synthesis and enzymatic hydrolysis of glutamic acid ... · boxy y-ethyl-l-glutamate anhydride which...

SYNTHESIS AND ENZYMATIC HYDROLYSIS OF GLUTAMIC ACID POLYPEPTIDES*

BY MAURICE GREEN AND MARK A. STAHMANN

(From the Department of Biochemistry, College of Agriculture, University of Wisconsin, Madison, Wisconsin)

(Received for publication, March 31, 1952)

Synthetic lysine polypeptides of high molecular weight (1) have been shown to reduce virus infectivity (2, 3), inhibit bacterial growth and respi- ration (4), agglutinate virus particles, bacterial cells, and erythrocytes (4-6), and to prolong blood clotting time (7). It has been suggested that most of these phenomena are associated with the formation of many ionic bonds between the positively charged lysine polypeptide and the appro- priate negatively charged receptors on the virus particles, bacterial or red blood cells.

In view of the above effects produced by the basic lysine polypeptides it was of interest to synthesize high molecular weight acidic glutamic acid polypeptides and to study their biological effects. This paper presents the synthesis and properties of L-glutamic acid polypeptides and their susceptibility to hydrolysis by proteolytic enzymes.

When this work was undertaken, the only published reference to synthetic glutamic acid polypeptides was a brief report by Hanby et al. (8). Subsequently and during the course of this work a more complete descrip- tion of the synthesis of glutamic acid polypeptides was published by the same authors (9) and also by Coleman (10). Although the synthesis described in this paper is somewhat similar to that reported by these authors, it differs in the method used to prepare the N-carboxy anhydride, in the technique of polymerization which allows control of the polypeptide size, and in the method for hydrolysis of polyglutamic ethyl ester which avoids racemization and gives an analytically pure polyglutamic acid.

synthesis-Polyglutamic acid was prepared by polymerization of N-carboxy y-ethyl-L-glutamate anhydride which was synthesized as shown by structures I to V.

The polymerization, which was carried out in dry dioxane at room temperature with varying amounts of anhydrous ammonia as initiator (l), yielded polypeptide preparations of different average molecular weights. The results are consistent with the polymerization scheme (V to VIII).

* Published with the approval of the Director of the Wisconsin Agricultural

Experimental Station. Supported in part by a research grant from the National Institutes of Health, United States Public Health Service.

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772 GLUTAMIC ACID POLYPEPTIDES

The monomer (V) reacts with the ammonia to open the lactone ring, forming the N-carboxy amide (VI), which decarboxylates to the amino

COOH COOH I I CH-NH* CH-NHt.HCl I + CtHsOH + dry HCl room~H~f;eratur% 1

(CHde

COOH COOC,Hs I. dXutsmic II. yEthy gluta-

acid mate hydrochloride

II + CH,=CH--CH,.~.CO.C~ pH7 to 8, -.------) &SO.

COOH I

CH-NH-CO.O.CH&H=CHI pcl I -%

(~Hzh 0”

COOCnHs III. Carboallyloxy r-ethyl

glutamate co-a I

CH--NH--CO.~.CHG.ZH=~H~ I -(CH,_C%-CH,C$

COOCzHr IV. T-Ethyl eater carboallyloxy

glutamic acid chloride

/O\ co co I I

CH---NH

COOCzHs V. N-Carboxy -pethyl-r,

glutamate anhydride

amide (VII). This, in turn, is acylated by another molecule of monomer V, and subsequent repetitions of the decarboxylation and acylation reac- tions at the amino end of the growing peptide chain result in the linear polypeptide, y-ethyl-L-polyglutamate (VIII).


