AMINO ACID COMPOSITION OF a-CHYMOTRYPSINOGEN, INCLUDING ESTIMATION OF ASPARAGINE

AND GLUTAMINE

BY PHILIP E. WILCOX, ELAINE COHEN, AND WEN TAN

(From the Department of Biochemistry, University of Washington, Seattle, Washington)

(Received for publication, April 3, 1957)

A general attack upon the problem of the structure of bovine cr-chymo- trypsinogen is being carried forward from several quarters. Work from this laboratory has been concerned with the mechanism by which chymo- trypsinogen is converted to the various chymotrypsins (l-3), with the preparation and properties of chymotrypsinogen derivatives (4, 5), with the hydrogen ion equilibria as revealed by potentiometric and spectropho- tometric titrations, and with the physical properties of the protein (6, 7). Basic to all of these studies of the protein are data on amino acid composi- tion derived by the most dependable and accurate methods available. In a recent paper (6) we have presented and compared values for the molec- ular weight of or-chymotrypsinogen derived by a variety of methods, in- cluding amino acid analysis. Taken as a whole, the values converge to- ward a molecular weight of 25,000.

Samples of cr-chymotrypsinogen may be obtained with a homogeneity that justifies extensive investigations of their composition. The high degree of homogeneity of the best preparations is well attested by solu- bility studies (8), ion exchange chromatography (9), electrophoresis (3), and sedimentation (3, 10).

The purpose of the present paper is to present in detail data on the amino acid composition of a homogeneous sample of cr-chymotrypsinogen, deter- mined largely by the chromatographic methods of Moore and Stein (11). Also included are a determination of cysteic acid in chymotrypsinogen which has been oxidized with performic acid and determinations of amide nitrogen as indicated by the release of ammonia during acidic or basic hydrolysis.

The titration and isoionic point of chymotrypsinogen (12) indicate that the number of groups which add hydrogen ions below pH 7 is greater than the number calculated from analytical values for amide groups and total glutamic and aspartic residues. Because reliable estimates of the number of free carboxyl groups in chymotrypsinogen are necessary for the inter- pretation of hydrogen ion equilibria in relation to structure, we have re- invest,igated a method for the determination of amide groups which is

999

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1000 COMPOSITION OF a(-CHYMOTHYPSINOGEN

independent of the estimation of ammonia. This is the method studied by Chibnall and Rees (13) and later by Grassmann et al. (14) whereby free carboxyl groups are esterified, the ester groups reduced with lithium boro- hydride, and the acid hydrolysate of the reduced protein is analyzed. Glutamic acid and aspartic acid in the hydrolysate represent glutamine and asparagine residues, respectively, except those which might occur as unblocked C-terminal residues. We have found that the estimation of glutamic acid and aspartic acid could be facilitated by the use of ion ex- change chromatography on Dowex 2. The analytical results not only provide data on the number of glutamine and asparagine residues in chy- motrypsinogen, but also corroborate the total amide-NH3 analyses which are based on the release of ammonia by acid or base.

EXPERIMENTAL

Materials

a+Chymotrypsinogen was obtained from the Worthington Biochemical Corporation, Freehold, New Jersey, as a once crystallized filter cake, and was crystallized seven times with ammonium sulfate (15) and twice with alcohol (16). As previously reported (3), this preparation was electro- phoretically nearly homogeneous at pH 4.97. After 7 hours of electrolysis, less than 5 per cent of the protein appeared outside the main peak. When tested against acetyl-L-tyrosine ethyl ester as substrate, the zymogen was found to contain 0.02 per cent active chymotrypsin.

Before each analysis, the sample of protein was dissolved in 0.001 M

hydrochloric acid and the solution was passed over a column of Amberlite IR-120,20 to 50 mesh, on the H+ cycle in order to remove ammonium and metallic ions or peptide impurities which might be present. The amount of nitrogen in chymotrypsinogen was determined on a sample which had been freed from all small ions by passage over a column of mixed bed resin, 20 to 50 mesh, composed of Amberlite In-120 on the H+ cycle and Amber- lite IRA-400 on the OH- cycle. Repeated dry weight measurements’ and Kjeldahl nitrogen determinations on the isoionic protein gave an average value of 16.5 per cent nitrogen. There was no ash, and no counter-ion corrections are necessary. The extinction coefficient of the maximum at 282 rnM, determined on the isoionic protein, was found to be 20.0 for a 1.00 per cent solution.

Dowex 50-X8 and Dowex 2-X10 ion exchange resins, 200 to 400 mesh, were obtained from The Dow Chemical Company, Midland, Michigan. Amberlite In-120 and IRA-400 ion exchange resins, 20 t,o 50 mesh, ana- lytical grade, were obtained from R.ohm and Haas Company, Philadelphia,

1 Constant weight was attained after about 3 hours at 100” under a high vacuum.

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P. E. WILCOX, E. COHEN, AND W. TAN 1001

and ninhydrin and hydrindantin were purchased from Dougherty Chemi- cals, Richmond Hill, New York. Amino acids were purchased commer- cially as analytically pure compounds, some of which were recrystallized by Dr. Earl W. Davie, and samples were stored in a vacuum desiccator over phosphorus pentoxide. BRIJ 35 detergent was obtained from the Atlas Powder Company, Wilmington, Delaware, and lithium borohydride was purchased from Metal Hydrides, Inc., Beverley, Massachusetts. Tetrahydrofuran was obtained as a commercial grade solvent from E. I. du Pont de Nemours and Company, Inc., and was purified by being refluxed over sodium ribbon and then by being distilled from sodium. The purified solvent was stored over sodium in the dark. All other chemicals were purchased commercially and were either chemically pure or reagent grade.

