can be accounted for O K the ~ basis of its maximum coordination number of 6. The analysis of such inhibition curves has been based 011 the implied assumption that these metal chelate complexes (E. L. Smith and Hanson, 1949) are analogous to those observed in simple systems (Martell and Calvin, 1952), an assumption for which little experimental justification exists. It should be emphasized that a metalloenzyme inhibited by binding of a chelating agent to a metal in situ represents a mixed complex, consisting of two different ligands combined with one metal. Studies of the physical chemistry of simple complexes of this type are as yet few (Klotz and Loh-Ming, 1954; de Witt and Watters, 1954). The data on mixed complexes available to date are apparently not adequate to justify extrapolations to the interpretation of inhibition of metalloenzymes (Coryell, 1955, personal communication). Thus, on the basis of data presently available, it is not possible to decide whether or not these inhibition curves imply a ‘Lcooperativeeffect” on the part of the zinc atoms of [(ADH)Zn4], each inhibitor molecule binding more strongly to a zinc atom than the previous one. The interpretation is further complicated by the known properties of simple zinc complexes (vide infra) . Since extensive data on the physical chemistry of zinc-OP complexes are available (McClure and Banks, 1951; Kolthoff, Leussing, and Lee, 1951; Brandt, Dwyer, and Gyarfas, 1954), OP was studied most intensively. The association constants, obtained by spectrophotometry, are shown in Table VIIT. Similar data have been obtained by potentiometric titrations (Hara,

384

BERT L . VALLEE

Snell, Hoch, and Vallee, unpublished experiments). OP has been employed for the analytical determination of zinc in the absence of iron. The absence of iron from purified ADH (Tables IX and X) and the inhibition of ADH activity by OP support the analytical data. Figure 6a shows data on the effect of OP on reduction of acetaldehyde. The enzyme was preincubated with varying concentrations of OP a t pI-1 6.5. The concentration curve is very similar in shape and close in absolute values t o that obtained for the effect of OP on the oxidation of ethanol (see Fig. 6). As has been indicated, iiihibition does not occur without preincubation with the agents studied. Figure 7 shows ADH activity as a function of time in the presence and absence of 2 X lW3 M OP a t 0" C., pII 7.5. In 5 hours, activity of the uninhibited enzyme falls slowly to 80 % of the original value, whereas in the presence of OP it falls to 17.5% as measured by the rate of oxidation of ethanol (DPN ---f DPKH). During the same period, activity of the uninhibited enzyme falls to 72.5%, and in the presence of 200

700

180

630

I60

560

I40

490

z120

420

&

-2 100 .-

350

2

z E

E

E ,

0

2 0

p 80

280

4

4

60

210

40

140

20

70

0

0 0

I 2

3 4 5 0

I

2

3

4

5

t (HOURS)

FIG.7 . Effect of time of preincubation on inhibition of ADH activity by OF'. Preincubation conditions: ADH f 2 X 10P M OP, pH 7.5, 0°C. Activity measurements: aliquots are removed from the same preincubstion mixture a t the times indicated. The rate of DPN D P N H a t pH 8.8, 23"C., or the rate of DI'NH -+ D P N a t pH 6.5, 23"C., is measured. [From: Vallee and Hoch, 1955133 -+

365

ZINC AND METALLOENZYMES

OP to 18.2% of the original rate of reduction of acetaldehyde (DPNH -+ DPN). I n the presence of OP, the activity of ADH falls much more rapidly during the first few hours than without it. Thereafter, the rates of inactivation of control and inhibited enzyme proceed nearly parallel. Since aliquots vf the same enzyme-OP mixture were used for the experiments in Fig. 7, it would appear that OP acts upon [(ADH)Zn4]arid not upon other components of the reaction mixture. This is also supported by the data shown in Fig. 6 when compared with Fig. 5-inhibition with OP is of almost identical magnitude whether the forward or backward reaction be measured, even though the reaction rates are examined a t different pH. At 0" C., activity of the OP-inhibited enzyme decreases slowly and linearly for 180 minutes, whereas there is little change in the activity of the control (Fig. 8). At 25" C. or a t 38" C., the rates of decrease in activity are so rapid that their accurate measurement is difficult and, particularly at 38" C., any comparison with the rapidly deteriorating controls becomes difficult. Accurate measurements could be made conveniently with a 1-hour preincubation period a t 0" C., arid this was adopted as a standard procedure. M Zn++ (always as ZnCls) is added a t the time a t which When 2 X the OP-inhibited enzyme still exhibits 18% of initial activity, an immediate return t o 59% of the uninhibited enzymatic activity is observed (Fig. 9). 5 X 10-3 M Zn++ does not reverse the inhibition further. The addition of

0

FIG.8. Effect of temperature and time of preincubation on inhibition of ADH activity by OP. All open symbols and broken lines represent rates of the control reaction in the absence of OP; all solid symbols and solid lines represent rates of the reaction in t,he presence of OF. Different temperatures are represented by t h e shape of symbols as indicated. Preincubation conditions: ADH f 2 X 10-3 M OP, p H 7.5. Act,ivity measurements: aliquots are removed from each preincubation mixture at the times indicated, and the rate of D P N + D P N H , p1-I 8.8, 23°C. is determined. [From: Vallee and Hoch, 1955131

BEIZT L. VALLEE 240.

200

I60

I

n U

0

$

IZO

\

0

:so

w

4

40

0 0

20

40

60

so

I00

120

I 0

t (MINUTES)

FIG.I).Reversal of inhibition of ADH activity by 01' on addition of Zn++ (as ZnCl?). All broken lines represent rates of the control reaction in the absence of OP; all solid lines represent rates of the reaction in the presence of OP. The addition of Zn++ is marked by arrows, and rates in the presenre of Zn++ are shown as square symbols. Preincubation conditions: ADH f 2 X 10-3 M OP, pH 7.5, 25°C. Activity measurements: aliquots are removed from each preincubation mixture a t the times indicated, and the rate of DPN 4 DPNH, pH 8.8, 23"C., is ascertained. [From: Vallee and Hoch, 1955bl

2 X lop3III Zn++ to ADH prodiices a decrease to 2G% of control activity i i i 18 minutes. Reversibility of the reactioii has also beeti demonstrated by the removal of OP by dialysis ~ i t subsequent h restoration of activity. M OP, first exposed for 5 minutes to Addition t o the enzyme of 5 X the same concentration of Zn++, results in 96% of normal activity, whilc llil OP inhibits ADII to 30 % of normal activity. 5X Therefore, the addition of [Zn(OP),]* or [Cu(OP),,]++to the enzyine does not inhibit it, indicating that the -N=C-C=N-group (ferroine) of OP (Smith, 1954) is responsible for the inhibitory action of this compound, since it is the known site of c~helationof these ions (Brandt, Divyer, a i d Gyarfas, 1954). Experiments on the kinetics of the effect of OP on ADH activity also demonstrate that OP is a dissociahle inhibitor (Hoch and Vallee, unpublished data). The inhibition is competitive with DPN, when the coenzyme and OP are both preincubated with yeast ADH. Since OP acts upon the zinc atoms of [(ADH)Zn4],it appears that they are the sites of attachment of each of the four DPN molecules associated with ADH.

367

ZINC AND METALLOENZYMES

4. Yeast ADH as a Zinc Metalloenzyme Analytical and enzymological data lead to the conclusion that zinc is a structural and functional component of ADH, and that it participates in the mechanism of its enzymatic action (Vallee and Hoch, 195513). The four metal atoms are firmly bound to the protein apoenzyme. Pending the results of investigation in progress, this discussion will assume that the four zinc atoms are bound to the protein in an equivalent manner, and that each atom of zinc acts independently of the other three in the catalytic action of alcohol dehydrogenase. This seems the more acceptable view at present, since Hayes and Velick (1954) found the four binding sites of DPN t o be equivalent. One molecule of yeast ADH has been shown to bind four molecules of DPN (Hayes and Velick, 1954). It is thought, therefore, that each zinc atom serves as a locus of reversible attachment for a molecule of the coenzyme, DPN. The general interaction is formulated as follows:

[(ADH)Zn4] (DPN)4 is the active complex composed of the apoenzyme, zinc, and the coenzyme, the brackets denoting structural association (vide supra). Enzymatic activity is decreased by incubation of the metalloenzyme with an inhibitor, I. [(ADH)Zna]

+ 41 @ [(ADH)ZnalL

(2)

n moles of I are bound through covalent and/or ionic bonds t o each zinc atom. To facilitate the presentation of the reactions, n has been set equal to 1, though there is no conclusive evidence concerning the number of moles of inhibitor bound in different cases. This reaction should result in an inactive, reversibly dissociable ZnI complex. Combinations of Eqs. 1 and 2 depict the complete reaction in the presence of an inhibitor. 2[(ADH)Zn4]

+ 4(DPN) + 41

+ [(ADH)Zn4]14

[(ADH)Znr](DPN)4

(3)

The existence of the two equilibria should lead to competitive inhibition between DPN and I, and experimental evidence indicates this to be the case (vide supra), lending support to the formulation of the reaction as shown in Eqs. 1 to 3. Apparently, chelate ligands exert their effects and are bound t o [(ADH)Zn4]in a manner similar to that employed for DPN.

368

BERT L. YALLEE

These considerations imply that the association between the zinc moiety of ADH and an inhibitor is reversible. This hypothesis is based on the thought that the zinc-protein bond is much stronger than any formed between zinc and an inhibitor. If it were postulated that the zinc-protein bond is broken by a ligand of high affinity for zinc, the reaction should result in the removal of zinc from the protein, and activity would be lost: [(ADH)Zn,]

+ 41 7(ADH) + 4ZnI

(4)

Activity could be restored only by addition of Zn++ to (ADH) to reconstitute the original compound. Experience with most other metalloenzymes (vide supra) makes this appear tenuous. The mechanism of inhibition, therefore, depends upon the equilibrium constants of Eqs. 2 and 4 for any given inhibitor. The inhibition of alcohol dehydrogenase activity by O P has been reversible under experimental conditions employed thus far. With OP as the inhibitor, Eq. 2 apparently prevails. I n view of the high association constant3 of the [Zn(OP),]++complexes (Kolthoff, Leussing, and Lee, 1951), the corresponding constants of the [(ADH)Zn4]complex must be very high indeed. Since zinc is bound so firmly to the protein-but the groups to which it is bound have not been verified-one can only speculate at this time concerning their identity. It has been postulated that sulfhydryl groups bind DPN to liver ADH (Theorell and Bonnichsen, 1951a). The existence of some 22 free SH groups in yeast ADH has been reported (Barron and Levine, 1952). The zinc atom itself may be bound to ADH through a thiol bond. The present findings do not rule out the existence of two linkages between ADH and DPN-one of them is definitely through a zinc atom of [(ADH)Znd. The experiment described in Fig. 9 was designed to test further the formulations of the reactions given in Eqs. 1-4. Zinc and OP form complexes in solution: Zn++

+ n(OP)

[Zn(OP),]++

(5)

Such complexes should form when Zn* is added to [(ADH)Znd] (OP), (Eq. 2), competing for OP bound to the metalloenzyme. The competition of the equilibrium shown in Eq. 5 with that of Eq. 2 should result in restoration of activity: [(ADH)Zndj(OP)r

+ 4Zn++ + [(ADH)Znr]+ 4[Zn(OP)]++

(6)

Figure 0 demonstrates the operation of this mechanism. The addition

ZINC AND METALLOENZYMES

369

of Znff rapidly restores and maintains activity a t 59% of the control level. Enzymatic activity is not restored completely, however, under the conditions of the experiment,. The following possibilities have been considered to explain the inconiplete restoration of activity: (a) The inactive metalloenzyme-inhibitor complex may be dissociated incompletely (Eq. G ) . (b) Zinc may be removed from the aopenzyme (Eq. 4). ( c ) OP may first remove some zinc from the metalloenzyme. Zinc ions, thereafter, only partially reconstitute the [(ADH)Zn4] complex. (d) Zinc ions may combine with DPN, making it unavailable. ( e ) Zinc ions may interact with enzymatically reactive end groups of the apoenzyme. The irreversible removal of zinc ( b ) cannot be ruled out, though experiments specifically designed t o remove zinc from the apoenzyme have not been successful thus far (b and c ) . For this reason, the reactivation of zinc-free (ADH) by Zn++ has not lent itself to experimental verification. The incomplete dissociation of [(ADH)Zn4] (OP)4 (a) is a t present hypothesized to be the cause for the incomplete restoration of activity. Since zinc ions themselves inhibit the enzymatic reaction (Fig. 9), the incomplete restoration of activity may be accounted for in part by the mechanisms proposed under (d) and ( e ) , which are believed to be responsible for the effect of Zn* on [ (ADH)Zn41, It has been reported that Neurospora crassa, grown on zinc-deficient media, was devoid of ADH activity, which was not restored by the addition of Zxi++. The s t r ~ ~ u r association al of zinc with the ADH apoenzyme was not considered as a possible explanation for the resultant functional derangements (Nason, Kaplan, and Colowick, 1951), which were attributed to “ail indirect influence on the synthesis of the apoenzyme” (McElroy and Nason, 1954). I n the absence of zinc, this organism is apparently unable to form the functional metalloenzyme molecule. It would seem that the ADH of Neurospora, like the ADH of yeast, is a zinc metalloenzyme. Since the molecular weight of ADH of horse liver has been reported to be 73,000 and two molecules of DPN per molecule of protein are bound, it was speculated (Vallee and Hoch, 1955), on the basis of molar proportions of protein to cofactor, that the presence of 2 moles of zinc per mole of protein may be expected in the horse liver enzyme. The known high zinc content of liver of which ADH has been stated t o be 1% of the total protein weight (Leiner and Leiner, 1941) lent support to this assumption. Two moles of zinc per tnole of liver ADH have recently been found (Theorell, personal communication, in print in Acta Chem. Scand., 1955). It has been reported (Leiner and Leiner, 1944) and confirmed (Bowness et al., 1952) that retina contains high concentrations of zinc. Horse liver ADH oxidizes vitamin A1 and reduces retinene, probably being identical \\-ith retinene reductase (Bliss, 1949; Theorell and Bonnichsen, 1951a).

370

I ~ X TL. VALLEE

Since mammalian A D H has hecn found to be a zinc enzyme, eiizyniologic*al m d analytical observations on liver, retina and those of yeast AD11 m:iy he shown t o have an analogous structural denominator. This is the first instance in which a c~oeazymc-dependeritdehydrogcnaso has been shown t o hc a metalloenzymr. The implication that the mettil is the site of binding for the coenzyme makes this ail interesting fitiding, thcl potentialities of which are heightened if one assumes a ternary ciomplex between apoenzyme, coenzyme, and substrate as essential to catalysis. Zinc ions aiid complexes do not absorb radiation in the visible region of the spectrum, and therefore do not draw attention t o themselves. The absence of changes in valence coupled with the stability and unusual characteristics of its complexes (vide infra) may explain both why the preseucc of ziiic has been overlooked aiid what its potential function may he-to serve as an “organizer” of reversible attachments between apoenzyme, coenzyme, and substrate. It is to be expected that other dehydrogenases will be found t o have similar or identical mechanisms of action.6

XIV. COO~~DINATION CHEMISTRY OF ZINC 1. General

The author is indebted to Dr. S. James Adelstein for his cw~peratioiii i i the preparation of this section. The recognition of metals as integral parts of enzyme systems has led to investigatioiis aimed a t elucidating their functional role in the catalytic. process. Of the transition metals, iron has been best studied in this regard. This stems from several circumstances: first, the characteristic color of the heme pigments early called attention to this element; second, the conceiitration of iron in liver, muscle, and red cells is high, relative t o the other transition elements; third, the functional heme unit, c.an be isolated with X o l e added i n p r o o j . Gh~tutirieI>ehydi oyennse. Recent spectrographic examinations of crystalline preparations of glutamic dehydrogenase of beef liver have shown this DPN dependent enzyme to contain about 300 pg of zinc per gram dry weight of protein (Vallee el a l . , 1955). Based on a molecular weight of 1,000,000 (Olson and Anfinsen, 1952), 4- t o 5-gram atoms of zinc per mole of apoenzyme have been found in several preparations. Fractionation of beef liver shows an u!ggregation of zinc as glutamic dehydrogenase activity to protein ratio reaches its maximum value. Simultaneously, the metal protein ratio of all other elcments studied decreases with increasing enzyme purity. The zinc/protein and activity/protein ratios become maximal in the third crystallization. The rate of conversion of DPN t o DPSH a t pH 7.7 in the presence of glutamate is inhibited significantly when the enzyme is preincubated with a number of metalbinding agents, such as sodium sulfide, sodium diethyldithiocarbamate and 1,lOphenanthroline. Glutamic dehydrogenase of beef liver apparently is a zinc metalloenzyme.

371

ZINC AND METALLOENZYMES

comparative ease-its chemistry and structure and that of its derivatives have been studied in detail; fourth, the electronic structure of iron (four or five unpaired 3d electrons) endows it with certain unusual properties, including the capacity to form stable metal-ligand bonds by the pairing of electrons and the utilization of unoccupied 3d orbitals, a high paramagnetic moment facilitating studies of the interaction of the metal with ligands and 0 2 , a characteristic spectrum which has allowed detailed studies of the kinetics of heme enzymes, and two stable valericies permitting oxidation and reduction of the ion in solution. Knowledge of the functional role of zinc as a component of metalloenzymes is much less complete. Zinc differs from iron in several aspects. The kiiown zinc enzymes are colorless, and the doubly ionized atom, having 110 unpaired electrons, has no paramagnetic moment. The metal has but one stable oxidatioii state in solution, is tightly bound to the apoenzyme, and can be removed only with scvrrc chemical treatment. In 110 instance, so far, has the group binding the metal to enzyme been established with certainty. The known chemical and physical properties of this element may be examined as a first approximation to an understanding of its role in hiological phenomena. Its ability to form strong complexes with the radicals aiid polar groups containing oxygen, nitrogen, and sulfur is one striking characteristic of Zn++. This property will be examined in (*loserdetail as it may relate to the role of the metal in the zinc metalloenzymes. 2. Successive Stability Conslants

In general, the reaction of a metal M with a ligand A may be written:

MA,-i(HzO)p

+A

F)t

MAn(H20)p-q

+ qHzO

Iiicorporating the water of hydration into 1;’ =

[IIIA ] n-* [M.L-il[Al

’

In many instances the thermodynamic constant 1;’ has not been deteriniried but rather the stability coefficient li at a given ionic strength, where y = activity coefficient, and therefore

k’ =

YMAn

YMA,,-~YA

-

(MA,) YMA. (M-~,-I)(A)’YILIA~,-~Y 4

To obtain the thermodynamic constant, (1) measurements arc made a t

372

BERT L. VALLEE

nearly infinite dilution (e.g., by conductivity methods), ( 2 ) k is determined at several values of ionic strength and extrapolated to infinite dilution; ( 3 ) activity is calculated from theory (e.g., Debye-Huckel). The methods for determining stability constants have been reviewed (Martell and Calvin, 1952). Bjerrum (1941) has indicated the significance of the consecutive stability constants for the following sequence of reactions:

If I n the absence of other factors, these stability constants should bear a certain relation to each other by virtue of the statistical association between metal and ligand formulated as follows: Let the probability of a complex MA, acquiring an additional ligand be proportional to ( N - n ) and the probability of losing a ligand be proportional to n. Then: -ten- - ( N - + ’)(% -I- ’) for monodentate ligands and when all cokn+l ( N - n)(n> ordination sites are equivalent and idential. For bidentate ligands ockl k ‘L cupying adjacent positions of an octahedron: log - = 0.682 and log - = k, 1CZ 0.972. Diff erences between the successive stability constants or ligaiid effect :ire designated by Hjerrum:

Ln,n+1 =

Sn.n+l

+

h’n,n+I

+

&,n+l

(

where Sn,n+lis the statistical factor mentioned above for monodentate li+ ‘1); E,,,+I represents electrostatic effects n>(n> for the interaction between charged ligands; and R,,,+] ,“the residual effect,” which is determined by other factors. The successive complexes of zinc tend to show a more negative residual

gands

=

log ( N - rL

(N

-

373

ZINC AND METALLOENZYMES

effect, R,,,,+I , than do those of other metals, so that the consecutive stability constants approach equality and a t times increase-in opposition to the decrease predicted on the basis of statistical factors alone. This is well illustrated by contrasting the ammonia complexes of Cu" and Zn (Schwarzenbach, 1954): CUT'

s,.,,,

log ki

log k2

log ka

4.12

3.48

2.87

0.43 0.21

Rr',,,! Zn

2.27

0.36 0.25 2.34

0.43 -0.50

Sn.n+l

R*,n+t

log

k4

2.11 0.43 0.33

2.40 0.36 -0.42

2.05 0.43 -0.08

For the copper complexes the residual effect is positive and the successive stability constants decrease in regular order. For the zinc-ammonia complexes, the residual effect is negative and the first three stability constants are about equal. The observations of Klotz and Loh-Ming (1954) on the binding of pyridine-2-azo-dimethylaniline to protein through a cation bridge indicate that a similar behavior of zinc occurs in mixed protein-small molecule complexes. They have measured the stability constants of the dye with cations bound to protein (pepsin and albumin) and free in solution. For Zn and Cu" specifically : log ki

+

Metal dye Pepsin - metal

+ dye

CU"

Zn

5.11 3.8

2.36 3.6

The binding by protein has an opposite effect on the affinity of the two metal ions for the dye. For Cu", the affinity is decreased; for Zn, it is increased and becomes nearly equal to that of Cu" in comparison to the large difference observed in the free ionic state. This observation indicates that zinc may have an especial advantage in mediating the linkage of small molecules to protein; this is of interest in view of the finding that each zinc of yeast alcohol dehydrogenase is probably a site of binding for a single DPN molecule and that it is also part of the carboxypeptidase molecule (vide supra). Since DPN molecules are bound to four separate metal sites on the ADH molecule, the influence of the occupany of one site on the successive occupancy on the others is to be considered. Taking into account statistical factors, the probability of association of coenzyme with enzyme should decrease with increasing occupancy; but the binding of ions and molecules to proteins frequently violates this statistical prediction, e.g., in the binding

374

1 3 E I W L . VALLEE

of Cu" t o conalbumin, the second Cu" ion is bound more tightly than the first (Wartier and Weber, 1953). The binding of successive 0 2 molecules t o hemoglobin, which, like ADH, contains four metal atonis per molecule, is facilitated by the prior occupancy of other sites (Adair, 1925), and a structural meahanisin for this "(~ooperative" phenomenon has been proposed by St. George and Pauling (1951). The relationship between the successive affinity coilstants in the binding of DPN molecules to ADH is as yet unknowi, although Hayes and Velick (1951) have suggested the equivalence of the four sites. Their relation to the inherent properties of the metal atom, to the interactions between sites, and t o configurational changes in the protein, are yet to be determined. 3. The Entropy Eflect

The thermodynamic stability constant k' which represents the free energy of complex formation (AFO = - RT In k ' ) can be subdivided into heat and entropy terms (118'" = A H o - TAS"). The entropy of complex formation has been discussed elsewhere (Cobble, 1953; Schwarzenbach, 1954; Williams, 1954). The factors involved include: (1) the size and geometry of metal ions and ligand molecules; ( 2 ) the change in the number of molecules in the system on complex formation as they affect translational freedom; (5') restrictions o n the freedom of rings imposed by chelation arid other restrictions of internal rotation; and (4) the entropy of hydration for the water molecules displaced by ligands. Charles (1954) has compared the entropy of ethylenediamine-tetraacetate (EDTA) complex formation for several elements, including zinc, and finds the entropy change to be a linear function of the partial molal entropy of the complexed metal. In Fig. 10, the entropy changes in the formation of ammonia (Williams, 1954), ethylenediamine (En) (Davies et al., 1954), and EDTA (Charles, 1954) complexes of Zn, Cu", and Cd are plotted as a reciprocal of the ionic radius minus a term coutaiiiing the molecular weight (Powell arid Latimer, 1951), as a measure of the partial molal entropy of the cations. The ASo values plotted are for the reaction:

M

(H&l)p-q

+ nA

=

MAn(HzO)p-q,

viz the published entropy values for the reaction

M(HaO),

+ nA = MA,(H2O),-, + gHzO

have been corrected for the entropy of hydration, assuming one ligand donor atom displaces one molecule of water. As the figure indicates, the entropy of complex formation for Zn is, in general, greater than that for Cd and less than that for Cu", its horizontal and vertical neighbors in the periodic table. Moreover, the entropy contribution to complex stability

375

ZINC AND METALLOENZYMES

40

t

Rln M

-31

FIQ.10. Entropy of complexation (AS") for Cu, Zn,and Cd complexes of ammonia, ethylenediamine (En), and ethylenediamine tetra-acetate (EDTA).

with ring formatioil increases markedly as one goes from the ammonia to the ethylenediamine (En) to the EDTA complexes of a given metal. The great stability of EDTA complexes in which the formation of several rings severely restricts the degree of internal rotation (L'wrap around complexes") derives in large measure from this entropy effect. The effect of chelate formation on the stability of complexes has been studied by Spike and Parry (1953), who investigated the reaction M (NH3)2 En = M (En) 2 NH3for Zn, Cu", and Cd. Their results are shown in the following table (in kcal. per mole) :

+

+

-AH"

z11

CU"

1.55 4.30

Cd

1.20

-0.1 f 0.1 2.6 f 0.1 -0.1 f 0.1

- TAS" -1.6 f 0.1 -1.7 f 0.1 -1.3 f 0.1

For the Zn arid Cd complexes, the increase in stability with ring formation derives almost completely from the entropy factor, whereas the marked increase for the CU" complexes derives from both the entropy and heat term equally. The entropy factor can be, therefore, of great importance in the stability of complexes, particularly those of the "wrap around" type. This may

376

BERT L. VALLEE

account for the stability of certain Zn and Cu'* metalloenzymes as compared with protein complexes (e.g., albumin and insulin). The groups responsible for the binding of these ions may be the same in both instances and the binding energy (AH") approximately equal. Configurational differences at the site of binding, however, could be the basis of a large entropy change and, hence, significantly affect the stability.6 4. The Coordinate Bond Zinc, which stands at the head of the vertical column of I I b elements in the periodic table, also follows the iron group of transition elements in horizontal sequence. I n the ground state, the doubly ionized atom has a closed shell of paired 3d electrons. I n general, the enhanced deformability of the underlying 18-electron arrangement renders all compounds of the Group I I b elements more covalent than those of Group I I a (Moeller, 1952). For the more common 4-coordinate complexes, sp3hybridization imparts tetrahedral geometry, whereas the octahedral sp3d2hybridization is assumed for the less common 6-coordinate complexes. The general order of stability for bivalent transition element complexes, which increases from left to right across the periodic table, reaches a maximum a t copper, falling sharply to zinc (see Fig. 11) (Irving and Williams, 1953). Data for nitrogen (ethylenediamine = En), oxygen (salicylaldehyde = S), and oxygen-nitrogen (glycine = G1) ligands are shown. No regular relation between the complexes of the I I b elements Zn, Cd, and Hg has been described (see also Fig. ll),but many of the Hg chelates and coniplexes are highly insoluble and data for them insufficient for comparison. The classical model of the metallic complex (Sidgwick, 1927) was of a spherical, central positive ion attracting spherical ligands to itself by electrostatic forces. This simple electrostatic model was found inadequate to explain the behavior of transition and group I I b elements. It has been replaced by the partial ionic-valence bond (Pauling, 1940; Coryell, 1954) or, in the language of molecular orbital theory, by an ordinary covalent bond possessing considerable polarity (Nyholm, 1954; Craig et al., 1954). Several authors have attempted to correlate observed bond energies with a number of parameters (viz., ionization potential, electronegativity, ionic charge and radius) representing the covalent and ionic character of the coordinate bond. Van Uitert and Fernelius (1954) assume that the bond energy AH" can be represented by: AHo = f(X', X ' o , X', - X ' D , $,,) where X', and X'n are the effective electronegativities of metal and ligand 6 I t is t o be appreciated t h a t the apparent stability of the metalloenzyme complexes may reflect a large energy of activation ( A F I ) between the associated and dissociated form and riot necessarily an unusually increased affinity of metal for urotein.

