THE JOURNAL OF BIOLO~KAL CHEMISTRY Vol. 237, No. 2, February 1962

Printed in U.S.A.

The Complete Enzymic Hydrolysis of Proteins*

ROBERT L. HrLLt AND WILLIAM R. SCHMIDT~

From the Laboratory for the Study of Hereditary and Metabolic Disorders and the Departments of Biological Chemistry and Medicine, University of Utah College of Medicine, Salt Lake City, Utah

(Received for publication, May 19, 1961)

The strong acids that are required for complete hydrolysis of proteins destroy some amino acids, such as tryptophan, aspara- gine, and glutamine. Other amino acids are destroyed to a lesser extent or in certain cases are released incompletely from peptide linkage. It is evident that these difficulties, as well as others, could be overcome by employing proteolytic enzymes as the sole agents for hydrolysis.

The complete enzymic degradation of proteins was studied by several investigators in the late nineteenth and early twentieth century. Meissner (2), Kiihne and Chittenden (3, 4), Chitten- den and Goodwin (5), and Neumeister (6, 7) demonstrated that pepsin and trypsin hydrolyzed proteins to produce a complex mixture of peptides called proteoses and peptones. These were considered to be the ultimate end products of proteolysis until 1901, when Cohnheim (8) showed that intestinal extracts (erepsin) hydrolyzed peptones to amino acids. Subsequent studies, in particular those of Frankel (9), showed that the combined action of pepsin, trypsin, and erepsin resulted in cleavage of over 90% of the peptide bonds in several purified proteins.1 Although it was evident from these early investiga- tions that proteins could be degraded by enzymes to amino acids, later investigators did not develop a system for complete enzymic hydrolysis. This can be attributed to a variety of causes, but two major problems which only recently have been satisfactorily solved presented the most difficulty. Micro- methods for the precise analysis and characterization of amino acids and peptides were not available, and secondly, purified proteolytic enzymes were not characterized sufficiently to be used as hydrolytic agents.

With these considerations, we have developed methods that employ proteolytic enzymes for hydrolysis of proteins. Our studies demonstrate that digestion of a protein by papain (12), followed by treatment with the purified kidney peptidases, leucine aminopeptidase (13), and prolidase (14) results in es- sentially complete hydrolysis of all peptide bonds and gives high yields of tryptophan, glutamine, and asparagine.

* This investigation was aided by research grants from the National Institutes of Health, United States Public Health Serv- ice. A preliminary report of this work has been presented (1).

t Present address, Department of Biochemistry, Duke Uni- versity, Durham, North Carolina.

.t United States Public Health Service Student Summer Re- search Fellow, 1959. National Science Foundation Student Sum- mer Research Program, 1958.

1 Excellent reviews, which discuss the earlier research in this area, have been presented by Vickery and Osborne (10) and Mann (11).

EXPERIMENTAL PROCEDURE

Materials and Methods

Enzymes-Leucine aminopeptidase was prepared from swine kidney (15). Several precautions were observed in its use in addition to those already published (16, 17). Only electro- phoretically purified preparations with a C1 B 35 were em- ployed. Prior to use, all preparations were dialyzed at 5” for several hours against 100 volumes of 0.005 M Tris buffer, pH 8.5, containing 0.005 M MgCIs. Dialysis removed the small quanti- ties of amino acids that often contaminate preparations that have been stored frozen ( - 10’) and thawed several times.

Prolidase was purified from swine kidney by the method of Davis and Smith (14). It is conveniently prepared from the prolidase-rich fraction that is normally discarded in purification of leucine aminopeptidase (15). The preparations which are obtained at Step 2 (Cl = 10 to 20) are sufficiently pure for use in the experiments described here. Lyophilization of prolidase of this purity was omitted because of losses in activity. The enzyme is most stable at pH 6 to 8 in the presence of 0.01 M

MnClz and should be stored frozen ( -10”). Considerable quantities of protein often precipitate after freezing and thawing, and some losses of enzymic activity occur. The precipitates contain no prolidase activity and can be removed by centrifuga- tion. As noted previously, one-half of the prolidase activity in preparations of this purity is lost in approximately 1 month (14). Additional purification is unnecessary if all of the insoluble ma- terial is removed and the preparation is dialyzed before use.

Prolidase prepared in this manner contains other enzymic activities. Hydrolysis of prolylglycine2 and leucinamide was found, indicative of the presence of iminodipeptidase (18) and leucine aminopeptidase, respectively. These activities were less than 5% of the prolidase activity. No activity could be found toward cr-benzoylargininamide, N-acetyltyrosinamide, carbobenzoxyglycylphenylalanine, and carbobenzoxyglutamyl- tyrosine.

Crystalline papain or mercuripapain was prepared from dried papaya latex (19). Both forms behave identically when em- ployed for enzymic hydrolysis. Only preparations with a C1 = 1.2 were used. Bacterial proteinase (Nagarse) was a commerical preparation obtained from the Biddle-Sawyer Corporation. Crystalline trypsin and chymotrypsin were commercial prepara- tions (Worthington Biochemical Corporation).