Methods and Results

Amino Acid Analysis on Dowex 50 Columns-Three times distilled con- stant boiling hydrochloric acid was used for hydrolysis of the protein. About 50 mg. of protein in 2 ml. of dilute acid were placed in a 15 ml. Pyrex tube, 12 ml. of constant boiling acid were added, and the tube was thoroughly evacuated with a high vacuum pump and then sealed. The final concentration of acid was about 5 N. Separate hydrolyses were carried out in an autoclave held at 110” f lo for 12, 24, 36, 48, and 72 hours. All of the hydrolysates became first light yellow and then darken- ing shades of purple. The contents of each tube were repeatedly taken to dryness in vacua and dissolved in water. Small amounts of humin were removed by centrifugation. Tests by Kjeldahl analysis showed that the humin contained negligible amounts of nitrogen. The nitrogen content of the hydrolysates was determined by duplicate micro-Kjeldahl analyses.

The preparation of columns of Dowex 50-X8 resin (250 to 400 mesh), the preparation of buffers, and the operation of the columns in the chro- matography of the hydrolysates were carried out as nearly as possible in the manner described by Moore and Stein (11). Effluent fractions were collected with a Technicon automatic fraction collector which was set to collect 1.0 ml. aliquots by drop count. The quantities of the various amino acids in the effluent fractions were estimated by the calorimetric ninhydrin method (17), and proline was estimated by the acid ninhydrin method of Chinard (18, 19). Color yields for all of the amino acids were determined for our reagents and recrystallized samples of amino acids. A Beckman model B spectrophotometer was used in all measurements of optical density.

The 100 cm. column was used for the acidic and neutral amino acids, and the 15 cm. column for the basic amino acids. The separation for one of the hydrolysates is shown in Figs. 1 and 2. Individual amino acids

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1002 COMPOSITION OF a-CHYMOTRYPSINOGEN

emerged from the columns at effluent volumes which were similar to those reported by Moore and Stein (11).

0 20 40 60 80 100 120 140 160 181 k k pH 3.42.37.5” pH 3.42,3X5”

EFFLUENT FRACTIONS EFFLUENT FRACTIONS

0 20 40 60 80 100 120 140 160 181

SER 3.0 B 2.6-

k 2.2-

5 I& B -- 3 I .4-

c LO- Q - Q .6-

.2-

ASP THR 1

GLY ALA

GLU

g 14 8 .

VAL

.I 1.0 3 A

LEU

R

TYR PHE

s .6-

$ .2- ?

r.. , ,~ 180 200 220 240 260 280 300 320 340 360

-pH 4.25, 50”-pH 4.25,?5”+

EFFLUENT FRACTIONS

FIG. 1. Amino acids of a 36 hour hydrolysate of Lu-chymotrypsinogen, separated on a 0.9 cm. X 100 cm. column of Dowex 50-X8. Optical density at 570 rnp is plotted on the ordinate for each effluent fraction after treatment with ninhydrin reagent. The dashed curve for proline gives optical density at 490 rnp after reaction with ninhydrin by the method of Chinard. All optical densities have been corrected for base-line absorption. The amount of hydrolysate placed on the column corresponds to 4.65 mg. of protein. The pH of buffers and temperatures used for elution are indicated along the abscissa. The contractions used are as follows: ASP, aspartic acid; THR, threonine; SER, serine; GLU, glutamic acid; PRO, proline; GLY, gly- tine; ALA, alanine; CYS, cystine; VAL, valine; MIST, methionine; ITZTJ, isoleueine; LEU, lencine; TYR, tyrosine; PHE, phenylalanine.

A summary of the analytical values is presented in Table I. When the results for the differing times of hydrolyses were compared, three amino acids were found to increase significantly with time from 24 and 72 hours.

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P. E. WILCOX, E. COHEN, AND W. TAN 1003

Proline increased 8 per cent, valine 12 per cent, and isoleucine 17 per cent (Fig. 3). Therefore, the values in Table I for these three amino acids are the averages of two runs at 72 hours of hydrolysis. On the other hand, between 12 and 48 hours, threonine decreased 7 per cent and serine 23 per cent. Data for these two amino acids were extrapolated to zero time by the method of least squares. A graph of the extrapolated data is given in Fig. 4.

One may question the validity of the assumption that during the initial stages of protein hydrolysis the destruction of an amino acid follows simple kinetics, either first order (22) or zero order, as indicated in Fig. 4. The

* 2.0- k. -TYR+PHE

ARG

IO 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190200 PpH 5- pH 6.8 PHOSPHATE - pH 6.5 CITRATE -----+I

EFFLUENT FRACTIONS

FIG. 2. Basic amino acids and ammonia of a 36 hour hydrolysate of a-chymotryp- sinogen, separated on a 0.9 cm. X 15 cm. column of Dowex 50-X8. Optical density at 570 rnp is plotted on the ordinate for each effluent fraction after treatment with ninhydrin reagent. All optical densities have been corrected for base-line absorp- tion of fractions on either side of each peak. The amount of hydrolysate placed on the column corresponds to 4.50 mg. of protein. The contractions used are HIS, histidine; LYS, lysine; ARG, arginine.

analytical value for serine, which decomposes so extensively, should be accepted with reservations.

The amount of methionine found in the different hydrolysates varied erratically. In some, especially after long times of hydrolysis, the yield was as low as 0.5 gm. per 100 gm. of protein. Either methionine was de- stroyed to a variable extent during hydrolysis or different amounts were lost on the column even though thiodiglycol was used as an antioxidant (20). The chromatograms showed no traces of methionine oxides, which would emerge just before aspartic acid. In view of this uncertainty, the value for methionine in Table I is an average of four similar values at 24 and 36 hours of hydrolysis. Maximal recoveries were found at these times. Furthermore, the values have been corrected upward on the assumption that only 90 per cent of the methionine in the hydrolysates was recovered from the column (20).

Destruction of cystine was extensive, and the loss of tryptophan was

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1004 COMPOSITION OF a!-CHYMOTRYPSINOGBN

TABLE I

Amino Acid Composition of a-Chymotrypsinogen As Determined by Chromatography of Acid Hydrolysates on Columns of Dowex 50

Amino acid

Aspartic acid. ................. Threonine ..................... Serine ......................... Glutamic acid. ................ Proline ........................ Glycine ........................ Alanine ........................ Valine ......................... Methionine .................... Isoleucine ..................... Leucine ....................... Tyrosine ...................... Phenylalanine ................. Histidine ...................... Lysine. ........................ Arginine .......................

