PREPARATION AND ENZYMATIC HYDROLYSIS OF POLY-y-ETHYLGLUTAMYL BOVINE

PLASMA ALBUMIN*

BY MAURICE GREEN+ AND MARK A. STAHMANN

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

(Received for publication, May 17, 1954)

One of the most interesting methods for modifying proteins, which has only recently been discovered, is the addition through peptide bond forma- tion of amino acids and peptides to native proteins by reaction with N- carboxyamino acid anhydrides. This was introduced by Stahmann and Becker (1, 2) who allowed buffered aqueous solutions of bovine plasma albumin and chymotrypsin to react with N-carboxyglycine anhydride, whereby the molecular weights of both proteins were increased by about 12 per cent. It was surprising that, despite the large increase in glycine content, there was no appreciable change in several of the physiological properties of the modified proteins; the chymotrypsin derivative still re- tained enzymatic activity, and the polyglycyl albumin was precipitated by an antiserum prepared against normal plasma albumin.

Since the properties of proteins are known to depend to a large extent upon polar groups in the molecule provided by acidic and basic amino acids, it was of interest to study the addition of glutamic acid peptides to a protein. In the work reported here, a-bonded glutamic acid peptides containing esterified y-carboxyl groups were attached to bovine plasma albumin by means of peptide bonds formed upon reaction of the protein with the N-carboxy anhydride of r-ethyl glutamate, and some of the phys- ical and chemical properties of the resulting modified protein were studied. The susceptibility to the action of several proteolytic enzymes of this modi- fied protein, as well as polyglycyl albumin, was also investigated.

EXPERIMENTAL

Preparation of Poly-y-ethyl+glutamyl Bovine Plasma Albumin-5 gm. of crystalline bovine plasma albumin* (BPA) were dissolved in 500 ml. of

* Published with the approval of the Director of the Wisconsin Agricultural Experiment Stat,ion. Supported in part by a research grant (No. E-101) from the National Microbiological Institute of the National Institutes of Health, United States Public Health Service.

t Present address, Research Department, the Children’s llospital of I’hil:rtlelphia, Philadelphia, Pennsylvania.

1 Purchased from Armour and Company, Chicago, Illinois.

259

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260 POLY-“/-ETHYLGLUTAMYL PLASMA ALBUMIN

~1/13 phosphate buffer, pH 7.3, and cooled to 3”. 5 gm. of N-carboxy-y- et’hyl-L-glutamate anhydride (3) were added with stirring, the solution becoming turbid and carbon dioxide being released. The reaction mixture was shaken for 4 hours at room temperature and kept at 3” for 20 addi- tional hours. The turbid reaction mixture was centrifuged at 17,000 X g for 30 minutes to remove insoluble polymerization products and then dialyzed with stirring against several changes of distilled water at 3” for 4 days to remove r-ethyl glutamate and soluble peptides. The poly-y-ethyl- glutamyl BPA was recovered from the dialysate by lyophilization to yield 5.0 gm. of white, fluffy solid. Control experiments were made in which no anhydride was added and also in which the anhydride was polymerized in buffer and t,hen mixed mit.h BPA.

Glutamic Acid Analysis-The amount of r-ethyl glutamate added to BPA was determined by analysis of the protein hydrolysate for the in- creased glutamic acid content with a glutamic acid decarboxylase prepara- tion. The COp released from the glutamic acid was measured in a War- burg respirometer. The enzyme was prepared from Escherichia c&i, NCTC strain 4157, as described by Umbreit and Gunsalus (4). All analyses were computed on a dry weight basis (constant weight at 110”) and a molecular weight of BI’A of 69,000. Appropriate cwrrect,ions were npplicd for low recovery from glutamic acid (‘ontrols (86 to 89 per cent) and highcr t,han theoretical amount’s of CO, evolved from 13PA hydrolysates (114 to 119 per cent).