377

ZINC AND METALLOENZYMES

22

i

i”

18

h -I

14

i 10

6

2l ’

Mn Fe Co Ni Cu Zn Zn Cd Hg FIG. 11. Relative stability of the Transition Group (Mn-Cu) and Group I1 b (Zn-Hg) complexes of ethylenediamine (En), glycine (Gl), and salicylaldehyde (S). (After Irving and Williams, 1953.)

donor atoms, respectively. The electronegativity product XIMXIDis taken as a measure of the covalent bond strength, and the electronegativity difference X M - X D as a measure of the dipole character of the bond. The +O term represents the influence of nuclear repulsion on the wave function overlap, and tends to weaken the bond for high electronegativity difference. For the stability series with a given ligand, the expression AHo = f’(X’,) can represent the situation, since +O is principally dependent upon the ligand groups. Substituting, f’(X’,) = f ) ( X M V J , )where , X I , is the efeclive electronegativity, and X , the characteristic electronegativity (Pauling, 1940; Haissinsky, 1946) of the metal ion and V M represents the angular dependence of the bond strength calculated by Pauling (1940) for the various hybrid orbitals, these authors have found that the relation: log K

=

V,X,

+ const.

holds for a number of chelates, including those of zinc. Charles (1954), on the other hand, finds a better correlation between the heat of formation of EDTA complexes and xX’,,,r where z = ionic charge and r = ionic radius of the central metal ion. It should be noted that Van Uitert et al. do not consider entropy changes, but that these may be important. Williams (1954) has proposed that bond energy may be represented by the linear combination of terms representing electrostatic, covalent, and nuclear repulsive interactions, AHo = a ( z / r ) b 102 - c ( l / r 3 ) , where 102 = the 2nd ionization potential of the gaseous metal atom in KcalJmol.

+

378

BERT L. VALLEE

and r in Angstroms. He has calculated the coefficients for the heat of hydration of a number of divalent cations and finds that the ohscrvc1cl values are closely approximated by n = 150, b = 0.3, and G = 40. Ahrens (1954) has suggested that the term Se,, = ~ z ~ . d;, ~ ~ repre/ I ~ ~ senting the nuclear. shielding efficieiicy of the cation electrons, should I)(> inversely related to the relative stability of metal complexes. It is to he expected that further theoretical and semienipirical studies of the coordinate bond will elucidate similarities and differences hetween zinc and other metallic cations.

ACKNOWLEDGMENTS I itm much indebted t o my colleagues, Doctors S. J. Adelstein, 1C. ( 2 . I%sll,C:. D. Coryell, F. I,. Hoch, H. Neurath, and R . J. P. Williams for many stimulating discussions and suggestions and t o the staff of the Biophysics Research ILahornt.ory for its enthusiastic support. REFERENCES Adair, G. S. (1925). J . Biol. Chem. 63, 529. Agner, K . (1938). Biocheni. J . 32, 1702. Agner, B. (1941). A c f a Physiol. Scand. 2, Suppl. 8. .4hrens, I,. H. (1950). “Spectrorhemical Analysis.” Addison-Wesley, ( h m bridge. 5. Ahreris, I,. H. (1954). A’ature 174, 644. 6. Albert, A. (1953). Biochein. J . 64, 646. 7 . Allen, T. H., and Bodine, J. H. (1941). Science 94, 443. 8. Anderson, 13. (1934). z. physiol. (;hem. 236, 57. 9. Anson, M. I,. (1937a). J . Gen. Physiol. 20, 663. 10. Anson, M. I,. (1937b). J . Gen. Physiol. 20,777. 11. Babko, A. K . , and Philipenko, A . T. (1947). Zhrcr. Anal. Khinr. 2, 33; (1949) (‘hem. Abst. 43, 5344. 12. Barron, 15. S. G., and Levine, 8 . (1952). Arch. Biocheni. and Biophys. 41, 175. 13. Barron, E. S. C . , and Singer, T . P. (1945). J . Biol. C h e t n . 167, 221. 14. Ujerrum, J . (1941). “Metal Ammine Format,ion in Aqueous Solution.” Haase, Copenhagen. 15. Bliss, A. F. (1949). Biol. Rrcll. 97, 221. 16. Uoltz, D. F., ed. (1952). “Selected Topics in Instrumentnl Analysis.” l’rentice-Hall, New York. 17. Ijonnichsen, It. K. (1950). Acta C h e m . S c a n d . 4, 714. 18. Bonnichsen, R . K. (1953). 4 f h PoZioq. Ges. physiol. (‘hen!., p . 151. 19. Bonnichsen, It. I<., and Wassen, A. M. (1948). Arch. Biocheni. 18, 361. 20. Bowness, J. M., Morton, R . A . , Shakir, M. I f . , arid Stubbs, A. I,. (1952). H i o chevti. J . 61, 521. 21. Boyer, P. D., Shaw, J. H . , and Phillips, 1’. H . (1942). J . B i d . Chem. 143, 417. 22. Brandt, W . W., Duyer, F. P., and Gyarfas, E. C. (1954). (‘hem. Revs. 64, 059. 23. Brinkman, R., Margaria, R . , Meldrum, N. IJ., and Roughton, F. J. W. (1932). J . Physiol. (London) 76, 3. 24. Calvin, M. (1954). In “The Mechanisms of Enzyme Action” (W. D . McHroy and H , Glass, eds.), p. 221. Johns Hopkins Univ. Press, Baltimore. 1. 2. 3. 4.

ZINC AND METALLOENZYMES

379

25. Chance, 13. (1951). I n “The Enzymes” (J. B. Sumner and K. Myrback, eds.), Vol. 11, Part I, p. 428. Academic Press, New York. 26. Charles, R . G. (1954). J. A m . Chem. Soc. 76, 5854. 27. Cloetens, R . (1941a). Biochem. 2. 307, 352. 28. Cloetens, R. (1941b). Biochem. 2 . 3 0 8 , 3 7 . 29. Cloetens, R. (1942). Biochem. Z. 310, 42. 30. Cobble, J. W. (1953). J. Chem. Phys. 21, 1443,1446,1451. 31. Cori, G. T., Colowick, S. P., and Cori, C. F. (1938). J. Biol. Cheni. 124, 543. 32. Coryell, C. D . (1954). In “Chemical Specificity in Biological Interactions” (F. R . N . Gurd, ed.), p. 90. Academic Press, New York. 33. Coryell, C. D. (1955). Personal communication. 34. Craig, D . P., Maccoll, A., Nyholm, R . S., Orgel, L. E., and Sutton, L. E. (1954). J . Chem. Soc., p. 332. 35. Davenport, H. W. (1939). J . Physiol. (London) 97,32. 36. Davies, T., Singer, S. S., and Stanley, L. A. K . (1954). J . Chem. Sac., p. 2304. 37. Davis, R. 1’. (1955). Personal communication. 38. Dawson, C. It. (1950). I n “A Symposium on Copper Metabolism” (W. D . McElroy and B. Glass, eds.), Johns Hopkins Univ. Press, Baltimore. 39. Dawson, C. R., and Mallette, M. F. (1945). Advances in Protein Chem. 2, 179. 40. Dawson, C. R . , and Tarpley, W. B. (1951). In “The Enzymes” (J.B. Sumner and K. Myrback, eds.), Vol. 11, P a r t I , p. 454. Academic Press, New York. 41. Day, R., and Franklin, J. (1946). Science 104,363. 42. Delahay, 1’. (1954). “New Instrumental Methods in Electrochemistry.” Interscience, New York. 43. De Renzo, E. C., Kaleita, E., Heytler, P. G., Oleson, J. J., Hutchings, B. I,., and Williams, J. H. (1953). Arch. Biochem. and Biophys. 46, 247. 44. De Renzo, E. C., Heytler, P. C., and Kaleita, E. (1954a). Arch. Biochem. and Biophys. 49,242. 45. De Renzo, E . C., Kaleita, E., Heytler, P., Oleson, J. J . , Hutchings, U . L., and Williams, J. H . (1954b). Ann. N. Y. Acad. S c i . 67,905. 46. de Witt, R., and Watters, J. I. (1954). J . A m . Chem. SOC.76, 3810. 47. Dills, W. J,., and Nelson, J. M. (1942). J. Am. Chem. SOC.64, 1616. 48. Dixon, M . (1937). Nature 140, 806. 49. Drabkin, D . (1951). Physiol. Revs. 31, 345. 50. Dunn, F. J., and Dawson, C. R. (1951). J. Biol. (:hem. 189,485. 51. Edlbacher, S., and Zeller, A. (1936). Z. physiol. Chem. 246, 65. 52. Edsall, J. T., ed. (1951). “Enzymes and Enzyme Systems Their States in S a t u r e . ” Harvard Univ. Press, Cambridge. 53. Eirich, F. R., and Rideal, E. K . (1940). Nature 146, 541. 54. Feigl, F . (1949). ‘Chemistry of Specific, Selective, and Sensitive Reactions.” Academic Press, New York. 55. Feldherg, W., Keilin, D., and Mann, T. (1940). Nulure 146, 651. 56. Feldman, C. (1949). ,4nd Chem. 21, 1041. 57. Gibson, J. G., 11, Vallee, B. L., FIuhart.y, R . G., and NeIson, J. E. (1950). Acta (’onfra(‘aticerzm 6, 1102. 58. Granick, S. (1946). Chem. Revs. 38, 379. 59. Granick, S. (1947). Advances in Enzyniol. 7,305. 60. Green, D. E. (1941). Advances i n Enzymol. 1, 177. 61. Green, D. E., and Beinert, H . (1953). Biochini. et Biophys. Acla 11, 599. 62. Green, D. E., Mii, S., Mahler, H. R., and Bock, R. M. (1954). .I. Biol. Che,m. 206, 1 .

380

BERT L. VALLEE

63. Green, N . M., and Neurath, H. (1954). I n “The Proteins” (H. Neurath and K . Bailey, eds.), Vol. 2, part B, Chapter 25. Academic Press, New York. 64. Gurd, F. R. N., ed. (1954). “Chemical Specificity in Biological Interactions.” Academic Press, New York. 65. Gurd, F. R. N., and Goodman, D. S. (1952). J . Am. Chem. Soc. 74,670. 66. Haissinsky, M. (1946). J. Phys. Radium 7,7. 67. Hall, C. (1953). “Introduction t o Electron Microscopy.” McGraw-Hill, New York. 68. H a m , R., Snell, F., Hoch, F. L., and Vallee, B. L. I n preparation for publication. 69. Harrison, G. R., Lord, R. C., and I,oofbourow, J. It. (1948). “Practical Spectroscopy.” Prentice-Hall, New York. 70. Haugaard, N . (1946). J. Biol. Chem. 164, 265. 71. Haurowitz, F., and Hardin, R . L. (1954). I n “The Proteins” (H. Neurath and K . Bailey, eds.), Vol. 11, Part A, Chapter 14. Academic Press, New York. 72. Hayes, J. E., Jr., and Velick, S. F. (1954). J . B i d . Chem. 207,225. 73. Hellerman, L., and Stock, C. C. (1938). J . Biol. Chem. 126,771. 74, Hernler, F., and Philippi, E. (1933). 2. physioZ. Chem. 218, 110. 75. Hoch, F . L., and Vallee, B. L. (1949). J. Bid. Chem. 181,295. 76. Hoch, F. L., andVallee, B. L . (1952). J . Biol. Chem. 196,531. 77. Hoch, F. I,., and Vallee, B. I,. (1953). Anal. Chem. 26, 317. 78. Holmherg, C. G., and Laurell, C. B. (1948). Acla. Chem. Scand. 2, 550. 79. Horecker, 13. I,,, Stotz, E., and Hughes, T. R . (1939). J. Biol. Chem. 128, 251. 80. Hove, E., Elvehjem, C. A., and Hart, E. B. (1940). J . Biol. Chem. 136, 425. 81. Irving, H., and Williams, R. J. P. (1953). J . C h e m . Soc., p. 3192. 82. James, W. 0. (1953). Ann. Rev. Plant Physiol. 4, 59. 83. Keilin, D., and Mann, T. (1939). Nature 144, 442. 84. Keilin, D., and Mann, T . (1940a). Biochem. J. 34,1163. 85. Keilin, D., and Mann, T. (1940b). Nature 146, 304. 86. Keilin, D., and Mann, T. (1944). Nature 163, 107. 87. Keller, H . (1955). Biochem. 2. 229, 104. 88. Kiese, M., and Hastings, A. B. (1940). J . B i d . Chem. 132, 281. 89. Klotz, I. M. (1954). In “The Mechanism of Enzyme Action” (W. D. McElroy and B. Glass, eds.), p. 257. Johns Hopkins Univ. Press, Baltimore. 90. Klotz, I . M., and Loh-Ming, W. C. (1954). J . Am. Chem. SOC.74,805. 91. Koechlin, 13. A. (1952). b. Am. Chem. SOC.74,2649. 92. Kolthoff, I. M., Leussing, D. I,., and Lee, T . S. (1951). J . Am. Chenz. SOC.73, 390. 93. Kolthoff, I. M., and Lingane, J. J . (1952). “Polarography,” Vols. 1 and 2. Interscience, New k’ork. 94. Kornberg, A,, Ochoa, S., and Mehler, A. H. (1948). J. Biol. Chem. 174, 159. 95. Kubowitz, F . (1937). Biochem. 2. 292, 221. 96. Kubowitz, F. (1938). Biochem. 2. 229, 32. 97. Lardy, H. (1951). In ‘(Phosphorous Metabolism” (W. D. McElroy and B. Glass, eds.), Vol. 1, Chapter 8. Johns Hopkins Univ. Press, Baltimore. 98. Laufberger, M. (1937). BuEl. Soc. Chim. Biol. 19, 1575. 99. Lauger, P. G., Fnllab, S., andErlenmeyer, H . (1955). Helv. Chinz. Acta38,92. 100. Lechartier, G., and Bellamy, F. (1877). Compt. rend. 84, 687. 101. Lederer, E., and Lederer, M. (1953). “Chromnkography, A Review of Principles and Applications.” Elsevier, New York. 102. Lehninger, A. 1,. (1950). Physiol. Revs. 30, 393. 103. Leiner, M., and Leiner, G . (1940). Riol. Zenlr. 60, 449.

ZINC AND METALLOENZYMES

381

104. I,einer, h l . , and Leiner, G. (1941). NaturwissenschaSterL 29, 763. 105. Leiner, M., and Leiner, G. (1944). Biol. Zentr. 64, 293. 106. Lemberg, R . , and Legge, J . W. (1947). “Hematin Compounds and Bile Pigments.” Interscience, New York. 107. Lemberg, R., and Legge, J. W. (1950). Ann. Rev. Biochem. 19, 431. 108. Lerner, A. B., Fitspatrick, T. B., Calkins, E., and Summerson, W. H. (1950). J . Biol. Chem. 187, 793. 109. I h g a n e , J. J. (1953). “Electroanalytical Chemistry.” Interscience, New York. 110. Lohmann, K., and Meyerhof, 0. (1934). Biocheni. Z . 273,60. 111. Lutwak-Mann, C. (1938). Biochem. J . 32, 1364. 112. Mackler, B., Mahler, H. R., and Green, D. E . (1954). J . Biol. C h e w 210, 149. 113. Mahler, H . R. (1954). J. B i d . Chem. 206, 13. 114. Mahler, H. R., and Elowe, D . G. (1954). J. Biol. Chem. 210, 165. 115. Mahler, H. R., Baum, H., and Huebscher, G . (1955a). Federation Proc. 14, 806. 116. Mahler, H. R., Fairhurst, A. S., and Mackler, B. (1955b). J . Am. Chem. Soc. 77. 1514. 117. Mahler, H . R., and Green, D. E. (1954). Science 120, 3105. 118. Mahler, H. R., Mackler, B., Green, D. E., and Bock, R . M. (1954). J . Biol. Chem 210.465. 119. Mahler, H., Sarker, N., Vernon, 1,. l’., and Alberty, R . A. (1952). J . Biol. Chem. 199, 598. 120. Mallette, M. F., and Dawson, C. It. (1949). Arch. Biochem. 23, 29. 121. Malmstrom, B. G. (1953). ’4rch. Riocheni. and Biophys. 46,345. 122. Malmstrom, B. G. (1954). Arch. Biochem. and Biophys. 49,335. 122a. Malmstrom, B. G. (1955). Personal communication. 122b. Malmstrom, B. G. (1955). Arch. Biochem. and Biophys. 68, 381. 123. Mann, T., and Keilin, D. (1938). Proc. Roy. SOC.Bl26,303. 124. Mann, T., and Keilin, D. (1940). Nature 146, 164. 125. Martell, A. E., and Calvin, M. (1952). “Chemistry of t h e Metal Chelate Compounds.” I’rentice-Hall, New York. 126. McClure, J. G., and Banks, C. V . (1951). U. S. Atomic Energy Commission Bull. ISC-164. 127. McElroy, W. D. (1953). I n “A Symposium on Nutrition,” p. 262. Johns Hopkins Univ. Press, Baltimore. 128. McElroy, W. D . , and Glass, B., eds. (1950). “A Symposium on Copper Metabolism.” Johns Hopkins Univ. Press, Baltimore. 129. McElroy, W. D., and Nason, A. (1954). Ann. Rev. Plnnt Physzol. 6. 1. 130. Meikkjohn, G T., and Stewart, C. 1’. (1941). Biochem. J . 36, 755. 131. Meldrum, N. U., and Roughton, F. J . W. (1932a). J. Physiol. (London) 76.151’. 132. Meldrum, N . U., and Roughton, F. J . W. (193%). J . Physiol. (London) 76,3P. 133. Meldrum, X. U., and Roughton, F. J. W. (1933). J.Physiol. (London) 80, 113. 134. Mellon, M. G., ed. (1950). “Analytical Absorption Spectroscopy.” Wiley, New York. 135. Moeller, T. (1952). “Inorganic Chemistry- An Advanced Textbook.” Wiley, New York. 136. Mohamed, M. S., and Grcenbcrg, D . M. (1945). Arch. Biochem. 8, 349. 137. Mohamed, M. S., and Greenberg, D. M. (1954). J . Gen. Physiol. 37, 433. 138. Montgomery, M. L., Sheline, G. F., and Chaikoff, I. L. (1943). J. Exptl. M e d . 78, 51.

352

BERT L. VALLEE

139. Muller, 0 . (1951). “The Polarographic Method of Analysis.” Chemical Education Publishing Co., Easton, Pennsylvania. 140. Nachtrieb, N . (1950). “Principles and Practice of Spectrochemical Analysis.” McGraw-Hill, New York. 141. Najjar, V. A. (1951). I n “Phosphorous Metabolism” (W. D. McElroy arid I3. Glass, eds.), Vol. 1, Chapter 8. Johns Hopkins Univ. Press, Baltimore. 142. Nason, A., and Evans, H. J. (1953). J . Biol. Chem. 202, 655. 143. Nason, A., Kaplan, N . O., and Colowick, S. 1’. (1951). J. B i d . (,”hem.188,397. 144. Negelein, E., and Wulff, H. J. (1937). Biochem. 2,293,351. 144a. Neilands, J. (1952). J . Biol. Chem. 197, 701. 145. Nelson, J. M., and Dawson, C. R . (1944). Advances i n Enzynrol. 4, 99. 146. Neumann, H. (1949). Biochini. et Biophys. A c f a 3, 117. 147. Neurath, H . (1955). I n “Methods in Enzymology” (S. Colowick and N . 0. Kaplan, eds.), Vol. 11, p. 77. Academic Press, New York. 148. Neurath, H . , and De Maria, C:. (1950). J. Biol. Chem. 186,653. 149. Neurath, H., and Schwert, G. W. (1950). C‘hem. Revs. 46, 69. 150. Nicholas, D. J. D., and Nason, A. (1954). J. B i d . Chem. 207, 353. 151. Nicholas, D. J . D., Nason, A., and McElroy, W. D. (1954). J. Biol. C l e m . 207, 341. 152. Nyholm, It. S. (1954). Revs. Pure and A p p l . Cheni. 4, 15. 152a. Olsen, J. A., and Anfinsen, C. B. (1952). J . Biol. Chern. 197, 67. 153. Oppenheimer, C., and Stern, I<. G. (1939). “Biological Oxidation.” J u n k , The Hague. 154. Paul, K. 0 . (1951). In “The Enzymes” (J. 13. Sumner and K. Myrbiick, eds.), Vol. 11, Part 1, p . 357. Academic Press, New Tork. 155. Pauling, L. (1940). “The Nature of the Chemical Bond and the Structure of Molecules and Crystals,” 2nd ed. Cornell Univ. Press, Ithaca. 156. Petermann, &I. N., and Hakala, N. V. (1942). .J. Biol. Chem. 146, 701. 157. Philpot, J. St. 1,. (1939). Biochent. J . 33, 1707. 158. Powell, It., and I.atimer, W. M. (1951). J. Chem. P h y s . 19, 1139. 159. Putnam, F. W., and Neurath, H. (1946). J . Biol. (‘hem. 166,603. 160. Racker, E. (1950). J . Biol. Chem. 184,313. 161. Racker, E. (1955). Physiol. Revs. 36, 1. 162. Itaulin, J. (1869). Ann. sci. nat. Botan. el biol. vegetale 11, 93. 163. Richards, M. M., and Hellerman, L. (1940). J. Biol. Chem. 134, 237. 1631,. Richert, D. A., and Westcrfeld, W. W. (1951). J . BioZ. Chenz. 2C9, 179. 164. Itoche, J . (1935). “Pigments Itespirntoires.” Masson et Cie, Pnriu. 165. Roche, J., and Fox, H. M. (1933). Proc. Roy. SOC.Bl14, 161. 166. Itoughton, F. J. W. (1943). Harvey L e c f w e s Ser. 39. 96. 167. Roughton, F.J . W., and Booth, 1‘.H. (1946). Biochem. J. 40,309. 168. Roughton, F. J . W., and Clark, A. 11. (1951). I n “The Enzymes” (J. 13. Suniner and K. Myrbiick, eds.), 1701. I, Part 2, p . 1250. Academic Press, New York. 169. Samuelson, 0. (1953). “Ion Exchangers in Analytical Chemistry.” Wiley, New Yo&. l i 0 . Sandell, E. €3. (1950). “Colorimetric Dctcrminntiou of Traces of I\letalx,” 2nd ed. Interscience, New York. 171. Schlenk, F. (1951). In “The Enzymes” (J. B. Sumner and K . Mrybiick, eds.), Vol. 11,Part 1, p. 250. Academic Press, New Tork. 172. Sch\\-arzenl)ach, C. (1954). I n “Chemical Specificity in Biological In1 C P actions” (F. R . N. Curd, ed.), p. 164. Academic Prew, New York. 173. Scott,, D. A , , and Fisher, A . M. (1942). , J . Biol. Chern. 144, 371. 174. Scott, D. A., n n d Mendive, J. It. (1941%). .I. Riol. Cham. 139,661.