Sum. .......................

Half cystine ................. Tryptophan. ................. Ammonia .....................

Total. ......................

P 11

_-

_-

--

-

hnino acid per IO gm. protein’

gm.

11.56 10.89$ 12.601

8.328 4.0011 6.97 7.71

10.4511 1.147 5.1711 9.85 2.95 4.29 1.17 7.72 2.77

L I

.-

_-

-

Amino acid residue per 100

gm. protein as per cent

total N

:&&ted No. If residues per n&c&, mol. wt. = zs,1oot

km.

9.99 9.24

10.44 7.30 3.37 5.29 6.15 8.84 1.00 4.46 8.50 2.65 3.82 1.03 6.76 2.48

7.37 7.76

10.17 4.80 2.95 7.88 7.34 7.57 0.65 3.34 6.71 1.38 2.20 1.91 8.96 5.40

21.8 23.0 30.1 14.2

8.7 23.3 21.7 22.4

1.9 9.9

18.8 4.1 6.5 1.9

13.2 4.0

91.32

4.07** 5.19tt

86.39

3.38** 4.73tt S.ll$$

C of 0

r

--

--

225.5

10.0 7.0

100.58 102.61 242.5

* Average of six analyses, two runs each at 24,36, and 48 hours of hydrolysis, un- less otherwise indicated.

t The calculation of the molecular weight of chymotrypsinogen from amino acid composition and the comparison of this value with data from physical measurements have been presented elsewhere (6).

1 Data for 12, 24, 36, and 48 hours extrapolated to zero time. 8 Corrected for 3 per cent loss of glutamic acid on the column (II). 11 Average of two runs at 72 hours of hydrolysis. 7 Average of four runs, two at 24 hours and two at 36 hours, corrected for 10

per cent loss of methionine on the column (20). ** Calculated according to the assumption of 10 half cystine residues per molecule

as indicated by analysis for cysteic acid in oxidized protein (see the text). tt Calculated according to the assumption of 7 residues per molecule (21). $$ Calculated according to 1.34 per cent amide nitrogen (see the text).

essentially complete. Although present in chymotrypsinogen, these two amino acids are not included in the main body of analytical results. None

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P. E. WILCOX, E. COHtiN, AND W. TAN 1005

of the ten remaining amino acids listed in Table I showed a trend either upward or downward with the time of hydrolysis. The mean deviation of the values which have been averaged for each of these ten amino acids varies from 1.4 per cent (alanine) to 3.9 per cent (hi&dine). The final

$ 10.5- A’ ,-m---p-

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/’ f$ 9.0 - A ,

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7 $j 5.0 _ ISOLEUCINE .----r-------g--

: 4.5- *) r/-

/@ . PROLINE f$ 4.0 - ~~~--~~--~----&--“---8--8--

? I I I

s 0 12 24 36 48 60 72

HOURS OF HYDROLYSIS

FIG. 3. Amounts of valine, isoleucine, and proline in acid hydrolysates of LY- chymotrypsinogen as a function of the duration of hydrolysis. Dashed curves have been added only to relate experimental points.

13.0-

9.0 - I I t

0 12 24 36 48 HOURS OF HYDROLYSIS

FIG. 4. Amounts of serine (0) and threonine (0) in acid hydrolysates of CY- chymotrypsinogen as a function of the duration of hydrolysis. Each straight line represents the best fit to the data as determined by the method of least squares, assuming a linear decrease of each amino acid with time.

value for glutamic acid has been corrected upward by 3 per cent to take account of the loss which is believed to occur on the long column (11).

A few comments on the chromatograms themselves are appropriate.

The typical chromatogram presented in Fig. 1 shows a peak emerging with

the solvent front at tube 25. We have labeled this peak “cysteic acid.” It is commonly believed that this acidic amino acid found in protein hy-

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1006 COMPOSITION OF a-CHYMOTRYPSINOGEN

drolysates arises from cystine by decomposition, although the peak could also be cysteine sulfinic acid. Based on the color yield of cysteic acid, the amount of the acidic material corresponds to 1.0 f 0.2 residues per molecule of chymotrypsinogen for all times of hydrolysis above 12 hours. There were no indications that the amount varied significantly with time of hydrolysis.

A small, unidentified peak appears about twenty effluent fractions.after phenylalanine in the chromatograms from both the 100 cm. column and the 15 cm. column (Fig. 1 and Fig. 2). The amount of this material is difficult to estimate because the peak is broad and varies from run to run. Based on the color yield for leucine, the material corresponds to somewhat less than 1 residue per molecule. We have been inclined to ascribe this peak to residual tryptophan or decomposition products of tryptophan (chymotrypsinogen contains about 5 per cent tryptophan). The unmodi- fied amino acid should emerge from the 15 cm. column near Fraction 55 (11).

At the bottom of Table I have been added the analytical values for cys- tine, tryptophan, and amide nitrogen. Cystine content has been based upon determinations of cysteic acid in oxidized protein which are described below. It has been assumed that there are an integral number of residues and that each half cystine residue yields approximately 1 cysteic acid residue. The value for the amide nitrogen is also calculated from the results presented in this paper, whereas the content of tryptophan has been taken to be 7 residues per molecule as reported by Brand and Kassel (21). The accumulated totals for amino acid residue weights and for nitrogen as per cent of total nitrogen show that we have accounted quantitatively for all of the protein and all of the nitrogen within experimental error.

Release of Ammonia by Basic and Acidic Hydrolyses-The amount of amide nitrogen in chymotrypsinogen was estimated by following the re- lease of ammonia under the following three different conditions: 1.0 N

NaOH at 25”, 5 N HCI at llO”, and concentrated HCI at 37”. The protein used in these experiments was in the form of deionized solutions whose concentrations were determined by measurements of optical density (E:.?‘cm. = 20.0 at 282 mp).