Alkaline Saponification of E’olll-r-ethylglu,tam~~l Alb?lmtn-Poly--y-cthyl- glutamyl BPA n-as treated with dilute alkali under nitrogen for difl’erentj periods of time. All reagents employed were CO%-free. The number of ester groups saponified as revealed by alkali consumption was determined by back-titrating the alkaline reaction mixture of poly-r-ethylglutamyl BPA under nitrogen with HCl to pH 6.0 by using an ultramicro burette and a Beckman model G pH meter equipped with a glass elect,rode from pH 0 to 11. The extent of ester hydrolysis was calculated from the differ- ence between the amounts of acid required t’o back-titrate poly-y-ethyl- glutamyl BPA and native BPh.

Electrophorctic LAnal?Jsis2-This was performed on the Spinco model H electrophoresis-diffusion apparatus equipped with a Tiselius cell of 11 ml. capacity in a bath ah 1”.

ICnzymafic ITj/&ol!Jsis oj Jfotlified Proteins-Appropriate buffered vol- umes of freshly prepared solutions of protein and enzyme were mixed and incubated at 37”. Suitable aliquots were removed at zero time and at, convenient intervals and analyzed to determine the extent of hydrolysis. Control experiments were included with no substrat,e and with no enzyme.

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M. GREEN AND M. A. STAHMANN 261

Papain and pancreas extract solutions were prepared as previously de- scribed (3). Crystalline trypsin,’ chymotrypsiql and pepsin’ were dis- solved in buffer for use. The crude peptidase preparation consisted of a glycerol ext,ract of hog intestinal mucosa prepared according to Johnson et al. (5).

The polyglycyl BPA used in the enzyme studies was kindly supplied by Dr. R. R. Becker. The molecular weight of this preparation was calcu- lated to be 81,000 from microbiological assay for the glycine content of the protein and a molecular weight of 69,000 for native BPA.

Triglycine was kindly provided by Dr. M. J. Johnson. The ninhydrin color yield of the tripeptide on a molar basis relative to leucine was found to be 0.92. The color yields of diglycine and glycine have been reported as 0.89 and 1.01, respectively (6).

The ninhydrin color procedure (6) for amino nitrogen was used in follow- ing the hydrolysis of the modified proteins by trypsin, chymotrypsin, pep- sin, and the peptidase preparation. This method has the advantage of great sensitivity which is useful in following hydrolysis in small samples. Although the ninhydrin color yields given by free amino acids may not be the same as when these amino acids are situated at the amino elld of pep- tide chains, such as in an enzymat’ic hydrolysate, a useful comparison can be made of t,he colors given by the enzymatic hydrolysates obtained by the action of an enzyme on the native and modified proteins. In soveral cases (7, 8), results obtained by the ninhydrill color procedure on proteins or protein hydrolysates were shown to be in agreement with the results of a-amino nitrogen analysis. a-Amino nitrogen analysis was used here in several experiments to verify further the results obt,ained with the nin- hydrin procedure. Good agreement was found between the t8mo methods for the per cent reduction in the hydrolysis of poly-y-ethylglutamyl BPA as compared to unmodified BPA by trypsin and chymotrypsin.

Triplicate 0.100 ml. aliquots of buffer, stsndard leucine, native BPA in- cubation mixture, and modified BPA incubation mixtures were pipetted into a series of photometer tubes containing 1 ml. of ninhydrin reagent, the color was developed as described by Moore and Stein (B), and readings were taken on the Beckman model R spectrophotometer at 570 rnp. The re- sults were expressed in terms of the equivalent,s of leucine standard cor- responding t#o the given caolor value. A straight line relationship was found bet,meen opt’ical density and standard leucine concentration under the condition employed up to 12.2 mmoles of leucine.