ZINC AND METALLOENZYMES

383

175. Scott, L). .I.,and Mendive, J. It. (1941b). J. Biol. Chew. 140, 445. 176. Sebrell, W. H., Jr., and Harris, It. S.,eds. (1954). “The Vitamins,” Vol. 1, Chapter 3. Academic Press, New York. 177. Seminova, D. 1’. (1946). Compt. rend. acad. sci. U . S. S. R. 61, 683. 178. Sidgwick, X. V. (1927). “The Electronic Theory of Valency.” Oxford IJniv. Press, New York. 17!). Singer, T. l’,, and Kearney, 12. B. (1954a). I n “The Proteins” (H. Neurath and K. Bailey, eds.), Vol. 11, P a r t A, Chapter 13. Academic Press, New York. 180. Singer, T. P., and Kearney, E. B. (1954b). Advances in Enzymol. 16,79. 181. Smith, I< . C. B. (1940). Biocheni. J. 34, 1176. 182. Smith, E. L. (1949a). Federation Proc. 8, 581. 1 S . Smith, E. 1,. (1949b). Ann. Rev. Biochem. 18,35. 184. Smith, I<. 1,. (1951a). I n “Enzymes and Enzyme Systems” (J. T. Edsall, ed.), p. 47. I-Iarvard Univ. Press, Cambridge. 185. Smith, E. 1,. (1951b). I n “The Enzymes” (J. 3.Sumner and K. Myrbiick, eds.), Vol. I, Part 2, p. 793. Academic Press, New York. 186. Smith, E. L. (1951~). Advances in Enzymol. 12, 191. 187. Smith, E. L. (1954). I n “TheMechanisms of Enzyme Action” (W. D. McElroy and B. Glass, eds.), p. 252. Johns Hopkins Univ. Press, Baltimore. 188. Smith, E. L., Brown, D . M., and Hanson, H. T. (1949). J . Biol. Chenz. 180,33. 189. Smith, 33. L., and Hanson, H . T. (1948). J. B i d . Chem. 176,997. 190. Smith, E. L., and Hanson, H. T. (1949). J. Biol. Chem. 179, 803. 191. Smith, E. I,. and Lumry, R. (1949). Cold Spring Harbor Symposia Quanl. Biol. 14, 168. 192. Smith, E. L., and Stockell, A. (1954). J. Biol. Chem. 207, 501. 193. Smith, G. F. (1954). Anal. Chem. 26,1934. 194. Smith, 0. C. (1953). “Inorganic Chromatography.” Van Nostrand, New York. 195. Snell, F., and Snell, C. T. (1949). “Colorimetric Methods of Analysis,” Vols. 1 and 2. Van Nostrand, New York. 196. Speck, J. F. (1947). J. Biol. Chem. 168,403. 197. Speck, J. F. (1948). J. Biol. Chem. 178, 315. 198. Spike, C. G., and Parry, R. W. (1953). J. Am. Chem. Soc. 76, 3770. 199. Stadie, W. C., and O’Brien, H. (1933). J . Biol. Chem. 103,521. 200. S t . George, It. C. G., and Pauling, L. (1951). Science 114, 629. 201. Stickland, L. H. (1949). Biochem. J . 44, 190. 202. Sumner, J . B., and Gralen, N. (1938). J. BioZ. Chem. 126,33. 203. Ssorenyi, E. T., Dvornikovtt, P. D . , and Degtyar, R. G . (1949). Doktady A k a d . N a u k S. S . S. R. 67,341; (1949). Chem. Abstr. 43,8410i. 204. Tanford, C. (1952). J . A,m. Chem. SOC.74,211. 205. Tanford, C., and Epstein, J. (1954). J . Am. Chem. SOC.76, 2170. 206. Tenenbaum, L. E., and Jensen, H . (1943). J. Biol. Chem. 147, 27. 207. Theorell, H . (1932). Biochena. 2.262, 1. 208. Theorell, H. (1942). Arkiu. Kenai Minerol. Geol. A16, No. 2. 209. Theorell, H. (1947). Advances in Enzymol. 7,265. 210. Theorell, H. (1951). In “The Enzymes” (J. B. Sumner and K. Myrbiick, eds.), Vol. 11, Part 1, p. 335. Academic Press, New York. 210a. Theorell, H. (1940). Arkiu. K e m i , Mineral. Geol. B14, No. 20. 211. Theorell, H., and Akeson, A. (1939). Science 90, 67. 211a. Theorell, H., and Maehly, A. C. (1950). Acta Chenz. Scand. 6,422. 212. Theorell, H., and Bonnichsen, R. (1951a). Acta Chenz. Scand. 6,1105. 213. Theorell, H., and Bonnichsen, R. (1951b). Acta Chem. Scand. 6, 329.

384

BERT L. VALLEE

214. Theorell, II., and Chance 13. (1951). Actn Cheni. Scand. 6, 1127. 21.5. Theorell, I I . , and I’edersen, I<. 0. (1944). I n “Svedberg 18841944’’ (A. Tiselius and I<. 0. l’edersen, eds.). Almquist and Wiksells, Uppsala. 216. Tissibres, A. (1948). Nali~re162, 341. 217. Tobias, C. A., and Dunn, It. A. (1949). Science 109,100. 218. Tsuchihashi, M. (1923). Biochem. 2.140,63. 219. Tupper, R., Watts, A . , and Wormall, R. W. E. (1951). Biochem. J. 60,429. 220. Vallee, B. L. (1951). Sci. Monthly 62, 368. 221. Vallee, B. I,. (1952). Il’ulrition Revs. 10,65. 222. Vallce, 13. I,. (1954). I n “Principles of Internal Medicine” (T. Harrison, ed.). Blakiston, Philadelphia. 223. Vallee, B. 1,. I n preparation for publication. 224. Vallee, B. L. Unpublished. 224a. Vallee, B. L., Adelstein, S. J., and Olsen, J. A. (1955). J. Am. Chem. SOC. 77, 5196. 225. Vallee, B. L., and Altschule, M. D. (1949). Physiol. Revs. 29, 370. 226. Vallee, B. L., and Coombs, T. L. Unpublished data. 227. Vallee, B. I,., Fluharty, It. G., and Gibson, J. G., 11. (1949). Actn U n i o Intern. (‘ontra Cancrum 6, 869. 228. Vallee, B. L., and Gibson, J. G., 11. (1948a). J. Biol. Chem. 176,435. 229. Vallee, B. L., and Gibson, J . G., 11. (1948b). J. Biol. Chem. 176, 445. 230. Vallee, B. L., and Gibson, J. G., 11. (1949). Blood 4, 455. 231. Vallee, 13. I,., and Hoch, F. I,. (1955%). J. Am. Chem. Soc. 77, 821. 232. Vallee, B. I,., and Hoch, F. I,. (1955b). Proc. Aratl. Acad. Sci. ( U . S.) 41,327. 233. Vallee, B. L., Hoch, F . I,., and Hughes, W. L., Jr. (1954). Arch. Biochem. and Biophys. 48, 347. 234. Vallee, B. I,., Lewis, H. D., Altschule, M. D., and Gibson, J. G., (11) (1949). Blood 4,467. 235. Vallee, B. L., and Neurath, H. (1954). J . Am. Chem. SOC.76, 5006. 236. Vallee, B. L., and Neurath, H. (1955). J. Biol. Chem. I n press. 237. Vallee, B. L., and Peattie, R. W. (1952). Anal. Chem. 24, 434. 238. Van Goor, H. (1934). Thesis, Groningen. 239. Van Goor, H . (1945). Rec. trav. china. 64, 313. 240. Van Goor, H . (1948). Enzymologia 13, 73. 240b. Van Uitert, 1,. G., and Fernelius, W. C. (1954). J . Am. Chem. SOC.76, 379. 241. Velick, S. F. (1953). J . BioE. Chem. 203, 563. 242. Velick, S. F. (1954). I n “Mechanisms of Enzyme Action” (W. D. McElroy and B. Glass, eds.), Chapter 5. Johns Hopkins Univ. Press, Baltimore. 243. Velick, S. F., Hayes, J. E., Jr., andHarting, J. (1953). J. Biol.Chem. 208,527. 244. von Euler, H., and Adler, E. (1935). 2. physiol. Chem. 232, 10. 245. Wagner-Jauregg, T., and Moller, E. (1935). 2. physiol. Chem. 236, 222. 246. Warburg, 0. (1949). “Heavy Metal Prosthetic Groups and Enzyme Action.” Oxford Univ. Press, New York. 247. Warburg, O., and Christian, W. (1941). h.’aturzuissenscha&n 29, 589. 248. Warner, R. C., and Weber, I. (1953). J . Am. Cheni. SOC.76,5094. 249. Webb, E C., and Van Heyingen, R. (1947). Biochem. J . 41, 74. 250. Weier, T. E., and Stocking, C. R. (1952). Botan. Revs. 18, 14. 251. Weissherger, A., ed. (1954). “Physical Methods of Organic Chemistry,” 2nd ed. Parts 1 and 2 (1949). Part 3, (1954). Interscience, New York. 252. Williams, R. J. 1’. (1953). Biol. Revs. 28,4. 253. Williams, R. J. P. (1954). J . Phys. Chem. 68, 121. 264. Zinoffsky, 0. (1880). 2. physiol. Chem. 10, 16.

Author Index Numbers in parentheses are reference numbers atid are included to assist in locating references i n which the authors' names are not mentioned in the text. Numbers in italics indicate the page on which the reference is listed.

A Abderhalden, E., 48,124 Abraham, E. P., 81(370), 133 Abraham, S., 220,225 Adair, G. S., 263,288, 374, 378 Adamkiewicz, A , , 35,124 Adelberg, E. A., 41, 91(600, 601), 139, 124 Adelstein, S. J., 370,384 Adler, E., 359, 360, 384 Agner, K., 322,323, 331, 378 Agren, G., 2 , 2 r Ahrens, L. H., 320, 378, 378 Airan, J. W., 231, 282 h e s o n , A., 172,226,322, $83 Akiba, T., 40(5), 124 Albanese, A. A., 35(7), 124 Albert, A , , 71(8), 124, 352,353,378 Albert, P. W., 81(9), 97(9), 1% Albert, S., 25(32), 28 Alberty, R. A., 174,179,221, 224,330,381 Alderton, G., 6(2), 27 Alejo, L. G., 101(113b), 127 Allen, T. H., 324, 378 Alsberg, C., 6, 28 Alston, E. J., 66(297), 133 illtman, K. I., 76(330a, 776a), 96(10), 124, 182,144

Altschule, M. D., 320,333,335,340,384 Amano, K., 233, 238, 269, 270, 282 Amano, T., 84(11), 124 Amberson, W. R., 242, 282 Anagnostopulos, C., 5(3), 16(3), 28 Anderson, B . , 353, $78 Anderson, D. N., 233, 282 Anderson, E. G., 113(862), 147 Anderson, J. O., 91 (12), 124 Anfinsen, C. B., 370,382

l~.,23 (4), 28, 201, 221, 224, 343, 344, 378 Arai, IC., 96(571a), 159 Arai, Y.,312,316 Arloing, S., 228, 286 Armitage, P., 102(223), 130 Armstrong, M. D., 35(14,15,15a),54(17), 55(16, 17a, 17c), 109(17), 124 Armstrong, S. H., Jr., 177,179,226 Arnow, L. E., 68(18), 124 Asano, M., 231,288 Asero, B., 104(19,238, 239), 124, 130 Asimov, I., 231, 234, 288 Atkinson, D. 15., 40(20), 124 Auerbach, V. H., 83(492), 85(484), 136, 137 Augustinsson, K. B., 272, 282 Autret, M., 516 Avener, J., 110(918), 148 Axelrod, B., 8(5), 26(6), 28 Axelrod, H. E., 80(21j, 124 Axelrod, J . , 63(22, 231, 64(106, 881, 882), 68(106), 126, 147 .411SOI1,1~.

B Baba, H., 238, 272, 286 Babko, A. I<.,353,378 Aaddiley, J., 43(25), 76(26), 91(21), 124 Baer, J. T., 66(888), 68(888), 147 Blrwald, L., 118(334), I33 Bailey, K., 198, 221, 233, 235, 236, 237, 253, 255, 257, 263, 264, 265, 266, 267, 268, 281, 882, 288 Baker, J. W., 110(28), 111(27), 124 Baker, L. E., 23(7), 88 Baker, R. S., 114(285), 131 Baldes, K., 46(225), 48(225), sO(225)1 ) 130 Baldwin. H. R.. 35(29), 12.4 Baldwin; W. H:, 311

385

386

AUTHOR INDEX

Balk, M. E., 54(179a), 128 Balken, K., 309,516 Ball, C. D., 121(132), 127 Ball, E. G., 271, 287 Bancrjee, S., 103(143), 127 Rrtngn, I., 235, 282 Iluriks, A., 238,271,282 Banks, C. V., 361, 363, 581 Baranowski, T., 235, 282 Barbit-ri, J., 34, 144 Bard, R. C., 40(702), I42 Barer, It., 229, 282 Barets, A , , 230,231,282 Barger, G., 72(355), 76, 124, 139 Barker, S. B.,71(31), 75(476), 124, f36 Barron, IC. S. G., 356,360,368,578 B a t h , I,., 34,124 Barthel, W. F., 116(526), 197 Bnshford, M., 41(686b), 142 Basinski, D. H., 49(792), 52(792), 60(33), 124, 144 Bate Smith, E. C., 234,269,282 Bateson, W., 48, 126 Bauer, C. D., 35(35), 126 Baum, €I., 322, 581 Baumnnn, C. A , , 40(785), 88(599),94(599) 169, 144 Barimann, E. A. G., 34,47,71,76,125,149 Bayerle, C., 118(776), 144 Bayliss, W. hI., 1(74), 99 Beach, E. F.,4(8), 28 Beadle, G. W., 40(855), 70(40), 80(41), 81(90, 92), 126, 126, 146 Bean, H. W., 231,234,286 Bean, R . S., 167, 222 Bear, J., 46(12), 125 Bear, It. S., 278,282 Becker, E., 80(125, 126), 12Y Beer, R. J. S., 116(43,44),126 Beerstecher, E., 40(45, 48), 42, 110(46), 125 Behrmann, J. S., 94(823), 145 Beiler, J. M., 106(49), I26 Beinert, H., 379 Bell, G. H., 81(50), 126 Bellnmy, F., 318,580 Bellamy, W. D., 76(326,327), 132 Benaglia, A. E., 219, 220, 225 Benassi, C. A., 95(619,619a), 240 Bendall, J. R., 269, 282 Bender, A. E., 300,315

Benesch, R., 173, 221 Benesch, R. E., 173,291 Bentley, J. A., 115(51, 452), 126, 196 Berezov, T. T., 35(52), 125 Berg, C. I’., 35(29, 35, 53, 54, 55, 139), 80(457), 84(584), 90(585), 124, 125, 127, 156, 139 Berg, I,. R., 300, 315 Berger, F., 118(819), 146 Berggrcn, R. E. L., 4(15), 5(15), 28 Bcrgmann, E. D., 41(56), 42,126 Bergninnn, M., 3, 7,28 Bergren, W. It., 110(330), 182 Bernhard, H., 68(777), 144 Bernhard, K., 76(59), 126 Bernhart, F. W., 185, 221 Bernheim, F., 55, 126 Bernheim, M. C. I,., 55(61), 126 Bernstein. S. S., 4(8), 28 Bessey, 0. A., 101(113b), 127 Bessman, S. l’., 54, 1-47 Bethell, F. H., 60(846,847), I46 Beveridge, J. M. R., 278,282,314,615 Beyer, K. H., 66(62), 67(63), 125 Bickel, A. S., 115(51), 125 Birkel, H., 35(65), 54(64,65), 55, 126, 1% Bicknell, R . L., 101(113h), 127 Biekert, E., 88(120, 121, 122, 122a), 127 Biely, J., 300, 315 Bier, M., 154, 167, 178, 219, 220, 222, 223, 224 Bigwood, E. J., 231, 282 Bilinski, E., 234, 282 Biorck, G., 104(870), f47, 231, 286 Birch, A. J., 116, 125 Bissett, H. M., 271,288 Bito, ILL, 233, 238, 269, 270,282 Bjerrum, J., 321, 372,978 Bjorkman, G., 104(870), 147 Black, E. S., 177,226 Black, W. A. P., 303,312,516 Blake, C. O., 117(67), I85 Blakwood, J. H., 7, 28 Blnnchard, M. H., 59(68, 69), 126, 168, 170, 181, 187,222 Blaschko, H., 51(80, 83), 66(72, 73, 74, 75), 67(70, 71, 78, 195a), 69(77), 77 (77), 107(76,77,79), 125,128, 129 Bliss, A. F., 369, 578 Bloch, K., 43, 191 Block, B., 48(1), 184

A4UTHOIt INDEX

387

Briggs, F. N., 75(859), f47 Briggs, G. M., 91(12, 315), 124, 152 Brignon, J., 277, 288 Brimley, It. C., 232, 233, 287 Brinkman, R., 333, 878 Hrock, J. I?., 315 Erodie, B. U.,63(22,23), 64(106,881, R82), 68(106), 124, 126, 147 Broomfield, 1%. J., 112(364), I33 Brown, D. >I., 344, 383 Brown, J. B., 115(107), 126 Brown, S. A , , 117(107a), 126 Mruce, J. M., 70(108), 126 Brull, I,., 273, 274, 283 Bruns, F., 76(330a), 182 Bubl, E. C., 35(109), 126 Buchs, S., 683 h d d e c k e , E., 05(908), 148 Bucher, T., 52(471), 136 Biihn, W., 229,230, 231, 283 Hiilbring, E., 67(110, l l l ) , 123 Buffa, A , , 309, 315 Bulgarelli, I<., 89(622), 140 149 Banner, J., 114(285, 378, 921), 131, lcq4, Bull, H. B., 174,185,221,222 I3u’Lock, J . D., 69(112, 113), 70, 110(362), 148 126,127,139 Boone, I. U., 101(95), 126 Uonnichsen, It. K., 353,354,355,360,368. Burch, H. R., 101(113b), 127 Bumpus, F. M., 107(113a), f 2 7 369, 378, 383 Burger, M., 40(474), 45(473), 186 Booth, N., 185, 221 Booth, V. M., 336, 382 Ruri, A,, 187, 223 Boretti, G., 108(240), 130 Burlr, X. F., 5, 21(12), 28 Burn, J. H., 66(114), 67(78, l l l ) , 126, 127 Bosch, M. W., 240, 242, 283 Bosrott, R . J., 55,60(99), 63(96), 78(100), Burris, R . H., 10(470), 118(931), 136, 148 126 Burton, I. F., 60(846), f 4 6 Butenandt, A , , 79, 80(116, 125, 126), 81 Bouchilloux, S., 113(100a), 126 (115, 128), 84(117), 88(120, 121, 122, Bourland, K., 5(3), 16(3), 28 122a), 89(123, 124), 96(118, 119), 98 Bowden, K., 117(101), 126 Bowes, J. H., 154,167,168,199,221 (123), 127 Hutts, J. S., 35(109), 46(130, 131), 49 Bowness, J. M., 369, 378 Boyd, W. C., 231,234,288 (130), 126, 127 Boyer, P. D., 271,283, 319, 378 Byerrurn, R . U., 121(132), 127 Boyle, F. P., 115(396), 13.4 Braaten, K., 49(150), 53(150), 128 Bramstedt, F., 238, 287 Cable, R. S., 17,28 Cain, C. K., 116(368), 133 Brandt, W. W., 363,366,378 Calkins, 15.. 324,381 Braunshtein, A. Ti;., 89, 126 Callow, H. J., 110(401), f3.4 Bray, H. G., 96(103), 100(104), 126 Calvin, PI.,321, 325, 326, 336, 353, 363, Bricas, E., 234, 283 372, 378, 381 Hrieger, L., 113(105), 126 Carnmarata, P. S., 59(133, 134), 127 Brierley, J. M., 60(791), 144 Campbell, G. F., 6(62), 1 1 , 2 9 Briggs, A. P., 97(810), 146 Block, It. J., 53(450), 78(450), 185, 277, 283, 304, 305, 308, 316 Blum, I,., 46(42), 48,126, 126 Blurnenthal, F., 112(85), 126 Bock, 13. G., 58(257), 59(257), 130 Bock, R . M., 323,330,379, 681 Bodine, J . II., 324,378 Boedeker, H., 47, 126 Bohrn, F., 112(87), 126 Bohr, D. F., 94(311), 132 Boissonnas, It. A., 67(460), 136 Bokman, A. H., 98(89), 99(88, 89, 608, 676), 126, 140, 141 IJolk, L., 228,283 Uolling, D., 53(450), 78(450), 135, 277, 283, 304, 305,308, 616 Boltz, D. F., 329, 378 Bond, H. W., 81(427), 103(426), 135 Aonner, D. M., 40(93, 663, 854, 855), 41(663, 853, 854), 81(90, 92, 970), 88(698), 94(439), 95(970), 96(663), 97, 99(971), 126, 135, 137, 141, f42, 146,

388

AUTHOR INDEX

Ciotti, M. M., 101(974), 150 Clancy, C. W., 87(140), 127 Clark, A.M., 333,335,336,382 Clark, C. T., 64(881, 882), 66(888), 68 [SSS), 104(883), 105(885), 106(148, 883, 884), 108(883), 128, 147 Clark, I., 81(675), 88(674), 141 Clarke, K., 116(43), 126 Clerc-Bory, M., 104(149), 128 Cloetens, R., 319, 379 Closs, K., 49(150, 273), 53(150, 151, 274), 128, 131 Cobble, J. W., 374, 379 Cohen, A. I., 103(797), 146 Cohen, 1'. P., 59(133,134), 197 Cohn, E. J., 4(15), 5(15), 28, 35(152), 128, 153, 157, 163, 164, 168, 170, 181, 187, 222 Cole, A. S., 35(135), 128 Cole, S.W., 34,35(410), 109(412), 110, 296 381 Colo, V., 104(19), 124 Chan, F. L., 87(140), 127 Colonick, S. P., 41(628), 101, 102(683), Chance, B., 333,355,379,383 l 4 O , l 4 l , 160,319,369,379,582 Chandler, J. P., 67(569), 199 Colvin, J. R., 275, 277, 283 Channing, D. M., 303,325 Combs, G . F., 91(12), 124 Chapman, I,. M., 174,222 Connell, J. J., 235, 238, 239,240, 244, 247, Chapman, V. J., 312, 916 252,261, 263,264,273, 280,281, 2885 Charconnet-Harding, F., 59(141), 86 Connors, W. M., 65(154), 128 (142), 87(142), 96(142), 127 Chargaff, E., 6(14), 28, 66(297), 182, 267, Conochie, J., 113(155), 128 Conway, E. J., 234,283 284 Cooke, W. T., 60(99), 186 Chari, S. T., 305, 313,316 Coombs, T. L., 351,353,984 Charles, R. G., 374, 377, 879 Coon, M. J., 35(732a, 732b), 14.9 Chattopadhyay, D., 103(143), 127 Cooper, A. R., 231,234,284 Chattopadhyay, H., 103(143), 127 Cooper, J. R., 54, 58, 66(888), 68(888), Chaudhuri, D. R., 154, 171, 222 76(887), 147 Cheldelin, V. H., 44(866), 147 Cooper, M., 174,222 Chen, K. K., 107(444), 196 Cooper, O., 271, 28Y Chen, T. T., 185, 226 Coppini, D., 88(620), 94(155a, 621), 100 Cherkes, G. A., 102(144), 128 (624), 128, 140 Cherrington, M. E., 60(952), 149 Chiancone, F. M., 88(620), 94(145), 128, Corfield, M. C., 276,283 Cori, C. F., 277,288,319,979 140 Cori, G. T., 44(861), 147, 319, 979 Chiari, H. H., 113(146), 128 Cornforth, J. W., 83(156, 157), 116(157, Chikano, M., 78(502), 237 Chinn, B., 242,282 158), 128 Cornforth, R. H., 83(156), 128 Chou, C., 185,222,226 Corse, J., 116(432), 196 Christensen, B. E., 44(866), 147 Christensen, H. N . , llO(147, 704), 128, Corson, 11.1.H., 103(542), 138 Coryell, C. D., 169,196,222,352,363,376, 142 979 Christensen, I,. R., 220, 222 Costabile, L., 101(159), 128 Christian, W., 319, 384