Enzyme assays were performed by the titrimetric method of

2 All amino acids except glycine and all amino acid derivatives used in this study were the L isomers.

389

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390 Hydrolysis of Proteins Vol. 237, So. 2

Grassmann and Heyde (20). Specific activities (Ci) were calculated for each enzyme from the first order rate constants obtained with the appropriate substrate (15). Enzyme concen- trations were estimated by the method of Bticher (21).

Substrates-c+Corticotropin, obtained from bovine pituitary glands (22), was generously supplied by Dr. C. H. Li. We would like to thank Dr. B. G. Malmstriim for his gift of the crystallline, yeast enolase (23). Carboxypeptidase recrystallized five times (kindly supplied by Dr. E. L. Smith) was prepared from beef pancreas (24). Ribonuclease A was prepared from commercial beef pancreatic ribonuclease by the chromatographic method of Him, Moore, and Stein (25) and was oxidized with performic acid as previously described (26). Crystalline pepsin (kindly sup- plied by Dr. E. L. Smith) was prepared from hog stomachmucosa (27). Human fibrinogen was prepared from human plasma by the method of Cohn et al. (28) and was a gift from Dr. J. N. Ash- worth of the American Red Cross. Bovine fibrinogen was a gift from Mr. W. White of Armour Research Laboratories. Casein (Bacto-Difco) was not purified before use. We are grateful to Dr. H. 0. Tallan for a sample of synthetic tyrosine-O-sulfate.

GZycyZproZyZZeucine-Leucine ethyl ester HCl (3.9 g) in 50 ml of dry pyridine was treated with 0.87 ml of PC& at -10”. The mixture was warmed to 30” and allowed to stand for 20 minutes. Carbobenzoxyglycyl-L-proline (6.13 g) (29) was added and the mixture heated for 3 hours on a steam bath. When concentrated to dryness in a vacuum, a syrupy residue was obtained which was suspended in 10 ml of water and extracted five times with 50 ml of ethyl acetate. The extracts were combined, washed suc- cessively with 1 N HCl, 1 N NaOH, and water, and then dried over sodium sulfate. The ethyl acetate was removed by con- centration in a vacuum, and the resulting oil was dissolved in 110 ml of dioxane-water (50: 50) and saponified by addition of 12 ml

I I I I I 1

6 12 18 24 Time -hours

FIG. 1. Hydrolysis of heat-denatured papain by pepsin, papain, trypsin and chymotrypsin, and bacterial proteinase. Aliquots (2 ml) of a solution of papain containing 20.5 mg (1 pmole) were heated in a boiling water bath (94”) for 2 minutes. The solutions were cooled to 40”, and the appropriate enzymes, activators or buffers were added as follows. Pensin. 0.2 ml of 0.01 N HCl and 1 mg of pepsin. Papain, 0.3 ml of 6.1 N’NaCN (acidified to pH 7), 0.2 ml of sodium acetate buffer, pH 5.2, and 1 mg of papain. Tryp- sin and chymotrypsin, 0.2 ml of 0.5 M Tris buffer, pH 8, and 1 mg each of trypsin and chymotrypsin. Bacterial proteinuse, 0.1 ml of 0.1 M sodium phosphate buffer, pH 7.0, and 1 mg of proteinase. The final volume of each mixture was 2.5 ml. Aliquots of each digest, 0.01 ml, were removed at intervals and treated with nin- hydrin reagent, and the resulting absorbancy was measured at 570 rnp.

of 2 N NaOH. After 23 hours at 30”, the mixture was brought to pH 2 with 1 N HCl and concentrated to dryness. The re- sulting oil was extracted into ethyl acetate and the extracts dried over sodium sulfate. The extracts were concentrated to dryness in a vacuum, and the resulting residue was dissolved in 100 ml of methanol containing 0.5 ml of glacial acetic acid. One g of I’d0 was added, and the mixture was hydrogenated at atmos- pheric pressure for 1 hour at room temperature. The mixture was diluted with 100 ml of water and filtered, and the filtrate was concentrated to dryness in a vacuum. The resulting solid was extracted repeatedly with methanol. The residue was crystallized from water with ethanol (yield, 1.1 g; m.p., 18% 189” (decomposition point)).

C13H23NaOh (285.33) Calculated: C 54.7, H 8.1, N 14.7 Found : C 53.6, H 8.3, N 14.6 w0 - 127.1” (loj, in 0.01 N HCl)

Chromatography of the compound on Whatman No. 1 paper in butanol-acetic acid-water (200 : 30: 75) revealed a single ninhydrin-reactive spot, RF = 0.7. After hydrolysis for 24 hours in 6 N HCl at 110” under reduced pressure, 1 pmole of the compound yielded 0.98 pmole of glycine, 0.95 pmole of proline, and 1.05 pmolcs of leucine, as determined by quantitative amino acid analysis (30).

