Determination of Tryptophan in Proteins Joseph R. Spies Dairy Products Laboratory, Eastern Utilization Research and Development Division, Agricultural Research Seroice, U.S . Department of Agriculture, Washington, D. C . 20250
Two methods are described for the determination of tryptophan in proteins which eliminate inaccuracies inherent in the previously described intact protein method. In one method, protein is hydrolyzed in oxygen-free sodium hydroxide with basic lead acetate and histidine. In the other method protein is hydrolyzed with the enzyme Pronase. Both hydrolyzates are analyzed spectrophotometrically by previously described methods. Interference in spectrophotometric analysis of alkaline hydrolyzates by sulfur-containing compounds is eliminated with silver sulfate. I n general, the results obtained by the two methods of hydrolysis are in satisfactory agreement.
hydroxide containing basic lead acetate and histidine; Pronase hydrolysis and the intact protein method ( I ) . Objectives (a) and (b) were accomplished by application of the following previous observations : that histidine added to the alkaline hydrolytic solution prevented destruction of tryptophan by serine when heated together at 150' C ( I ) ; that basic lead acetate in the hydrolytic solution afforded some protection of tryptophan from cystine, and diminished interference by sulfur-containing decomposition products in the spectrophotometric analysis ( I ) and that silver sulfate did not interfere in the spectrophotometric analysis of tryptophan (5).
IN OUR PREVIOUS method for the determination of tryptophan in proteins, unhydrolyzed or intact proteins were used because of the partial destruction of tryptophan caused by cystine, cysteine, lanthionine, serine, and threonine during alkaline hydrolysis of proteins ( I ) . Maximum absorption of the colors formed with tryptophan and p-dimethylaminobenzaldehyde (DAB) in the intact protein method ranged from 580 to 620 mp (I) as compared with 590-600 mp for free tryptophan (2). Recently, Harrison and Hofmann (3) reported four proteins-liver alcohol dehydrogenase, apoferritin, diaphorase, and human serum albumin-that gave abnormal colors with absorption maxima of 545 to 560 when analyzed by our intact protein method. These authors eliminated this difficulty and obtained normal color when these proteins were first denatured, then partially digested with a mixture of trypsin and chymotrypsin before analysis. The observations of Harrison and Hofmann were confirmed in this laboratory on three of these proteins except that the enzyme, Pronase (4, was used on the undenatured proteins. Moreover, we observed that when /3-lactoglobulin was digested with Pronase before analysis, the tryptophan content was lower than that obtained by the intact protein method, even though normal color was obtained in both cases. This observation indicated that some of the tryptophan values obtained on the intact proteins, as previously reported ( I ) , might be too high. This possibility prompted reinvestigation of the determination of tryptophan in proteins to eliminate these inaccuracies of the intact protein method. The analysis of free tryptophan in the presence of the other free amino acids found in proteins presents no difficulty. Accordingly, the objectives of the present study were: (a) to devise a method for preventing destruction of tryptophan during alkaline hydrolysis of proteins; (b) to eliminate interference by sulfur-containing decomposition products in the spectrophotometric analysis of alkaline hydrolyzates; (c) to devise a milder method of hydrolysis with Pronase amenable to spectrophotometric analysis, and (d) to compare the tryptophan contents of various proteins as determined by : hydrolysis with sodium hydroxide; hydrolysis with sodium
EXPERIMENTAL
(1) J. R. Spies and D. C . Chambers, ANAL.CHEM., 21, 1249 (1949). (2) Zbid., 20, 30 (1948). (3) P. M. Harrison and T. Hofmann, Biochem. J., 80, 38P (1961). (4) M. Nomoto, Y . Narahashi, and M. Murakami, J. Biochem. (Tokyo),48, 593 (1960).