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M. GREEN AND M. A. STAHMANN 773

Average molecular weights were determined by end-group analysis for the a-amino nitrogen content of y-ethyl polyglutamate. The data in Table I show that the average chain lengths of the polypeptides depend upon the

/O\ co co I I

CH-p KH + HNHr

I (C&h

COOGHs

V. N-Carboxy yethyl glutamate anhydride

4

CO--NH* CO-NH*

CH-NH-C00H I cH-NH2 +(n + 1)V

-+I (CHZ)Z

-(n + 1)COT

I COOCLHs COOCzHs

VI. N-Carboxy amide VII. Amino amide

CO-NH*

I

VIII. yEthyl-kpolyglutamate

ratio of anhydride (V) to ammonia used in the polymerization. The chain lengths were in general somewhat higher than the ratio of anhydride to ammonia. This is believed to be due to removal of part of the ammonia by reaction with the y-ethyl groups. Amide analysis of the polypeptide ester supported this view.

Conditions were worked out by which y-ethyl+polyglutamate could be saponified completely to free poly+glutamic acid. However, polyglutamic acid could not be used for chain length determinations, since only negligible amounts of nitrogen were liberated upon reaction with nitrous acid. This was due to cyclization of the terminal amino group with the terminal y-carboethoxy group to form a pyrrolidonecarboxylic acid residue. This cyclization occurred during the alkaline saponification of the polyester to the polyacid. In support of this it was shown that y-ethyl-L-glutamate rapidly cyclized in alkaline solution to form pyrrolidonecarboxylic acid. That such cyclization would occur has been indi- catcd by a study of kinetics of glutamic acid lactam formation (11). Thus the glutamic acid polypeptido consists of a linear chain of glutamic acid residues linked through peptide bonds between the a-amino and a-carboxyl groups. I’yrrolidonecarboxylic acid residues are present at the “amino” end of most of the molecules and free carboxyl or amide groups at the other end.


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Since peptides and proteins are readily racemized under alkaline conditions (12), it was of interest to determine whether such a racemization had occurred during the alkaline saponification of r-ethyl-L-polyglutamate. This was investigated by hydrolyzing polyglutamic ester and polyglutamic acid to the parent amino acid and comparing the specific rotation of each hydrolysate with L-glutamic acid similarly treated. It is apparent from the data in Table II that some racemization occurred during the acid hydrolysis of the samples. However, the results reveal that no appreciable

TABLE I

Chain Lengths of Polyglutamic Ethyl h’ster Preparations

Ratio of anhydride to I initiator

Chain length by a-amino N determination Average mol. wt. of polyglutamic acid

20 36.8 4,750 36.8

50 29.4 10,300

80.1 100 119.8 15,600

I 122.1

TABLE II

Specific Rotation of Hydrolysates of Polyglutamic Acid and Polyglutamic Ester

Polypeptides were hydrolyzed to the parent amino acid by autoclaving in 12 N

HCl for 5 hours. The specific rotations were determined in 6 N HCl.

StUUple bl; ~.- _._

degrees

L-Glutamic acid. . +26.5 Polyglutamic ethyl ester.. . . . +26.1 Polyglutamic acid.. . . . . . . . . +26.1 L-Glutamic acid (not autoclaved) . . . . . . . . +29.8

racemization occurred during the alkaline saponification of y-ethyl-n-polyglutamate. In contrast, the preparations of Hanby et al. (9) were cxten- sively racemized.

The rate of racemization of polyglutamic acid of molecular weight 10,300 in 0.73 N potassium hydroxide was determined. The straight line shown in Fig. 1 for the plot of log specific rotation against time indicates that the racemization followed first order kinetics. After treatment for 136 hours with alkali, the specific rotation decreased to half its initial value, from -76.2” to -36.1”, while at the same time only 2.5 per cent hydrolysis of peptidc bonds occurred. The change in rotation attributed to the hy- drolyses of the peptide bonds would be less than 3 per cent of the observed


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M. QREEN AND hi. A. STAHMANN 775

rotation. In 4 N alkali the rate of racemization was much more rapid, half the optical activity being lost in 3 hours.