Analytical Methods-Ninhydrin determinations were per- formed by the method of Moore and Stein (31). Tyrosine-O- sulfate was estimated by the chromatographic method of Tallan et al. (32). Amino acid analyses of enzymic hydrolysates were made by the procedure of Moore and Stein (33), as modified by Kimmel and Smith (34), or by the method of Spackman, Stein, and Moore (30, 35) with the aid of the Spinco model MS amino acid analyzer. Qualitative analyses of amino acids and peptides were performed by unidimensional paper chromatography on Whatman 3MM paper with the propanol-pyrophosphate system of Dixon, Kauffman, and Neurath (36), or with n-butanol- acetic acid-water (200: 30 : 75). The combined electrophoretic chromatographic method of Ingram (37) was used for analysis of peptides as well as amino acids.

RESULTS

Action of Endopeptidases on Proteins-For complete enzymic hydrolysis an endopeptidase is required which will degrade a protein extensively into small peptides. Enzymes that are available include trypsin, chymotrypsin, papain, bacterial protease (subtilisin), and pepsin. The ability of each of these enzymes to hydrolyze heat-denatured crystalline papain (Fig. 1) demonstrates that papain is most effective. From the measure- ments of the increase in ninhydrin color, it is estimated that 65 to 68 bonds per mole of substrate were broken. A lesser number of bonds were hydrolyzed by the other enzymes, 50 with bac- terial proteinase, 25 with pepsin, and 25 by the combined action of trypsin and chymotrypsin3 Other proteins that were tested in a similar manner also were degraded to a greater extent by papain. These results are not unexpect.ed when the specificity of each enzyme is considered (38).

An additional means of judging the extent of hydrolysis is

3 When trypsin and chymotrypsin are used at higher concentra- tions, papain is hydrolyzed more extensively. Presumably, the peptides released from papain during the initial stages of diges- tion strongly inhibit further hydrolysis.

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February 1962 R. L. Hill and W. R. Schmidt 391

provided by comparing the peptide patterns of the above digests. Fifty-four ninhydrin-reactive spots were found in the papain hydrolysate, 42 in the bacterial proteinase hydrolysate, and only 24 in the peptic hydrolysate. Amino acids, such as aspartic acid, glutamic acid, arginine, lysine, glycine, leucine, and iso- leucine were present in the papain digests and, to a lesser extent, in the bacterial proteinase digest. No free amino acids were identified in the peptic digest.

Action of Leucine Aminopeptidase and Prolidase on Proline Peptides-It is apparent that all of the peptides that are produced by hydrolysis with papain can be degraded to amino acids by leucine aminopeptidase except those that contain the imino nitrogen of proline in peptide linkage, e.g. glycylproline. Pre- vious studies with synthetic substrates (39) showed that peptides of this type are resistant to the action of leucine aminopeptidase, although more recent studies (40, 41) on proteins and poly- peptides indicate that proline can be liberated from peptide linkage at high ratios of enzyme to substrate. In order to understand the behavior of leucine aminopeptidase toward peptides containing proline, several synthetic peptides were tested as substrates.

When the hydrolysis of glycylproline, glycylhydroxyproline, and phenylalanylhydroxyproline was measured by the Grass- mann-Heyde method, no detectable cleaving was found at low to moderately high concentrations of aminopeptidase. At high concentrations of enzyme, a small but significant rate of hy- drolysis was observed. Table I shows the rate of hydrolysis of glycylproline. The C1 for this substrate is 7.4 x 1O-4 whereas the same enzyme preparation has a C1 = 45 with leucinamide. If the rate for leucinamide is assigned a value of 100, then the relative rate of hydrolysis of glycylproline is 0.0016. Thus, L- leucinamide is hydrolyzed at a rate approximately 60,000 times greater than the rate of proline peptides. Glycylhydroxyproline and phenylalanylhydroxyproline were hydrolyzed at slower rates. Hydrolysis was demonstrated by the fact that hydroxyproline and glycine or phenylalanine were observed on paper chro- matographic analysis of the hydrolytic products.

In order to check whether the hydrolysis of proline peptides by leucine aminopeptidase was the result of contaminating amounts of prolidase, glycylprolylleucine was tested as a substrate for prolidase as well as for aminopeptidase. Prolidase should be unable to hydrolyze a peptide with this structure (14), and leucine aminopeptidase would cleave it no faster than the proline dipeptides discussed above. Table II shows the course of hydrolysis of glycylprolylleucine by prolidase, by aminopeptidase, and by a mixture of both enzymes. These data show that prolidase has an average Ci of 3 x 10e4 for this substrate. If the rate of hydrolysis of glycylproline by this preparation is assigned a value of 100, then the relative rate of hydrolysis of glycylprolylleucine is 0.015. When quantities of leucine amino- peptidase, which cleave glycylprolylleucine at a rate only 5% that of prolidase, are added to prolidase, then a slightly greater rate of hydrolysis is observed. From these data it is apparent that the prolidase preparations, when used in conjunction with leucine aminopeptidase, should be able to hydrolyze all peptides that contain proline. The enzymes can be used together without difficulty. Both are activated by Mn++ and are highly active between pH 8 and 8.5. When incubated at 40” for 24 hours at pH 8.5, in 0.005 M MnC&, only small losses (10 to 20%) in the activity of both enzymes were found. In addition, neither enzyme digests the other under these conditions.