Synthesis-Poly-y-ethyl-L-glutamyl bovine plasma albumin was prepared by treatment of BPA with an equal weight of y-ethyl-L-glutamate anhy- dride in phosphate buffer at pH 7.3. As coalculated from the amount of

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262 POLY-‘y-ETHYLGLUTAMYL PLASMA ALBUMIN

r-ethyl glutamate added to BPA (Table I), the glutamic acid content of the protein was increased by 110 per cent, and the molecular weight of the protein increased by 19.5 per cent. The glutamic acid content of the dialyzed poly-y-ethylglutamyl BPA was not decreased by exhaustive elec- trodialysis, thus providing evidence that the amino acid was chemically attached and not just reversibly bound to the protein.

Treatment with Alkali-Preliminary experiments were made treating polyy-ethylglutamyl BPA and native BPA with dilute alkali for different periods of time in order to investigate conditions for the hydrolysis of the y-ethyl ester groups to free carboxyl groups. The number of free carboxyl groups formed and the solubility of the resulting protein derivative between pH 2 and 11 are summarized in Table II. It is seen that treatment with

TABLE I

Addition of y-Ethyl Glutamate to BPA

Protein

y-Ethyl glutamate added per 100 gm. protein

Mol. wt. Gl;;rm$o~id

Added, +-;

protein P mo e protein

gm. moles WWkS

BPA 69,000 77* Poly-7.ethylglutamyl BPA.. 19.5 82,500t 163 86

“ “ $. 19.5 82,500t 163 86

* Calculated from data of Stein and Moore (9). t Calculated from the increase in r-ethyl glutamate content. $ This preparation, in addition to being dialyzed, was exhaustively electrodi-

alyzed.

0.05 N NaOH resulted in a maximum of 80 per cent hydrolysis of the ester groups in 1 to 2 hours.

Native BPA and poly-y-ethylglutamyl BPA were completely soluble from pH 2 to 11 (Table II) under the conditions tested. Treatment of native BPA with 0.05 N NaOH for 1 or 2 hours did not convert it to an insoluble protein. However, poly-y-ethylglutamyl BPA, upon treatment with 0.05 N NaOH for as little as 30 minutes, became insoluble in the pH region 5 to 2.5, which encompasses the estimated isoelectric region of the new modified protein (10). It cannot be said whether this decreased solu- bility is due to a change in the parent structure caused by the alkali or to the large increase in the number of charged y-carboxyl groups on the pro- tein molecule. In this respect, it is pointed out that chemical modifica- tion of proteins by reaction with organic compounds under mild conditions may produce changes in the physical properties of the protein without denaturation (11).

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M. GREEN AND M. A. STAHMANN 263

Electrophoretic Analysis-Electrophoresis studies were made on poly-y- ethylglutamyl BPA and its alkali-treated product. The electrophoretic patterns of the protein derivatives are shown in Fig. 1 and the correspond- ing calculated mobilities are given in Table III. Poly-y-ethylglutamyl BPA showed a single peak with a 9 per cent increase in mobility over BPA in Verona1 buffer at pH 8.6. An increase in mobility would be expected,

TABLE II

Treatment of BPA and Poly-y-ethylglutamyl with Alkali

Protein Treatment*

BPA “ “ ‘I

Poly-r-ethylglu- tamyl BPA

‘I “

‘I “

‘I I‘

“ “

‘I “

No treatment 0.05 N NaOH, 1 hr. 0.05 “ “ 2 hrs. 0.1 “ “ 19 “ $

No treatment

0.05 N NaOH, 30 min.

0.05 ‘I “ 1 hr.

0.05 “ “ 2 hrs.