For basic hydrolysis, procedures similar to those studied by Warner and Cannan (23) were carried out. The reaction was performed in a series of Conway dishes which had been given water-repellent surfaces by treat- ment with Desicote. Into each of the dishes, 1.00 ml. of protein solution and 1.0 ml. of 2 N NaOH were placed on opposite sides of the outer ring. 1.0 ml. of 0.1 N H&04 was pipetted into each center well, the dish was closed, and the base and protein were mixed. The final protein concen- tration was about 5 mg. per ml. Control distillations were carried out

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P. E. WILCOX, E. COHEN, AND W. TAN 1007

with a standard solution of ammonium sulfate substituted for the protein solution. At scheduled time intervals, the liquid in the center well of a dish was neutralized, and the ammonia content was determined by the calorimetric ninhydrin method of Moore and Stein (17). The agreement between two separate series was close, as shown in Fig. 5. The course of the release of ammonia is similar to that described for P-lactoglobulin by Warner and Cannan (23). Extrapolation of the nearly linear portion of the curve, between 30 and 80 hours, by the method of least squares, gives a zero time intercept of 1.37 per cent amide nitrogen.

HOURS FIG. 5. Ammonia obtained from chymotrypsinogen by the action of base or acid

as a function of time. Closed and open circles represent two separate runs in 1.0 N NaOH at 25” % 2”. Closed and open triangles represent two separate runs in 5 N HCl at 110” & 1”. The method of least squares has been used to extrapolate the data to the ordinate, where values for amide nitrogen as per cent of total protein may be read.

Hydrolysis with three times distilled hydrochloric acid was carried out in evacuated and sealed Pyrex tubes at 110’ f lo by procedures which have been described above for amino acid analyses. About 50 mg. of protein were hydrolyzed with 12 ml. of acid. After scheduled time inter- vals, individual hydrolysates were evaporated to dryness, the residues were taken up in water, and samples were assayed for ammonia by a combination of distillation in Conway dishes (24) and calorimetry with ninhydrin rea- gent (17). For distillation, 1.00 ml. of sample was mixed with 1 ml. of 1 M Na2C03. With sodium carbonate, 4 hours are required for complete distillation of ammonia. Sodium carbonate was preferred over sodium hydroxide because it was found that the release of ammonia from a hy- drolysate by the weaker base is inappreciable after 4 hours, whereas am- monia continues to be released at a measurable rate with sodium hydroxide (cf. Conway and Byrne (24)). The results of two separate series of acid

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1008 COMPOSITION OF a-CHYMOTRYPSINOGEN

hydrolyses are shown in Fig. 5. Extrapolation by the method of least squares gives a zero time intercept of 1.35 per cent amide nitrogen. The scatter of data about the fitted line is greater than in the case of base hy- drolysis, and could be expected from the results of Jacobsen (25) in work with P-lactoglobulin. It was shown that trace impurities in the acid used for hydrolysis affected the rate at which ammonia was released.

The use of concentrated hydrochloric acid for the determination of amide nitrogen has been described by Rees (26). In our experiments, 5.00 ml. of deionized protein solution were added to 40 ml. of concentrated HCl at 37”, and the volume was made up to 50.0 ml. with acid. The final protein concentration was about 2.5 mg. per ml. The solution was stored at 37”, and samples of 1.00 ml. were removed at scheduled time intervals. Each sample was pipetted into the outer ring of a Conway dish and was evaporated to dryness in a vacuum desiccator. Ammonia was determined

TABLE II

Ammonia Obtained by Hydrolysis in Concentrated Hydrochloric Acid at 37” Expressed As Per Cent of Total Nitrogen

I Hydrolysis

72 hrs. 120 hrs. 168 hrs. 291 hrs. 341 hrs. 408 hrs. ~--- --

cu-Chymotrypsinogen (N, 16.5%) 0.61 6.85 7.20 7.90 7.94 8.15 7.94 7.98 8.05

p-Lactoglobulin (N, 15.6%) 0.67 6.14 7.00 7.02 6.95 7.10 6.76

138 hrs.

8.00

7.98

7.03 7.00

by distillation from 1 M Na2C03 into 0.1 N HzS04 and then by calorimetry with a ninhydrin reagent, as described above for hydrolysates from 5 N

HCl. For purposes of comparison, a sample of &lactoglobulin, which had been prepared and deionized in this laboratory, was assayed by the same procedure. The results of the two assays are presented in Table II. The release of ammonia follows a similar course for both proteins; the values remain essentially constant after 290 hours. The final value for /3-lacto- globulin corresponds closely to the total amide nitrogen reported by Rees (26). However, the ammonia found before 120 hours is much less than that found by Rees at similar times of hydrolysis. The difference may be due to the different ways of estimating ammonia or to the use of somewhat different concentrations of hydrochloric acid.2 If the results for chymo-

2 In the earlier work (26)) dry protein was dissolved in concentrated hydrochloric acid. Ammonia was determined by neutralizing a sample with sodium hydroxide and distilling the ammonia from the sample mixed with buffer at pH 10 in a Kjeldahl flask.

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P. E. WILCOX, E. COHEN, AND W. TAN 1009

trypsinogen are averaged for times longer than 290 hours, a value of 1.32 per cent amide nitrogen is obtained.

Xummary of Values for Amide Groups As Determined by Release of Am- monia-The results for amide nitrogen obtained by three different methods of hydrolysis are in close agreement. Small differences may be ascribed to experimental errors, which we estimate to be of the order of 2 to 3 per cent. In terms of a molecular weight of 25,100, the results for the three methods correspond to 24.1, 24.4, and 23.7 amide groups per molecule, respectively, for hydrolysis with 1 N NaOH, 5 N HCl, and concentrated HCl.