TABLE I

Hydrolysis of glycyl-L-proline by leucine aminopeptidase

Hydrolysis was followed by the titrimetric method (20). The reaction mixture contained 0.05 M glycylproline, 0.05 M Tris buffer, pH 8.5, 0.0025 M MnCl2, and leucine aminopeptidase, 0.375 mg of N per m1.a

Time Hydrolysis Cl

min. % 120 9 9.2 x IO-* 240 13.5 7.2 X lo-* 510 24 6.4 X 1O-4 950 42 6.7 X lo-*

1500* Average: 7.4 X lo-4

- a With L-leucinamide this preparation of aminopeptidase gave

a Cl = 45. b Hydrolysis was incomplete as judged by chromatographic

identification of glycylproline, in addition to glycine and proline.

TABLE II

Hydrolysis of glycylprolylleucine by prolidase and leucine aminopeptidase

Each reaction mixture contained 0.05 M substrate, 0.05 M Tris buffer, pH 8.5, and 0.0025 M MnC12. Hydrolysis was followed by the titrimetric method (20).

Enzyme Time

Prolidaseb . . . . . .

Leucine aminopep- tidaseb

Prolidasec + leu- tine aminopepti- dased...........

ROWS % 1 6 4 22

10 50 24 77

10 6 24 11

1 4

11 31

10 56 24 83

- H [ydrol

ysisO Cl

2.9 x 10-4 2.8 X lo-* 3.2 X lo-* 2.8 x 10-4

5.1 x 10-S

5.3 x 10-4’ 6.7 X lo-*

6.0 x lo-* 3.3 x 10-4

Concentration of enzyme

mg protein ‘v/ml

1.6

0.7

1.6 (prolidase) 0.7 (leucine ami-

nopeptidase)

Q Calculated on the basis that hydrolysis of one bond is equal to 100%.

b Prolidase used in this experiment had a Cl = 2 with glycyl- proline as substrate, whereas the aminopeptidase had a Cl = 35 with leucinamide as substrate.

c C1 values calculated from concentration of prolidase. d Complete hydrolysis was demonstrated after 48 hours as

judged by chromatography of the reaction mixture on paper.

Hydrolysis of Polypeptides and Prote&s-The demonstration that a mixture of leucine aminopeptidase and purified prolidase can be used to hydrolyze peptides containing proline eliminated the most serious problem in the use of enzymes for the complete hydrolysis of polypeptides and proteins. Complete enzymic hydrolysates of corticotropin, performate-oxidized ribonuclease

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392 Hydrolysis of Proteins Vol. 237, No. 2

TABLE III Analysis of enzymic h: yd

Amino acid

Lysine .............. Histidine ........... Ammonia. .......... Arginine ............ Tryptophan. ........ Aspartic acid. ...... Threonine. .......... Serine + aspara-

gine + glutamine. Serine .............. Glutamic acid ....... Proline ............. Glycine ............. Alanine ............. Valine .............. Methionine ......... Isoleucine. .......... Leucine ........ . .... Tyrosine. ........... Phenylalanine .......

-

h

-

Enzymic ydrolysis

residues/ mole

5.4 1.0 0.2 2.6 1.2 1.8 0.34

3.2

4.5 4.2 2.9 2.9 2.9 1.0 0.27 1.4 0.6 3.0

rolysates of corticotropin

residues/ mole

4.7 1.0 2.7 2.2

1.9 0.30

2.1 0.33

2.1 0.04

3.2 3.1 3.0 3.9 4.7 5.4 4.1 4.1 4.3 3.2 3.2 3.2 3.0 3.1 3.1 3.0 3.0 3.0 1.4 1.2 1.2 0.28 0.26 0.07 1.5 1.4 1.4 1.5 1.3 1.3 2.8 2.7 2.7

Theory w

4 1 3 3 1 2 0

3 5 4 3 3 3 1 0 1 2 3

A,4 papain, enolase, and carboxypeptidase have been prepared as follows.

One to two pmoles of protein are dissolved in sufficient water to give a 0.1 to 0.5% solution. After adjusting to pH 7 with dilute acid or alkali, the solution is heated for 3 minutes at 95” in a boiling water bath.5 The solution is cooled and 0.2 M sodium acetate6 buffer (pH 5.2) is added to give a final concentration of 0.03 to 0.05 M. The solution is incubated at 40” for 10 minutes and then made 0.01 M in cyanide by the addition of 0.1 M sodium cyanide which had been neutralized to pH 7.0. Sufficient papain (C, = 1.2 to 1.5) in 0.5 to 1.0% solution is added in an amount representing 1 to 5% of the weight of the protein sub- strate. The resulting mixture is incubated in a stoppered flask at 40” for 18 to 24 hours. At the end of the digestion, the pH is adjusted to 2 in order to inactivate the papain. Hydrolysis is estimated most conveniently by analysis of aliquots of the reac- tion mixture with ninhydrin (31). A more precise means for estimating the extent of hydrolysis is unnecessary in that these analyses are used only to judge when hydrolysis is complete. At 40” most proteins are completely digested by papain within a 2- to a-hour period, but to insure maximal degradation, hydrol- ysis is continued for 15 to 24 hours. At the end of the digestion,