0.05 I‘ “ 4 “

0.1 ‘( “ 22 “ 8

1

I -

No. free carboxyl

i%%%d

P er

no ecule

37 45

50 60

67 80

66 80

81 95

Per cent ester

1 hydro- lysis

Solubilityt after treatment, pH

range 2-l 1

Soluble “ “

Insoluble, pH 5.0-3.3

Soluble

Insoluble, pll 4.5-3.0

Insoluble, pII 4.G2.7

Insoluble, pH 4.7-2.7

Insoluble, pH 4.7-2.5

Insoluble, pH 4.9-2.0

* Room temperature (25-28”) unless otherwise specified. t Protein concentration approximately 0.6 per cent in about 0.15 M NaCl solution. $ Treatment at 3”. Q Treatment at 3” for 19 hours and then at 23” for 3 hours.

since, in the formation of poly-y-ethylglutamyl BPA, E-amino groups of lysine (pK = 9.4 (13)) in BPA are acylated by reaction with N-carboxy -y-ethyl glutamate anhydride and replaced by the terminal a-amino groups (pK = 8.0 (13)) of the added glutamic acid peptides. The result of this exchange of e-amino for a-amino groups is a decrease in positive charge and thus an increase in mobility of the negatively charged protein at pH 8.6.

The electrophoretic pattern of alkali-treated poly-r-ethylglutamyl BPA showed a slight shoulder and revealed an increase in mobility of 1.8 X lo+

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264 POLY-‘y-ETHYLGLUTAMYL PLASMA ALBUMIN

mobility unit wit)h respect to untreated poly-y-ethylglutamyl BPA (a 25 per cent increase in mobility). Since this preparation contained 60 newly formed completely ionized r-carboxyl groups (at pH 8.6), it was of interest

FIG. 1. Electrophoretic patterns in descending limb at pF1 8.6 in Verona1 buffer, 0.1 ionic strength. A, poly--,-ethylglutamyl BPA at 131 minutes. B, poly-r- ethylglutamyl BPA (treated with 0.05 N NaOH for 1 hour) at 119 minutes. C, BPA (treated with 0.05 N NaOH for 1 hour) at 199 minutes.

TABLE III

~llobilities of BPA Derivatives at pH 8.6 in Verona1 Buffer, 0.1 Ionic Strength

j hhbility X lo-” cm.2 per

volt per sec. ir desirer

BPA Poly-r-ethylglutamyl BPA (Fig. 1, A)

“ “ 1 hr. in 0.05 N SaOH$ (Fig. 1, B)

BPA, 1 hr. in 0.05 N NaOH (Fig. 1, C)

-6.501 -7.07 -8.83

-5.40 -6.70

* Compared with unmodified 13PA. t From data of Albertp (12). $ Contained 60 new -y-carboxyl groups.

3ange in mobility*

-

+9 +36

-17

$3

Remarks

Single peak ‘< ‘I

Slight shoulder

Main component 10% of total

__~~- -

to calculate the increase in mobility that would be expected from such a large increase in negative charge. On employing the value of 0.2 X 10e5 cm.2 per volt per second per charge for bovine plasma albumin (lo), one would expect from 60 added carboxyl groups an increase in mobility of 60 X 0.2 X 1O-5 = 12 X 10e5 cm.2 per volt per second. An increase of only 1.8 X 10e5 was observed, which is only 15 per cent of that calculated above.

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M. GREEN AND M. A. STAHMANN 265

Native BPA, after treatment with alkali, showed two components upon electrophoretic analysis. The main component had a 17 per cent decrease in mobility and may represent a soluble, alkali-denatured form of the pro- tein. The small amount of faster moving component (about 10 per cent) had about the same mobility as native BPA and may be a small residue of undenatured BPA.

Enzymatic Hydrolysis of Modijied Proteins-The modified proteins used in a study of their susceptibility to the action of several proteolytic en-

TABLE IV Hydrolysis of Poly-r-ethylglutamyl and Polyglycyl BPA by Papain

and Pancreas Extract Incubation mixtures were made up in 0.2 M acetate buffer, pH 5.0, and contained

9 mg. of protein substrate per ml. and 10 per cent of the enzyme extract. Papain mixtures contained 9 mg. of sodium thioglycolate activator per ml. The extent of hydrolysis was determined by a-amino nitrogen analysis on 2.0 ml. aliquots of the reaction mixture.