EsteriJication, Reduction, and Hydrolysis of Chymotrypsinogen-The rea- gent chosen for the esterification of chymotrypsinogen was a mixture of acetic anhydride and absolute methanol. This reagent had been used previously by Blackburn and Phillips (27) and more recently by Grassmann et al. (14). The latter authors have reviewed various methods for the esterification o proteins. It is especially pertinent to the present study that methanol-acetic anhydride does not appear to cause loss of amide nitrogen from protein. We may add that this reagent has the further advantage that free amino groups are blocked by acetylation. We have observed that serum albumin esterified with diaxoacetamide (28) becomes less soluble upon aging and shows a decreasing amount of free amino ni- trogen as determined by the Van Slyke analysis. The possibility of reac- tion between ester groups and amino groups while protein is suspended with lithium borohydride in refluxing tetrahydrofuran must be considered; acetylation prevents such a side reaction.

Preliminary experiments showed that when chymotrypsinogen in the form of a lyophilized powder was treated with methanol-acetic anhydride, and the product was reduced in refluxing tetrahydrofuran, the reactions were not complete. It appeared that some portion of the insoluble pro- tein was impervious to the reagents. Therefore, in order to begin with a finely divided amorphous material, the protein was precipitated from a deionized solution with methanol.

In a typical procedure, 2.0 gm. of deionized chymotrypsinogen in 50 ml. of 0.001 N HCl were precipitated with 200 ml. of methanol containing 2 gm. of lithium chloride. The precipitate was centrifuged and washed three times with absolute methanol. The gelatinous protein was sus- pended in 100 ml. of absolute methanol, 20 ml. of acetic anhydride were added, and the mixture was allowed to stand at room temperature with occasional stirring. After scheduled intervals of time, the product was recovered and was washed thoroughly with methanol, all operations being carried out in a centrifuge. The final product was dried in a vacuum des- iccator, ground to a fine powder, and stored over phosphorus pentoxide.

In each reduction, the following procedure was followed: 50 mg. of es-

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1010 COMPOSITION OF a-CHYMOTRYPSINOGEN

terified protein were placed in a dry flask into which were distilled about 30 ml. of pure tetrahydrofuran. 50 mg. of lithium borohydride were transferred quickly to the flask and the solvent was heated to a reflux on a sand bath. The condenser was protected from moisture with a tube of calcium chloride.

After scheduled time intervals, each reduction mixture was cooled and treated with 2.0 ml. of 6 N HCl in order to destroy excess borohydride. Boric acid formed from the reducing agent was removed by distillation with methanol. Absolute methanol was added in four successive portions of 25 ml. each, and, after each addition, the solvent was removed by heating the mixture under a vacuum. A test of the final vapors showed that all free boric acid was removed by this procedure.

The reduced protein was hydrolyzed by adding 30 ml. of constant boil- ing hydrochloric acid to the residue in the reaction flask and refluxing the mixture for 24 hours, and the acid was removed from the hydrolysate by evaporation in a vacuum desiccator over sodium hydroxide pellets. The residue was taken up in 10 ml. of water, the humin was removed by cen- trifugation, and samples of the solution were used for determinations of total nitrogen and estimation of glutamic and aspartic acids.

Determination of Glutamic and Aspartic Acids in Protein Hydrolysates-A chromatographic method has been developed by which glutamic acid and aspartic acid may be separated from protein hydrolysates with columns of Dowex 2 ion exchange resin. No other amino acids appear in the chro- matogram.

The resin and the column are prepared as follows: Dowex 2-X10, 200 to 400 mesh, is washed and cycled twice with 1 N NaOH and 1 N HCl. About 500 ml. of the washed resin are suspended in 2 liters of water in a graduate cylinder. After a settling time of 1 hour, the fines in suspension are decanted and discarded. A suspension of the prepared resin is used to fill a chromatographic tube, 9 mm. in diameter, to a height of 15 cm. The column is washed with the acetate buffer at pH 5, which is described below. A hydrostatic head is produced such that the flow rate is about 20 ml. per hour. 3 hours are sufficient to saturate the column with ace- tate ion.

The three buffers used for washing the column and for elution are the following: (a) acetate buffer, 1.0 M, pH 5.0, which is prepared by dissolving 272 gm. of NaOAc .3Hz0 in 800 ml. of water, adding 50.0 ml. of glacial acetic acid, and diluting to 2.00 liters, and (b) pyridine-acetate buffer, pH 5.0, which is prepared by adding 4.5 ml. of pyridine and 3.0 ml. of glacial acetic acid to 2.00 liters of water. It is sometimes necessary to adjust the pH to 5.0 f 0.1 with pyridine or acetic acid. (c) Pyridine-acetate buffer, pH 4.2, is made from the buffer at pH 5.0. Glacial acetic acid is

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P. E. WILCOX, E. COHEN, AND W. TAN 1011

added to 500 ml. of buffer until the pH is between 4.1 and 4.2. About 3 ml. of acid are required. All buffers are made up to contain 0.4 per cent BRIJ 35 detergent.

Before each analysis, the column is equilibrated with pyridine-acetate buffer, pH 5.0, by passing 50 ml. of the buffer through the column at a flow rate of about 20 ml. per hour under a moderate hydrostatic head. All liquid is then removed from the top of the column and 1.0 ml. of a 0.24 per cent solution of pyridine in water is placed above the resin. A 1.00 ml. sample of protein hydrolysate is mixed into the pyridine solution on the column. The most accurate analyses are obtained if the sample contains from 2 to 4 pmoles of each amino acid. The 2 ml. of liquid are allowed to enter the resin by gravity. Two 0.5 ml. portions of pyridine-acetate buffer, pH 5.0, are used to wash the last portions of the sample into the resin.

With the sample absorbed at the top of the column, the elution of the neutral and basic amino acids is begun by passing about 12 ml. of pyridine- acetate buffer, pH 5.0, through the column at a flow rate of 6 ml. per hour. Only a small hydrostatic head is required for this rate. The separation and elution of glutamic and aspartic acids are obtained by a pH gradient elution. The change from the reservoir of buffer at pH 5.0 to a mixing vessel is made by turning a three-way stopcock in the three-way line. The mixing vessel contains initially 50 ml. of pyridine-acetate buffer at pH 5.0 in a closed system and is agitated with a magnetic stirrer. Into this vessel is introduced the buffer at pH 4.2 under a small hydrostatic head. The flow rate is maintained at 6 ml. per hour. A fraction collector is used for the collection of 1.00 ml. fractions after gradient elution has begun. Fractions 50 to 150 are used for determination of the acidic amino acids.