4 The first enzymic hydrolysates of ribonuclease did not give the same amino acid composition as that established (42). The greatest discrepancies were found in the yield of cystine and those amino acids which are adjacent to the disulfide bridges. Similar results were obtained with insulin. Thus, only ribonuclease oxidized with performic acid was used in the present studies.

6 Papain hydrolyzes insoluble proteins but acts more rapidly when proteins are dissolved. Heat denaturation as employed here often gives precipitates that dissolve during the initial stages of the digestion.

6 All reagents contained a crystal of thymol as a preservative. Growth of microorganisms during the long digestion periods was eliminated by this means.

an accurately measured aliquot of the reaction mixture is lyophilized in the vessel in which the succeeding hydrolysis is to occur.

The lyophilized digest is dissolved in 1 to 2 ml of water and adjusted to pH 8.4 to 8.6 with dilute NaOH. Tris buffer (0.5 M)

at pH 8.5, and MnClz (0.025 M) are added in amounts to give each a final concentration of 0.005 M. Leucine aminopeptidase equivalent to 4 to 6 mg of a preparation with a C1 = 88 and prolidase equivalent to 0.5 to 1.0 mg with a C1 = 20 are added, and the mixture is incubated at 40” for 15 to 24 hours. The enzymes are inactivated by adjusting the digest to pH 2 with 1 N HCl, and the amino acids are removed from an aliquot by dialysis or picric acid deproteinization. Each method yields identical hydrolysates. The dialysis apparatus is modified from that described by Craig, King, and Stracher (43) in that the glass tube which fits into the dialysis bag has a diameter that is 4 mm less than Visking 23/32 dialysis casing. This accom- modates 5 ml of a digestion mixture in a tube that is 20 cm in length. If the outside tube for collecting the dialysates has a radius that is 5 mm greater than the dialysis casing, the assembly will contain 25 to 30 ml of dialysate. When dialysates are changed eight times at 30-minute intervals, all amino acids are removed quantitatively. The dialysates are evaporated to dryness in a vacuum and redissolved in a measured volume of water or 0.2 M sodium citrate buffer, pH 2.2. The picrate method of Stein and Moore (44) was used, but 1 ml of 1% picric acid per ml of reaction mixture was adequate for deproteinization. Hydrolysates were kept frozen ( - 10”) until just before quantita- tive amino acid analysis.

For determination of the amide content, aliquots of the en- zymic hydrolysate, containing 0.25 to 1.0 pmole of glutamine and asparagine, are taken to dryness, dissolved in 3 ml of 2 N HCI (prepared from three times glass-distilled 6 N HCl) and heated under reduced pressure at 110” for 2 hours. The acid is removed under reduced pressure, and the dried sample is dissolved in 0.2 M citrate buffer, pH 2.2, and analyzed. Asparagine and gluta- mine, which emerge with serine from the chromatographic columns, are converted by this procedure to aspartic acid and glutamic acid, respectively. Thus, the increase of these two amino acids after acid treatment gives an estimate of the two amino acid amides. Serine can be estimated only after glutamine and asparagine are hydrolyzed.

Control experiments in which enzymic digestion was performed in the absence of the substrate were made with each combination of papain, leucine aminopeptidase, and prolidase used in this study. Several amino acids were detected in these digests, but they were not present in amounts that would alter the final amino acid composition by more than 5 to 15% of one residue of any amino acid in the substrates tested. In light of these re- sults, no corrections were applied to the amino acid compositions of the proteins hydrolyzed.

Tables III, IV, and V present the amino acid composition of the enzymic hydrolysates prepared by these methods. The data are expressed in moles of amino acid per mole of protein. These values were calculated in some cases on the basis of the exact weight (corrected for moisture) of the protein hydrolyzed. Alternatively, the micromole value for one residue was obtained by dividing the yield of each amino acid by the theoretical value obtained from analysis of acid hydrolysates. The values calculated by either of these means were within 10% of one another.

