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ADVANCES IN PROTEIN CHEMISTRY VOLUME V

This Page Intentionally Left Blank

ADVANCES IN PROTEIN CHEMISTRY EDITED BY M. L. ANSON

JOHN T. EDSAU

Continental Foods, Inc., Hoboken, New Jersey

Harvard Medical School; Boston, Mascrchurettr

Associate Editor for the British Isles KENNETH BAILEY Trinity Cdlege, Cambridge, Enghnd

VOLUME V

1949 ACADEMIC PRESS INC., PUBLISHERS NEW YORK, N. Y.

COPYRIGHT Cc)

l(.)#) BY ACADEMIC PRESS

INC

ALL ~ I ( : € I T s RESERVED NO PART OF T H I S BOOK MAY BE REPKODUCED IN ANY FORM BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM T H E PIIIILISlIERS.

ACADEMIC PRESS INC 1 I I FIFTHAVENUE

NEWYORK3,N. Y .

llnited Kingdom Edition Puhlished by ACADEMIC PRESS INC. ( L O N D O N ) LTD. BERKELEY SQUARE HOUSE.LONDONw. I

First Printing, 1949 Second Printing, 1963

I’RINTED IN T H E lJNITED STATES OF AMERICA

CONTRIBUTORS TO VOLUME V

JAMESB. ALLISON,Rutgers University, New Brunswick, New Jersey ALBERTCLAUDE,The Rockefeller Institute for Medical Research, New York, New York* JOSEPHS. FRUTON,Department of Physiological Chemistry, Yale University, New Haven, Connecticut K . H. GUSTAVSON,Swedish Tanning Research Institute, Stockholm, Sweden J . W. H . Luaa, Department of Biochemistry, University of Melbourne, Melbourne, Victoria, Australia HAROLDP. LUNDQREN,Western Regional Research Laboratory, U . S. Department of Agriculture, Albany, California THOMAS L. MCMEEKIN,Eastern Regional Research Laboratory, U. S. Department of Agriculture, Philadelphia, Pennsylvania B. DAVIDPOLIS,Eastern Regional Research Laboratory, U. S. Departm m t of Agriculture, Philadelphia, Pennsylvania G. R. TRISTRAM, S i r William D u n n Institute of Biochemistry, University of Cambridge, Cambridge, England * Present Addreee: Institute Jules Bordet, Bruxelles, Belgium.

V


CONTENTS CONTRIBUTORS TO VOLUMEV .

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

v

The Synthesis of Peptider BY JOSEPES FRUTON. Department of Physiolopkul Chemistry. Y a k University. New R a m . Connedicvt 1. Introduction and Nomenclature . . . . . . . . . . . . . . . . . . . 1 I1. General Methods of Peptide Synthesis . . . . . . . . . . . . . . . . 6 I11. Special Aspecte of Peptide Synthesis . . . . . . . . . . . . . . . . . 34 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

.

Amino Acid Composition of Purided Proteins BY G . R . TRISTRAM. Sir William Dunn Znatituk, qf Biochemistry. University of Cambridge. Cambridge. England I Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 I1. Purposes of Amino Acid Analysis . . . . . . . . . . . . . . . . . . 86 I11. Methods of Analysis . . . . . . . . . . . . . . . . . . . . . . . . 86 IV Establishment of Accuracy and Specificity of Methods of Analyaie. . . . 99 V Comparison of Analytical Methods . . . . . . . . . . . . . . . . . 106 VI . Structure of Proteha aa Revealed by Amino Acid Analysis . . . . . . . 125 VII . Amino Acid Composition of Certain Proteins . . . . . . . . . . . . . 129 VIII . General Discussion . . . . . . . . . . . . . . . . . . . . . . . . 141 I X . Conclusion and Summary . . . . . . . . . . . . . . . . . . . . . . 148 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

.

. .

Biological Evaluation of Proteins BY JAMES B . ALLISON. Rutgers Universily. New Brunewick. New Jersey

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 I1. Evaluation through Nitrogen Balance . . . . . . . . . . . . . . . . 167 I11. Evaluation through Growth . . . . . . . . . . . . . . . . . . . . . 173 IV. Evaluation through Tissue Regeneration . . . . . . . . . . . . . . . 180 V. Evaluation through Amino Acid Analysis . . . . . . . . . . . . . . 192 VI . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

Milk Proteins

.

.

BY THOMAS L MCMEEKINAND B DAVIDPOLIS. Eastern Regional Rcsearch Laboratory. Philadelphia. Pennsylvania

I . Introduction . . . . . . . . . . . . . . . I1. Protein Distribution in Milk . . . . . . . I11. Separation and Properties of Milk Proteins . IV. Proteins of Whey . . . . . . . . . . . . . vii

. . . .

. . . .

. . . .

. . . .

. . . .

. . . . . . . 202 . . . . . . . . 202 . . . . . . . . 203 . . . . . . . 210

viii

CONTENTS

.

V Amino Acid Composition of Milk Proteins . . . . . . . . . . . . . . 219 VI . Encymee in Milk . . . . . . . . . . . . . . . . . . . . . . . . . 219 VII . Relationship of Milk Proteins to Serum Proteins . . . . . . . . . . . 223 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 Plant Proteins

BY J . W . H . LUOO,Department

of Biochemistry, University of Melbourne, Melbourne, Victoria, Australia

I. I1. I11. IV

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 Bulk Proteins of Plants, Plant Organs, etc . . . . . . . . . . . . . . . 232 “Individual” Proteins of Plants . . . . . . . . . . . . . . . . . . . 259 Modes of Occurrence of Protein in Plants . . . . . . . . . . . . . . . 265 V . Protein Metabolism in Plants . . . . . . . . . . . . . . . . . . . . 269 VI . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

.

Synthetic Fibers Made from Proteins

BY HAROLD P. LUNDOREN, Weetern Regional Research Laboratory, U . S. Department

of

Agriculture, Albany, California

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 I1. General Considerations of Proteins Chain Behavior . . . . . . . . . . 307 I11. Preparation of Fibers from Proteins . . . . . . . . . . . . . . . . . 311

IV . Molecular Basis for Mechanical Properties of Fibers Made from Proteins 327 V. Summary and Conclusion . . . . . . . . . . . . . . . . . . . . . . 345 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 Some Protein-Chemical Aspects of Tanning Processes BY K . H . GUSTAVSON. Swedish Tanning Research Institute. Stockholm. Sweden

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 354 I1. Chemistry of Collagen . . . . . . . . . . . . . . . . . . . . . . . 356 I11. Keratolysis and Action of Alkali on Hide . . . . . . . . . . . . . . . 375

IV . General Aspects of Tanning . . . . . . . . . . . . . . . . . . . . . 378 V Reaction of Chromium Compounds with Collagen (Chrome Tanning) . . 379 VI . Vegetable-Tanning Process . . . . . . . . . . . . . . . . . . . . . 394 VII . Reaction of Condensed Sulfo Acids (Syntans) with Collagen . . . . . . 402 VIII . Tanning Power of Aldehydes . . . . . . . . . . . . . . . . . . . . 405 I X . Quinone Tannage . . . . . . . . . . . . . . . . . . . . . . . . . 411 X General Comments . . . . . . . . . . . . . . . . . . . . . . . . 413 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415

.

.

Proteinr, Lipids, and Nucleic Acids in Cell Structure8 and Functions

BY ALBERT CLAUDE,The Rockefeller Znatitute for Medical Research, New York, New York I . Introduction . . . . . . . . . . . . . . . . I1. TheCell . . . . . . . . . . . . . . . . . . I11. The Nucleus . . . . . . . . . . . . . . . . IV The Cytoplasm . . . . . . . . . . . . . . . V Constitution and Duplication of Living Matter

. .

. . . . .

. . . . .

. . . . .

. . . . .

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

423 425 426 428 433

ix

CONTENTS

VI . Phospholipids in Cell Structures and Functions . . . . . . . . . . . VII . Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : . VIII . Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

.

434 436 437 439

AUTHOR INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

441

SLIBJECT INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

466


The Synthesis of Peptides

BY JOSEPH 5. FRUTON Department of Physiological Chemistry, Yale University, New Haven, Connectiml

CONTENTS

pooc

I. Introduction and Nomenclature.. . . . . . . . . . . . . . . . . . . . . . ............ 11. General Methods of Peptide Synthesis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Synthesis of Dipeptides by Partial Hydrolysis of Diketopiperasines 2. Condensation of Peptide Esters... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Synthesis of Peptide Derivatives by Means of Acylamino Acid Chlorides and Aeides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Use of Amino Acid Chloridea in Peptide Synthesis.. . . . . . . . . . . . 5. Use of a-Halogen Acyl Halides in Peptide Synthesis. . . . . . . . . . . . . . . 6. “Adactone” Method for Syntheeb of Peptides 7. Condeneetion of Keto Acids and Amides.. . . . . 8. Use of N-Carbonic Acid Anhydrides of Amino Acids in Peptide Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Use of Toluenesulfonylamino Acids in Peptide Synthesis. . . . . . . . . 10. Use of Aeidoacyl Chlorides in Peptide Synthesis.. . . . . . . . . . . . . . . 11. “Carbobenmxy” Method of Peptide Synthesis.. . . . . . . . . . . . . . . . . . . 12. Use of Phthalylamino Acids in Peptide Synthesis. . . . . . . . . . . . . . . . . . 13. Enzymatic Synthesis of Peptide Derivatives.. . . . . . . . . . . . . . . . . . . . . 111. Special Aspects of Peptide Synthesis.. . . . . . . . . . . . . . . . . . . . . . 1. Peptides of Glycine, Alanine, Valine, Leucine, and Isole 2. Peptides of Aspartic and Glutamic Acids.. . . . . . . . . . . . . . . . . . 3. Peptides of Phenylalanine and Tyrosine. . . . . . . . . . . . . . 4. Peptides of Cystine and Cysteine. . . . . . . . . . . . . . . . . . . . 5. Peptidesof Serine.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Peptides of Lysine and Other Diamino Acids. . . . . . . . . . . . . . . . . . . . . 7. Peptidesof Arginine.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Peptides of Histidine.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Peptides of Proline and Hydroxyproline.. . . . . . . . . . . . . . . . . . . . . . 10. Peptidesof Tryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Peptides of Methionine. . . . . . . . . . . . . . . . . . . . . . . . . ............. 12. Peptides of Other Amino Acids.. . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................

1 5 5

6 9 11

21

25 32

33

66 68 62 66 67 72 73 73

I. INTRODUCTION AND NOMENCLATURE The objectives of the search for satisfactory methods of peptide eynthesis were clearly stated by Fischer and Fourneau (149) in the 1

2

JOSEPH

a.

FRUTON

memorable paper which initiated the systematic exploration of this field of study : “Der Gedanke, die aus den Proteinstoffen durch Hydrolyse entstehenden Aminoskuren durch Anhydridbildung wieder eu g r h e r e n Komplexen eu vereinigen, ist schon seit lkngerer &it von verschiedenen Forachern experimentell behandelt worden, W k emnern nur a n die Anhydride von Schaal (262), ihre Verwandlung einerseits in den kolloidalen Polyaaparaginharnstoff von Grimaux (199), anderseita in die Polyaspartakuren von H. Schiff (263), ferner an die Verauche von Schtitzenberger (270) nber die Vereinigung verschiedener Aminoshuren (Leucine und Leuceine) mit Harnstoff durch Erhiteen rnit Phosphorsiiureanhydrid, an die iihnlichen Beobachtungen Lilienfelds (238) tiber die Wirkung von Kaliumbisulfat, Formaldehyd und andereo Kondensationsmitteln auf ein Gemiach von Aminostiureestern und endlich an die Angaben von Balbiano und Trasciatti (39) tiber die Verwandlung dea Glycocolls in ein hornartigea Anhydrid durch Erhiteen mit Glycerin. Aber alle von ihnen beschriebenen Produkte sind amorphe, schwer characteresierbare Substanzen, tiber deren Struktur man ebensowenig wie tiber den Grad ihrer Verwandtschaft mit den natblichen Proteinstoffen etwas sagen kann. “Will man auf diesem achwierigen Gebiete xu sicheren Resultaten kommen, BO wird man zuerst eine Methode finden m h e n , welche es gestattet, aucceeaive und mit definierbaren Zwiachenstufen die Molektlle verachiedener Aminoakuren anhydridartig aneinander eu reihen.”

Since the enunciation of this view, it has become abundantly clear that one of the principal contributions of the organic chemist to the study of the structure and reactions of proteins has been indeed the development of several techniques for the synthesis of compounds in which amino acids are joined to one another “anhydridartig” by means of acid amide linkages. The services of peptide synthesis to protein chemistry have been many and various. The finding of synthetic peptides and peptide derivatives which are hydrolyzek by crystalline enzymes specifically adapted to the hydrolysis of proteins has buttressed the theory, first expressed by Hofmeister (217) and Fischer (126), that in proteins the peptide bond represents the most general type of linkage between the individual amino acid residues.* In addition, modern methods of peptide synthesis have made possible the preparation of special peptides of low molecular weight and known chemical structure In stressing the importance of the peptide bond in the structure of proteins, i t is not intended to exclude the possibility that other types of covalent linkage may also play a significant role in the architecture of the protein molecule (99,136). Of particular interest in connection with the search for labile bonds which may be involved in the phenomena associated with protein denaturation ia the recent suggeution of Linderatr0m-Lang and Jacobsen (239) that cysteine residues of proteins might participate in the formation of thiaeoline rings. Similar consideration might be given to the participation of serine (or threonine) residues in oxaeoline groupings (46). The recent synthesis, by Ehrentwhd and Davideeohn (119), of labile peptides with thioiminoether linkages is a further experimental approach in this direction.

SYNTHE8IB OF PEPTIDES

3

for use as models in the examination of several physical and chemical properties of proteins. Thus, the use of synthetic peptides has notably facilitated the interpretation of data on the acid-base relationships of proteins (101). Furthermore, in the study of the reactions of proteins with various chemical reagents, parallel experiments with peptides or peptide derivatives have frequently clarified the results observed with proteins. Of the numerous examples in the recent literature one may cite the study of the iodination of tyrosine-containing proteins (212), or the investigation of the action of mustard gas (250) and the nitrogen mustard gases (191) on proteins. Apart from their importance in providing simple models for study of the enzymatic degradation , physical properties, or chemical reactions of proteins, the newer techniques of peptide synthesis have been invaluable in the final proof of the chemical structure of several physiologically important substances, such as glutathione (202) and carnosine (272). The discovery that the antibiotics gramicidin, tyrocidine, and gramicidin S are peptides (219,282) and the recent reports that there is, in pancreatic hydrolyzates of several proteins, a factor (or factors) of peptide nature which promotes the growth of certain microorganisms and the rat (299,302) offer further opportunity for the fruitful use of the methods of peptide synthesis to establish the chemical structure and to study the physiological action of peptides of biological interest. The development, in recent years, of new methods for the separation of amino acids and peptides (103,242) has led to i renewed interest in the products of the partial hydrolysis of proteins (281). It is clear, however, that, whatever methods are used to separate and identify peptides obtained from proteins, the conclusive evidence for the identity of such peptides must come from the comparison of the isolated material with synthetic peptides of known structure. This is the procedure that allowed Fischer and Luniak (159) to establish definitely that the peptide isolated by Osborne and Clapp (253) from a gliadin hydrolyzate was indeed L-prolyl-L-phenylalanine. In a similar manner, Stein, Moore, and Bergmann (278) demonstrated the presence of glycyl-L-alanine and L-alanylglycine in partial hydrolyzates of silk fibroin. Enough has been said in the foregoing to justify the importance attached to peptide synthesis as a tool in the study of proteins. The purpose of this review is to survey the available methods for the synthesis of peptides. These methods will be evaluated, whenever possible, with regard to their relative difficulty, their adaptability to meet the many problems encountered in amino acid chemistry, and the yield and purity of the products of synthesis. Although the preparation of peptides containing nonprotein amino acids will occasionally be men-

4

JOSEPH 8. FRUTON

tioned, primary emphasis will be placed on the synthesis of peptides of amino acids definitely known to be formed upon protein hydrolysis. In what follows, the configuration of the amino acids will be given in accordance with the report of the Editorial Board of the Journal of Biological Chemielty (284). T h e amino acid residue6 will, in general, be designated by adding the suffix “yl” to the roots of the names of the free amino acids. “hue, for glycine, the term will be glycyl; for proline, prolyl; for tyrosine, tyrosyl; and 80 forth. Several departures from this rule will be noted, however. The peptides of cysteine will not be designated (‘cysteyl” but “cysteinyl” peptides, and for the cystine peptidee, the designation will be “cy& tinyl” rather than “cyetyl.” In the caw of peptides of glutamine, the amino acid reaidue will be termed “glutaminyl” to differentiate it from “glutamyl,” which refers to glutamic acid. Similarly, for asparagine peptides, the term will be “asparagiuyl” and, for aapartic acid peptides, it will be “mpartyl.” Jn addition, the term “tryptophyl” will be used to deeignate the amino acid residue of tryptophan. Since thia nomenclature impliee that an amino acid has been converted to an acyl group, it follows that the amino acid residues in a polypeptide should be listed in the sequence of substitution at the amino group of the adjacent amino acid. The tripeptide glycyl-calanyl-cleucine has, therefore, the following formula:

CHa NHrCH,CO---NdHCO--NH

tH9 HCOOH

A brief discussion of the configurational relationships of peptides appears necessary at this point since frequent mention will be found in the literature of racemic peptides which contain more than one optically active amino acid. It is clear that, in the case of a racemic dipeptide containing two optically active amino acids, i e . , Dcleucyl-Dcalanine, four isomers are possible: (a) D-leucyl-Palanine, (b) cleucyl-L-alanine, (c) cleucyl-D-alanine, and (d) mleucyl-calanine. Two racemates may be expected; one composed of forms a and b, and another composed of forms c and d. Separation of the two racemates may sometimes be achieved by taking advantage of their differences in solubility. It is then customary to designate the less soluble form by the letter “A” and the more soluble racemate by the letter “B.” Reports will be encountered in the literature of the synthesis of peptides containing more than one optically active amino acid and in which one amino acid residue is present in a single configuration, while another is given as the DL form, e.g., DL-alanylglycyl-L-glutamic acid. Such preparations are clearly mixtures of diastereoisomers. When attempts to separate the two forms are not successful, three possibilities may be envisaged: (a) the two isomers have very similar solubilities, thus making their separation by fractional crystallization difficult; (6)

5

SYNTHESIS O F PEPTIDES

they are isomorphic and form mixed crystals; or (c) they form addition compounds in stoichiometric proportions. Since i t cannot be predicted beforehand whether, after the synthesis of a mixture of such diastereoisomers, a pure peptide can be isolated, it appears preferable, whenever possible, to perform peptide syntheses with optically active amino acids under conditions where racemization is avoided. Furthermore, in the study of the specificity of proteolytic enzymes or of the properties of model compounds related t o proteins, it is usually desirable to have peptides containing optically active, rather than the racemic, forms of the amino acids. Greater interest attaches itself, therefore, to methods which permit the synthesis of such optically active peptides.

11. GENERALMETHODSOF PEPTIDE SYNTEIESIS 1. Synthesis of Dipeptides by Partial Hydrolysis of Diketopiperan'nes

In 1888 Abenius and Widmark (38) found that ditolyldiketopiperazine could be partially cleaved by acid hydrolysis : /CH1-Co CHaCsHdN

\

CO-CHI

\

/

CsH4CHa

I

NCsHdCHr -+ CH,C~HINHCH~CO-NCHICOOH

An analogous reaction of aliphatic compounds was not observed until 1901, when Fischer and Fourneau (149) hydrolyzed glycine anhydride by heating it briefly with concentrated hydrochloric acid and thus obtained glycylglycine, the simplest representative of the group of substances under discussion in this review. The method used by Fischer and Fourneau still is a convenient method for making this dipeptide, especially in view of the recent development of an excellent procedure for the synthesis of glycine anhydride from glycine (261). Fischer noted, however, that brief acid hydrolysis of DL-alanine anhydride and of obleucine anhydride did not yield the expected dipeptides (125). Fischer later found that brief hydrolysis with dilute sodium hydroxide a t room temperature would also yield dipeptides from diketopiperazines (132). In addition to glycylglycine, one of the two possible racemic forms of Dtalanyl-DL-alanine was prepared in this way. It soon became clear, however, that the method was not generally applicable to the synthesis of dipeptides containing optically active amino acids, for, when Fischer attempted to prepare L-alanyl-L-alanine from the corresponding anhydride, the product which resulted was partially racemic (153). Similarly, treatment of L-tyrosine anhydride with alkali caused appreciable racemiaation (166). As was shown by the later systematic studies of Levene (237) and Bergmann (82), among others, the racemization of optically

0

JOSEPH 8. FRUTON

active diketopiperazines is favored by alkali. I t is obvious, therefore, that this procedure is essentially limited to work with glycine anhydride. In fact, it is frequently convenient to treat glycine anhydride with sodium hydroxide and to use the resulting solution of the sodium salt of glycylglycine for condensation reactions involving the amino group of the dipeptide. The racemization of diketopiperazines, so pronounced in alkaline media, is notably less in acid. In some cases, it is possible, therefore, to achieve a satisfactory synthesis of optically active dipeptides by cautious treatment with hydrochloric acid. Thus, Greenstein (195) was able to make tcysteinyl-L-cysteine from L-cysteine anhydride by hydrolysis of the latter with concentrated hydrochloric acid a t room temperature. It must be added, however, that some diketopiperazines, such as histidine anhydride and tyrosine anhydride, are fairly'stable when they are heated with strong acid, while alanine anhydride and leucine anhydride require prolonged treatment with hot concentrated acid to effect cleavage of the ring. To the difficulties encountered in the partial cleavage of diketopiperazines containing like amino acid residues, must be added those met in the splitting of diketopiperazines derived from two different amino acids. In the latter case, two different dipeptides may result, as in the hydrolysis of glycyl-DL-leucine diketopiperazine, which yielded a mixture of glycyl-DL-leucine and DL-leucylglycine (166). The separation of such mixtures is usually difficult. If, however, the formation of only one of the two possible dipeptides is favored, this difficulty may be eluded. One of several instances in which a mixed dipeptide could be prepared in pure form by hydrolysis of a diketopiperazine was described by Bergmann and Tietzman (68), who obtained L-prolyl-L-phenylalaninefrom L-prolyltphenylalanine diketopiperazine. The dipeptide had the same rotation as that of the product obtained by Fischer and Luniak (159), who coupled L-prolyl chloride with L-phenylalanine ethyl ester, and then saponified the dipeptide ester. As the first available method for the synthesis of free peptides, the procedure discussed in this section has considerable historical interest. It is clear from the foregoing, however, that it has many limitations. Despite the occasional successes of this method, and its value as an adjunct to other synthetic procedures, it may be expected that, even for the synthesis of dipeptides, preference will be given to other methods. 2 . Condensation of Peptide Esters Curtius noted in 1883 (105) that glycine ethyl ester undergoes spontaneous transformation to yield glycine anhydride and a substance

7

SYNTHESIS OF PEPTIDES

which gives a positive biuret test. For this reason, the latter product was termed “biuret base.” Later studies by Curtius (107) showed that, if moisture is excluded, the formation of the anhydride is suppressed and the “ biuret base,” which he formulated as (triglycy1)glycine ethyl ester, is the chief product. In a similar manner, Fischer (134) converted (diglycy1)glycine methyl ester to (pentaglycy1)glycine methyl ester by heating the former at 100”. 2 NH&H&O-NHCH&O-NHCH&OOCH:

+

NH&H&O-(NHCHrCO)r-NHCH&OOCH~

+ CHtOH

More recently Pacsu and Wilson (254) and Frankel and Katchalski (176) have shown that, under suitable conditions, long-chain polycondensation products may be obtained from amino acid and peptide esters. The materials obtained represented mixtures of homologous peptide esters containing 20 to 100 amino acid units. It may be noted a t this point that, in general, amino acid esters and dipeptide esters readily yield diketopiperazines rather than polycondensation products. In fact, when a diketopiperazine composed of like amino acid residues is desired, it is most convenient to prepare it from the corresponding amino acid ester with ammonia in alcohol. In this manner, there have been synthesized a variety of diketopiperazines, such as histidine anhydride, lysine anhydride, and serine anhydride (172). Proline methyl ester, in particular, cyclizes with great ease (223). For the preparation of diketopiperazines derived from two different amino acids, treatment of the dipeptide ester with ammonia in alcohol usually leads to the desired product. Examples of this procedure are, among others, the synthesis of kleucyl-L-alanine diketopiperazine from L-leucylL-alanine methyl ester (136) and glycyl-L-valine diketopiperazine from glycyl-L-valine methyl ester (164). A modification of this method, employing a lithium hydroxide solution saturated with carbon dioxide, was useful for the preparation of L-glutamylglycine diketopiperazine from a-L-glutamylglycine ethyl ester (78). Fischer’s first attempts to develop a general method of peptide synthesis led him to prepare the carbethoxy derivative of glycylglycine ethyl ester by treatment of the dipeptide ester with ethyl chlorocarbonate (149). C rH ,OCOCl

+ NH 2CH $30-N

HCH zCOOC zH i -+ C~HsOCO-NHCH&O-NHCH&OOCrH‘

It was his hope, in this way, to introduce a substituent which would protect the reactive amino group from further attack in the course of condensation reactions and which could also be removed without hydrolysis

8

JOSEPH 6. FRUTON

of the peptide bonds of the synthetic product. When the carbethoxy peptide noted above was heated for 36 hours with m-leucine ethyl ester, ethyl alcohol was eliminated, and the resulting product was carbethoxyglycylglycylleucine ethyl ester (124). c4H1 I

CIHrOCO-NHCHICO-NHCH

lCOOCIH6

+ NHdHCOOC aH

L ---t

C4H9

I

C~H~OCO-NHCH~CO-NHCH~CO-NHC?HCOOC~H,

Fischer soon realized, however, that such condensation reactions are increasingly difficult as the peptide chain is lengthened and that even in the preparation of smaller peptides the yields are low. For this reason he turned his efforts t o the development of other methods of peptide synthesis. Of some interest in this connection is the behavior of carbethoxyglycylglycine ethyl ester on prolonged hydrolysis with alkali. Fischer (124) obtained a product which he formulated as glycylglycine carbamino acid : HOOC-NHCHaCO-NHCH&OOH

I t appeared, therefore, that the carbethoxy group could be removed from the acylated peptide ester without cleavage of the peptide bond. However, the complex nature of the reaction led Fischer t o abandon further study of its mechanism. It remained for Wessely (296) t o show that the product obtained by Fischer was actually carbonyl-bis-glycine, and the following sequence of reactions was suggested t o explain its formation:

It may be added that, in the same paper in which he described the hydrolysis of the carbethoxyglycylglycine ethyl ester, Fischer also reported the synthesis of carbonyl-bis-glycylglycine by the treatment of the dipeptide ester with phosgene (in toluene), followed by saponification : C1

/ co \

NH&H&O-NHCHiCOOCiHr

+ Cl

/

4

NH2CH1CO-NHCHaCOOCzHr

NHCH1CO-NHCHZCOOC~HK

co

‘NHCHICO--NHCH1COOC2Hr

9

SYNTHESIB OF PEPTIDES

Such compounds may also be prepared by the reaction of the sodium salts of amino acids or peptides with phosgene.

3 . Synthesis of Peptide Derivatives by Means of Acylamino Acid Chlorides and Azides While working on the synthesis of hippuric acid, Curtius found, in 1881 (104), that one of the products of the interaction of benzoyl chloride and glycine silver was the substance benzoylglycylglycine. As pointed out by Fischer (129) in the course of a polemic with Curtius, this reaction is rather complex in nature, and, although it may be considered to represent the first recorded synthesis of a well-defined peptide derivative, the method is not suitable for general application. Curtius’ studies, during the period 1890-1900, on the reactions of hydrazides and azides, led him to use these in the synthesis of peptide derivatives. After the report by Fischer and Fourneau (149) that dipeptides could be made by the partial hydrolysis of diketopiperazines, Curtius (106) described the use of azides of benzoylamino acids or peptides according t o the following reaction, illustrated for the case of the synthesis of benzoylglycylglycylglycine: C,HrCO-NHCH*CON,

+ NHsCHrCO-NHCHtCOOH

+

+ HNr

CoH‘CO-NHCH&O-NHCH&O-NHCH~COOH

In order to obtain the aside, the corresponding ester was treated with hydrazine hydrate, thus yielding a hydrazide, which was in turn converted to the azide by means of nitrous acid:

R I

C~H~CO-NH~HCON,

These reactions proceeded smoothly and, frequently, with excellent yield. Curtius and Levy (111) were able to make benzoyl(tetraglycy1)glycine ethyl ester by the condensation of benzoyl(diglycy1)glycine azide with glycylglycine ethyl ester, and with his collaborators, Curtius extended this method to the synthesis of benzoylated peptides containing alanine (110), aspartic acid (108), and aminobutyric acid (109). As was noted in the previous section of this review, Fischer’s first attempts to develop a general method of peptide synthesis led him to prepare the carbethoxy derivative of glycylglycine ethyl ester (144). When he conoluded that condensation of such esters with esters of amino acids was not feasible as a general procedure, he decided to convert the carbethoxyamino acids to the corresponding acid chlorides by means of

10

JOSEPH 8. FRUTON

thionyl chloride (127), a reaction which had been found by Meyer (246) to be suitable for the preparation of the acid chloride of pyridine carboxylic acid :

By warming carbethoxyglycine or carbethoxyglycylglycine with thionyl chloride at 35-10', noncrystalline products were obtained which were used directly for coupling in ethereal or in chloroform solution with amino acid or peptide esters. From the resulting carbethoxy peptide esters, the corresponding acids could be prepared by saponification. In several cases, these acids could be converted to acid chlorides with thionyl chloride, and the peptide chain lengthened by coupling with amino acid or peptide esters. In principle, the above methods of Curtius and Fischer provide the basis for the further development of the techniques of peptide synthesis. All the subsequent procedures for lengthening the peptide chain have involved the conversion of the carboxyl group of an amino acid into forms which permit reaction with the amino group of another amino acid. Of the various derivatives of carboxylic acids which have proved useful for this purpose, the aeides and chlorides have been of the most general value. Indeed, in many cases, the older azide method of Curtius is preferable to the chloride method, particularly in coupling reactions involving acyl peptides (cf. page 26). Although Fischer showed in 1905 (132) that it was possible to convert free amino acids to acid chlorides, it was realized that, in order to permit smooth coupling reactions, the amino group had to be blocked by acylation, or otherwise modified to avoid complicated side reactions in the course of the conversion of the carboxyl group to an acid chloride. In addition to the carbethoxy and beneoyl groups mentioned above, a variety of acyl substituents were introduced into peptide chemistry. In addition to others to be discussed later may be mentioned the naphthalenesulfonyl (144), phenylureido (170), benaenesulfonyl (145), and methanesulfonyl (207) groups. However, as long as it was necessary to remove an acyl substituent by hydrolysis, i t could not be used in the synthesis of free peptides, since attempts to eliminate the acyl group in thismanner invariably led to either partial or complete cleavage of the linkages between the amino acids. The essential problem of peptide synthesis thus became the development of methods which would obviate the necessity for the hydrolytic removal of an acyl substituent a t the end of a series of coupling reactions.


11

It should be added, however, that the methods diecussed in this section have been of considerable value in the preparation of benzoyl peptides, as well as amides of benzoylamino acids and benroyl peptides, which were required for studies of the specificity of proteolytic enzymes 4. Use of Amino Acid Chlorides in Peptide Syntheds

As noted earlier, Fischer, in 1905 (132), described a method of peptide synthesis which obviated the necessity for blocking the amino group of an amino acid prior to the conversion of the carboxyl group to an acid chloride. This method takes advantage of the fact that several amino acids, when shaken with phosphorus pentachloride and acetyl chloride, are readily converted into crystalline hydrochlorides of the amino acid chlorides. In this manner, Fischer prepared the acid chlorides of Dcleucine, Lalanine, DLphenylalanine, and cproline. Peptide synthesis was effected, in the case of balanylglycine (133), by the addition of calanyl chloride hydrochloride to a solution of glycine ethyl ester in dry chloroform, neutralization of the hydrochloric acid by the addition of sodium methylate, and the saponification of the dipeptide ester with alkali. The yields obtained by this procedure are not too satisfactory, since, as can readily be seen, one of the possible side reactions is the forester. Only isolated mation of a diketopiperazine from the -tide instances of its application can therefore be cited. Fischer and Luniak (159) used cprolyl chloride to make cprolyl-cphenylalanine and Abderhalden and Kempe (19) made ctryptophylglycine by means of Ltryptophyl chloride. Havestadt and Fricke (206) have reported the synthesis of Dcalanyl-thistidine through the coupling of Dcalanyl chloride with Ghistidine methyl ester but, as pointed out by Hunt and du Vigneaud (220), the identity of their product is open to some doubt. It is clear, therefore, that, except for a few cases, the amino acid chlorides are not suitable reagents in peptide synthesis. 5. Use of u-Halogen A w l Halides i n Peptide Synthesis

The greatest of Fischer’s many important contributions to protein chemistry may be said to have been the invention of the first of several methods now available to circumvent the difficulty encountered in the removal, by hydrolysis, of an acyl substituent of a peptide without cleavage of the peptide itself. The first report of this method came in 1903, when Fischer and Otto (160) described the synthesis of glycylglycylglycine. Chloroacetyl chloride and glycylglycine ethyl ester were coupled, and the product was saponified to yield chloroacetylglycylglycine. The next step was the introduction of the free amino group

12

JOSEPH 8. FRUTON

of the peptide by treatment of the chloroacetyl dipeptide with 25% ammonia at 100':

+ NHaCHaCO-NHCHsCOOCsHa -+ ClCHaCO-NHCHaCO-NHCHaCOOCsHr + HCl ClCHaCO-NHCHsCO-NHCHaCOOH + NH, ClCHaCOCl

NHaCH&O-NHCHrCO-NHCHaCOOH

It was later noted that the amination could be carried out more effectively by allowing the reaction mixture to stand at room temperature for two to three days. This ingenious method was applied in a similar manner to the condensation of a-bromopropionyl bromide with glycylglycine ethyl ester, thus leading to the synthesis of Dbalanylglycylglycine (128). In rapid succession, there followed a memorable series of papers which described the extension of this method to the synthesis of peptides of Dbleucine (132),Drcproline (169), Dbphenylahine (130), and other amino acids. In Table I are listed the various halogen acyl halides used in connection with these syntheses. TABLE I Halogen Acyl Halides Used in Peptide Sytthcsia

Amino acid residue

Halogen acyl halide

Glycyl . . . . . . . . . . . . . . . . . Chloroacetyl chloride Bromoacetyl bromide Alanyl. . . . . . . . . . . . . . . . . a-Bromopropionyl bromide (or ohloride) Valyl . . . . . . . . . . . . . . . . . . a-Bromoiaovaleryl chloride Leucyl . . . . . . . . . . . . . . . . . a-Bromoiaocaproyl ohloride Isoleucyl . . . . . . . . . . . . . . . a-Bromo-&methyl-mthylpropionyl ohloride Phenylalanyl . . . . . . . . . . . . a-Bromo-pphenylpropionyl chloride Prolyl . . . . . . . . . . . . . . . . . . a,6-Dibromovaleryl chloride

One of the principal difficulties of this method arose from the fact that those members of the group of a-halogen acyl halides in which the acyl group waa propionyl or larger could occur in at least two stereoisomeric forms (135). Thus, if the racemate were to be used in a reaction with an optically active amino acid or peptide, there would be produced a mixture of diastereoisomera. Except in rare instances, where the differences in the rates of reaction for the two isomeric acid halides are considerable, approximately equal amounts of the diastereoisomers would result, thus making the separation by fractional crystallization a laborious operation of dubious outcome. Accordingly, Fischer set about to prepare halogen acyl halides from optically active a-halogen acids. With Warburg (131) he prepared the optically active 2-bromopropionyl

13


chloride by treatment of levorotatory a-bromopropionic acid with thionyl chloride. This acid was obtained in two ways: either by resolution of the synthetic dl acid, or by treatment of talanine with nitrosyl bromide according to the method of Walden (293). Fischer assumed that a change of configuration (“ Walden inversion”) had taken place in the course of the latter reaction. Subsequent work (180) showed, however, that the 1-bromopropionic acid obtained from Galanine retains the configuration of the amino acid. The coupling of the l-bromopropionyl chloride with glycine ethyl ester, followed by saponification of the coupling product, gave a substance which was then subjected to amination to yield the peptide now recognized to be D-alanylglycine. Actually Walden inversion had occurred during the amination of the bromopropionylglycine rather than during the synthesis of the bromo acid, as Fischer had assumed (136, 252a). Despite this error, Fischer was, in general, correct in the conclusion that, in order to synthesize peptides containing amino acids of the L series (“natural” amino acids) it wm necessary, in the halogen acyl halide method, to prepare halogen acids from amino acids of the D-series (“unnatural” amino acids). These pamino acids could be obtained only by the resolution of synthetic racemic products, and Fischer and his collaborators devised a number of excellent, albeit time-consuming, procedures for achieving this end. The synthesis of D-alanylglycine from Galanine and glycine ethyl ester may be summarized in the following scheme: CHa NHIAHCOOH L-Alanine

+NOBr

CH8 BrhHCOCI

+ NH&H&OOC:H,

8“’

CH,

+sOCli

Br AHCOOH Z-Bromopropionic acid

Br HCO-NHCHgCOOH I-Bmmopropionylglycme

XH’ - 1”’ + Br

NHI

CHa

BrbHCOCI

HCO-NHCHICOOCXH,

NsOH

NHx HCO-NHCHXCOOH D- AhIlylgly Che

With the aid of several optically active a-halogen acyl halides, Fischer, and later Abderhalden, succeeded in synthesizing an impressive aeries of peptides,. in which all the components were either glycine or higher amino acids of the L or D series. The most notable achievements in this regard were the synthesis of the octadecapeptide bleucyl(triglycyl)-~-leucyl(triglycyl)-~-leucyl(octaglycyl)glycine which Fischer made in 1907 (138) and of a nonadecapeptide prepared by Abderhalden and Fodor in 1916 (10). I n the preparation of long peptides of this type, special care had to be taken in the conversion of halogen acyl peptides to

14

JOBEPH 8. FRUTON

the corresponding chlorides. Fischer effected this reaction by means of phosphorus pentachloride and acetyl chloride, as in the case of a-d-bromoisocapronylglycylglycylglycine. The resulting chloride could then be coupled with (pentaglycj 1)glycine in alkaline solution. The bromoisocapronylpeptide was then aminated with liquid ammonia, since aqueous ammonia was not effective, and the decapeptide was coupled with a-d-bromoisocapronylglycylglycylglycylchloride. Needless to say, as the chain length increased, the experimental difficulties became more serious. Furthermore, by coupling optically active halogen acyl halides with amino acids such as cphenylalanine, Ltyrosine, L-histidine, L-cystine, etc., or peptides of the L series such as glycyl-Ltyrosine, a large variety of interesting products were obtained by Fischer and Abderhalden, among others, and used for the study of the specificity of proteolytic enzymes. Despite the ingenuity and wide applicability of this method, its many difficulties greatly limit its general application. In addition to the labor involved in the synthesis of optically active peptides, the method has disadvantages in several other respects. First, the reaction of N-(halogen acyl) hydroxyamino acids with phosphorus pentachloride is complicated and, even if the hydroxyl group is blocked, as in the case of 0-carbomethoxy-N-chloracetyl-ctyrosine,chlorination of the carboxyl group may result in complete racemization (141). Surprising deviations from the expected result were also observed in. the course of the amination of several halogen acylamino acids. Thus, when Fischer attempted to prepare L-leucyl-cproline he found ,that, on treatment of a-d-bromoisocapronyl-tproline with ammonia, there resulted, not the expected dipeptide, but rather the substance a-hydroxyisocapronyl-L-prolinamide (162) : CHI

-

CHI

‘ck

lNH4

COOH

\ck \CHI

CONHI

AH*

AH-CH,

CHa

bH-cHa

SHa Br HCO-N

:1. CHI-

b

HO HCO-N Ha

< I CHI-

Hi

Later, it was found that bromoisocapronyl-N-phenylglycineundergoes a similar reaction on treatment with ammonia (151): CHI

‘ck

CHI

CHa \CH/

CH,

15


Another instance of anomalous behavior on amination was noted in the case of a-bromo-8-phenylpropionylglycine,from which, on amination, hydrogen bromide is eliminated to yield a derivative of cinnamic acid (146):

-

C6Hr

C6Hr

INHI

AH*

&

Br HCO-NHCHZCOOH

AH ISHCO--NHCH,COOH

Finally, it must be mentioned that the optical purity of many of the peptides prepared by the halogen acyl halide method is open to doubt. While the values for the rotation of the peptides described by Fischer have, in most cases, been confirmed by the application of newer methods of peptide synthesis, the synthetic products described from other laboratories have frequently been found to be partially racemic. One of the many examples to illustrate this is the recent report of Schott et al. (267) that the optical rotation of Gleucylglycylglycine is actually 12 to 15’ higher than that reported by Abderhalden and Fodor (lo),who prepared this peptide by amination of the product obtained by the reaction of ad-bromoisocapronyl chloride with glycylglycine.

6. “ Azlactone ” Method for Synthesis of Peptides In the classical methods of Curtius and Fischer, discussed in preceeding sections of this review, individual amino acids or peptides were linked to one another by first converting the carboxyl group of one of the reactants to an acid chloride or to an azide, which would then react readily with the amino group of the other reactant. The work of Mohr and Strohschein (249),in 1909,showed that, in place of such acylamino acid chlorides or asides, it was possible to use, for such reactions, the azlactones (also termed oxazolones) of benzoylamino acids. In particular, they prepared benzoyl-Dcalanine azlactone by the reaction of benzoylalanine with acetic anhydride, and coupled the product with glycine ethyl ester: CH:

C6H,CO--NH~HCOOH

-

CHS-CH-CO

(CH aC0) 2

0

A\c/ d I

+ NHICHICOOCIH~

__

F

CsHsCO-NH

A-

HCO-NHCH&OOCZH~

The wider application of this method to the synthesis of peptides did not develop until 1926, when Bergmann and his collaborators reported

16

JOSEPH 8. FRTlTON

eleqant procedures for the synthesis of phenylalanyl and tyrosyl peptides (60,67,90). In their work, use was made of the azlactones of a-ace& aminocinnamic acid (described by Erlenmeyer and Frllstiick, 121) and of a-acetamino-p-coumaric acid (the corresponding benzoyl compound was described by Erlenmeyer and Halsey, 122). For example, it was possible to synthesize cphenylalanyl-cglutamic acid and Pphenylalanyl-cglutamic acid (67) by the following series of operations:

co

C,HbCH=C-

rs\,/b +

COOH

&Ha

NHJXCOOH

AHr

COOH

+

AHt

CoH,

AH,

AH

bHt

CHaCO-NH&CO-NH

I

Ha __*

CJ%

bHt

&HI

AH1

catslyat

A

c:

HCOOH

COOH

COOH

-

CHaCO-NHAHCO-NH HCOOH Acetyl-cphenylalanyl-cglutamio acid Acetyl-D-phenylalanyl-~glutamic acid

mild hydrol.