Enzymatic Studies-Although considerable work has been carried out on the enzymatic hydrolysis of synthetic peptides of low molecular weight, relatively little attention has been given to the action of proteolytic enzymes on synthetic polypeptides of high molecular weight. Since these high molecular weight polypeptides have some features in common with proteins which the smaller peptides lack, it is of interest to determine the

I I I I L 6 I I I 40 a0 120 I60 200 240

TIME (HOURS) Fro. 1. Rate of racemization of polyglutamic acid in alkaline solution. The log

specific rotation is plotted against time. The solvent was 0.73 N KOH. The concentration of polyglutamic acid, molecular weight 10,300, was 0.0280 gm. per cc. of solution.

action of proteolytic enzymes on synthetic high molecular weight polypeptides.

Go and Tani (13) showed that the water-insoluble polyglycine and poly- glycylleucine were not hydrolyzed by erepsin, papain, or trypsin. Brand and Katchalski (cf. Katchalski (14)) reported that. polylysine is rapidly hydrolyzed by trypsin.

Experiments were carried out to determine the extent of hydrolysis of polyglutamic acid by pancreas extract, papain, and crystalline pepsin, trypsin, chymotrypsin, and carboxypeptidase. The results obtained with a 2.5 per cent pancreas powder extract at pH 4.0, 5.0, and 6.0 are presented in Fig. 2. These data show that maximum hydrolysis occurred at pH 5, at which 68 per cent of the polyglutamic acid was hydrolyzed within 50 hours. No appreciable hydrolysis was observed at pH 4, while at pH 6 only 24 per cent hydrolysis occurred.


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Papain hydrolyzed polyglutamic acid to the extent of 34 per cent within 48 hours. Polyglutamic acid was not hydrolyzed to any significant extent

70

IO 20 30 40 50 60 70 TIME (HOURS)

FIG. 2. Hydrolysis of polyglutsmic acid by pancreas extract. 0, pH 5.0, 0.1 x acetate buffer; A, pH 6.0,0.05 M citrate buffer; Cl, pH 4.0,0.15 acetate buffer. En- zyme concentration, 0.56 mg. of N per cc. of test solution. Substrate concentration, 1.6 mg. of polyglutamic acid (mol. wt. 10,300) nitrogen per cc. of test solution. Incu- bation temperature 37”. Per cent hydrolysis determined by a-amino 6 analysis with the microvolumetric Van Slyke apparatus.

TABLE III

Hydrolysis of Polyglutamic Acid by Carbozypeptidase

Polyglutamic acid of molecular weight 10,300 was incubated at 37’ for 24 hours. The per cent hydrolysis was determined by a-amino nitrogen analysis.

PH

7.3, phosphate buffer, 0.03 M.. . 7.5, Verona1 buffer, 0.04 M.. 5.0, acetate buffer, 0.1 M.. 5.0, “ “ 01“. 5.0, “ ‘( 0:1 “. :. . .

-.-

m.c. N per cc. mg. N per cc.

0.057 0.74 0.23 0.71 0.072 0.75 0.23 0.77 0.46 0.87

Per cent hydrolysis

0 0

25

40 50

by pepsin at pH 5.0, 4.0, and 2.0, nor by trypsin or chymotrypsin at pH 5.0 and 7.5.

Table III shows that polyglutamic acid was hydrolyzed by carboxypeptidase at pH 5.0, but no hydrolysis was observed at pH 7.5, which is near the pH optimum for this enzyme. It was shown by paper chromatography


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M. GREEN .4ND M. A. STAHMANN 777

that glutamic acid was the only product of this enzymatic hydrolysis. Considerably more carboxypeptidase was required for polyglutamic acid hydrolysis than is usually employed for other substrates. This higher enzyme requirement was not due to enzyme denaturation or product inhibition. That no substrate inhibition by polyglutamic acid at pH 7.5 occurs is indicated by its failure to reduce the zero order rate of hydrolysis of carbobenzoxyglycyl-L-tryptophan by carboxypeptidase.

EXPERIMENTAL’

y-Ethyl Glutamate Hydrochloride (II)-This compound was prepared from L-glutamic acid by esterification of the y-carboxyl group. The method of Bergmann and Zcrvas (15) was not satisfactory for more than 5 gm. of starting material. The following modification gave good yields (75 per cent) with up to 200 gm. of starting material.