The amino acid content of the fractions is determined by the colori- metric ninhydrin method with acetate as the buffer in the ninhydrin reagent.3 Factors by which optical density readings are converted to micromoles of glutamic acid or aspartic acid have been determined for pure samples of each amino acid in the pyridine-acetate buffers. Yields of the known amounts of the two amino acids recovered from the column have been found to be 100 & 2 per cent. A typical separation from a hydrolysate of esterified and reduced chymotrypsinogen is shown in Fig. 6.

3 We have obtained the most satisfactory and precise results with a ninhydrin reagent which differs somewhat from that described by Moore and Stein (29). The final concentrations in our reagent are ninhydrin, 8.0 gm. per lit,er; hydrindantin, 1.6 gm. per liter; acetate buffer, I .O M, pH 5.5; and Cellosolve, 700 ml. per liter. This reagent gives a low blank of optical density around 0.030 and color yields for most of the amino acids between 90 and 95 per cent of theoretical (cf. Connell et aZ.(30)).

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1012 COMPOSITION OF a-CHYMOTRYPSINOGEN

The amounts of glutamic and aspartic acids obtained from unmodified chymotrypsinogen corresponded closely to the amounts found in the com- plete amino acid analyses described above.

Analyses of samples of chymotrypsinogen which had been esterified and treated with lithium borohydride for various periods of time are summarized in Table III. It will be noticed that, for the periods shown in Table III, the amount of glutamic acid shows no significant trend as a function of time of reaction. The variations which appear are probably within the limits of experimental error, except for some analyses of samples which were reduced for 48 hours. The results for aspartic acid do appear to

I. I s, 1.0 GLU

40 50 60 70 80 90 100 110 120 130 140 150 -pH 5.0 PYRIDINE-ACETATE BUFFER GRADIENT ptl4.24

EFFLUENT FRACTIONS

FIG. 6. Separation of glutamic acid and aspartic acid from an acid hydrolysate of esterified and reduced chymotrypsinogen by chromatography on Dowex 2-X10. Protein had been esterified for 187 hours and reduced for 48 hours. The amount of hydrolysate placed on the column corresponds to 4.01 mg. of chymotrypsinogen. Optical density at 570 rnF is plotted on the ordinate for each 1 ml. of effluent fraction after treatment with ninhydrin reagent. All optical densities have been corrected for base-line absorption.

show a significant downward trend from 24 to 72 hours of reduction, but show no significant change with time of esterification. It, therefore, appears that esterification and reduction of chymotrypsinogen lower the glutamic acid which appears in a hydrolysate from 14 to 11 residues per mole and lower aspartic acid from 22 to 15 or 14 residues per mole.

Determinations of Cysteic Acid in Hydrolysates of Oxidized Chymotrypsin- ogen-The procedure used for the oxidation of the protein with performic acid was essentially that described by Schram, Moore, and Bigwood (31). Two different reaction times were used as a test of the course of the reac- tion. Difficulties were encountered in obtaining reproducible yields of cysteic acid when the formic acid solution was evaporated in a rotary evaporator. The results were more reproducible when the reaction

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P. E. WILCOX, E. COHEN, AND W. TAN 1013

mixture was diluted with 10 volumes of ice water, and the solution was immediately frozen and lyophiliaed in the usual manner. The dry residue of oxidized protein was divided into 50 mg. portions for hydrolysis with constant boiling HCl at 110” in evacuated and sealed tubes. Hydroly- sates were prepared for chromatographic analysis by procedures which have been described above for complete amino acid analysis.

The amount of cysteic acid found in each analysis needs to be related to the original amount of protein which corresponds to the sample placed on the column. Total nitrogen analyses by the Kjeldahl method are used for this purpose since it is desirable to avoid errors which may arise

TABLE III

Aspartic Acid and Glutamic Acid Obtained by Hydrolysis of EsteriJied and Reduced Chymotrypsinogen

The results are expressed in terms of gm. of amino acid per 100 gm. of protein, and as numbers of residues (in parentheses) per molecule of weight 25,000.

Aspartic acid Glutamic acid

Reduction time Reduction time

24 hrs. 48 hrs. 72 hrs. I I I 24 hrs. 48 hrs. 12 hrs.

Esterification time, 33 hrs

Esterification time, 187 hrs.

8.14 (15.3) 7.40 (13.9) 7.35 (13.8) 6.58 (11.2) 6.60 (11.2) 6.65 (11.3) ~ 7.82 (14.7) 17.30 (13.7) j 16.93 (11.8) 16.54 (11.1)

in transfer of materials. Calculations based on the weight of protein used for analysis are unreliable when the protein is lyophilized from a large volume of solution. Small portions of the protein are sometimes blown into the condenser. The use of total nitrogen as reference presup- poses that no nitrogen is lost from the protein during the preparation of the analytical samples; for example, by hydrolysis of amide groups by formic acid and sublimation of ammonium formate. If all amide nitrogen were lost in this way from chymotrypsinogen, which is improbable, the maximal error introduced into the calculations would be 8 per cent. This question was investigated by letting chymotrypsinogen stand in formic acid for 4 hours at room temperature and evaporating all of the solvent under a high vacuum in a rotary evaporator. No ammonia was found in

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1014 COMPOSITION OF cr-CHYMOTRYPSINOGEN

the condensate, although the experiment was designed to detect a loss of 0.2 per cent of the total protein nitrogen. We believe that total nitrogen is an accurate measure of protein in each analysis for cysteic acid.