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February 1962 R. L. Hill and W. R. Schmidt

With corticotropin, theoretical yields of most amino acids were obtained. Because additional amino acids were not detected after the enzymic digest was hydrolyzed for 24 hours with 6 N

HCl, enzymic hydrolysis was judged to be complete. Proline, which is difhcult to liberate enzymically from peptide linkage, is quantitatively released. The yields of all other residues were those expected with the exception of lysine, glutamine, and asparagine. Enzymic hydrolysis reveals five rather than the expected four residues of lysine per mole. Lack of corticotropin precluded a check of this result. In early experiments on enzymic hydrolysis, lysine recoveries were somewhat high as the result of excessive quantities of Tris buffer which were employed during digestion with aminopeptidase and prolidase. Tris, which emerges near lysine from the chromatographic columns, has a very low color yield with ninhydrin and in small amounts re- mains undetected. When present in large amounts, it can cause

TABLE IV

Amino acid composition of carboxypeptidase and enolase determined by anal YSt is of enzymic hydrolysates

- 7

Carboxypeptidase

Amino acid Found’” Theory

(42) Foundb T(p$Y

__ moles per molt+ moles per moleC

Tryptophan. ......... Lysine ................ Histidine ............. Arginine. ............. Threonine. ........... Serine ................ Proline ............... Glycine ............... Alanine. .............. Valine ................ Methionined. ......... Isoleucine ............ Leucine .............. Tyrosine ............. Phenylalanine ........ Half cystine. ......... Serine + aspara

gine + glutamine. .. Aspartic acid ......... Aspartic acid + as-

paragine. ........... Glutamic acid. ....... Glutamic acid + glu-

tamine, ............ Asparagine .......... Glutamine. ...........

5.8 f 0 17.7 f 0.7

8.3 f 0.2 10.0 f 0.1 25.0 f 0.5 32.9 f 0.1 10.5 f 0.5 24.6 f 0.4 21.0 f 0.3 17.5 f 0.2

3.2 f 0.3 20.1 f 0.7 24.8 f 0.5 17.5 f 0.8 15.8 f 0.3

1.3 f 0.2

42.0 f 1.6 18.2 f 0.6

6 7.2 f 0.1 5 18 50.8 f 1.6 53

8 15.1 f 0.2 14 10 18.6 f 0.9 18 27 29.2 f 0.6 31 33 50.7 f 1.3 54 11 21.6 f 0.1 19 23 56.2 f 0.7 55 20 77.8 f 1.7 88 16 48.5 f 0.5 47

3” 8.2 f 0.3 8 20 29.4 f 0.8 29 25 56.8 f 1.9 60 20 14.5 f 0.7 15 15 22.8 f 0.5 24

2* 0.0 0

29.2 f 0.2 15.9 & 0.4

23.3 f 0.1 11

7

30

25

76.3 f 1.6 20.4 f 1.8

65.2 f 1.0 32.8 f 0.8

47.3 & 1.0 35 14

Total amide. ..... 18 19 49 56 -

Enolase

77

50

Q Average values from five experiments with caiboxypeptidase. b Average values from three experiments with enolase. c Average deviations. d Includes the yield of methionine plus the small amounts of its

oxidation products, methionine sulfoxide and methionine sulfone. e These values are different from those originally reported (46).

Analysis of acid hydrolysates of carboxypeptidase which was oxi- dized with performic acid revealed three methionine and two half- cystine residues per mole, values that agree with the known sulfur content (5 atoms per mole) previously established.

TABLE V

Amino acid composition of papain and ribonuclease determined by analysis of enzymic hydrolysates

Amino acid -

moles per moleC

Tryptophan. ......... Lysine ............... Histidine ............. Arginine. ............. Threonine. ........... Serine ................ Proline. .............. Glycine ............... Alanine. .............. Valine. ............... Methionine ........... Isoleucine ............ Leucine .............. Tyrosine ............. Phenylalanine ........ Half cystine .......... Serine + aspara-

gine + glutamine. .. Aspartic acid. ........ Aspartic acid + as-

paragine. ........... Glutamic acid. ....... Glutamic acid + glu-

tamine. ............ Asparagine ........... Glutamine. ...........

4.2 f 0.4 5 9.0 f 0.2 9 2.0 f 0.1 2 9.6 f 0.4 10 7.3 f 0.1 7

11.0 f 0.5 11 6.9 f 0.4 9

22.4 f 0.8 23 12.9 f 0.3 13 14.5 f 0.6 15

0 0 10.0 f 0.2 10 10.0 f 0.3 10 13.6 f 0.9 17

3.8 f 0.2 4 3.7 f 0.6 6

19.9 f 0.8 6.3 f 0.2

15.9 f 0.6 9.7 f 1.0

17.4 f 0.6 10

7

Total amide. ..... 17 -

17

17

17

Ribonuclease

Foundb Theory (48)

I moles per moleC

0 10.2 f 0.2

3.8 f 0.2 3.7 f 0.1

10.2 f 0.1 14.7 f 0.1

4.3 f 0.2 5.0 f 0.1

11.6 f 0.4 9.1 f 0.2 3.6 f O.ld 3.4 f 0.2 3.6 f 0.2 5.7 f 0.2 3.5 f 0.1 7.5 f 0.2

20.3 f 0.1 5.1 f 0.4

12.7 f 0.2 5.7 f 0.2

10.8 f 0.2 8 4

12

0 10

4 4

10 15

4 3

12 9 4 3 2 6 3 8

26 4

15 6

12 11

6

17

a Average values from eight experiments with papain. . . _

b Average values from five experiments with ribonuclease. c Identified as cysteic acid. d Identified as methionine sulfoxide. c The values for lysine, histidine, arginine, isoleucine, and leu-

tine differ from those originally reported (47). The correct values, given here, have been established in unpublished experi- ments of J. R. Kimmel and E. L. Smith.

apparent increases in the lysine values. Its unique brownish color with ninhydrin produces an unsymmetrical 440 rnp tracing on the leading edge of the lysine peak, and inordinate quantities are readily detected in this manner. In the experiments re- ported here, the quantities of Tris in hydrolysates were insuf- ficient to cause the observed increase in lysine.