156 gm. (1.05 M) of L-glutamic acid were shaken 5 minutes with 1500 cc. of absolute ethanol containing 94 gm. (2.58 -M) of dry HCI. The solution was concentrated in uacuo at 40” to a white solid. The solid was suspended in 300 cc. of hot absolute ethanol, shaken for 5 minutes, filtered rapidly by suction, and 1500 cc. of absolute ether were immediately added to precipitate the y-ethyl glutamate hydrochloride. After standing in the refrigerator overnight, the product was filtered, washed with absolute ether, and dried over 1’206, soda lime, and paraffin in a vacuum desiccator; yield 166 gm., 74 per cent of theory; m.p. 129-132”. After recrystallization from alcohol-ether, m.p. 134-135”.

Carboallyloxy y-Ethyl Glutamate (IIf)-This was prepared by the acylation of y-ethyl glutamate hydrochloride with ally1 chloroformate (Hooker Electrochemical Company, Xagara Falls, New York) under Schotten- Raumann conditions. This derivative proved superior to the carbobenz- oxy derivative for the preparation of the N-carboxy anhydride.

A solut,ion of 163 gm. (0.77 M) of y-ethyl glutamate hydrochloride in 800 cc. of water was brought to pH 5.0 with 4 N NaOH. 67 gm. (0.80 M) of KaHC03 and 41 gm. (1.0 M) of MgO were added, the mixture placed in an ice bath and stirred with a mechanical stirrer, and 130 gm. (1.1 M) of ally1 chloroformate were added dropwise over a period of 15 minutes. After standing for 1 hour in an ice bath and 3 hour at room temperature, the excess MgO was filtered off by suction and the filt.rate extracted with ether to remove excess ally1 chloroformate. The aqueous solution was made acid to Congo red with 5 N H&XI4 and the product separated out as an oil. This was extracted with ether, and the ether extracts were washed with water and dried with MgSOr. The ether solution was filtered and

1 All melting points are uncorrected.


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concentrated in vanu, to a thick syrup, redissolved in absolute ether, and concentrated to a clear colorless syrup with the careful exclusion of moisture. Yield 150.5 gm., 76 per cent of theory. The product was used as such for the next step.

N-Carboxy y-Ethyl Glutamate Anhydride (V)-This was prepared from carboallyloxy y-ethyl glutamate via the acid chloride (IV) which was not isolated.

144.7 gm. (0.56 M) of carboallyloxy -y-ethyl g1utamat.e were dissolved in 600 cc. absolute ether and placed in an ice bath, and 125 gm. (0.60 M) of PC16 were added with shaking. The solution was shaken intermittently for 14 hours in an ice bath and for 1 hour at room temperature, the excess PC16 filtered off, and the ether solution concentrated in vucuo to a syrup. The syrup was taken up in anhydrous benzene and concentrated in vaeuo at 4045” to a white solid. The product was crushed to a fine powder in Skellysolve A to remove acid chlorides, filtered, and recrystallized from benzene-Skellysolve A. Yield 108 gm., 96 per cent of theory. After two recrystallizations the white crystalline product melted at 72-73” with evolu- tion of carbon dioxide and gave a negative chloride test with silver nitrate and a precipitate of BaCO3 with aqueous Ra(O11)2.

Polymerization of N-Carboxy y-Ethyl Glutamute Anhydride-12.004 gm. (59.8 mM) of N-carboxy y-ethyl glutamate anhydride were dissolved in carefully dried and purified dioxane and placed in a round bottom flask equipped with a Hershberg stirrer and a drying tube to maintain anhydrous conditions. 0.598 mM of anhydrous ammonia in dioxane was added, making the total volume of the reaction mixture 120 cc. The ratio of anhydride to initiator was 100. The solution became turbid after 10 minutes and became very viscous as the reaction proceeded. After 20 hours, 125 cc. of dilute acid were added to the viscous translucent reaction mixture to decompose any unchanged anhydride. A white precipitate of y-ethyl-L-polyglutamate immediately formed. The reaction mixture was stirred for 4 hours. The white solid was centrifuged, suspended in water, and recentrifugcd several times. The product was lyophilized, to give 9.3 gm. of white fluffy solid, 99 per cent of theory. An average chain length

of 121 units was calculated from a-amino N analysis.