Camurri, M., 94(155a), 128 Csnnan, R. K., 14(13), 28, 155, 156, 157, 158, 163, 165, 167, 169, 172, 178, 185, 217, 220, 222 Cannon, P. R., 315 Cantoni, G. L., 103,127 Canzanelli, A., 72(137), 127 Carpenter, D. C., 5,26(91), 30 Carpenter, K. J., 110(494), 137,300, 916 Carpenter, 1,. M., 5, 26(91), 30 Carretero, It., 80(817), 145 Carrigan, E. J., 309,916 Carroll, C. J., 116(368), 133 Carroll, W. R., 264,285 Casini, E., 81(623), 140 Cawte, J. E., 53(138), 54(138), 127 Celander, D. R., 35(139), 127 Chaikoff, I. L., 72(612,625,665,666,860), 75(859, 860a, 873), 140, 141, 147, 353,

AUTHOR INDEX

Couceiro, A., 273, 283 Coursaget, J., 85(314a), 132 Couteaux, R., 229,283 Coyne,B. A., 110(147,704), 128,142 Craig, D . P., 376, 979 Craig, J. P., Jr., 174, 222 Crammer, J. L., 172, 185, 221, 222 Crandall, D. I., 55(695), 57, 58(160, 161, 164), 60(160), 64, 65(160, 161, 163, 164), 99(162), 128, 1.42 Credner, K., 66(404,405), 68(403), 134 Crepax, P., 240,242,243, 245,281,283 Crerc’h, P. V., 231, 234, 285 Crester, J. H., 4(30), 28 Crokaert, R., 234, 282, 283 Cromartie, R. I. T., 88(122a), 105(165), 127, 128 Crom, N., 67(569), 139 Cubiles, R., 9(86), 30 Custer, J. H., 154,174,224 Cutinelli, C., 45(166), 128 Cutting, C. L., 231, 234, 238, 287 Cuypers, Y., 274, 283

D Daimler, B. H., 275, 283 Dagley, S., 40(167), 78(167), 128 Dakin, H. D., 46(169, 902), 66(168), 128, 148 Dalgliesh, C. E., 54(174), 55, 59(141), 64 (174), 67(177), 68(174), 80(170), 83 (156, 157, 170), 84(176), 85, 86(142, 171, 173, 176), 87(142, 170, 173), 89 (171, 176), 94(171), 95(170, 171, 173, 178), 96 (142, 171, 174), 98(171), 100 (178), 106(179), 107(179), 112(175), 116(157), 127, 128 Damodaran, M., 5, 28 Dancis, J., 54(179a), 128 Dangschat, G., 36(268), 191 Dann, M., 53(180), 128 Dann, W. J., 102(181), 168 Darby, W. J., 60(182,952,953), 103(249a), 189,19O,ld9 Dassow, J. A., 309, 516 Davenport, H. W., 336,3Y9 David, P., 35(911), 148 Davidson, J. R., 25(17), ?8 Davies, T., 374, 379 Davis, B. D., 36, 37, 38(191a, 192), 39, 42 (187), 45(456a, 45613, 805, 805a, 823a),

389

46, 54(187, 188), 129, 136, 1.60, 1,@, I&, 148 Davis, R. P., 334, 337, 379 Davis, V. I., 35(7), 124 Davison, J. A., 270,289 Dawes, E. A., 110(194), 129 Dawson, C. R., 320, 322, 324, 330, 333, 379,381, 382 Dawson, R . F., 117(195), 129 Day, R., 334, 379 Dayton, A., 35(911), 148 de Almeida, D. F., 273, 283 Deas, C. P., 237,283 de Gouveia, A. J. A., 305,316 de Gouveia, A. P., 305, 316 Degtyar, R . G., 323,383 Dekker, C. A., 10,28 Delahay, P., 329, 379 de la Huerga, J., 110(517), 137 de Lange, D. J., 80(399), 103(398), 184 De Las Heras, A. R., 231, 285 Della Monica, E. S., 154,174,224 della Pietra, G., 97(685), 142 Delluva, A. M., 66(328), 132 D e Maria, G., 350,382 Dennis, D . J., 67(195a), 129 Deolalkar, S. T., 269, 283 D e Renzo, E. C., 333, 379 de Ropp, R . S., 110(918), 148 Derrien, Y., 254, 287 Deuel, H. J., 80(430), 81(9, 50), 89(768), 97(9), 12.4, 125, 135, 144 Deulofeu, V., 107(196, 197), 129 Deuticke, H. J., 241, 252, 283 Deutsch, H . F., 273,274,279,285 de Verdier, C. H., 2,3,27,30 De Vincentiis, M., 271, 289 Devine, J., 68(198), 129 Dewnr, E. T., 303, 315 de Witt, R., 352, 363, 379 Diaz, C. J., 49(199), 129 Dills, W. L., 322, 379 Dingle, J. R., 239, 240, 283 Dinning, J. S., 91(200), 129 Dirr-Kaltenbnch, TI.,55(262), 151 Dische, R., 57, 129 Dixon, M., 360,379 Doherty, M. E., 220,293 Doisy, E. A., 116(368), 153 Dolby, D. E., 111(202), 129 Donovan, F. W., 116(66), 225

390

AUTHOR INDEX

Dorfman, .4.,220, 224 Dowling, M. T., 42(808), 145 Drabkin, D., 333, 879, Drechsel, E., 75, 128 Drilhon, A . , 273,288 Dubeck, &I.,121(204), 129 Dubuisson-Brouha, A , , 240, 280, 281, 288 Dubuisson, M., 154, 169, 222, 234, 235, 243, 245, 249, 263, 265, 280, 289 D tic kwor th , J., 300,-315 Duerr, J. D., 256,283 Dugal, I,. C., 312, 815 Duke, J. A., 154, 167, 178,219,222 D ul i he, W. I,., 66(205), 69(205), 129 Dunc:tn, A l . , 101(206), 129 Dunn, F. J., 322, 330, 979 Dunn, bf. S., 35(207), 16(130, 131), 49 (130), 127, 129,169,222 I h n n , I{. A . , 329, 384 Duprat, E., 107(197), 129 I h r f e e , A. I<., 115(396), 154 du Vigneaiid, V., 35(895), 67(460, 569), 196, 199,l48

Ihornikov:t, 1’. D., 323,583 Ihyyer, F. P . , 363, 366,378 Dycr, W. J., 228, 235, 236, 237, 239, 243, 244, 245, 255, 256, 271, 885,284

E Eagles, L). I<., 239, 240, 283 ICbisuzaki, I<., 101(519), 137 Edlbacher, S., 319, 379 Edmonds, 13. J., 110(46), 125 Edmunds, M. E., 52(961), 149 ICdsall, J . T . , 35(152), 128, 153, 157, 163, 164,I68,170,181,222,235,284,321, 379 Iklson, N. I,., 46(208), 48, 55, 129 ICtlwxrds, H. W., 62(20!)a), 65(209, 4X5), 139, 135

l<:fimochink:i, 1,:. F,,85(210), 129 IiCggstein, M., 99(374), Ehrenberg, A., 154, 225, 254, 288 IShrensvard, G . , 39(856), 41(68611), 43, 44(211), 45(166), 46(G86:t, 686h), 124, 128, 129, 149, l.&

b h l i r h , F., 78(214), 10ti(214), fd9 Eijkmann, J. F., 36(21.5), 129 Eirich, F. R., 334, 879 I’isentxrg, II., 163, 223 l~:isenbcrg,M. A , , 216, 922 I~Xlelnian,l<.S., 281, 286

PX, A., 84(216), 91(216), 105(217, 218), 106(217), 129 I’lnm, D. W., 273, 286 I
F Falwr, X1 , 66(297), 132 Fairhurst, A. S., 333, 381 Fajn, F. S., 269,288 Vdronirri, G., 117(241), 150 l:allab, S., 352, 353, 580 F d t x . W., 47(638), 48(250), 150, 140

39 1

AUTHOR INDEX

Faur6-Freniiet, B., 277, 284 Fawcett, D. M., 75(251, 252, 253), 13c Fearon, W. It., 272,284 Feigclson, P., lOO(924, 025), 101(254, 255, 926), 130, 148 Feigl, F., 329, 379 Feinstein, R. N., 22(20), 26(20), 28 Feldberg, W., 106(256), 130, 336, ,979 Feldman, C., 344,379 Felix, K., 51, 52(258, 259, 260), 55, 57, 58, 59(257), 130, 131, 136, 228, 274, 275, 276,284,287 Fellers, C. R., 233,282 Ferguson-Wood, 15. J., 272, 284 Fernelius, W. C., 376, 384 Ferri, & G., I.114(921), 1.48 Ferry, J. D., 154, 222, 226 Ferry, R . M., 170,222 Feuer, G., 264, 284 Fevold, H. L., 6, 27, 28 Fewster, PYI. E., 40(167), 78(167), 128 Figiltaua, K., 238, 284 Fildes, P , 40(263, 264), 55(264), 131 Fink, I<., 72(265), 191 Fink, R. M., 72(265), 131 Finn, D. B., 291,316 Fischer, E., 34(267), 131 Fischer, F. E., 104(341), 193 Fischer, H. 0. I,., 36(268), 131 Fisher, A. RI., 331, 334, 382 Fisher, H., 274, 275, 284 Fitzpatrick, T. B., 69(260, 554), 131, 139, 324, 381 Flscher, F., 66(270), 131 Flamand, C., 35,110(222), 130 Flaschentriger, B., 60(271), 131 Flavin, M., 15, 25(22), 28 Fletcher, L. I., 232, 233,287 Florkin, M., 273, 284 Fluharty, G. R . , 339, 379, 384 Fiilling, A , , 49(273), 52, 53(151, 274), 128, 191

Folsome, C. I(:.,94(823), 145 Fonci, A . , 8,28 Foote, M. W., 22(24), 26(24), 28 Forbes, R. M., 231,234, 284 Forrest, H. S., 87(275), f3f Forrest, W. W., 211, 222 Foster, D. I)., 272, 284 Foster, G. I,., 72(276), 131 Foster, J . F., 218, 226

Foster, &I.,68(277), 131 Fougere, H., 256, 284 Fox, H. M., 322, 382 Fox, S. W., 40(20), 124 Fraenkel-Conrat, H., 167, 174, 222 Franklin, J., 334, 379 Frazer, S. C., 25(17), 28 Fredericq, E., 154, 163, 170, 222 Freire, J. R. C., 273, 283 French, D., 168,222 French, H. V., 228,235,236, 237,243,241, 255, 284 Freund, H. A., 60(33), 124 Freyburger, W. A , , 131 Friedberg, F., 57(933), 148 Frieden, E. H., 169, 222 Friedman, B. K., 106(977), 150 Friedmann, E., 48, 56, 131 Frohlich, H. G., 154,165,229 Fromageot, C., 173,222,234,283 Fruton, J. S., 7,28,40(809), 146 Fryth, P. W., 116(903), 148 Fuchs, H., 89(939, 940), 149 Fugitt, C. H., 153, 154, 160, 161, 166, 169, 172, 177, 178, 199,226 Fujiki, T., 95(573), 139 Fujimaki, M., 269, 284 Fujito, S., 88(468), 136 Fukui, G., 238, 286 Funk, C., 65(280), 131

G Gaby, W. I,.,116(368), 133 Gad, I., 68(281), 131 Gaddum, J. H., 104, 131 Galdston, R'I., 49(788), 144 Gale, E. F., 76, 109, 124, 131 Galston, A. W., 114(285, 296, 805h), 131, 132, 145 Gammes, T., 53(274), 181 Gardner, F. H., 60(723), 143 Gardner, M. J., 80(782), 144 Garnjobst, L., 39(856), 146 Garrett, A. G., 174,222 Garrod, A. E., 47, 110(287), 191 Gautheret, R., 113(521), 137 Geiger, E., 228, 277,278,284 Geratz, D., 58(257), 59(257), 130 German, B., 195,222 German, H. I,., 80(782), 144 Gerrard, J., 35(65), 54(64, 65)) 126

392

AUTHOR INDEX

Uhosh, H., 178, 222 Ghosh, N. (>.,103(143), 127 Gibbs, 11. J , , 215, 216, 219, 220, 222, 227, 226

Gibson, .I. (i.,[ I , 835. M!), 340, 345, 379, S84 Gibson, ( 2 . I I . , 35(28X), I:?/ Gillis, J., 163, 223 Gilvarg, C . , 37(964a), 30(288a, 907), 43, 131, 148, 149 Ginoulhiac, E., 86(292), 88(620), P9(293), 131, 140 Glass, B., 333, 381 Glassman, H. N., 167,174,223 Glasstone, S., 214, 223 Glazer, H . S.,94(294), 181 Gleysteeu, L.F., 165, 167, 168, 172, 225 Glynn, I,. E., 49(205), 52(295), 131 Glomset, J., 2,27 Goldblith, S. A , , 272, 286, 287 Golder, Ti. H., 281, 286 Goppert, R., 228, 283 Goldacre, 1’. J,., 114(296), 132 Goldbloom, A , , 60(611), 140 Goldenberg, RI., 66(297), 162 Goldsmith, G . A , , 80(755), I43 Goldstein, A., 220, 223 Goodall, MrC., 66(298, 299), 132 Gooder, €I., 92(300), 111 ( N O ) , 132 Goodland, It. l,,, 55(789), 57, 60(791), 144 Goodloe, RI. 13., 273,279, 283 Goodman, D. S., 173,223, 320, 380 Goodwin, S., 63(946), 99(946), 14.9 Gorbman, A4.,75(718), 142 Gordon, H. H., 52(5,55, 556, 557), 60 (648a), 138, 141 Gordon, &I., 39(301), 41(302), 132 Gordon, 9. A . , 111(303, 304), 132 Gordon, W. G., 17, 28 Gorini, I,., $25 Gortner, W. A , , 114(305), 132 Goryachenkovo, I+:. V . , X9(102), 01 (306), 126, 132

Gots, J. S., 40(307), 132 Goutarel, It., 119(308), 1% Govan, C. D., Jr., 60(648a), 141 Govier, W. M., 107(278), 131 Grabar, P., 5(3), 16(3), 28 Graham, B. IC., 107(278), 131 Gralcn, N., 194, 223, 253, 284, 331, 383 Granick, S., 320, 333, 379

Graser, G., 85(947), 149 Grnu, C . R . , 59(309), 132 Green, A . A , , 44(861), IOJ(B!)3), / 4 2 , 141, 181, 187, 222 ({repn, D. E., 59(G8, 69), 77, 126, /&‘, 3 l ! l . 323,324,330,333,879, S R / Green, H., 26(58), 29 Green, X. M., 343,344,380 Green, NI. M., 85(310), I36 Greenberg, D. M., 5, 21(12), 28, 57(752, 933), 59(523, 560), 61, 04(523), 137, 138, 146, 148, 174, 222, 223, 251, 285, 319, 322, 343, 381 Greenberg, L. D., 94(311), 132 Greenberg, S., 78(100), 126 Greene, C. W., 228,284 Greenstein, J. P., 65(592), 139, 181, 185, 214,216, 217, 219, 220, 224 Grifliths, R , 54(961a), 149 Gripenberg, J., 116(312), 132 Gros, H., 52, 58, 232 Gros, P., 85(314a), 132 Groschke, A. C., 91(12,315), 124, 132 Gross, J., 74, 75(320, 321), 132 Gross, O., 48, 139 Gross, S. R., 39(856), 1.46’ Grossman, W. I., 83(492), 102, 135, 137 Groves, M. L.,4(30), 18(31), 28, 154, 217, 220, 223 Guba, F., 237,245,257,258,284 Gudaitis, H., 94(898, 899, 900), 148 Guggenheim, E. A., 202,223 Cuggenheim, M., 65, 66(324), 107(325), 132

Gullberg, M. E., 81(371), 133 Gunsalus, I. C., 41(890), 76(326,327), 111 (890, 95i), 132,14r, 149 Gurd, F. R. N., 173,266,320,325,326, 58F Gurin, S., 55,66(328), 132, 144 Gustafson, F. G., 113(329), 132 Gustafsson, K., 100(423), 165 Gustavson, K. H., 277, 278, 279, 284 Gutfreund, H., 218,219,223,267,28? Gyarfas, E C., 363,366,378

H Haan, A. M. F. H., 240,252,284 Haagen-Smit, A. J., 79(551), 80(857), 110(330), 113(496, 497), 132, 137, 138, 147 Haberland, G. L., 76(330a), 132

AUTHOR INDEX

Hackman, R. H., 71(331, 332), 132 Haddox, C. H., 39, 133 Hahn, G., 118(334,335,336,337,338), 133 Haines, W. J., 35(728, 729), 143 Haissinsky, M., 377, 380 Hakala, N. V., 334, 382 Hakim, A. A . , 111(339,310),155 Halawani, A., 60(271), 13f Halikis, D. N., 58(164), 65(161), 158 Hall, C., 329, 380 Hall, I). A., 111(202), 129 Hallman, I,. P., 46(130), 49(130), 167 Halsey, J. T., 34(227), 130 Hamberg, U., 66(245), 130 Hameed, K. A., 104(283), 131 Hamill, R. I,., 121(132), 127 Hamilton, A., 60(588), 139 Hamilton, T. S., 231, 234,286 Hamlin, K. E., 104(341), 133 Hammarsten, O., 4, 28 Hamoir, G., 154, 222, 235, 237, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 253, 254, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 277, 280, 281, 284 Handler, P., 101(342, 533), 103(429), 133, 135, 138

Hanke, hI. T., 76(495), 77(495), 137 Hankes, L. V., 80(381), 81(343), 97(344), 133, 134

Hansel, A., 118(335), 133 Hanser, G.,84(117),89(124),106(124), 127 Hanson, H. T., 344,349,350,352,363,383 Hanson, J., 228, 285 Happold, F. C . , 10(167), 76(346), 78(167, 346), 92(300), Il0(28), 111, f.24, 128, 129,138, 193

Hara, >I., 52(852), 146 Hara, R., 364, 367, 380 Hardin, R. I’., 320,333,380 Harington, C. R., 71, 72, 73, 74, 127, 133 Harley-Mason, J., 41, 68(359), 69(112, 113), 70, 91(361), 104(363), 105(165, 360), 107(363), 110(362), 126,187, 128, 133 Harpur, E. R., 60(611), 140 Harris, D. L., 22(27), 26(27), 28 Harris, 1,. J., 110(494), 137 Harris, M . , 153, 154, 160, 161, 165, 166, 167, 168, 169, 172, 177, 178, 199, 223, 235

393

Harris, R. S., 228, 269, 284, 320, 383 Harrison, G. A , , 112(364), 193 Harrison, G. R., 329, 380 Harshman, S., 15(83), 30 Hart, E. B., 334,336,380 Harting, J., 355, 384 Harvey, H. W., 291, 315 Hashimoto, K., 255, 286 Iiasltins, F. A , , 39(301), 40, 12(650), 132, 133, 141

Hasselbach, W., 236, 237, 244, 284 Hastings, A. B., 336,380 Ha ta , M., 231, 288 Hatz, F., 88(941), I49 Haugaard, N., 334, 380 Haurowitz, F., 320,333,380 Hawkins, V. R., 94(294), 131 Hayaishi, O., 78(826), 83(366), 84(743), 85(366), 91 (367), 108(826, 827, 828, 874), 133, 143, 146, 147 Hayes, J. E., Jr., 331, 354, 355, 357, 35S, 363,367, 374,380, 384 Hays, E. E., 116(368), I S 3 Ileden, C. G., 41(686b), I & Hegsted, D. Rf., 100(562), 138 Heidelberger, C., 81(369, 370, 371), 133 Heidelberger, AT., 181, 185, 217, 223 Heilmeyer, L., 196, 223 Heimberg, hI., 88(372), I33 Heinrich, W. D., 94(400), 134 Heinsen, H. A., 67(373), 68(373), 134 Heinzelmann, It. V., 104(821), 145 Heise, It., 66(406), 134 Hellerman, L., 319, 327, 380, 382 HelImann, H., 84(117), 87, 89(376), 98 (376), 99(374), 127, 134, 149 Hellmann, K., 107(79), 226 Hellner, S., 66(245), 130 Helwig, H. L., 254, 285 Hempelman, I,.H., 76(776a), f 4 4 Henbest, H. B., 114(377), 115(107, 377, 452), 126, 134, 135 Henderson, J. AT., 114(378), I34 Henderson, L. M., 80(381), 81(387), 86 (385,387),89(387), 91 (384), 97,98(382, 763), 99(386, 564), 100(564), 110(517), 134, 137, 138, 154 Hendrix, B. M., 185,023 Henrotte, J. G., 241, 252, 253, 285 Henschen, G. E., 23 Henze, M., 75(388), 134

394

AUTHOR INDEX

Hermanns, J,., 104(389),134 Hernler, F., 322, 380 Herriott, R. kl., 22(28), 24, 28, 168, 170, 171, 223 Herter, C. A., 110, 134 I-Ierter, 15., 76(38), 125 Hesse, G . , 107(913),148 Heymanil, €I., 87(140), 127 Heytler, P G., 333, 379 Hibiki, S.,308, 316 Hickman, E. R l . , 35(65), 54(64, 65), 125 Hicks, C. S., 196, 223 Hill, D. W.,3, 15(38), 28 Hill, H. N., !)8(382), 09(564), 100(564), 134, 138 Himsworth, H. l’., 49(295), 52(295), 131 Hingerty, D., 234,283 Hinsvark, 0. N., 109(417),135 Hipp, N. J., 4, 18(31), 28, 154, 217, 220, 223 Hirai, K., 78(391), 134 Hirata, Y., 81(392), 134 Hird, F. J. It., 59(393), 134 Hirs, C. H. W., 53(832), 146 Hirsrh, H. RI., 07(383),134 Hishikawa, M., 94(509), 1.97 Hitchcock, D. C., 198,199,223 Hoagland, C. I,., 101(3!?4),f34 Hoch, F. I,., 320, 323, 324, 325, 328, 331, 333, 339, 340, 341, 342, 345, 354, 356, 358, 360, 361, 362, 364, 365, 366, 367, 369, 380, 384 Hoffman, M., 113(956),149 Hoffman, 0 . D., 4(8), 28 Hogben, L., 48(395), 134 Hogness, D. S., 103(542), 138 Hogness, J. R., 103(738),143 Holden, H. F., 196, 201, 223 Holley, A. D., 115(396),134 Holley, R. W., 115(396),184 Holman, W. I. M., 80(399), 97(397), 103 (397,398), 134 Holmberg, C. G., 322, 330, 380 Holt, L. E., Jr., 54(816a), 80(817), 145 Holt, P. F., 110(401),134 Holton, P., 50(80), 126 Holtz, P., 66(402, 404, 405, 406, 407), 68 (4031, 194 Homer, A., 79(408), 110(409), 112, 113, 134, 135

Honsley, S.,115(51), 185 Hopkins, F. G., 34,35(410), 109(412),110, 131, 135 Hoppe-Seyler, F., 112(413),135 Hoppe-Seyler, G., 111(414), 135 Horecker, B. I,., 44(415), 136, 327, 380 Horwith, M. K., 277, 289 Hoshino, T , 107(416), 136 Houff, W. H., 109(417), 135 Hough. A., 165, 169, 223 Hove, E., 334, 336, 380 Hoyle, I,., 110, 133 Hsia, D. Y. Y., 62(209a), 129 Huebscher, G., 322,381 Huff, J. W., 80(731), 81(732), 88(732), 89 (732), 94(732), 102(181),128, 135, 143 Hughes, D. E., 101, 103(420), 155 Hughes, G. K., 117(422),135 Hughes, T. R., 327,380 Hughes, W. Id., Jr., 173,223,320,323,328, 333, 341, 342, 384 Hultin, T., 100(423), 135 Humphrey, J. H., 106, 135 Hundley, J. M., 81(427), 103(426),135 Hunt, C . R., Jr., 40(307), 192 Hunter, S.F., 59(428), 103(429), 136 Hurt, W. W.,80(430), 135 Hutchings, B. TA., 116(903),148,333,879 Hutchison, W. C., 25(17), 28 Huys, J. V., 253, 285 Huxley, H. E., 228, 285

I Ichihara, K., 60(878), 61, 62,78(502, 654, 879), 89(434), 111(742, 877), 136, 196, 137, 141, 143, 14Y Ihle, J. E. W., 228, 285 Ikawa, &I.,78(593),91(593), 139 Inada, T., 94(503, 504, 505, 506), 137 Inagami, K., 71(431), 86(575), 155] 133 Ingraham, J. L., 78(568,829), 139,lqS Ingraham, L. L., 116(432),136 Inouye, A,, 96(510), i3Y Irai, I., 40(5), 1.94 Irisawa, A. F., 273,285 Irisawa, H., 273,286 Iritani, H., 84(11), 12.4 Irving, H., 376, 377, 380 Ito, F., 98(433), 156 Ito, T., 89(434), 136

395

AUTHOIt INDEX

Itoh, F., 97(572), 08(572), 139 I~vno,J., 79!507), 137 Izquirrdo, ,J. A , , 112(435), 135