1412
ANALYTICAL CHEMISTRY
Apparatus and Materials. BOMB. The bomb with Teflon gasket was obtained from Parr Instrument Co. (No. 4760; volume 310 ml). The head has a connecting tube to which was attached a 100-mm length of silicone tubing (wall thickness, 3 mm, bore, 6 mm) for closure. CRUCIBLE HOLDER. The crucible holder is a solid brass cylinder, 60 mm in diameter and 95 mm high. Nine holes are bored near the periphery on the top of the cylinder and 1 hole was bored in the center of the top. The holes are 14 rnm in diameter and 16 mm deep. STANDARD TRYPTOPHAN. L-Tryptophan was recrystallized once from 6 5 z ethanol. The product was spectrophotometrically equivalent to Sample I1 ( 2 ) . PROTEINS. Sources of the proteins are given in the Tables. Apoferritin was prepared by the method of Behrens and Taubert (6). REAGENTS,The purest commercial reagent chemicals available were used, Basic lead acetate was the type used for sugar analysis containing 70-73% lead. Nitrogen was 99.999 pure. Nitrates, nitrites, other oxidizing agents, and reducing agents are impurities that must be avoided. Methods. WATERAND ASH. Water was determined by heating protein for 3 hours in an evacuated dryer at 110" C. Ash was determined by ignition in a platinum boat at 750850' C. TRYPTOPHAN STANDARD CURVES. Procedure H ( 2 ) was used for the standard curve of tryptophan for analysis of sodium hydroxide-containing solutions. Tryptophan was dissolved in 0.5N sodium hydroxide. Procedure G ( 2 ) was used for the standard curve for analysis of water or phosphate buffer solutions of tryptophan. PROCEDURE U (hydrolysis of proteins in oxygen-free, 5N sodium hydroxide saturated with basic lead acetate containing histidine, 50 mg per ml). The hydrolytic solution was freshly prepared as follows: 2.5 ml of 5N sodium hydroxide was added to 250 mg of basic lead acetate in a glass-stoppered flask. The suspension was shaken for 15-30 minutes and clarified by centrifugation. Seventy-five mg of histidine was dissolved in 1.5 ml of this solution. A 1- to 6-mg sample of pulverized protein, estimated to contain 10 to 100 pg of tryptophan, was weighed into a 1.3-ml standard platinum micro crucible, T o the sample was ( 5 ) J. R. Spies, ANAL.CHEM., 22, 1447 (1950). (6) M. Behrens and M. Taubert, Hoppe Seylers 2.Physiol. Chem., 290, 156 (1952).
z
added 100 p1 of the hydrolytic solution and 5 pl of a 1 solution of ketone-free, 2-octanol in toluene. Two procedures for conducting the alkaline hydrolysis are described. Strict adherence to directions is essential to avoid loss of sample by foaming. SEALED-IN-GLASS PROCEDURE. The charged crucible was placed in an 18 X 150-mm borosilicate glass, mediumwalled test tube containing 0.025 ml of water. The tube was constricted 60 mm above the crucible and then cooled at -20" C for 30 minutes. This temperature turns the hydrolytic mixture to slush-it should not be frozen solid. The cold tube, alternately evacuated to 25- to 35-mm pressure and filled with nitrogen three times, was sealed under the reduced pressure. Evacuation to lower pressures may cause foaming. Sealed tubes were placed in a covered metal container in an electric oven at room temperature. The oven was turned on and heated slowly up to a constant temperature in the range of 125-50" C. Ten hours after starting the heating, the oven was automatically turned off and allowed to cool slowly to room temperature without disturbing the tubes. T o open the tubes, air was admitted by gently pressing a 16 gauge (BStS) platinum wire, held with a hemostatic forceps, against the constricted part of the glass while heating the wire about 15 mm from the glass with a small oxygen flame. The small hole formed allowed slow admission of air. This technique