(C,HAN),-,~I. Calculated, C 53.5, H 7.0; found, C 53.7, H 7.0

Poly-a-L-Glutamic Acid-100 cc. of 0.74 N alcoholic KOH were added to 4 gm. of r-ethyl-r,-polyglutamate and the mixture stirred rapidly. White granules of potassium polyglutamato were formed within a few minutes. .4fter 100 minutes 100 cc. of water were added to the suspension to give a clear solution of potassium polyglutamate. After 45 minutes of stirring, the alkaline solution was made acid t.o Congo red with concentrated HCI


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M. GREEN AND hf. A. STAHMANN 779

and concentrated in vacua at 40” to about 60 cc. The white precipitate of polyglutamic acid after standing in the refrigerator for 4 hours was centrifuged, washed several times with water, and dialyzed against distilled water in the cold until free of chloride. The product was then lyophilized to a fine white powder. Yield 3.1 gm., 94 per cent of theory. Micro- scopic observation revealed the product to be a cluster of regularly shaped spheres. The polypeptide gave a positive biuret test. The neutral equivalent was determined by dissolving a sample of the polypeptide in excess standard alkali and back-titrating with standard HCl; a neutral equivalent of 130 was found, theory 129.

Wd%ON.-m. Calculated, C 46.5, H 5.5; found, C 46.5, H 5.7

End-Group Analysis-To determine the chain length, samples of r-ethyl polyglutamate were dried to constant weight at 57” over I’206 and analyzed for a-amino N by means of the reaction with nitrous acid in the auxiliary chamber of the manometric Van Slyke apparatus (16). Although-about 90 per cent of the nitrogen was liberated in the first 5 minutes, the gas was collected and measured at intervals until nitrogen liberation had ceased. The chain length could be calculated from the ratio of total N to or-amino N.

Optical Rot&m oj Glutumic Acid PolypeptidesThe optical rotations of glutamic acid polypeptides of chain lengths 37,80, and 121 were determined in 4 N alkali. The samples were made up to 5 cc. with alkali and the rotations read after 15 minutes in a 2 dm. tube on the Schmidt and Haensch polarimeter. The results indicated in Table IV show that there is relatively little change in specific rotation with a large change in molecular weight.

Racemization Rate of Glutamic Acid Polypeptides-0.2602 gm. of polyglutamic acid (mol. wt. 10,300) was made up to 10 cc. volume with 0.73 N

KOH. Optical rotations were read periodically and the specific rotation calculated. The extent of hydrolysis was determined by Van Slyke cr-amino N analysis.

Optical Purity of Glutumic Acid PolypeptidesSamples of polyglutamic ester, polyglutamic acid, and L-glutamic acid were autoclaved for 5 hours in 12 N HCl in sealed test-tubes. The resulting hydrolysates were diluted to 6 N acid concentration and the optical rotation determined. Total nitrogen (Kjeldahl) and a-amino nitrogen (Van Slyke) analyses indicated complete hydrolysis. Long periods in the autoclave resulted in increased racemization of all three samples.

Enzymatic Hydrolysis of Polyglutamic Acid-Weighed amounts of polyglutamic acid were dissolved in an equivalent amount of alkali, adjusted to pH, buffer and enzyme added, and the solution made up to volume with buffer and placed in an incubator at 37”. Aliquots were taken at suitable


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780 OLUTAMIC ACID POLYPEPTIDES

intervals and analyzed for a-amino nitrogen with the Van Slyke microvolumetric apparatus. Control runs were made with no substrate and with no enzyme.

The pancreas extract was prepared by shaking 500 mg. of desiccated and dehydrated pancreas powder (VioBin Corporation, Monticello, Illi- nois) with 20 cc. of water for 3 hour and filtering off the suspended material by suction.