Cysteic acid was separated from each sample by chromatography on columns of Dowex 2 with chloroacetic acid as the eluting agent (31). Calorimetric ninhydrin assays of the fractions gave the results shown in Table IV. Neither time of oxidation between 4 and 24 hours nor time of hydrolysis between 16 and 32 hours significantly affected the amount of cysteic acid found in the hydrolysates. The values have not been corrected in any way for losses which may occur during treatment of the protein (cf. Schram et al. (31)). Recovery of cysteic acid from our columns was quantitative, and yields of cysteic acid from oxidized cystine ranged

TABLE IV

Cysteic Acid Content of Acid Hydrolysates of Oxidized Chymotrypsinogen

The results are expressed in terms of gm. of amino acid per 100 gm. of unoxidized protein, and as numbers of residues (in parentheses) per molecule of weight 25,000.

Time of oxidation

4 hrs. I

24 hrs.

Hydrolyzed 16 hrs.

6.64 (9.8) I

6.39 (9.4)

Hydrolyzed 32 hrs.

6.49 (9.6) I

6.56 (9.7)

between 90 and 95 per cent. However, yields of cysteic acid from chy- motrypsinogen varied 10 per cent with small changes in technique in an unpredictable fashion. No controls have been found adequate. The results presented in Table IV were obtained in one particular series of runs which gave the highest yields. If we assume that this cysteic acid all arose from half cystine residues in native chymotrypsinogen, it appears that 10 such residues are present in each protein molecule.

DISCUSSION

The values for the amino acid composition of bovine oc-chymotrypsinogen, as determined in the present investigation, may be compared with the values reported by Brand in 1948 (32) and by Lewis et al. in 1950 (33). These earlier assays employed for the most part microbiological methods, but also a few chemical and spectrophotometric methods. Values for

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P. E. WILCOX, E. COHEN, AND W. TAN 1015

the basic amino acids agree within 5 per cent for all laboratories, and threonine and valine may also be added to this list. Of the remaining amino acids, agreement within 5 per cent is found between the present and earlier determinations for aspartic acid, proline, isoleucine, leucine, and tyrosine, but in each case only one of the two earlier values agrees with the present results.

It should be noted that the analytical results expressed as number of residues per molecule of chymotrypsinogen (see Table I) lie close to integral values for most of the amino acids. The exceptions are glycine, alanine, and valine, all of which occur in numbers larger than 20, and phenylalanine, which remains unexplained.

A comparison of cY-chymotrypsinogen with other proteins in regard to the rates of liberation and destruction of various amino acids shows that each protein has a distinctive pattern of behavior in acid hydrolysis. Decreases in aspartic acid, glutamic acid, arginine, and lysine during the acid hydrolysis of carboxypeptidase or papain have been reported by Smith and his coworkers (34, 35). Hirs, Stein, and Moore have reported decreases in tyrosine, aspartic acid, glutamic acid, proline, and arginine during the acid hydrolysis of ribonuclease (22). In the present study of chymotrypsinogen, none of these amino acids was found to decrease at between 24 and 72 hours of hydrolysis, at least within the limits of precision of about 2 per cent. On the contrary, proline was found to increase, along with increases in valine and isoleucine. This result suggests a bond between proline and one of these two latter amino acids which are known to form peptide bonds resistant to hydrolysis (cf. Harfenist (19) ; Smith and Stockell (34)). The decreases in serine, threonine, and cystine are in line with what has been found with other proteins; one would expect cystine to be extensively destroyed because of the high content of trypto- phan in chymotrypsinogen (36).

In turning to the question of amide nitrogen, chymotrypsinogen may be compared with ,&lactoglobulin and insulin, two proteins whose behavior toward hydrolysis has been carefully studied by several methods (13, 23, 25, 26). The release of ammonia from chymotrypsinogen was in all respects similar to the release of ammonia from these other two proteins. Three different methods of estimating the amide nitrogen as ammonia give close to twenty-four groups per molecule of chymotrypsinogen. That these determinations truly measure amide groups is indicated by an entirely independent method. A total of 25 or 26 residues of glutamic and aspartic acids is unaffected by esterification with methanol-acetic anhydride fol- lowed by reduction with lithium borohydride.

The uncertainties in the interpretation of results obtained by esterifica- tion and reduction with a borohydride must be recognized. There is the

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1016 COMPOSITION OF a-CHYMOTRYPSINOGEN

possibility that esterification of free carboxyl groups is not complete. On the other hand, amide nitrogen may be lost slowly from the protein during esterification, and the newly liberated carhoxyl groups may themselves be esterified. Our results appear to rule out these two possibilities. Very long and widely spaced times of reaction, 33 and 187 hours, were chosen with a view to establish this point.

The side reactions which may accompany the reduction of ester groups in esterified peptides and proteins have been discussed by Bailey (37), by Grassmann et al. (14), by Hormann et al. (38), and by Crawhall and Elliott (39). Vigorous conditions may result in the reduction of peptide bonds to secondary amine groupings. Experiments with model peptides cited by the above authors would indicate that reduction of peptide bonds is unlikely under our conditions; namely, refluxing tetrahydrofuran, 0.1 M

lithium borohydride, and protein as an insoluble phase. A more serious complication has been studied by Hiirmann et al. (38).

Treatment of glycyl-phenylalanyl-serine with methanol-acetic anhydride led to partial migration of the glycyl-phenylalanyl group to the hydroxyl group of serine. If glutaminyl or asparaginyl bonds in the protein were involved in such a shift during esterification, one would expect the lithium borohydride to reduce the new ester bonds of serine or threonine. Conse- quently, the amount of glutamic or aspartic acid found in the hydrolysate would be lowered by an amount corresponding to the acyl shift.

A similar loss would occur if the glutaminyl or asparaginyl bond were involved in reductive cleavage of the kind reported by Crawhall and Elliott (39). Experiments with oligopeptides of silk fibroin and with lysozyme gave evidence that certain residues, particularly glycine and alanine, were reduced to amino alcohols with a concomitant cleavage of the chain. However, Hijrmann et al. (38) could find no evidence for reductive cleavage in model experiments with a limited number of pure peptide esters. The results which we have obtained do not support the idea that glutamine was destroyed by reductive cleavage under our condi- tions, since extended reduction from 24 to 72 hours did not result in a significant decrease of glutamic acid in the hydrolysate. The decrease in aspartic acid might have been due to slow reductive cleavage of aspara- gine residues. On the other hand, it may have been due to slow reduction of one or more of the esterified carboxyl groups.