prevented loss of solution from the crucible which occurred when the tip of the glass was broken off in conventional manner. Test for no loss of sample by foaming was done by adding phenolphthalein solution to the opened glass tube after removal of the crucible. BOMBHYDROLYSIS PROCEDURE.The bomb containing the crucible holder, but without the head, was cooled to - 20" C. T o the bomb was added 0.06 ml of water. The charged platinum crucibles were placed in the holes in the holder and each was covered with a perforated Teflon disk. A nickel cover, 62 mm in diameter, was placed on top of the crucible holder. The charged bomb then was cooled at -20" C for 30 minutes. The hydrolytic mixture should be slushy but not frozen solid. The capped bomb was alternately evacuated to 25- to 35-mm pressure and filled with nitrogen (4 pounds pressure) three times. The evacuated bomb, sealed by clamping the silicone tubing, then was placed in an electric oven at room temperature. The slow heating and slow cooling of the bomb was similar to that of the glass tubes described above. Test for no loss of sample by foaming was done by dropping each Teflon disk into dilute phenolphthalein solution. ANALYSISOF HYDROLYZATES. The platinum crucible containing hydrolyzate obtained by either method was set upright in a 25-ml glass-stoppered Erlenmeyer flask containing 9.0 ml of 21.2N sulfuric acid plus 30 mg of DAB. To the crucible was added 0.9 ml of a water solution containing 6 mg per ml of silver sulfate (5). The suspension was mixed with the acid solution by quickly tipping the crucible and swirling (this sequence is essential). The flask was cooled to room temperature in water and then agitated gently, mechanically, for 10 minutes at room temperature is subdued light (7). The suspension was transferred to a glass-stoppered tube and centrifuged at 25 O C. for 25 minutes at approximately 2600 X G. The clear supernatant solution was decanted into a 25-ml flask and placed in the dark at 25" C. Sixty minutes after mixing the hydrolyzate with acid, 0.1 ml of 0 . 0 4 5 z sodium nitrite solution was added and mixed to develop the color. After 40-45 minutes at room temperature, the transmittance was read at 590 mp. A controlled hydrolysis, without protein, served as a blank. Platinum crucibles were cleaned by brief boiling in dilute nitric acid, and ignition. (7) J. R Spies and D. C . Chambers, J. Am. Chem. SOC.,70, 1682 (1948).
Table I. Analysis of Tryptophan in Various 5N Sodium Hydroxide Solutions Containing 50 Mg Tryptophan per 100 kcrl Additional components of hydrolytic solution
Recovery, %a ProUnheated Heated (150" C ) cedure
99.8 i 0 . 9 (7) None Cystine, 1 mg serine, 1 mg 89.9 =tl.0(3) Histidine, 5 mg basic lead acetate, saturated 101.1 f 0 . 1 (3) Cystine, 1 mg serine 1 mg + histidine, 5 mg 96.7 & 0 . 8 (3) Cystine, 1 mg serine, 1 mg basic lead ace94.5 i 0.6 (3) tate, saturated Cystine, 1 mg serine, 1 mg histidine, 5 mg basic lead acetate, satu5 p1 rated 1 :100 2-octanol (ketone-free) in 99.4 & 0 . 3 (3) toluene
+ +
+
+ +
+ +
97.6 i 1.0(7)
V
=t1.9 (3)
Vb
101.2 i 0.3 (3)
U
98.4 =!= 0.0 (3)
U
92.6 i 0 . 5 (3)
U
100.9 i 0 . 4 (3)
U
62.7
+
+
a The number of determinations is in parentheses. Precision is expressed as average deviation of the mean. * Silver sulfate used as in Procedure U.