J ,J:tckoki~K ~ ~. lA , ,,

78(214), 106(214), 12s Jitckson, A. H . , 104(363), 107(363), fS.9 ,J:lckson, Philip., 299, 315 ,Jackson, It. W., 35(436), 135 Jac-ob, J., 235, 237, 239, 240, 252, 280,283, 285 Jscol,s, B., 313,311,615 J a c o b s ~F. A , , 116(368), 133 Jacobs, hl. B., 304, 312, 315 Jacohsen, C. F., 170, 171, 823, 224 Jacobsen, E., 68(281), 131 Jacoby, T. F., 185, 226 J a k o b y , W. B., 85, 01(438, 439), 94(439), 135 Jacquot, It., 231, 234, 285 .Jnisle, F . ,257, 285 .James, A. T., 116(158), 128 James, W. O. , 117(440), 135, 319,321, 336, 380 Jamieson, G . S., 75(910), 14!l Janisch, H., 66(407), 134 Janot, AX. M., 119(308), 132 .Jaques, I<., 104(441), 106(424), 136 Jagson, G . G., 84(442), 135 Jean-Blain, h l . , 231, 285 Jennings, B. E., ll6(44), 125 Jerisen, H., 107(443,444), 136, 324, 383 Jervis, G. .4., 53, 54, 78(450), 135 Johnson, B. C . , 103(561), 138 Johnson, P., 250, 261, 264, 285 Johnson, H . M., 25(32), 28 Johnson, T. H., 72, 135 Jones, E. It. H., 114(377), 115(107, 377, 452), 126, 134, 195 Jones, G . I., 300,314,316 Jones, J . D., 76(453), 136 Jones, K. K., 68(13), 124 Jones, K. M., 103(454), 136 Jones, I,. R., 116(368), 153 Jones, K . It., 233, 272, 285 Joshi, J. V., 231,282 Jouanneteau, d . , 60(596), 139 dunqueira, 1’. B., 81(45.5), 136 Jutisz, hf., 276, 286

K I
Kawaguchi, K., 95(573), 139 I i a , ~H.D., , 5,6(79), 8(79), 15(79), 16(70), 29 Kenrney, 13. B., 101, 102(812a), l@, 199, 212, 220,225,320,333,354, 383 Keilin, D., 320,321,322,323,324,325,331, 333, 334, 335, 336, 337, 339, 340, 379, 380,381 Keller, E. B., 67(460,569), 136, 139 Keller, H., 335, 380 Kendall, E. C., 71, 136 Kennedy, F:. P., 25(33, 96), 28 Kennedy, J., 68(764), 144 Kent, M., 114(305), 132 Kenten, R . H., 114, 118(464), 136, 154, 167, 168, 199, 221 Kerekjarto, B. V., 258, 285 Kern, M., 64(465), 136 Kertess, D., 69(466), f36 Ketran, K. C., 80(817), 145 Khan, M. R., 238,271, t85 Khorana, H. G., 116(43), f26 Kihrick, A., 14(13), 28, 155, 156, 157,158, 163, 165, 167, 169, 172, 178, 185, 217, 220, 222 Kiese, M., 336, 380 Kilikawa, H., 80(467), 88(467, 468), 81 (392), 96(796), i34, 136, 145

396

AUTHOR INDEX

Kilby, 13. 78(469), 136 Kind, C. A , , 22(24), 26(24), 28 King, C G . , 101(113b), 127 King, Ii.W . , 40($70),f$6 liirbcrgcr, E , 52(471), 136 Kirkwood, S., i5(251, 252, 253), 116(538, 586), 121(204, 586, 786), 129, 150, 139, 144 Kirnherger, I<.J., 52,58, 132 Iiishinouye, K., 228, 239, 253, 286 Kissman, A , , 84(216), 91(216), 129 Iiistler, l'., 187, 223 Kjarr, A., 116(472), 136 Klein, I<.,43(25), 124 Kleinxeller, A., 40(474), 45(-173, 475), 136 Klitgnard, H. nf., 75(476), 136 Klotz, I. M., 157, 177, 223, 320, 352, 363, 373, 380 Knoll, P., 228, 885 Knox, W. E., 57, 59(482, 489), 60(489), 62,64(489),65,83,84(176,490), 85, 86 (li6). 87,8!3,90,95, 102,128,129,136,

Iiotake, Y., Jr., 94, 95(515), 96(510, 5131, 137

Krebs, 1:. G., 277, 255 Krrhl, W . A., 80(518), 97(51G), 110(517), 137

Iirekels, A . , 274,275,276,284 Krcy, J., 290, 315 Krimsky, I., 61(686), f42 Kring, J. P., 101(519,520), 137 Kroepelin, H., 257, 286 Kriiger, P., 228, 229, 285 Kubie, G., 40(474), 45(473, 475), 106 Kubowitz, F., 324, 380 Kudrjashov, J. B., 269,288 Kiichel, C. C., 228,235,236,987 Kulescha, Z . , 113(521), 137 Kung, H. C., lOl(113b) , 127 Kunitz, M., 10(35), 19(35), 28, 212, 216, 223

L

Lacombe, It., 74(720a), 143 Ladd Prosser, C., 253, 254, 274, 285 Kobayashi, T., 107(416), 135 L a D u , €3. N., 57, 59(523), 60(522), 61, 62, 64(523), 137 Iiobayashi, Y., 107(767), 144 Kodama, S.,4, 16, 29 La Forge, F. B., 116(526), 137 Koechlin, 13. A., 320, 380 I,aidl:tw, P. P., 68(247), 107(249), lo!) Koepp, W., 66(404), la4 (248), 110(249), 130 Koeppe, 0. J., 91(384), 134 Lituger, 1'. G., 352,353, $80 Koerner, J. F., 10(87), 19(87), 30 Laidler, K . J., 214, 223 Kodicek, E., 97(G96), l00(696), 302(493), Lake, H. J., 96(103), I26 103(696), 110(404), 137, 148 Laki, K., 165, 169, 283,264, 886 Lambert, C. F., 35(732a, 732b), 143 Kijgl, F., 113, 137 Landolt, H. R., 259,261,264,285 Koessler, K. K., 76(495), i7(495), 137 JAangemann,H., 67(78), f86 Kofranyi, I<., 276, 288 I,nngham, W. TI., 97(541), 103(738), 138, Iiogure, T., 238, 286 Iioh, W. Y,,40(307), 132 141, 143 Kohn, H. I., 101(342), 1SS Langstein, L., 48(250), 130 Kojo, K., 269, 284 Idanham,W. B., 314, 315 Iiolthoff, 1.M., 329,352,353,363,368,380 Lankester, I<. It., 228,253, 286 Lansimaki, T. A , , 229, 286 Komine, D. R., 165, 169, 223 Konz, W., 107(914), 148 I,anyar, F., 49(527, 528), 50, 138 Kornberg, A , , 9, 98, 319, 380 Lardy, H., 326, 380 Lnrsen, P., 114(530), 138 Iiornmann, P., 88[122), 127 Jmkowski, M., Jr., 160,223 Tiorzuev, P. A , , 274, 286 Latimer, W. M., 374, 382 Koski, R . JC., 86[385), 98(382), 1S4 Laufberger, &I., 322, 380 Kosscl, A., 228, 285 k'oste~manns,D. G . F. It., 113(499), 137 Lauffer, M. A,, 220, 293 I m n e l l , C. H., 322, 330, 380 Koialte, M., 81(500), 83(49'3), 137 Iiotake, Y., 52(511), 78(502), 79(507), 89, Laurent, P., 272, 285 J,avocat, A., 228, 886 94(509), 96(510, 514), 137 13Y, 138, 139

AUTHOR INDEX

Lswrie, R. A . , 228, 213, 271, 285 Laxer, G . , 70(531,532), 198 Leach, 13. E., 35(732b), 143 Leblond, C. P., 72(576), 1S.9 Lechartier, G., 318, 380 Leder, I. G., 88(372), 101(533), 133, 138 Lederer, E., 76(535), 87, 138, 329, 38G I’ederer, M., 329, 380 Lee, N. D., 85(535a, 536, 537), 138 Lee, T. S., 352, 353,363, 368, 380 Leech, W. D., 110(330), 132 Leeper, L. C., 40(605), 58, 140 Leete, E., 117(538,539,540), 121(540), 138 Legge, J. W., 196, 223, 253, 286, 320, 333, 381

Lehninger, A. I,., 319,326,380 I,eifer, E., 41(652), 97(541), 103(542, 738), 138, 141, 143 Lein, J., 40(602), 139 Lein, M., 35(7), 124 Leiner, G., 334, 336, 369, 380, 381 Leiner, M . , 334, 336, 369, 380, 381 J.e May-Knox, M., 57, 59(489), 60(489), 64(489), 136, 138

Lembeck, F., 104(544,545), 138 Lemberg, R . , 196, 223, 253, 286, 320, 333, 381

Lemon, J . M., 314,316 Lepkovsky, S., 79, 80(21), 81(370, 371), 88(698), 124, 133, 138, 142, 274, 286

Leonhardi, G., 51(549), 52(258, 259, 546), 69(547,548, 548a), 131, 138 I,erner, A. B., 46(552), 55, 69(269, 553, 554), 131, 138, 324, 381

Leussing, D. I,., 352, 353, 363, 368, 380 Levanidov, I . P., 313, 315 Levene, P. A., 2, 3, 6, 7, 15(38), 28,29 Levine, S., 356, 360, 368, 378 Levine, S. Z., 52, 53(180), 128, f38 Levitas, N., 80(667), 141 Levy, M., 168, 218, 219,220, 223 Lewis, H. B., 49(662), 141 Lewis, H. D., 335, 340,384 Lewis, P. S., 185, 224 Lewis, U. J., 197, 224 Li, Si-Oh, 2(40), 29 Lichtenstein, I., 170, 171, 224 Liebig, J., 34, 79(559), 138 Lien, 0. G., 59(560), 138 Lifson, S., 163,164,223, 224 Lin, P-H., 103(561), 138

397

Lindenblad, G. E,, 141 I,[email protected], I<., 3 , 4, 12, 16, 2.9, 157,159, 168, 170,224

Jindvall, S.,100(423), 135 Lineweaver, €I., 167, 222 l i n g , C. T., 100(562), 138 Lingane, .J. J., 329, 381 Linko, R. R., 237,239,243,246,256,286 Linnell, I,., 69(563), 138 Linstead, R. P., 78(246), 130 Lipman, F., 2, 3(44, 46), 7(44), 26(46), 29

Lipner, H. J., 75(476), 136 Lipp, A., 34(228,229), 130 Lisovskaya, N . P . , 25(19), 28 Lissitsky, S., 74(714, 715, 717), 75(716, 718, 720), 14.2

Littlejohn, J. M., 91(811), 97(810), 99 1 8 W , 145

Liu, S. C . , 185, 226 I,loyd, B. B., 60(806), 145 Lobel, S., 94(901), f48 Lockaert, E. E., 272, 287 Loeffler, W., 107(325), 132 J,bfgren, T., 5, 29 Logan, &I. A., 278,286 Lohmann, K., 3(57), 29, 319, 381 J,oh-Ming, W. C., 352, 363, 373, 380 Long, C. L., 99(564), 100(564), 138 Longenecker, ,J. B., 67(594), 94(564a), 138,139

T.ongsworth, 1,. G., 12(48), 29, 159, 178, 224, 251, 268, 186 Idontie,R., 198, 22.4 J,oofbourow, J. R., 329, 380 Lord, R. C., 329, 380 I,oughlin, W. J., 185, 224 Lowry, 0. H., 101(113b), 127 J,OWY,R. S., 94(733, 823), 143, 146 J,ubosch, W . , 228, 283 Lucas, C. C., 278, 282 J,uckwill, I,. C., 114(565), 138 Ludes, H., 242, 282 Ludewig, H., 118(336), 133 Luduefia, F. P., 66(849, 850), 146 Ludwig, W., 72(566), 138 Ludtke, K., 66(406), 134 Lumry, R., 194, 350, 224, 383 Lundgren, H. P., 174, 224 Lusk, G , 46(706), 1.42 1,utwak-hlann, C., 360, 381

398

AUTHOH INDEX

Ly11IaI1, It. 81(343), 139 Lynen, F.,79(567), IS9 Id.,

M McClure, J . G . , 361, 363, S81 Maccoll, A . , 376, 979 Macdonald, 1).I,., 78(568), 139 McElroy, W. D., 326, 327, 333, 369, 581, 382

McGanity, W. J., 60(182), 129 McGrath, I$., 94(311), 132 Xlacheboeuf, XI., 12(49), 29, 174, 224 MticInnes, D. A., 251, 268, 286 SIacLenzie, C. G., 67(569), 189 Mackler, 13.) 323, 333, 981 hlarlagan, N . F., 46(923), 75(570, 571, 923), 139, 148

Xlclaren, A. D., 277, 286 McMeekin, T. I,., 4, 18(31), 28, 29, 154, 174, 217, 220, 223, 224

AlcShan, W. H., 273,274,279,283 hfacVicrtr, It., 35(867), 147 Macy, I. C . , 4(8), 28 Madsen, ?i. I< ., 277, 286 Maehly, A. C., 217, 224, 330, S83 Maetz, J., 273, 286 Mager, A . , 276,284 Mahler, 13. It., 322, 323, 324, 330, 333, 379,381

Makamura, K., 114(964), 149 Makino, K., 86(575), 95(573), 96(571a), 97(572), !)9(572), 106(574), 139

Nallette, h1. F., 320, 322, 330, 379, 381 hIalmstrom, 13. G., 321, 326, 381 Mnnn, F. G., 67(177), 128 PtInnn, 1'. .J. G., 114, 118(464), 136 X h n n , T.,320,321,322,323,324,325,331, 333, 334, 335, 336, 337, 330, 340, 379, S80, S81 Mann, W., 72(576), 1S9 hlanske, R . H., 117(.577), 139 March, 13. I!;., 300, 316 hlargaria, R . , 333, 378 Marion, I,., 117(101, 538, 539, 540, 578, 586), 121(540, 586), 126, 138, 139 Murplev, 15., 52(555, 656, 557), 53(180), 128, 1S8 nlarsh, 13. 13., 260, 286 Martell, A. Is:., 321, 326, 336, 353, 363, 372, 381 Martin, F., 257, 286

Martin, G. J., 106(49), 126 Martinek, W. A., 313, 314, ,916 Marvel, C. S., 35(55), f26 Marvin, €I. H., Jr., 179, 221 hlasai, I*., 52(511), 137 Maser, J . , 228, 230, 886 Mason, H. S.,69, 70, f27, 139 Mason, hl., 84(584), 86(582), 90(583,583n, 585), 96(582), 139

Afassey, V . , 174,224 M a s t , G. W., 113(956), 149 Mlttchett, T. J., 116(586), 121(586), 139 hhthews, M. 13., 220, 224 Mathies, J. C., 273, 286 Matsuniura, Y., 94(506), 1S7 Matsuura, F., 238, 255, 272, 286 Matuura, F., 238, 286 Matsuoka, Z., 79(587), 139 Max-Moeller, K . , 9, 29 May, C. D., 60(588, 589, 747, 748, 7491, 139,14s

Merham, D. I<., 7, 25(53), 29 Medes, G., 51,66(590), 1S9 Mehler, A. H., 83(490, 591), 84(4!10), 85, 100(590a), 137, 139, 319, 380 hleister, A . , 05(592), 78(739), 109(739), 139,143

Meiklejohn, G. T., 324, 381 illeldrum, N. U., 333, 334, 336, 341, S78, 381

Ptlellander, O., 5, 16, 29 Mellon, M. G., 329, 381 Mendel; Isla, hl. C., 231, 283 Xiendive, J. R., 334,335, 382,38S Xlendoza, €1. C . , 49(199), 129 hlenefee, S. G., 4(56), 29 Menon, I<. R., 273, 274, 286 hfentzer, C., 104(149), 128 hfetzler, D. E., 67(594), 58(593), 91(503, 595), 139

Meunier, P., 60(596), 1S9 AIeyer, F., 48, 56, 159 bleyer, K., 235,236, 241, 288 Meyerhof, O . , 3(57), 26(58), 29, 319, 381 Mchel, O., 75(718, 720), 132 hlirhel, It., 74(714, 715, 717, 719, 720a), 75(716, 718, 720, 721, 721a), 142, 143

Rliekeley, A., 3, 98 Miescher, F., 227, 286 hlihtilyi, E., 154, 162, 165, 167, 169, 171, 172, 178, 999, 994, 263, 486

AUTHOR INDEX

399

Moss, €3. L., 312, 315 M i , S., 330, 379 Mikuta, E. T., 85(868, 869), 147 Motzel, W., 108(919), 148 Milch, H., 48(598), 139 Mouneyrat, A,, 34(267), 131 Milch, R. A , , 48(598), 139 Moyer, 1,. S., 181, 185, 224 bfueller, J. F., 94(294), 13L Miller, D. Y., 300, 315 Mullcr, &I.,81(944), !)7(943, 944), 149 Miller, 1'. C , 88(599), 94(599), 13.9 &filler, I<:., 281, 286 Muir, It. I)., 116(368), 133 Miller, G . , %(lo), 124 Muir, R. hi., 113(929), 114(!122), 148 Miller, G . I.., 281, 286 Muller, O., 329, 382 Munro Fox, II., 274, ,086 hliller, 1. I , . , !)1(600, 601), 139 Alilliiigton, 11. II., 46(905), 55, 148 Munro, T. A., 53(615), 140 Afingioli, 11;. S., 37(906), 38(192), 3!)(!)07), Alusajo, I,., 79(616,617), 81(623), S8(620), 129,148 89(622), 04(618, 621), !)5(618, 61!1, 619a), 100(624), 113(618), 140 hlirsky,A.15.,201,221,2,02,04 Mitchell, H. I I . , 231, 234, 284, 286 hlussett, M. V.,75(624a), fa0 Mitchell, H. I<., 39(301), 40(602), 41(302, Rlyholm, R. S., 376, 379 652), 42(650), 80, 81(603, 604, 651), N 87(275), 97(541, 603, 604), 125, 131, 132, 133, 138, 139, 141 Nabeh, I., 60(271), 131 bfitoma, C., 40(605), 58, 1.40 Nachtrieb, N., 329, 382 37(606), 39(607), 140 Naganumu, II., 238, 284 Mitsuhashi, S., Najjttr, V. A,, 321, 326, 382 Mittasch, H., 107(913, 914), 148 Nakanishi, K., 81(392), 134 hliwa, T., 83(399), 137 Miyake, A., 99(608), 140 Nakayamu, Y., S9(512), 137 Nanninga, I,. B., 163, 165, 167, 169, 171, Aloller, I<., 359, 384 Moeller, T., 376, 381 224 Moon, A., 309,315 Nason, A , , 41(628), 64(629), 113(626, 627), 140,327,333,369,38ft 382 Aloe\\us, F., 116(66), 195 Mohamed, hI. S., 31!1, 322, 343, 381 Nasset, E. S., 35(630, 631), 140 Mohr, R., 274,284 Neale, F. C., 96(103), 226 Neelin, J. M., 239,240,283 Moir, G . F. J., 116(609), 140 Negelein, E., 353, 354, 355, 359,382 Molnar, D. M., 167, 171, 228 Neilands, J., 330, 382 hIolnar, F., 264, 284 Mommaerts, W. F. €1. &I., 235, 241, 263, Neish, A. C., 117(107a), 126 286 Nelson, J. E., 339,379 Nelson, E. N., 60(589), 139 Moncrieff, A , , 54(961a), 149 Monier, R., 276,286 Nelson, J. M., 322, 333, 379, 382 Nencki, hi., 109, 110(632), 140 Monsonyi, I,., 49(610), 140 Montgomery, M.I,., 353, 381 Nero, K., 83(492), 137 Moore, D. H., 273, 286 Neubauer, O . , 47(638), 48(636), 49, 51 Moore, S., 53(832), 146 (635), 56, 62, 79, 140 Morgan, A. F., 80(2l), 81(371), 124, 133 Neuberg, C., 112(639), 140 Mori, T., 238, 272, 286 Neuberger, A., 35(640), 48(643), 49, 50, Mori, Y., 52(511), 137 52(295, 644), 62, 73, 83(156, 157), Morrie, J. E., 60(611), 140 84(176), 86(142, 176), 87(142), 89 Morris, M., 17, 28 (176), 116(157), 127, 128, 131, 140, Morrison, P. R., 154, 222 168, 170, 172, 185, 221, 222, 224 Morton, NI. E., 72(612, 625, 665, 666), R. E., 278, 286 Neuman, 140, 141 Neumann, H., 319, 382 Morton, R. A , , 369, 378 Neumeister, R., 31, 140 Moss, A. R., 33, 54,58,140 11:.

400

*iUTHOR INDEX

Neurath, H., 174,181,185,214,216,217, 218,219,220,222,224,272,287,320, 323,324,328,333,343,344,345,347, 348,350,351,363,380,382,384 Newey, H., 35(288), 131 Nichol, C. A , , 60(646), l4U Sicholas, D. J. D., 333,389 Nickcrson, J . T.I t . , 272,286,287 Nicolai, €I., 112(647), 141 Xielsen, 15., 79(551), 138,291, 315 Niemnnn, C., 74(648), 141 Nierstrasz, I f . F.,228,285 Nieva, F.S.,114(303,304), 13:‘ Nikkila, 0 . IC., 237,239, 243, 246,256, 272,286 Silson, I€. W., 313,314,315 Nishi, K., 97(572), 90(572), 139 Nishi, S.,229,286 Nitowsky, €I., 60(648n), l4l Nitschmann, lr., 187, 223 Nizet, E.,273,283 Nocito, V., 59(68, 69), 125 Nogami, I<.,96(513), 1.37 Nomura, T., 231, 288 Norberg, B., 22(59), 26(59), 29 Kord, F.I?., 151,167,174,178, 219, 240, 222,,923,224,225 Norris, E. R . , 273,286 Yorthrop, J. H., 22(60,61),29,168,171, 223 Sorton, P., 51(816a), 145 Novello, N.J . , 112(649), 141 Nunez, J., 74(720a), 148 Nyc, J. F., 41(652), 42,80, Xl(603, 604, 651), 97(541,603, 604), 125,138,139, 141 Xyholm, R. S.,376,38.6

0 O’Urien, If., 336,383 Ochoa, S.,319,380 Oesterling, hf. J., 35(653), 141 Ogita, Z., 88(468), 136 Ogston, A. G., 267,282 Ohigashi, K., 78(654), 141 Oka, I., 103(655), 141 Olcott, H.S., 7,25(53), 29 Oleson, J. J., 333,379 Olsen, J. A . , 370, 382,384 Olson, R . A , , 114(655a),141 Ongel, I,. E., 376,379

Oppenheimer, C., 319,382 Osborne, T.B., 6(62), 11, 29 Osborne, W. A . , 47(657), 141 Ord, W. M . , 113(656), 141 Oshima, A , , 78(658), l4l Osterberg, A. E.,71(463), 136 Otsuka, J., lOn(756,757),143,144 Ottesen, M., 12, 29 Overman, 0 . R., 4(56), 29 Owen, It.D., 81(604), 97(604),139 Oya, J . , 272,286

P Pace, J . , 185,224 l’acheco, I€., 104(149), 110(659), f98, 141 Pacht, M., 5(3), 16(3), 28 Page, I.H., 104(660,693),107(113n), 127, 141,142 Painter, H. A,, 57(661), 60(661), 141 Pait, C. F.,240,287 Paladini, A. C., 53(832), 146 Palmer, A . H., 14(13), 28,155,156,157, 158,163,165,167,169,172,178,185, 217,220,222 Palmer, W.W . , 217,225 Panier, A., 198,224 Panzer, F., 40(783), 144 Papageorge, C. W. H., 49(662), 141 Parpajola, A., 95(619,619a),140 Parrish, R. G., 264,286 Parry, R . W., 375,383 I’artmann, W., 233, 234,269, 270, 271, 272,$86 Partridge, C. I f . W . , 40(93, 663), 41, 96(663), 126,141 Partridge, S. M., 23’2, Y33,287 Pashkina, T.S.,X9(102), 126 Passman, J. M., 24,30 Patrick, J . B.,84(216), 91r216),129 Paul, K. G., 333,382 Pauling, L.,196,222,374,376,377,382, 383

Pearson, P. I]., 81(784), 102(668), 141,144 Peattie, R.W., 329,384 Pedersen, K.O., 5,29,181,185,217,229, 254,274,279,687,288,323,330, 384 Penrose, 1,. S.,53(663a), 141 Perkinson, J. D., 49(792), 52(792), 144 Perlman, I., 72(665, 666),l4l Perlmann, G. E.,3(72), 8(72), 11, 12,14 (64,69,72),15(68,72),16,17(66), 18,