Substrnte Increase in a-amino N,

I pmoles a-NH%-N per Per cent reduction mg. substrate in hydrolysis

Papain, 26 hrs.

BPA 4.37 Poly-r-ethylglutamyl BPA. 3.31 Polyglycyl BPA. 3.43

Pancreas extract, 39 hrs.

25 20

BPA Poly-7.ethylglutamyl BPA.. Polyglycyl BPA.

zymes were poly-y-ethylglutamyl BPA of molecular weight 82,500 and polyglycyl BPA of molecular weight 81,000. The proteolytic enzyme preparations employed include papain, pancreas extract, a crude peptidase preparation, and crystalline trypsin, chymotrypsin, and pepsin.

Table IV illustrates the hydrolysis of modified proteins by papain and pancreas extract. With both enzyme preparations, there is a decrease in the extent of hydrolysis of 20 t,o 25 per cent for both modified proteins as compared to nat’ive BPA. Since these enzyme preparations contain vari- ous proteolytic enzymes of different specificities, it is likely that the glu- tamic acid and glycine residues attached to the protein were hydrolyzed to some extent. In this respect, it is pointed out that polyglutamic acid is hydrolyzed by both pancreas extract and papain (3).

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266 POLY--f-ETHYLGLUTAMYL PLASMA ALBUMIN

TABLE V

liydrolysis of Poly-r-ethylglutamyl and Polyglycyl BPA by Crystalline Trypsin, Chymotrypsin, and Pepsin

Trypsin and chymotrypsin incubation mixtures were prepared in ~/15 phosphate buffer, pH 7.3, and contained 8 mg. of protein substrate per ml. Trypsin reaction mixtures had 0.05 mg. of enzyme per ml. and chymotrypsin mixtures 0.03 mg. per ml. Pepsin reaction mixtures consisted of 8 mg. of protein substrate and 0.18 mg. of pep- sin per ml. of 0.2 M phosphate buffer, pH 2.0. Hydrolysis determined by photo- metric ninhydrin analysis.

Substrate Increase in ninhydrin

color,’ pm&s per mg. substrate

Trypsin, 17 hrs.

Per cent reduction in hydrolysis

BPA ....................................... Poly-y-ethylglutamyl BPA .................. Polyglycyl BPA. ...........................

0.429 0.170 60 0.172 60

Chymotrypsin, 23 hrs.

BPA Polyq-ethylglutamyl BPA.. Polyglycyl BPA. . .

~

Pepsin, 24 hrs.

BPA ....................................... Poly-y-ethylglutamyl BPA .................. Polyglycyl BPA. ...........................

* Ninhydrin color expressed in terms of micromoles of leucine standard.

TABLE VI

Effect of Crude Peptidase Preparation on Poly-r-ethylglutamyl and Polyglycyl BPA

Incubation mixtures contained 0.3 mg. of triglycine per ml. or 4 mg. of protein substrate per ml. and 20 per cent peptidase preparation in ~/15 phosphate buffer, pH 8.0. Hydrolysis determined by photometric ninhydrin analysis.

Substrate Time Per cent hydrolysis

hrs.

Triglycine....... 1 140* “ . . . . . . . . . . . . . . . . . . . . . .

BPA. :::::::::: 16 200* 24 0

Poly-r-ethylglutamyl BPA.. 24 0 Polyglycyl BPA. 24 0

* Complete hydrolysis of the two peptide bonds is 200 per cent.

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M. GREEN AND M. A. STAHMANN 267

Table V presents data obtained on the hydrolysis of modified proteins by the crystalline enzymes trypsin, chymotrypsin, and pepsin. With tryp- sin and chymotrypsin there is a decrease of 55 to 70 per cent in the extent of hydrolysis of both modified proteins as compared to unmodified BPA. However, with pepsin, there appears to be the same degree of hydrolysis of modified albumins and native BPA.