I

CaHr

bHt

AHa

AH,

NHl&HCO-NH HCOOH cPhenylalany1-cglutamic acid D-Phenylalanyl-cglutamicacid

With regard to the first step, it may be mentioned that the azlactone can be coupled either with the amino acid ester in an organic solvent or with the sodium salt of the amino acid in aqueous solution. The product of the reaction is an acetyldehydrophenylalanylamino acid (or its ester). In many cases, the coupling also proceeds in good yield with the sodium salt of the amino acid in a mixture of acetone and water. If acetyldehydrophenylalanylglycine is treated with benzaldehyde and acetic anhydride, it is converted to the acetyldehydrophenylalanyldehydrophenylalanine aslactone (51) : CaHr AH CH,CO-NH!!CO-NHCHaCOOH

+ CaHrCHO + (CHaC0)iO

+

CHaCO-NH

CaHr CaHr I I

r8" C=N

CO

The coupling of this aslactone with an amino acid permits one to lengthen the peptide chain and, in this manner, a notable variety of long-chain acetyldehydrophenylalanyl peptides have been prepared by Doherty et d.(115). I n these studies, aalactones were also made by the action of

17


acetic anhydride on acetyldehydrophenylalanylpeptides in which trana8-phenyl-Dcsenne was the terminal amino acid :

CHSCO-NH

CcHi

C~HI

AH

bHoH

II CO-NH

c:

-

C d h C&

(CHC0)sO

HCOOH

AH

b

CHaCO-NH C=N

AH

eCO

Ld

In passing, mention may be made of two aspects of the chemistry of the acyldehydroamino acids and peptides. The first of these concerns the conversion of acetyldehydrophenylalanylamino acids, by mild acid hydrolysis, to the corresponding phenylpyruvylamino acids (189) : CeH6 AH CH,CO--NHIICO--NH

x

CeH, AH,

H c o o H -, bCO--NH

x

HCOOH

The second point of interest lies in the use of the chloroacetyl group as an acyl substituent in dehydroamino acids and peptides. Thus, from the azlactone of chloroacetyldehydrophenylalanine (prepared from chloroacetylphenylserine, 7), there may be obtained, on hydrolysis, an acylamino acid which, on amination by the method of Fischer, yields glycyldehydrophenylalanine (65). The last-named substance hasproved to be a substrate for the enzyme dehydropeptidase, present in animal tissues, which is specifically adapted to the hydrolysis of dehydropeptides (64,304). If, instead of hydrolyzing the chloroacetylazlactone, it is coupled with an amino acid such as eglutamic acid, tripeptides such as glycyldehydrophenylalanyl-Lglutamicacid may be prepared (65). Returning now to the reaction scheme for the synthesis of phenylalanylglutamic acid by the azlactone method, the acetyl dehydropeptide must next be converted to a saturated compound. This is readily effected by catalytic hydrogenation at low pressure with palladium black (298) or other related catalysts. In the case of acetyldehydrophenylalanyl-cglutamic acid, two diastereoisomers are formed. Under favorable circumstances, as in the example under discussion, the two isomers may be separated by fractional crystallization. Sometimes, however, such ready separation cannot be achieved. Thus, the diastereoisomeric beneoyl-D- and cphenylalanyl-carginines appear to have considerable affinity for one another, and all attempts to effect a resolution have hitherto been unsuccessful (186). This factor is a severe drawback of the azlactone method if optically active peptides or peptide derivatives are desired. The final step in the synthesis of phenylalanylglutamic acid, the

18

JOSEPH 8. FRUTON

removal of the acetyl group, is effected by hydrolysis with dilute acid. This operation entails considerable loss, however, owing to the formation of the diketopiperazine. Although the application of the azlactone method to peptide synthesis has involved primarily the use of the well-crystallized azlactones of acylaminocinnamic acid derivatives, the use of azlactones of other amino acids for this purpose has also been described. As noted earlier, Mohr and Strohschein (249) used the azlactones of benzoylamino acids, but it must be added that, in general, the azlactones of saturated acylamino acids are difficult to purify (236). Additional examples may be found in the work of Carter el al. (96), who prepared benzoylaminocrotonic acid azlactone by the treatment of benzoyl-Dballothreonine with benzoyl chloride and pyridine, or with two equivalents of acetic anhydride (98). This reaction is an extension to the aliphatic hydroxyamino acids of the reaction mentioned earlier for phenylserine. Another azlactone of an aliphatic acyldehydroamino acid is that of acetyldehydroleucine, which was prepared by Doherty et al. (115) from chloroacetyl-c leucine by treatment with acetic anhydride: CH,

CH,

CHI

‘Ck

CHa

‘Ck +

AH

CHIC=N/!CO

The synthesis of several acyldehydroamino acids from the corresponding halogen acylamino acids was described by Bergmann and coworkers in 1926 (58,66). Treatment of the halogen acids with acetic anhydride yielded azlactones from which there could be prepared, by hydrolysis, acetyldehydrophenylalanine,acetyldehydrotyrosine, and propionyldehydroaspartic acid. An excellent review of the chemistry of the azlactones has been provided recently by Carter (95), who has also made important contributions to the study of the stereochemistry of these substances (97).* Recently, there has been described (103s) the synthesis of an extensive series of thiazolones, the sulfur analogues of the azlactones, and it

* For a report of the extensive work on adactones studied in connection with the investigation of the chemistry of penicillin, see the chapter by J. W. Cornforth in ( l O a ) . Among the many important findings mentioned is the demonstration that some of the compounds previously thought to be a-acylamino acyl halides (e.g., hippuryl chloride) are actually hydrohalides of azlactones.

19


may be expected that these will prove to be valuable new reagents in peptide synthesis.

7. Condensation of Keto Acids and Amides Reactions that involve the condensation of keto acids with amides are of interest in peptide synthesis, not primarily because of their preparative value, but because of their possible relationship to the biological synthesis of peptide bonds (54). Bergmann and Grafe showed in 1930 (56) that pyruvic acid and acetamide readily react to form u,a-diacetaminopropionic acid, which, on treatment with acetic acid, is converted to a-acetaminoacrylic acid (acetyldehydroalanine). Hydrogenation of the acetyldehydroamino acid with palladium black as the catalyst gives acetyl-m-alanine.

CHaCOCOOH

+ 2 ClFaCONH*+ CHIlHCOCHa COOH

-

hCOCHI CHa CHICO-NHECOOH

2

It will be recalled that acyldehydroamino acids have also been prepared by the treatment of halogen acylamino acids with acetic anhydride (66). If, instead of acetamide, chloroacetamide is used for the condensation with pyruvic acid, a-chloroacetaminoacrylic acid is obtained, * and a subsequent amination leads to the synthesis of glycyldehydroalanine, which, on hydrogenation, gives glycyl-Dcalanine. Glycyldehydroalanine, like glycyldehydrophenylalanine, is hydrolyzed by the enzyme dehydropeptidase (64,198) : R

R AH NH&HaCO-NHbCOOH

-+

NHaCH,COOH

+ NHa +

A representative of an interesting group of substances was obtained when a,a-diacetaminopropionic acid was converted to an azlactone by treatment with acetic anhydride: A more satisfactory method for the synthesis of chloroacetyldehydroalanine has been described recently by Price and Greenstein (255b). The procedure involves the reaction of chloroacetonitrile with pyruvic acid in the presence of dry hydrogen chloride.

20

JOBEPH 8. FRUTON

This azlactone reacts rapidly with amino acids (e.g., glycine, alanine, phenylalanine) in alkaline solution to give diacetaminopropionylamino acids, which, upon mild hydrolysis, give pyruvylamino acids (57,271) : NHCOCH, R CHJCO-NH

I NHCOCHz

A

HCOOH + CHaCOCO-NH

1

HCOOH

Mention was made earlier in this review of the synthesis of phenylpyruvylamino acids by the hydrolysis of acetyldehydrophenylalanylamino acids (189). Shemin and Herbst (271) have shown that the oximes of the pyruvylamino acids may be reduced with Adams' platinum oxide catalyst to yield dipeptides, in analogy to the synthesis of amino acids from the oximes of keto acids (205) : RCH,LO-NH

r

NHz

HCOOH

2RCHAHCO-NH

x

HCOOH

In recent years, much interest has been evinced in the possibility that ketoacylamino acids (e.g., pyruvylalanine) might serve as acceptors of amino groups in enzyme-catalyzed transamination reactions similar to those discovered by Braunstein and Kritzman (93,209) in the case of pyruvic and a-ketoglutaric acids. Although this possibility has not received experimental support thus far, it should be mentioned that Herbst and Shemin (211) have shown that a transamination reaction can be effected by heating pyruvyl-DL-alanine with a-aminophenylacetic acid. The products of the reaction are both racemic Dtalanyl-DLalanines, benzaldehyde, and carbon dioxide. The last two products arise from the decomposition of phenylglyoxylic acid : CH,COCO-NH

XH'

HCOOH

NHa

+ CaH,AHCOOH --+

bH' X"'

+

CHI HCO-NH HCOOH CaHrCOCOOH -+ CaH'CHO + CO,

This reaction is analogous to the transamination between keto acids and aromatic amino acids studied by Herbst and Engel (208,210).

21


8. Use of N-Carbonic Acid Anhydrides of Amino Acids

It will be recalled that Fischer and Otto prepared several carbethoxyamino acids, which could be converted to acid chlorides by treatment with thionyl chloride (160). Coupling of the carbethoxyamino acid chlorides with esters of amino acids or peptides gave carbethoxy peptide esters (cf. page 10). Leuchs and collaborators (228-230) continued the study of the carbethoxyamino acid chlorides, and showed that these substances readily form N-carbonic acid anhydrides with the elimination of ethyl chloride. Thus, carbethoxyglycyl chloride gave rise to glycine N-carbonic acid anhydride: C*HrOCO-NHCH&OCI

+ CO-NHCHrCO

L

+ CIH~CI

A

In the presence of water, this anhydride, and those of other amino acids (112), rapidly opened with the elimination of carbon dioxide and the regeneration of the amino acid. In concentrated solution, the evolution of carbon dioxide was accompanied by the deposition of an insoluble polymer. Leuchs showed that this polycondensation could also be effected by warming a solution of an N-carbonic acid anhydride in organic solvents containing small amounts of water. On treatment with hydrogen chloride in alcohol, the carbonic acid anhydrides were converted into the hydrochlorides of the corresponding amino acid esters. These findings were confirmed and extended by Curtius (112), Fuchs (192), Wessely (295), and Woodward and Schramm (301), among others. The formation of the anhydrides has been effected with several carboalkyloxy derivatives of amino acids. Although the carbomethoxy derivatives (prepared with methyl chlorocarbonate) are especially suitable for this purpose, carbethoxy (from ethyl chlorocarbonate) and carbobenzoxy (from benzyl chlorocarbonate, 74) derivatives are converted readily into N-carbonic acid anhydrides. The N-carbonic acid anhydrides react not only with water and alcohol, as noted above, but also with amines and, in particular, the amino groups of amino acids and peptides. The formation, in moist organic solvents, of polymeric products is believed to be the result of a chain reaction in which a molecule of anhydride is opened by water and the amino group thus liberated is made available for reaction with another molecule of the anhydride. In this manner, there is formed a dipeptide which can react with a third molecule of anhydride to give a tripeptide, and so forth:

22

JOSEPH 8. FRUTON

R qO-NH

Lo-l

c:

HFO

+

+ COl

4

R +I8

L

O

I

1

(A)

(A) R

R

CO--NHbHCO P

NHICHCO-(NH

R

HCO).-NH

I:

HCOOH

+ n CO,

Polymers of glycine and sarcosine have been prepared by Wessely (297) and, more recently, Frankel and Katchalski (176a) have obtained polymers of t-carbobenzoxylysine (cf. page 59), which, upon hydrogenation (223a), gave polymers of lysine. In addition, Woodward and Bchramm (301) have made polymers of phenylalanine or leucine, as well as products arising from the interaction of the N-carbonic acid anhydrides of both these amino acids. In the presence of a n excess of an amino acid (sodium salt) in aqueous solution (273) (or an amino acid ester in a n organic solvent, 220), the N-carbonic acid anhydride will react to give a moderate yield of the expected dipeptide (or dipeptide ester). Sigmund and Wessely (273) made DL-phenylalanylglycinein this manner: CIH,

CSI

~ H Z

~ H s

CO-NH

HCO

+ NHiCHzCOOH

4

L

NH, HCO-NHCH1COOH

L 3 - J

With glycylglycine, the tripeptide DL-phenylalanylglycylglycine was obtained. * It will be clear from the foregoing, however, that, in the case of a reaction between a N-carbonic acid anhydride and an amino acid or peptide, there is considerable possibility of side reactions involving polymerization or hydrolysis. It may be questioned, therefore, whether it will be feasible to apply this method generally to the synthesis of peptides of precisely known structure, since the separation of the desired peptide from the polymeric product and the amino acid formed on hydrolysis may be attended with some difficulty. The usefulness of the N-carbonic acid anhydrides in several special aspects of peptide synthesis should be emphasized, however. For example, they have proved to be It has recently been reported (J. L. Bailey, International Biochemical Congress, Cambridge, 1949) that N-carbonic acid anhydrides may be used successfully for the synthesis of a variety of peptides by allowing an anhydride to react either with two equivalents of amino acid ester or with one equivalent of amino acid ester plus a tertiary base. At the present writing, details of this important extension of the Weeeely method are not available.

23


of considerable value in the synthesis of iysine and ornithine peptides (cf. page 59).

9. Uae of ToluenesuljonylaminoAcids in Peptide Synthesis It was noted earlier in this review that, prior to the introduction of the halogen acyl halides into peptide chemistry, Fischer had attempted to use acylamino acid chlorides for peptide synthesis in the hope that an acyl substituent might be found which could be selectively removed by mild hydrolysis. The carbethoxy group was found to be unsuitable for this purpose, and although the later work of Bergmann et al. (67) showed that some acetyl dipeptides could be deacetylated with dilute acid, other studies emphasized the fact that the selective hydrolysis of even the acetyl group could not be relied upon in all cases. There was need, therefore, for methods in which acyl substituents could be removed from peptides by procedures not involving hydrolysis with strong acids. * The first such method was proposed by Schoenheimer (266), who took advantage of the discovery of Fischer (142) that p-toluenesulfonylamino acids could be converted to the parent amino acid by reduction with a mixture of hydriodic acid and phosphonium iodide: R

c:

CHJCOHSOZ-NH HCOOH

HI

PHd

R

+

I

CHJCOH&~HNH&HCOOH

Schoenheimer showed that, under the conditions of this reduction, the hydriodic acid does not hydrolyze peptide linkages to an appreciable extent. By employing the azides of toluenesulfonylamino acids, he was able to synthesize several peptides by the following series of reactions, illustrated for the case of glycyl-Dbalanine: CH~C*H~SO-NHCH&ONJ

+ NHz

iH*-

CHJC~H~SOZ-NHCH&O-NH HCOOH

HI

PHJ

NHzCH*CO-NH

IHa

HCOOH

As was shown by du Vigneaud and Behrens (287), the toluenesulfonyl group may be removed from an amino acid derivative by reduction with sodium in liquid ammonia. Recently, Ehrensvlird (1 18s) has described the use of phenylthiocarbonyl chloride as a reagent for the protection of amino groups in peptide synthesis. The phenylthiocarbonyl group is stable to acids, but may readily be split off with dilute alkali in the preeence of lead hydroxide or lead carbonate. At the present writing, only a preliminary account of this method is available.

24

JOSIPPH 8. FEUTON

I n addition to the Curtius aeide method, the Fischer method for the conversion of acylamino acids to the corresponding acid chlorides (with thionyl chloride or phosphorus pentachloride) also serves satisfactorily for the toluenesulfonylamino acids and obviates the necessity for the preparation of the ester and the hydradde prior to the coupling. As noted earlier in the case of other acyl peptides, however, when toluenesulfonyl peptides are to be used for coupling reactions to lengthen the peptide chain, the azide method may be expected to give better yields. It is regrettable that insufficient information is available in the literature regarding the applicability of the Schoenheimer method to the synthesis of peptides containing some of the more complex amino acids. Woolley (303) has recently reported the synthesis of a mixture of the diastereoisomeric D and cserylglycyl-cglutamic acids by the use of this method. 10. Use of Azidoacyl Halides in Peptide Synthesis

An interesting modification of the halogen acyl halide method for peptide synthesis was developed by Bertho and Maier (91) and by Freudenberg, Eichel, and Leutert (179), who used a-azidoacyl halides for coupling reactions with amino acids. It had been shown by Forster and Fierz (175) that the reduction of a-azido acids with aluminum amalgam led to the formation of amino acids. In a similar manner, reduction of an azidoacylamino acid, e.g., dl-a-azidopropionylglycine gives DG alanylglycine. CHI N8bHcoa

+ NHlCHaCOOH

iH'

NI HCO-NHCH&OOH

Hi

+ CH8

NHIAHCO--NHCHlcOOH

The reduction may be effected not only with aluminum amalgam but also by catalytic hydrogenation in the presence of platinum or palladium (91). Since the azidoacyl halides are usually prepared by treatment of the corresponding chloro or bromo compounds with sodium ande, optically active halogen acids would be required for the synthesis of optically active peptides. Mention has been made previously of the difficulties which attend the preparation of such acids. It is not surprising, therefore, that the use of the azidoacyl halides has not assumed an important place among the methods for the synthesis of peptides. Although this method does not offer any decided advantages from a preparative standpoint, it is of interest in the historical development of the subject under


25

review because of the use of reduction, rather than hydrolysis or amination, as the final step in the peptide synthesis.

11.

“

Carbobenzoxy ” Method of Peptide Synthesis

Of the numerous contributions to the development of methods for the synthesis of ‘peptides, two are outstanding in their importance. The first of these was the use, by Fischer, of the halogen acyl halides (cf. page ll), and the second was the invention by Bergmann and Zervas (74) of the procedure which they termed the “carbobenzoxy” method. The latter has now assumed the pre-eminent place among the techniques of peptide synthesis. The potentialities of the carbobenzoxy method are indicated by its recent use for the synthesis of the pentapeptide valylornithylleucylphenylalanylproline, by Harris and Work (204a). In their search for acyl substituents which could be removed from peptides without resorting to hydrolysis, Bergmann and Zervas were led to consider the possibility of introducing the benzyl group into such an acyl moiety, for it had been shown by Rothemund and Zetzsche (259), Freudenberg et al. (178),and Fischer and Baer (173)that benzyl groups attached to oxygen or nitrogen atoms could be removed readily by catalytic hydrogenation. Accordingly, Bergmann and Zervas prepared the benzyl analog of ethyl chlorocarbonate (used by Fischer as a reagent for amino acids) by the reaction of benzyl alcohol with phosgene: C6HrCHsOH

+ ClCOCl + CaHrCHsOCOCl + HC1

The product, benzyl chlorocarbonate, was called carbobenzoxy chloride and, in what follows, it will be referred to by the latter term.* Carbobenzoxy chloride can be condensed with all amino acids and yields excellent crystalline derivatives with most of them. Exceptions are tleucine, L-isoleucine and L-proline,for which crystalline carbobenzoxy derivatives have not been described as yet. Of particular advantage is the fact that, during the reaction of carbobenzoxy chloride with the sodium salts of optically active amino acids, no appreciable racemization is observed, in contrast to the result noted with acylating agents such as bensoyl chloride (71),acetic anhydride (71,290),or ketene (94). For the conversion of the carbobenzoxyamino acids into compounds which will react with the amino group of an amino acid or peptide, the procedures of most general application are the Fischer acid chloride method (with phosphorus pentachloride or thionyl chloride) and the Curtius azide method. The choice between these two methods will be At a recent meeting of the American Chemical Society (September, 1947), Stevens and Mdne have reported the use of ally1 chlorocarbonate in place of the benzyl compound.

26

JOSEPH 8. FRUTON

determined, in large part, by the nature of the carbobenzoxy compound to be used. For certain carbobenzoxyamino acids, such as carbobenzoxyglycine (74) or carbobenzoxy-cphenylalanine (85), the conversion to the acid chloride, when conducted under proper conditions, proceeds smoothly and in good yield. On the other hand, the preparation of carbobenzoxybleucyl chloride is not entirely satisfactory, and for better yields, the conversion of carbobenzoxy-Lleucine methyl ester to the hydrazide and azide appears preferable (80). Clearly, in the case of carbobenzoxyserine (and presumably threonine) , treatment with phosphorus pentachloride would be complicated by the replacement of the hydroxyl group by chlorine, and here the azide method is the method of choice (181). Where a-peptides of glutamic acid or aspartic acid are desired, neither the acid chloride nor the azide method can be used, since this would yield, upon coupling with amino acids, disubstitution products of the dicarboxylic amino acids. For these amino acids, special methods are required, and these will be discussed in a later section (cf. page 41). As noted previously in the case of other acyl peptides, when carbobenzoxy peptides are to be coupled with amino acids or peptides t o lengthen the chain, the azide method is to be preferred, since the acid chloride procedure apparently is accompanied by side reactions of the halogenating agent with peptide bonds (254). An additional point of some importance relating to the carbobenzoxyamino acid chlorides is their tendency to form N-carbonic acid anhydrides, referred to previously (cf. page 21). This is a troublesome side reaction and, to be kept at a minimum, requires rapid operation under anhydrous conditions and strong chilling of the reaction mixture during the halogenation and subsequent coupling. In the case of acylamino acid azides, an unwelcome side reaction is the occasional tendency for the occurrence of the Curtius rearrangement to isocyanate derivatives, which then react with amino groups to form ureido compounds, or with hydroxy groups to form urethanes (77,181). In the synthesis of peptides by the carbobenzoxy method, the carbobenzoxyamino acid chloride or azide is preferably allowed t o react in an organic solvent (e.g., ethyl acetate, ether) with the appropriate amino acid or peptide ester: R

c:

C6HrCHtOCO-NH HCOCl

R'

+ NHsCJ HCOOCH, -+ CaH,CHsOCO-NH

!

HCO-NH

R'

c:

HCOOCHI

+ HCl

The free esters of amino acids or peptides may be prepared from the corresponding hydrochlorides by either of the two methods devised by

27


Fischer. The first of these involves the neutralization of the amino acid ester hydrochloride with concentrated alkali in the presence of solid potassium carbonate, and immediate extraction of the free base with ether or ethyl acetate (149). In the case of peptide ester hydrochlorides, the concentrated alkali usually is omitted, and only carbonate is used. The second method, which is preferable when the amino acid ester has an appreciable solubility in water (e.g. , histidine methyl ester) , entails the neutralization of an alcoholic solution of the hydrochloride with the calculated amount of sodium methylate (172). Since, in the coupling reaction of an acid chloride with a base, one molar equivalent of hydrogen chloride is formed, two molar equivalents of base are needed for the complete utilization of a carbobenzoxyamino acid chloride. When, however, the ester of a peptide or of a costly amino acid is used in the coupling reaction, the second molar equivalent of base may be provided by shaking the reaction mixture with an aqueous solution of potassium bicarbonate. In the case of amino acid esters which are not too expensive (Le., those of glycine, glutamic acid, tyrosine), the use of two molar equivalents of ester is to be recommended, since this obviates the possibility of the concomitant hydrolysis of the acid chloride by the aqueous bicarbonate. Furthermore, the ester hydrochloride formed during the coupling usually crystallizes out and may be recovered for future use. Although the Schotten-Baumann reaction of the carbobenzoxyamino acid chlorides or azides with the sodium salts of amino acids or peptides is occasionally successful, in many cases it yields products which are difficult to purify owing to the hydrolysis of appreciable quantities of the chloride (or azide) to the corresponding carbobenzoxyamino acid. The separation of such by-products from the carbobenzoxy peptides formed during the coupling reaction is frequently a laborious operation and markedly reduces the yield of the desired material. When coupling amino acid or peptide esters with the azides of carbobenzoxy peptides, only one molar equivalent of ester is required, since the hydrazoic acid liberated during the reaction passes off as a gas.

CIHICHSOCO-NH

R l

R’ I

CIHrCHrOCO-NHkHCO-NHCHCOOCHa

+ HNa

In contrast to the reaction between acid chlorides and amino acid esters, which proceeds quite rapidly (15-60 minutes are usually sufficient to

28

JOSEPH 8. FRUTON

ensure complete reaction), the azides react with amino acid esters much more slowly. For this reason, it is customary to allow the mixture to stand overnight a t room temperature. After the coupling reaction is completed, the solution of the carbobenzoxy peptide ester in ether or ethyl acetate is freed of any remaining free base and of carbobenzoxyamino acid (or carbobenzoxy peptide) arising as a result of hydrolysis, by successive extraction with dilute hydrochloric acid, dilute bicarbonate solution, and water. This procedure may require modification in special cases, however. For example, in the synthesis of carbobenzoxyglutamyl peptide esters in which the ycarboxyl is unsubstituted, extraction with bicarbonate must be avoided and the solution should be washed with hydrochloric acid, followed by several extractions with water. After the extraction of possible impurities has been achieved, the solution of the coupling product may be dried over sodium sulfate and concentrated under reduced pressure. In most cases, the carbobenzoxy peptide ester crystallizes out directly or may be induced to crystallize by the addition of petroleum ether. It is important that the purity of the carbobenzoxy peptide ester be established before proceeding to the next step in the synthesis, since, in the later stages of the carbobenzoxy method, the purification of the products is generally more difficult than in the case of the carbobenzoxy peptide esters. In order to convert the carbobenzoxy peptide esters to the corresponding carbobensoxy peptides, the former are saponified in acetone-water solution with slightly more than one molar equivalent of normal alkali. The product is then isolated by acidification of the solution and removal of the acetone under reduced pressure. Usually, crystalline products are obtained if the eater was pure; if an oily carbobenzoxy peptide results, however, it may be extracted with ethyl acetate, and the ethyl acetate solution then extracted with dilute bicarbonate. Acidification of the bicarbonate solution will precipitate the carbobenzoxy peptide and crystallization may sometimes be induced. Before proceeding to the discussion of the removal of the carbobenzoxy group from the acylated peptides, some comment may be inserted regarding the conversion of the carbobenzoxy peptide esters into products other than the corresponding carboxylic acids. I n particular, their reactions with hydrazine or ammonia have been used extensively in peptide chemistry. As shown by Curtius (cf. page 9), for the benzoyl peptide eaters, the reaction between carbobenzoxy peptide esters with hydrazine in absolute alcohol leads to the formation of well-crystallized hydrazides, which may, in turn, be converted to azides, thus permitting one to lengthen the peptide chain by means of further coupling reactions:

29

SYNTHES18 OF PEPTIDES

R'

R CaH,CH,OCO-NH

HCO-NH&HCOOCH~ R

C~HICHIOCO-NH

NHtNHi

R'

-

I:HCO-NH I:HCONHNH,R R CeH,CHaOCO-NH bHCO-NRI:HCON HNOI

I

The interaction of carbobensoxy peptide esters with dry ammonia in absolute methanol gives the corresponding amides, except in isolated cases, which will be discussed in a later section (cf. page 48). Such carbobensoxy peptide amides have proved to be useful where studies on the specificity of proteolytic enzymes have required substrates in which the terminal carboxyl group of the peptide chain is blocked: R C,H,CH,OCO-NH

R'

-

I:H C ~ N H A H C O O c H a

NHI

:

'R'

A

C~H~CHIOCO-NH H C S N H HCONH,

The final step in the carbobenzoxy method is the removal of the acyl substituent by catalytic hydrogenation with palladium black (298) or related catalysts a t pressures of somewhat more than one atmosphere. In the course of this hydrogenation, toluene and carbon dioxide are split off and the amino group is set free:

1

C~H,CH~OCO-NH HCO-NHR'2

CeH'CH.

+ COI + NH,

!

HCO-NHR'

The hydrogenation may be conducted in any of a variety of apparatus available commercially or which may be readily assembled in the laboratory. Whatever the form of the equipment, however, provision must be made for passing a stream of dry hydrogen through the system, and one must be able to determine from time to time whether carbon dioxide is still being evolved. As noted before, this operation represents the most important feature of the method, since the conditions are so mild as to preclude the scission of the peptide bonds. What is more, in the course of the development of the carbobensoxy method, substituents have been found for the polar side chain groups of several amino acids (e.g., the nitro group for the guanido group of arginine, the bensyl group for the sulfhydryl group of cysteine or for the imidasole group of histidine), which also may be removed by catalytic hydrogenation. Discussion of these special modifications of the carbobensoxy method will be found in subsequent sections of this review. A final attribute of the csrbobensoxy method which confers upon it

30

JOSEPH S. FRUTON

unusual advantages is the fact that, following the hydrogenation of a carbobenzoxy peptide, the solution contains only the free peptide and toluene. The catalyst is then removed by filtration, and the solvent (usually methanol plus a few drops of glacial acetic acid) and toluene may be removed completely by evaporation a t reduced pressure. The remaining product is the desired peptide uncontaminated with inorganic salts which may accompany peptides prepared by the halogen acyl halide method unless special treatment (addition of silver oxide, followed by hydrogen sulfide) is applied. If, in the course of the hydrogenation of a carbobenzoxy peptide in methanol, the free peptide separates, the addition of water prior to the removal of the catalyst will usually bring the peptide back into solution. It is obvious that the hydrogenation method may also be applied to carbobenzoxy peptide esters, and, in this case, it is necessary to add one molar equivalent of acid to neutralize the amino group which appears during the hydrogenation. In this manner, it is frequently possible to prepare peptide esters for coupling reactions. Thus, the compound carbobenzoxyglycyl-L-glutamyl-Ltyrosine ethyl ester was made by coupling carbobenzoxyglycyl chloride with cglutamyl-ctyrosine ethyl ester, the latter reactant having been obtained by the hydrogenation of the corresponding carbobenzoxy derivative (187) : CHiCOOH CsHdOH CaHrCH,OCO-NHCH&OCl

+

AH1 AH2 I I NHaCHCO-NHCHCOOCiHr-+ CHiCOOH CaHdOH

I"* I

CaHrCH*OCO-NHCHiCO-NH

HCO-NH

AHs HCOOCrHr

It may be added that the hydrogenation procedure has also been applied with good results to the preparation of amides of peptides, as well as amino acid amides. As in the case of the hydrogenation of the carbobenzoxy peptide esters, one molar equivalent of acid must be added to neutralize the amino group set free during the hydrogenation. It has been noted that glacial acetic acid is especially suitable for this purpose since the acetates of the amino acid or peptide amides crystallize more satisfactorily than do the corresponding hydrochlorides (188). Mention was made at the start of the discussion of the carbobenzoxy method that it is possible to remove, by catalytic hydrogenation, benzyl groups attached either to nitrogen or to oxygen atoms. It follows, therefore, that, if in a coupling reaction the benzyl ester of an amino acid ia used, the resulting carbobenzoxy peptide benzyl ester does not require

31

SYNTHESIS. OF PEPTIDES

prior saponification to yield the free peptide. The hydrogenation will remove both the N-carbobenzoxy and the 0-benzyl groups, thus eliminating one step in the peptide synthesis and frequently improving the over-all yield. An example is the coupling of carbobenzoxy-calanylglycyl azide with glycine benzyl ester, followed by the hydrogenation of the product to yield talanylglycylglycine (49). CHa CaH&HIOCO-NH

c:

+ NHnCH&OOCHsCaHr -+

HCO-NHCHzCONa

CaHbCH2OCO-NH

8"'

HCO-NHCHzCO-NHCHzCOOCH,CsH, CHa

HZ +

L

NHZ HCO-NHCHICO-NHCHiCOOH

One of the difficulties with the benzyl ester hydrochlorides of amino acids is that they cannot, in general, be prepared in high yield by the treatment of the amino acid with benzyl alcohol and hydrogen chloride, although examples of this procedure may be found in the literature (276). A better yield is usually obtained if the benzyl ester hydrochloride is made by the reaction of an amino acid N-carbonic acid anhydride with benzyl alcohoi and hydrogen chloride (86). Glycine benzyl ester hydrochloride has also been made from glycyl chloride hydrochloride and benzyl alcohol (203). It was noted in an earlier section that ptoluenesulfonylamino acids and peptides may be reduced by means of phosphonium iodide and hydriodic acid. Harington and Mead (202) showed that phosphonium iodide in acetic acid at, 45-50" would also eliminate the carbobenzoxy group, benzyl iodide being formed instead of toluene, as in the case of catalytic hydrogenation: R CtH,CHzOCO-NH

c:

HCOOH

PHd

CaHhCHJ

1

+ COz + NHI

HCOOH

The introduction of pbosphonium iodide as a reducing agent made it possible for Harington and Mead to adapt the carbobenzoxy method to the synthesis of glutathione since the metallic catalysts are readily inactivated by sulfur compounds. Of even greater importance for the synthesis of peptides of cystine, as well as of other amino acids, was the finding of Sifferd and du Vigneaud (272) that carbobenzoxy or benzyl groups could be removed smoothly by treatment with metallic sodium in liquid ammonia. The numerous fruitful applications of this reduction method will be discussed in later sections of this review. It has been noted (41) that, in some cases, carbobensoxy groups may

32

JOSBPH 8. FRUTON

be removed by treatment with absolute ethanol and dry HC1 a t 0'. Thus, carbobenzoxytyrosyltyrosine,when treated in this manner, gave an appreciable quantity of tyrosyltyrosine ethyl ester hydrochloride. This valuable observation has not received the further study it clearly merits. 12. Use of Phthalylamino Acida in Peptide Synlhe& Recently, Kidd and King have reported, in a preliminary communication (223b), a new method of peptide synthesis which takes advantage of the fact that the phthalyl group of phthalyl peptides may be removed by treatment with hydrazine, without scission of the peptide bonds. This ingenious technique is based on the observation of Ing and M a d e (221s) that N-alkyl phthalimides readily react with hydrarine to give phthalhydraride and the corresponding amine. Kidd and King have described the application of the new procedure to the synthesis of peptides of Lglutamic acid, using phthalyl-cglutamic acid anhydride as the acylating agent. At the present writing, the details of this work are not yet available. A more complete account of the phthalyl method has been published by Sheehan and Frank (270a), whose work was done independently of the English investigators, and who have applied it successfully to the eynthesis of glycyl-Dbphenylalanine, glycylglycine, glycyl-L-cysteine, and Dbphenyldanylglycylglycine. The salient features of the method are given in the following series of reactions for the synthesis of glycylphenylalanine.

The phthalyl method promises to be a valuable addition to the available techniques of peptide synthesis, and its further applications will be awaited with interest.


33

13. Enzymatic Synthesis of Peptide Derivatives

Recent experiments have given unequivocal proof for the expectation that, like other catalysts, the proteolytic enzymes may, under suitable conditions, cause the synthesis of peptide bonds aa well as their hydrolysis (62,M). Several examples of such enzymecatalyzed peptide synthesis are the following:

-

+ cleucinanilide papain benzoyl-cfeucyl-cleucinanilide(249) papain Benzoyl-cleucine + glycinadide benzoyl-cleucylglycinanilide(249) Obymotrypsin Benzoyl-ctyrosine + glycinanilide benzoyl-ctyrosylglycinanilide (40) papain Carbobenzoxy-cphenylalanylglycine+ ctyrosinamide Benzoyl-cleucine

r

carbobenzoxy-cphenyldanylglycyl-ctyrosie (101)

This type of peptide synthesis produces insoluble compounds, and , indeed, the insolubility of the products is an essential attribute of the method. This follows from the fact that the energy necessary for the synthesis of a peptide bond (about 3000 cal. per mole, 92) is provided by the removal, by crystallization, of the synthetic product from the solution. In the cases cited above, the solubility of the synthetic peptide derivative is less than its equilibrium concentration; consequently, in order to restore the balance of the equilibrium reaction, synthesis occurs, which in turn, causes more of the synthetic product to crystallize (54). At the present stage of its development, this method cannot be compared in usefulness for peptide synthesis with the procedures discussed earlier in this review. One of the principal difficulties lies in the removal of the substituent groups. While the carbobenzoxy group may be removed by hydrogenation, the scission of the amide or anilide linkage requires hydrolysis, with the attendant destruction of the peptide. Attempts have been made to effect enzymatic synthesis of peptide derivatives in which the terminal amino group is carbobenzoxylated and the terminal carboxyl group is linked with a benzyl group. While initial efforts (186)in this direction have not met with success, further exploration of such possibilities appears desirable. A further limitation of the enzymatic method becomes apparent if one considers the extreme specificity of action of the proteolytic enzymes. None of these enzymes acts a t peptide bonds indiscriminately, and each enzyme hydrolyzes or synthesizes only such peptide bonds as are present in the substrate in a certain structural setting. Extensive discussion of the specificity of proteolytic enzymes may be found in several recent review articles (47,52,182). One application of the synthetic capacity of the proteolytic enzymes,

34

JOSEPH 8. FRUTON

which takes advantage of the stereochemical specificity of enzyme action, has proved of considerable indirect value in peptide synthesis. As was noted in the Introduction, it is usually desirable t o employ, for the synthesis of peptides, either the t or D-amino acids, rather than the racemic forms. It has been shown that the stereochemical specificity of the proteolytic enzymes permits them to serve as agents in the resolution of Dtamino acids (190). This is illustrated below for the resolution of m-glutamic acid by activated papain: Carbobenroxy-Dcglutamic acid

+ aniline

Ipaprin

Carbobenroxy-ln-lllutamicacid Carbobenzoxy-Lglutsmic acid anilide Ohydrogenation D-

lutamic acid

1hydrol.

cGlutamic acid

T h e further exploitation of this method has provided procedures for the preparation of tamino acids from synthetic Dtamino acids, where the latter are less costly than are the amino acids isolated from protein hydrolyzates (cf. 112s). Furthermore, the enzymatic resolution represents an additional convenient method for, the preparation of Pamino acids.

111. SPECIAL ASPECTSOF PEPTIDE SYNTHESIS In what follows, there will be discussed some of the problems encountered in the synthesis of peptides containing particular protein amino acids, and attention will be given to the manner in which the general methods discussed earlier have been modified to meet these special problems. For the convenience of those who may wish t o discover rapidly whether a particular peptide or useful intermediate has been synthesized, tables have been prepared to accompany the sections on the individual amino acids.* One series of tables lists some of the more useful or interesting derivatives, their melting points, and references to papers in which their synthesis is described. In addition, another set of tables is devoted t o free peptides and their optical rotation (when available), since the majority of the peptides listed contain optically active amino acid residues. The bibliographic citations in the tables for the peptides give the literature sources for the values of the rotation listed in the tables. Where several references are cited for a single peptide, the rotation which is included in the table is, in general, the largest positive or negative value reported. In view of the large number of investigators whose data have been used in the preparation of these tablee, no assurance can be given of the accuracy of the melting points or The author ia greatly indebted to Dr. Sofia Simmonds for invaluable assistance in the preparation of the tablee and of the bibliography.

SYNTHESIB OF PEPTIDES

35

optical rotations as listed. The reader is urged to examine the original papers for details. It should be stressed that the material in the tables represents a selection from the extensive literature on peptide synthesis, and no attempt has been made to provide a complete listing of all peptides and peptide derivatives which have been described. Such a task, it was felt, did not accord with the purpose of this review. Although an effort has been made to list the peptides .and their derivatives according to some rational order, which will be readily apparent upon inspection of the tables, the compounds are grouped arbitrarily according to the particular amino acid residue which appears to be of primary interest. Thus, glycyl-btyrosine will be found in the table which lists the tyrosine peptides and not in the table for the glycine peptides. In order to conserve space, each peptide or peptide derivative is listed only once in the tables. In searching for a reference to a complex peptide or peptide derivative composed of several different amino acid residues, the reader is advised to examine more than one table. 1. Peplides of Glycine, Alanine, Vdine, Leucine, and Isoleucine

This group of amino acids may be treated as a unit since the side chains do not contain special reactive groups. The ester hydrochlorides of the optically active forms of all of them have been prepared and may readily be converted to the free base by treatment with alkali in the presence of an organic solvent (cf. page 27). The free esters may then be coupled with a variety of reactive amino acid derivatives. It is appropriate to mention a t this point that, although Galanine and Gleucine are currently readily available, this cannot be said for the corresponding optically active forms of valine and isoleucine. As has been noted elsewhere (185), however, the two last-named amino acids are now accessible in DL form, and the development of resolution methods based on the stereochemical specificity of proteolytic enzymes seems feasible (cf. page 33). The attachment of a glycyl residue to the amino group of an amino acid or peptide presents no especial difficulty. This can readily be achieved either by means of chloroacetyl chloride (or bromoacetyl bromide), followed by amination, or, preferably, by coupling with carbobenzoxyglycyl chloride, with subsequent hydrogenation. In general, if simple glycyl dipeptides are desired, the chloroacetyl chloride method will be found satisfactory. For the preparation of longer peptides or of derivatives with glycine a t the amino end of the peptide chain the use of carbobenzoxyglycyl chloride is to be preferred. The latter reagent may

36

.

JOSEPH 8 FBUTON

TABLE I1 Derivatives of cflyn'ne and Alanine Compound

M . p., "C. (ref.)