401

AUTHOR INDEX

10(71), 20, 21(71, 72), 22(67), 25(70, 71), 23, 165, 174, 224 l’dzweig, W. A , , SO(667, 731), 81 ( 7 3 2 ) , 88(372, 732), S9(732), !‘4(732), 102 1668), 110(734), 133, 141, 143 Perry, S. V., 228, 235, 236, 257, 282, 287 Petermann, M. N., 334, 982 Pettko, E., 264, 284 Pflanz, L., 275, 276, 288 Philipenko, A. T., 353, 378 Philippi, E., 322, 380 Phillips, D. M. P . , 277, 287 Phillips, P. H., 319, 378 Philpot, F. J., 107(81), 126 Philpot, J. St. I,., 172, 224, 331, 582 Pictet, A., 117, l 4 l Pitt-Rivers, R . V., 71(673), 72, 74, 75 (320, 321, 624a, 863a), 1.92, 133, 1.40, 141,147 Plapinger, R. E., 3,23 Plimmer, R . H. A , , 1(74), 29 Plum, C. M., 68(281), 191 Polis, B. D., 4(51), 29, 154, 174,224 l’ollaczek, H., 3, 4(77), 5(78), 6, 8 ( 7 8 ) , 15(77, i s ) ,23 Polonovski, J., 174, 224 Polonovski, M., 234, 287 Porter, C. 81(675), 88(674), 141 Porter, R. R., 182, 183, 224 I’ortzehl, H., 250, 257, 259, 261, 263, 264, 265, 287, 288 Posternak, S., 5, 7, 23 Posternak, T., 3, 4(77), 5(76, 78), 6, 7 , 8(78), 15(77, 78), 23 Potgieter, M., 35(54), 125 Potter, G. D., 75(860a), 147 Pottinger, S. R., 311 Powell, R., 374, 382 Prelog, V., 119(308), 142 Price, J. M., 141 Prideaux, E. B. R., 185, 224 Priest, It. E., 99(676), 141 Proctor, B. E., 272, 286, 287 Pryor, M. G. RI., 71(332, 677, 678, 679, 680), 88(678), 192, 141 Pugh, C. E. M., 69(682), 107(681), 1.41 Pullmann, hl. E., 102(683), l 4 l Pummerer, R ,72,141 Putnam, F. W., 174, 181, 185, 214, 216, 217,218,219, 220, 224, 343, 344, 582

c.,

Q Qungliarello, I;., 97(685), 142 Quastel, J. H., 53, 107(681), f4f Qnreshi, hf. I?., 308, 313, 316

R Rachele, J. It., 67(569), 133 Racker, E., 61(686), 64(465), 136, 142, 354, 355,358,360,382 Radhakrishnamurty, R., 86(844), 103 (843), 146 Rafelson, 11.E., 41(68Ga, b ) , 46(686~,I)), 142 Raffel, S.,240, 287 R a l p h , B . J . , 116(609,687), lgO,i42 Ramachandran, B. U., 5, 28 Ramasarma, G. B., 81(387), 89(387), 97 (386), 99 (386), 134 Rand, M., 104(699), 142 Randall, S. S.,72(137, 357). 127, 13.9 Ranke, E., 238,287 Ranvier, I,., 228, 287 Raper, H. S., 66(205, 688), 68(690), 69, 129, 138, 141, 142 Rapkine, I,., 220, 224 Rasmussen, K. E., 168, 224 Ratner, S., 59(68, 69, 774), 126, 144 Rauen, H. M., 275, 276, 284, 287 Raulin, J., 318, 382 Itavdin, R. G., 55(695), 57, 64,142 Ray, P. M., 114(836), 146 Reay, G. A . , 228, 231, 233, 234, 235, 236, 238, 269, 271, 287 Reber, E., 35(867), 147 Reddi, K. K., 97(696), 100(696), 103(696), 142 Redemann, C. T., 114(697), 1.42 Redfield, A. C., 290, 316 Reid, D. F., 88(698), 142 Reid, G., 104(699), 142 Reindel, W., 68(777), 144 Reinits, Ii. E., 57(700), 142 Reio, I,., 41(686b), 43(25), 44, 45(166), 124, 128, 129, 142 Renard, S., 240, 253, 254, 280, 287 Renner, U., 96(119), 197 Reti, I,., 117(701), 142 Rhuland, L. E., 40(702), 142 Richards, A. G., 270, 283 Richards, M. M., 319, 327, 882 Richert, D. A., 323, 331, 333, 382

402

.4UTHOI1 INDEX

Richter, D., 107(82), 126 Itidesl, 1:. I<., 334, 379 Riggs, D. S., 71(703), 141 Itiggy, T.R., 110(147, 704), 128, 142 Rimniington, C., 5, 6(79, SO), 8(7!)), 15 (79, 80), l6(79), 29, 48(643), 40(643), 50(643), 113(705), 140, 142 Itinehnrt, J . F., 94(311), 152 Ringer, .4.J., 46(706), 141 Riou, I,., 312, 316 Ititchie, I<., 117(422), 136 Itittenberg, D., 57, 59(774), 129, 144 Ilobcrts, E. C.,116(368), 153 Roberts, G. L. Jr., 160, 173, 218, 226 Itohertson, A., 116(43, 44, 687), 126, 142 Robinson, J., 80(667), 141 Itobinson, I(. S., 54(17), 55(16, 17c), 109 (171, 124 Itobinson, It., 36(707), 117, 118(710), 148 Itoboz, E., 79(551), 138 Robson, A . , 276,283 liohson, W., 35(135), 128 Itoche, J., 74(714, 715, 717, 719, 720a), 75, 113(100n), 126, 148, 143, 192, 195, 224, 254, 287, 322, 382 Rockland, L. B., 35(207), 129 Rodney, G., 60(722), 145 Rodriguez, J. S., 49(199), 129 Itoester, U., 95(908), 148 Rower, F.,118(909), 148 Rogers, W. F., 60(723), 148 Ttoka, IA., 58(257, 724), 59(257), 130, 143 Ilollett, A , , 228, 230, 887 Rona, l’., 48(1), 184 Itoos, I<., 71(39), 186 Rose, W. C., 35(55, 653, 725, 726, 727, 728, 729, 730, 732a, 732b, 950), 186, 141, 143, 149 Itoseri, F.,80, 81(732), 88(372, 732): 89 (732), 94(732, 733), 102(668), 110 (734), 133, 141,143 Rosenblueth, A., 66(743), 149 Rosenfeld, F., 112(85), 186 Rosin, TI., 110, 113(736), 143 Ross, V . , 277, 287 Rossi, A., 254,287 Roth, E., 237, 245, 257, 258, 263, 287 Roth, 1,. J., 103(542, 738), 138, 1.43 Roughton, F. J. W., 333, 334, 336, 336, 341, 378, 581, 582 Itoule, I,., 253,287

Itousselot, A., 154, 22.4 Rowsell, IC. V . , 59(303), 154 Itubinstein, A., 116(472), 136 Rutlmm, D., 78(739), 109(73!l), 143 Rumpf, F.,118(337), 135 Russell, A., 143 Itussell, 1’. B., 71 (67!),680), l 4 i Ruud, J., 274, 287 Rydon, 11. N., 40(741), 143 S

Sable, €1. Z.,103(797), 146 Sachs, P., 104(389), 134 St. George, R . C. G., 374, 383 Sakai, S., 312, 316 Sakamoto, Y.,60(878), 61, 62, lll(7.12, 877), 143, 147 Sslran, T., 81(500), 83(499), 84(743), 94 (509), 98(795), 157, 143, 144

Balamon, I. I., 37(744), 143 Salkouski, E.,110(745), 145 Salkowski, €I., 110(745), 143 Salmon, It. J., W(589,717, 748, 749), 139,143

Salmon, W. D., 35(750, 757n), 143, 144 Salomon, I I . , 46(226), 48(226), 130 Salter, W. T., 71(358, 751), 72(358), 133, 143, 170, 212 Saluste, I.:., 41(686b), 43(25), 45(166), 124, 128, 14.2 Samuelson, O., 329, 382 Sanadi, J. It., 57(752), 143 Sandell, E. 13., 329, 382 Sang, C. €I., 44(866), 147 Sanger, F., 15, 30, 182, 224, 226, 276, 287 Sarett, 1-1. l’., 80(755), 07(754), 101, 110 (753), 129,148 Surker, K . , 330, 381 Sarma, 1’. S., 17, 21(90), 22(88, 89, no), 26(88, CO), SO, 80(518), S5(800), 86 (844, 845), 103(843). 113(708, 799), 157, 146, 146 Sasaki, T., 109(756 757), 143, 14.4 Sato, R., 39(459), 136 Satoh, K., 86(575), 95(573), 139 Sauherlich, H. E., 35(757a), 40(785), 144 Scala. I(., 101(159), 188 Scardi, V . , 101(159), 128 Scatchard, G., 177, 179, 226 Schachter, M., 104(441), 135 SchutTer, N. K., 15(83), 30

AUTHOR INDEX Schales, O., 66(758), 118(334), 188, f44 Schales, S. S., 66(758), 1.64 Schayer, R. W., 35(759), 67(762), 68, 81 (759), 98(763), 107(767), 144 Scheer, B. T., 80(430), 81(9, 50), 89(768), 97(9), 124, 126, 156, 144 Scheinberg, I. H., 177, 179, 226 Schepartz, B., 55, 57(769), 59(769), 64, 65(770,771), 144 Scheraga, H. A., 160, 223 Schiedt, U., 88(120, 121, 122, 122a), 127 Schlenk, F., 354,582 Schirokow, N . W., 272, 287 Schlossberger, H., 81(125), 89(123), 98 (123), 127 Schlossman, H., 107(82), 126 Schmid, K., 254,287 Schmidt, C. 1,. A., 35, 148, 174, 222, 226 Schmidt, F., 46(226), 48(226), 180 Schmidt, G., 3, 5(85), 9(85, 86), 16(85), 30, 116(920), 148 Schneider, G., 236,237, 244,284 Schnek, G., 173, 282 Schoenheimer, R., 33,54, 58,59(774), 140, 144 Schopf, C., 117(775), 118(776), 144 Scholes, G., 84(142), 136 Schormuller, A., 2(37, 39), 28, 29 Schramm, G., 250, 257, 259,261,263, 264, 287 Schreier, K., 56(776a), 144 Schuler, W., 68(777,778), 144 Schulz, G., 89(124), 106(124), 127 Schulee, E., 34, 144 Schwarting, A. F., 40(876), 147 Srhnarzenbach, G., 373, 374,882 Schneigert, 13. S., 40(783, 785), 80(782), 81(455, 784), !)8(89), 99(88, 8!), 608, 676), 126, 136, 140, 141, 144 Schwenck, E., 112(639), f4O Schwert, G. W , 216, 222, 343, 344, 882 Scott, D. A , , 331, 334, 335, 882, 383 hi., 2(9s), 30, 220, 223 Scott, Scribney, &I., 121(786), 144 Seagran, €1. I,., 309, 316 Sedock, R. R., 35(895), 49(787, 788, 792, 794),52(792,794), 55,57,60(791,963), 144, 148, 149 Seay, P. If., 107(278), 131 Sebrell, W. H., Jr., 320, 383 Seki, T., 96(796), 146 14;.

403

Sell, H. M., 10(J(417), 114(697), f36, I42 Senimett, W. F., 17, 28 Seminova, D. l’., 329, 383 Senoh, S., 81(500), 96(796), 1‘8(795), 187, 144,146 Sensi, P., 86(292), 131 Seraydarian, M. W., 103(797), 146 Shahinian, S. S., 100(925), 101(926), 148 Shakir, M. H., 369, 578 Shank, R. E., 101(394), 134 Shanmugasundaram, E. R. B., 85(800), 86(845), 113(798, 799), 146, 146 Shavit, N., 163, 228 Shaw, E., 104, 149 Shaw., J . H., 319, 378 Shaw, K. N. F., 55(17c), 124 Sheline, G. F., 353, 381 Shemin, D., 41, C4(858), 147, 148 Shepherd, D. hl., 66(802), 68(801, 803), 146 Sherwin. C. I’., 112(649), l4f Shenan, J. &I., 231, 232, 233, 234, 238, 269, 271, 287, 308, 316 Shiguera, €I., 43(804), 45(804, 805, 805a), 146 ShimodairtL, K., 107(416), 186 Shirai, Y.,!‘6(514), 157 Shive, W., 40(48), 42, 126 Shore, P. A , , 64(106), 68(106), 126 Shore, W. S., 154, 160, 167, 218,226 Shrimpton, D. IT., 300, 516 Shugar, D., 220,284 Shulman, S., 154, 226 Sibata, S., 308, 316 Sicher, S., 41(56), 42(57, 58), 166 Sidgwick, N . V., 376, 383 Sieber, N.,109, 140 Siege], S. M., 114(805), 146 Silber, R. I-I., 81(675), 88(674), 141 Silherstein, H. 15., 49(794), 52(794), 1-44 Siliciano, A. M., 35(631), 140 Sikorski, J., 70(531), 138 Simic, B. S., 60(806),146 Simidu, W., 308,316 Simnionds, S., 40(809), 42, 146 Simonovitch, I,., 220,224 Sinclair, H. hf., 60(806), 146 Siiignl, S. A . , 91(811), 97(810), 99(812), 146 Singer, S. S., 374, 379

404

AUTHOR INDEX

Singer, T. P.,101, 102(812a), 146, 199, 212, 220, 223, 320, 333, 354, 360, 978, 383

Sinnhuber, It. O., 46(131), 127 Sinsheimer, It. I.., 10(87), 19(87), 30 Sistrom, W. It , 78(813), 145 Sizer, I. W . , 69(814), 145 Sjostrom, A. G . M., 303, 313,315 Skoog, F., 114(815), 145 Sloane-Stanely, G. H . , 50(80, 83), 126 Small, P. A., 172, 224 Smiley, R I , . , 68(764, 765, 766), 107(767),

144 Smith, 1%. 8. W , i(i(453), 78(246), 130, 136

Smith, E. C., 235, 236, 237, 246, 287 Smith, E. C . B., 334, 383 Smith, E. I,., 343, 344, 349, 350, 352, 363, 366,383 Smith, G. F., 114(377), 115(377, 452), 134, 136, 383

Smith, 0. C., 329, 383 Smith, S. W., 25(33), 28 Smorodinzew, I. A., 272, 287 Smyrniotis, P. Z., 44(415), 135 Smyth, D. H . , 35(288), 131 Snell, C. T., 329, 383 Snell, E. E . , 40(816), 671594), 78(593), 91(593, 595), 94(564a), 158, 139, 245 h e l l , F . , 329,364, 367,880,383 Snellman, O., 259, 261, 263, 264, 287 Snoke, J. E . , 272, 287 Snow, J. M . , 228, 235, 236, 237, 243, 244, 255, 256, 284, 287 Snyderman, S. E., 54616a), 80(817), 145 Sbrensen, M . , 12,29 Sbrensen, S. 1'. I,., 12,29 Sohonie, I<.,269, 983 Sonenberg, M . , 177, 223 Sorm, F., 275, 276, 287 Sormova, Z., 275, 276, 287 Spacek, M . , 95(818n), 145 Sptlda, .4.,81(623), 89(622), 100(624), 140 Spath, I+:., 118(810), 145 Speck, J . I?., 319, $83 Spector, H., 80(820), 145 Speeter, hl. E . , 304(831), 1.45 Spicer, J . S., 116(822), 145 Spikc, C. G., 375, 383 Spitnik, P., 161, 223 Sprecher, M . , 45(805:t, X24), 145, 146

Sprince, I$., 94(733, 823), 143, 145 Sprinson, D . H . , 39(456b, 823a), 43(804), 45, 136, 145, 146 Sprissler, G. P., 273, 287 Sprott, W. E., 75(570), 139 Sreenivasan, A , , 60, 61, 148 Srinivasan, 1'. R., 39(456b, 823a), 45 (456a, 456h, 805a, 823a, 824), 136, 145,146

Stadie, W. C., 336, 383 Stamm, W., 275, 287, 288 Stamp, I,. I)., 316 Stanbury, J. B., 75(963u), 149 Stander, I t . S . , 35(911), 148 Stanier, It. Y.,78(568, 813, 825, 826, X2!)), 83(366), 85(366), 91(367), 108, 133, 199, 145, 146, 147

Stanley, I.. A. K., 374, 379 Stsnsby, hl. E., 233,887 Stare, F. J., 100(562), 138 Steele, J . M., 49(788), 144 Steele, R., 59(309), I32 Steggerda, F. R . , 231, 234, 286 Stein, G . , 103(831), 146 Stein, W . €I., 53(832). 146 Steinbach, H. U., 270, 287 Steinhardt, J., 153, 154, 160, 161, 165, 166, 169, 172, 177, 178, 181, 185, 186, 187, 192, 194, 197, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 213, 214, 215, 216, 218, 210, 220, 225, 226 Stephenson, M . , 76(833), 146 Stern, J . A , , 272, 287 Stern, K . G . , 319, 382 Stcn:wt, C. l'., 324, 381 Stewart, C. T., 60(588), 139 Stickland, J,. H . , 319, 383 Stirling, W . , 228, 287 Stjernholm, R., 45(166), 128 Stock, C. C . , 42(458), 136, 31!), 580 Stockell, A. K.,60(182, !)52), 128, f@, 344,383 Stocking, C . R., 333, 384 Stolz, F . , G6(834), 1.417 Stone, I)., 42(808), 145 Stoppani, A. 0 A l , , IE(435, h85), 1&7, 146

Storck, R., 40(012), 78(912), 10!1(01'2), 148

Storvick, C . L4.,101(113t)),127 Stotz, E., 65(154), 128, 327, 380

AUTHOIZ INDEX

405

Takemoto, Y., 78(879), 147 Ta lna r, G. P., 85(314a), 132 Tanaka, T., 52(852), 64(842), 146 Straub, F. n.,264,284 Tanford, C., 154, 159, 160, 163, 166, 167, Strecker, F. J., 95(908), 148 169, 170, 172, 173, 176, 178, 184, 104, Strickland, J. I). li., Il6(822), 145 218, 226, 320, 383 Strittmstter, c'. F., 271, 287 Tani, S., 94(515), 95(515), I37 Struyvenherg, A,, 110(348), 13.3 Tarpley, W . 13., 333, 379 Stubbs, A. L., 369, 378 Tarr, I€. L. A , , 237, 269, 271, 272, 278, Stumpf, P. I<.,7 7 , 146 283, 288, 300, 316 Subba Rao, G. N., 228,235,236,255,263, Ta ta , J., 75(721,72la), 143 288 Suda, M., 64(840, 841, 842), 65(841), 76 Tatarskaja, R . I., 269, 288 Tatum, 15. I>., 39(856), 40(809, 854, 855), (840), 146 41, 88(698), 94(858), 142, 146, 146, 147 Shllmann, H., 99(897), 148 Taurog, A , , 72(860), 75(859, 860a, 873), Sujishi, K., 52(852), 64(842), 146 80(857), 147 Sumerwell, W. N., 60(791), 144 Taylor, €1. F., 300, 316 Summerson, W. H., 324, 381 Taylor, 1. F., 44(861), 147 Sumner, J. 13., 331, 383 Sundararajan, T . A., 17, 21(90), 22(88, Taylor, W. I., 119(308), I32 Teague, I). M., 4(8), 28 89, 90), 26(88, UO), 3G Sundaram, T . K., 86(844, 845), 103(843), Teas, H. J., 113(862), 147 Tekman, S., 05(178), 100(178), 128 146 Surtevant, J . M., 211, 218, 210, 222, 223 Tenconi, I,. T., 89(293), 131 Tenenhaum, 1,. E., 324, 383 Sutton, I,. E., 376, 379 Tenow, ill., 259, 287 Suzuki, S., 60(878), 61, 147 Teply, 1,. J., 80(518), 137 Suzuki, Y., 231, 288 Terrell, A. J . , 64(629), 140 Svedberg, T., 5, 26(91), 30, 254, 288 Terry, M. C., 240,287 Svensson, H., 23, 240, 288 Teske, R., 52(260), 131 Swallow, A. J., 103(831), f 4 6 Teubner, F. G., 114(863), 147 Swanson, A. I,., 60(722), 143 Swenseid, M. E., 60(722, 846, 847), 14.9, Tewkesbury, 1,. R., Jr., 72, 136 Thannhauser, S . J , , 5(85), 9(85, 86), 16 146 Swoap, 0. F., 107(278), 131 (851, 30 Sydenstricker, lr.l'., 91(8ll), 07(810), Thayer, 6. A., 116(368), 133 Theis, E. R., 185, 226 99(812), 146 Theorell, H., 154, 172, 226, 253, 254, 288, Symonds, l'., 165, 169, 223 322, 323, 330, 331, 333, 354, 355, 360, Szent-Gyorgyi, A , , 235, 282, 288 368, 369, 383, 384 Szorenyi, E. T., 323,383 Thibault, O., 75(863a), 147 T Thimann, I<. Ir,,114(836, 837, 838, 864), Tabone, D., 96(848), 146 115(838, 865), 146, 147 Tabone, J . , 96(848), 146 Thomas, It. C . , 44, 147 Tainter, M. I,., 66(849, 850), 146 Thomassey, S., 96(848), 146 Takahashi, H., 86(575), 106(574), f39, Thompson, C., 94(294), 131 Takahashi, I., 231, 288 Thompson, C. &I., 35(867), 1-47 Takahashi, T., 278,288 Thompson, E. 0 . P., 15(82), 30 Takamine, J., 66, 146 Thompson, J . F., S5(868, 869), 147 Takana, T . , 278, 288 Thorpe, W. V., 96(103), 100(104), 126 Takeda, K., 308, 316 Takeda, P.,52(852), 64(840, 841, 842), Thorson, A., 104(870), 147 Timasheff, S. N., 174, 226 65(841), 76(840), 146 Stowe, B. B., 114(836,837,838), 115(838), 146

406

AXITHOlt I N D E X

Tirunarayanan, RI. O., 85(800), 113(799), 146 Tiselius, A., 23 TissiBres, A,, 324, 384 Titus, E., 104(883), 105(885), 106(883, 884), 107(871), 108(883), 147 Tobias, C. A., 329, 384 Todd, A. It., 8, 30, 71(332, 679, 680), 132, 141 Toh, C. C., 106(179, 256, 872), 107(179), 128, 190, 196, 147 Tong, W., 72(860), 75(873), 147 Tonster, O., 103(249n), 190 Torii, M., 84(11), 124 Tracy, P. H., 4(56), 29 Travia, L., 5,30, 254, 287 Trier, G., 117, 149 Tristram, G. R., 164, 191, 193, 226, 276, 288 Tsao, T. C., 261, 263, 264, 267, 288 Tschuchida, M., 91(601), lOS(828, 830, 874), 199, 146, 147 Tsuchihashi, M., 334,389 Tsuchiya, Y., 231, 288 Tsunetoshi, A , , 78(654), l 4 l Tullar, B. F., 66(850, 875), 146, 147 Tupper, R., 335,341,384 Tuppy, H., 15, 90 Turney, D . F., 101(95), 126 Tuttle, W. W., 234, 288 Tyler, F. H., 54(17b), 124 Tyler, V. E., 40(876), 147

U Uchida, M., 60, 61, 62, 78 (654, 879), 89 (434), 111, 136, 141, 143, 147 Udenfriend, S., 54, 58, 63(22, 23), 64(106, 881, 882), 66(888), 68(106, 888), 76 (887), 104(883), 105, 106, 107(871, 889), 108(883), 124, 126, 128, 147, 273, 275, 276, 277, 281, 288 Uematsu, H., 230, 288 Umbreit, W. W., 41(890), 76(327), 111 (890, 951), 132, 147, 149 Urivetsky, M., 97(344), 193

V Vallee, B. L., 318, 319, 320, 323, 324, 325, 326, 328, 329, 331, 333, 335, 339. 340, 341, 342, 344, 345, 347, 348, 350, 351,

353, 354, 356, 358, 360, 361, 362, 363, 364, 365, 366, 367, 369, 370, 379, 380, 384 van Arman, C. G., 68(13), 124 Vandelli, I., 94(891), 147 Vander Brook, M. J., 107(278), 191 von Derjugin, W., 79(127, 129), 127 van Elten, C., 35(895), 148 van Eys, J., 103(249a), 190 Van Goor, H., 333, 335, 336, 337, 339, 984 Van Heyingen, R., 336, 384 Van Kempen, 1’. N., 228, 285 Van Uitert, I,. G., 376,984 van Veen, A. G., 301, 316 Van Wyk, G. F., 231, 233, 238, 288 Velick, S. F., 178, 226, 273, 275, 276, 277, 281, 288, 331, 354, 355, 357, 358, 363, 367, 374, 380, 984 Venkataraman, R., 305, 313, 316 Vercellone, A,, 104(19), 124 Vergara, A,, 316 Vernon, 1., P., 330, 381 Veronese, A., 5, 30 Versluys, J., 228, 285 Vescia, A,, 254, 287 Vialleton, I,., 228, 288 Vialli, M., 104(892, 893), 108(242), 190, 147 Vickery, €I. 13., 35, 148 Vieil, M., 254, 287 Vilter, R. W., 94(294), 191 Vinet, A., 68(896), 148 Viollier, G., 81(944), 89(945), 97(943, 944),99(897), 148,149 Virgilo, A,, 101(159), 128 Vocke, F., 107(915), 148 Volcani, B. E., 42(57, 58), 126 Vollr, M. E., 22(20), Z6(20), 28 von Euler, A., 359, 360, 984 von Euler, U. S., 66(243, 244, 245), 130 von Glasenapp, I., 51(549), 52(258, 259), 58(257), 59(257), 130, 131, 138 von Holt, C., 94(400), 13.4 von Holt, L., 94(400), 134 von Mutsenbecher, P., 72,138,140 Vuilleumier, J., 76(59), I26