Crystalline trypsin, chymotrypsin, and pepsin (Armour and Company, Chicago, Illinois) were dissolved directly in buffer for use.

The papain solution was prepared by shaking 10 gm. of papain with 25 cc. of 0.2 M acetate buffer, pH 5.0, for 1 hour and filtering off the undissolved material by suction. The enzyme concentration was 14.9 mg. of papain nitrogen per cc. of test solution; substrate concentration, 1.7 mg. of poly-

TABLE IV

Optical Rotations of Glutamic Acid Polypeptidea in 4 N Alkali

Rotation

SXllple Weight .~.-

Observed specilic, [ml; ~- -

chain length gm. gcr 100 cc. degrees degrees

37 2.014 -1.84 -45.7 80 1.962 -1.83 -46.7

121 1.906 -1.93 -50.6 - -. .~

peptide nitrogen per cc. of test solution; concentration of sodium thio- glycolate activator, 9 mg. per cc. of test solution. J Carboxypeptidase crystallized three times (Worthington Biochemical Sales Company, Freehold, New Jersey) was dissolved (or suspended) in buffer for use. Carbobenzoxyglycyl-L-tryptophan was prepared according to the method of Smith (17).

DISCUSSION

L-Glutamic acid polypept.ides of different average molecular weights were synthesized by the polymerization of N-carboxy y-ethyl-L-glutamate anhydride with varying amounts of ammonia as the initiator and subsequent saponification of y-ethyl-L-polyglutamate to remove the ester groups. The slow rate of racemization of poly-L-glutamic acid in alkali indicated that racemization occurring during saponification was insignificant. The optical activity of the polypeptide hydrolysates demonstrated that there was no appreciable racemization during this synthesis. In contrast, Hanby et al. (9) obtained glutamic acid polypeptides which were extensively racemized and in addition contained residual y-ester groups. Coleman (10) noted irreversible gel formation in his preparations of polyglutamic acid.


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M. GREEN AND M. A. STAHMANN 781

We have also observed insoluble gel formation in preparations which were not rapidly prepared and isolated under mild conditions. It seems that the rapid polymerization and the mild conditions used in the isolation of the products avoid complications cncount.cred by other workers.

Studies of the enzymatic hydrolysis of poly-L-glutamic acid by a pancreas extract and also by crystalline pancreatic enzymes revealed its susceptibility to the action of the crude extract as well as crystalline carboxypeptidase. Pancreas extract hydrolyzed polyglutamic acid to the extent of 24 per cent at pH 6.0 and 68 per cent at pH 5.0. No hydrolysis was detected at pH 4.0, owing probably to the insolubility of polyglutamic acid at this low pII. Crystalline carboxypeptidase produced 50 per cent hydrolysis at pH 5.0, no such hydrolysis being observed at pH 7.5. Furthermore the concentration of carboxypeptidase required for 50 per cent hydrolysis was 0.46 mg. of N per cc. as compared to a concentration of only 0.56 mg. of N per cc. of pancreas extract necessary to produce 68 per cent hydrolysis. This would suggest that the hydrolysis produced by the crude extract was not due entirely to its carboxypeptidase content. Since poly-cglutamic acid was not split by trypsin or by chymotrypsin, it. would appear that the hydrolysis by the pancreas extract was produced by enzymes other than the three known crystalline pancreatic proteolytic enzymes. Poly-L-glutamic acid was not hydrolyzed by crystalline pepsin; it was split by papain.