Glycine anhydride. . . . . . . . . . . . . . . . . . . . . . . . . . . ca . . 305 (261) Glycine benryl ester HC1 . . . . . . . . . . . . . . . . 139-140 (203,260) Glycine-N-carbonic acid anhydride. . . . . . . . - (228) Barcosine-N-carbonic acid anhydride. . . . . . . . . . . 99-100 (273) Glycylglycine ethyl ester HCI . . . . . . . . . . . . . . . . . . 182d (149) Glycyldehydroalanine. . . . . . . . . . . . 192-193 (56) Bencoylglycinamide . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 (132) Bensoylglycyl chloride . . . . . . . . . . . . . . . . . . . . . . . - (132) Beneoylglycylglycine . . . . . . . . . . . . . . . . . . . . . . . 208 (132) Carbobenzoxyglycine. . . . . . . . . . . . . . . . . . . . . 120 (74) Carbobenzoxyglycyl chloride . . . . . . . . . . . . . . . . . . 43 (74) Carbobenzoxyglycyl-a-aminoisobutyric acid . . . . . . 164 (80) Carbobensoxyglycylglycine. . . . . . . . . . . . . . . . . . . . 178. (74) Carbobenroxyglycylglycinamide. . . . . . . . . . . . 179-181 (188) Carbobenzoxyglycylglycinhydrazide. . . . . . . . . . . . 166 (258) Carbobenzoxy(diglycy1)glycine . . . . . . . . . . . . . . . . 196(78) Carbobenroxy(diglycy1)glycinamide. . . . . . . . . . . . . 220 ( M a ) Carbobenroxy(triglycy1)glycine . . . . . . . . . . . . . . . . 230 (78) Carbobenroxyglycylsarcosine . . . . . . . . . . . . . . . . . 102 (59) Carbobenzoxyglycyl-Lalanine. . . . . . . . . . . . . . . 135 (24, 49) Carbobensoxyglycyl-Galaninhydraride. . . . . . . . . 133 (77) Chloroacetylglycine. . . . . . . . . . . . . . . . . . . . . . . . . . . 98-100 (235) Chloroacetylglycylglycine. . . . . . . . . . . . . . . . . . . . 178-180 (136, 160) Cbloroacetylsarcosine. . . . . . . . . . . . . . . . . . . . . . . . 95-98 (235) Phenylpyruvylglycine . . . . . . . . . . . . . . . . . . . . . . . . . 167-168 (189) Pyruvylglycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 (56) Toluenesulfonylglycinhydraride. . . . . . . . . . . . . . 155.5 (266) Toluenesulfonylglycylglycine. . . . . . . . . . . . . . . . . . 178.5 (266)

.

cAlanine anhydride. . . . . . . . . . . . . . . . . . . . . . . . . . 297 (134) D-Alanine-N-carbonic acid anhydride . . . . . . . . . . . 89 (220) Bencoyl-calaninamide . . . . . . . . . . . . . . . . . . . . . . 235-240 (201s) Bensoyl-Dcalaninamide . . . . . . . . . . . . . . . . . . . . 229-230 (244) Carbobenzoxy-calanine . . . . . . . . . . . . . . . . 84 (74) Carbobenmxy-obalanine . . . . . . . . . . . . . . . . . . 114-1 15 (74) 106 (272) Carbobenzoxy-Falanine. . . . . . . . . . . . . Carbobenaoxy-Saleninhydrazide. . . . . . . . . . . . . . . 143 (272) Carbobencoxy-bslanylglycine. . . . . . . . . . . . . . . . . 132-133 (278) Carbobeneoxy-balanylglycinhydrazide. . . . . . . 157 (49, 276) Carbobenaoxy-calanyl-Lalanine. . . . . . . . . . . . 152-153 (278) Chloroacetyl-Lalanine . . . . . . . . . . . . . . . . . . . 93.5-94.5 (6,167) Toluenmlfonyl-balanine . . . . . . . . . . . . . . . . . . 134-135 (158) Toluenmlfonyl-Dtalaninhydraeide . . . . . . . . . . . 171 (266) Toluenesulfonyl-DLalanylglycine. . . . . . . . . . . . . . . 147 (266)

37


TABLE I11 Dctivalivcs of Valine. W n e . and Isoleucine Compound Carbobenzoxy-cvaline . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chloroacetyl-cvaline.. . . . . . . . . . . . . . . . . . . . . . . . .

M. p., "C. (ref.) 64-65 (282a) 113-115 (164)

125-126 (43) chucinamide acetate. . . . . . . . . . . . . . . . . . . . . . . . . . . . chucinamide HCl. . . . . . . . ... . 230-237 (276a) chucine-N-carbonic acid anhydride. . . . . . . . . . . . . . . 76. Ei-78 (301) Dchuche-N-carbonic acid anhydride. . . . . . . . . . . . . . 48-50 (229) Acetyldehydroleucine. . . . . . . Acetyldehydroleucylglycine. . . . . . . . . . . . 130-132 (115) Bencoyl-cleucinamide. ..... Benzoyl-Dcleucinamide. . . . . . ............. 171 (244) Bencoyl-cleucinhydrazide. . Bencoyl-cleucyl-cleucine . . Carboethoxy-cleucyl-cleucine.. . . . . . . . . . . . . . . . . . . . 147-148 (136) Carbobenzoxy-cleucinamide.. . . . . . . . . . . . . . . . . . . . . . 122-123 (43) Carbobenzoxy-cleucinhydrazide.. . . . . . . . . . . . . . . . . . 121 (80) Carbobenzoxy-cleucylglycine. . . . . . . . . . . . . . . . . . . . . . 115 (78) Carbobenzoxy-cleucylglycylglycine. . . . . . . . . . . . . . . . 144 (78) Carbobenzoxyglycyl-calanyl-cleucinhydrazide ...... 186 (77) Carbobenzoxyglycyl-cleuci ne . . . . . . . . . . . . . . . . . . . . . . 141- 142 (277) Carbobenzoxyglycyl-cleacinamide. . . . . . . . . . . . . . . . . 123-124 (276s) .................. 136 (168) Chloroacetyl-cleucine. . . . . Toluenesulfonyl-Dbleucinhydrazide ........ 146 (266) Tolueneaultonyl-Dcleucylglycine.. . ........ 121.5 (266) Toluenesulfonyl-calanyl-cleucine. ................. 186 (266) Carbobenzoxyglycyl-cisoleucine. . . . . . . . . . . . . . . . . . . Chloroacetyl-cisoleucine . . . . . . . . . . . . . . . . . . . . . . . . . .

114-1 15 (277) 74-75 (16)

be obtained in crystalline form by the procedure described by Bergmann and Zervas (74), although some workers have encountered difficulty in crystallizing this substance without inoculation with seed crystals. The chloride is best used immediately after its preparation, a small sample being set aside under petroleum ether for the inoculation of the next preparation. With regard to the attachment of an alanyl residue to an amino acid or peptide, the bromopropionyl bromide (or chloride) method of Fischer is feasible only when Dcalanyl peptides are desired. The preparation of optically active a-bromopropionic acid, as was noted previously, is a tedious operation, and consequently, for the attachment of t or D-alanine residues, the carbobenzoxy method appears preferable. Unfortunately, carbobenzoxy-calanyl chloride is accessible only as an oil, and although

38

JOSEPH 9. FRUTON

TABLE IV Peptides of Cflyn'ne,Alanine, Valine, Leucine, and Ieoleucine P

Peptide Glycy lglycine ( Diglycy1)glycine (Triglycy1)glycine (Tetraglycy1)glycine (Pentaglycy1)glycine (Diglycyl)-D-leucine (Diglycy1)-Lleucine (Triglycy1)-~leucylglycine (Diglycy 1)-walanylglycine (Diglycy1)-Lleucylglycine Gly cyl-calanine Glycyl dehydroalanine Glycy I-~alanylglycine Glycyl (di-D-alanyl)glycine Glycy 1-talanyl-tleuciiie

...

... ...

... ...

II

4-27.5 -28.0 -28.4 +53.7 -43.2 -50

...

-64.3 4-104.8 -88.0

Temp., "C.

Concn. and solvent

...

... ... ...

...

...

... 26 26 24 20 24 20 *.. 20 20 22

...

D- Alanylglycine

LAlanylglycine D- Alany lglycy lglycine LAlanylglycylglycine LAlanyl (diglycy1)glycine LAlanyl(diglycy1)-balanylglycylglycine LAlanylglycy 1-Lalanine cAlanylglycy 1- leuc cine D- Alanyl-Lalanine LAlany I-D-alanine LAlanyl-halanine (Di-calanyl)-Lalsnine

-50.7 $50.2 -31.6 $32.4 $27.0 $13.2 -19.5 -11.2 -68.5 $68.9 -21.6 -72.2

20 20 25 20 21.5 26 24 20 20 20

20 20 20 20 23 20 25

190s 190a

2%, HzO 2%, Ha0 2.5%, HsO 3%, HzO 2.5%, HzO 4%, HzO

...

4.3%, HtO 2.9%, HIO 1.1%, HzO eq. HCI

Glycyl-Lalan yl-cleucyl-tiso-80.6 leucine Glycyl-Lvaline -19.7 Glycy 1-D-leucine 4-35.7 Glycy 1- leuc cine -35.1 Sarcosyl-Lleucine -30.4 Cly cy1-wleucylglycine $42.6 Glycyl-Lleucylglycine -41.2 Glycyl-~leucylglycyl-~leucinc -51 .O Glycyl-Lleucyl-talanine -59.0 Glycyl-~leucy1- leuc cine -67.0 Glycyl(di-Lleucyl)-Lleucine -78.6 Glycyl(tri-Lleucy1)-Lleucine -118.1 GIycyl-o-isoleucine $13.6 Glycyl-~isoleucine -14.7

149 128 129 129 134

...

...

Ref.

+1

78 233 78 24,30,167 66,255b 140 234 9,264

0.9%, N HC1

15

lo%,

164,231 275 168 275b 190a 78 34

Hi0

3 % ~HzO 4.2%, HsO 2.2%, HzO 2%, H i 0 2.5%, HtO 2.3%, 10% HCl 2.5%, Hz0 2.9%, EtOH 2%, N NaOH 2.5%, N NaOH 4.1%, HzO 4.1%, HzO

9 8 8 8

28,36 16,36

4.3%, HzO lo%, HtO lo%, HzO lo%, Hz0 3%, H i 0

80 133,140 32,49 49,136,140 17

8 % , Hz0

136 24 9 161 161 134,237,278 . . 12

3.7%, H20 2.3%, HzO 8.7%, HIO 7.4%, HzO 5%) HzO 3.5%, 2 N HCI

39


Temp.,

TABLE IV.-(Continued) Peptide (Tri-calany1)-Lalanine (Tetra-Lalany1)-talanine (Di-D-alanyl)glycine G Alanyl-cvaline L Alanyl-Lleucine G Alanyl-~leuc ylglycine ~ A l a n y l - ~ - l e yu1-~valine c D- Alany 1-D-leuc y 1-D-leucine GAlanyl-Lleuc yl-~isoleucine &Alanyl- leuc cine LAlanyl-~isoleucine G Alanyl-a-aminoisobutyric acid LAlanylsarcosy lglycine

[alD

-120.5 -136.4 +47.2 -5.9 -17.3 -30.4 -60.2 +62 .O -24.9 -31 .O +6.1

"C. 20 20 20 20 20 20 19 25 20 26 20

Concn. and solvent 2.2%, 2 N HCl 1.9%, 2 N HCl 3%, HzO

lo%, Ha0 5%, HzO 2.1%, HzO 4.4%, HzO 3.2%, N NaOH 4.975,N HCl 1.5%, H i 0 3.8%, N HC1

Ref. 12 12 234 164 138,266 9 30 237 15 201a

16

+34.5 +10.8

20 25

2%, H i 0 5%, H i 0

80 49

LValylglycine D-Valyl-Lvaline tValyl-Lvaline

+93.6 -74.0 -54

20 20 20

lo%, Ha0 lo%, H i 0

164 164 33

GLeucylgly cine GLeucylglycylglycine bLeucyl(diglycy1)glycine cLeucyl(pentaglycy1)glycine

+85.8 +57.7 +45.9 +5.2

24 25 20 20

GLeucyl (hexaglycy1)glycine

+6.3

20

LLeucyl (triglycy1)-Lleucine L-Leucylglycy1-Galanine L-Leucylglycyl-r.-lcucine L-Leucylgly cy 1-D-isoleucine ~ L e u c y l gycyl-~isoleucine l ~ L e uyl-D-alanine c L-Leucyl-Lalanine ~ L e uycl - ~ a l a ylglycine n LLeuc y 1-p-alanine xAeucyl-Lvaline D-Leucy 1-D-leucine D-Leucyl-Lleucine cLeucy 1-D-leucine GLeucyl-Gleucine (Di-D-leucyl )+-leucine (Di-L-leucy1)- leuc cine (Tri-cleucyl)-Lleucine ~ L e u c 1-D-isoleucine y LLeucyl-Lisoleucine

+21.3 +20.3 +6.0 +41.2 +26.5 +76.0 +23.5 -17.3 +28 .O +18.0 +13.9 -68.0 +68.9 -13.4 +46.0 -51.4 -90.0 +53.1 +25.7

20 20 20 20 20 22 20 20 26 20 25 20 20 20 25 20 20 20 20

LIsoleucylglycine

+33.6

20 -

4.3%, HzO 2,4%, HnO 5%, Ha0 9.6%, Hn0 3.7%, 0.1 N NaOH 6%, HzO 1 eq. NaOH 2.4%, HzO lo%, Hz0 3.6%, 10% HCl 2 % , 0.2 N HCl 3%, Hz0 2.5%, HIO 5%, MeOH 1.2%, HnO 5 % , HIO LO%, Hz0 1.5%, N NaOH 3.4%, N HC1 3.7%, N HCl 3.1%, N NaOH 3.5%, N NaOH 3.1%, N NaOH 7.6%, N NaOH 1.8%, HzO 3 % , N HCl

+

78,136 10,267 136 10 138 168 6,168 34

37 28,37 80 80,136 9 201s

30,164 154,237 154 154 136 237 B B

28 15,16

16

40

JOSEPH 8. FRUTON

it may be used in this form for peptide synthesis (53,220), the tendency of such noncrystalline carbobenzoxyamino acid chlorides to form N-carbonic acid anhydrides is appreciable. For this reason, the use of carbobenzoxy-Galanyl azide will, in many cases, be found to give somewhat better yields in coupling reactions. The considerations noted above in the cam of alanine apply with even greater force in the synthesis of leucyl peptides. The synthesis of L- and wleucyl derivatives, for which Fischer used optically active a-bromoisocapronyl chlorides, is now accomplished more readily by coupling reactions which involve the use of carbobenzoxy-c or D-leucyl azide. Carbobenzoxyleucyl chloride is an oil, and coupling reactions in which it is employed frequently do not proceed satisfactorily. The very large number of known synthetic peptides containing glycyl, alanyl, or leucyl residues attests to the relative ease with which these peptides may be prepared by means of currently available methods. Several of these peptides have proved invaluable as standard substrates for studies of proteolytic enzymes. Thus, glycylglycylglycine, calanylglycylglycine, and Gleucylglycylglycine have been used widely in the study of bacterial enzymes (243) and of the peptidases of intestinal mucosa (276), animal tissues and fluids (184). In addition, cleucinamide acetate has been recommended as a standard substrate for the manganese-activatable aminopeptidase of animal tissues (184). In this connection, it may be stressed once more that, for enzyme studies, the use of racemic peptides is to be avoided since there are several reports in the literature to the effect that the presence of the D or L antipode may markedly inhibit the rate of hydrolysis of an optically active peptide by proteolytic enzymes (117,277). In the synthesis of valyl and isoleucyl peptides, only the halogen acyl halide method has been used extensively, and insufficient information is available a t present regarding the application of the carbobenzoxy method to the synthesis of such peptides. There is good reason to believe, however, that, whenever peptides involving the optically active forms of valine or isoleucine are desired, this method will be found to be more suitable than the classical halogen acyl halide synthesis. Recently, Synge (282a) has reported the synthesis of a-(cvaly1)-Gornithine by the use of the carbobenzoxy method. It may be added that a large variety of peptides of nonprotein aliphatic amino acids have also been described. Thus, a-aminoisobutyryl peptides have been prepared both by the halogen acyl halide method (26) and the carbobenzoxy method (80), and a-amino-n-butyryl (14,27) as well as alloisoleucyl (36) peptides have been made by the halogen acyl halide procedure.

41


2. Peptides of Aapartic and Glutumic Acids

Although the coupling of the diesters (or the disodium salts) of these amino acids with N-substituted amino acid chlorides or asides, or with azlactones, presents no special problems, the synthesis of peptides involving the carboxyl groups of aspartic or glutamic acid frequently offers some difficulty. Fischer and Koenigs (155) described the synthesis of a Dcaspartylmonoglycine by the condensation of fumarylchloride with glycine ethyl ester. The resulting fumaryldiglycine ethyl ester was saponified and, upon aminatiQn of the fumaryldiglycine, the above dipeptide was obtained. It was not established, however, whether the product contained an a- or a 8-amide linkage.

Since L-aspartic acid may readily be prepared from Gasparagine by the method of Vickery and Pucher (2851, methods involving the use of the carbobenzoxyamino acid are preferable to the older procedure of Fischer. If disubstitution products are desired, carbobenzoxy-caspartic acid dichloride may be coupled with an amino acid ester in the usual manner (193). If monosubstitution products are sought, however, a different procedure must be employed. This special method depends on the fact that N-acylaspartic acid upon treatment with acetic anhydride gives an acid anhydride (cf. 67,203a). CHiCOOH

-

CHIC0

(CH8CO):O

RCO-NH

c:

HCOOH

In the case of the carbobenzoxy derivative, this reaction is not accompanied by racemization (74), unless sodium acetate also is present (186). The resulting carbobenzoxy-L-aspartic acid anhydride may be used for coupling reactions with alcohols to give monoesters and with amino acid esters to give carbobenzoxy-caspartyl peptide esters in which only one carboxyl group of aspartic acid is bound in peptide linkage.

42

JOSEPH 8. FFtUTON

CH&O

I

' 0

CsHrCHaOCO-NH

R

+ NHibHCOOCHa

-t

IHiCooHb

CeHrCHiOCO-NH H C O - N H HCOOCHa or

CHSCO-NHCHCOOCHa CaH'CHiOCO-NH

bHCOOH k

As indicated above, in the reaction with amino acid esters, carbobenzoxy-baspartic acid anhydride may give rise either to a- or to 8-peptide linkages. For example, in the coupling with glycine ethyl ester, the major product isolated is the a compound; thus, caspartyl-aglycine may readily be prepared (193). Similarly, in the reaction with whistidine methyl ester, there is formed the a-peptide derivative (197). On the other hand, the coupling with L-tyrosine ethyl ester leads to the isolation of the p-peptide derivative (88). A general method is available, however, for the synthesis of p-peptides of aspartic acid. This may be effected by taking advantage of the reaction of carbobenzoxy-baspartic acid anhydride with benzyl alcohol to give the a-benzyl ester (87). The 8-carboxyl may then be converted to an acid chloride with phosphorus pentachloride. L-Aspartyl-8-glycine (203), caspartyl-@-ctyrosine(88), and L-aspartyl-j3-L-histidine (288) may be prepared by coupling this acid chloride with the appropriate amino acid eater:

CsHrCHrOCO-NH

x

R

HCOOCH&sHr

+ NHabHCOOCH&aHr -+ R CHiCO-NH

CsHrCHsOCO-NH

bHCOOCH&sH'

bHCOOCHiCsHr

It may be added that carbobenzoxy-baspartic acid anhydride reacts with ammonia to give the a-amide, carbobenzoxyiso-L-asparagine (74). The synthesis of basparagine by the carbobenzoxy method has been effected through the reaction of carbobenzoxy-L-aspartyl-a-benzyl ester /%chloride with ammonia, followed by hydrogenation of the product (87). In the 'case of the synthesis of glutamyl peptides, the same general procedures may be used as for the aspartyl peptides. Carbobenzoxy-c glutamic acid readily gives the optically active anhydride on treatment with acetic anhydride, and this product reacts with glycine

43

8YNTHE818 OF PEPTIDES

TABLE V Derivatives of Aspartic and Glutamic Acids Compound

M. p., "C. (ref.)

a-chpartylglycine ethyl ester . . . . . . . . . . . . . . . . . . . 232 (78) Acetyl-caapartic acid anhydride . . . . . . . . . . . . . . . . . 141 (67) Carbobenzoxy-caspartic acid .................... 116 (74) Carbobeneoxy-casparagine...................... 165 (74) Carbobenroxy.cisoasparagine .................... 164 (74) Carbobeneoxy-caspartic acid anhydride ........... 84 (74) Carbobencoxy-a-csepartic acid benzyl ester ....... 84-85 (87) Carbobeneoxy-caspartic acid dichloride ........... 46 (193) Carbobeneoxy-a-baspartylglycine. . . . . . . . . . . . . . . . 171 (78, 193) Carbobenzoxy.8.caspartylglycine . . . . . . . . . . . . . . . . 154 (193,203) Carbobeneoxy.8.caspartyl.ctyrosine . . . . . . . . . . . . . 110 (74) Carbobeneoxy-pcaspartyl-8-benaylcysteinylglycine168-170 (248) Carbobeneoxy.a.casparty1.chistidine . . . . . . . . . . . . . 171 (197) Chloroacetyl-caspartic acid ..................... 142-143 (148) Chloroacetyl.basparagine....................... 148-149 (155) 130 (76) YcGlutamic acid ethyl ester .................... 151 (78) a-cGlutamylglycine ethyl ester .................. a-cGlutamyl.cleucinamide ...................... 175-177 (276a) a-cGlutamyl-ctyrosine ethyl ester ............... 144 (187) Carbobenaoxy-eglutamic acid ................... 120 (74) Carbobeneoxy.cglutamine ....................... 137 (74) Carbobenroxy-Lglutamic acid anhydride .......... 94 (74) Carbobeneoxy.eisog1utamine .................... 175 (74) Carbobenzoxy--y.cglutamic acid hydrazide ........ 178-179 (206a, 227a) Carbobene0xy.a-cglutamylglycine ................ 143 (193) Carbobenzoxy-7-cglutamylglycine . . . . . . . . . . . . . . . 159-161 (227a) Carbobeneoxy.a-cglutamylglycylglycine . . . . . . . . . . 142 (79) Carbobensoxy.a-cglutamyl.cleucinamide . . . . . . . . . 165-169 (276a) Carbobenroxy-a-cglutamyl-eglutamicacid ........ 176 (74) Carbobene0xy.a-cglutamyl.D.phenylalanine . . . . . . . 122 (187) Carbobencoxy.a-cglutamyl.cphenylalanine . . . . . . . 162 (187) Carbobeneoxy-a-cglutamyl-~rphenylalaninamide . . 185-187 (215) Carbobenzoxy.a-cglutamyl.btyrosine . . . . . . . . . . . . 185 (88) Csrbobeneoxy.a-cglutamy1.ctyrosinamide ........ 181 (187) Carbobeneoxy-a-bglutamyl-btyrosinhydraaide.... 194 (187) Carbobeneoxy-ycglutamyl-tcysteinylglycine ..... 163 (202) Carbobeneoxy-a-cglutaminyl-cphenylalanine..... 180 (187) Carbobenroxyglycyl-Lglutamicacid . . . . . . . . . . . . . . 160-162 (215) Carbobenzoxyglycyl.Licisoglutamine ............... 185 (78) Carbobensoxyglycyl.scglutamylg1ycine . . . . . . . . . . 98-100 (78) Carbobenzoxyglycyl.a.Lglutamylglycinamide ...... 175 (50) Chloroacetyl-cglutamic acid ..................... 143 (157) Chloroacetyl-cglutamine ........................ 130- 132 (283) Glycyl-cglutamic acid diketopiperazine ........... 240 (78) Phenylpyruvyl-cglutamic acid ................... 142-143 (189)

44

JOSEPH 8. FRTJTON

TABLE VI Pevtides t

-

Aspartic and Gluiumic Acids remr "C.

Peptide Glycyl-baspartic acid Glycyl-caaparagine Glycyl-baaparagin y 1-cleucine bleucylglycyl-baapartic acid cLeucyl-baspartic acid D-Leucyl-baaparagine cLeucy1-baaparagine a-chpartylglycine &cAapartylglycine a,B-cAapart yldiglycine &tAspartyl-btyroaine 8-c Aspartyl-Lcysteinylgly cine a-bAspartyl-D-histidine &cAspartyl-chistidine

+11.1 -6.4 -46.8 +55.3 +27.1 -53.8 +17.8 +36.7 +7.2 +33.8 +SO. 1 -29.0 -6 .O 4-38

Glycyl-r,-alanyl-cleucyl-~ ... glutamic acid Glycyl-calanyl-bglutamic acid -69.8 Glycyl-cglutamic acid -6.3 Glycyl-bglu tamine -2.4 Glycyl-a-bglutam ylglycine ... Glycyl-a-bglutamyl-ctyrosine ... Glycyl-a-cglu taminylglycine -28.4 D- Alanyl-eglutamine -20.1 cAlanyl-cglutamine +9.3 cleucyl-bglutamic acid +10.6 cleucyl-Lglutamine +12.6 a-cGlutamylglycine +80.3 y-cGlutam ylglycine +11.1 a-cGlutamyl-cglutamic acid +19.9 a-cGlutam yl-cphen ylalanine +27.0 a-LGlutamyl-ctyrosine +30.1 a-cGlutam yl-ccysteine 4-13 .65 a-cGlu tam yl-ccysteinylglycine +2.5 y-D-Glutamy 1-ccyeteinylglycine -34.6 y-LGlutamyl-ccysteinylglycine -21.3 Bia (y-bglutamyl)-bcystine - 120" bGlu taminylglycine 76 cGlutaminy1-cglutamic acid +I5 bGlu taminyl-tcysteine -9.8" Bia(bglutaminy1)-ccystine -119"

+

" A = 546 mp.

Concn. and solvent

20 20 20 20 20

D%, Hi0 7%, H i 0 4.8%, N HCl 5%, HIO B%, HtO

20

5%, Hi0 5.4%, Hz0 1.974, H 2 0 2.4%, H,O 2.2%, H,O 2.3%, HzO

20 23 22 23 18 25 30 27

... 20 20 19

...

... 19 16 18 20 18 22 14 18 25 19

... 25 16 27

... 18 18

... ...

I%, H i 0 I%, H s 0 I%, H i 0

+ 1eq. HC + 1eq. HC + 1eq. HC + 1eq. HC

Ref. 148 166 166 148 148 156 156 193 193 193 74 248 197 288 77 204 157 283 78 187 283 283 283 139,276a 283 193 227a 74 274

88 202 289 224 202,291 202 245 245 203 203

45

SYNTHESIS OF PEPTIDEB

ethyl ester to give the a rather than the y compound as the principal product. CHrCO &HI

I

C~H'CHXOCO-NHCHCO

'+

0 '

NHxCHXCOOCIH'+ CHrCOOH ~ H x CeHrCHaOCO-NH

HCO-NHCHaCOOCxH'

It is of interest that, in the reaction with Gtyrosine ethyl ester (88) Gglutamic acid diethyl ester (74), or Gcysteine benzyl ester (203), the major products are also derivatives of the a-peptides. It would appear, therefore, that the tendency to yield the a substitution product is more general with carbobenzoxy-cglutamic acid anhydride than it is with the aspartic acid analog (cf. also 245). The preparation of y-peptides of glutamic acid may be accomplished by a method similar to that noted above for the p-aspartyl peptides. Carbobenzoxy-cglutamic acid anhydride gives the a-benzyl ester with benzyl alcohol, and subsequent treatment of the y-carboxyl with phosphorus pentachloride yields the yacid chloride (87). This method has been used recently for the synthesis of derivatives of y-glutamylglutamic acid (91a,250a). With the aid of this chloride, there has been effected a synthesis of Gglutamine (87). Furthermore, Melville (245) has used the y-acid chlorides of carbobenzoxyglutamyl peptide esters to make several cglutaminyl peptides. Carbobenzoxyq-cglutamic acid hydraside has also been employed for the synthesis of y-glutamyl peptides (206aJ2!27a). The phthalyl method, developed recently, has also been applied to this purpose (22313). Recently, a synthesis of cglutamine has been described (183) in which carbobenzoxy-cglutamic acid diamide (prepared from the diethyl ester with ammonia) is selectively hydrolyzed at the a-amide linkage with cysteine-activated papain to yield carbobensoxy-cglutamine, from which L-glutamine may be obtained by catalytic hydrogenation. To date, the possibility of using this method for the synthesis of y-peptides of glutamic acid has not been explored experimentally. It may be added that, by the use of the carbobenzoxy method, Schneider (265) has prepared .several peptides of the dicarboxylic amino acid aminomalonic acid. Interest in the physiological role of peptides of glutamic acid has been heightened by the isolation of a polyglutamic acid peptide from the capsule of Bacillus anthracis (201,222), and of a glutamic acid peptide from sea weed of the genus Pelvetia (200), as well as the demonstration that glutsmic acid is bound in peptide linkage in folic acid (280).

46

JOSEPH 8. FRUTON

3. Peptides of Phenylalanine and Tyrosine

A large number of peptides containing one or both of these amino acids has been prepared, and by a variety of methods. Treatment of the esters of Lphenylalanine or Ltyrosine with halogen acyl halides has led to the synthesis, by Fischer and later workers, of numerous peptides such as glycyl-Lphenylalanine, glycyl->tyrosine, calanyl-c tyrosine, etc. Except for the glycyl dipeptides, peptides in which the amino group of phenylalanine or tyrosine is linked to another amino acid can be made more readily by means of the carbobenzoxy method. In general, the coupling of carbobenzoxyamino acid chlorides, azides, or anhydrides with the esters of phenylalanine or tyrosine proceeds without unusual difficulty. An indication of the advantages of the carbobenzoxy method for the synthesis of optically active peptides containing a tyrosine residue is provided by a comparison of the optical rotation of glycyl-L alanylglycyl-Ltyrosine as prepared by Abderhalden et al. (2) , who used this method, and as prepared by Fischer (140) by means of the halogen acyl halide method. The material obtained by the former workers had an [ a ]of~ 18.4', while Fischer's preparation had an [aIDof +4.0". For the synthesis of peptides in which the carboxyl group of the aromatic amino acid is involved in peptide linkage, at least three methods are available. Historically, the first of these was the use by Fischer (130) of a-bromo-8-phenylpropionyl chloride as the coupling agent; subsequent amination of the coupling product led to the synthesis of DGphenylalanyl peptides:

+

CHtCdh BrLHCOC1

+ NHsCHICO-NHCHSCOOH CHsCdIr

Br~HCO-NHCHtCO-NHCHtCOOH CHaCdh

NHa

NHS~HC~NHCHSCO-NHC~Scoo~

A comparable application of the halogen acyl halide method has not been described for the synthesis of Dbtyrosyl peptides. As a consequence of the search for more satisfactory procedures of peptide synthesis, a second method, involving the use of azlactones (cf. page 75), was introduced by Bergmann el al. (67). This method made possible the synthesis of peptides such as cphenylalanyl-Lglutamic acid (67) and mtyrosyl-carginine (90), among others. In the face of the potentialities of the carbobenzoxy method for the synthesis of phenylalanyl and tyrosyl peptides, however, both the


47

halogen acyl halide and azlactone methods must now be considered to be of subsidiary importance. The carbobenzoxy method is especially suitable for the synthesis of such peptides, not only because of its general advantages, but also because the carbobenzoxy derivatives of phenylalanine and tyrosine give excellent crystalline acid chlorides. In the case of the chlorination of carbobenzoxy-ctyrosine, the phenolic hydroxyl group must be protected; either an acetyl (88) or a carbobenzoxy (3) group may be used for this purpose. There are numerous examples of the use of carbobenzoxy-cphenylalanyl chloride (85) or of 0-acetyl-N-carbobenzoxy-L-tyrosylchloride (88) for peptide synthesis. Of particular interest is the synthesis by Barkdoll and Ross (41) of L-tyrosyl-ctyrosyl-L-tyrosine by successively building up the peptide chain through coupling reactions involving 0-aoetyl-N-carbobenzoxy-ctyrosyl chloride:

The fact that tyrosyl peptides may be made equally well by the use of carbobenzoxy-L-tyrosine azide is illustrated by the synthesis of N-carbobenzoxy-L-tyrosylglycylglycine ethyl ester (50) and of N-carbobenzoxy-Ltyrosyl-S-benzylcysteineethyl ester (204). Peptides of the aromatic amino acids have become of considerable interest in recent years because of the finding that some of them (or their derivatives) are hydrolyzed by crystalline pepsin (187,204) or by crystalline chymotrypsin (50). Thus, pepsin hydrolyzes carbobenzoxycglutamyl-ctyrosine (187) and, as shown by Harington and Pitt Rivers (204), even free peptides, notably ccysteinyl-ttyrosine. For the preparation of synthetic substrates for chymotrypsin, it has been frequently necessary to convert the terminal carboxyl group of a phenylalanine or tyrosine peptide to the corresponding amide. This usually proceeds smoothly by the treatment of the appropriate carbobenzoxy peptide ester with ammonia in methanol. In this manner,

48

J0SE)PH 8. FRUTON

carbobensoxy-ctyrosylglycine ethyl ester was tramformed into the amide (50). When carbobensoxy-Irphenylalanylglycine ethyl ester was treated in the Bame way, however, the reaction took an unexpected course, and the resulting product was not the carbobenzoxy peptide amide. Instead, there occurred the elimination of bensyl alcohol and the formation of the amide of 5-bensylhydantoin-3-aceticacid (188) :

-

CHaCdI, C:H,CHaOCO-NH

bHCO-NHCHaCOOCaH, NH CH~CIHI CO-NH bHCO-NCHaCONHa + C'H&HaOH a

U

Of a large number of other carbobenzoxy dipeptide esters subjected to treatment with ammonia in methanol, only two other cases of hydantoin formation have been reported thus far. In one of these, carbobenzoxyGleucylglycine ethyl ester yielded the amide of 5-isobutylhydaritoin-3acetic acid (112b):

NHi

CeH'CHaOCO-NH CH,

CH:

\Ck

CO-NHbHCO-NCHaCONHa

I

+ CeH'CHaOH

The other case involves an exactly analogous reaction involving carbobensoxy-cmethionylglycine ethyl ester (112b). It would appear that these reactions are closely related to that postulated in the conversion of carboethoxyglycylglycine ethyl ester to carbonyl-bis-glycine by means of alkali (cf. p. S), which has been explained by the assumption that there occurs the formation of hydantoin-3-acetic acid aa a transient intermediate (296). This is in accord with the observation that such hydantoin derivatives are readily hydrolyzed by alkali (1924. To avoid the difficulty presented by the occurrence of hydantoin formation, it is possible to couple carbobenzoxy-cphenylalanyl chloride directly with glycinamide to give the desired dipeptide amide (188).

49


TABLE VII Dcrivdives of Phyldcmins

M. p., "C. (ref.)

Compound

GPhenylalaninamide acetate. . . . . . . . . DcPhenylalanine-N-car~nicacid anh cPhenylalany1-bphenylalaninamide.. . . LPhenylslanyl-ctyroain~ide. . . . . . . Acetyl-wphenylalanyl-bleucine.. . . . . . . . . . . . . . . . . 183-184 (44) Acetyl-cphenylalanyl-bleucine. . . . . . . . . . . . . . . . . . 191-193 (44) Acetyl-bphenylalanyl-cglutamicacid. . . . . . . . . . . . . 140 (67) Acetyl-D-phenylalanyl-btyroaine. Acetyldehydrophenylalanine. . . . . a-Acetaminocinnamic acid arlactone. Acetyldehydrophenylalanyl-calanine.

. . . . . . . 196-196 (116)

Acetyldehydrophenylalanyl-btyroaine.. .....

217-218 (67,116)

Acetyldehydrophenylalanyl-carginine. . . . . . . . . . . . . 192193 (60) Acetyldehydrophenylalanyl-bproline... . . . . . . . . . . . 140-142 (44) Carbobenroxy-cphenylalanine.. . . . . . . . . . . . . . . . . . 126-128 (86) Carbobencoxy-Dcphenylalanine . . . . . . . . . . . . . 103 (74) Carbobencoxy-bphenylalanylglycine. . . . . . . . . . . . . . 161-162 (44,188) Carbobensoxy-bphenylanylglycinamide. Carbobenmxy-cphenylanyl-cgluta Carbobenzoxyglycyl-bphenylalanine.. Carbobenroxyglycyl-bpphenylalanina Carbobenzoxyglycyl-bphenylalanylg Carbobenroxy-balanyl-x.-phenylalanine. . . . . . . . . . . 68-68 (63) Carbobeneoxy-x.-leucyl-D-phenylaninhydrazide. . . 170 (2044 Chloroacetyl-cphenylalanine.................... 126 (165) F'yruvyl-Dbphenyhhnbe. . . . . . . . . . . . . . . . . . . . . . . 94 (66) Tolueneeulfonyl-L-pltenylalanine.. . . . . . . . . . . . . . . . . 184-166 (168)

In discutming the synthesis of peptides of the aromatic amino acids, it is appropriate to add that peptides of 3,bdiiodo-ctyrosine may be prepared either by the iodination of a tyrosine peptide with iodine-potassium iodide in alkaline solution or by coupling reactions involving diiodotyrosine (13,187). It may be added that the carbobenzoxy method has been applied to the synthesis of peptides of @-phenyl-8-alanine(116). 4. Peptides of Cystine and Cysleine

The classical methods of Fischer permitted the synthesis of cystine peptideg in which the amino groups of the cystine moiety are linked to

JOSEPH 8. FRUTON

TABLE VIII Den'vdaves of Turoa'nc

M. p., "C. (ref.)

Compound

N-Carbobenzoxy-ctyrosinhydrazide... N-Carbobensoxy-ctyrosylglycine.. . . . . . . . . . . . . . . . . 100 (50) N-Carbobenzoxy-btyrosylglycinamide.. . . . . . . . . . . . 116 (50) N-Carbobeneoxy-ctyrosylglycylglycine.. . . . . . . . . . . 213-215 (2) N-Carbobenzoxy-btyrosylglycylglycinamide... . . . . . 218 (50) N-Carbobencoxy-L-tyrosyl-Lcysteine . . . . . . . . . . . . . . 120 d. (204) N-Carbobenzoxy-btyrosyl-S-benzyl-Lcysteine. . . . . . 166 (204) Bis(N-carbobenzoxy-ctyrosy1)-Lcystine.... N-Carbobenzoxy-btyrosyl-ctyrosine.. . . . . . N-Carbobenzoxy-ctyrosyl-ctyrosinamide.. . . . . . . . . 187-189 (188) N-Carbobensoxy (tri-btyrosy1)-ctyrosine

... .....

224-225 (41) 107 (50)

Carbobenzoxy-calanyl+tyrosine. . . . . . . . . . . . . . . . . . 149-150 (53) Carbobenzoxy-calanylglycyl-btyrosine.. . . . . . . . . . . 128 (2) 0,N-Dicarbomethoxy-btyrosine. . . . . . . . . . . . . . . . . . . 97 (206) N-Chloroacetyl-btyrosine........................ 86-87 (129) Glycyl-Ltyrosine diketopiperazine . . . . . . . . . . . . . . . . . 295 (1 66) N-Toluenesulfonyl-btyrosine...

glycyl (171), alanyl, or leucyl (150) residues.* In order to prepare the corresponding cysteine peptides, Pirie (255) reduced the disulfide group with zinc and acid and isolated the sulfhydryl peptide as a cuprous mercaptide. The advent of the carbobenzoxy method wa8 decisive in the develop

* In naming peptidea in which both amino groups of cystine are substituted by similar amino acid residues, it appeara desirable to use the prefix "bis" as in bisglycylL-cystine. Similarly, where both carboxyl groups of cystine are linked to similar

61


TABLE IX Peptides of Phmylalanine and Tyosine reml "C.

Peptide Glycyl-D-phenylalanhe Glycyl-bphenylalanine Glycy1-D-phenylalanyl-cglutamic acid Glycyl-bphenylalanyl-bglutamii acid Glycyldehydrophenylalanine Glycyldehydrophenylalanylglycine DbAlanyldehydrophenylalanine cLeucyl-~-phenylalanine.2H~O LPhenylalanylglycine D-Phenylalanyl-D-leucine LPhenylalanyl-cglutamic acid Glycylglycyl-ctyrosine Glycyl-balanylglycyl-ctyrosine Glycyl-balenyl-Ltyrosine Glycyl-ctyrosine Glyc y l - ~yrosylglycine t Glycyl-diiodo-ctyrosine cAlanylglycy1-ctyrosine L-Alanyl-ctyrosine L- Alenyl-diiodo-ctyrosine bLeucy1-ctyroeine cTyroaylglycine >Tyros ylglycylglycine cTyroeyl-t-aspartic acid L-Tyrosyl-~tyromne cTyrosyl-Lcysteine Bis(ctyrosy1)-Lcystine D-Tyrosyl-carginine

-41.7 4-42,a -11.3

21 20 20

-4.7

20

... ...

.. . ...

...

...

274 165 65

... ...

65 65

...

...

17 20

...

2.4%, HAc 2.4%) HzO

+20.3

20

4.8%, HzO

+42.3 +18.4 -4.8 4-43.7 +24.1 +52.7 4-41.9 4-43.1 4-62.9 +10.4 4-83.5 4-42.8 4-20.4 4-30.1 +22.6 -70.8 -105.7

20 20 20 25 20 20 20 20 20 20 20 20 19 19 23 23 23

1.6%, HzO 2.6%) HYO 4.3%, HzO 2%, H20 1 eq. HCl 4.1%, Ha0 5%, N&OH 4.6%, Ha0 2%, HzO 7.8%, NHdOH 2%, HzO 2.2%, HzO 2.7%, 20% HCl 3.6%, HzO 1 eq. HC 4%, HzO 1 eq. HCl 5%, N HCl 5%, N HCl 6.4%, 0.2 N HCI

4-25 4-54.2

Ref.

Concn. and solvent

198a 2828 165 276 07

...

+

+

+

~

2 2,140 17 129,274 2 13 2,139 17 17 17 2,3,274 2 88 88 204

204 90

_ I

ment of new methods for the synthesis of cystine and cysteine peptides. The need for the synthesis of such peptides became urgent with the formulation of glutathione as 7-cglutamyl-tcysteinylglycine (218). Final proof of this structure was provided by Harington and Mead (202), who synthesized the tripeptide by means of the carbobenzoxy method, amino acid residues (e.~., glycine) the peptide should be named ccystinylbisglycine. In this manner, it may be possible to avoid the ambiguity inherent in names such a8 diglycyl-rccyetine, ccystinyldiglycine, or ccystinylglycine.

52

JOBBIPH 8. FaUTON

which they modified to eliminate the carbobenzoxy group by treatment with phosphonium iodide in acetic acid at 45-50'. This modification was necessitated by the poisoning effect which sulfur compounds exert on palladium or platinum catalysts for the hydrogenation reaction.