Glutamine and asparagine were not found in the enzymic hydrolysates of corticotropin. Other methods of analysis show that one to two amides are present, one of which is ‘probably glutamine (46). The absence of glutamine or asparagine deamidase activities in our enzyme preparations precludes destruction of amides by this means.

Threonine and isoleucine, which are not present in cortico- tropin, were present in nonstoichiometric amounts in the enzymic hydrolysates. Although these amino acids were not found to this extent in control experiments, they probably were liberated from the protein in the enzyme preparations employed.

It is evident that the compositions of carboxypeptidase,

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3!34 Hydrolysis of Proteins Vol. 237, No. 2

Arginine

40 80 120 Volume~ml

FIG. 2. Chromatographic analysis of a complete enzymic hy- drolysate of carboxypeptidase. The hydrolysate was prepared as described in the text.

Tyrosine-O-Sulfate

VOLUME-ml

FIG. 3. Chromatographic analysis of synthetic tyrosine-O- sulfate and an enzymic hydrolysate of bovine fibrinogen. Both samples were analyzed on the same column in different experi- ments.

enolase, papain, and ribonculease, as revealed by enzymic hydrolysis, agree with results based on analyses of acid hy- drolysates. Whereas this provides one kind of evidence for judging that enzymic hydrolysis is complete, additional evidence was also obtained. No ninhydrin-reactive substances were detected which emerged from the chromatographic columns at positions other than those established for the known amino acids. This is demonstrated in Fig. 2, which shows a typical chro- matogram of an analysis of carboxypeptidase. Finally, no peptides were observed when the enzymic digests were analyzed on paper by a two-dimensional electrophoretic-chromatographic technique employed for detection of peptides.

Certain small variations are noted between the compositions revealed by enzymic and acid hydrolysis. Enolase is shown to contain seven rather than the expected four residues of trypto- phan. The excellent recovery of this amino acid from carboxy- peptidase, papain, and corticotropin (Table III), as well as from glucagon (41) and cytochrome-c,7 suggests that reanalysis of

7 R. L. Hill, E. Margoliash, J. R. Kimmel, and E. L. Smith, unpublished observations.

enolase may be desirable. The yields of glycine and leucine from ribonuclease are higher than expected, and although con- trol experiments did not show that these amino acids were derived from the enzymes, this explanation should not be ex- cluded. Of particular concern is the low recovery of alanine from enolase. This is difficult to understand in that no special problem is ordinarily encountered with this amino acid in other proteins. It is evident that tyrosine is generally obtained in yields lower than expected. The oxidation of this amino acid by Mn++ during the enzymic hydrolysis might explain these results,

Although glutamine and asparagine are present in all of the enzymic hydrolysates, the yields of these amino acids are low. It should be possible to obtain a direct estimate of the total serine, glutamine, and asparagine content since these amino acids emerge together on the chromatographic column. This is seldom pos- sible because considerable quantities of glutamine are converted to pyrrolidone carboxylic acid, which does not yield a colored product with ninhydrin. The only model protein used in these studies in which the total quantity of each of these amino acids is known, is ribonuclease. It is noted that the yield of serine, asparagine, and glutamine is 20 residues per mole, an amount exactly equal to the expected value if all glutamine were con- verted to pyrrolidone carboxylic acid. Similar situations proba- bly exist with carboxypeptidase and enolase. Despite this, it is possible to arrive at amide values which are only within 10 to 15% of the expected values. In certain cases, e.g. carboxy- peptidase, the analysis appears to give an answer in good agree- ment with previous measurements (42), whereas with ribonuclease, the total amide content is not in close accord with values previously established (48).

Complete Enzymic Hydrolysis of Fibrinogen, Pepsin, and Casein-In order to determine the sensitivity of the methods for complete enzymic hydrolysis, studies were made to determine the yield of tyrosine-O-sulfate obtained from bovine fibrinogen. At least one residue of this amino acid ester is present in the fibrino- peptides released during the thrombin-catalyzed transformation of fibrinogen to fibrin (49), but it is not clear if additional resi- dues are present in other parts of the molecule. Enzymic hydrolysates of bovine fibrinogen were prepared as described above and analyzed chromatographically on Dowex 2. Fig. 3 shows the single peak that emerged as well as the peak given by authentic tyrosine-O-sulfate in a separate experiment on the same column. From these data it was estimated that 0.85 mole of tyrosine-O-sulfate was obtained per mole of fibrinogen (mol. wt. = 330,000). The results suggest that bovine fibrinogen contains

only 1 mole of tyrosine-O-sulfate. Thus, the enzymic methods allow detection of one unique amino acid in a hydrolysate con- taining nearly 2000 amino acids. Enzymic digests of papain, human fibrinogen, and pepsin contained no substances that behaved like tyrosine-O-sulfate.