It was surprising that crystalline carboxypeptidase should hydrolyze poly-L-glutamic acid at pH 5.0 but not at pH 7.5, which is near the reported optimum for this enzyme (18). It was shown that polyglutamic acid did not affect the hydrolysis of carbobenzoxyglycyl-L-tryptophan by carboxypept,idase at pH 7.5. Therefore it must be concluded that the failure of carboxypeptidase to act on polyglutamic acid at pH 7.5 was not due to an inactivation of the enzyme. It is suggested that at pH 7.5 the clcctrostatic repulsion of the negatively charged carboxypeptidase by the highly negatively charged glutamic acid polypeptide prevents enzyme- substrate interaction. This view is supported by the observation that the introduction of a glut,amyl residue for glycine or alanine in the substrates for carboxypeptidase markedly decreases the hydrolysis rate (18). This decrease has been attributed to the retarding influence of the negatively charged y-carboxyl group (18). Carboxypeptidase is isoelectric at pH 5.95 (19); therefore at pH 5 it carries a net positive charge which permits interaction with t.he negatively charged poly-L-glutamic acid and thus facilitates enzymatic hydrolysis.

Our thanks are due to Dr. R. R. Becker for generous advice and to Mrs. 15. C. Wood for technical assistance in the preparation of y-ethyl glutamate hydrochloride and carboallyloxy y-ethyl glutamate.


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SUMMARY

A method is described for the synthesis of L-glutamic acid polypeptides of varying molecular weights composed of cY-peptide linkages. The synthesis involves the preparation of t,he N-carboxy anhydride of r-ethyl glutamate and the controlled polymerization of the anhydride monomer to yield y-ethyl polyglutamate, which was saponified to free polyglutamic acid. The product obtained was not appreciably racemized by this procedure. Molecular weights ranging from 4700 to 15,600 were obtained.

Polyglutamic acid was hydrolyzed by pancreas extract, crystalline carboxypeptidase, and papain. Crystalline pepsin, trypsin, and chymotrypsin had no hydrolytic action on the polypeptide.

BIBLIOORAPRY

1. Becker, R. R., and Stahmann, M. A., J. Am. Chem. Sot., 74,38 (1952). 2. Stahmann, M. A., Graf, L. H., Patterson, E. L., Walker, J. C., and Watson,

D. W., J. Biol. Chem., 189, 45 (1951). 3. Rubini, J. R., Rasmussen, A. F., Jr., and Stahmann, M. A., Proc. Sot. Ezp. Biol.

and Med., 78, 662 (1961). 4. Burger, W. C., and Stahmann, M. A., Arch. Biochem. and Biophys., in press. 5. Burger, W. C., and Stahmann, M. A., J. Biol. Chem., 198, 13 (1951). 6. Rubini, J. R., Stahmann, M. A., and Rasmussen, A. F., Jr., Proc. Sot. Exp. Riol.

and Med., 78, 659 (1951). 7. De Vries, A., Schwager, A., and Katchalski, E., B&hem. J., 49. 10 (1951). 8. Hanby, W. E., Waley, S. G., and Watson, J., Nature, 181, 132 (1948). 9. Hanby, W. E., Waley, S. G., and Watson, J., J. Chem. Sot., 3239 (1950).

10. Coleman, D., J. Chem. Sot., 2294 (1951). 11. Cleaves, D. W., J. Biol. Chem., 188, 163 (1950). 12. Levene, P. A., and Bass, L. W., J. Biol. Chem., 8a, 171 (1929). 13. Go, V., and Tani, H., Bull. Chem. Sot. Japan, 14,510 (1939). 14. Katchalski, E., Advances in Protein Chem., 8, 160 (1951). 15. Bergmann, M., and Zervas, L., Z. physiol. Chem., 221, 51 (1933). 16. Doherty, D. C., and Ogg, C. L., Znd. and Eng. Chem., Anal. Ed., 16. 751 (1943). 17. Smith, E. L., J. Biol. Ckm., 176, 39 (1948). 18. Neurath, H., and Schwert, G. W., Chem. Rev., 48, 69 (1950). 19. Putnam, F. W., and Neurath, H., J. Biol. Chem., 188, 603 (1946).


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Maurice Green and Mark A. StahmannPOLYPEPTIDES

HYDROLYSIS OF GLUTAMIC ACID SYNTHESIS AND ENZYMATIC

1952, 197:771-782.J. Biol. Chem.

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synthesis and enzymatic hydrolysis of glutamic acid ... · boxy y-ethyl-l-glutamate anhydride which...

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