[f'+

3

HNH-OCOCHlC~Hr O-NHCHaCOOCaHr

I

NH-OCOCHLhH I

CHlSH

+C1COCHrCH1CBCOOCHI

~HNHI

b

ho--NHCHICOOCIHr NH4COCHsCeHr

I:Z:--COCHaCH*

c:

saponification

HCOOCHl

____)

reduction

bo-NHCH,COOC1Hr CHaSH AHNH-cOCHICH1

IH1

HCOOH

L-NHCH,COOH

Despite its considerable value in the initial synthesis of glutathione, this reduction method has given way to the procedure of Sifferd and du Vigneaud (272), who introduced the use of sodium in liquid ammonia at -60' (286) for the elimination of the carbobenzoxy group. The latter method gives better yields, and its use by du Vigneaud and collaborators, as well as by Harington, has led to the synthesis of a variety of interesting cystine and cysteine peptides. In the synthesis of cysteinyl peptides, it is frequently necessary to protect the sulfhydryl group in the course of the synthetic operations. This may be done by treatment of the cysteine moiety with benzyl chloride to give the S-benzyl thioether. S-benzylcysteine itself may be prepared by the reduction of cystine with sodium in liquid ammonia, followed by the discharge of the color due to the sodium ion with ammonium chloride and treatment with benzyl chloride (300). This procedure is also suitable for the preparation of S-beneylcysteinyl peptides (240) : CHT-S--5-CHI LNH1

hNH1

L N H R

hO-NHR

CHaSH

-

Na 2h N H I lis. NHI hO--NHR

benayl

CHaSCH&.H,

2 bHNHl

L---NHR

As may be expected, the S-benzyl group can be removed by reduction with sodium in liquid ammonia, so that this substituent is eliminated in the same operation which reduces the carbobenzoxy group. The application of the modified carbobenzoxy method for the synthesis of cysteine peptides is well illustrated by the sequence of reactiona

53


employed by du Vigneaud and Miller (291) for the synthesis of glutathione (cf. also 206s) : CHBCHxCaHt

k

NH4COCHiCeHr

~

~

+ ClCOCHaCHrbHCOOCH:

I

~

~

~

+

NH4COCHrCeHr

LHCOOCH: ~ H &

~

LO-NHCHaCOOCH:

H

~

isponification

r s

reduction

I:::-- lHx c o c H , c H I HCOOH

LO-NHCHxCOOH

By an analogous series of reactions, Kogl and M e r m a n (224) have synthesized 7-D-glutamyl-ccysteinylglycine, which these authors term epi-g-glutat hione. The aspartic acid analog of glutathione, 8-caspartyl-ccysteinylglycine (asparthione) has been synthesized by the coupling of carbobenzoxy-caspartyl-a-benzyl ester @-chloridewith S-benzyl-ccysteinylglycine methyl ester, followed by saponification and reduction of the coupling product (248). It is of interest that, when S-benzylcysteinylglycine was coupled with carbobenzoxy-cglutamic acid anhydride, the condensation product TABLE X Derivatives of cy8h*neand Cy8tine ~~

Compound

M.p., "C. (ref.)

S-Beneyl-ccysteine. . . . . . . . . . . . . . . . . . . . . . . . . . S-Bencyl-mysteine ethyl eater HC1. . . . . . . . . . . . S-Beneyl-mysteinylglycine . . . . . . . . . . . . . . . . . . . . Carbobeneoxy-ccysteinyl-L-tyroshe.. . . . . . . . . . . Carbobencoxy-S-benryl-ccystehe.. . . . . . . . . . . . . Carbobencoxy-S-benzyl-mystebhydraeide. . . . . . Carbobeneoxy-S-benzyl-ccysteinyl-ttyroshe. . .

216-218 d. (300) 156-157 (204) 166-167 (240) 160-162 (204) 93-95 (203) 133-134 (204) 198.200 (204)

Bis(carbobensoxyglycy1)-ccystbe. . . . . . . . . . . . . . 130-132 (222s) Biscarbobensoxy-ccystine. . . . . . . . . . . . . . . . . . . . . 123 (74) Biscarbobeneoxy-bcystinylbisglycine. . . . . . . . . . . 182-183 (240) 210 (196) Biscarbobeneoxy-Lcystinylhis(glycylg1ycine). . . . Bkarbobeneoxy-ccystinylbis-btyrosine.. . . . . . . 158 (204) Monochloroacetyl-mysthe. . . . . . . . . . . . . . . . . . . . 185-190 d. (35,150) Bbchloroacetyl-ccystbe... . . . . . . . . . . . . . . . . . . . 136 (171,255)

l

54

JOSEPH 8. FRUTON

represented the a-peptide derivative of glutamic acid, rather than the y-peptide derivative required for the synthesis of glutathione (cf. also page 42). du Vigneaud et al. (289) took advantage of this result to prepare a-r.,-glutamyl-ccysteinylglycine (isoglutathione) . TABLE XI Peptidea of Cvsteine and Cvstine Peptide Glycyl-bcysteine HCl r,-Cysteinyl-cglutamine L-Cyateinyl-btyrosine L-Cysteinyl-ccy steine Bie(glycy1-cleucy1)-ccyetine Bisglycyl-bcystine Bin(o-alanyl)-bcystine Bis(talany1)-kcystine Bie(bleucylglycy1)-ccystine Bis(bleucyl-qalanyl)-ccystine Bin (cleucyl)-ccystine bCystinylbisglycine bCystinylbk(glycylg1ycine) bCystinylbia(Ltyrosine) -~ a

I

Ref.

+2.4" +6.6@ +15.2 +35 -108.9 -104.3 -227.9 -137.4 -72.2 -115.3 -136.6 -86.0 -55 -50.8

X = 546 mp.

To the list of cysteinyl peptides prepared by means of the carbobenzoxy method there may be added ccysteinyl-cglutamine (203) and ccysteinyl-ctyrosine (204). It may be noted in this connection that either 8-benzyl-N-carbobenzoxy-ccysteinylchloride or the corresponding aside may be employed for the coupling reactions, although the use of the azide appears to give somewhat better yields. For the preparation of cystinyl peptides, the cysteinyl peptides may be oxidized by aeration (204). I n addition, Loring and du Vigneaud (240) made bcystinylbisglycine by treatment of its dicarbobenzoxy derivative with sodium and liquid ammonia and reoxidation by aeration of the sulfhydryl group which appeared during the reduction. In a similar manner, Greenstein (196) prepared ccystinylbis(glycylg1ycine). Although no reports of the synthesis of homocystine or homocysteine peptides have come to our attention, it may be expected that these can be made by the use of the methods described above. In addition, it should be of interest to explore further the possibilities of homocysteine thiolactone as a coupling agent in peptide synthesis in view of the finding


55

of du Vigneaud el d.(292) that this substance, on treatment with alkali, forms a diketopiperazine: S-CHI

It may be added that White (297a) has described the synthesis of glycyltaurine and glycylcysteic acid. 5. Peptides of Serine

Prior to the application of the carbobenzoxy method to the synthesis of serine peptides, two procedures had been employed for this purpose. Fischer and Roesner (163) made glycyl-Dcserine and optically inactive alanylserine from the corresponding chloracetyl and bromopropionyl derivatives. Fischer (137) obtained a partially racemized preparation of Gseryl-cserine by partial hydrolysis of Gserine anhydride, which had been isolated from a n acid hydrolyzate of wool. The hydrolysis of synthetic diketopiperazines containing seryl residues also was employed by Bergmann and Miekeley (62), who described the synthesis of optically inactive phenylalanylserine, and by Abderhalden and Bahn (4) , who reported the preparation of Dcserylglycine and optically inactive serylleucine. The latter workers have claimed that the treatment, with acid, of diketopiperazines composed of a serine residue and another amino acid residue yields seryl dipeptides, while treatment with alkali gives aminoacylserine dipeptides : acid

HN

/ \

CO-CH

R

alkali

Abderhalden and Bahn (4) have prepared tyrosylserine by the reaction of 0,N-dicarbobenzoxy-L-tyrosylchloride (3) with Dkserine, followed by hydrogenation of the coupling product. Esterification of this peptide, followed by treatment with ammonia in methanol gave the corresponding diketopiperazine, which, on partial hydrolysis with acid,

56

JOSEPH 8. FRUTON

yielded a noncrystalline mixture of the diastereoisomeric D- and cserylctyrosines. The use of the carbobenzoxy method has recently permitted the synthesis of a number of cseryl peptides (181). The procedure involved the coupling of carbobenzoxy-cserine azide with an amino acid ester, followed by saponification and hydrogenation: R

CHIOH

I

I

hHNH-OCOCHzCaH'

+ NHkHCOOCH: -+

In this manner, there have been prepared L-serylglycine, L-seryl-calanine, cseryl-cglutamic acid, and cseryl-cserine. Furthermore, Lserylglycyl-cglutamic acid has been made by the carbobenzoxy method as follows (186) : CHzCOOCzHs

CHIOH

AH*

t:

C~H'CH~OCO-NH H +

AO-NHCH;CON: CHzOH I

+

AHNH, AOOCZHI

CHzCOOCzHr I

60-NHCH~CO-NH~H I

C'OOH

An analogous series of reactions has led to the synthesis of L-seryl-b alanyl-cglutamic acid. It will be noted that, in the syntheses just cited, the azides have been used in preference to the acid chlorides. This was made necessary by the presence of the 8-hydroxyl group of serine, although it may be expected that the protection of this group by conversion to a benzyl

57


ether would make it possible to make the desired acid chloride for coupling reactions. In conducting the coupling reaction between carbobenzoxy-cserine azide and an amino acid ester, it is desirable to maintain the temperature below 25", since the azide, when warmed, may undergo an intramolecular condensation to form 4-carbobenzoxyaminooxazolidone-2 (181). CHiOH

CsHrCHzOCO-NH

A

CHIO

+

HCONj

I

CsHsCHzOCO-NH

A'

'co

HNH

A similar reaction had been observed by Schroeter (268) on heating the azide of 8-phenylhydracrylic acid. A recent report by Woolley (303) describes the use of p-toluenesulfonyl-m-serine azide in coupling reactions with amino acid esters. Attention may be called to the excellent method for the isolation of relatively large quantities of L-serine from hydrolyzates of silk fibroin (279). This obviates the need for the resolution of synthetic serine according to the method of Fischer and Jacobs (152). The value of optically pure Lserine and its derivatives in the study of protein metabolism is brought out by the recent findings of Fishman and Artom (174) and of Dent (113). Little can be said a t present concerning the synthesis of peptides of D- or Lthreonine or of D- or Lallothreonine, except that it is to be anticipated that the knowledge gained in the synthesis of serine peptides will TABLE XI1

-

Derivatives of Serine

Compound

M. p., "C. (ref.)

LSerine anhydride. ..................... 0-Benzoyl-Dbserhe ........... Carbobenzoxy-Lserine . . . . . . . . Carbobeneoxy-tserinamide. . . . .

. . . . 132-133 (181)

Carbobenaoxy-bser ylglycine . . . . . . . . . . . . . . . . . . . 131 (181) Carbobenzoxy-Lserylglycinhydrazide. . . . . . . . . . . . . . . . 181-182 (186) Carbobeneoxy-tseryl-Lalanine . . . . . . . . . . . . . . 161-162 (181) Carbobenaoxy-bser yl-Gglu tarn . . . . . . . . . . . . . . . 152-153 (181)

Toluenesulfonyl-Dr,serinhydrazide. . . . . . . . . . . . . . Toluenesulfonyl-Dbserylglycinhydrazide.. . . . . . . . . . . . . 215-216 (303)

58

JOSEPH 8. FRUTON

prove useful in their preparation. It may be hoped that this gap will be filled in the not too distant future, and that threonine peptides and related derivatives will become available for studies of enzyme specificity and intermediary metabolism. TABLE XI11 Peptides of Serine Concn. and solvent

Pcptide

+30.2 -3 0 . 4

~ S eylglycine r L-Seryl-talanine tSeryl-tglutamic acid L-Seryl-Lserine

-9.4 f14.2

26 26 25

25

1

6%, N HCl 6%, N H C I 6%, N HCI 7%, N HCl

Ref. 181 181

181 181

6. Peptides of Lysine and Other Diamino Acids

As in the case of several other amino acids with polar side chain groups, only isolated cases of the synthesis of lysine peptides were described prior to the invention of the carbobenzoxy method. Fischer and Suzuki (172) prepared optically inactive lysyllysine (as a picrate) by partial hydrolysis of the diketopiperaaine. Abderhalden and Sickel (31) obtained peptides of Dclysine, in which both the a- and c-amino groups were involved in peptide linkage, by the treatment of the amino acid with halogen acyl halides, followed by amination of the coupling products. An interesting application of the Schoenheimer method (cf. page 23) was the preparation, by Enger (120),of eglycyl-Dclysine by the conversion of ebenzoyllysine to the a-toluenesulfonyl derivative, removal of the e-beneoyl group by partial hydrolysis with alkali, and treatment of the resulting product with toluenesulfonylglycyl chloride. The toluenesulfonyl groups were then removed by reduction with phosphonium iodide and hydriodic acid and the dipeptide, which was difficult to purify, was obtained as a crystalline picrolonate:

lH1

NH-COC6H'

c:

( Ha14

( Hl)4

+CHaC~H~SOi-NHCH~COCI

__ ____)

AHNH-SO~C~H~CHI ~ H N H - S O ~ C ~ . H & H I

AOOH

AOOH NH-COCH2NH-SO2CaHdCHa

AI

(

Hi),

CHNH-SOIC~H~CHI

AOOH

NH-COCH~NHI PHiI (

+ HI

A

H1)4

LNH2 LOOH

59

BYNTHEBIB OF PEPTIDEB

Turning now to the application of the carbobenzoxy method to the synthesis of lysine peptides, it may be well to discuss first the preparation of clysyl peptides. This presents no special difficulties, since cqa-dicarbobenzoxy-clysine azide may readily be coupled with amino acid or peptide esters in the usual manner: C~H'CHZOCO-NH

CoHsCHnOCONH

I

I

R I

60N:

60-NHbHCOOCH:

For example, Bergmann et al. (813 4 ) made clysylglycine, clysyl-c glutamic acid, clysyl-caspartic acid, and clysyl-ehistidine by the saponification of the appropriate coupling product, followed by hydrogenation in the presence of palladium. I n view of the basic character of clysylglycine and clysyl-chistidine, these dipeptides were isolated as sulfates. Prelog and Wieland (255a) made clysylglycylglycyl-c glutamic acid by an analogous procedure. For the synthesis, by the carbobenzoxy method, of peptides in which both of the amino groups of lysine participate in peptide linkage, special methods are not required, since the reaction of a carbobenzoxyamino acid chloride (or azide) with Glysine methyl ester will lead to the synthesis of ale-lysine peptides. When, however, peptides are desired in which the a-amino group of lysine is bound in peptide linkage and the eamino group is free, the modification introduced by Bergmann et al. (86) must be used. This procedure takes advantage of the property of carbobenzoxyamino acid chlorides to eliminate bensyl chloride and to form the corresponding N-carbonic acid anhydrides (cf. page 27). Thus, when dicarbobenzoxy-clysyl chloride is heated to 50-60°, an anhydride is formed which, on treatment with water, yields ecarbobensoxy-clysine. On treatment with methanol and hydrochloric acid, the anhydride gives a-carbobenzoxy-clysine methyl ester hydrochloride : CsHICH:OCO-YH CeHBCHxOCO-NH

L

( Hi14

CeH'CHxOCO-NH

A

H

bOCl

OC

'I

'0-

0

60

JOSEPH 5. FRUTON

The monocarbobenzoxy ester may be coupled with the chloride or azide of a carbobenzoxyamino acid or peptide to give a dicarbobenzoxy peptide ester which, in turn, may be converted to a hydrazide and azide, thus making it possible to lengthen the peptide chain by a further coupling reaction with an amino acid or peptide ester. After saponification of the coupling product, both carbobenzoxy groups may be removed by catalytic hydrogenation to yield the free peptide: C,H,CH*OCO-NH

+

AHR

Aoa

AOOCHa

LOOH

Bergmann et al. (86) have used this method to prepare a series of lysine derivatives, some of which were later found to be substrates for crystalline pancreatic trypsin (214). For example, a-hippuryl-Llysinamide hydrochloride was made as follows (63,214) :

+ CsH,CO-NHCHzCOCl+

A practical note of some importance in carrying through the conversion of dicarbobenzoxy-Llysine to the chloride (preparatory to the synthesis of the N-carbonic acid anhydride) is the desirability of using the syrupy dicarbobenzoxyamino acid rather than the crystalline derivative. The latter, because of its insolubility in ether, reacts with phosphorus pentachloride extremely slowly. As shown by Synge (282a), the methods outlined above for the synthesis of lysine peptides are applicable t o bornithine a8 well. This

61


TABLE XIV Derivatives of Lysine, Ornithine, Arginine, and Hietidine Compound

M. p., "C. (ref.)

200-202 (214) a-Benzoyl-Llysinamide HCl . . . . . . . . . . . . . . . . . . . . . . t-Carbobenzoxy-Llysine methyl ester HCl cCarbobenzoxy-Llysine-N-carbonicacid a a,cDicarbobenzoxy-Llysine. ...................... 150 (86) a,cDicarbobenzoxy-Llysinamide... . . . . . . . . . . 155 (86) a,t-Dicarbobenzoxy-~lysinhydrazide ... . . . . . . . 159 (81) a,tDicarbobenzoxy-L-lysylglycine. . . . . . . . . . . . . . . . . 158-159 (84) a,cDicarbobenzoxy-Llysyl-cglutamic acid.. . . . . . . . 130 (81) a,t -Carbobenzoxygl ycyl-t-carbobenzoxy-~lysinhydrazide.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 (86) a-Glycyl-Llysine methyl ester 2HC1. ...... pHippury1-clysinamide HCl . . . . . . . . . . . . . a-Hippuryl-ccarbobensoxy-Irlysine.. . . . . . . . . . . . . . . 148-149 (214)

d-Carbobenzoxy-cornithine - (282a) d-Carbobenzoxy-Lornithine-N-carbonic acid anhy86-88 (282a) dride. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a,&Dicarbobenzoxy-~ornithine.. . . . . . . . . . . . . . . . . . 112-114 (282a) ~ , b D i c a r b o b e n ~ o x y - ~ o m i t h y l - ~ l e u c i n e ~ 2..... H ~ O .63-64 . (282s) a-(Carbobenzoxy-Lvaly1)-bcarbobenzoxy-L ornithine . . . . . . . . . . . . . . 193-195 (282s) LArgininamide 2HC1. ............................ Triacetylan h ydro-D~arginine. . . . . . . . . . . . . . . . . . Benzoyl-Largininamide HCl . . . . . . . . . . . . . . . . . . . . . . Monocarbobenzoxy-Larginirie. . . . . . . . . . . . . Carbobenzoxynitro-~arginine. . . . . . . . . . . . . Carbobenzoxyglyc ylnitro-~arginine Hippurylnitro-~arginine. . . . . . . . . . . . . . . . Hippurylnitro-Largininamidc.. . . . . . . . . . . . . . . . . . . .

148-152 (216)

GHistidine anhydride. . . . . . Bensoyl-Lhistidinamide. . . . Carbobenzoxy-Lhistidine. . . . . . . . . . . . . . 209 (74) Carbobensox y gly cy 1-thistid Carbobenzoxy-Lalanyl-&his Carbobenzoxy-j3-alanyl-Lhistidine.. . . . . . . . . . . . . . . . 160-161 (272)

investigator has also found that 6-carbobenzoxy-cornithine may be prepared conveniently by the reaction of carbobenzoxy chloride with the copper derivative of ornithine. Bergmann and Koster (60) showed that phenylalanylarginine (cf. page 17), on treatment with acetic anhydride, yields phenylalanylornithine :

62

NH~~HCO-NHC~HCOOH ThiB method has many disadvantages, however, not the least of which is the extensive racemization caused by the acetic anhydride. It may be added that the methods developed by nergmann et al. (86) for the synthesis of lysine peptides have been applied with success by Schneider (265) in the synthesis of peptides of a,p-diaminopropionic acid. TABLE XV Peptides of Lysine, Ornithine, Arginine, and Histidine

-

GLysylglycine sulfate ~ L y s y(diglycy1)-bglutamic l acid cLysyl-bglutamic acid cLysyl-baspartic acid cLysyl-bhistidine sulfate a-(cValyl)-bornit.hine HCl.%H,O cOmithyl-cleucine HCI.jf;HaO

Concn. and solvent

22 20 19 20 20

1.5%, H20 1%, HzO 3.1%, HzO 1.3%, Hz0 1.3%, H20

84 255a 81 84 81

+2

19 18

2%, Hz0 2.1%, H20

282a 282a

...

...

...

...

...

+25 -2.5 +27.0 -20.4 +20.5 +12.3 +32.1

26 24 27 28 25 30 18

+

30 +33.5 +22.9 +23 +35.3

+19

Glycyl-barginine sulfate Glycylglycyl-bhistidine sulfate Glycyl-bhistidine HCI D-Alanyl-bhistidine GAlany 1-bhistidine p-Alanyl-D-histidine p- Alany 1-bhistidine p-Alanyl-1-methyl-bhietidine L-Leucyl-thistidine

I

Temp. "C.

Peptide

-

... 1%, HzO 1%, Hz0 I%, HtO 2%, H2O 2%, HZ0 5%, Hz0 5%, Hz0

1

Ref.

~

.83 258 221 220 220 240 272 45 147

P

7. Peptides of Arginine Of the protein amino acids, arginine presents perhaps the greatest difliculties in peptide synthesis. This is due, in large part, to the strongly


63

basic character of the guanido group. Because of the physiological importance of arginine and its derivatives, numerous attempts have been made to overcome these difficulties and to solve the problems encouutered in the synthesis of arginine peptides. Fitxher and Suzuki (172) obtained, by the autocondensation of Garginine methyl ester, a substance which they designated tentatively as “arginylarginine.” Experiments by these and later workers (118, 226), however, cast considerable doubt on this formulation of the reaction product, but it remained for Zervas and Bergmann (305) to show that the material actually was the anhydride of a,6-bisguanido-n-valeric acid. What is more, it was found that, in the course of the reaction, ornithine methyl ester is formed. It became clear, therefore, that the autocondensation of Garginine methyl ester leads to a dismutation of arginine into ornithine and bisguanidovaleric acid : NHz

NH,

A=NH

L=NH

ILH

AH

AI

( Hz)a

CHNHl COOCHI I

NHz

I

(CHI):

‘ b Hi): NH + L N H Z (

LHNHJNH

b0-

LOOCHI

I

Some progrem in the synthesis of arginine peptides came with the demonstration by Bergmann and Koster (60) that acetylaminocinnamic acid azlactone reacts readily with carginine in alkaline solution to give acetyldehydrophenylalanyl-carginhe. On hydrogenation in the presence of palladium black, there resulted a mixture of diastereoisomers which were hydrolyred with dilute hydrochloric acid to remove the acetyl groups. By the use of salicylaldehyde (an excellent precipitant for arginine and other amino acids, 70), these workers succeeded in isolating D-phenylalanyl-carginine, but were unable to obtain the isomeric cphenylalanyl-carginine in pure form. An analogous sequence of reactions, using acetylamino-p-acetoxycinnamic acid azlactone, led to the synthesis of D-tyrosyl-1,-arginine (90). The limited possibilities of the azlactone method for the synthesis of arginine peptides led to the application of the carbobenzoxy method when it became available. The principal problem was to find a substituent for the guanido group which would mask its basic properties and which also could be removed by catalytic hydrogenation at the end of the peptide synthesis. Bergmann et al. (83) showed that the nitro group was suitable for this purpose, since nitro-carginine (225) could be. con-

64

JOSEPH 8. FRUTON

verted to carginine by reduction with hydrogen in the presence of palladium black. It was noted, however, that, at normal temperature and pressure, the hydrogenation is slow. Nitro-L-arginine was used by Bergmann et al. for the synthesis of glycyl-barginine sulfate.

dOOH

In later studies by Bergmann and collaborators, it was shown that crystalline pancreatic trypsin hydrolyzes certain arginine derivatives (55), some of which were prepared by the method outlined above. Thus, hippuryl-L-argininamide hydrochloride was synthesized by the coupling of hippuryl chloride with nitro-carginine methyl ester, followed by treatment with ammonia and hydrogenation of the nitro group (216). It may be added that a typical substrate for crystalline trypsin, benzoylcargininamide hydrochloride, may be prepared from the corresponding methyl ester by treatment with ammonia (55,114). Thus far, no well-defined peptides have been synthesized in which the carboxyl group of arginine is linked to another amino acid. The substituents employed thus far to block the guanido group are not sufficient to mask its reactivity, and, as a result, attempts to convert the carboxyl group of carbobenzoxynitro-carginine or dibenzoyl-carginine to an acid chloride or an aside have not been successful. Nor has it been possible to prepare dicarbobenzoxy-carginine (186) by the methods which yield other disubstitution products of arginine, such as dibenzoylcarginine (123) or dibenzenesulfonyl-L-arginine (100). Another possible approach to the synthesis of arginyl peptides, which remains to be explored, may be the prior synthesis of the corresponding ornithyl peptides by the method discussed previously for lysine peptides, followed by the conversion of the &amino group to a guanido group by treatment with cyanamide (42), guanidine (256), S-methylisothiourea or O-methylisourea (196a,269).

65


8. Peptides of Histidine The preparation of peptides in which the amino group of Ghistidine is linked to another amino acid may readily be achieved by the coupling of histidine methyl ester with acid chlorides or azides. Histidine methyl ester is best prepared from the dihydrochloride by neutralization with sodium methylate according to the directions of Fischer and Cone (147). The reaction of d-a-bromoisocapronyl chloride with L-histidine methyl ester yields a crystalline product which, upon saponification and amination, gives Lleucyl-chistidine (147). In a similar manner, Abderhalden and Geidel (11) prepared glycyl-chistidine and related dipeptides of histidine. I n general, the yields in these syntheses were unsatisfactory. The introduction of the cnrbobenzoxy method made it possible to synthesize a large variety of histidine peptides in good yield. du Vigneaud and collaborators have used this method with marked success in the syntliesis of glycyl-L-histidine (221), calanyl-L-histidine (220), 8 - e aspartyl-L-histidine (288), as well as several other similar peptides. The procedure usually employed was to allow the appropriate carbobenzoxyamino acid chloride to react with Ghistidine methyl ester, and the carbobenzoxy peptide ester was saponified and hydrogenated. In the synthesis of a-caspartyl-D-histidine, Greenstein and Klemperer (197) used carbobenzoxy-Laspartic acid anhydride for the coupling reaction. In the synthesis of p-alanyl-chistidine (carnosine) or 8-alanyl-1-methyl-c histidine (anserine), du Vigneaud and associates condensed carbobenzoxy8-alanyl aside with the appropriate ester (45,272):

CaH,CH*OCO-NHCH,CH&ONa

+ NHn

I

I

N \Ck

NH--OCOCH&sHr AH*

NH

ssDonification

NHzCH&H&O-NH

COOH

bHCHzC===C

H

A practical detail which may be noted in connection with syntheses of the type mentioned in the preceding paragraph is that, because of the basic character of the imino group of the imidazole ring, acid is to be

66

JOSEPH 8. FRUTON

omitted in the extraction of the reaction mixture (cf. page 28). Instead, several extractions with water are substituted. In the course of their studies on the synthesis of amino-N-methy1-Lhistidine, du Vigneaud and Behrens (287) found that the imino group of the imidazole ring, after treatment with sodium in liquid ammonia, would react with benzyl chloride to give a benzyl derivative. The benzyl group may be removed by reduction with sodium in liquid ammonia, as in the case of carbobenzoxy and S-benzyl groups. In this manner, it was possible to block the imino group while methylating the a-toluenesulfonylamino group according to the method of Fischer and Bergmann (145). It may be added that du Vigneaud and Behrens also found that the toluenesulfonyl group may be removed by reduction with sodium and liquid ammonia. There are relatively few examples of the synthesis of histidyl pepti,des. The first of these was the preparation of histidylhistidine from L-histidine anhydride (22,172); no data are available, however, to indicate the extent of racemization of the dipeptide. Another method for the synthesis of Dthistidyl peptides was described by Bergmann and Zervas (73), who showed that the treatment of acetyl-Dthistidine with acetic anhydride gives rise to a product (not isolated, but assumed to have the azlactone structure indicated below) which reacts wkh glycine ethyl ester to give acetyl-Dthistidylglycine ethyl ester. Brief hydrolysis with dilute hydrochloric acid gave the desired peptide, although the yield in this method is an extremely modest one. CH==CCH&HCOOH

I

NH

I

N

\C
iss:rtation, Technische Ilochschule, Darmstadt. 220. Bowes, J. €I., and Kenten, R. I f . (1949). Riochem. J . 44, 142. 221. Weir, C. E. (1949). J . Am. Leather Chemists’ Assoc. 44, 108. 222, Weir, C.E. (1949). J . A m . Leather Chemists’ Assoc. 44. 142.


Proteins, Lipids, and Nucleic Acids in,Cell Structures and Functions BY ALBERT CLAUDE The Rockefeller Institute for Medical Research, New York, New York

CONTENTS Page

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. The Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. The Nucleus.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Chromosomes and Chromatin. . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Nucleolus. ...................................... IV. The Cytoplasm. ...................................... 1. Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Microsomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Fibrous Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Constitution and Duplication of Living Matter.. . . . . . . . . . . . . . . . . . . . . . . VI. Phospholipids in Cell Structures and Functions.. . . . . . . . . . . . . . . . . . . . . . . VII. Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Nucleic Acids .............................................. References . . . . . . . . . . . . .. .. . . . . . . . .

428 428 430 432 432 433 434 436 437 . 439

I. INTRODUCTION Proteins are products and constituents of cells. I n the past, chemists have been increasingly successful in isolating proteins from their cellular environment, and in determining some of the specific properties that these compounds exhibit in vitro. There is no doubt that these properties are utilized by the cell, but it is not known to what extent such properties are modified by virtue of chemical association with other cellular constituents, and by the influence of other associated or competing biochemical systems. It is no more plausible to assume that the properties exerted by a protein in the cell are just those exhibited by the isolated protein in vitro than to consider th at the properties of amino acids separated from a protein hydrolyzate can entirely explain the properties of the original protein. In the cell, proteins are parts of structures of considerable complexity, of ten in association with phospholipids or nucleic acids. I n recent years, methods have become available which permit the separation of certain morphological constituents of the cell, in quantities sufficient for biochemical analysis. The present paper deals 423

424

ALBERT CLAUDE

with a n approach to the direct study of the nature of a number of cellular complexes, and the integration of biochemical functions. The reactivity of chemical compounds is determined not only by their elementary composition but, t o a much greater extent, by the spatial disposition of the individual atoms within the molecule, and by the configuration of the molecule as a whole. This property, which allows for considerable variations, is fully utilized by biological systems and is the basis of the specificity of biochemical reactions. The sensitivity of biological systems to molecular configuration was vividly demonstrated by Pasteur when he showed that Yenicillium glaucum could utilize the d but not the I form of tartaric acid (42). In recent years observations have been rapidly accumulating which show the importance of structural variations in the control and conduct of essential biological processes; minor changes, as slight as the rotation of a single carbon atom in the prosthetic group of an antigen, affect the reactivity of the entire protein complex, and the nature of the immunological response (2,31); analogs and isomers are found to play decisive roles in problems of nutrition and growth, and in the limitation and invasiveness of infectious diseases (27,62). Isomeric variations may prove to be sufficient to determine the genetic specificity of certain nuclear constituents, for example, in the case of the nucleic acids isolated by Avery and coworkers (3,34). At a higher level of organization, genetic studies have repeatedly shown the dependence of gene effect on the position of the gene along thc chromosome (26,55). It is clear that the larger the molecule or the molecular aggregate, the greater will be the opportunity for structural variations and for reciprocal influences between the various parts of the complex, and for ultimate effect on the properties of the complex as a whole. Active cellular components such as enzyme proteins, coenzymes, hormones, and vitamins are being isolated in increasing number, and the kinetics of their specific properties determined in vitro. In vitro, however, isolated chemical reactions follow a uniform course and eventually reach an equilibrium which, for the cell, means death. In living protoplasm active chemical constituents are linked to form three-dimensional systems of varying, and probably considerable, complexity ; substances known to be readily diffusible in water are held in place and consequently are not permitted to react freely. I n this manner, chemical processes can be channelled through preferential paths, slowed down, stopped, or speeded up, according to needs or to conditions established possibly at a distance rcmote from the locus where the particular reaction is taking place. It is in this structural arrangement, which permits a particular system t o determine and control the timing, order, and sequence of a

PROTEINB, LIPIDS, NUCLEIC ACIDS IN CELLS

425

great variety of individual chemical reactions that probably resides, for the biologist, the ultimate challenge of living matter. Morphological and cytochemical studies, especially in recent years, have indicated that important cellular functions are localized in definite areas of the cell. This is the case for the aerobic respiration (14,28,47) and the fatty-acid oxidation systems (30) which involve, among others, the cytochrome-linked enzyme structures. By means of the method of differential centrifugation (15,29), now finding wide application in cytological and histological studies, it is likely that our knowledge of the topography of intracellular functions will be considerably extended and that i t will be possible to map the distribution in the cell of the major biochemical processes. It will be the further task of modern cytology to investigate the constitution and architecture of the various integrated cell structures, a t the molecular level, and to discover in what way structure conditions biological processes and insures the continuance of biochemical cycles.

11. THE CELL Differentiated areas first recognized in cells were the nucleus and the region surrounding it, the cytoplasm. Late'r, delicate structures were found in both: in the nucleus, the nucleolus and the chromosomes; in the cytoplasm, mitochondria, centrospheres and centrioles, specialized vacuoles, and fat bodies. More recently submicroscopic constituents were discovered or postulated, namely, the microsomes (10,13) and the fibrous protein system which, through viscosity changes, is presumably responsible for certain movements of cytoplasm (51). The elements just mentioned can be detected either under the light microscope, directly, or indirectly with the help of special techniques (13,19); the electron microscope so far has added little, except for morphological details, to this usual complement of protoplasm. The fact t ha t many of the cell constituents could be stained differentially for examination under the microscope led to numerous attempts to adapt the sensitivity of chemical and biochemical tests to the dimensions of the cell, and t o conduct the analysis of the various cell structures a t the microscopic level. The methods that were tried suffered considerable limitations and uncertainties: the scope and application of the tests were generally restricted to those which would yield colored products of a density sufficient for detection under the microscope; furthermore, the reactions had t o be carried out in a cellular environment of unknown complexity, already profoundly modified by the preceding fixation. The validity of most histochemical tests so far proposed has repeatedly been

426

ALBERT CLAUDE

challenged and to date only a few, such as the Feulgen reaction for desoxyribonucleic acid, continue to be considered specific. Thanks to the method of differential centrifugation, perfected And systematically applied to cell studies during the past few years, it is now possible to separate a number of the morphological cell structures, namely, those which originally are of different sizes or densities, and to obtain distinct cell fractions in practically unlimited amounts (15). This fact a t once liberates the cytochemist from the limitations of the microscopic techniques and offers the possibility to subject the formed cellular constituents to wide and exhaustive biochemical analysis. By adaptations of this method, 2 to 3 g. of purified chromatin strands can be readily prepared in the course of 2 to 3 hours (21), 5 to 6 g. of purified mitochondria or microsomes in 4 to 6 hours (15). 111. THENUCLEUS 1. Chromosomes and Chromatin

Chromatin threads from the resting nucleus were first isolated by Claude and Potter (12,21)) and their method was later adopted, with slight modifications, by Mirsky and associates for the study of nucleoprotein complexes (37-39). ' The chromatin strands separated by differential centrifugation were found to contain, in per cent of dry weight: nitrogen, 15.57; phosphorus, 3.72; carbon, 45.60; and sulfur, 1.67. The amount of phosphorus, and chemical tests, indicated that as much as 40% of the chromatin complex was represented by nucleic acids of the desoxyribose type. Histological studies and chemical analysis have shown that cells are rich in lipids, principally phospholipids, which are found mostly as constituents of cytoplasmic structures (mitochondria and microsomes) and of the cellular and nuclear membranes. Lipids appear to be rare or absent in chromosomes and the only nuclear structure in which it has been detected, besides the nuclear membrane, is the nucleolus. The small amount of lipids recovered from the chromatin threads, 2.3% or less, may have been derived from cytoplasmic contaminants or from a certain proportion of the nucleoli which have been found to remain attached to some of the chromatin strands.* From cytological tests, and the direct chemical analysis of chromatin threads, it would appear that chromosomes are predominantly composed of nucleoproteins

* Isolated chromatin strands in dilute methyl green or methyl green-pyronine aolutions appear stained a bright green; frequently a relatively large, bean-shaped body staining pink or red, presumably the nucleolus,~isfound attached at one point on B ohrometin strand.

PROTEINS, LIPIDS, NUCLEIC ACIDS IN CELLS

427

extremely rich in desoxyribonucleic acids. There is evidence, especially based on the study of salivary gland chromosomes, that chromosomes are not structurally homogeneous, and it must be assumed that other substances are also present, possibly proteins of a different nature, such as the chromosomin of Stedman and Stedman (54),a finding confirmed later by Mirsky. Because of their abundance in the nucleus, their particular arrangement in chromosome structures, and their participation in the mitotic cycle, desoxyribose nucleoproteins have been postulated to play a leading role in the economy of the cell. This view has been consistently sustained by the continuous accumulation of indirect evidence which indicates that chromosomes are the repository of genetic characters, i.e., exert a controlling influence on the morphology and the functions of the cell. The way by which this influence is mediated to other parts of the cell is not known. It has been suggested that chromosomes are represented by systematic arrays of enzymes or proenzymes, representatives of which migrate to the cytoplasm where they take part in, or direct, metabolic processes (52,53,63,64). A definite example of this kind of action may be found in the type transformation of pneumococci in which a desoxyribonucleic acid has been shown to be the determinant and specific factor (3,34). The most concrete evidence of a functional relation between nucleus and cytoplasm appears in the striking observation of McClintock where a terminal segment of a chromosome in the somatic cells of maize (twothirds of the short arm of chromosome 9) becomes detached during mitosis and lags behind in the cytoplasm, becomes pycnotic, and is finally eliminated. This event is accompanied, in the direct descendants of this cell, by the loss of the tendency, or the ability, to synthesize chlorophyll (35). This case seems to provide visual demonstration of the dependence of plastids, elements which are essentially cytoplasmic, on chromatin. If chromatin normally penetrates the cytoplasm, it must be in a subtle manner, and in minute amounts, since histochemical tests have consistently failed to demonstrate desoxyribonucleic acid in the cytoplasm. These observations were confirmed in experiments in which the main cell constituents were separated by differential centrifugation, and analysis carried out in vitro. The results indicated that practically the entire desoxyribonucleic acid of the cell could be recovered with the nuclear fraction, while tests with the cytoplasmic fractions remained negative (15,46,47). Chromosomes, and their chief chemical constituents, nucleoproteins and nucleic acids, have received continued attention since they were discovered, 60 to 70 years ago. It is noteworthy that, in spite of this sustained interest, the biochemical function of nucleoproteins and

428

ALBERT CLAUDE

nucleic acids is still unknown, and that no tests exist whereby their action could be measured in vitro. The fact that “native” chromatin can be obtained readily in large quantities should encourage research in this field. 2. Nucleolus

The nucleolus, another distinctive constituent of the nucleus, contrasts in morphology and properties with the chromosomes and in many respects resembles cytoplasmic components, namely, mitochondria. This is demonstrated by similar staining properties and by the fact that both nucleoli and mitochondria contain appreciable amounts of lipids and nucleic acid of the ribose type. The size of the nucleolus may vary greatly from cell to cell, or in the course of the mitotic cycle, and morphologically, is not an homogeneous structure; light-microscope pictures frequently show the nucleolus to be composed of granules or globules of uniform size; this appearance is confirmed in electron micrographs of sections of guinea pig liver (unpublished observations) and of cells from tissue culture (16). Histochemical tests indicate that the nucleolus is probably rich in alkaline phosphatase (58). As in the case of other cell constituents, it is probable that the function of the nucleolus will not be fully understood until a way is found whereby nucleoli can be isolated and prepared in bulk, thus making possible a systematic study of their constitution and biochemical properties.

IV. THE CYTOPLASM Since the discovery of chromosomes and the initial work of Strasburger and Flemming (60), the attention and ingenuity of cytologists has been directed in large part toward the study of the phenomenon of mitosis, and only sporadically has it been devoted to the problems concerning the morphology and functions of cytoplasm. No doubt this interest first arose from the attractive orderliness of the mitotic process, and later received further encouragement and impetus from the science of genetics; these developments must have detracted research workers from the less rewarding investigations on cytoplasm. The countless fixatives empirically compounded year after year aimed at the fixation or, at least, the precipitation of chromatin; most of them featured acids or alcohols, two types of reagents which have destructive and dissolving effects on cytoplasm and their almost universal use led to misconceptions regarding the composition and structure of the cell. Experience has now shown that no single fixative can a t the same time preserve both nuclear and cytoplasmic structures, and fixatives found to be satisfactory either far the nucleus or for the cytoplasm are generally exclusive.

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Since the studies by Altmann (l), it was realized that acids and alcohols should be avoided, and that reagents with strong oxidative or reducing properties were good fixatives of cytoplasm. The best fixatives known for the preservation of cytoplasmic inclusions are neutral or only slightly acid and contain, alone or in combination, dichromates, osmium tetroxide, and formalin. This particular behavior toward fixatives can be understood from the knowledge of the chemical constitution of those formed elements of the cytoplasm which, by their abundance, contribute most to the microscopic picture, namely, mitochondria and microsomes. Each of these components represent 15 to 20% of the cell mass; they have been separated by differential centrifugation under various conditions and, in the isolated state, subjected t o chemical and biochemical analysis. Both mitochondria and microsomes have been found to be complex structures composed in large part of phospholipids, proteins, and ribose nucleotides especially in the form of nucleic acid (10,11,13,15). Mitochondria and microsomes react similarly toward acidification of the medium : increasing acidification produces slight agglutination to massive clumping; a t pH 3.5 both elements disintegrate with concomitant denaturation of proteins, separation of phospholipids, and solubilization of ribose nucleotides and nucleic acid (15). Similar events take place on the alkaline side in the neighborhood of p H 12. On the other hand, strong alcohol, especially if acid is present, will denature the proteins, dissolve the phospholipids, and reprecipitate nucleic acid. It is clear that the same destructive effects must obtain in the cell, and that the choice of chemically suited fixatives is of paramount importance in studies dealing with cell structures and the distribution of substances within the cytoplasm and the nucleus. In recent years extensive studies concerned with the microscopic detection of nucleotides by means of characteristic light absorption in the ultraviolet have given rise to far-reaching conclusions regarding the distribution and possible interchange of nucleic acids among the various cell structures (4-6,8,50). It must be noted that measurements by Caspersson and his followers have frequently been conducted on tissues fixed in mixtures containing large proportions of both acid and alcohol. Although such treatment produces preparations which are conveniently transparent, i t would be gratifying if the observations could be confirmed on material subjected to fixatives known to preserve best the morphology of the cell structures involved, namely, the nucleolus, microsomes, and mitochondria. It has been suggested that the pattern of ultraviolet absorption ascribed by these authors to nucleic acids, especially that in the nucleiis, is apparent only after cell injury (32).