W Wachstein, M., 94(898, 899,900), 148 Wade, N. J., 116(368), 193

AUTHOR INDEX

Wagner, M. I,., 154, 160, 163, 167, 172, 173,226 Wagner-Jauregg, T., 3, 29, 359,884 Wagreich, H., 220,226 Wakeman, A. J., 46(902), 148 Walderistrom, J., 104(870), 147 Waldi, D., 89(945), 149 Waldschmidt-Leita, 15., 275, 276, 288 Waldt, I,., 277, 286 Walker, I3. S., 231, 231, 288 Waller, C. W., 116(903), 148 Walpole, G . S.,76, 124 Walter, H., 66(405), 134 Wandruff, B., 60(847), 146‘ Warburg, O . , 319, 333, 384 Ward, F. W., 109(904), 148 Ward, S. hf., 101(394), 134 Warner, D. T., 35(728, 729), 148 Warner, R. C., 4, 5(95), 17(95), 21(95), SO, 199, 217, 218, 219, 220, 223, 825, 226, 320, 374, 884 Warren, 8. I,., 72(576), 139 WassBn, A. M . , 353, 378 Watters, J. I., 352, 363, 879 Watts, A., 335, 341, 384 Webb, 1C. C., 336, 884 Weber, H. H., 235,236, 241, 250, 257, 259, 261, 263, 264, 265, 287, 288 Weber, I., 320, 374, 384 Webster, T. A., 49,52(644), 1.40 Weichert, R., 79(129), 127 Weidel, W., 79(127, 129), XO(125, I%), 81(128), 127 Weier, T. E., 333, 384 Weinhouse, S., 46(905), 55, 148 Weinstock, I. XI., 81(387), 86(387), 89 (387), 95(382), 99(564), 100(564), 134, 138

Weintraub, R. I,., 114(296), 181 Weisblatt, D. I., 104(821), 146 Weisenberger, K., 58(257), 59(257), 130 Weiss, J., 84(442), 136 Weiss, U., 37(193, 906), 39(193, 907), 129, 148 Weissbach, H., 106(148, 889), 107(889), 128,147

Weissberger, A., 329, 384 Weitsel, G., 95(908), 148 Welch, A. D., 60(646), 67(195a), 129, 140 Weller, 1,. I<;., 100(417), 136

407

Wells, I. C., 116(368), 133 Werle, E., 118(909), 148 Werner, H., 115(338), 138 West, G. B., 66(802), 68(80l, 803), 146 Westerfeld, W. W., 323, 331, 333, 382 Whaler, 13. C., 35(288), 131 Wheeler, II. I, . , 75(910), 148 Wheeler, J. F. G., 291, 816 Whewell, C. S., 70(531, 532), 138 White, J., 35(911), 148 Whitehair, C. K., 35(867), 147 Wiame, J . M., 40(912), i8(912), 109(912), 148

Wiedemann, A,, 68(778), 144 Wielantl, €I., 79(917), 107, 148 Wielund, 0. P., 110(918), 148 Wieland, T., 107(916), 108(919), 116(920), 148

Wieland, U., 79(567), 139 Wildman, S. G., 113(922), 114(921, 922), 148

Wilkinson, J. IT., 46(923), 75(571, 923), 189,148

Williams-Ashman, H.G., 25(96), 30 Williams, H. B., 174, 222 Williams, J . D., 60(33), 124 Williums, J. H.,116(903), 148, 333, 379 Williams, J . N . , Jr., 60, 61, 100(924,925), 101(254, 255, 519, 520, 926), 180, 137, 148 Williams, R. H., 85(536, 537), 138 Williams, R. J. P.,319, 325,326,374,376, 377, 380, 384 Williams, R. T., 51(930), 148 Williamson, RI. B . , 24, 30 Wilson, D. G., 40(470), 118(931), 136, 148 Wilson, J. hl. G., 48(643), 49(643), 50 (6431, 140 Wilson, V., 185, 223 Wiltshire, G. H., 83(932), 86(932), 113, 148

Winnick, T., 2(98), 3U, 57(933), 75(476), 136,148

Winterstein, E., 117,149 Wintrobe, Pvf. M., 107(935), 149 Wishart, G . M., 7, 28 Wiss, O., Sl(94-41, 87, 89, 90, 97(943, 9441, 98(376), 99(938a), 183, 149 Witkop, B., 63, 79(917), 84, 85(947), 91

408

AUTHOR INDEX

(216), 99(946), 105(217, 218), 106 (217), 129, 148, 149 Wittwer, S . H., 109(417), 114(697), 136, 142

Wolf, F. T., 114(948), I49 Wolf, W., 112(649), 141 Wolff, R,, 277, 288 Wolkow, M. &I., 47, 149 Womack, L.,35(730), 143 Womack, M., 149(950), 149 Wood, 1'. B., 96(103), 100(10J), 126 Wood, W. A., 41(890), lll(890, 951), 147, 149

Woodruff, C. W.,60(182, 952, 953), 129, $49

Woods, D. D., 110, 149 Woods, D. E., 185, 224 Woods, 1-1. J., 70(531), 138 Woodward, F. N., 303, 316 Woodward, I(.T., 101(95), 126 Woodward, R. 13., 118,119,149 Wooldridge, W. E., 113(956), f49 Woolf, I,. I., 52((361), 53(960), 54(961a), 149

Woolley, I).W., 80(957), 104?149 Work, T. S., 106(179), 107(179), 198 Wormall, 11. W. E., 335, 341, 384 Worrall, R. I,., 48(395), f34 Wosilait, W. D., 64(629), 140 Wretlind, K. A. J., 35(962), 149 'Nu, H., 185, 222, 226 Wu, K. Ti. T., 107(767), f & Wu, hl. C. C., 60(1363), 149 Wulff, H. J., 353, 354, 355, 359, 382 Wyman, J., JT., 167, 171, 172, 191, 195, 222, 226, 274, 288 Wyngaarden, J. B., 75(963a), 149

Y Yamada, S., 255, 286 Yamaguchi, M., 94(509), 137 Yamaki, T., 114(964), 149 Yang, J. T., 218, 226 \i:tniv, H., 37(964a), f49 fanofsky, C., ~ ( 6 6 3 ) ,41, 81(Y70), ! 5 (870), !i6(663), 97, 09(971), f26, 187, 141

Yoshida, Z.I., 87(972), 150 Yoshimatsu, N . , 79(587), 139 Young, G. P., 303,315

Z Zahn, It. K., 275, $88 Zaiser, 11:. M., 154, 166, 177 181, 192, 194, 197, 199, 200, 201, 202, 204, 205, 206, 207, 209, 213, 214, 216, 218, 226, 226 Zannoni, V., 62, 187 Zatman, 1,. J., lOl(S73, Y74), I50 Zeller, A., 319, 379 Zieve, I., 43(395), 134 Zillig, W., 96(118), f27 Zilva, S. S., 57(661), 60(661, 975), 150 Zimmer, G . TI., 276, 284 Zimmerman, H. H., 91(384), 134 %inoffsky, O . , 322, 384 Zoethout, %Ir. D., 234, 288 Zorn, I<., 55, 150 Zorn, W., 55, 131 Zucker, 11.1. B., 106(977,978), f50 Zwingelstein, G., 60(596), 139 Zyganoua, 1'. W., 272, 287

186, 203, 215,

f4f,

Subject Index A ADH, see Alcohol dehydrogenase Nn-Acetylkyniirenine, occurrence, 95

Adrenochrome, 68 effect on maturation of reticulocytes,

68

Acid-base dissociations, of native pro. Albinism, defective 3,4-dihydroxyphenplalanine teins, 153-180 metabolism in, 47 Actin, possible metabolir block in, 70 fish, 262,263,264-265 isolation, 262 Albumin(s), carp, electrophoretic pattern, 239,241 properties, 263 F-Ac tin, ultracentrifugal pattern, 241 reactivity Of prototropic groups in naelectrophoretic mobilities of fish, 247 tive and denatured, 182 properties, 263,265 serum, binding of anions by, 177,178 G-Actin, denaturation, 219,220 fish, electrophoretic mobilities, 247 kinetics of, 218 polymerization to F-actin, 264-265 intrinsic dissociation constants of properties, 263, 265 Actomyosin, human, 163 curves~ extractability, effect of miiscle ATP in white fish muscles, 236,239 content on, 243 Alcohol dehydrogenase(s), fish, 255-261 association of D P N and D P N H with, comparison of rabbit and, 256,257,

258,259,263

355,373,374

liver, inhibitors of, 360 isolation, 353 as zinc metalloenzyme, 369-370 numher of metal atoms per molecule,

deiiaturation, 255-256 electrophoretic behavior, 256-257 solubility, 256 ultracentrifugal d a t a , 258-261 viscosity, 257-258

374

sediment a t ion, 258-261 effect of concentration on, 259, 260 Adenosinetriphosphatase, activity in muscle, 270-271 species differences in, 270 Adrenaline, aromatic amino acids as precursors of,

33 biosynthesis, 66-68, 123 pathway, 67 precursors, 33,66,67 tyrosine as intermediate in, 66 degradation, metabolic, 68 structure, 67 Adrenals, tyrosine metabolism and, 60

409

occurrence, 323 yeast, 333,353-370 activity, D P N and, 353,367 zinc content and, 357-359 chemical composition, 334, 336,

355-359 empirical formula for, 328,331 in hi bition, 359-367 factors affecting, 360-363 mechanism of, 363 reversal of, 368 isolation, 353,354 metal content, 323,331,356-359 methods used in study of, 337-339 molecular weight, 331 physical properties, 353-355 as zinc metalloenzyme, 356,367-370

410

SUBJECT INDEX

Algae, protein contcnt of, 303-304 factors affccting, 312-313 Alkaloids, biogenesis, from amino acids, 115-121, 123 theories of, 117-121 Alkaptonuria, 47-51 experimental, 49 metabolic defects in, 48ff vitamin BI? and, 60 Amanita m a p p a , bufotenin in, 108 Amino acids, see also individual cwnipounds in algae, 303-304 aromatic, biosynthesis, 36-46 from glucose, 38, 39, 43-45 “common precursor” pathway of, 36-39 essential, 35 “straight-chain” pathway of, 42 metabolism of, 31-150 natural products probably related to, 115-121, 123 physicochemical properties, 35 as precursors of biologically active substances, 33, 117-121, 123 species differences in availability of, D-, 35 in carbonic anhydrase, 334 in carboxypeptidase, 344 decarboxylation, in microorganisms, 76-77 essential, in fish, 310-311 in shellfish, 310-311 in fish protamines, 276 in fish proteins, 308-309 in muscles of marine invertebrates, 305 pyridosal phosphate as coenzyme in reactions involving, 91-94 sequence in peptides, 15, 23, 24 titration curves, effect of formaldehyde on, 168-169 l-Amino oxidase, snake venom, denaturation, 220 Aminopeptidase, intestinal, effect on phosphopeptones, 6 Amphibia, methylation products of enteramine in, 107-108 Anserinase, in fish muscle, 272 Anthranilic acid, accumulation in plants, 113

conjugation, !I6 escretion, in congenititl hypoplastic anemia, 96 formation, in inscct mutants, 96 metabolism, 96 as precursor of tryptophan, 40 Antibiotics, derived from aromatic amino acids, 115-116 Arginase, 319 of fish muscles, 238, 272 Arginine transphosphorylase, manganese content, 323 source, 323 Aromatic compounds, degradation, microbial, 78 Aromatic rings, origin, 36 Arterenol, see Noradrenaline Ascorbic acid, biological functions, 121 metabolism of homogentisic acid and, 49 of tyrosine and, 49 rolc in p-hydroxyphenylpyruvir acid metabolism, 52 in tyrosine metabolism, 60, 63-64 Ascorlic acid oxidase, copper content, 322, 330 empirical formula, 330 molecular weight, 330 source, 322 Aspergillus niger, zinc as growth factor for, 318 Auxins, indolic, 114-115 origin of, 115

B Barley, germinating, aromatic amino acids in, 117 Benzylisoquinoline alkaloids, biosynthesis, 117-118 Blood, fish, proteins of, 273-274 Blood platelets, 5-hydroxytryptamine in, 106-107 Bufotenin, isolation, 107 occurrence, 107, 108 structure, 108 Butyryl CoA dehydrogenase, copper content, 322, 330 empirical formula, 330

SUBJECT INDEX

molecular weight, 330 source, 322 C

Cancer, 5-hydroxytryptamine and, 104 Candicine, 117 Carbohydrate metabolism, in fish, 271 Carbon, organic, yearly production by land and sea, 290 Carbonic anhydrase, 333-337 empirical formula, 331 in fish tissues, 273 inhibition, by metals and zinc complexing agents, 336-337 molecular weight, 331, 334 occurrence, 323 physical properties, 333-334 purification, 333-334 separation from other zinc containing fractions in leucocytes, 342 stability, 334 tripeptide in, 335-336 zinc content, 323, 331, 334 activity and, 335 species differences in, 335 Carbonylhemoglobin, unmasking of masked groups in, effect on heme-globin linkage, 198 relations of kinetics of denaturation to, 199-201 titrimetric criteria for, 186, 187, 189 Carboxypeptidase, pancreatic, 333, 343353 activity, metal content and, 347, 349, 350,353 bond between zinc and protein in, 349 chemical composition, 344-349 empirical formula for, 327, 331 homogeneity, 343 inhibitors of, 349-353 isolation, 343 metal content, 344-349 methods used in study of, 337-339 molecular weight, 331, 343 occurrence, 323 physical properties, 343-344 zinc content, 323, 331 as zinc metalloenzyme, 349 Casein (8) , 4-5 components of, 16, 27

411

dephosphorylation, 5 enzymatic, 16-22 electrophoretic pattern, 16 homogeneity, 4-5 iodination products, 72 molecular weight, 5 phosphopeptones from. 5-6 titration curves, 154 a-Casein, 16 composition, 17, 21 enzymatic dephosphorylation, 17, 18, 19,20 molecular weight, 21 nature of phosphate groups in, 17, 18, 19, 20, 21, 27 physicochemical properties, 16, 21 @-Casein, 16 composition, 17 enzymatic dephosphorylation, 17, 1922 nature of phosphorus linkages in, 27 y-Casein, 16 Catalase, empirical formula, 328, 331 iron content, 322 source, 322 Cathepsin, in stomach of fish, 273 Cernloplasmin copper content, 322, 330 empirical formula, 330 molecular weight, 330 source, 322 Chelating agents, inhibition of carboxypeptidase, 350, 351 mechanism of, 352 Chemicals, trace, sea as source of essentinl, 280 Chlorocruorin, iron content, 322 source, 322 Cholinesterase, in fish tissue, 273 human plasma, denaturation, 220 Chromatography, in fractionation of ovalbumin, 14 Chromogens, urinary, 109-110 Chymotrypsin, effect on ovalbumin, 14 Cinchonamine, biosynthesis, 120 Cinchonine, biosynthesis, 120

412

SUBJECT INDEX

Clupein, 275 composition, 276 Cobalt, in vitamin Bl2 , 320 Collagen(s), compnrntive studies on, 281-282 denaturation, 221 fish, 277, 278, see also Elastoidin, Ichthyocol comparison with mammalian, 278, 279 denaturation, 278 shrinkage temperature, 278 mammalian, comparison with fish, 278, 279 masked groups in, 199 stabilizers for, 279 titration curves, 154 Compound Z 1 , 38 nature of, 38 Compound Za , nature of, 38 Conalbumin, denaturation, 220 masked groups in, 220 ummasking of, 217 titrimetric evidence for, 199 Copper, metalloenzymes containing, 330, 332333 in proteins, 322 Copper enzymes, reactivation, 324 Copper proteins, 322, see also individual compounds Cortisalin, 116 Cozymase, bacterial synthesis, 101 Cytochrome c, empirical formula, 330 in fish muscles, 255 iron content, 322, 330 niolecular weight, 330

D DPN, see Diphosphopyridine nucleotide DPNH, see Diphosphopyridine nucleotide, retlucwl ~cliydrol~ufotcr~iri, 107 5-Drh yd roqui niv :tc i tl , 3s metabolism, 38, 30 its precursor of 5-dehydroshikimic acid, 37, 38 6-Dehydroshiliimic acid, 37, 38 as precursor of shikimic acid, 37, 38

5-Dehydroshikimic reductase, cofactor of, 37 Denaturation, of proteins, effect on reactivity of prototropic groups, 181, 182 on susceptibility to proteolytir enzymes, 181 Diabetes, xanthurenic acid and, 94 Diesterases, snake venom, 10 effect on caseins, 19,20,21 on ovalbumin, 13 on pepsin, 23, 25 on phosphoserylglutamic acid, 4 3,4-Dihydroxybenzoic acid, in insects, 71 5,6-Dihydroxyindole, as precursor of melanin, 69,70 from tyrosine, 69 3,4-Dihydroxyphenylacetic arid, in insects, 71 2,5-Dihydroxyphenylalanine, conversion t o 2,5-dihydroxyphenylethylamine, 51 formation of homogentisic acid from, 50 3,4-Dihydroxyphenylalanirie, biological importance, 65 in insects, 71 occurrence, 65 2,5-Dihydroxyphenylpyruvic acid, formation from p-hydroxyphenylpyruvic acid, 56, 59 3,5Diiodotyrosine, in thyroid, 72 Diphosphopyridinenucleotidase, action, 101

Diphosphopyridine nucleotide, associaition with alcohol dehj drogenases, 355,367,368,369 binding of zinc by, 367 as coenzyme of glutamic dehydrogenme, 370 DPNII-cytochrome reductase, cmpirieal formula, 328, 330 iron content, 322, 330 m o l e d a r weight, 330 sourre, 322 u ,a'-Dipyridyl, inhibition of carboxypcptiditse hy, 350 Dopa, see 3,4-Dihydrou~.phenylalaniric Dopachrome, formation, 69

413

SUBJECT INDEX

E Edestan, formation, 198 Edestin, denaturation, 198 masked groups in, 220 tihimetric evidence for unmasking of, 198-199 Egg, phosphoproteins from, 6-9 Ehrlich ascites tumor, phosphoserine in, 25 Elastoidin, 278 Electrostatic factor w,in the analysis of protein titration curves, 15Off Emetine, biogenesis, pathways of, 118119 Enolase, 319, 321 Enteramine, see also 5-Hydroxytryptamine, occurrence, 103-104 Enteramine I, Erspamer'B, 107 Enzymes, action, configuration and, 4 association of trace metals with, 318319 dephosphorylation of phoflphoproteins by, 9-25 fish, 269-273, see also Thiaminase hydrolysis of phosphopeptones by, 6 metallo-, see Metalloensymes and individual compounds participating in adrenaline biosynthesis, 66 in conversion of hydroxyanthranilic t o nicotinic acid, 9&99 of p-hydroxyphenylpyruvic acid to homogentisic acid, 62-64 of tryptophan to kynurenine, 83 formation of homogentisic acid, 6162

in homogentisic acid degradation, 64-65 proteolytic, action on phosphopeptides, 4 specificity, molecular ronfiguration and. 10, 11, 15 phosphate linkage and, 4, 6, 7 relation between metals and, 319, 321 specificity for certain peptide linkages, 6,7 tryptic, in pyloric caeca of fish, 272 Epinephrine, see Adrenaline

Erythrocytes, as source of carbonic anhydrase, 323,334,335 Esherichin coli, biosynthesis of aromatic amino arids in, 36-39, 42 Eserine, structure, 117

F Factor S, sources of, 300 Ferrihemoglobin (Methemoglobin) denaturation, energy activation, 212215 and equilibria rates of, 201-205 reversal of, kinetics and thermodynamics of, 215-216 thermodynamic functions, 211-212 effect on heme-globin linkage, 196197 unmasking of, denaturation and, 194, 201-215 model of the reaction, 210-211 titrimetric criteria for, 186, 188, 189,190 Ferritin, iron content, 322 source, 322 Fertilizers, soluble, sea a s sourcc of, 289 Fibrin, titration curves, 154 Fibrinogen rlotting, mechanism of, deduced from differential titrations, 171 titration curves, 154 Fish, carbohydrate metabolism in, 271 connective tissues, proteins of, 277-279 enzymes in, 269-273 muscles, different protein composition of red and white, 242-213, 245 enzymes in, 270ff glycolysis in, 271 lipolysis in, 271 nitrogen content of, 306-307 oil content of, 309 clualities of commerrially valuable, 203 protarnines of, 274-277 composition, 275-276 isolation, 271-275 proteins of, see Fish proteins species, number of, 292

414

SUBJECT INDEX

Fish meal, acceptability of dishes made with, 301-303 nutritive value, 300,301 quality, factors affecting, 300 species variations in, 300 protein of, 309 use as animal feed, 300 as source of factor S, 300 Fish proteins, 227-287, 305, 306, 30Pr311 amino acid composition, 308-309 biological value, 313-314 comparative, 314 blood, 273-274 comparative biochemistry of, 279-282 of connective tissue, 277-279 history, 227-228 in muscle, 228-269 differences between, and other muscle proteins, 237-238 electrophoretic pattern of chief components, 247-251 extractability of, 243-245 factors influencing, 243-245 extracts of high ionic strength, composition of, 245-251 extracts of low ionic strength, composition 238-243 electrophoretic behavior, 239243 fractions of, 236-237 isolation, 247, 251ff structure, separation, 251 Fisheries, geography of, 297, 298 Flavonoids, derived from aromatic amino acids, 116 Folling’s syndrome, see I’henylketonuria Formaldehyde, effect on titration curves of amino acids :ind proteins, 168-169 Formylnsc, occurrencr, 85 Formylkynurenine, formation, 83 intermediates, 83-84 Fumarylacetoacetic acid, from homogentisic acid, 56, 64 Fungi, biosynthesis of indoleacetic acid in, 114 metabolites derived from aromatir amino acids in, 116 tryptophnn synthesis in, precursors of, 40

G Gelatin, titration curves, 154 Globin, titration curves, 154 Globulins, reactivity of prototropic groups in native and denatured, 182,183 in fish muscles, 236 Glucose, as precursor oe aromatic amino acids, 4345, 122 Glutamic acid, phosphorylated peptide of, 3 Glutamic dehydrogenase, liver, as zinc metalloenzyme, 370 Gramine, structure, 117

H Hemerythrin, iron content, 322 source, 322 Hemocuprein, copper content, 322 source, 322 Hemocyanin, copper content, 322 sources, 322 Hemoglobin(s), composition, comparative studies of, 279-280 denaturation, 220 fish, 274 iron content, 322 masked groups in, 186-198 unmasking of, relation to heme splitting, 196-198 unmasking of, titrimrtric cvidenco for, 186-193 trigger groups initiating, 195 number of metal atoms per molerrile, 374 occurrence, 322 reactivity of prototropic groups in native and denatured, 182, 183 separation of heme and globin, 196 structure of, differential titrations and elucidation of, 171 titration curves, 154 trigger groups, 220 in unmasking reactions, I95

415

SUBJECT INDEX

Hemoglobin-I) binding of hematin by, 197, 198 formation, 194-195, 198 Hepatocuprein, copper content, 322 Heteroauxin (indole-3-acetic acid), occurrence, 113 plant hormone activity, 114 Homogentisic acid, conversion of p-hydroxyphenylpyruvic acid to, 56, 60 degradation of, 56, 64-65 excretion in alcaptonuria, 47fl formation, pathways of of, 48,50 Homogentisic ase, 64 inhibitors of, 65 occurrence, 65 Hordenine, 117 Hormones, amino acids as parent substances of, 33 Horseradish peroxidase, empirical formula, 330 iron content, 330 molecular weight, 330 titration curves, 154 unmasking of masked groups in, 217, 220 Hyaluronidase, denaturation, 220 Hydroxyanthranilic acid, conjugated, 96 conversion to nictoinic acid, 97-99 mechanism of, 98-99, 101 formation, 81, 82,96 phosphate, as nicotinic acid precursor, 87 p-Hydroxybenzoic acid, a s bacterial growth factor, 37 5-HydroxyindoIeacetic acid, excretion in cancer, 104 formation, 105, 107, 121 Hydroxykynurenine, derivatives, interrelationships with kynurenine derivatives, 90 excretion of conjugated, 95-96 in human pathologiral conditions, 94, 95 formation, 86-87 in insects, 71 as precursor of eye pigments in, 87 isolation, 81 occurrence, 86 structure, 82

p-Hydrosyphenylethanolamine (octopamine) occurrence, 67 structure, 68 p-Hydroxyphenylpyruvate oxidase, 62 p-Hydroxyphenylpyruvic acid conversion to 2,5-dihydroxgphenylpyruvic acid, 56, 59 t o honiogentisic acid, 56, 6044 enzymes participating in, 61-62 pathways of, 61-62 of tyrosine to, 56, 59 excretion, pathological, 51,52 metabolism, ascorbic acid and, 52 as precursor of tyrosine, 40 Hydroxyproline, stabilizing activity in collagen structure, 279 5-Hydroxytryptamine, see also Enteramine aromatic amino arids as precursors of, 33 hiologicsl activity, 104 biosynthesis, 104-107 pathways, 105-106 precursors, 105 in blood platelets, 106 conjugation of, 107 as constituent of venoms, 104 degradation, 107 formation, 123 methylation products of, 107-108 distribution, 107, 108 pharmacology, 108 properties, 104 in tumors, 104 a-Hydroxytryptophan, 79 as precursor of formylkynurenine, 83 as “prokynurenine”, 80 structure, 83 5-Hydroxytryptophan formation, 105 in venom of tropical toad, 106 p-Hydroxy-X-tryptophan, 84 5-Hydrosytryptophan decarboxylase, 106, 107 1