When the digest of fibrinogen was examined on paper by the two-dimensional electrophoretic-chromatographic method, one unknown, ninhydrin-reactive component was observed in addition to amino acids (including tyrosine-O-sulfate). The component was slightly basic and migrated electrophoretically at pH 6.5 like histidine, but chromatographically somewhat slower. When eluted from paper, the unknown material gave an orcinol reaction of greater intensity than that given by eluates of other parts of the paper. On this basis, it was concluded that the material is the carbohydrate moiety of fibrinogen. No studies were made to determine the amino acid composition of this

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February 1962 R. L. Hill and W. R. Schmidt 395

glycopeptide, but this result demonstrates the application of the complete enzymic hydrolytic methods for isolation of covalently linked prosthetic groups in proteins. Glycopeptides from other proteins have been isolated recently from enzymic hydrolysates (47).

Enzymic digests of crystalline pepsin and crude casein were prepared as described above and examined by the two- dimensional electrophoretic chromatography method. Phospho- serine was found in the digest of pepsin, and both phosphoserine and phosphothreonine were detected in the digest of casein. These results are also indicative of the ability of the enzymic methods to detect components of an unusual nature in proteins. No quantitative studies were made on these digests.

1)ISCUSSION

The data presented here indicate that complete hydrolysis of proteins can be achieved by employing only proteolytic enzymes. This method of hydrolysis has advantages over the acid hydro- lytic procedures that are ordinarily used. Acid labile amino acids such as tryptophan, asparagine, glutamine, and tyrosine- O-sulfate are liberated in close to theoretical amounts. Threo- nine and serine, which are destroyed partially by acid, as well as valine, leucine, and isoleucine, which often are released in- completely from peptide linkage by acids (50), are obtained in the expected quantities.

Despite these advantages, it is apparent that enzymic hy- drolysis cannot replace acid hydrolysis in protein analysis. The data of Table IV indicate that those amino acids that are lib- erated from peptide linkage without destruction can be estimated more accurately through the use of acid hydrolysis. In addition, analysis of acid hydrolysates reveals most of the composition of a protein without the operational difficulties that can be encoun- tered with the use of enzymes. Thus, enzymic hydrolysis should be a valuable supplement to existing hydrolytic methods and can be used advantageously when acid hydrolytic methods can- not be employed.

The methods that were developed for complete enzymic hydrolysis depend largely on the properties of the proteolytic enzymes that are used. Because papain generally degrades proteins more extensively than other endopeptidases (Fig. 1) and can be prepared without difficulty in highly pure form, it appears to be the ideal choice as the initial hydrolytic agent. Leucine aminopeptidase and prolidase are employed because no other combination of pure or partially purified enzymes is capable of completely hydrolyzing peptides to amino acids. Peptides which do not contain proline are easily degraded by amino- peptidase, whereas prolidase must be used for the complete hy- drolysis of peptides containing proline. Other specific peptidases have not been helpful in enzymic hydrolysis. When carboxy- peptidase was employed along with aminopeptidase and prolidase no beneficial effects could be noted. However, other peptidases might be helpful, particularly for hydrolysis of proteins which contain large amounts of cystine. The resistance of proteins of this type to enzymic hydrolysis suggests that a peptidase which has a high specificity for hydrolysis of peptides containing cystine would be useful.

SUMMARY

1. Methods have been developed for the complete enzymic hydrolysis of proteins. When a protein is digested by papain,

the resulting peptides are hydrolyzed completely to amino acids by the combined action of leucine aminopeptidase and prolidase.

2. Although most peptides can be degraded completely by leucine aminopeptidase, hydrolysis of peptides containing proline is facilitated by prolidase.

3. Carboxypeptidase, enolase, papain, oxidized ribonuclease, and crb-corticotropin were submitted to complete enzymic hydrolysis. The composition of the resulting hydrolysates was in close accord with those previously determined by analyses of acid hydrolysates.

4. Asparagine, glutamine, and tryptophan, which are ordi- narily destroyed by acid, can be obtained in close-to-theoretical yields from enzymic hydrolysates of these proteins.

5. The sensitivity of the enzymic methods was demonstrated by the observation that a single residue of tyrosine-O-sulfate can be obtained in close-to-theoretical yield from enzymic hydro- lysates of bovine fibrinogen.

Acknowledgments-The authors wish to express their apprecia- tion to Drs. Emil L. Smith and J. R. Kimmel for their interest in this work, and to Mr. Boyd Lythgoe for performing many of the amino acid analyses reported here.

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Robert L. Hill and William R. SchmidtThe Complete Enzymic Hydrolysis of Proteins

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