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As already noted, analysis reveals similar chemical constituents in mitochondria and microsomes, especially ribonucleic acid and phospholipids; lipositol has been found t o occur in equal proportions in both elements, i.e., 12% of the total lipids (13). It is not known whether the presence of similar substances in both elements indicates the existence of common properties. On the other hand, differences between mitochondria and microsomes are varied and numerous; in size, organization, functions, and degree of complexity they undoubtedly constitute two different classes of cytoplasmic entities. 1. Mitochondria

Mitochondria are relatively large elements, about 0.5 to 1 p in diameter, and therefore are detectable under the light microscope. They may vary considerably in shape, depending on the cell type, from short rods in liver cells to slender filaments several times the length of the cell in cxtcnded fibroblasts; the width, however, although variable aecording t o organs or cell species, is remarkably uniform and constant for a given cell type. In contrast to chromosomes which duplicate according to their length, mitochondria grow by elongation; in certain cells, for example, in the germ cells of Scorpio (59,61) and of the grasshopper,* they can be seen to be passively cut in apparently equal portions by the constricting furrow at the time of cell division. The major chemical constituents so far detected in mitochondria are lipids (25 to 30%)-two-thirds being represented by phospholipids-nucleotides and flavins, and ribonucleic acid ( 1 3 ~ 5 ) . A characteristic feature of mitochondria is the presence of a limiting membrane possessing semipermeable properties. Mitochondria respond osmotically to changes in the salt concentration of the medium; in hypotonic solution they round up and swell, depending on the degree of dilution; in water they may reach the size of a mammalian red cell, and finally disintegrate (I 3,15). Under physiological conditions, when osmotic variations are probably slight and localized, this selective membrane must play an important role in the exchange of fluid and metabolites between the functioning mitochondrion and the surrounding cytoplasm. The most significant development in recent years has been the demonstration that important cell functions are segregated in mitochondria and homologous “large granules” (14). It has been shown by quantitative measurements that the respiratory system which utilizes

* In the germ cells of the grasshopper, filamentous mitochrondria become arranged in bundles alongside the spindle at mctaphase, instead of in the ring formation characteristic of Scorpio, and are divided during cell division so that opposite halves arc retained by the two daughter cells.


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molecular oxygen is localized in these elements, probably exclusively. This is the case for important members of the system, namely, cytochrome oxidase, succinoxidase (28,47), and cytochrome c (49). Likewise, D-amino acid oxidase activity has been found exclusively in the mitochondria, or large granule fraction (13). Recently Kennedy and Lehninger (30) have shown that other important groups of enzymes, namely, the fatty-acid oxidase system and members of the Krebs cycle, are also segregated in mitochondria. Thus, these distinct cytoplasmic elements appear to constitute the power plants of the cell, where the energy of molecular oxygen is transferred and utilized and, in addition, are probably the site of active metabolic and synthetic processes. A mitochondrion, rod-shaped as in mammalian liver, 2 p long and 0.5 p in diameter, would have a volume of 0.4 p 3 and, for a density of 1.2, a corresponding net weight of 4.8 X lo-' y ; if the dry weight of mitochondria is not greater than th at found for the whole liver, i.e., 30% of the wet weight, the solid matter present in a single mitochondrion would amount to 1.4 X y. It has been shown that lipids account for 25 to 30 % of the mitochondrial substance (11,13); if we assume that another 20 t o 25% of the dry weight is represented by inorganic matter and compounds of low molecular weight, it appears that proteins may contribute approximately one-half of the mitochondrial body, or 7 x 10-8 y of the bulk. It follows that a mitochondrion of the size considered could accommodate a t least one million protein molecules, a figure calculated on the basis of proteins of average molecular weight of 35,000 (absolute weight, 6 x lO-'4 y). If we venture the further assumption th a t one mitochondrion has a complement of, let us say, 25 different enzymatic systems, such as those already identified (the cytochrome-linked system, the fatty-acid oxidase system, the so-called Krebs cycle) each system being composed of 20 different protein molecules, it is apparent that there could exist, simultaneously in the same mitochondrial unit, as many as 2000 duplicates of each of the 25 enzyme systems postulated. The possibility has already been discussed (17,lS) that special activities may be segregated in different cytoplasmic granules, thus allowing for an even greater variety of functions. The manner in which the various enzyme systems are integrated in the mitochondrial structure is not known. The type of growth by elongation and transverse subdivision of mitochondria suggests th a t the different enzyme systems present must be uniformly distributed, since uneven arrangement along the rod or filament would eventually produce a segregation of the various systems in divergent cell lines, in the course of successive cell division, and result in the progressive loss of functiohs. In the investigation and further understanding of the fine structure of

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mitochondria it is probable that the biochemical methods, in conjunction with cell fractionation, will play a role similar to that being played by the science of genetics in the understanding of the organization and functions of chromosomes. 2. Microsomes The microsomes are small elements ranging in size approximately from 60 to 250 mp, and therefore not detectable under the light microscope. They were discovered through their concentration and isolation from tissue extracts, by means of centrifugation a t high speed (10); later evidence established their derivation from the cytoplasm and demonstrated that they constituted the basophilic component of the ground substance (13). In contrast to mitochondria no limiting membrane has been demonstrated around these elements although they are affected by changes in the salt concentration of the medium, and become more highly hydrated when in hypotonic solutions (15) ; in electron micrographs of osmic-acid-fixed preparations, the microsomes appear as discrete vesicles generally swollen to various degrees as a result of their passage through distilled water, during the washing to which they are subjected in the course of their preparation for electron microscopy (20,44). Present evidence does not permit one to decide whether this vesiculation of the microsomes is conditioned by a pre-existing semipermeable membrane or is an artifact of osmic acid fixation. Chemical analysis has shown that the microsomes are complex structures composed in large part of lipids and nucleoproteins; as much as 40% of the mass is represented by lipids, two-thirds of which are phospholipids, and 10 to 12% of which is lipositol; ribose nucleotides of low molecular weight have not been detected in appreciable amount but the microsomes have been found to be especially rich in ribonucleic acid. At least 60% of the ribonucleic acid of the cell has been recovered with the microsome fraction (15,29,47,48). The function of the microsomes in the cell is still obscure; biochemical tests so far have failed to detect in the microsome fraction characteristic and exclusive biochemical activities (14,28,47). Recent reports suggest that the microsome fraction may be rich in esterase (41). The thromboplastic activity of tissues appears t o reside in the microsomal fraction (9,15). Their relative abundance-they constitute 15 to 20% of the cell mass-and their high content of ribonucleic acid indicate that the microsomes must have an important share in the normal physiology of the cell. 3. Fibrous Matrix

Another morphological component of importame, long recognized in cells, is a fibrous framework associated with the motility and plasticity


433

of protoplasm, and intracellular movements. In order to account for the peculiarities of the plasma gel layer, changes in cell shape, the activity and direction of cytoplasmic currents, and the formation of asters and spindles, a fibrous protein was postulated, and variations in the degree and type of aggregation of elongated molecules were assumed to he responsible for the properties of reversible gelation displayed by living cytoplasm (51). In some of the events mentioned, especially the production of asters and spindles, it is apparent that the centrosomes have an important and directing influence. The existence of a fibrous protein system is confirmed by the precipitation in the cytoplasm of an abundant fibrous network under the action of a variety of fixatives. The fibrous nature of the network is even more apparent in electron micrographs of cells fixed by acids, and by osmic acid and alcohol (19,44).

V. CONSTITUTION AND DUPLICATION OF LIVINQMATTER This brief review indicates that a cell is composed essentially of six different systems of structures, namely, chromosomes, nucleoli, mitochrondria, microsomes, a fibrous framework, and the centrioles. It must be noted that the chief cell components mentioned are fundamentally distinct in their chemical constitution, their morphology, and their biochemical functions. A living cell, therefore, is not a biochemical continuum but a composite entity, the sum of the interactions of associated elements which appear to be heterogeneous, if not autonomous. In this respect it is probable t b t , as the mechanisms of cellular synthesis are better understood, the concept of “self-duplication ” will be enlarged and extended to other components of the cell (45). In recent years, attention has been centered almost exclusively on the problem of reduplication of gene substance, although it is obvious that all the other essential cell structures are likewise reduplicated during cell growth, or a t the time of cell division. The template theory, elaborated to account for the supposedly unique process of gene reproduction assumes that the gene or the chromosome serves as a mold for the systematic apposition of new substance. This operation, if possible, would result in the production of a negative image, which would be unusable, and could even constitute a danger for the survival of the cell in view of the competing and interfering power that analogs and isomers are known to possess. For exact reproduction by the template process we would have to postulate the preliminary construction of an intermediate replica, to be discarded later. The template theory appears even less workable when viewed a t the molecular level, taking into account the numerous and diverse steps undoubtedly involved even in relatively simple reactions, such as the assembly of a variety of

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amino acids into a specific protein. In discussing this type of reproduction it is usually forgotten that chromosomes are of considerable thickness and complexity, made up of numerous molecular layers, and it is difficult to visualize how template reduplication could thus operate in depth. Finally, the mode of growth by accretion would presuppose the production of the various building stones in excess so that enough of them may find their proper place, in time. This aspect of synthesis by statistical method also appears impractical and unsafe. I n the light of biochemical processes already known, it is conceivable that the duplication of essential and characteristic cell substances is the end result of a series of rigidly ordered chains of reactions, the final product in turn taking part a t some point, and thereby directing the specificity, of the same or other, interlocked, biochemical cycles. The specificity of a gene is not more striking, and probably not more difficult to achieve, than the structural and functional specificity of a proteolytic enzyme, or that of a polysaccharide. Thus the term “self-duplication” appears meaningless when applied t o those complex but highly organized cyclic biochemical processes leading to the production of new cell substances, and t o the reciprocal action that these may exert on the system that produced them. From chemical analysis and histochemical tests three groups of substances, namely, proteins, lipids, and nucleic acids, appear as the most conspicuous and most abundant constituents of cells. Among these, proteins and nucleic acids have already beqn assigned important functions in cellular physiology; although equally abundant, lipids have only occasionally attracted the attention of cytologists and the consensus seems to be that they play a role less essential than proteins or the nucleic acids in the economy of the cell.

VI. PHOSPHOLIPIDS IN CELLSTRUCTURES AND FUNCTIONS Phospholipids are known to take an important part in the formation of cellular membranes, and have been uniformly detected in the limiting niembrttnes of most morphological cell structures, i.e., the nucleus, mitochondria, vacuoles, secretory granules, and the cell itself. This property of phospholipids to constitute membranes has been beautifully investigated by Nageotte (40),and more recently by Dervichian (25). Even in their simple form, phosphatide films represent filters of high sensitivity, capable of concentrating selectively certain ions etc.; in vivo, they may be reinforced by oriented protein films or, as has been suggested, certain types of membranes may be composed of alternating patterns of phosphatides and proteins. Through their ability to form semipermeable membranes phospholipids have a selective role in the interchange of

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water and solutes between nucleus and mitochondria, and the cytoplasmic fluid. About one-third of the total cell mass is made u p of lipids (36); a s already mentioned, the lipid content of mitochondria and microsomes is about 25 and 40%, respectively (13,15). I t has been found that the phospholipids present in these cell inclusions occur in firm chemical association with the rest of the structure since they cannot be removed even by prolonged extraction with organic solvents, such a s ether, benzene, or pentane, unless the complex is severely disrupted (unpublished obaervations). Release of phospholipids, as a rule concomitant with denaturation of the associated proteins and solubilization of ribonucleic acid and ribose nucleotides, is accomplished by a variety of conditions such as heating a t 55°C. for 30 minutes,* hydrogen ion concentrations of pH 3.0 and 12.0, treatment with chloroform, alcohol, or acetone, drying, and repeated freezing and thawing (15,22). The high lipid content of certain structures, for example, mitochondria, cannot be accounted for in the constitution of the membranes alone since upon lysis of the large granules in distilled water a residue is left which is richer in phospholipids than the intact elements; it may be significant t hat the original ribonucleic acid complement of mitochondria is found in this residue in association with the phospholipids, and in proportion close to th at existing in the microsomes (14). Lipids are commonly thought of as substances particularly adapted for supply and storage of energy, or as relatively static constituents of structures such a s membranes, the covering of nerve fibers, etc. This position of lipids is not unique; proteins, essential structural and specifically active constituents of cells, can be utilized as a source of energy if supplied in excess, and constitute many comparatively inert body structures. The abundance of phospholipids in the cell, their occurrence in constant proportion in highly active elements such as mitochondria, their presence in nucleoli and microsomes, in all cases in apparent chemical combination with proteins, ribonucleic acids, and ribose nucleotides (6,8,13,15), suggest that they are integral parts of metabolically active structures. Besides their potential value in the supply and transfer of energy, i t is probable that phospholipids have a definite role in the conduct of biochemical reactions; thus it is possible that, thanks to their

* It is interesting t h a t the lethal points for animal cells are found in the range 37-42°C. in vim, and 47-50°C. in uilro, when the phospholipid-nucleoprotein complexes are known to disintegrate (15). T h e first injury noted microscopically is in the mitochondria (33,43), but it is possible that other phospholipid-nucleoprotein structures may also be affected.

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dual hydrophilic and hydrophobic (oleophilic) property, phospholipids may condition the physical state of certain biochemical systems. Under ordinary conditions, cytochrome oxidase and succinoxidase are not soluble in salt solutions or water, and i t has been suggested that cytochrome oxidase may exist as a lipoprotein complex (57). On the other hand, there is evidence that certain components of the cytochrome system which are readily soluble in water, such as cytochrome c, are not freely diffusible in the living cell, nor in isolated mitochondria (49). It is apparent that phospholipids, by their chemical association with native protein aggregates, may restrain the dispersion of the latter into the aqueous medium. On the other hand, phospholipids, partly through their hydrophilic properties, may help t o regulate the transfer and exchange of water not only through membranes, but also through the mesh of biochemical structures. VII. PROTEINS

It is probable that the high and often rigid specificity of biological reactions has its physical counterpart in equally rigid spatial configuration of the corresponding biochemical systems. Evidence for this is most abundant in the field of amino acid and protein chemistry where a narrow correspondence is often required between a specific enzyme and the configuration of the substrate. du Vigneaud and coworkers have shown that the rat could not utilize acetyl-D-tryptophan for growth purposes, whereas excellent growth was achieved with acetyl-L-tryptophan. The apparent reason is that the cells of the rat are lacking in the specific enzyme capable of hydrolyzing the acetyl group when the latter is attached to the unnatural isomer. When free D-tryptophan was provided in place of ctryptophan, excellent growth resulted, indicating that the body was equipped for the conversion of the compound into its Zevo-isomer (56). Obviously, high specificity limits the reactivity of the system and the freedom of reaction must decrease correspondingly with an increase in the complexity of the structure. Thus it is understandable that, in the construction of biological systems, an analog or isomer cannot substitute for another, since it would result in spatial distortion and therefore in a profound vitiation of the normal biochemical processes. It is clear that, at an early stage of evolution, a choice had to be made between the various chemical configurations available. It would be of interest to know if option for the Zevo-eniantiomorph entailed some definite advantages for the biological systems and for the organisms evolved on this selected pattern.


437

VIII. NUCLEIC ACIDS The nucleic acids, so far, have not disclosed the variety of configurations characteristic of amino acids and proteins, although the possibility for variation exists and structural differentiation must be postulated, a t least in the case of desoxyribonucleic acid, if the latter has to account for the wide range of genetic potentialities generally ascribed to it. The hypothesis that ribo- and desoxyribonucleic acids can be converted one into the other during growth (4,6,7,50) is based on the simple assumption that the molecular differences between these compounds is slight, and perhaps not greater than the chemical difference observed between their sugars, and between uracil and thymine. Experience has shown that, given the proper tests, substances which a t first appeared elementarily alike, for example, polysaccharides or proteins from the same or from different cell species, could be differentiated into numerous isomers or homologs, each distinguished by physical or by highly specific biological properties. Likewise, it would be reasonable to suspect th a t structural differences will be found not only between ribo- and desoxyribonucleic acid molecules, but also among molecules of the same type of nucleic acid. If conversion or interchange is taking place in the cell between the two main types of nucleic acids, it would be more in keeping with the known specificity of physiological processes to expect it to occur through a complete breakdown of one, followed by synthesis of the other. Recent experiments of Cohen seem to clarify this point, so far as the growth of certain bacterial viruses is concerned (23,24). The observations dealing with the multiplication of TB bacteriophage in Escherichia coli B seem to show conclusively that the desoxyribonucleic acid of the newly formed bacteriophage particles was essentially built from the inorganic phosphorus of the medium. I n addition, it was found that the ribonucleic acid of the host cells remained inert, showing a very low, if any, turnover during the growth of the virus. Thus it would appear that the desoxyribonucleic acid of the bacteriophage was the product of an independent synthetic process and that the nucleic acids of the infected cells did not play a n active part in the reaction. The two types of nucleic acids have a different distribution in cells and i t can be inferred that, in these different locations, they perform different functions. I n animal cells, ribonucleic acid is found in formed elements, i e . , in nucleoli, in mitochondria, and in the microsomes (6,13,15,50); i t may be physiologically significant th a t in each case it occurs in association with appreciable amounts of phospholipids. On the basis of ultraviolet absorption studies and characteristic staining reactions, Caspersson and Brachet have endeavored to correlate

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the presence of ribonucleic acid in the cell with protein synthesis (6-8,50). It has been pointed out in preceding papers (17,18)that the role attributed t o ribonucleic acid as a factor in protein synthesis is not the only hypothesis t ha t could be held to account for the abundance of this substance in certain cells. So far, the evidence produced has been by association, the abundance of ribonucleic acid being correlated with a presumed high protein requirement by the cell. Proteins and, more recently, nucleic acids remain the most popular structural constituents of the cell. It should not be forgotten, however, that lipids may account for as much as one-third of the cell mass, and th at growing and metabolically active cells have a high phospholipid turnover. Moreover, phospholipids and ribonucleic acid are frequently found together in the cell, being espxially concentrated in microsomes, mitochondria, and nucleoli. If topographical association is significant, it would be reasonable to expect that phospholipids and ribonucleic acid are also related metabolically. It should be pointed out that large quantities of ribonucleic acid in the cytoplavm coincide with another outstanding property of these cells. In general, the cells that have been found to have a high ribonuc!eic acid content, for example, embryonic and tumor cells, have been shown by the work of Warburg and followers to possess t o a high degree the power of anaerobic glycolysis. Thus, the abundance of ribonucleic acid in these cells may prove to be related in some way to their captLcity for anaerobic respiration. Mono- and dinucleotides of various types are known to be involved in energy transfer during metabolic processes and to take part in a variety of enzymatic reactions. It is conceivable th a t ribonucleic acid may play a comparable role in energetic reactions taking place in the course of anaerobic respiration. This view would seem to be supported by the concurrence of large amounts of ribonucleic acid and of active fermentative processes, in cells such as yeast and certain bacteria. It has been demonstrated that the power of aerobic respiration, ie., the transfer and utilization of molecular oxygen through the cytochromelinked system, is localized exclusively in the cytoplasm, precisely, in the large granules and mitochondria (14,28,47). From these findings, and from considerations such as those just presented, it appears possible that the two complementary respiratory mechanisms, a t least in animal cells, are segregated in separate cellular regions, the aerobic respiration being restricted, as already shown, t o the mitochondria, and the energy derived from anaerobic glycolysis being mediated through ribonucleicacid-containing elements. I n the cytoplasm, this function might be assumed, in the main, by the microsome system, a situation which would explain the highly basophilic character of the ground substance of cells placed in an environment where the supply of oxygen is failing, as in

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certain tumors, or naturally inadequate, as in young embryos. The nucleus, lacking the cytochrome-linked mechanism necessary for aerobic respiration, might be expected to obtain at least part of the energy for its growth from an anaerobic mechanism, possibly through the agency of the nucleolus.

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Amen, Iowa. 52. Spiegelman, 8. (1946). Cold Spring Harbor Symposia Quant. Biol. 11, 256. 53. Spiegelman, S., and Kamen, M. D. (1947). Cold Spring Harbor Symposia Quant. Biol. 12, 211. 54. Stedman, E., and Stedman, E. (1947). Cold Spring Harbor Symposia Quant. Biol. 12, 224. 55. Sturtevant, A. H., and Beadle, G. W. (1939). An Introduction to Genetics.

Saunders, Philadelphia. 56. du Vigneaud, V., Sealock, R. R., and Van Etten, C. (1932). J . B i d . Chem. 98, 565. 57. Wainio, W. W., Cooperstein, 9. J., Kollen, S., and Eichel, B. (1948). J . Biol. Chem. 173, 145. 58. Wicklund, E. (1948). Nalure 181, 556. 59. Wilson, E. B. (1916). Proc. Natl. Acad. Sci. U.S.2, 321. 60. Wilson, E. B. (1925). The Cell. 3rd ed., Macmillan, New York. 61. Wilson, E. B. (1931). J. Morphol. andphysiol. 62,429. 62. Woolley, D. W. (1947). Physiol. Reus. 27, 308; (1945-46). Haruey Lectures 41, 189. 63. Wright, S. (1941). Phyuiol. Reus. 21, 487. 64. Wright, 9. (1945). Am. Naturalist 79, 289.

Author Index Numbers in parentheses are reference numbers. They are included to assist in locating references in which the authors’ names are not mentioned in the text. Names in parentheses indicate coauthors. Numbers in italics indicate the page on which the reference is listed. Ezample: Akkerman, A. M., 44 (ref. 224),53, 80 meana that this author’s article is reference 224 on p. 44, that it is mentioned on p. 53, and is lkted on p. 80 at the end of the article. Allison, J. B., 158, 160, 161, 162, 163, 164, 166, 167, 168, 169, 170, 171, Abderhalden, E.,11, 13, 14, 15, 17 (ref. 177, 181, 182, 185, 187, 188, 189, 196,197 7), 36 (ref. 6,24), 37 (ref. 8, 16), 38 (ref. 8, 9, 12, 15, 16, 17, 24, 28, 30, Almquist, H. J., 244, ,996 32, 34,36), 39 (ref. 6,8,9, 10,12, 15, Altman, R. F. A., 241, 296 16,28,30, 33,34,37), 40 (ref. 14,26, Altmann, R.,429, 489 27, 36), 46, 47 (ref. 3), 49 (ref. 13), Alvaree-Tostado, C.,212 (ref. 60),226 50 (ref. 2, 3),51 (ref. 2,3, 13, 17),53 Anderson, E.G., 158 (see Borman), 197 (ref. 35), 54 (ref. 21, 35), 55, 57 Anderson, J. A., 158, 160, 161, 163, 164, 167, 168, 170 (see Allison), 171, 177, (ref. 7), 58, 61 (ref. 22),65,66 (ref. 181, 185, 187, 188, 196, 197 22), 67, 68, 69 (ref. 20, 25), 70 (ref. Anslow, W.K., 373 (ref. l),416 23, 25), 71 (ref. 18), 72, 76,76,78 Abderhalden, R., 46 (ref. 2), 50 (ref. 2), Anson, M.L., 139, 141, 148, 161,346 Archibald, R. M.,85, 148 51 (ref. 2), 76 Arenz, B., 279, 296 Abeniue, P. W., 5, 76 Abitz, W.,308 (ref. 57),328 (ref. 57),948 Arhimo, A. A., 271, 303 Armstrong, W.D., 182, 197 Ackerman, H.,268, 303 Arnold, C.,219, 222 (ref. 2),296 Adair, G.S., 128, 134, 148, 163 Adair, M. E., 128, 134,2 4 8 , 163 Artom, C.,57, 79 Asenjo, C. F., 198 Addis, T., 182, 187, 196 Adler, E.,271, 297 Astbury, W.T., 141, 143, 148, 306 (ref. Agatov, P.,240,294, ,996,299 11, 12, 15),308 (ref. 9, 12), 309, 310 Agner, K., 222, 226 (ref. 14, 30), 311 (ref. 8, 14), 316 h e s o n , A., 89, 163, 219 (ref. 135), 222 (ref. 4, 5), 326, 328 (ref. lo), 329 (ref. 135), (ref. 6), 346, $47, 359, 360, 369 Akkerman, A. M., 44 (ref. 224),53,80 (ref. 3), 395 (ref. 3), 416 Albanese, A. A., 89, 118, 119, 120, 148, Atkin, W.R., 369 (ref. 16),372 (ref. 16), 395 (ref. 185), 400, 426,4.90 158, 171 (see Cox), 176, 196, 198, Audrieth, L. F., 52 (ref. 286),82 234, 243, 296 Albaum, H. G., 276, ,996 Auerbach, G.,67 (ref. 194), 68 (ref. 194), 79 Alcock, R. S., 276, ,996 Avery, 0. T., 424 (ref. 2, 3), 427 (ref. 3), Alekseeva, T. S., 255,303 Alfrey, T., 316 (ref. 3), 327, 332 (ref. 439 Ayres, M. M.,180 (see Bosshardt), 197 1, 2), 3qs Alge, A., 362 (ref. 114),428 B AUing, E. L., 181 (see Zeldis), 184, 198, Bach, S., 406 (ref. 47),426 199, 2m Bacon, J. S. D., 240, $99 Allison, F. E., 274, 296, ,998 441 A

442

AUTHOR INDEX

Bawden, F. C., 254,255, 293,296 Baer, E., 25, 79 Beach, E.F., 105, 148, 202 (ref. 8), 203 Baernstein, H.D., 96, 103: 105, 148 (ref. 8),218 (ref. 8),226 Baertich, E.,46 (ref. 2), 50 (ref. 2), 51 Beadle, G. W., 424 (ref. 55),4.40 (ref. 2),76 Bahn, A,, 47 (ref. 3), 60 (ref. 3), 51 (ref. Beadles, J. R., 176, 197, 199 Bear, R. S., 306 (ref. 19, 20), 346, 360, 3), 65,76 359,416 Bailey, C. H., 262,303 Beatty, W.A., 67 (ref. 232),80 Bailey, J. L., 22 Bailey, K.,96,97,103,105, 106,108,112, Beek, J., Jr., 362 (ref. 8), 363 (ref. 9), 367,401,416 115, 133, 134, 136, 140, 141, 142, 147, 148, 148,218 (ref. 3), 826, 233, Beeson, W.M., 244, 304 247, 257, 296, 896, 306 (ref. 16), 309 Behrens, M.,265,298 Behrens, 0.K., 23, 37 (ref. 43), 43 (ref. (ref. 7),310 (ref. 30), 346, 347 248), 44 (ref. 248), 49 (ref. 44), 60 Bain, J. A., 215 (ref. 4), 826 (ref. 43), 63 (ref. 248), 62 (ref. 45), Baker, W.O., 328 (ref. 17, 18, 56), 348, 65 (ref. 45), 66, 76, 81, 82 343 Bell, F., 311 (ref. 8),346 Balbiano, L., 2,76 Belozersky, A. N., 240,265,268,296, %09 Baldwin, E.,285,295, 296 Baldwin, M. E., 384 (ref. 4, 199, 200), Belt, A. E., 182, 199 Benditt, E.P., 190, 197 395 (ref. 4),416, 420 Balfe, M. P., 394 (ref. 5a), 395 (ref. 5), Benedict, F. G., 264, 296 Berg, J. L., 182,197 400 (ref. 5),416 Ball, E. G.,219 (ref. 5), 220, 221 (ref. Bergell, P.,9 (ref. 144), 10 (ref. 144), 78 Berger, L.,264,206 log),2%6,2.27 Berggren, R.E. L., 204 (ref. 22),207,226 Ballou, G.A., 217 (ref. 6),226 Balls, A. K., 33 (ref. 40), 73, 76, 222 Bcrgmann, M.,2 (ref. 46), 3,6,6, 7 (ref. 78), 10 (ref. 145),11 (ref. 50), 15, 16 (ref. 7),226, 292, 293, 300 (ref. 51, 60,67,90, 115), 17 (ref. 64, Baly, E.C. C., 270,296 65, 189),18, 19, 20 (ref. 57, 189),21 Barkdoll, A. E., 31 (ref. 41),47, 60 (ref. (ref. 74), 23, 25, 26 (ref. 74, 77, 80, 41),76 85), 30 (ref. 187, 188), 31 (ref. 49, Barmore, M. A., 262, 296 86,276),33 (ref. 47, 62,64),34 (ref. Barnes, R. H., 162, 173, 174, 175, 180, 190), 36 (ref. 49, 56, 74, 77, 80, 89, 197 188, 189, 276, 278), 37, 38 (ref. 49, Barnum, C. P., 432 (ref. 41),440 56, 78, 80,278), 39 (ref. 49, 78, 80), Barth, K.,381 (ref. 175),420 40 (ref. 53, 80, 276, 277), 41 (ref. Bartholomew, E.T., 241,302 67, 74), 42 (ref. 74, 87,88), 43 (ref. Bartner, E.,1 1 1 (see Smith, E.L.), 112 60, 67, 74, 76, 78, 79, 87, 88, 187, (see Smith, E. L.), 113 (see Smith, 189, 216), 44 (ref. 74, 77, 78, 88, E. L.), 117 (see Smith, E.L.), 162, 187),45 (ref. 74,87,88), 46, 47 (ref. 212 (ref. 123), 213 (ref. 123), 218 50, 85,88, 187),48 (ref. 50, 188), 49 (ref. 123),2%8,263, SO2 (ref. 44,50, 53,56,60,62,66,67,74, Barton, R. W., 171 (see Cox),108 85, 115, 187, 188, 215), 50 (ref. 43, Bass, L. A., 38 (ref. 231), 80 60, 51, 53, 66, 74, 88, 188),51 (ref. Bates, M.J., 173, 174 (see Barnes), 175 65, 67, 88, 90), 63 (ref. 74), 55, 57 (see Barnes), 197 (ref. 81,65, 74, 279), 59, 60,61,62, Baudisch, O., 270,296 63, 64,66,68,69, 70, 71,74, 76, 77, Baudouy, C.,371 (ref. 37), 416 78, 79, 80, 88, 148, 277, 896, 357 Baumann, E.,64 (ref. 42),76 (ref. lo),360,400 (ref. 12),416, 416 Baumann, L.,72 (ref. 5), 76 Bernal, J. D., 133, 149, 254, 896 Baur, H., 40 (ref. 117), 78

AUTHOR INDEX

Bernhart, F. W., 191, 200 Bernstein, S. S., 105 (see Bench), 148,202 (ref. 8), 203 (ref. 8), 218 (ref. 8), 226 Berridge, N. J., 209, 226 Bertho, A., 24, 77 Best, R. J., 237, 254, 255, 256, 293, 294, 296, 300

Bezer, A. E., 263, 299 Billimoria, M. C . , 288, 301 Birch, T.W., 406 (ref. 13), 416 Birkhofer, L., 96, 103 (see Kuhn), 105 (see Kuhn), 139 (see Kuhn), 151 Bivshich, N., 240, 299 Bjorksth, J., 272, 296 Black, A., 117, 160, 244, 298 Black, H. C., 158 (see Borman), 197 Blake, M. A., 271, 303 Blank, P., 15 (ref. 146), Y8 Blaxter, 162, 197 Blish, M. J., 262, 296, 301 Block, H., 93, 103 (see Dunn), 110 (see Dunn), 160 Block, R. J., 97, 101, 102, 103, 105, 109, 110, 111, 112, 113, 114, 116, 117, 118, 119, 120, 129, 130, 14.9, 169, 192, 193, 194, 197, 199, 236, 237, 243, 244, 245, 246, 247, 250, 263, 296, 300 Blom, J., 274, 296 Blotter, L., 108 (see Lyman), 112 (see Kuiken), 113 (see Kuiken), 119 (see Lyman), 138 (see Lyman), 161, 235, 244, ,999 Blum, A. E., 110 (see Horn), 120 (see Horn), 161, 263, 298 Blum, W. A,, 399 (ref. 197), 409 (ref. 1971, &O Blumenthal, D., 96, 103, 105, 149, 247, ,996 Boivin, A., 268, 296 Bolin, D. W., 244, 301, 304 Bollenbach, G . N., 424 (ref. 27), 439 Bolling, D., 97, 102, 103, 105, 109, 110, 111, 112, 113, 114, 116, 117, 118, 119, 120, 129, 130, 149, 237, 243, 244, 245, 246, 250, 263, 296 Bondy, C., 241, 296 Bonem, P., 63 (ref. 118), 78 Bonner, D. M., 273, 303 Bonner, J., 260, 267, $04

443

Boothe, J. H., 45 (ref. 91a), 77 Borasky, R., 306 (ref. 133), 360,359 (ref. 162), 360 (ref. 162), 419 Borman, A., 158, 197 Borodin, J., 278, 283, 296 Borsook, H., 33 (ref. 92), 77 Bosshardt, D. K., 162, 173, 174, 175, 180, 197, 216, 226

Bot, G. M., 267, 296 Bourque, J. E., 184, 199 Boussingault, J. B., 277, 279, 296 Bower, F. O., 257, 296' Bowes, J. H., 143, 149, 357, 377 (ref. 14), 391 (ref. 13a), 406 (ref. 15), 414, 416, 421

Bowles, L. L., 182 (see Berg; Hall, W. R.), 197, 198 Bowman, D. E., 263, 296 Boyd, G. L., 211, 226 Boyd, M. J., 109, 149 Boyer, P. D., 217 (ref. 6), 226 Boyer, R. A., 311 (ref. 21), 315 (ref. 21), 347

Boyland, E., 253, 297 Brachet, J., 429 (ref. 4-6) 435 (ref. 6), 437, 438 (ref. 6, 7), 439 Brand, E., 85, 87, 88, 93, 95, 96, 100, 101, 102, 103, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 128, 130, 132, 133, 135, 136, 139, 142, 145, 149, 161, 162, 216, 218 (ref. 13, 61), 223, 226, 226 Brandon, B. A., 307 (ref. 49), 34'7 Branscombe, D. J., 235, 902 Brauns, F., 411, 412, 418 Braunstein, A. E., 20, 77, 271, 273, 296 Braybrooks, W. E., 369 (ref. 16), 372 (ref. 16), 400, 416 Brazier, M. A. B., 248, 296 Breusch, F. L., 285, 296 Bricker, M. L., 163, 197 Briefer, M., 367, 416 Brierley, P., 293, 296 Briggs, D. R., 217, 226 Brockmann, H., 36 (ref. 6), 39 (ref. 6), 76 Brown, A. E., 38 (ref. 275), 51 (ref. 275), 81, 307 (ref. 22), 328 (ref. 70), 331 (ref. 70), 347, 348 Brown, E. B., 250, 299

444

AUTHOR INDEX

Brown, J. H., 162, 163, 167, 168, 169, 170 (see Allison), 171, 182, 185, 189, 197 Brown, R., 287,296 Briickner, V., 45 (ref. 222), 80 Bruhre, P., 263, 301 Brumberg, E. M., 429 (ref. 32), 4.39 Bruah, M. K., 171, 172, 197, 200 Buadze, S., 17 (ref. 7), 57 (ref. 7), 76 Buchheimer, K., 362 (ref. 127), 418 Buehler, H. J., 124, 125, 143, 149 Bull, H. B., 128, 149, 215 (ref. 15), 226, 309 (ref. 23), 330, 335 (ref. 24, 25), 547

Bungenberg de Jong, H. G., 395 (ref. 18), 416

Burack, E., 185, 198 Burk, D., 274, 998 Burk, N. F., 128, 134, 1.69, 207, 226, 416 Burnett, R. S., 311 (ref. 26), 315 (ref. 261,347

Burris, R. H., 250, 251, 254, 274, 275, 296, 997,304

Burstram, H., 270, 29'7 Burton, I. F., 25 (ref. 94), 77 Busse, W. T., 335, 547 Butler, B., 103 (see Lyman), 161 von Buzagh, A., 311 (ref. 28), 325, 347 C

Cable, R. S., 223 (ref. 147), 226, 228 Cahill, W. M., 25 (ref. 94), 77 Caldwell, T. D., 311 (ref. 138), 314 (ref. 138), 560

Cameron, D. H., 387 (ref. 146), 400 (ref. 150), 419

Camien, M. N., 93, 101 (see Shankman), 103 (see Dunn), 108 (see Dunn), 110 (see Dunn), 111 (see Dunn), 119 (see Dunn), 120 (see Dunn), 1.69, 160, 162, 218 (ref. 115), 228, 250, 251, 252, 253, 254, 297

Campbell, R. M., 182, 187, 197, 198 Cannan, R. K., 85, 88, 93 (see Keston), 94, 100, 101 (see Keston), 106, 108, 114 (see Keston), 126 (see Longsworth), 135, 136, 138, 1.69, 161, 168, 214 (ref. 17), 216 (ref. 17), 218 (ref. 66), 826, 226, 263

Cannon, P. R., 158 (see Frazier), 189, 190, 191, 192, 194 (see Block), 197, 198, 200

Carman, G. G., 165, 174, 199 Carothers, W. H., 310, 347 Carpenter, C. M., 268, 303 Carpenter, D. C., 207, 228 Carpenter, L. M., 207, 228 Carter, H. E., 18, 77, 89, 149, 249, 297 Carter, J. R., 183 (see Madden), 198 Caspersmn, T., 265, 277, 297, 429, 435 (ref. 8), 437, 438 (ref. 8), 439 Caswell, M. C., 103 (see Stokes), 110 (see Stokes), 111 (see Stokes), 112 (see Stokes), 113 (see Stokes), 117 (see Stokes), 120 (see Stokes), 165, 256, 303 Cederquist, D. C., 245, 297 Chambard, P., 400 (ref. 20), 416 Chanutin, A., 207 (ref. 18), 226 de Chardonnet, Count, 310, 311 Chargaff, E., 266, 268, 297, 432 (ref. 9), 439

Cherbuliez, E., 205, 226, 369 (ref. 21), 416

Chernov, N. W., 362, 416 Chevalier, R., 221 (ref. 43), 226 Chibnall, A. C., 2 (ref. 99), 77, 85, 87, 93, 97, 101, 104, 105, 106, 107, 108, 113, 114, 115 (see Bailey), 116, 118, 119, 120, 130, 131, 132, 133, 134, 135, 136, 138, 139, 143, 147, 148, 149, 218 (ref. 3), 226, 233, 234, 235, 237, 238, 239, 241, 253, 257, 264, 267, 270, 271, 272, 277, 279, 281, 283, 284, 285, 297, 300, 310, 347, 357, 358, 359, 360, 416 Chick, H., 179, 197, 240, 297 Chou, C., 57 (ref. 279), 82 Chow, B. F., 125, 149, 181, 187, 197 Chuan Chti, P., 398 (ref. 51), 427 Christensen, L. K., 217 (ref. 59), 218, 226 Christian, W., 264 Chrzaszcz, T., 219 (ref. 21), 226 Circle, S. J., 262, 502 Clandinin, D. R., 193, 194, 197, 199 Clapp, 5. H., 3, 67 (ref. 253), 68, 70, 81 Clark, H., 200 Clark, L. C., 170 (see Murlin), 171 (see Murlin), 198, 199

445

AUTHOR INDEX

Clarke, H. T., 18 (ref. lOOa), 64 (ref. loo), 75 (ref. l a , 257), 77, 81, 96, 103, 105, 1.69, 233, 246, 247, 248, 249, 257, 296,300,303 Claude, A., 425 (ref. 10, 13-15, 28), 426, 427 (ref. 15), 428 (ref. 16), 429 (ref. 10, 11, 13, 15),430 (ref. 13-15), 431 (ref. 11, 13, 17, 18, 28, 49), 432 (ref. 10, 13-15, 20, 28, 44), 433 (ref. 19, 44), 435 (ref. 13-15, 22), 436 (ref. 49), 437 (ref. 13, 15), 438 (ref. 14, 17, 18,281, 439,440 Coghill, R. D., 250,297 Cohen, P.P.,276, 286,2996,297 Cohen, S. S., 437,4.99 Cohn, E. J., 3 (ref. 101), 33 (ref. 101), 77, 133, 144, 149, 204 (ref. 22), 207, 215 (ref, 33), ,926, 263, 297, 341 (ref. 31),347, 363 (ref. 25), 365, 416 Cole, W. H., 181, 187 (see Chow), 197 Coleman, D., 109, 110, 114, 116, 119, 149, 311 (ref. 32, 33), 324, 325,347 Colowick, S. P.,264, 296 Cone, L. H., 62 (ref. 147), 65,78 Consden, R.,3 (ref. 103),77,97, 136,1.49 Cook, A. H., 18 (ref. 103a),77 Cooper, M., 347, 409 (ref. 40), 410 (ref. 401,416 Cooperstein, S. J., 436 (ref. 57), 4-40 Copley, M. J., 311 (ref. 131, 132), 313 (ref. 151), 325 (ref. 131), 327, 328 (ref. 151),349,360 Copping, A. M., 179,197 Corbet, A. S., 270,897 Cori, C. F., 264, 296 Cornforth, J. W., 18 Corran, H.S.,220, 221 (ref. 23), 226 Cougny, A., 371 (ref. 38),41.6 Cowgill, G. R., 158, 159, 174, 185, 198 Cox, W.M., Jr., 159, 171,198 Coy, N. H., 212 (ref. 120),228 von Cramm, E., 43 (ref. 283), 44 (ref. 283),82 Cravens, W. W., 193, 194,197, 199 Crisp, D. J., 311 (ref. 34),347 Croad, R. B., 403, 416 Crook, E.M., 234, 255, ,997 Croston, C. B., 311 (ref. 35, 37), 314, 347 Crowfoot, D., 128, 133, 1.69, 215, 2.26

Crowther, C., 202,210, 212, 223 (ref. 25), 996 Cruickshank, D. H.,281, 282, 286, 287, 289,304 Csonka, F. A., 103, 149, 243, 249, 262, 297 Currie, B.T.,215 (ref. 15),226 Curtius, H., 9 (ref. 108), 77 Curtius, T.,6,7,9, 10, 15,21, 28, 77 Custer, J. H., 126 (see McMeekin), 127 (see McMeekin), 135 (see McMeekin), 161, 212 (ref. 80), 217 (ref. 79,81, 82), 227 Cuthbertson, W. R., 376, 416 Cutting, M. E. M., 240, 2997