Table VI summarizes the results of experiments on the susceptibility of the modified albumins to the action of a crude peptidase preparation ob- tained from hog intestinal mucosa. Triglycine was hydrolyzed completely to free glycine, indicating that the enzyme preparation had high tripepti- dase and dipeptidase activity for glycine peptides. However, no hydroly- sis was observed on polyglycyl BPA, poly-y-ethylglutamyl BPA, or un- modified BPA. The lack of hydrolysis of polyglycyl BPA discloses that glycine peptides when attached to BPA are not split by this active pep- tidase preparation.

DISCUSSION

Becker and Stahmann (2) demonstrated that, in their preparation of polyglycyl albumin which contained 171 moles of added glycine per mole of protein, one-third of the free amino groups of albumin had been acylated. This indicated that the glycine was attached in the form of peptides having an average length of 7 residues, assuming that only the amino groups of the protein had reacted. The preparation of polyglycyl albumin employed in the enzymatic studies was prepared under similar conditions except that a large ratio of anhydride to protein was used, thus resulting in more gly- tine being added to the albumin. The reaction of bovine plasma albumin with an equal weight of N-carboxy y-ethyl glutamate resulted in the addi- tion of 86 moles of y-ethyl glutamate per mole of albumin. If it is assumed that only amino groups of the protein have reacted and that one-third of these has been acylated, it can then be calculated that the glutamic acid is attached on the average as tetrapeptides. Although there is sufficient glutamic acid added (86 moles per mole of protein) to react with all the free amino groups of the protein, it is unlikely that each amino group is acylated because of the great reactivity of the anhydride molecule. It is more probable that the more sterically available amino groups are first acylated, the added r-ethyl glutamate residues being subsequently further acylated in a stepwise manner by anhydride resulting in an elongation of the peptide chain. The albumin molecule is thus modified by the attach- ment of a number of peptide chains at various loci.

Although it would not be anticipated that protein denaturation would occur during the mild conditions employed to prepare polyy-ethylglu- tamyl BPA, some of the physical properties of this derivative were ex-

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M. GREEN AND M. A. STAHMANN 269

BIBLIOGRAPHY

1. Stahmann, M. A., and Becker, R. R., J. Am. Chem. Sot., 74,2695 (1952). 2. Becker, R. R., and Stahmann, M. A., J. Biol. Chem., 204,745 (1953). 3. Green, M., and Stahmann, M. A., J. Biol. Chem., 197,771 (1952). 4. Umbreit, W. W., and Gunsalus, I. Cl., J. Biol. Chem., 169, 333 (1945). 5. Johnson, M. J., Johnson, G. H., and Peterson, W. H., J. Biol. Chem., 116, 515

(1936). 6. Moore, S., and Stein, W. H., J. BioZ. Chem., 176, 367 (1948). 7. Harding, V. J., and MacLean, R. M., J. BioZ. Chem., 24, 503 (1916). 8. Maurer, P. H., and Heidelberger, hl., J. Am. Chem. Sot., 73, 2070 (1951). 9. Stein, W. H., and Moore, S., J. BioZ. Chem., 176, 79 (1949).

10. Longsworth, L. G., and Jacobsen, C. F., J. Phys. and Colloid Chem., 63, 126 (1949).

11. Putnam, F. W., in Neurath, H., and Bailey, K., The proteins, New York, 1, pt. B, 807 (1953).

12. Alberty, R. A., J. Chem. Education, 26, 426 (1948). 13. Tanford, C., J. Am. Chem. SOL, 72, 441 (1950). 14. Levene, P. A., and Bass, I,. W., J. BioZ. Chem., 62, 171 (1929).

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Maurice Green and Mark A. StahmannALBUMIN

-ETHYLGLUTAMYL BOVINE PLASMAγHYDROLYSIS OF POLY-

PREPARATION AND ENZYMATIC

1955, 213:259-269.J. Biol. Chem. 

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