The fluctuations in the estimations for glutamic acid and aspartic acid in the hydrolysates of reduced protein (see Table III) are not surprising in view of the above discussion and in view of the fact that the reaction is carried out with the protein as an insoluble phase. Therefore, the results must be accepted with reservation, but we believe that they are significant to within 1 residue as a measure of glutamine and asparagine. Of course,

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P. E. WILCOX, E. COHEN, AND W. TAN 1017

glutamic acid or aspartic acid in the hydrolysates may arise from isomeric forms of the residues, either isoglutamine or isoasparagine, if any exist in the protein.

Oxidation of chymotrypsinogen with performic acid, hydrolysis, and chromatography led to an average value for cysteic acid of 9.6 residues per mole. This value is in close agreement with the amount of cystine and cysteine found by Brand and Kassel (21) in hydrolysates of chy- motrypsinogen; the sum of these amino acids corresponded to 9.55 half cystine residues. All of our efforts and those of others (34) to detect sulfhydryl groups in denatured chymotrypsinogen have failed; therefore, residues of cysteine are not present in the protein. The recovery of sulfur in methionine from hydrolysates at 24 and 36 hours corresponds to 0.22 per cent of the protein, and the sulfur recovered in cysteic acid cor- responds to 1.23 per cent. Brand and Kassel (21) reported the sulfur content of sulfate-free chymotrypsinogen to be 1.47 per cent. It appears that all protein sulfur is divided between methionine and residues which give cysteic acid upon oxidation with performic acid.

SUMMARY

The amino acid composition of a-chymotrypsinogen has been deter- mined by chromatography on columns of Dowex 50. Changes in the amounts of the various amino acids in the acid hydrolysates were followed for 12, 24, 36, 48, and 72 hours of hydrolysis. Half cystine residues were separately determined as cysteic acid after oxidation of the protein with performic acid. Three different methods for the determination of amide nitrogen as ammonia were compared. Hydrolyses with 1 N NaOH, 5 N HCl, and concentrated HCl led to concordant results which indicated close to twenty-four amide groups per molecule.

For most of the amino acids, the analyses correspond to integral values for the number of residues in a molecule of weight 25,100. Essentially all of the weight and all of the nitrogen are accounted for by the recovered amino acids and amide nitrogen, if one adds also the content of tryptophan as reported by other investigators.

A study has been made of the estimation of glutamine and asparagine residues in chymotrypsinogen by esterification of free carboxyl groups with methanol-acetic anhydride, reduction of the ester groups with lithium borohydride, and hydrolysis of the reduced protein. Glutamine and asparagine appear in the hydrolysate as glutamic acid and aspartic acid, which are determined by a new chromatographic method. The significance and reliability of the results are discussed; it may be concluded that the results corroborate the amide nitrogen analyses and that each molecule of

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1018 COMPOSITION OF a-CHYMOTRYPSINOGEN

chymotrypsinogen contains 11 f 1 residues of glutamine and 14 f 1 residues of asparagine, including any isomeric forms present.

Thanks are due to Miss Anne Cederyuist for technical assistance in chromatographic analyses. Facilities for chromatographic analyses were generously provided by Dr. Hans Neurath. His advice and interest are gratefully acknowledged. The work reported in this paper has been supported by a grant from the National Science Foundation (No. G-1108) and a grant from the Initiative 171 Research Fund of the State of Washing- ton.

BIBLIOGRAPHY

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59, 145 (1955). 4. Chervenka, C. H., and Wilcox, I’. E., J. Biol. Chem., 222, 621 (1956). 5. Chervenka, C. H., and Wilcox, 1’. E., J. Biol. Chem., 222, 635 (1956). 6. Wilcox, P. E., Kraut, J., Wade, 12. D., and Neurath, H., Biochim. et bioph,ys.

acta, 24, 72 (1957). 7. Neurath, H., Rupley, J. A., and Dreyer, W. J., Arch. Biochem. and Biophys.,

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102 (1955). 15. Northrop, J. H., Kunitz, M., and Herriott, R. M., Crystalline enzymes, New

York, 2nd edition (1948). 16. Kunitz, M., J. Gen. Physiol., 32, 265 (1948). 17. Moore, S., and Stein, W. H., J. Biol. Chem., 1’76, 367 (1948). 18. Chinard, F. P., J. Riot. Chem., 199, 91 (1952). 19. Harfenist, E. J., J. Am. Chem. Sot., 75, 5528 (1953). 20. Moore, S., and Stein, W. H., J. Biol. Chem., 211, 893 (1954). 21. Brand, E., and Kassel, B., J. Gen. Physiol., 25, 167 (1941). 22. Hirs, C. H. W., Stein, W. H., and Moore, S., J. Biol. Chem., 211, 941 (1954). 23. Warner, R. C., and Cannan, R. K., J. Biol Chem., 142, 725 (1942). 24. Conway, E. J., and Byrne, A., Biochem. J., 27, 419 (1933). 25. Jacobsen, C. F., Compt. rend. trav. Lab. Cadsberg, S&rie chim., 26, 455 (1949). 26. Rees, M. W., Biochem. J., 40, 632 (1946). 27. Blackburn, S., and Phillips, H., Biochem. J., 38, 171 (1944). 28. Wilcox, P. E., Abstracts, XIIth International Congress of Pure and Applied

Chemistry, New York, Sept. (1951). 29. Moore, S., and Stein, W. H., J. Biol. Chem., 211, 907 (1954). 30. Connell, G. E., Dixon, G. H., and Hanes, C. S., Canad. J. Biochem. and Physiol.,

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Philip E. Wilcox, Elaine Cohen and Wen TanGLUTAMINE

ESTIMATION OF ASPARAGINE AND -CHYMOTRYPSINOGEN, INCLUDING

αAMINO ACID COMPOSITION OF

1957, 228:999-1019.J. Biol. Chem. 

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