D Dahlberg, A. C., 222,228 Dakin, H.D., 357 (ref. 28),416 Damodaran, M., 208, 226, 280, 297 Danielason, C. E., 395 (ref. 29),416 Darmon, 5. E., 112, 117, 1.69 Das, N. B., 271, 297 Davidson, J. N., 266, 277,297 Davidssohn, B.,2,78 Davies, W.L., 240, 297 Davis, B. D.,217,226 De, P. K., 274, 698 Dekker, C. A., 34 (ref. 112a), 48 (ref. 112b), 71 (ref. 112a, 112b), 72 (ref. 112b),73 (ref. 112b), 77 Delaporte, B., 265, ,997 De Ley, J., 259,304 Della Monica, E. S., 126 (see McMeekin), 127 (see McMeekin), 135 (see McMeekin), 161, 212 (ref. 80), 217 (ref. 79, 81, 82),227 Denes, K., 205 (ref. 42),226 Dent, C.E., 57, 77, 271, 252, 288, 297 Denton, C. A., 103,149 Derksen, J. C., 361 (ref. 115),418 Dervichian, D., 434,439 Desveaux, R.,270, 299 Deuel, H.J., Jr., 171,198 Deutsch, H. F., 202, 215 (ref. 4), 223 (ref. 28), 226 Dewan, J. G., 220 (ref. 23), 221 (ref. 23), 2.96 Dexter, S. T.,367, 418

446

AUTHOR INDEX

Dickineon, S., 309 (ref. 7), 3.bs Dirr, K., 61 (ref. 114), 64 (ref. 114, 123), 77, 78 Diskant, E. M., 100 (see Brand), 112 (see Brand), 149 Dittrich, W., 271, 297 Dixon, M., 220,S'86 Dobzhansky, R.,424 (ref. 26), 439 Doherty, D. G . , 16, 18, 37 (ref. 115), 49 (ref. 44, 115),76, 77, 886 Doty, D. M., 243,897 Doty, P.M., 332 (ref. 2),546 Douglas, G. W.,394 (ref. 30),416 Driscoll, P. E., 36 (ref. 19Oa), 38 (ref. Nos), 79 Dubs, R. J., 217, 886, 268 (see Robinow), 302 Dubuiseon, M., 140, 146,f@ Du Buy, H. G., 294,304 D b r , W.,79 DufrBnoy, J . , 294, 297 Dunn, M. S., 15 (ref. 267), 39 (ref. 267), 81, 85, 91, 92, 93, 100, 101, 103, 108, 110, 111, 119, 120, 149, 160, 168, 218 (ref. 115), 888, 250, 251, 252, 253, 254, 297 Duspiva, F., 376, 429 Dwyer, I. M., 103 (see Stokes), 110 (see Stokes), 111 (see Stokes), 112 (see Stokes), 113 (see Stokes), 116 (see Gunness), 117 (see Stokes), 120 (see Stokes), 160, 163, 256, 303 Dyer, E., 49 (ref. 116), 78

Edwards, L. E., 170 (see Murlin), 171 (see Murlin), 198, 199 Ehrensviird, G., 2, 23, 78 Eichel, B., 436 (ref. 57), 4-40 Eichel, H., 24, 79 Elam, D. W., 317 (ref. 94), 349 Eldred, N. R., 194,198 Ellenbogen, 128, 132 Ellison, H.L.,400 (ref. 150),419 Elman, R.,171 (see Cox), 187, 198 Elad, E., 367 (ref. 33),387, 416 Elsden, 5. R., 95, 160 Elvehjem, C. A., 103 (see Riesen), 162, 193, 194,197, 199 Engel, H., 211 (ref. 32), 826 Engel, L. L., 20,80 Enger, R.,58, 78 Engler, 231 Eppling, F. J., 274,296 Erickson, J. O . , 97 (see Neurath), 161, 309 (ref. 130), 311 (ref. 130), 315 (ref. 130),316 (ref. 130),349 Erlenmeyer, E., 16, 49 (ref. 121), 78 Erxleben, H.,253, 999 Essig, K.A., 349 von Euler, H., 271, 997 Evans, C.D., 311 (ref. 35, 37), 314, 347 Evans, R.J . , 193, 198 Everson, G. J . , 171 (see Swanson, P.), 199, 245, 897 Ewald, A.,369 (ref. 34, 35), 371 (ref. 35), 411,416 Eyring, H.,308 (ref. 38),332 (ref. 3941, 161), 333,335,347, 5.48,560

E F Eckerson, S. H., 270, 997 Fankuchen, I., 215 (ref. 33), 826, 254, Eckstein, H.C . , 250, 897 896 Eddy, C. R., 311 (ref. 152), 325 (ref. FaurB-Fremiet, E., 371,411 (ref. 36), 4f6 152),360 Edlbacher, S.,40 (ref. 117),63 (ref. 118), Fearing, D. F., 314 (ref. 168),361 Felix, K.,64 (ref. 123),78 78 Edaall, J. T., 3 (ref. 101), 33 (ref. 101), Ferguson, F. P., 162, 166, 182 (see Allison), 197 77, 85, 93, 95, 105, 107, 128, 133, 142, 144,14.9, 160, 207, 215 (ref. 33), Ferri, C.,335 (ref. 121), 34.9, 411, 419 216, ,926, 263, 897, 307 (ref. 54), 311 Ferry, E. L.,175,199 (ref. 36), 316 (ref. 36),341 (ref. 31), Ferry, J. D., 217 (ref. 34), 826, 306 (ref. 42), 311 (ref. 42, 43), 332 (ref. 42), 347, 363 (ref. 25), 365,405 (ref. 43), 406,407,413,416 347

AUTHOR INDEX

Feulgen, R., 265,,998 Fevold, H. L., 249,,999 Ficken, K.,390 (ref. 137), 429 Fiedler, A., 43 (ref. 148), 44 (ref. 148),78 Fiere, H. E., 24, 79 Fink, H., 250, ,998 Fischer, E.,1, 2,3,5,6,7,8,9,10,11, 12, 13, 14,15, 17,21,23,25,27,36 (ref. 132,134, 136,149, 158,160, 167),37, 38 (ref. 128, 129, 133, 134, 136, 140, 149, 161, 164, 167, 1681,39 (ref. 136, 138, 154, 164, 168), 40, 41, 43 (ref. 148,155,157),44 (ref. 139,148,155157), 46, 49, 50 (ref. 129, 150, 158, 166. 171),51 (ref. 129,139,140,165), 53 (ref. 150, 171), 54 (ref. 150, 171), 55,57,58,61 (ref. 172),62 (ref. 147), 63, 65, 66, 67, 69 (ref. 162), 70, 75 (ref. 133), 78,79,307 Fischer, H. 0. L., 25,79 Fisher, A. M., 132,262 Fishman, W.H., 57, 79 Fishmann, M. M., 267, 298,300 Fleiachmann, R., 37 (ref. 8), 38 (ref. 8), 39 (ref. 8),76 Flemming, 428 Fling, M., 424 (ref. 27), 4.39 Flory, P. J., 312 (ref. 44), 328 (ref. 45, 46),335,S47 Fo6, C., 311 (ref. 47),347 Fodor, A., 13, 15, 38 (ref. 9), 39 (ref. 9, lo), 76 Fogg, G. E.,274, ,998 Folin, 98,247 Foltzer, J., 310 (ref. 48),347 Fontaine, T.D., 262, 298,299 Foreman, F. W., 97,260 Forster, M.O., 24, 79 Foster, G. L., 85, 90, 93, 94, 101, 106, 108, 111, 112, 114, 116, 120, 122, 260,262 Foster, S. B., 373, 384 (ref. 201), 385 (ref. 201), 386 (ref. 201), 395 (ref. 202),397 (ref. 204),420 Fourcroy, A. F., 233, 698 Fourneau, E., 1, 5, 7 (ref. 149), 9, 27 (ref. 149),36 (ref. 149),38 (ref. 149), 78 Fourt, L., 331 (ref. 71),348 Fox, 8.W., 424 (ref. 27), 439

447

Fraenkel, G., 412, 426 Fraenkel-Conrat, A , 96, 119, 121, 129, 133, 135, 145,262 Fraenkel-Conrat, H., 76, 307 (ref. 49-52, 134),347,360,407, 409 (ref. 40,41), 410,426 Fraenkl, W., 120 (see Dunn), 160 Frampton, V. L., 235, 260, 293, 294, ,998 Frank, V. S., 32,54 (ref. 270a),81 Frankel, M.,7, 22, 67 (ref. 177), 79,80, 307 (ref. 53),347 Frazier, L. E., 158, 189, 191, 194 (see Block), 197,298 Freeland, J. C., 250, 251, 252, 254, 276, ,998 Freeman, S., 182, 298 French, D., 307 (ref. 54), 347, 405 (ref. 43),406, 407,416 Freudenberg, K.,13 (ref. 180), 24, 25, 79,397,426 Freund, E. H., 308 (ref. 55),348 Freundlich, H.,241, ,996 Fricke, R.,11, 50 (ref. 206), 80 Fried, S.,299 Friedes, R., 108 (see Hier), 110 (see Hier), 111 (see Hier), 112 (see Hier), 113 (see Hier), 116 (see Hier), 118 (see Hier), 119 (see Hier), 120 (see Hier), 260 Fritsch, F. E., 274,298 Frost, D. V., 159,298,199 Frllstllck, E., 16,49 (ref. 121), 78 Fruton, J. S.,3 (ref. 191,250),7 (ref. 78), 11 (ref. 50), 16 (ref. 51), 17 (ref. 186, 189, 304), 19 (ref. 54), 20 (ref. 189), 26 (ref. 80, 181), 30 (ref. 187, 188),31 (ref. 49),33 (ref. 52,54, 182, 186),34 (ref. 112a,190),35 (ref. 185), 36 (ref. 49, 80, 188, 189, 19Oa), 37 (ref. 78-80, 277),38 (ref. 49, 78, 80, 19Oa), 39 (ref. 49, 78, 80), 40 (ref. 53, 80, 184, 277), 41 (ref. 186), 43 (ref. 50, 78, 79, 187, 189), 44 (ref. 78, 187, 274), 45 (ref. 183),47 (ref. 50, 187), 48 (ref. 50, 112b, 188), 49 (ref. 50,53, 187, 188),50 (ref. 50,51, 53, 188), 51 (ref. 274), 56 (ref. 181, 186),57 (ref. 181,186),58 (ref. 181), 61 (ref. 55), 64 (ref. 55, 186), 68 (ref. 273a), 69 (ref. 273a), 70 (ref.

448

AUTHOR INDEX

273a), 71 (ref. 112a, 112b, 18h, 188), 72 (ref. 112b, 18h), 73 (ref. 112b),76, 77, 79, 82, 82, 277, f?96 Fuchs, F., 21,79 Fugitt, C. H., 363 (ref. l90), 364 (ref. 1911,MO Fuld, M.,307 (ref. 123), 308 (ref. 123), wQ

Fullam, E. F., 425 (ref. 19), 432 (ref. 44), 433 (ref. 19, 44),@9, 440 Fuller, C. S., 328 (ref. 17, 18, 56), 346, 34R G

Gale, E. F., 90, 108, 116, 118, 119, 120, 122, 130,260,218 (ref. 35),$26, 250, 251,252, 253,254, 276, $98 Gall, E. C.,307 (ref. 22),347 Gallacher, A. M., 295 Gallun, E.A., 384 (ref. 213),482 Calvin, J. A., 328 (ref. 137),360 Garrod, M.,335 (ref. 89),348 Caw, H. Z.,256, $98 Geidel, W.,65,76 Geiger, W.,21 (ref. 229),37 (ref. 229),49 (ref. 229), 80, 307 (ref. 87),348 Gerngross, O., 50 (ref. 150),53 (ref. 150), 54 (ref. 150), 78, 308 (ref. 57), 328 (ref. 57),348, 405 (ref. 46), 406,426 Gibbs, J. W., 335 (ref. 58), 348 Gibbs, W.,335,34.8 Giffhorn, A,, 220, 226 Gillespie, H. B., 64 (ref. loo),77 Glasstone, S.,333 (ref. 59),34.8 Gliek, D.,432 (ref. 41),440 Gluud, W.,14 (ref. 151), 78 Go, Y.,307 (ref. 120), 311 (ref. 60),315 (ref. 60),348, 349 Goebel, W.F., 424 (ref. 2), 439 Goettsch, E.,186 (see Weeeh), 200 Gohdes, W., 38 (ref. 12), 39 (ref. 12), 76

Goldberg, 8.C., 108 (see Dunn), 149 Goldwater, W. H., 116 (see Brand), 120 (see Brand), 149, 216 (ref. 13), 218 (ref. 13), 286 Goodloe, M. B., 202 (ref. 28), 223 (ref. 28), 286 Goralowna, C.,219 (ref. 21),$26

Gordon, A. H., 3 (ref. 103), 77, 88, 97, 109, 110, 136, 249, 160, 220 (ref. 23), 221 (ref. 23), 226 Gordon, 8.A., 267, 268, 298, SO4 Gordon, W. G., 223, $26, 228, 307 (ref. 2% 347 Gorter, E., 311 (ref. 8, 61), 346, 4148 Gortner, R. A., 257, 262, 298 von Gorup-Besanez, F., 278, $98 Gould, B. S., 220 (ref. 38),226 Gould, S. P., 314 (ref. 171),4162 Goyco, J. A., 298 Grbacher, C., 48 (ref. 192a),79 Grafe, K.,19,20 (ref. 57),36 (ref. 56),38 (ref. 56), 49 (ref. 56), 76 Graham, C. E., 108 (see Hier), 110 (see Hier), 111 (see Hier), 112 (see Hier), 113 (see Hier), 116 (see Hier), 118 (see Hier), 119 (see Hier), 120 (see Hier), 121,260 Graham, W. R., 221 (ref. 39),226 Graniek, S.,234, 267, 298 Grantham, J., 139, 2 4 9 Grassmann, W.,41 (ref. 193), 42 (ref. 193),43 (ref. 193),44 (ref. 193),67 (ref. 194), 68, 79, 360, 362, 363 (ref. 126), 364 (ref. 61), 366 (ref. 126),368 (ref. 126),369 (ref. 35, 50), 370,371 (ref. 35), 378 (ref. 66), 383 (ref. 66),385 (ref. 66), 386 (ref. 66), 388 (ref. 66), 389 (ref. el), 392 (ref. 61), 393 (ref. 61, 66), 398, 405 (ref. 46), 406 (ref. 46),415 (ref. 50), 416, 417, 418

Grau, C. R., 244, 296 Graves, G. D., 326,348 Green, D.E., 33 (ref. 182), 79, 220 (ref. 23), 221 (ref. 23),226, 273, 298 Greenberg, D. M., 128, 134, 249, 207, 286, 426

Greene, R. A., 250, 298 Greene, R. D., 92,95, 99, 102, 103, 105, 110, 111 (see Smith, E.L.), 112, 113 (see Smith, E. L.), 117, 118, 119, 120, 138, 260, 262, 212 (ref. 121123), 213, 214 (ref. 121), 218 (ref. 121-123), 828, 244, 263, 264, $98, 302 Greenstein, J. P., 6, 19, 38 (ref. 255b), 42 (ref. 197), 43 (ref. 197), 44 (ref.

449

AUTHOR INDEX

197), 51 (ref. 198a), 53 (ref. 196), 54, 59 (ref. 81), 61 (ref. 81), 62 (ref. Sl), 64 (ref. 196a), 65, 76, 79, 81,97, 105, 134, 139, 141, 160, 161, 255, 257, 266, 298, 309 (ref. 130), 311 (ref. 130), 315 (ref. 130), 316 (ref. 130), 3.49, 365, 417 Gregory, F. G., 283, 287, 289, 298 Grimaux, E., 2, 79 Grimmer, W., 222, 826 Grindley, H. S., 234, 242, 898 Granwall, A., 126, 135, 160,216, 217, 886 Groh, J., 205, 886 Gromyko, E., 294, 301 Groot, E. H., 240, 298 Grossfeld, I., 22 (ref. 223a), 80 Groves, M. L., 223 (ref. 148), 828 Griinert, H., 10 (ref. 207), 80 Gilnther, G., 271, 297 Guggenheim, M., 49 (ref. 14, 51 (ref. 13),76 Guggisberg, H., 207, 227 Guillermond, A., 266, 298 Guiltonneau, G., 221, 2.26 Guirard, B. M., 91, 108, 113, 118, 119, 120, 160,162 Gulick, A., 277, 298 Gumlich, O., 9 (ref. log), 77 Gunnese, M., 91, 103 (see Stokes), 106, 109, 110 (see Stokes), 111 (see Stokes), 112 (see Stokes), 113 (see Stokes), 116, 117 (see Stokes), 118 (see Stokes), 119 (see Stokes), 120 (see Stokes), 122, 160,162,163,248, 249, 250, 251, 252, 253, 254, 256, 258, 259, 303 Gunsalus, I. C., 273, 300 Curd, F. N., 189, 198,200 Gustavson, I(. H., 308 (ref. 63), 309 (ref. 63), 948 Gustavson, K. H., 361 (ref. 68, 71, 93), 362 (ref. 63), 363 (ref. 94), 364 (ref. 61, 74), 365 (ref. 82), 368 (ref. 71), 370 (ref. 71, 79, 82), 371 (ref. 71, 72, 75, 91), 372 (ref. 68, 71), 373 (ref. 55, 71), 374 (ref. 55, 71, 74), 375 (ref. 71, 74), 378 (ref. 66), 379 (ref. 77, 80, 81), 380 (ref. 66), 381 (ref. 194), 382 (ref. 77, 80, 81, 97), 383 (ref. 66, 68, @4,97), 384 (ref. 59, 65,

81), 385, 386 (ref. 66), 387 (ref. 56, 57, 87, 88), 388 (ref. 57, 66, 71, 84), 389 (ref. 61), 390 (ref. 54, 62), 391 (ref. 60, 68), 392 (ref. 61, 64,94), 393 (ref. 61, 65, 66, 68), 394 (ref. 70, 83), 397 (ref. 70), 398 (ref. 70, 85, 92, 94), 399 (ref. 55, 70, 71, 79, 83, 85), 400 (ref. 75), 401 (ref. 61), 402 (ref. 94), 403 (ref. 76, 78, 96), 404 (ref. 76, 78, 94, 95), 405 (ref. 67, 89, 90, 94), 406 (ref. 67), 407 (ref. 69, 73, 86), 408 (ref. 69, 73, 93), 409 (ref. 70, 73), 410 (ref. 67), 411 (ref. 67), 412 (ref. 93), 414 (ref. 68), 415 (ref. 79), 417,418,480 Gutfreund, H., 128, 132, 160 Guth, E., 335, 3.48 Gutmann, M., 330 (ref. 25), 335 (ref. 25), 347

H Haag, J. R., 236, 298 Haas, P., 45 (ref. 200), 79, 220 (ref. 44), 2.26 Haase, E., 40 (ref. 14), 76 Hac, L. R.,106, 108, 138, 160, 218 (ref. 45), 226,264, 298 Hadley, P., 92, I60 Hadorn, H., 406, 407, 410 (ref. 161), 419 Hafner, F. H., 244, 298 Hakala, M., 275, 304 Hale, F., 103 (see Lyman), 108 (see Lyman), 112 (see Kuiken), 113 (see Kuiken), 119 (see Lyman), 138 (see Lyman), 161,235, 244, 299 Hale, W. S., 222 (ref. 7),226 Hall, C. E.,960, 359 (ref. 98, 180), 418, 4.90 Hall, C.P., 400 (ref. 150), 419 Hall, D. H.,233, 897 Hall, L., 175 (see Zucker, L.), 179 (see Zucker, L.), 180 (see Zucker, L.), 800 Hall, W. K., 182, 197,198,199 Halpin, J. G.,193, 197 Halsey, G., 332 (ref. 39-41), 333, 335 (ref. 66), 947,344 360 Halsey, J. T., 16, 78 Halwer, M., 311 (ref. 132), 327, 360 Hamilton, T. S., 199,234, 242, 263, 298, 300

450

AUTHOR INDEX

Hammarsten, O., 202 (ref. 46),886 Hamoir, G.,140, 146,149 Hanby, W.E.,45 (ref. 201), 79,265,298 Handler, P.,18 (ref. 96),77 Hanke, M. T., 119,160 Hansen, R. G., 102, 103, 106, 108, 110, 111, 112, 113, 117, 118, 119, 120, 160, 213, 218 (ref. 48), 223 (ref. 47, 48), 826 Haneon, E. A., 234,267,898 Haneon, H. T., 36 (ref. 201a), 39 (ref. 201a),70 (ref. 201a),80 Harden, A., 222, 226 Harington, C. R., 3 (ref. 202), 31, 36 (ref. 203), 41 (ref. 203a), 42 (ref. 203),43 (ref. 202, 203), 44 (ref. 202, 203), 45 (ref. 203),47, 50 (ref. 204), 61, 52, 53 (ref. 203, 204), 54 (ref. 203,204),80, 100, 132, 133, I60 Harmon, K. M., 50 (ref. 240a),81 Harris, G., 18 (ref. 103a),77 Harris, J. I., 25, 49 (ref. 204a),80 Harris, L. J., 406 (ref. 13,99),416,418 Harris, M., 308 (ref. 69), 312 (ref. 155), 328, 329, 348, 360, 363 (ref. 190), 364 (ref. 191),420 Harris, R. H., 262,298 Harrison, H. C.,182, 188,198 Harrison, W.,329 (ref. 721,348 Harte, R. A., 176, 192, 198 Hartig, T.,260, 298 Hartree, E.F., 220,226 Hartung, W.H., 20 (ref. 205),80 Ham, E.,264 Havestadt, L., 11, 50 (ref. 206),80 Hawley, E. E., 170 (see Murlin), 171 (see Murlin), 198,199 Haydee, E., 182,197 Hayward, J. W., 244, 298 Hegediie, B.,43 (ref. 206a),45 (ref. 206a), 53 (ref. 206a), 80 Hegsted, D. M., 99, 111, 113, 160, 179, 198,246,9102 Heidelberger, M., 263, 299 Heiduschka, A., 220 (ref. 501, 226 Heilbron, I. M., 18 (ref. 103a), 77,270, d96 Heinsen, J., 198 Helferich, B., 10 (ref. 2071,80

Hellbach, R., 311 (ref. 138), 314 (ref. 138), 360 Hemingway, A., 285,304 Henderson, L.M.,102,103,106,108,110, 111, 112, 113, 116, 117, 118, 119, 120, 160, 218 (ref. 51), 826 Hendry, J. L., 149 Henze, E., 36 (ref. 260),81 Herbst, R. M., 20,80,81,276, 298 Hermann, K.,308 (ref. 57), 328 (ref. 57), 348 Herriott, R. M., 3 (ref. 212),80,125,126, 145, 160, 307 (ref. 73), 548 Hem, W.C.,72 (ref. 213),73,80,96,103, 105, 111, 117, 160, 163, 218 (ref. 129), 828,256,262, 298,300 Hetler, D. M., 250,298 Hevesy, G.,283, 298 Hewitt, F. O., 109, 110, 114, 116, 119, 149 Hier, 9. W., 108, 110, 111, 112, 113, 116, 118, 119,120, 121 (see Graham), 160 High, L. M., 311 (ref. 166),317 (ref. 166), 360 Highberger, J. H., 361 (ref. 101), 362, 363 (ref. loo), 367, 368, 372 (ref. 101), 376 (ref. 147), 377 (ref. 104), 406 (ref. 102), 407, 408 (ref. 101), 409, 410 (ref. 101), 411, 412, 418, 419,P O Hill, D. W., 208, 286 Hill, T.G., 45 (ref. 200),79,220 (ref. 44), 826

Hilpert, S., 411, 412, 418 Hipp, N.J., 223 (ref. 148),228,311 (ref. 138),314 (ref. 138),360 Hippius, A., 219 (ref. 52), 221, 226' Hird, F. J. R., 295 Hirsch, P.,37 (ref. 16), 38 (ref. 15, 16), 39 (ref. 15, 16), 76 Hirsch, R. R., 193 (see Russell), 199 Hirszowski, A., 38 (ref. 17), 51 (ref. 17), 76 von Hochstctter, H., 79 Hock, C. W., 182 (see Berg; Hall, W. R.), 297, 198 Hoffman, 0.D.,202 (ref. 8), 203 (ref. 8), 218 (ref. 8),226 Hoffman, W.F., 262, 298

451

AUTHOR INDEX

Hofmann, K., 43 (ref. 21b), 49 (ref. 215), 60 (ref. 214), 61 (ref. 214, 216), 64 (ref. 216), 80 Hofmeister, F., 2, 80, 373, 418 Hogeboom, G. H., 425 (ref. 28, 29), 431 (ref. 28, 49), 432 (ref. 28, 29), 436 (ref. 49), 438 (ref. 28), 4.99, GO Holland, H. C., 361 (ref. 107), 393 (ref. 169), 418, 419 Holman, R. L., 182, 185, 186, 198 Holt, L. E., Jr., 171 (see Cox), 198 Holter, H., 209, 226 Hooke, R., 310, 348 Hoover, S. R., 218 (ref. 88), 227, 274, 296

Hopkins, F. G., 51 (ref. 218), 80, 220, 827

Horn, M. J., 110, 120, 151, 263, 298 Homer, C. K., 274, 298 Horowite, N. I€., 273, 302 Hotchkiss, R. D., 3 (ref. 219), 80, 217 (ref. 75), 226, 280, 299, 425 (ref. 28), 431 (ref. 28), 432 (ref. 28), 438 (ref. 28), 4.39 Houston, J., 159 (see Kade), 198 Howe, E. E., 166, 199 Howe, P. E., 202, 211, 226, 227 Howitt, F. O., 311 (ref. 32, 33), 324, 325, 3447,348

Huddleson, I. F., 223 (ref. 110), 227 Hudson, D. P., 270, 296 Huffman, H. M., 33 (ref. 92), 77 Huggins, M. L., 308 (ref. 76), 328 (ref. 76), 348, [360, 361 (ref. log), 401, 418 Hughes, A. H., 311 (ref. 77), 348 Hull, R., 217, 225 Hulme, A. C . , 241, 286, 299 Hummel, F. C . , 105 (see Bectch), 148 Humphreys, E. M., 190, 197 Humphreys, F. E., 394 (ref. 30, 110), 416, 418

Hunt, M., 11, 22 (ref. 220), 36 (ref. 220), 40 (ref. 220), 42 (ref. 288), 44 (ref. 288), 55 (ref. 292), 61 (ref. 220, 221), 62 (ref. 220, 221), 65 (ref. 220, 221, 288), 80, 82 Hurwits, S. II., 182, 198 Hutchinson, J. C . D., 240, 299

I Ing, H. R., 32, 80 Irving, G. W., Jr., 34 (ref. 190), 7'9,262, 298, 899

Ivanovics, G., 45 (ref. 222), 80

J Jackson, R. W.,307 (ref. 22), 311 (ref. 110, 138), 314 (ref. 138), 347, 34.9, 360 Jacobs, W. A., 57, 78 Jacobsen, C. F., 2, 53 (ref. 222a), 80, 81, 100, 126, 132, 133, 135, 161, 217 (ref. 59), 218, 226 Jakus, M. A., 360, 359 (ref. 98, 180), 418, 420

Jameson, E., 212, 226 Jander, G., 381 (ref. l l l ) , 418 Janisch, R., 260, 299 Jansen, E. F., 342 (ref. 78),348 Jarrouse, H., 221 (ref. 43), 826 Jeannerat, J., 311 (ref. 122), 34.9, 369 (ref. 21), 416 Jensen, H., 105 (see du Vigneaud), 163 Jirgensons, B., 316 (ref. 79), 348 Johansen, D., 280, 299 Johansen, G., 217 (ref. 75), 226 Johanson, R., 244, 252, 299 Johns, C. O., 262, 299 Johnson, J. R., 18 (ref. lOOa), 75 (ref. lOOa), 77 Johnson, M., 262, 298 Johnson, R. M., 171, 198 Johnson, T. B., 250, 299 Johnson, W. A., 285, 299 Jones, D. B., 110 (see Horn), 120 (see Horn), 161, 244, 247, 262, 263, 297, 298, 299

Jones, M. J., 273, 299 Jordan Lloyd, D., 355, 418 Jovanovits, J. A., 362, 418 Just, F., 250, 298

K Kabat, E. A., 263, 299 Kade, C. F., Jr., 159, 198 Kamen, M. D., 277, 302, 427 (ref. 53), 440

Kann, E., 18 (ref. 58), 76

452

AUTHOR INDEX

Kapfhammer, J., 7 (ref. 223),80 Kardos, E.,205 (ref. 42), 926 Kariher, D.H., 183,199 Karrer, E.,335,348 Kassell, B.,87, 96, 103, 105, 109, 110, 116, 117, 118, 120 (see Brand), 135, 149, 161, 216 (ref. 13), 218 (ref. 13, 61),226, 226 Katchalski, E., 7, 22, 79, 80, 307 (ref. 531,347 Kattus, A. A., Jr., 183 (see Madden), 198 Katz, J. R., 361 (ref. 115, 116),418 Katz, S.,348 Kautzsch, K., 5 (ref. 153), 78 Kay, H. D., 208, 221 (ref. 39, 62), 226, 227 Kaye, M. A. G., 409 (ref. 217),421 Keilin, D.,220, 226, 275, 299 Kekwick, R. A., 135, 138,161, 214 Kelly, M. W.,391 (ref. 206), 396 (ref. 205),411, 42f Kelly, P. L., 221 (ref. 64, 65),226 Kemm, E.,8 (ref. 296), 48 (ref. 296), 88 Kemmerer, K. S., 171 (see Cox), 198 Kemp, A. R., 241,299 Kempe, M.,11, 71 (ref. 18), 72,76 Kennaway, E.I,., 63 (ref. 225),80 Kennedy, E. P., 425 (ref. 30),431, 439 Kenten, R. H., 143, 149, 357, 414, 416, 481

Kenyon, A. E., 270, 303 Kern, E.J., 384 (ref. 214), 491 Kerr, W.J., 182, 198 Keston, A. S., 93,94, 101, 114, 161, 218 (ref. 66),996,283, 298 Kibrick, A. C., 106, 108, 135 (see Cannan), 136 (see Cannan), 138, 149, f61,214 (ref. 17),216 (ref. 17),226 Kidd, D. E., 32, 45 (ref. 223b), 80 Kiesel, A., 240,299 King, F.E., 32,45 (ref. 223b), 80 King, H., 373 (ref. l), 416 Kinsman, G. M., 163,197 Kinzer, R.,383, 418 Kirkwood, J. G., 341 (ref. 82),348, 360 Klein, D., 108 (see Hier), 110 (see Hier), 111 (see Hier), 112 (see Hier), 118 (see Hier), 116 (see Hier), 118 (see Hier), 119 (see Hier), 120 (see Hier), 121 (see Graham), 160

Klein, G., 280, 299 Klemm, O., 307 (ref. 123), 308 (ref. 123), 349

Klemperer, F. W.,42 (ref. 197), 43 (ref. 197), 44 (ref. 197),65, 79 Klose, A. A., 249, 299 Klotz, I. M., 207, 226 Knight, C. A., 101, 102, 103, 105, 106, 108, 109, 110, 111, 112, 113, 114, 116, 117, 118, 119, 120, f61, 255, 256, 259,299 Kodama, S., 204, 227 Kogl, F., 441 (ref. 224),53, 80, 253, 999 KBhler, E.,260, 299 Kohler, F.,33 (ref. 40),73, 76 Koelker, A. H., 39 (ref. 154), 78 Koenigs, E.,41, 43 (ref. 155), 44 (ref. 155, 166),78 Koppel, W., 54 (ref. 21), 69 (ref. 20), 76 Koessler, K. K., 119, 160 Koster, H., 5 (ref. 82), 16 (ref. 60), 49 (ref. 60),61, 63, 76' Kollen, S.,436 (ref. 57),440 Kolyakova, G.E., 420 Komm, E., 220 (ref. 50),226 Koning, C. J., 219, 226 Kooper, W.D., 222 (ref. 69),226 Koorn, V. M., 311 (ref. loo), 316 (ref. loo), 336 (ref. loo), 345 (ref. loo),

349

Korn, A. H., 218 (ref. 88),227 Kossel, A,, 63 (ref. 225, 226),80 Kosterlitz, H. W., 182, 187, 197, 198 Kraemer, E. O., 316 (ref. 83), 348, 367, 418

Kraft, W. M., 80 Kratky, O., 306 (ref. 84),348 Kratzer, F. H., 244,296 Krauel, K., 159 (see Kade), 198 Krauss, B. H., 271,302 Krebs, H. A., 285, 299 Kritzinger, C.C., 377, 418 Kritzman, 20 Kritzmann, M. G.,271, 273, 296 Kropp, W.,43 (ref. 157),44 (ref. 157),78 Kuchel, R. H., 281,304 Klihne, E.,369 (ref. 35), 371 (ref. 35), 416

KUntzel, A., 360, 362 (ref. 127, 129),363 (ref. 126), 366 (ref. 126), 368, 369

AUTHOR INDEX

(ref. 120, 121, 123), 372 (ref. 123), 375 (ref. 123), 377, 380 (ref. 122), 383, 386 (ref. 130), 390, 393 (ref. 131), 403, 411, 418, 419 Kuhlmann, A. G., 262, 299 Kuhn, R., 96, 103, 105, 139, 161 Kuhn, W., 321 (ref. 85), 348, 369, 418 Kuiken, K. A., 93, 108 (aee Lyman), 112, 113, 115, 119 (see Lyman), 138 (see Lyman), 161, 235, 244, 299 Kuk, S.,67 (ref. 177), 79 Kulka, J. P., 181 (see Zeldis), 200 Kunitz, M., 126, 145, 161, 162, 263, 264, 299 Kuttner, A., 211, 226

L Laidler, K. J., 333 (ref. 59), 348 Laine, T., 273, 275, 304 Lamanna, C., 124 (see Buehler), 125 (see Buehler), 143 (see Buehler),

1.69 Lambotte, E., 9 (ref. 110), 77 Lampen, J. O., 273, 299 Lampman, C. E., 244, 301 Lams, M., 412 (ref. 198), 420 Landolt, H., 48 (ref. 192a), 79 Landsteiner, K., 424 (ref. 31), 439 Lane-Claypon, J. E., 222, 226 Larionov, L. P., 429 (ref. 32), 439 Larkin, J. B., 15 (ref. 267), 39 (ref. 267), 81

Larsmn, A., 404 (ref. 95), 418 Latimer, W. M., 393, 419 Laufer, M. A., 162 Lavine, T. F., 103, 161 Leach, M. F., 250, 299 Leaderman, H., 331 (ref. 86), 332 (ref. 861, 348

Leavenworth, C. S., 233, 235, 273, 281, 284, 288, Sol,303 Leeper, G. W., 270, 299 Lehmann, W., 209, 227 Lehninger, A. L., 425 (ref. 30), 431, @9 Lehoult, Y., 268, 303 Leinert, F., 36 (ref. 89), 61 (ref. 22), 66 (ref. 22), 68 (ref. 89), 69 (ref. 89), 70 (ref. 89), 76, 77 Leloir, L. F., 273, 298

453

Lemoigne, M., 270, 299 Leplat, G., 371 (ref. 134), 419 Le Quesne, W. J., 43 (ref. 227a), 44 (ref. 227a), 45 (ref. 227a), 80 Leuchs, H., 21, 36 (ref. 228), 37 (ref. 229), 49 (ref. 229), 80, 307 (ref. 87, 881, 348 Leutert, F., 24, 79 Leuthardt, F. M., 19 (ref. 198), 51 (ref. 198a), 79, 257, 298, 373 (ref. 135), 419

Levene, P. A., 5, 18 (ref. 236), 36 (ref. 235), 38 (ref. 231, 233, 234, 237), 39 (ref. 234, 237), 67 (ref. 232), 80, 208, 226

Levitt, J., 241, 299 LCVY, L., 9, 77 LCW,W., 182, 187, 196 Lewis, J. C., 108, 138, 161 Lewis, J. H., 211, 226 Ley, H., 390, 419 Li, C. H., 126, 127, 135, 161, 216, 226 Li, Tsan-Wen, 182, 198 Lilienfeld, L., 2, 80 Linderstrgim-Lang, K., 2, 81, 89, 100, 132, 133, 161, 204, 205, 209, 217 (ref. 75), 226, 227, 276, 280, 283, 898, 299, 376, 419 Linkola, H., 275, 304 Lintcel, W., 240, 299 Lipmann, F., 208, 227,'286, 299 Lipschitz, E., 36 (ref. 158), 49 (ref. 158), 50 (ref. 158), 78 Little, R. B., 211, 228 Lloyd, D. J., 335 (ref. 89), 348 Loeb, J., 366, 367 (ref. 139), 419 Lowe, H., 406 (ref. 48), 416 Logan, M. A., 109, 149 Long, C. N. H., 182, 188, 198 Longsworth, L. G., 126, 138, 161 Loo, Y. H., 89, 1.69 Loring, H. S., 44 (ref. 289), 52 (ref. 240, 286), 53 (ref. 240), 54, 62 (ref. 240), 81, 82 Lowe, B., 306 (ref. go), 3.48 Lowndes, J., 218 (ref. 102), 227 Luck, J. M., 217 (ref. 6), 226 Ludewig, S., 207 (ref. 18), 226 Lugg, J. W. H., 98, 103, 105, 116, 117, 119, 121, 161, 218 (ref. 78), 227, 233,

454

AUTHOR INDEX

234, 236, 236, 237, 238, 239, 240, 243, 244, 245, 240, 247, 248, 265, 268, 267, 258, 279, 293, 294, 298, 299,900 Lum, F. G., 217 (ref. 6),926 Lundgren, H. P., 306 (ref. 99, 101), 311 (ref. 96, 96, 98, 100, 166), 313 (ref. 96), 315 (ref. 97, lOl), 316 (ref. 91, 92, loo), 317 (ref. 93-96, 166), 323 (ref. 96), 336 (ref. 100), 346 (ref. 100, 101),349,360 Luniak, A., 3, 6, 11, 68,70, 78 Lyman, C. M., 93, 103, 108, 112 (see Kuiken), 113 (see Kuiken), 115, 119, 138, 161, 236, 244, 299

M Maack, J. E., 173, 174 (see Barnes), 176 (see Barnes), 197 MacAllister, R. V., 50 (ref. 240a), 81 MacArthur, I., 306 (ref. 102, 103),349 McCalla, A. G., 262,302 McCandlish, D.,369 (ref. 16), 372 (ref. 16), 400,416 MacCardle, R. C., 435 (ref. 33),439 McCarty, M., 424 (ref. 3, 34), 427 (ref. 3,341, 439 McClintock, B., 427, 440 McCoord, A. B., 181 (see Zeldis), 600 McDonald, M. R., 264,299 MacFarlane, A. S . , 135,161 McGavack, T.H., 171,199 McGinnis, J., 193, 198 McGowan, J. C., 235,30% Macheboeuf, M., 435 (ref. 36), 440 MacInnes, D.A,, 126 (see Longsworth), 138, 161 McIntyre, J. M., 168 McKinney, H. H., 292,293,SO0 McLaughlin, G. D., 372 (ref. 148), 376, 387, 392 (ref. 148),419 MeLennan, E.,295 MacLeod, C. M., 424 (ref. 3), 427 (ref. 3), 439 McMahan, J. R., 113, 118,161 McMeekin, T. L., 126, 127, 135, 161, 212 (ref. 80), 215, 217 (ref. 79, 81, 82), 223 (ref. 148), 867, 698, 311 (ref. 109, 110),325,349

McNaught, J. B., 183, 198 MacPherson, H. T., 88, 89, 90, 95, 98, 104, 110, 118, 119, 120, 122, 130, 161, 218 (ref. 84),227 Macrae, T. F., 240, 699 Macy, I. G., 105 (see Beach), 148, 202 (ref. 8),203 (ref. 81,218 (ref. 8), 926 Madden, 8. C., 183, 187,198 Miirkert, L., 13 (ref. 180), 79 Magnant, C.,434 (ref. 25), 439 Mahadevan, S., 280, 297 Mahdihassan, S., 265, d98 Mahoney, E. B., 182, 185, 187 (see Holman), 198 Maier, J., 24, 77 Manasse, W.,21 (ref. 230), 80,307 (ref. 881, w Manske, R. H. F., 32, 80 Marais, J. S . C., 236,SO0 March, M. E., 172 (see Murlin), 174 (see Murlin), 199 Marenzi, 247 Mark, H., 307, 308 (ref. 55, 104, 105), 312 (ref. 106), 313, 328 (ref. 106), 330, 332 (ref. 106), 335 (ref. 64), 348, 349, 359 (ref. 156))419 Marker, R. E., 5 (ref. 237),38 (ref. 237), 39 (ref. 237),80 Markley, A. L., 221,667 Marriott, It. H., 355 (ref. 112),360, 369 (ref. 141), 375, 376 (ref. 140, 143), 418, 419 Martell, A. E., 81 Martin, A. J. P., 3 (ref. 103, 242), 77, 81, 84,88,94,97,110, 123, 136,149, 160, 161, 237, 300,360,419 Martin, L. F., 292, 293,SO0 Maschke, O.,260,300 Maschrnann, E.,40 (ref. 243),81 Masket, A. V., 207 (ref. 18),866 Mason, T.C., 284,300,SO1 Massart, L., 221, 267 Matthes, K.,7 (ref. 223),80 Mauersberger, H.R., 311 (ref. 107),314 (ref. 107),349 Max, J., 11 (ref. 244), 36 (ref. 244), 37 (ref. 244),81 Maxwell, J. C., 332,349 Maycr, J., 8 (ref. 296), 48 (ref. 296),8.2 Mazur, A., 246, 247, 248, 249, 257, 300

465

AUTHOR INDEX

Mead, T. H., 3 (ref. 202), 31, 36 (ref. 203), 42 (ref. 203), 43 (ref. 202, 203), 44 (ref. 202, 203), 45 (ref. 203),

Miller, G. L., 43 (ref. 248), 44 (ref. 248, 289, 291), 53, 54 (ref. 289), 81, 89,