Ichthyocol, 278 amino acid composition, 278 mammalian collagen and, 278

416

SUBJECT INDEX

Imidazole groups, combination with zinc, 173 Indican, 111-113 relation to indigo ant1 indigo deriv:itives of marine organisms, 113 urinary, formation, 112 nature of, 111 Indigo, in marine organisms, 113 Indigo stones, 113 Indirubin, urinary, 113 Indole, bacterial degradation, 111 conversion t o tryptophan, 40,41 role of tryptophan desmolase in, 41 detoxication, 112 formation, 110 pharmacology, 112 Indoleacetaldehyde, occurrence in plants 115 Indoleacetic acid, aromatic amino acids a s precursor of, 33 t)iosynthesis, 123 by plant parasites, I14 in plants, 114 metabolism, in plants, 114 urinary, 109, 110 origin, 110 Indoleacetic acid oxidase, 114 nature of, 114 Indoleacetonitrile occurrence in plants, 115 Indoleaceturic acid (indole-3), as urin:iry chromogen, 110 Indolepyruvic acid, from tryptophan, 109 Influenza A virus hemagglutinin, denaturation, 220 Insects, catechol pltthway in, 71 eggs, pigments of, 88 eye-pigments, precursors of, 80, 87-88 Insulin, intrinsic dissociation constants, 163 titration curves, 154 effect of comhination with zinc on, 175-176 Invertebrates, marine, amino acids in muscle of, 305 proteins of. 304-305. 308 Iodogorgoic acid, 75

Iron, functional role in heme enzymes, 370 371 metalloenzymcs containing, 330, 831, 332-333 Iron proteins, 320, 322, 323

K Keratin, ~ 0 0 1 titration , curves, 153, 154 effect of ions on, 17i Kynurenic acid, antibiotic8 derived from, 116 formation, 79, 82 structure, 90 Kynureninase, 41, 87, 90-91 action, 82, 90 bacterial, 91 inhibitors of, 90-91 role in tryptophan metabolism, 82, 86, 89 Kynurenine, hacterial degradation, 109 conversion t o hydroxykynureninr, 8687 role of riboflavin in, 86 of tryptophan to, 79,80, 83-85 enzymes participating in, 83 derivatives, interrelations with hydroxykynurenine derivatives, !10 exrretion, fever and, 95 pyridoxine deficiency and, 88, 89 as precursor of insect eye-pigments, 87 of nicotinic acid, 80, 81, 82 Laccase, copper content, 322 source, 322 p-Lactoglobulin, denaturation, 220 dissociation, effect of added salt on, 156, 159, 160 intrinsic dissociation constants, 163 reactivity of prototropic groups in, 183 t.itration curve of dodecylsulfate complex of, 154 unmasking of masked groups in, 217218 Lactoperoxidase, empirical formula, 330

SUBJECT INDEX

iron content, 323, 330 molecular weight, 330 source, 323 Leukemia, effect on zinc content of human leukocytes, 339 Leukocyte zinc protein, 333, 339-343 chemical and enzymatic properties, 343 isolation and purification, 339-343 methods used in study of, 337-339 molecular weight, 343 source, 323 zinc content, 323 Leukocytes, human, zinc content, 339 effect of leukemia on, 339 Lignin, aromatic amino acids as precursors of, 116-117,123 Lipolysis, in fish muscle, 271 Lipoxidase system, fish, 271 Liver disease, p-hydroxyphenylpyruvic acid excretion in, 52 Lysozyme, intrinsic dissociation constants, 163 titration curves, 154

M Magnesium in carboxypeptidase, 344,345, 346, 350 in yeast alcohol dehydrogenase, 356, 357,358,359 Mammals, marine, protein content, 312 Manganese, in arginine trsnsphosphorylase, 323 tMarine organisms, halogenated tyrosines in, 75 proteins of, 303-312 biological value of, 313-314 variations in content, 312-313 Melanin, biosynthesis, 68-70, 123 pathways of, 69-70 precursors of, 65,69 properties of, 70 Mental disorders, hydroxytryptamine and, 104 L-Meromyosin, titration curves, effect of Mg on, 175 Meromyosins, titration curves, effect of salt on, 163 Metal-binding agents, as inhibitors of

417

zinc metalloenzymes, 349-353, 360366 Metal cheIates, 321 elements participating in the formation of, 321 Metal complexes, entropy effect, 374-376 successive stability constants, 371-374 Met a1-enzyme complexes, characteristics of, 325-327 difference between metalloenzymes and, 321,325-326 Metalloenz ymes, characteristics of, 325 difference between metal-enzyme complexes and, 321, 325-326 empirical formulas for, 327-328, 330, 331 inhibition of, 324 reversal of, 324 metals in, 332 role of, 324, 325 properties, 321-325 zinc, see also individual compounds inhibitors of, 318, 349, 350-353, 360366 Rletalloflavoproteins, 333 Metalloproteins, 32C321, 333-378, see also Metalloenzymes and individual compounds detection of metals in, 328-329, 332 metal content, 322-323 zinc, 333-378, see also individual compounds Metals , in carboxypeptidase, 344-349 enzymatic activity and, 347,349 detection in metalloproteins, 328-329, 332 as enzyme activators, 326-327 as inhibitors of yest alcohol dehydrogenase, 359, 360 in metal-enzyme complexes, 325ff role in enzyme activity of, 326 in metalloenzymes, 321ff, 332-333 binding to protein, 324 place in periodic system, 325 as reactive groups, 324,325 relation between enzymes and, 319,321 trace, see Trace metals Metaphosphoric acid, combination with proteins, 174

418

SUBJECT INDEX

N-Methylnicotinamide, a s metabolite of nicotinic acid, 102 0-Methyltyrosine, as constituent of antibiotics, 115 Microorganisms, biosynthesis of tyrosine in, 40 metabolic products related to aromatic amino rtcids, 115-116 mutants, biosynthesis of aromatic amino acids by, 36-46 production of, 36 phenylalanine metabolism in, pathways of, 76-78 tryptophan metabolism in, 108-113 pathways of, 1098 tyrosine metabolism in, pathways of, 76-78 Milk, casein content, 4 Molybdenum, metalloenzymes containing, 331, 332333 i n oxidases, 323,331 Muscle (a), extractability of struciure proteins of, 243-245 factors influencing, 243-244 extracts, comparative studies on elecphoretic behavior of, 280 on ultracentrifugal behavior, 280281 fish, ATPase activity in, 270, 271 composition, 231-235 species differences in 231-233, 240241 difference8 between other striated muscles and, 251 between red and white, 238 glycolysis in white, 271 histology, 228-231 lactic acid content, 269 lipolysis in, 271 &peak of extracts of, 246 proteins of, 228-269 proteolysis in, 272 red, 238,239, 241-243 electrophoretic pattern, 242 ultracentrifugal data, 241 white, 238, 239-241 electrophoretic pattern, 239-241 ultracentrifugal data, 241 frog, composition, 234

human, composition, 234 red, proteins of, 241-242 stability, species differences in, 244-245 Mywen, nature of, 235 Myogen, Henrotte’s, 252-253 distribution, 252 isolation, 252 properties, 253 Myoglobin(s), fish, 253-255 isolation, 253, occurrence, 253 properties,specirs differences in, 254255 iron content, 322 source, 322 titration cnrves, 154 Myosin, 235 extractability, effect of p H on, 244, 248 species differences in, 251 fish, 261-262 combination with F-actin, 262 rompared v i t h rabbit myosin, 262, 263 electrophoretic mobilities, 247 isolation, 262 properties, 263, 264 titration curves, 154 effect of salt on, 162

N Xervous system, central hydroxytryptamine in, 104 h’eurospora acetylkynurenine in, 95 biosynthesis of aromatic acids in, 30 of nicotinic acid in, 97, 98 kynureninase of, 91 Nicotinic acid, biosynthesis in plants, 113 conversion of hydroxyanthranilic acid to, 97-101 glucuronide, 103 metabolism, 101-103, 122 species differences in metabolites, 102 tryptophan as precursor o f , 33, 79, 80, 121 Nitrogen, in fish, 306-307, 308

SUBJECT INDEX

Xoradrenaline, aromatic amino acids a s precursors o f ) 33 biosynthesis, 66, 123 degradation, met itbolic, 68-69 methylation, 67 as precursor of adrenaline, 67 Nucleotropomyosin, carp, 266-267, 268

0 OF, see 1,IO-Phenanthroline Octopamine, 67 Octupus, noradrenuline synthesis in, 6768 Oils, essentials, derived from aromatic amino acids, 116 Oligophrenia, see Phenylketouuria Omnatins, nature of, 88 Omnins, 88 Omnochrome, see Insect, eye-pigments Ovalbumin, acid lmse dissociation of, 155-156 components, 11 electrophoretic behavior, 11-13 enzymatic dephosphorylation, 9-16 intrinsic dissociation constants, 163 nature of phosphorus bonds in, 11-16 phosphoserine in, 15 physicochemical properties, 11 Ovotyrine 8 2 , identity with vitellinic acid, 7 Ovotyrines, 6-7 composition, 7 Oxaloacetic decarboxylase, 319 Oxalosuccinic decarboxylase, 319 Oxindolylalanine, 83, 84 Oxyhemoglobin, masked groups in, titrimetric criteria for unmasking of, 187

P Parahemoglobin, function, 192 titration data, 19Z193 Pepsin, action on ovalbumin, 14 on vitellin, 7 denaturation, 219 dephosphorylation, enzymatic, 22-25 effect on electrophoretic behavior, 23, 24, 25

419

on proteolytic activity, 22 fish, 273 molecular weight, 22 nature of phosphorus linkage in, 22-25 physicochemical properties, 22, 23, 24 molecular weight, 22 nature of phosphorus linkage in, 22 Peptides, intrinsic dissociation constants, 163 Peroxidases, metal content, 323 sources, 323 I’halloidine, 79, 116 1,lO-Phenanthroline, as inhibitor of zinc metalloenzymes, 350-353, 363-366 Phenol oxidase, copper content, 330 empirical formula, 330 molecular weight, 330 Phenylacetic acid, as constituent of benzylpenicillin, 115 Phenylalanine, 33 biosynthesis, final stages in, 39 pathways, 38, 39-40 conversion to tyrosine, 53-54 enzymatic, 56, 58-59 in muscle and liver extracts, 59 degradation t o acetoacetate, 46 essentiality for higher organisms, 122 history, 34 a s hormone precursors, 33 metabolism, 123 defective, 47, 48ff microbial, 76-78 by decarboxylation and amine oxidation, 76-77 by oxidative deamination or transamination, 77-78 pathway of, 48,50,55,56-58 as precursor of adrenaline, 66 requirements of mammals, 35 Phe nyl ke tonu ria, 52-55 absence of pigmentation in, 54 mental symptoms in, 52,53 factors contributing to, 54 metabolic defects in, 47,53,54,55 l’henylpyruvic acid, DS precursor of phenylalanine, 39 of prephenic acid, 40 Phenylpyruvica, see Phenylketonuria

420

SUBJECT INDEX

Phosphatase, acid, of citrus fruit, effect on phosvitin, 8 of seminal plasma, effect on phosphate linkage, 9 alkaline, of intestinal mucosa, 319 bone, effect on phosphopeptones, 6 on phosvitin, 8 intestinal, effect on phosphopeptones, 6 on phosphorylserylglutamic acid, 4 potato, effect on ovalbumin, 11 on pepsin, 22,23 on pepsinogen, 22, 23 on phosphate linkages, 9,lO prostate, effect on casein and components, 17, 18, 19, 21, 20 on ovalbumin, 5, 11, 12, 13, 14, 15 on pepsin and pepsinogen, 22 on phosphate linkage, 4, 11 Phosphatases, action on pepsin and pepsinogen, 22,23 linkage specificity of, 9-11, 27 Phosphate group, stability, 2, 3, 8, 9, 14, 26 Phosphoamino acids, see ntso individual compounds chemical properties, effect of incorporation into peptide or protein on, 9, 26 from phospho proteins, 2-4 stability of, 9, 26 Phosphoarginine, 3 biological role, 3 Phosphoglucomutase, 319 Phosphomonoesterases, dephosphorylation of ovalbumin by, 9-16,27 Phosphopeptones, amino acid sequence of, 8 from casein, 5-6 composition of 5,6 enzymatic hydrolysis, A from vitellenin, 6 from vitellin, 6-7 Phosphoproteins, see nlso individual compounds a s active metabolites, 25 amino acid sequences in, 8, 15,26 classification, 1 diester and pyrophosphate bonds in, 10, 27

enzymatic transformation to tnonoesters bonds, 9, 10 maymatic dephosphorylation, 8, !b25 nature of phosphorus linkages in, 1-30 occurrence, 1, 25 phosphoamino acids from, 2-4 possible biological function, 25-28 of protein moiety, 26 stability, 1, 8, 26 as storage reservoir for phosphorus, 26 Phosphorus, bound to N, 2 , 3 , 8 , 9 , 15, 19, 20, 21, 22 to 0,2,9,10,19,20,22,24 Phosphorylserines, 2, 3, 7, 9, isolation of 0-,7 0-Phosphorylserylglutaniic acid, 3-4 structure, 4 Phosphorylthreonines, 3,9 Phosphoserine, 3, 26 in Ehrlich ascites tumor, 7 in ovalbumin, 15 in pepsin, 24 in vitellinic acid, 25 Phosvitin, 6, 7-9 dephosphorylation, 8 electrophoretic compoments, 8 homogeneity, 8 molecular weight, 8 Pigments, fungus, derived from aromatic amino acids, 116 Plankton, differences in composition, 299 distribution, 298-299 toxicity of some kinds of, 299 types of, 299 Plants, biogenesis of indoleacetic acid in, 114 growth factors, nature of, 114 indolic auxins in, 114-115 marine, amino acid composition, 304 metabolites derived from iiromatic amino acids in, 116 tryptophan metabolismin, 113-115,123 Pregnancy, xanthurmic acid excretion in human, 94 Prephenic acid, 39 Protamine sulfate, binding of organic ions by, 277 Protamines, see also individual cam pounds

SUBJECT INDEX

fish, 274-277 composition, 275-276 properties, 277 pharmacological activity, 277 Protein(s), animal, production in the sea, 291 anionic groups, combination with dyes and detergents, 174 cationic groups, anions combining with, 173-174 comparison of salt effect on soluble and insoluble, 160-164 configuration of, trigger groups and, 221 copper-containing, of liver, 322 of milk, 322 denaturation, see Denaturation of proteins electrophoretic behavior, effect of denaturation on, 185 end group analyses with D N P and FDNB, 181-183 fish, see Fish proteins food, sea as potential source of, 289316 ways of enlarging harvest of, 291292 hydrogen ion equilibria in active and denatured, 151-221 stoichiometry of, 164-176 interaction with ions, 179 stoichiometric combination, 173174 intrinsic dissociation constants, 163 from analysis of titratjion curves, 163-1 64 iodinated, titration curves of, 169-170 ion-binding behavior, variations in, 159 isoelectric points of native and denatured, 181 in marine organisms, 303-312 in algae, 303-304 biological value of, 313-314 in fish, 308-311 in invertebrates, 304-308 in marine mammals, 312 variations in, 312-313 metallo-, see Metalloproteins and individual groups and compounds

421

milk, copper-containing, 322 masked groups in, 221, see also prototropic group, unreactive denaturation and unmasking, 194 mechanism of masking, 194-195 muscle, see also individual compounds classification, 235 nomenclature, 235 number of cationic groups in, determination from amino acid analyses, 165-166 from titration curves, 164, 165166 prototropic groups of, conversion t o nonprototropic groups, 171-1 72 determination, 179-180 from amino acid assays, 167ff, 180 from titration curves, 166ff, 180 heats of ionization, 172 modification of dissociation constants of, 168-171 reactivity in native and denatured, 180-221 unreactive, 182ff titrimetric criteria for unmasking of, 184-199 reversible combination with cations, 320 structure, see Structure proteins and individual compounds titration curves, assignment of segments of, to specific groups, 166168 effect of concentration on, 159 of formaldehyde on, 168-169 of ion-protein interactions on, 174179 estimation of number of cationic and anionic groups from, 164-166 factors affecting, 156 general aspects of, 153-157 relation between electrophoretic d a t a and, 178-179 theoretical analysis oi, 157-164, I70 electrostatic factor 1u in, 159-160 Proteolysis, effect of denaturation on susceptibility to, 181 in fish muscle, 272

422

SUBJECT INDEX

Pseudomonas, tryptophan metabolism in, 108 I’terins, occurrence in insect eyes, 87 Pyridine nucleotides, 101 biosynthesis, 99-101 of human red blood cells, 101 nicotinic acid and, 99,100,101 6-Pyridone, a s metabolite of nicotinic acid, 102,103 Pyridoxal phosphate, I ~ Acoenzyme in amino acid reactions, 91-94 of tryptophanase, 110-111 kynureninase activity and, 89,90 role in tryptophan biosynthesis, 41 Pyridoxine deficiency, assay in man, 94 effect on tryptophan metabolism, 79,80 kynurenine excretion in, 88, 89 xanthurenic acid, excretion in, 88, 89 Pyrophosphatase, yeast, effect on casein, 19,20,21 on phosphate bonds, 10

Q Quercetin, biosynthesis in ChlamJldonzonas eugamelos, 116 Quinic acid as precursor of dehydroquinic acid, 39 Quinolinic acid, formation, 98, 99

R Itaffinase, yeast, denaturation, 220 Retina, zinc in, 369 Riboflavin, role in t ryptophan met alxlism, 86-87 Rilwse, post-mortem liherution in fish muscle, 271 Iiicin, denaturation, 219, 220

species of vertebrate and invertebrate animals used as, 293 Sedoheptulose-1,7-diphosphate, as intermediate in aromatic amino acid biosyntheses from glucose, 39, 45 Serine, derivatives of, 2, 3 in ovotyrine p2 , 7 in vitellinic acid, 7 Serotonin, see also Enteramine t t n d 5-Hydroxy tryptamine identity with enteramine, 104 isolation, 104 Shellfish, protein content, 304 Shikimic acid, formation, 122, 123 metabolism, 37-39 as precursor of aromatic amino acids, 36-39 of bacterial growth factors, 122 Silk fibroin titration curves, 154 Skatole (3-methylindole), formation, 113 from tryptophan, 109 Skatole red, nature of, 113 Skatoxyl, I13 Streptokinase, denaturation, 220 Structure proteins, see also individual compounds comparative studies on, 263, 281 muscle, extractability, 243-245 factors influencing, 243-244 Strychnine, biosynthesis, 120 Sturine, 275 Substance CP, as precursor of aromatic amino acids, 37, 38, 39 SulfhydryI groups, rombination with mercury and silver, 173 Sulfonamides, inhibition of rarboitic anhydrase by, 337

S

T

Salmines, composition, 276 peptides in, 277 Sea, as potential source of protein food, 289-316 Sen food, harvest of, limiting factors, 293-297 ways of enlarging, 299-300

Tetrahydropripaverine, biosynthesis, 118 Thiaminase, 228 occurrence, 269 Thyroglobulin, hog, unmasking of masked groups, 217, 221 Thyroid hormones, see also individual hormoncs

SUBJECT INDEX

conversion of tyrosine to, 71-75 metabolism, 75 Thyronine, 71 structure, 72 Thyroxine, 71 aromatic amino acids as precursors of, 33 biosynthesis, 72-73, 123 mechanism of, 72-74 iodine content, 71 metabolism, tyrosine metabolism and, 75 precursors, 72 structure, 72 Tissues, embryonic, phosphoproteins in, 25 Titration curves, of proteins, general aspects of, 153-157 Trace metals, association with enzymes, 318-319 terminology, 318 3,5,3’-Triiodo-~-thyronine, activity, 74 biosynthesis, 74-75, 123 occurrence, 72,74 structure, 74 Trimethylamine oxide-rrductnse, in fish muscle, 271 Triosephosphate dehydrogrnase, den:tturation, 220 Triphosphopyritline nucleotide, as cofactor of 5-dehydroshikimic reductase, 37 Tripeptide, of rnrhonir anhydrase, 335336 Tropomyosin, 235 electrophoretic mobilities of fish, 247 fish, 265-269 degradation, 268-269 fractions of, 266-268 isolation, 265 ff properties, 263 rabbit, properties of, 263 Trypsin, action on ovalbumin, 14 on vitellin, 7 denaturation, 219 titration curves, 154 effect of bivalent ions on, 175

423

Trypt amine occurrence in plants, 114 as precursor of indoleacetic ncid, 114 Tryptamine receptors, 10.1 Tryptophan, biosynthesis, final stages in, 40-42 conversion t o nicotinic acid, 79-103 mechanism of, 80 thiamine deficiency and, 85 history, 34-35 as hormone precursor, 33 metabolism, defective in phenylketonuria, 54 effect of vitamin B deficiencies on, 80, 81, 86 by enteramine-serotonin pathway, 103-108 in microorganism, 108-113 pathways, 121, 122 in plants, 113-115 pathways of, 113 metabolites, biologically active, 123 variants of, 95-96 as parent substance of plant hormones, 115, 123 as precursor of 5-hydroxytryptamine, 105-106 of indoleacetic acid, 114 of nicotinic acid, 33 requirements of mammals, 35 Tryptophan cycle, 41 Tryptophan decarboxylase, 109 Tryptophan desmolase (tryptophan synthetase), role in conversion of indole t o tryptophan, 41 of zinc in, 41 Tryptophan peroxidase-oxidase system, 83, 85 adaptation, 85-86 Tryptophanase, 110 action, mechanism, 111 adaptive formation, 111 coenzyme of, 110,111 Tyrian purple, 113 Tyrosinase, action of, 66 on 3,4-dihydroxyphenylalanine,66 copper content, 322 source, 322 6-Tryosinase, action on tyrosine, 77, 78

424

SUBJECT INDEX

Tyrosine, 33 biosynthesis, 122 final stages in, 40 conversion t o p-hydroxyphenylpyruvic acid, 59 of phenylalanine to, 53-54 enzymatic, 56, 58-59 t o thyroid hormones, 71-75 degradation t o acetoacetate, 46 by catechol pathwaj., 65-71 halogenated, see also individual compounds, in marine organisms, 75 history, 34 a s hormone precursors, 33 metabolism, 123 adrenals and, 60 ascorbic acid and, 59, 60, 61, 6 3 6 4 defective, 47, 49, 51-52 melanin as end product of, 71 microbial, 76-78 by decarboxylation and amine oxidation, 76-77 pathways of, 50,55,56-58 as precursor of adrenaline, 66 of 5-hydroxytryptamine, 105 spectrophotometric titration of phenoxyl groups of, 172-173 Tyrosine decarboxylase, bacterial, 76 Tyrosine oxidase, 75 Tyrosinosis, 51-52 Tyrosyluria (hydroxyphenyluria), 59-60 effect of hematopoietic factors on, 60

U Urease, in fish muscle, 272 Uricase, copper content, 322 heteroauxin in human, 113 source, 322

V Venoms, 5-hydroxytryptamine in, 104 5-hydroxytryptophan in, 106 Vibrio spp., phenylalanine metabolism in, 78 Vitamin Be , see also Pyridoxine effect on conversion of n- t o L-phenylalanine, 35

Vitamin € 3 1 ~, cobalt content, 320 Vitamins, effect on tyrosyluria, 60 Vitamins €3, see also individual members deficiency, effect on tryptophan mctabolism, 81, 86-87 Vitellenin, 6 Vitellin, 6 action of proteolytic enzymes on, 7 isolation, 6 ovotyrines from, 6, 7 phosphopeptones from, 5, 6-7 Vitellinic acid, 6 identity with ovotyrine B2 , 7 0-phosphorylserine in, 2, 7

W Wool, titration curves, effect of salt on, 160,161

X Xanthine oxidase, empirical formula, 331 metal content, 331 molecular weight, 331 Xanthurenic acid, excretion, in conjugated form, 96 in human pathological conditions, 94 pyridoxine deficiency and, 88, 89, 94 vitamin I3 deficiency and, 91 structure, 79,90 Xanthurenicase, 96

Y Yohimbine, biosynthesis, 118-119 E

Zinc, bond between protein of yeast alcohol dehydrogenase and, 367,368 in carboxypeptidase, 344, 349 enzyme activity and, 347, 349, 350, 353 combination with imidazole groups, 173, 176 complexes, successive stability constants, 371-374

SUBJECT INDEX

coordination chemistry of, 37e378 distribution in plants and animals, 318 isotopic, excretion in pancreatic juice, 353 in liver glutamic dehydrogenase, 370 metalloproteins containing, 333-378 ratio of protein to, in leukocytes, 340 reaction wit,h insulin, mechanism of, 176

425

with I , 10-phenanthroline, 363, 368369 in retina, 369 in yeast alcohol dehydrogenase, 356359, 367 Zinc chelates, association constants, 353 Zinc proteins, 320, 323, 333-378, see also Metalloproteins and individual compounds

This Page Intentionally Left Blank