51, 80, 100, 132, 160 Mecchi, E., 244, 296 Mecheels, O., 311 (ref. 112, 113), 949 Mehl, J. W., 171, 198 Mehrhof, T. G., 193 (see Russell), 199 Melchers, G., 255, 300 Mellander, O., 204, 205, 206, 208, 218 (ref. 87), 219 (ref. 87), 227 Mellon, E. F., 218 (ref. 88), 227 Melnick, D., 158, 159, 174, 185, 186, 193, 198 Melville, D. B., 18 (ref. 96), 77 Melville, J., 44 (ref. 245), 45, 81 Mendel, L. B., 175, 193, 199 Menefee, S. G., 202 (ref. 89), 203 (ref. 89), 227 Menke, W., 234, 239, 267, 900 Mercadante, M., 278, 300 Mercer, E. H., 330, 349 Mercer, F. V., 281, 304 Meridith, R. J., 331 (ref. 115), 349 Merkel, R., 67, 70 (ref. 231, 76 Merrifield, A. L., 311 (ref. 116), 315 (ref. 1161, 349 Merrill, H. B., 375, 387 (ref. 151), 400, 419 Meunier, L., 411, 412 (ref. 152), 419 Meyer, C. E., 25 (ref. 2901, 82 Meyer, F., 205, 226 Meyer, H., 10, 81 Meyer, K. H., 307, 308 (ref. 123, 124), 309 (ref. 124), 311 (ref. 122), 335, 349, 359 (ref. 156), 369, 378, 406 (ref. 153), 410, 411, 416, 419 Mez, 257 Mezey, E., 400 (ref. 20), 416' Michael, G., 281, 300 Michaelis, L., 376 (ref. 157), 419 Miekeley, A., 17 (ref. 65), 18 (ref. 58), 49 (ref. 62), 51 (ref. 65), 55, 57 (ref. 61, 65), 76 Miescher, K., 75 (ref. 252, 294), 81, 82 Migliardi, C., 68 (ref. 247), 70 (ref. 247), 81 Millar, A., 310, 311, 349 Miller, E. J., 233, 234, 238, 241, 29Y, 300 Miller, G., 265, 300

Miller, L. L., 166 (see Whipple, G. H.), 171, 183, 184, 185, 189, 194, 198, 199, 200 Milne, 25 Minard, F. N., 424 (ref. 27), 439 Mirsky, A. E., 139, 141, 148, 161, 308 (ref. 126), 311 (ref. 127, 140), 316 (ref. 5), 346, 349, 360, 369, 419, 426,

105, 161

427,

.bdo

Mitchell, H. H., 156, 158, 162, 163, 164, 165, 169, 171, 173, 174, 176, 192, 193, 194, 197, 199, 236, 263,-300 Miaell, L. R., 331 (ref. 71), 348 Moeller, O., 247, ,999 Mohr, E., 15, 18, 33 (ref. 249), 81 Moir, F., 252, 299 Moir, R. J., 244, SO3 Monguillon, P., 270, 999 Moore, D. H., 216, 226 Moore, E. K., 376 (ref. 147), 419 Moore, S., 3, 36 (ref. 278), 38 (ref. 278), 67 (ref. 279), 81, 88, 95, 101, 102, 111, 112, 113, 116, 122, 161 Morehouse, M. G., 171, 198 Morel, M., 290, 900 Morgan, E. J., 220, 227 Morneweg, W . , 46 (ref. 2), 50 (ref. 2), 61 (ref. 2), 76 Moro, E., 219 (ref. 91), 221, 227 Morris, H. J., 274, 296 Morris, M., 223 (ref. 147), 228 Moseley, O., 103 (see Lyman), 161 Mothes, K., 272, 283, 284, 300 Mourgue, M., 264,901 Mowat, J. H., 45 (ref. 250a), 81 Moxon, A. L., 272, 303 Moyer, L. S., 267, 998, 300 Mueller, A. J., 159, 171 (see Cox), 198 Murlin, J. R., 170,171,172,174,198,199

rl Nageotte, J., 371 (ref. 159), 419, 434, 4-40

Nakamura, F. L., 176 (see Beadles), 197 Naeset, E. S., 172 (see Murlin), 174 (see Murlin), 199

456

AUTHOR INDEX

Olcott, H. S., 96, 108, 119, 121, 129, 133, 135, 138, 145, 161, 162, 307 (ref. 49-52, 134), 347, 360, 407 (ref. 42), 409 (ref. 40, 41), 410, 416 Olofsson, B., 391 Olsen, C., 283, 298 Olsen, R. T . , 198 Omachi, A., 432 (ref. 41), 440 Oncley, J. I,., 128, 132, 144, 217 (ref. 34), 226 Onslow, M. W., 263, 300 Oparin, A. I., 289, SO0 Orcutt, M. L., 211, 887 van Ormondt, J., 311 (ref. 8), 346 Orten, A. U., 184, 199 Orten, J. M., 184, 199 Osborne, T. B., 3, 67 (ref. 253), 68, 70, 81, 134, 162, 175, 193, 199, 202, 205, 206 (ref. 97), 223, 897, 298, 233, 235, 260, 261, 263, 264, 296, 300, 301 Oser, B. L., 193, 198 416 Nienburg, H., 68, 69 (ref. 25), 70 (ref. Ott, E., 311 (ref. 135, 136), 324 (ref. 136), 360 25), 76 Otto, E., 11, 21, 36 (ref. 160), ‘78 Nier, A. O., 285, 304 Nitschmann, H., 207, 209, 867,406, 407, Otto, G., 364, 385 (ref. 164), 387 (ref. 164), 419 410, 419 Overhoff, J., 41 (ref. 203a), 80 Niven, C. F., 182, 199 Overman, 0. R., 202 (ref. 89), 203 (ref. Noack, K., 239, 300 89), 287 Nocito, V., 273, 298 Noguti, Z., 311 (ref. 60), 315 (ref. 60), Owen, E. C., 236, 301 348 P Norman, W. H., 112 (see Kuiken), 113 (see Kuiken), 161, 235, 244, g99 Pacsu, E., 7, 26 (ref. 254), 81 Northrop, J. H., 126, 145, 161, 168 Nutting, G. C., 306 (ref. 133), 311 (ref. Paech, K., 285, 289, 301 131, 132, 150), 313 (ref. 151), 325 Page, It. O., 360 (ref. 1681, 393 (ref. 169), 399 (ref. 168), 401, 419 (ref. 131, 150), 327, 328 (ref. 151), 349, 360, 359 (ref. 162), 360 (ref. Pallade, G. E., 425 (rcf. 29), 432 (ref. 29), 162), 419 439 Palmer, A. H., 127, 135, 136 (see Can0 nan), 138 (see Cannan), 149, 162, 214, 215, 216, 217, 826, 28’7, 298 O’Connell, R. A., 311 (ref. 95, loo), 316 Palmer, K. J., 323, 328 (ref. 137), 360 (ref. loo), 317 (ref. 95, 96), 336 (ref. Palmer, L. S., 241, 304 Palmes, E. D., 256, 298 loo), 345 (ref. loo), 349 O’Doherty, K., 197 Pankhurst, K. G . A., 370, 371 (ref. 170), 415 (ref. 170), 490 Oesterling, M. J., 158 (see Borman), 197 Ogston, A. H., 128, 160, 168, 360 (ref. Pape, N. R., 328 (ref. 18, 56), 346, $48 Parker, E. D., 311 (ref. 26), 315 (ref. 26), 163), 419 O’Kane, D. E., 273, 300 347

Neale, S. M., 391, 392, 419 Nedvidek, R. D., 241, 30.2 Needham, J., 266, 300 Negelein, E., 264, 271, 304 Neglia, F. J . , 262, 300 Neher, R., 75 (ref. 251, 252), 81 Nelson, R. E., 120, 163 Neuberger, A., 13 (ref. 252a), 81, 89, 133, 139, 160, 161 Neumann, A., 36 (ref. 24), 38 (ref. 24), 76 Neurath, H., 97, 144, 161, 309 (ref. 130), 311 (ref. 130), 315 (ref. 130), 316 (ref. 128, 130), 330, 349 Nevens, W. B., 234, 242, 298 Neville, H. E., 89, 149 Newcomer, E. H., 266, 900 Newell, G. W., 199 Nicolet, B. H., 88, 89, 109, 110, 115, 161, 168, 208, 227 Niemann, C., 50 (ref. 240a), 81, 148, 360,

AUTHOR INDEX

Parsons, H. T., 245,297 Pssteur, L.,424,440 Patterson, W.I., 55 (ref. 292),82 Paul, W.,197 Pauling, L., 308 (ref. 126),349,369,379, (ref. 171),419, 420 Pavlova, M., 240,299 Payne, D.S., 244,301 Pearsall, W.H., 288,301 Pedersen, K. O.,134, 135,162,163,202, 207, 214, 215, 216, 222, 2.27, 238, 263,303 Pedlow, C., 281,SO4 Peakett, G. R., 223,227 Peters, R. H., 391 (ref. 160),419 Peterson, C. F., 244, 301 Peterson, R. F., 311 (ref. 138), 314,360 Petrie, A. H. K., 271, 276,277, 284,285, 286,289,301,304 Petrik, F. G.,268,301 Pfaltz, M. H., 36 (ref. 235),38 (ref. 233, 234), 39 (ref. 234),80 Pfeffer, W.,278, 283, 285,301 Pfeiffer, P.,373,390, 397,420 Pfeiffer, S. E., 249,303 Phillips, G.E., 249,297 Phillips, H., 376, 416 Phillips, J., 362 (ref. 129),377 (ref. 129), 418 Phillips, P. H., 102 (see Hansen), 103 (see Hansen), 106 (see Hansen), 108 (see Hansen), 110 (see Hansen), 111 (see Hansen), 112 (see Hansen), 113 (see Hansen), 117 (see Hansen), 118 (see Hansen), 119 (see Hansen), 120 (see Hansen), 160, 213 (ref. 48), 218 (ref. 48), 223 (ref. 47,48),826 Phillis, E.,284, 300,301 Pickels, E. G.,267, 302, 432 (ref. 20), 439

Picken, L., 335 (ref. 121a),349 Pinner, 9. H., 306 (ref. 139), 360 Piria, R.,277,301 Pirie, N. W., 50, 53 (ref. 255), 54 (ref. 255),81,125 127,166,235,254,255, 293,296,301 Pitt Rivers, R. V., 47, 50 (ref. 204), 51 (ref. 204), 53 (ref. 204), 54 (ref. 204),80 Pleass, W. B., 406 (ref. 15), 416

457

Plimmer, R. H. A., 218 (ref. 102), 227 Plowe, J. Q., 560 Policard, A., 435 (ref. 43), 4 0 Polis, B. D., 126 (see McMeekin), 127 (see McMeekin), 136 (see McMeekm), 161, 217 (ref. 81, 82), 227 Polis, E., 204 (ref. 141), 208, 222, 2d8 Pollister, A. W.,311 (ref. 127, 140), 349, 360, 426 (ref. 38), 440 Pollok, H.,61 (ref. 55),64 (ref. 55), 76 Pomes, A. F., 311 (ref. 116), 315 (ref. 1161,549 Pommerenke, W.T., 183, 199 Poo, L. J., 182, 187, 196 Porter, C. W., 393,419 Porter, K. R., 432 (ref. 20, 44), 433 (ref. 441,439,440 Porter, R. R., 98,99, 113, 129, 134, 135, 136, 138, 139, 141, 147, 148,162 Posternak, S.,208,267 Potter, J. S., 426,4.99 Potter, R. L., 102 Tsee Hansen), 103 (see Hanaen), 106 (see Hansen), 108 (see Hansen), 110 (see Hansen), 111 (see Hansen), 112 (see Hansen), 113 (see Hansen), 117 (see Hansen), 118 (see Hansen), 119 (see Hansen), 120 (see Hansen), 160, 213 (ref. 48), 218 (ref. 48), 223 (ref. 48), 226' Powell, R. E., 332 (ref. 161), 335 (ref. 161), 560 Prantl, 231 Prelog, V., 59,62 (ref. 255a),81 Preston, C.,287, 292,302,303 Prianishnikov, D., 278, 279, 280, 289, 301

Prianishnikov, D. N., 281,301 Price, V. E., 19, 38 (ref. 255b), 51 (ref. 198a),79, 81 Price, W. C., 255,300, 301 Pringsheim, E. G.,268,301 F'rocter, H. R.,366,420 Pryor, M.G. M., 412, 0 0 Pucher, G.W., 41,82,273,279,281,282, 284,288,303 Pund, E. R., 182 (see Berg), 197 Putnam, F. W., 97 (see Neurath), 161, 309 (ref. 130), 311 (ref. la), 315 (ref. 130), 316 (ref. 130), 317, 349

458

AUTHOR INDEK

Rieaen, W. H., 103, 166, 194, ls’g Riess, c., 381 (ref. 175), 386 (ref. la), 390 (ref. 130), 393 (ref. 131), 418, Quackenbush, F. W., 96, 103 (see Kuhn), 105 (see Kuhn), 139 (see 40 Rietz, E., 342 (ref. 78),348 Kuhn), 161 Quisenberry, J. H., 176 (see Beadles), 197 Riley, D., 128,149 Rimington, C., 208, 227 R Rinke, H., 26 (ref. 85),36 (ref. 258), 47 (ref. 85),49 (ref. 85),59 (ref. a), 61 (ref. 83, 84), 62 (ref. 83, 84, 258), Raistrick, H., 202, 210, 212, 223 (ref. 63 (ref. 83), 74 (ref. 85), 78, 77, 81 25), 926 Ris, H.,426 (ref. 39), 440 Ramachandran, B. V., 208, 226 Rischkov, V.L., 294,301 Ramamurti, T.K., 287,SO2 Riaser, W.C.,18 (ref. 97),77, 159, 199 Ramaawamy, R., 280, 297 Rittenberg, D.,90, 101, 162, 156, 199, Ramdas, K., 280,297 282,239,301,303 Ramsay, H., 64 (ref. 25ci),81 Ramaden, W.,311 (ref. 141), 341 (ref. Ritter, A., 416 Rivers, T. M., 255 (see Stanley, W. M.), 141),360 256 (see Stanley, W.M.), 302 Ranefeld, A. N., 91,162 Roberta, E. J., 311 (ref. 26), 315 (ref. 26), Raske, K., 38 (ref. 161),78 Ratner, B., 211, 226 347 Robinow, C. F., 268, SO1 Ratner, S.,75 (ref. 257),81 Robinson, R., 18 (ref. lOOa), 75 (ref. Ratti, R., 36 (ref. 260), 81 100a), 77 Rautanen, N., 273,275,301,SO4 Robscheit-Robbins, F. S., 156 (see Ravdin, I. S., 189, 198 Whipple, G.H.), 183, 184, 185, 189, Rees, A. L. G.,330,349 194, 198,199,800 Reea, M. W.,87 (see Chibnall), 88, 89, 95,96,97, 100, 104, 106 (see Bailey; Roche, J., 264,SO1 Chibnall), 108 (see Bailey; Chib- Rockland, L. B., 15 (ref. 267), 39 (ref. 267), 81, 85, 91, 92, 100, 101, 103, nall), 109, 110, 114 (see Chibnall), 108 (see Dunn), 111 (see Dunn) , 115, 118 (see Chibnall), 119 (see 119, 120 (see Dunn), f@, 160 Chibnall), 120 (see Chibnall), 121, 122, 130, 132, 136, 138 (see Chib- Roddy, W. T., 400 (ref. 176),&!O nall), 139, 140, 148, l@, 168, 218 Rodkey, F.L., 221 (ref. 108), 927 Rodney, G.,194, 198 (ref. 3, 104),,826,227,253, 897 Roepke, R. R., 271,273, 899 Reeves, E. B., 186 (see Weech), 900 Rehner, J., Jr., 328 (ref. 461, 338 (ref. Roesner, H.,55, 57 (ref. 163),78 Ronzoni, E.,101, 102, 103, 105,106, 108, 461,347 109, 110, 111, 112, 113, 116, 117, Reid, K., 222 (ref. 105),287 118, 119, 120, 130, 140, 147, 163 Reid, T.S., 311 (ref. 1101,349 Reif, G.,14 (ref. l62), 67 (ref. lSZ), 68 Rose, W. C., 3 (ref. 299), 88, 85, 156, 158,185,194,197,199,$00 (ref. 162),69 (ref. 162), 78 Ross, A. F.,102, 109, 110, 111, 116, 117, Retzch, C. E., 406 (ref. 102),418 118, 169,256, 301 Rhoades, M.M., 433 (ref. 45), 440 Ross, W. F., 31 (ref. 41,86), 47, 50 (ref. Rice, E.E., 158, 194,199 41), 59 (ref. 86), 60 (ref. 63,86), 61 Rice, F. E., 221, 927 (ref. 86), 62 (ref. 86), 76, 77 Rich, C. E., 263,301 Rowenbeck, H., 265,998 Richards, F.J., 286,287, SO1 Rossner, E.,40 (ref. 26), 76 Rideal, E. K., 311 (ref. 77), 348 RothBs, F.,263,301 Riederle, K.,360,417

Q

AUTHOR INDm

Rothemund, K. W.,25, 81 Rothen, A., 277, 290, 301, 435 (ref. 22), 439

Rouelle, M., 233,234,301 Rowland, S.J., 202 (ref. log), 227 Rudall, K. M., 376, 377, 412, 413 (ref. 177), 415, 416, 420 Ruggli, P., 36 (ref. 260), 81 Ruhland, W.,279,901 Russell, W. C., 193,199 Ryan, F. J., 100 (see Brand), 102, 110, 112, 116 (see Brand), 117, 120 (see Brand), 149, 162, 216 (ref. 13), 218 (ref. 13),226 Rydon, H. N., 45 (ref. 201),79, 265, 698

S Sahyun, M., 159 (see Kade), 198 Saidel, L. J., 101, 102, 109, 112, 116 (see 216 Brand), 117, 119, 120, f@, (ref. 13),218 (ref. 13),226 St. John, J. L., 193,198 Saito, M.,40 (ref. 27), 76 Salcedo, I. S., 407, 411 (ref. 178), 418, @O Salle, A. J., 250,251, 252,253,254, 297 Salsmann, L., 42 (ref. 87, 88), 43 (ref. 87,88),44 (ref. 88), 45 (ref. 87,88), 47 (ref. 88),50 (ref. 88), 51 (ref. 88), 77

San Clemente, C. L., 223 (ref. 110),6.97 Sandstedt, R. M., 262, 296, 901 Sanger, F., 98, 99, 101, 113, 129, 133, 134, 135, 136, 139, 141, 146, 147, 148,16.2 Sanni6, C., 5 (ref. 261), 36 (ref. 261), 81 Sarich, P.,176, 198 Scarth, G. W., 306 (ref. 142),960 Scatchard, G.,360 Schaal, E., 2, 81 Schachowskoy, T.,387 (ref. 32),416 Schanderl, H.,274, 275, 302 Schardinger, F.,220, 927 Schauts, E. J., 124 (see Buehler), 125 (see Buehler), 143 (see Buehler),

149 Scheele, W., 381 (ref. lll),418 Scheibler, H.,7 (ref. 164),37 (ref. 164),38 (ref. 164),39 (ref. 164), 79 Sohein, A. H., 264, 306

469

Schels, H., 398 (ref. 51), 417 Schenck, J. R., 199 Schenk, R., 219 (ref. 112),228 Schiff, H.,2, 81 Schlag, H.,211 (ref. 32), 626 Schleich, H.,17 (ref. 64), 19 (ref. 64),26 (ref. 80, 85), 36 (ref. 80, 89), 37 (ref. SO), 38 (ref. SO), 39 (ref. 80), 40 (ref. SO), 42 (ref. 88), 43 (ref. 88). 44 (ref. 88), 45 (ref. 88), 47 (ref. 85, 88),49 (ref. 85), 50 (ref. a),51 (ref. 88),59 (ref. 84),61 (ref. 84),62 (ref. 84), 68 (ref. 89), 69 (ref. 89), 70 (ref. 89), 74 (ref. 85), 76, 77 Schmid, 121 Schmidt, C. L. A., 33 (ref. 92), 77, 133, 162, 263,902,311 (ref. 36),316 (ref. 36), 347, 358 (ref. 183), 389 (ref. la),393,@O Schmidt, H. L., Jr., 182 (see Sydenstricker), 199 Schmitt, F. O., 266, 302, 306 (ref. 144, 145), 325, 360, 359 (ref. 98, 179, 180),372 (ref. 179),418, 4.90 Schmitt, V., 17 (ref. 65), 51 (ref. 65),57 (ref. 65), 76 Schneider, F.,26 (ref. 80), 36 (ref. SO), 37 (ref. SO), 38 (ref. 80, 264), 39 (ref. SO), 40 (ref. SO),41 (ref. 193), 42 (ref. 193), 43 (ref. 193), 44 (ref. 193,264), 45,62,76. 79, 81 Schneider, M. L., 205,226 Schneider, W. C., 425 (ref. 29, 47), 427 (ref. 46, 47), 431 (ref. 47, 49), 432 (ref. 29, 47, 48), 436 (ref. 49), 438 (ref. 47), 439, 4.60 Schoenheimer, R., 23, 36 (ref. 266), 37 (ref. 266),39 (ref. 266), 81, 156,199, 282,303 Schoeller, W., 49 (ref. 165),511 (ref. 165), 79

Schoenebeck, O., 67 (ref. 194), 68 (ref. 194),79 Schofield, R. K., 306 (ref. 146), 960 Schott, H.F., 15,39 (ref. 267),81 Schramm, C. H., 21,22,37(ref. 301),82, 307 (ref. 174),961 Schrauth, W., 5 (ref. 166), 6 (ref. 166), 50 (ref. 166), 79 Schroeder, H., 387 (ref. 151), 419

460

AUTHOR INDEX

Schroehr, G., 57, 81 Schryver, 9. B., 233, 297 Rchutte, E., 64 (ref. 269), 81 Schuteenberger, P., 2 Schuler, J., 37 (ref. l6), 38 (ref. 16, 28), 39 (ref. 16, 28), 76 Schultr, J., 285, 697, 429 (ref. 8, 50),435 (ref. 8), 437 (ref. 50), 438 (ref. 8,

W , 439, 440 Schulee, A., 36 (ref. 167), 38 (ref. 167), 79 Schulze, E., 278, 279, 283, 308 Schwab, G., 272, 279, 902 Schwank, M., 403, 419 Schweigert, B. S., 92, 103 (see Riesen), 166

Schwimmer, D., 171, 199 Scott, D. A., 132, 166 Bcott, V. C., 183, 198 Scott Blair, G. W., 306 (ref. 147), 960 Scotti, H. C., 365, 480 Sealock, R. R., 436 (ref. 56), 440 Sebelien, J., 210, 268 Seeler, A. O., 166, 199 Seeley, R. D., 160, 161, 162, 164, 166, 167, 168, 170 (see Allison), 171, 181, 182, l 8 5 , i 8 6 , i 8 7 , i 9 r l 199 Seibert, F. B., 265, 902 Seifrie, W., 306 (ref. 148, 1491, 960,425 (ref. 51), 433 (ref. 51), 440 Sekora, A., 306 (ref. 841, 948 Seligsberger,Id., 405, 406 (ref. 181), 420 Semmett, W. F., 223 (ref. 147), 226, 228 Sen, P. K., 283, 287, 289, 298 Senti, F. R., 228, 311 (ref. 131, 132, 152), 313 (ref. 151), 325,327,328, 3@,360 Serenyi, V., 205 (ref. 421, 226 Seycwetz, A., 411, 412 (ref. 152), 419 Seymour-Jones, F. L., 392, 421 Shankman, S., 91, 93, 101, 103 (see Dunn), 108 (see Dunn), 110 (see Dunn), 111 (see Dunn), 117 (see Dunn), 1.69, 160, 166, 218 (ref. 115), 228

Shaw, G., 18 (ref. 103a), 77 Shedlovsky, T., 126, 162 Sheehan, J. C., 32, 54 (ref. 270a), 81 Sheffield, F. M. L., 255, 697 Shemin, D., 20, 80, 82, 85, 90, 93, 94, 101, 106, 108, 114, 116, 120, 122, 162, 276, 289, 298, 301

Sherman, J. V., 311 (ref. 163), 314 (ref. 153), 360 Sherman, M. S., 274, 698 Sherman, 9. L., 311 (ref. 153), 314 (ref. 153), 360 Sherman, W. C., 245, 306 Shinn, L. A., 88, 109, 110, 115, 161, 162, 208, $27 Shore, A,, 88, 100, 162 Shuttleworth, 5. G., 389 (ref. 182), 420 Sickel, H., 38 (ref. 30), 39 (ref. 30), 58, 72 (ref. 29), '76 Sideris, C. P., 271, 90% Sieber, W., 21 (ref. 112), 77 Siegmund, W., 367 (ref. 33), 416 Sifferd, R. H., 3 (ref. 2721, 31, 36 (ref. 272), 52, 61 (ref. 272), 62 (ref. 272), 65 (ref. 272), 81 Sigmund, F., 22, 36 (ref. 273), 49 (ref. 273), 81, 82 Sigurgiersson, T., 255, 902 Silber, R. H., 166, 199 Simmonds, S., 34, 44 (ref. 274), 51 (ref. 274), 68 (ref. 273a), 69 (ref. 273a), 70 (ref. 273a), 81 Simms, H. S., 36 (ref. 235), 80 Simpson, F., 199 Sinclair, W. B., 241, 262, 298, 902 Singer, W., 38 (ref. 32), 76 Sjogren, B., 214, 228 Skoog, F., 284, 502 Slack, E. B., 179, 197 Slade, R. E., 235, 302 Slavin, H. B., 183, 199 Slein, M. W., 264, 696 Slonim, N. B., 37 (ref. 276a), 43 (ref. 276a), 44 (ref. 276a), 88 Smith, A. H., 241, 302 Smith, A. K., 262, 302,311 (ref. 35), 314 (ref. 35), 347 Smith, A. M., 238, 302 Smith, C. S., 38 (ref. 275), 51 (ref. 275) 81

Smith, E. L., 31 (ref. 2761, 36 (ref. 201a, 276), 37 (ref. 276a), 38 (ref. 275b), 39 (ref. 201a), 40 (ref. 276), 43 (ref. 276a), 44 (ref. 276a), 68, 69, 70 (ref. 201a, 276), 71 (ref. 275a, 276), 72, 80, 81, 82, 92, 95, 99, 102, 103, 105, 110, 111, 112, 113, 117, 118,

AUTHOR INDEX

119, 120, 138, 162, 202, 210, 212, 213, 214, 218 (ref. 117, 118, 121123), 223, 228, 263. 264, 267, 308 Smith, E. P., 121 (see Graham), 160 Smith, H. Dew., 331 (ref. 154), 360 Smith, H. P., 182, 199 Smith, J., 314 (ref. 168), 361 Smith, J. D., 275, 899 Smith, T., 211, 828 Smith, V. A., 36 (ref. 19Oa), 38 (ref. 190a), 79 Smith, W. H., 286, 299 Smuts, D. B., 236, 300 Smythe, C. V., 358 (ref. 183), 389 (ref. 183),393, 420 Snell, E. E., 85, 90, 91, 92, 101, 102, 103, 104, 106, 108, 110, 111, 112, 113, 116, 117, 118, 119, 120, 138, 160, 161, 168, 218 (ref. 45, 51), 226, 264, 276, 298, 302 Sgirensen, M., 135, 162, 214, 215, 223 (ref. 127), 228 S@rensen, S. P. L., 214, 215, 223 (ref. 127), 228 Sokolov, S. J., 4-20 Somers, G. F., 263, SO3 Sookne, A. M., 312 (ref. 155), 360, 363 (ref. 9), 367, 416 Soule, M. H., 250, 297 Sourlangas, S. D., 395 (ref. 185), 4.80 Soutoulov, A. N., 280, 302 Spath, H., 61 (ref. 114), 64 (ref. 114), 77 Speakman, J. B., 308 (ref. 156), 331, 360, 376 (ref. 186), 420 Spiegelman, S., 277, 302, 427 (ref. 52, 53), 440

Spielman, M., 75 (ref. 251), 81 Srb, A. M., 273, 502 Stahlschmidt, A,, 43 (ref. 157), 44 (ref. 157), 78 Stahmann, M. A., 37 (ref. 277), 40 (ref. 277), 82 Stamberg, 0. E., 244, 301 Stamm, G., 57 (ref. 279), 82 Stanley, P., 238, 300 Stanley, W. M., 162, 254, 255, 256, 277, 292, 298, 299, 302 Stare, F. J., 246, 302 Staudinger, H., 406 (ref. 187), 420 Staudt, W., 63 (ref. 226), 80

46 1

Stearn, A. E., 308 (ref. 38), 347 Stecker, H. C., 367 (ref. 104), 368, 377 (ref. 104), 411, 412 (ref. 188), 418, 420

Stedman, E., 266, 302, 427, 440 Stedman, E., 266, 302, 427, 4.40 Steenbock, H., 245, 897 Steffee, C. H., Jr., 158 (see Frszier), 189, 190, 191, 194 (see Block), 197, 198, 8rn Steiger, R. E., 5 (ref. 237), 18 (ref. 236), 38 (ref. 231, 237), 39 (ref. 237), 80 Stein, A. M., 311 (ref. loo), 316 (ref. loo), 336 (ref. loo), 345 (ref. loo), $49 Stein, R., 333 (ref. 157), 360 Stein, W. H., 3, 36 (ref. 278), 38 (ref. 278), 57 (ref. 279), 79, 81, 82, 85, 95, 101, 102, 103, 104, 111, 112, 113, 116, 122, 161, 162 Steingroever, J., 37 (ref. 168), 38 (ref. 168), 39 (ref. 168), 79 Steinhardt, J., 363, 364, 420 Stekol, J. A., 73 (ref. 279a), 82 Stepka, W., 297 Stepto, R. L., 197 Stern, F., 16 (ref. 67), 18 (ref. 66), 19 (ref. 66), 23 (ref. 67), 41 (ref. 67), 43 (ref. 67), 46 (ref. 67), 49 (ref. 66, 67), 50 (ref. 66), 51 (ref. 67), 76 Stern, K. G., 264, $02 Stevens, C. M., 18 (ref. 98), 25, 77 Steward, F. C., 270, 271, 273, 276, 287, 288, 289, 292, 297, 302, 303 Stewart, A. M., 244, 303 Stewart, C P., 220, 667 Stewart, G. F., 171 (see Swanson, P.), 199, 306 (ref. go), 348 Stiasny, E., 365, 380 (ref. 193), 381 (ref. 193, 194), 383, 393 (ref. 193), 397, 406 (ref. 192), 418, 420 Stockelback, L. S., 262, 303 Stockinger, H. E., 268, 303 Stokes, J. L., 91, 103, 106, 109, 110, 111, 112, 113, 116 (see Gunness), 117, 118, 119, 120, 122, 160, 166, 163, 248,249, 250,251,252, 253, 254, 256, 258, 259, SO3 Stoklasa, J., 220, 888 Stout, P. R., 287, 303 Straitiff, W. G., 241, 299

462

AUTHOR INDEX

Strasburger, 428 Straube, R. L., 190, 194 (see Block), 197 Street, H. E., 270, 273, 276, 287, 289, 303 Strohschein, F., 15, 18, 33 (ref. 249), 81 Strong, F. M., 162 Stuart, L. S., 244, 301 Stuart, N., 293, 296 Stueck, G. J., 88 (see Shore), 100 (see Shore), 162 Sturtevant, A. H., 424 (ref. 55), 440 SubbaRow, Y., 45 (ref. 280), 82 Sukhov, K. S., 255,303 Sullivan, J. T., 235, 303 Sullivan, M. X., 72 (ref. 213), 73, 80, 96, 103, 105, 111, 117, 160, 163, 218 (ref. 129), 828,256, 262, 298,300 Sumner, J. B., 263, 303 Sutherland, G. B. B. M., 112 (see Darmon), 117, 149 Suzuki, U., 7 (ref. 172), 10 (ref. 170), 12 (ref. 169), 27 (ref. 172), 50 (ref. 171), 53 (ref. 171), 54 (ref. 171), 58, 61 (ref. 172), 63, 66 (ref. 172), 67 (ref. 169), 79 Svanberg, O., 220, 228 Svedberg, T., 134, 163, 207, 214, 228, 263, 303 Swallen, L. C., 311 (ref. 168), 360 Swaneon, P. P., 171, 172, 197,199,200 Sydenstricker, V. P., 182, 197,198,199 Synge, R. L. M., 3 (ref. 242, 281, 282), 37 (ref. ZSZa), 40, 51 (ref. 282a), 60, 61 (ref. 282a), 62 (ref. 282a), 68 (ref. 282a), 69 (ref. 282a), 70 (ref. 282a), 81,82,84, 88,94, 95, 110, 123, 160, 161,237, 259, 300, 303,360, 419 Szent-Gyorgyi, A., 306 (ref. 159), 311 (ref. 159), 360 Seymamki, T. A., 199

T Takahashi, W. N., 235,260,293,294,298 Tamura, S., 250, 303 Tanner, F. 249, 303 Tatum, E. L., 44 (ref. 274), 51 (ref. 274), 81,273, 303 TaubBck, K., 280, 299 Taylor, E. S., 276, 303 Taylor, G. L., 128, 163

w.,

Taylor, M. W., 193 (see Rumell), 199 Taylor, 8. P., 48 (ref. 112b), 71 (ref. 112b), 72 (ref. 112b), 73 (ref. 112b), 77 Teague, D. M., 202 (ref. a), 203 (ref. 8), 218 (ref. 8), 226 Templeman, W. G., 287, 301 Thatcher, R. W., 222, 228 Theis, E. R., 372 (ref. 148), 391, 392 (ref. 148), 399 (ref. 197), 409 (ref. 197), 412 (ref. 198), 419,420 Theorell, H., 89, 163,219 (ref. 135), 222, 228 Thiele, H., 400 (ref. 12), 416 Thierfelder, €I., 43 (ref. 283), 44 (ref. 283), 82 Thomas, A. W., 373, 384, 385 (ref. 201), 386 (ref. 201), 391 (ref. 206), 392, 395 (ref. 202), 396, 397, 411, 420,4x1 Thomas, K., 156, 164, 169, 200 Thomas, L. E., 116, 163 Thomson, R. H. K., 311 (ref. 160), 315 (ref. 160), 360 Thurlow, S., 220, 826 Tiedjens, V. A., 271, 303 Tieteman, J. E., 6, 16 (ref. 116), 18 (ref. 116), 37 (ref. 115), 49 (ref. 115), 69, 70 (ref. 68), 71, 76,77 Timm, E., 239, 300,303 Timmerman, W. A., 211, 228 Tincker, M. A. H., 284,303 Tiselius, A., 94, 163 Toboleky, A., 332 (ref. 161), 335 (ref. lei), 360 Tomarelli, R. M., 191, 800 Tomlineon, J., 403 (ref. 96), 418 Tracy, P. H., 202 (ref. 89), 203 (ref. 89), 227 Traill, D., 311 (ref. 162-164), 315 (ref. 162-164), 360 Trasciatti, 2 Travers, J. J., 176, 192, 198 Trikojus, V. M., 295 Tristram, G. R., 85, 94, 95, 101, 102, 104, 108, 109, 110, 111, 112, 113, 114, 116, 117, 118, 122, 124, 129, 130, 143, 149, 163, 218 (ref. 138), 228,234, 235, 237, 241, 247, 257,303 Turley, H. G., 375, 421 Turner, J. S., 295

AUTHOR INDEX

U Udenfriend, S., 93 (see Keston), 94, 101 (see Keston), 114 (see Keston), 161, 218 (ref. 66), 886 Umlauft, W., 278, 308 Underhill, 184 Underwood, E. J,, 252, 299

V

463

Waisbrot, S. W., 342 (ref. 78), 348 Wakeman, A. J., 205, 206 (ref. 97), 227, 233,235,273,281,284,288,301, 303 Walden, P., 13,82 Waldschmidt-Leitz, E.,17 (ref. 298), 29 (ref. 298),82 Walker, J. F., 411 (ref. 210),421 Walkley, J., 284, 304 Wall, F.T., 335,360 Wallerstein, J. S., 264, 302 Wang, T.,238, 306 Warburg, O.,12,264,271,304, 438 Ward, W. H.,311 (ref. 166), 317 (ref. 166),560 Warner, R. C., 88, 100, 163, 204, 205, 206, 208, 209, 215, 222, 887, 928, 262, 298, 999, 311 (ref. llO), 325 (ref. lll),349 Washburn, M. R., 182 (see Niven), 199 Watson, G. M., 270,303 Waugh, D.F., 314,361 Weber, H. H., 311 (ref. 169), 314 (ref. 169),361 Weber, L. E., 38 (ref. 34),39 (ref. 34), 76 Weech, A. A., 186,2W Weidinger, A., 361 (ref. 116),418 Weidle, H.,46 (ref. 2), 50 (ref. 2), 51 (ref. 2), 76 von Weimarn, P. P., 309 (ref. 170),311 (ref. 170), 316 (ref. 170), 361 Weir, C. E., 414,415, 481 Weiss, S., 193, 198 Weizmann, C.,22 (ref. 176a),79 Weller, R. A., 234, 236, 237, 238, 239, 243, 245, 279,300 Wells, H.G., 202, 211, 223, 226' Werkman, C.H., 285,304 Werner, L.H., 75 (ref. 251, 294),81, 88 Wessely, F., 8, 21, 22, 36 (ref. 273), 48 (ref. 296),49 (ref. 273), 81, 8.2 Westall, R. G., 233, 697 Wettstein, A., 75 (ref. 251, 252, 294), 81,

Valk6, E., 335 (ref. 119),349 Vandendriessche, L., 221, 227 Vandevelde, A. J. J., 220,228 Van Etten, C., 311 (ref. 37),314 (ref. 37), 347, 436 (ref. 561,440 Van Lanen, J. M., 249, 303 Vam, H.M., 189, 198, 200 Vaseel, B., 105, 163 Velick, 5.F., 101,102,103, 105, 106,108, 109, 110, 111, 112, 113, 116, 117, 118, 119, 120, 130, 140, 147, 163 Vendrely, R., 268,296, 303 Venkatesan, T. R., 280, 297 Vickery, H.B., 4 (ref. 284), 41, 82, 84, 105, 118, 119, 141, 163, 233, 234, 242, 262, 267, 271, 273, 279, 281, 282, 284, 288, 303 Viets, F. G., 272,303 du Vigneaud, V., 3 (ref. 272), 11, 16 (ref. QO), 22 (ref. no), 23, 25 (ref. 290), 31, 36 (ref. 220, 272), 40 (ref. 220), 42 (ref. 288), 43 (ref. 248), 44 (ref. 248, 288, 289, 291), 46 (ref. go), 51 (ref. QO), 52,53, 54, 55, 61 (ref. 220, 221, 272), 62 (ref. 45, 220, 221, 240, 272),63 (ref. go),65,66,71 (ref. 69), 76, 77, 80, 81, 88, 105, 132, 161, 163, 436,G O Virtanen, A. I., 259, 271, 273, 274, 275, 303,so4 Vischer, 266 Vivino, A. E., 241, 304 Vlassopoulos, V.,39 (ref. 33),76 88 Vogl, L.,400,401, 421 Wetzel, K., 279, 301 Von Susich, G., 335 (ref. 119),349 Whewell, C.S., 376 (ref. 186),420 Vorob'eva, M. N., 292,SO4 Whipple, G. H., 156, 180, 182, 183, 184, Vovk, A. M., 255, 303 185, 187, 189, 194, 198, 199, 9LW SV White, A., 105,163 Wahling, H. B., 274, 696 White, H.J., Jr., 333 (ref. 68),348 Wainio, W. W., 436 (ref. 57), 4 0 White, J., 55, 89

464

AUTHOR INDEX

White, J. I., 188, 196 Wintersteiner, O., 105 (see du Vigneaud), Whitehead, E. I., 272, 303 163 Whittier, E. O., 314 (ref. 171), 361 Wissler, R. W., 158 (see Frazier), 189, Wichmann, A., 214, 228 190, 191, 194 (see Block), 19Y, 198, Wicklund, E., 428 (ref. 58), 440 do0 Widen, P. J., 381 (ref. 97), 383 (ref. 97). Witte, C., 16 (ref. 67), 23 (ref. 67), 41 (ref. 67), 43 (ref. 67), 46 (ref. 67), 49 418 Widmark, O., 5, 76 (ref. 67), 51 (ref. 67), 76 Wielend, P., 59, 62 (ref. 255a), 81 WBhlisch, E., 335 (ref. 172), 361, 369, Wildman, S. G., 260, 207, SO4 370, 421 Wilkins, €1. L., 234, 304 Wolesensky, E., 403, 421 Williams, E. F., 87 (see Chibnall),' 97 Wolf, F. T., 249, 304 (see Bailey), 106 (see Bailey; Chib- Womack, M., 3 (ref. 299), 82, 158 (see nall), 108 (see Bailey; Chibnall), 114 Borman), 185, 194, 197, 200 (see Chibnall), 115 (see Bailey), 118 Wood, H. G., 285, 304 (see Chibnall), 119 (see Chibnall), Wood, J. G., 270, 271, 275, 276, 281, 282, 120 (see Chibnall), 136 (see Bailey), 283, 284, 285, 286, 287, 289, 301, 138 (see Chibnall), 148, 149, 218 304 (ref. 3), 826,253, 697 Wood, J. L., 52 (ref. 300), 53 (ref. 300), 82 Williams, G., 240, 304 Wood, S., 103 (see Lyman), 161 Williams, H. H., 105 (see Beach), 148 Wood, T. R., 158 (see Borman), 197 Williams, J. W., 316 (ref. 92), 349 Woodman, H. E., 212, 228 Williams, R. C., 255, 301 Woods, E., 244, SO4 Williams, R. F., 286, 287, 301, 30.4 Woods, F. M., 183, 198 Williams, R. J., 108 (see Hac), 113 (see Woods, H. J., 329 (ref. 6), 335 (ref. 173), 346, 361 Guirard), 118 (see Guirard), 160, 264, S98 Woods, M. W., 294, 304 Williamson, M. B., 218 (ref. 144), 219 Woodward, R. B., 21, 22, 37 (ref. 301), 82, 307 (ref. 154), 361 (ref. 144), 838 Willman, W., 171 (see Brush), 172, 197, Woolley, D. W., 3 (ref. 302), 24, 57, 82 194, 200,424 (rcf. 62), 440 $00 Willstlitter, R., 17 (ref. 298), 29 (ref. Woolridge, R. L., 158 (see Frazier), l%J, 190, 191, 194 (see Block), 197, 198, 298), 8,9 200 Wilson, E. B., 428 (ref. 60), 430 (ref. 59, Worcester, J., 179, 198 6l), 440 Worden, A. N., 240, 299 Wilson, E. J., 7, 26 (ref. 254), 81 Work, T. S., 25, 49 (rcf. 204a), 80, 290 Wilson, H., 88 (see Shore), 100 (see Wormell, R. L., 242, Y04, 409 (ref. 217), Shore), 168 431 Wilson, J. A., 354 (ref. 211), 366, 384, Wright, S., 427 (ref. 63, 64), 440 385 (ref. 212), 391 (ref. 212), 396 Wrinch, 132 (ref. 212), 398 (ref. 212), 400 (ref. Wulff, H. J., 264 211), 406 (ref. 212), 408 (ref. 212), Wybert, E., 54 (ref. 35), Y6 411 (ref. 212), 420, 421 Wyckoff, R. W. G., 255, SO1 Wilson, P. W., 274, 275, 296,297, 304 Wyrnan, J., 141, 163 Wiltshire, G., 97, 106, 108, 115, 122, 132, Wynd, F. L., 292, 304 130, 138 Y Windus, W., 375, 421 Winnick, T., 110,163 Ydse, L. C., 180 (see Bosshirrdt), 197 Winternitz, O., 119, 163 Yemm, E. W., 281,304

AUTHOR INDEX

Young, G. T., 43 (ref. 227a), 44 (ref. 227a), 45 (ref. 227a), 53 (ref. 35), 80 Young, H. Y., 271, 302 Young, M., 175 (see Zucker, L.), 179 (see Zucker, L.), 180 (see Zucker, L.), 800

Yudkin, W. H., 17 (ref. 304), 82 Z

Zaitschek, A., 219 (ref. 146), %28 Zamenhof, S., 268, 297 Zeisset, W., 38 (ref. 36), 39 (ref. 37), 40 (ref. 36), 76, 76 Zeldis, L. J., 181, 182, 200 Zervas, L., 5 (ref. 82), 7 (ref. 78), 16 (ref. go), 21 (ref. 74), 25, 26 (ref. 74, 77, 80, 85), 31 (ref. 86), 36 (ref. 74,

465

77, 80, 89), 37, 38 (ref. 78, 80), 39 (ref. 78, 80), 40 (ref. 80), 41 (ref. 74), 42 (ref. 74, 87, 88), 43 (ref. 74, 76, 78, 79, 87, 88), 44 (ref. 74, 77, 78, 88),45 (ref. 74, 87, 88), 46 (ref. go), 47 (ref. 85, 88), 49 (ref. 74, 85), 50 (ref. 74, 88), 51 (ref. 88, go), 53 (ref. 74), 57 (ref. 74), 59 (ref. 81, 84, 86), 60 (ref. 86), 61 (ref. 74, 81, 83, 84, 86), 62 (ref. 81, 83, 84, 86), 63, 66, 68 (ref. 89), 69 (ref. 89), 70 (ref. 89), 71 (ref. 69), 74, 76, 77, 8% Zetzsche, F., 25, 81 Zittle, C. A., 120, 163 Zucker, L., 175, 179, 180, 200 Zucker, T. F., 175 (see Zucker, L.), 179 (see Zucker, L.), 180, 800

Subject Index A

Aldehydes (see also Formaldehyde), as tanning agents, 405-41 1 Aldehydrase, see Xanthine oxidase Aldolase, see Myogen A

Acetyldehydroalanine, synthesis of, 19 Acetyldehydrophenylalanine, synthesis of, 18 AhW, Acetyldehydrotyrosine, synthesis of, 18 amino acid composition of proteins of, N-Acetyldiketopiperazine, 71 247-249, 258 Actinom ycetes, Alkalis, amino acid composition of, 252, 258 effect on collagen, 368,377 Acylamino acids, synthesis of peptide Amandin, derivatives from chlorides of, 9 molecular weight of, 263 Amino acids, see also under names of Acyldehydroamino acids, preparation of, 19 individual members, analysis in protein hydrolyzates, 83-124 synthesis of peptides from azlactones of, 18 accuracy of methods, 99-106 Acyl halides, a-halogeno, by colorimetric methods, 98 preparation of optically active, 12, 13 by electrodialysis, 89 synthesis of peptides from, 11-15 by enzymatic methods, 90 Aerobacter aerogenes, by isotope dilution, 93-94 amino acid composition of protein by microbiological assay, 90-93 from, 252 by partition chromatography, 94ff. Alanine, 9 availability of, 193 derivatives of, 36 chlorides of, estimation in protein hydrolyeates, 115 preparation of, 11 comparison of methods for, 96, 114 synthesis of peptides from, 1 1 peptides of, 35-40 in collagen, 357ff. optical rotation of, 38, 39 destruction during protein hydrolysis, synthesis of, 3, 5, 13, 19,24,35ff. 123ff. distribution in muscle proteins, 140 sources of,114,129,130, 131,137, 140, in plant proteins, 235-241, 243-246, 142, 143, 145 DxAlanine anhydride, 5 247,252 stability of, 6 in purified proteins, 83-148 8-Alanyl-chistidine, see carnosine effect on body weight, 185 8-Alanyl-l-methyl-bhistidine, see Anessential, determination of, 158 serine diet and, 158, 195, 196 Albumin, 181 egg, Bee Ovalbumin effect on appetite, 190 on body weight, 190 plasma, amino acid composition of, 142 on nitrogen equilibrium, 158, 159 erythrocytes and, 190 serum, amino acid composition of, 96, 101, nitrogen-sparing action of, 172 102, 103, 105, 106, 109, 110, 111, serum proteins and, 190 112, 113, 116, 117, 118 utilization of, 192 466

SUBJECT INDEX

estimation of dicarboxylic, 97-98, 114 of sulfur-containing, 95-97 fatigue and, 158 nervousnem and, 158 nitrogen balance and, 157-159 non-essential, 171 from proteins, 180 regeneration of plasma proteins and, 189 relation between protein levels and, in plants, 284 requirements, 176 resolution of optically inactive, by proteolytic enzymes, 34 specificity of methods for determination of, in protein hydrolyzates, 99-106 stores in animals, 180 synthesis in liver, 180 of peptides from N-carbonic acid anhydrides of, 21-23 in plant t k u e s , 271, 274 unnatural isomers of, 170 pAminobenzoic acid, as component of folic acid, 73 peptidee of, 73 Aminobutyric acid, 9 4-Amino-n-butyryl peptide, synthesis of, 40 a-Aminobbutyryl peptide, synthesis of, 40 Ammonia, protein synthesis from, in plants, 271274 role in nitrogen metabolism. 281 Amylase, in milk, 219 activity of, as criterion for quality of, 220 Angiosperms, isolation of proteins from leaves of, 233240 Anorexia, amino acids and, 158 Anserine, structure of, 65 Antibodies, transfer from mother to infant, 212 Arachm, 262

467

Arginine, derivatives of, 61 effect on body weight, 190 estimation in protein hydrolyzates, 90 comparison of methods for, 96, 118 hemoglobin output and, 185 isolation from soybean oil meal, 194 nitrogen metabolism and, 169, 185 peptides of, 46, 62-64 optical rotation of, 62 synthesis of, 63, 64 sources of, 118, 129, 130, 131, 132, 136, 140, 142, 143, 145 Ascomycetes, proteins of, 250-254 Asparagine, role in protein metabolism of plants, 278 synthesis of L-, 42 Aspartic acid, 9 derivatives of, 43 estimation in protein hydrolyzates, 114 comparison of methods for, 96, 106 peptides of, 41ff. optical rotation of, 44 synthesis of, 41, 42 sourcesof, 106, 124, 131, 132, 136, 137, 138, 140, 142, 143, 145 @-LAspartyl-Lcysteinylglycine (Asparthione), synthesis of, 53 Aspergillus niger, amino acid composition of protein from, 251 Aucuba virus, amino acid composition of green, 256 of yellow, 256 effect on protein metabolism of host, 292 Auxin, in chloroplasts, 267 Azides, synthesis of peptide derivatives from, 9 a-Azidoacyl halides, preparation of, 24 synthesis of peptides from, 24 Azlactones, 18 preparation of, 16, 17, 18 synthesis of peptides from, 15-19, 20, 46 Azotobacter, nitrogen assimilation, 274

468

SUBJECT INDEX

Azotobacter vinelandii, amino acid composition of protein from, 254

B Bacillus anthracia, capsular substance of, 251, 253, 259, 265,269 chemical nature of, 251, 265 function of, 253 as source of polyglutamic acid peptides, 45 Bacillua brevis, amino acid cornposition of protein from, 252 Bacillus subtilis, amino acid composition of protein from, 252 Bacteria, proteins of, 251-254, 268 amino acid compoRition of, 258 p-Benzoquinone, as tanning agent, 411-413 Benzoyl-Lleucylglycinanilidc, enzymatic synthesis of, 33 Benzoyl-cleucyl-bleucinanilide, enzymatic synthesis of, 33 Benzoyl-ctyrosy lglycinanilide, enzymatic synthesis of, 33 “Biuret base,” 7 Body weight, effect of amino acids on, 189 Bristles, made from casein powder, 325 a-d-Bromoisocaprony Iglycylgl ycylglycine, synthesis of, 14 Bryophytes, proteins of, 231 amino acid composition of, 258

C Cancer, and plasma proteins, 181 Carbethoxyglycylglycylleucine ethyl ester, synthesis of, 7, 8 Carbobenzoxy-balanyl azide, 40 Carbobenzoxyamino acids, synthesis of peptides from chlorides of, 25-32

4Carbobenroxyaminooxazolidone-2, 57 Carbobenzoxy-~glutamyl-~-tyrosine, hydrolysis by pepsin, 47 synthesis of ethyl ester of, 30 Carbohydrases, in milk, 219 Carbohydrates, effect on protein synthesis in plants, 285 Carbonyl-bis-gl ycine, synthesis of, 8 Carbonyl-bis-glycylglycine, synthesis of, 8 Carnosine, 3 structure of, 65 synthesis of, 65 Casein, 204-209 amino acid composition of, 101, 103, 105, 106, 109, 110, 111, 112, 113, 114, 116-120, 206, 218, 224 chemical difference between human and cow’s, 204, 206-208 heterogeneity of, 204 hydro1yzates, biological evaluation of, 159 plasma proteins and, 187 fibers made from, 310, 311, 314, 325 from mixtures of, and polyamides, 326 in milk, 202 molecular weight of, 207 phosphopeptones from, 208 composition of, 208 plasma proteins and, 186, 187 preparation of a-,205-206 of 8-, 206 regeneration of liver proteins and, 188 rennet, 209 separation of a- and 8-, 205 Catalase, in milk, 222 Caulerpa racemoaa, 248 Cells, constituents of, 423-439 distribution of proteins in, 265-267, 423-439 duplication of living, 433 localization of specific functions in, 425 role of nucleic acids in, 434 of phospholipids in, 434 of proteins in, 434

SUBJECT INDEX

Chenopodiacae, amino acid composition of proteins in leaves of, 237 Chitin, 251 Chloroacetyldehydrophenylalanine,17 Chloroplasts, auxin in, 267 proteins of, 267 Chondrus erispans, amino acid composition of protein of, 248 Chromatin, 428 composition of, 426 isolation from cell nucleus, 426 Chromium, complex formation with collagen, 388392 salts of, reaction with collagen, 382 structure of basic, 379-380 and tanning potency, 380 as tanning agents, 354, 379-392 factors governing effect of, 379 Chromosomes, 425, 433 composition of, 426, 427 structure of, 427 Chromosomin, 266, 427 Choline, nitrogen-sparing action of, 172 Chymotrypsin, hydrolysis of peptides by, 47 peptides as substrates for, 47 synthesis of peptide derivatives by,

33 Chymotrypsinogen, amino acid composition of, 145 Clostridium botulinum type A toxin, amino acid composition of crystalline, 124, 143 Codium fragile, 248 Collagen, 306, 355 acid- and base-binding capacity of, 362-365 amino acid composition of, 143, 357, 358 chemistry of, 356-360 complex formation with chromium, 388-392 denaturation of, 369

469

effect of alkali on, 368, 377 of lyotropic agents on, 372-375 of neutral salts on, 372ff. of pretreatment on reactivity, 374 fibers made from, 311, 325, 326, 335 heat shrinkage of, 369 isoelectric point of, 3666. modified, reaction with tanning agents, 414 molecular weight of, 358 reaction with aldehydes, 405 with chromium compounds, 379,328 with condensed sulfo acids (syntans), 402 with vegetable tannins, 3 9 5 4 0 0 factors governing, 396-400 Stabilization of, 360-362 structure of, 355, 359, 360 swelling of, 356, 365, 368 Colostrum, 210-214 chemical composition of, 212 immune properties of, 211-213 protein content of, 211 pseudoglobulin, amino acid composition of, 102, 103, 105, 106, 109, 110, 111, 112, 113, 117, 118-120 Conarachin, 262 Conglutin, heat of combustion, 264 Cottonseed, fibers made from proteins of, 311, 315 Cruciferae, amino acid composition of proteins in leaves of, 237 Cucumber 3(4) virus, amino acid composition of, 256 Cupriethylenediamine, complex formation with silk fibroin, 324 Cysteine, derivatives of, 53 determination in intact proteins, 96 in protein hydrolyzates, 96, 111-1 13 comparison of methods for, 105 peptides of, 6, 49-55 hydrolysis by pepsin, 47 optical rotation of, 54 synthesis of, 32, 49-54 sourcesof, 105, 124, 131, 132, 135, 137, 140, 142, 143, 145

470

SUBJECT INDEX

LCysteine anhydride, hydrolysis of, 6 Cystine, 147 derivatives of, 54 estimation in protein hydrolyzates, 111-113

comparison of methods for, 105 hematopoiesis and, 184 nitrogen-sparing action of, 172 peptides of, 496. optical rotation of, 54 synthesis of, 49, 54, 55 regeneration of liver proteins and, 189 sources of, 105, 124, 131, 132, 137, 140, 142, 143, 146

stability of keratin and, 376 Cysloseira osmundaceae, 248 Cytochrome c, 431, 436 role in aerobic cell respiration, 438,439 Cytochrome oxidasc, 431 nature of, 436 Cytoplasm, 428-433 aerobic cell respiration and, 438 constituents of, 425,426,427,429, 433, 438

functional relation between cell nucleus and, 427 labile liver, 187 ribonucleic acid content of, 438

D Decarboxylaaes, amino acid estimation in protein hydrolyaates with, 90 sources of, 90 Dehydrogenase, in milk, 220 Dehydropeptidasc, 17 action on peptides, 17, 19 Depilation, of animal hide, 375-378 Desoxyribonucleic acids, 265, 268, 427 biological conversion to ribonucleic acid, 43 distribution in cells, 437 location in cells, 437 Detergents, application to protein fiber preparation, 317-324 complex formation with proteins, 318320

Diet, amino acids and, 158 effect on plasma proteins, 182 Digestibility, of dietary nitrogen, 162, 165 3,5-Diiodo-btyrosine, synthesis of peptides of, 49 Diketopiperazines, racemization of, 5, 6 synthesis of, 7 of dipeptides from, 5-6, 9 &fl-Dirnethyl-D-cysteine, as component of penicillin, 74

5,5-Dimethylthiaaolidine4 carboxylic acid methyl ester, 75 Donnan effect, 365, 366

E Edestan, conversion of edeatin to, 1 3 4 Edestin, amino acid composition of, 98, 101, 102, 103,105,106,109, 110,111, 112, 113, 114, 116-120, 131, 134, 264 conversion to edestan, 134 fibers made from, 325 heat of combustion, 264 molecular weight of, 98, 128, 134, 263 structure of, 134-135, 148

Egg protein, see also Ovalbumin, effect on growth, 177 nitrogen equilibrium and, 164 Egregria wnziesii, 248 Elastin, fibers made from, 335 Enzymes, crystalline, 263, 264 amino acid composition of, 145 in milk, 31S223 in mitochondria, 431 protein metabolism in plants and, 288290

proteolytic, in milk, 222 peptides and, 11, 14, 33, 34, 40 resolution of Dbamino acids by, 34, 35

specificity of, 11, 33

47 1

SUBJECT INDEX

DrEpiglucoaaminic acid, configuration of, 74 synthesis of peptide of, 74 Escherichiu coli, amino acid composition of protein from, 252 Esterases, in milk, 221 Estrus cycle, effect on milk lipase activity, 221 Euglobulin, 210, 213 kr whey, 203 Euphorbiacae, amino acid composition of proteins in leaves of, 237 Excelsin, molecular weight of, 263

F Fatigue, and amino acids, 158 Feathers (see also Keratin), fiber-forming properties of, 315 protein nature of, 315 Fibers, synthetic, from proteins, 305-346 alkaline agents in preparation of, 313-315

crystallization in, 328 deformation behavior of, 332 effect of water on, 345 interpretation of stress-strain behavior of, 331-334 molecular basis for mechanical properties of, 327-336 experimental methods in the study of, 331 muscle protein as source of, 306, 311, 335

polypeptide chains of, 308 relation between, and mechanical properties of, 312, 327-336 properties of, 345 stabilizing bonds in, 308, 309, 316, 339ff., 344, 345 stability of, 308, 346 structure of, 308 Fibrin, amino acid composition of, 101, 102, 103, 106, 113, 116, 117, 118, 120

evaluation of hydrolyzates of, 159 stability of, 376 Fibrinogen, amino acid composition of, 142 fiber-forming properties of, 311 Fishes, fiber-forming properties of protein8 from, 311 Flavin adenine nucleotide, flavin moiety of milk xanthin oxidase and, 220 Flavoproteins, isolation from milk, 220 Folic acid, structure of, 45, 73 Formaldehyde, reaction with proteins, 405, 410 as tanning agent, 405-411 Fucus furcatus, 248 Fungi, proteins of, 249-250 amino acid composition of, 250, 251, 258

nutritive value of, 249 G

Gelstin, acid-binding capacity of, 365 amino acid composition of, 143 base-binding capacity of, 365 fibers made from, 310 from mixtures of polyamides and, 326

isoelectric point of, 367 proline peptides from, 67 swelling of, 356, 366 Gliadin, 262, 263 amino acid composition of, 143, 264 hydrolyzation products of, 3 proline peptides from, 67, 68, 70 regeneration of liver proteins and, 188 solubility of, 147 Globulin, colostrum, 210, 211 amino acid composition of, 210, 211, 218

fibers made from, of tobacco seeds, 325 milk, composition of, 210 preparation from whey, 203, 210 in plasma proteina, 181, 182

472

SUBJECT INDEX

u-Globulin, amino acid composition of, 112 7-Globdin, amino acid composition of, 101, 102, 103, 105, 106, 109, 110, 111, 113, 116-120, 142 immune properties of plaema, 213 Gloedrichia echinulda, 248

rr

peptides of, 3, 35-40 optical rotation of, 38 synthesis of, 5, 6, 7, 11-12, 32, 40 preparation of polymers of, 22 role in tho folding of proteins, 330 source8 of, 129, 130, 131, 137, 140, 142, 143, 145

Glycine anhydride, Glucosaminic acid, hydrolyzation products of, 5 melting point, 36 peptides of, 74 Glutamic acid, synthesis of, 5 Glycinin, 262 derivatives of, 43 estimation in protein hydrolyzates, 80, Glycyl-Lalanyl-cleucine, structure of, 4 114, 115 comparison of methods for, 96, 108 Glycyldehydroalanine, synthesis of, 19 molecular weight of D( -)-, 265 Glycyldeh ydrophen ylalaninc, peptides of as substrate for dehydropeptidasr, 17 physiological role of, 45 synthesis of, 17 optical rotation of, 44 Glycyldehydrophenylalanyl-I-glutamic sources of, 45 acid, synthesis of, 17 synthesia of, 16, 17,32, 43,44, 45,46 Glycyltaurine, 55 polypeptide of D( -)-, in capsular sub- Gramicidin, 259 stance of Bm'llu8 anlhracis, 251, peptide nature of, 3 253, 265,268 Gramicidin S, sources of, 108, 124, 131, 132, 136, 137, peptide nature of, 3 138, 140, 142, 143, 145 Graminae, LGlutamine, amino acid composition of proteins in synthesia of, 45 leaves of, 237 a-LGlu tamyl-Lcysteinylglycine Growth, (isoglutathione), 54 effect of diet on, 179 7-L-Glutamyl-Lcysteinylglycine, see and nitrogen retention, 173 Glutathione, Gymnosperms, 7-D-Glutamyl-Lcysteinylglycine(epi-gproteins of, 231 glutathione), 53 H Glutathione, 3 structure of, 51 synthesis of, 51, 52, 53 Hematopoiesis, Glutelin, 263 and isoleucine, 184 Hemoglobin, Gluten, wheat effect on growth, 177 amino acid composition of, 98, 101, on nitrogen equilibrium, 164 102, 103, 105, 106, 109, 110, 111, fractionation products of, 262 112,113, 114, 116, 117-120, 137, 139 nutritive value and plasma proteins, fibers made from, 325 molecular weight of, 98, 128, 139 187 Glutenin, 262 in root nodules, 275 GIycine, structure of, 139, 147 Hexokinase, 264 derivatives of, 36 effect on nitrogen balance, 159 Hide, see also Collagen, Leather estimation in protein hydrolyzates, 110 depilation of, 375-378 comparieon of methods for, 96, 101 stability of vegetable tanned, 399

473

SUBJECT INDEX

Hippuric acid, 9 Histidine, derivatives of, 61 effect on body weight, 190 on hemoglobin output, 185 on nitrogen excretion, 185 estimation in protein hydrolyzates, 90 comparison of methods for, 96, 119 peptides of, optical rotation, 62 synthesis, 65-67 sourcesof, 119, 131, 137, 140,142, 143, 145 Histidine anhydride, stability of, 6 synthesis of, 7 Holmes’ masked virus, amino acid composition of, 256 Holmes’ rib grass virus, amino acid composition of, 256 Homocysteine, peptides of, 54, 73 Homocystine, peptides of, 54 Hordein, heat of combustion, 264 Hormones, protein metabolism in leaves and, 284 Hydantoin, conversion of peptides to derivatives of, 48 Hydrogen ion concentration, role in chrome tanning, 386-388 in formaldhyde tanning, 408 in vegetable tanning, 396, 400-402 Hydroxylysine, sources of, 143 Hydroxyproline, derivatives of, 69 estimation in protein hydrolyzates, 109 peptides of, optical rotation, 70 synthesis of, 71,72 sources of, 109, 143

I

fibers from, 314 molecular weight of, 128, 132 structure of, 132-133 Isoleucine, derivatives of, 37 synthesis of, 35,38, 39 effect on body weight, 190 on nitrogen balance, 158 estimation in protein hydrolyzates. 115, 117-119 comparison of methods for, 96, 112 hematopoiesis and, 184 nitrogen excretion and, 185 peptides of, 35 optical rotation, 39 sources of, 112,131,137,140,142, 143, 145

J J 14 D1 virus, amino acid composition of, 256

K Keratin, 306 amino acid composition of wool, 143 chain interaction in fibers made from feather, 336-346 chemical structure and stability of, 376 fibers made from, 311,323,324 from mixtures of, and polyamides, 326 solubility of, 316, 345 structure of, 356 water uptake by, 356 Keto acids, synthesis of peptides from, 19,20 Krebs cycle, 431 1

Lactalbumin, isolation of, 214 Infection, probable identity of crystalline, with proteins and, 180, 181 p-lactoglobulin, 214 Insulin , regeneration of liver proteins and, 188 amino acid composition of, 98, 101, source of, 203 102, 105, 106, 109, 110, 111, 112, Lactase, 113, 114, 116, 118-120, 130-132 in milk, 220

474

SUBJECT INDIJX

LaetobaciUus s p p . , amino acid composition of protein from, 252, 253 Lactoglobulin, composition of immune, 213, 218 immune properties of colostral, 213 isolation from colostrum, 212 #-Lactoglobulin, 144, 214 amino acid composition of, 96, 98, 101, 102, 103,105, 106,109, 110, 111, 112, 113, 114, 116, 117-120, 131, 135, 136, 138, 216, 218 denaturation of, 217-219 molecular weight of, 98, 128, 129, 215 preparation of, 214-215

probable identity with lactalbumin, 214

properties of, 215 solubility of, 216 structure of, 135, 136 whey ae source of, 203 Lactomucin, 214 Lactoperoxidase, isolation of crystalline from milk, 222 molecular weight of, 222 physical properties of, 223 Laminaria sp., amino acid composition of protein of, 248

Latex, protein from, 241 Leather (see also Collagen, Hide), chrome, 392-394 Leaves, proteins of, amino acid composition of, 236-240, 271

isolation of, 233-240 metabolism of, 281-283 hormonal control of, 284 nutritive value of, 236 relation between levels of, and amino acids, 284 and water, 284 Legurnelin, heat of combustion, 264 Leguminoaae, amino acid composition of proteins in leaves of, 237 Lcssoniopeie littoralis, 248 Leucine, derivatives of, 37

effect on body weight, 190 on nitrogen balance, 158 estimation in protein hydrolyeates, 90, 115

comparison of methods for, 96, 112 nitrogen excretion and, 186 peptides of, 6, 35ff. optical rotation, 38, 39 synthesis of, 12, 35-40 sourcesof, 112, 124, 130, 131, 137, 140, 142, 143, 145

synthesis of polymers of, 22 Leucine anhydride, stability of, 6 DGLeucyl-Dbalanine, isomers of, 4 Lignosulfonic acid, molecular weight of, 403 reaction with collagen, 403, 404 Lipase, in milk, 221 pitocin and, 221 relation between activity of, and estrus cycle, 221 Lipides, 434 as cell constituents, 426, 431, 435, 438

protein deficiency and, 182 role in cellular physiology, 435 Lipositol, 432 Liver, effect on growth, 180 protein metabolism and, 189 Lyotropic agents, effect on animal skin, 372-375, 377 Lysine, availability of, 194 derivatives of, 61 aa substrate for pancreatic trypsin, 60 effect on body weight, 190 on digestibility of wheat gluten, 165 on growth, 158 on nitrogen balance, 158 hemoglobin output and, 185 isolation from soybean oil meal, 194 methoda for estimation of, 96, 120 nitrogen excretion and, 185 peptides of, 58ff. optical rotation, 62 synthesis of, 58-60

475

GUBJECT INDEX

preparation of polymers, 22 sources of, 120,124,131, 137,140,142, 143, 145 Lysine anhydride, synthesis of, 7

M Macrocystis pyrifera, 248 Maize, amino acid composition of protein from, 244 Malnutrition, plasma proteins and, 181 Mesonin, 262 Methionine, availability of, 193 derivatives of, 71 effect on body weight, 190 on digestibility of casein, 165 of fibrin hydrolyzates, 165 on nitrogen balance, 158 estimation in protein hydrolyzates, 96, 111 comparison of methods for, 96, 103 hematopoiesis and, 184 nitrogen excretion and, 184 nitrogen-sparing action of, 171 peptides of, 72ff. optical rotation, 72 synthesis of, 73 protein metabolism and, 169, 189 regeneration of liver proteins and, 189 sources of, 103, 131,137, 140,142 143,

145 Microsomes, 425, 432, 433 composition of, 429,430,432,435,437 function in cell, 432,438 isolation of, 432 size of, 432 Milk, enzymes in, 219-223 isolation of flavoproteins from, 220 proteins of (see also under name of individual members), 201-225 amino acid composition of, 218, 219 distribution of, 202-203 properties of, 203-210 relationship to serum proteins, 223 separation of, 203-210 serum, see Whey

Mitochondria, 425,430-432,433 aerobic cell respiration and, 438 composition of, 429, 430, 431, 435, 437 enzymes in, 431 size of, 430 Molybdenum, effect on nitrogen assimilation, 274 Muscle, protein, fibers made from, 311,335 water uptake by, 356 Mustard gas, effect on proteins, 3 Myogen A, amino acid compoaition of, 140 source of, 147 Myoglobin amino acid composition of horse, 98, 137 molecular weight of horse, 98 structure of, 148 Myosin, amino acid composition of, 105, 140 sources of, 147 stability of, 376

N ,9-Naphthalenesulfonic acid, reaction with collagen, 402 Nervousness, amino acids and, 158 Nitrate, assimilation in plants, 270ff. Nitrogen, assimilation in plants, 274ff. effect of metals on, 274 role of ammonia in, 281 balance, 157, 160, 161, 163, 164, 167 essential amino acids and, 158, 159 repletion of plasma proteins and, 186 digestion, 162, 163 excretion, 157, 162, 163, 166, 168, 169, 170, 171, 172, 180, 183 diet and, 170 protein stores and, 161 fecal, 162, 163, 180 requirement in dietary proteins, 158 retention of, and growth, 173 and protein efficiency, 176

476

SUBJECT INDEX

Nucleic acids, 428,434, 437439 Pelvetia, in chromosomes, 427 as source of glutamic acid peptides, 45 in cytoplasm, 429 Penibillin, Nucleolus, 425,428,433 B,B-dimethyl-D-cysteine as component constituents of, 428,437 of, 74 Nucleoproteina, 428 Penicillium notalum, bacteria as source of, 268 amino acid composition of protein composition of, 427, from, 251 as constituents of chromosomes, 426, Pepsin, 427 amino acid composition of, 103, 145 function in cell, 427 hydrolysis of peptides by, 47 Nucleotides, 429 Pcptides (see also under names of indiNucleus, 425 vidual amino acids), functional relation between cytoplasm biological activity of, 3 and, 427 configuration of, 4 isolation of chromatin threads from, conversion to hydantoin derivatives, 420 48 derivatives of, synthesis, 9-11, 33-34 0 nomenclature, 4 Oranges, synthesis of, 1-75 amino acid composition of protein from amino acid chlorides, llff, from, 241 azlactone method for, 15-19 Ornithine, 60 by the carbobenzoxy method, 25-32 derivatives of, 61 from N-carbonic acid anhydrides of estimation in protein hydrolyzates, 90 amino acids, 21-23 peptides of, 618. by condensation of keto acids and optical rotation of, 62 amides, 19-20 synthesis of, 60-62 of peptide esters, 6-9 Osmunda claytonianu, from u-halogeno acyl halides, 11-15 isolation of protein of, 246 by partial hydrolysis of diketopiperaamino acid composition of, 247 zines, 5, 6 Ovalbumin, from phthalylamino acids, 32-34 amino acid composition of, 98, 101, from toluenesulfonylamino acids, 102, 103, 105, 106, 109, 110, 111, 23-24 112, 113, 114, 116, 117-120, 137, Phaseolin, heat of combustion, 264 138, 139 Phenylalanine, fibers from, 310,313,315,323,325,326 derivatives of, 49 relation between applied stretch and effect on body weight, 190 mechanical properties of, 327 on nitrogen metabolism, 158, 184 molecular weight of, 98, 128,313 on plasma protein output, 185 structure of, 139, 141 estimation in protein hydrolyzates, 115 Oxidases, in milk, 222 comparison of methods for, 96, 11 1 peptides of, 3, 6,11, 12, 32, 46ff. P optical rotation, 51 Papain, synthesis of, 46ff.,49 resolution of Dbglutamic acid by, 34 polymers of, synthesis, 22 synthesis of peptide derivatives by, 33 sourcesof, 111, 124,131, 137, 140, 142, Peanuts, 143, 145 protein of, 244,262 Phenylpyruvylamino acids, fibers from, 311, 315 synthesis of, 20

BUBJECT INDEX

Phormidium valderianum, amino acid composition of protein of, 248 Phosphatase, in milk, 221 properties of, 221 Phospholipides, as constituents of cytoplasm, 426,429, 430, 431, 432 role of, in the formation of cell membranes, 4346. Phosphopeptones, from casein, 208 composition of, 208 Phosphoserylglutamic acid, isolation from casein digests, 208 Phthalylamino acids, synthesis of peptides from, 32-34 Pitocin, activation of milk lipase by, 221 Placenta, transfer of antibodies through, 212 Plants (see also under names of the various divisions) assimilation of nitrate in, 270-271 proteins of, 229-295 homogeneity of, 261-263 metabolism of, 26S294 amino acids and, 284 carbohydrates and, 285 enzymes and, 288-290 respiration and, 286-288 water and, 284 synthesis of amino acids in, 271-277 utilization of ammonia by, 271-274 Plasmapheresis, 182, 185, 186 Pollen, proteins of, 241 Polyphenols, tanning properties of, 403 Potatoes, amino acid composition of protein from, 240 Prolamin, see Gliadin Proline, derivatives of, 69 estimation in protein hydrolyzates, 110-111 comparison of methods for, 96, 102 peptides of, 67ff. optical rotation, 70 from protein hydrolyzates, 67 synthesis of, 12, 14, 67-71

477

sources of, 102, 129, 130, 131, 137, 140, 142, 143, 145 Propionyldehydroaspartic acid, synthesis of, 18 Protease, in milk, 222 Proteins, acid-base relationships of, 3 action of mustard gas on, 3 amino acid composition of muscle, 140 and nutritive value of, 192 of plasma, 142 of purified, 129-148 analysis of hydrolyzates of, 88-125 biological evaluation of, 155-200 cellular, 423439 enzyme nature of, 259 determination of free amino groups in, 99 dietary, bioassay of, 188, 190 biological value of, 169 unidentified substances in, 185 effect on body weight, 176, 179, 180, 185 of food processing on nutritive value of, 173 of urea on soluble, 399 efficiency ratios, 175 fibrous, 305-346 chemical composition and properties of, 316, 355, 356 coagulation of, 309 complex formation with detergents, 318-320 conversion of corpuscular to crystalline, 325 denaturating agents for, 315, 316 peptide chains of, 315-317, 330 relation between molecular weight and properties of, 313 solubilizers for, 316 stabilizing bonds in, 316, 317, 373 effect of lyotropic agents:on, 373, 375 structure of, 355 and solubility, 316 suitability for fiber preparation, 326 infection and, 180, 181 iodination of, 3 liver, repletion of, 187

478

SUBJECT INDEX

milk, 201-225 amino acid composition of, 218, 219 of colostral, 21Ck214 distribution of, 202-203 properties of, 203-210 separation of, 203-210 molecular weights of, 128-129 nutritive value of, 156, 170, 175 of plants, 229-295 metabolism of, 269-294 asparagine and, 278 phylogenetic aspects of composition Of,

257-259

relation between type and function of, 265 synthesis of, 271-277 plasma, albumin content of, 181 cancer and, 181 malnutrition and, 181 nitrogen balance and, 186 regeneration of, 182, 185, 186 tuberculosis and, 181 potency ratios, 186 purity of, definition of, 125 determination of, 125-127 reaction with formaldehyde, 405 requirements of rats, 174 ribonucleic and cellular synthesis of 438

stability of a-,376 structure, 355 and solubility, 144, 147, 316 tryph-inhibiting, in soy beans, 263 unidentified factora in, 194 Proleus vulgari8, amino acid composition of protein from, 252 Protoplasm, 306 structure of, 266 Pseudoglobulin, 210 in colostrum, 211 in whey, 203 Pteridophytes, proteina of, 231, 246 amino acid composition of, 247, 258 Pteridium aquilinum, amino acid composition of protein from, 246, 247

R Respiration, aerobic, cytoplasm and, 438 role of cytochrome in, 438, 439 anaerobic, role of ribonucleic acid in, 438 relation between, and protein metabolism in plants 286-288 Rhizopus nigricans, amino acid composition of protein of, 25 1

Rhoddwula ruber, amino acid composition of protein of, 25

Ribonucleaae, amino acid composition of, 145 Ribonucleic acid, 265, 266, 430, 431 biological conversion to desoxyribonucleic acid, 437 cellular protein synthesis and, 438 distribution in cells, 438 function in cells, 437 location in cells, 437, 438 in microsomes, 432 RiCin, molecular weight, 263 source of, 263 toxicity of, 263 Roots, amino acid composition of proteina from, 240 hemoglobin in nodules of, 275

s Saccharomyces spp., amino acid composition of protein from, 251 Salmine, amino acid cornposition of, 98, 101, 102, 109, 112, 113, 114, 118, 129, 130 molecular weight of, 98, 128, 313 peptide nature of, 129 Salts, neutral, action on soluble proteins, 373 role in chrome tanning, 373, 384-386 Sarcosine, preparation of polymers of, 22

SUBJECT INDEX

Satgassum $uuilana, amino acid composition of protein from, 248 Satgassum Mtana, amino acid composition of protein from, 248 Schardinger enzyme, see Xanthine oxidase

seeds, proteins of, 242-246, 267, 268 amino acid composition'of, 243-246, 263-264 and nutritive value, 243-245, 246 distribution of, 245 heat of combustion of, 264 homogeneity of, 261 isolation of, 242-243 metabolism of, 277-281 molecular weights of, 263 physical properties of, 263 stability of, 261 toxicity of, 263 trypain-inhibiting activity of, 263 transaminaae activity in, 276 Selaginella unecinda, protein of, amino acid cornposition of, 247 isolation of, 246 Serine, derivatives of, 57 estimation in protein hydrolyzates, 115, 122 comparison of methods for, 96, 109 isolation of G,57 peptides of, 55ff. optical rotation of, 58 synthesis of, 55-58 aources of, 109, 130, 131, 132, 137, 140, 142, 143, 145 Serine anhydride, synthesis of, 7

-

sew,

relationship of milk proteins to proteins of, 223 Shrinkage, of animal skins, 369ff., 383 Silk fibroin, 309,359 amino acid composition of, 101, 102, 103, 106, 109, 110, 112, 113, 114, 116, 118-120, 143,329

479

complex formation with cupriethylenediamine, 324ff. fibers made from, 311, 335 from, and polyamides, 329 hydrolyzation producta of, 3, 57 molecular weight, 324 solubility of, 147 water uptake by, 356 X-ray diffraction of, 307 Skin, animal, see also Hide shrinkage of, 369ff. species differences in, 371 structure of, 354-355 Solanacae, amino acid composition of proteins in leaves of, 237 Soybeans, protein of, amino acid composition of, 244 fibers made from, 311, 315 of mixtures of, and polyamides, 326 molecular weight of, 263 trypsin-inhibiting activity of, 263 Soybean oil meal, effect of heat on nutritive value, 193 aa source of amino acids, 193ff. Spermatophytes, proteins of, 231-246 amino acid composition of, 258 SlaphyEoeoceus autew, amino acid composition of protein from, 252 Stseptoeoecwr fecalis, amino acid composition of protein from, 252 Streptogenin, effect on nutrition of mice, 194 Streptomyces griSeua, 251 amino acid composition of protein from, 252 Succinoxidaae, 431 Sulfo acids, condensed, see Sydtans Sunflower, amino acid composition of seed proteins of, 244 Swelling of fibrous proteins, 356, 365, 366 Syntans, nature of, 402 as tshning agents, 402405

480

BTJBJECT INDEX

T

Transamination, 20 in green plants, 271, 273 Tanning, Triose phosphate dehydrogenase, definition of, 354 amino acid composition of, 140 general aspecta of, 378-379 source of, 147 protein chemistry of, 353416 Tropomyosin, role of micellar reactions in, 360 amino acid composition of, 140 theory of vegetable tanning, 400-402 source of, 147 Tanning agents, 354,379ff. Trypsin, aldehydes as, 405-411 amino acids as substrate for, 60,64 chromium compounds aa, 379-392 inhibitory effect of soybean protein on, condensed sulfo acids (syntans) as, 263 402404 Tryptophan , effect on tensile strength, 362 biological isomerization of d-, 436 quinones 88,411-413 derivatives of, 71 reaction with hide proteins, 414 effect on body weight, 190 with modified collagens, 413 on nitrogen balance, 158, 159 vegetable, 394-396 nitrogen excretion, 184 molecular weights of, 395 estimation in protein hydrolyzates, properties of, 394-396 119, 121 reaction with collagen, 395 comparison of methods for, 117 factors governing, 396-340 fortification of casein hydrolyzate by, sources of, 395 159 Tannins, see Tanning agents, vegetable isolation from soybean oil meal, 194 Thallophytes, peptides of proteins of, 231, 247ff.,264-265 optical rotation, 71,72 amino acid composition of, 258 synthesis of, 11, 71, 72. 73 Thiazolidine4carboxylic acid methyl plasma protein output and, 185 ester, 75 sourcesof, 117, 124, 131, 137,140, 142, Thiazolones, 18 143, 145 Threonine, Tuberculin protein, 265 effect on body weight, 190 Tuberculosis, and plasma proteins, 181 estimation in protein hydrolyzates, Turnips, 115, 122 amino acid composition of protein comparison of methods for, 96, 110 from, 240 nitrogen metabolism and, 158, 184 Tyrocidine, 259 plasma proteins output and, 185 pcptidc nature of, 3 sources of, 110,115, 124, 131, 132,137, Tyrosine, 140, 142, 143, 145 derivatives of, 49,50 Tissues, estimation in protein hydrolyzntcs, 90, dehydropeptase in animal, 17 119 effect of protein depletion on, 182 Comparison of mctjhods for, 96, 116 Tobacco mosaic virus, peptides of, 46ff amino acid composition of, 101, 102, optical rotation of, 4G,51 103, 105, 106, 109, 110, 111, 112, synthesis of, 46,47 113, 114, 116, 117-120, 255, 256 sourcesof, 116,131, 137, 140,142, 143, effect on protein metabolism of host, 145 292-294 Tyrosine anhydride, Toluenesulfonylamino acids, stability of, 6 synthesie of peptides from 23-24, 57

48 1

SUBJECT INDEX

U Ulva ladwa, amino acid composition of protein of,

248 Urea, effect on soluble proteins, 399

V Valine, derivatives of, 37 effect on body weight, 190 on nitrogen equilibrium, 158 excretion, 185 estimation in protein hydrolyzates,

115ff. comparison of methods for, 96, 113 peptides of, 35ff. optical rotation, 38, 39 synthesis of, 40 sources of, 130, 131, 137, 140, 142, 143 Vanadium, effect on nitrogen assimilation, 274 ViNeeS, nucleoprotein nature of, 254 phytopathogenic, 254-256 amino acid composition of, 255,256-

258 and activity of, 259 crystalline, 255 effect on protein metabolism of host,

292-294

W Water, relation between protein levels and, in plants, 284 uptake of by fibrous proteins, 356 Wheat, proteins from, 244, 262 amino acid composition of, 244 Whey, composition of, 203 proteins of, 210-219 Wool, structure of, 329

X Xanthine oxidase, in cow’s milk, 220-221 molecular weight, 221 properties of, 221

Y Yeast, crystalline proteins from, 264 Z

Zein, amino acid composition of, 143, 264 fibers made from, 311, 314, 315, 325,

326 from mixtures of, and polyamides, 326 regeneration of liver proteins and, 881 solubility of, 147


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