PROCEDURE V (hydrolysis of proteins in oxygen-free, 5N sodium hydroxide). This procedure was similar to Procedure U except that basic lead acetate and histidine were omitted from the hydrolytic solution. PROCEDURE W (determination of tryptophan in proteins after hydrolysis with Pronase). The Pronase hydrolytic solution contained 10 mg of Pronase per ml of 0.1M phosphate buffer, pH 7.5. Solutions were prepared on the day they were used. The Pronase suspension was shaken gently for 15 minutes and clarified by centrifugation. A 1- to 6-mg sample of pulverized protein, estimated to contain 10 to 100 pg of tryptophan, was weighed either into the 1.3-ml platinum crucible or a small glass vessel. T o the sample was added 100 p1 of Pronase solution. The suspension was stirred with a 23-mm platinum spatula which was left in the crucible. The crucible was placed in a vial, approximately 18 mm in diameter and 32 mm high, and one drop of toluene preservative was added. The vial was stoppered and then heated for 24 hours at 40" =+= 1 ' C. The cooled crucible, with spatula, was set upright in a 25-11-11glass-stoppered Erlenmeyer flask containing 9.0 ml of 21.2N sulfuric acid plus 30 mg of DAB. To the crucible was added 0.9 ml of 0.1M phosphate buffer solution. The crucible was tipped over and the contents quickly mixed by swirling (this sequence is essential). The flask was cooled to room temperature and placed in the dark at 25" C for 6 hours. Then 0.1 ml of 0.045x sodium nitrite was added. After 30 minutes, transmittances were read at 590 mp. The blank solution contained everything but protein and Pronase. Duplicate samples of the Pronase hydrolytic solution, without protein, were treated and analyzed as described above. The tryptophan content of the Pronase solution was subtracted from the total tryptophan. All of the insoluble proteins used in this study were digested to soluble products by the Pronase. VOL. 39, NO. 12, OCTOBER 1967
1413
Proteinb 0-Lactoglobulin, A B (5) 0-Lactoglobulin, A (4) fi-Lactoglobulin, B (4) &Lactoglobulin, C (4) 0-Lactoglobulin Goat 0-lactoglobulin (4) Sheep 0-lactoglobulin (4) Casein a.-Casein, A @,-Casein, B a,-Casein, C 0-Casein, A 0-Casein, B ,%Casein, C K-Casein, B a-Lactalbumin (3)
Table 11. Tryptophan Content of Some Milk Proteins0 Intact protein Wave length Pronase max. Proc. N abs. Alkaline hydrolysis hydrolysis Proc. Vc % Proc. Uc proc. Wd Z mp 1.98 f 0.06 2.11 f 0.04 1.99 f 0.01 2.31 590 1.92 f 0.01 2.03 & 0.03 2.09 f 0.01 1.92 f 0.04 2.11 f 0.03 1.98 f 0.01 2.11 f 0.01 2.05 f 0.00 2.13 f O . 0 1 1.98 f 0.02 2.12 f 0.03 2.09 f 0.04 2.14 f 0.03 2.14 f 0.05 1.30 f 0.01 1.22 i 0.02 1.26 i 0.02 1.62 610 1.56 f 0.02 1.95 1.62 f 0.02 1.45 f 0.00 610 1.47 f 0.01 1.59 f 0.02 1.42 f 0.01 1.46 f 0.02 1.57 f 0.02 1.44 f 0.01 0.828 f 0.014 0.858 0.876 f 0.008 0.851 f 0.001 580-590 0.794 f 0.001 0.832 f 0.001 0.818 f 0.001 0.854 f 0.003 0.812 =t0.012 0.827 f 0.003 1.01 f 0.00 1.25 610 1.15 f 0.00 4.90 f 0.06 5.26 f 0.09 5.17 f 0.01 620 6.02
z
z
Source Gordon Kalan Kalan Kalan (1)
Kalan & Basch Kalan (I)
Thompson Thompson Thompson Thompson Thompson Thompson Woychik Gordon
Tryptophan content is expressed as grams tryptophan per 100 grams of water and ash-free protein. Precision is expressed as the average deviation of the mean. Procedures V, U, and W are described in the text. Procedure N has been described previously ( I ) . * Number of recrystallizations in parentheses. c Average of triplicate, or more, determinations. Average of duplicate determinations.
Table 111. Tryptophan Content of Some Enzymes0
Proteinb a-Chymotrypsin (3) Emulsin Horse liver alcohol dehydrogenase (1) Lysozyme (4) Nagarse (crystalline) Pepsin (2) Trypsin (2) Ribonuclease A Ribonuclease B
Alkaline hydrolysis Proc. V,c % Proc. U,c % 5.28 i 0.03 6.14 i 0.05 1.09 i 0.01 1.06 f 0.01 1.06 i 0.01 7.08 f 0.02 1.74 2~ 0.02 2.42 f 0.04 3.10 f 0.06
1.09 f 0.01 7.76 f 0.07 1.98 f 0.03 2.75 1 0 . 0 4 3.45 f 0.03 0.01 0.04
Pronase hydrolysis proc. Wd, Z 5.95 f 0.05 1.03 f 0.01 1.03 f 0.03 7.66 i 0.16 1.94 f 0.03 2.67 f 0.01 3.35 f 0.03 -0.01
Intact protein Wave length max. abs., Proc. N, mfi 6.68 620 1.06 590-600
z
0.892 7.88 2.35 3.24 3.77
550 620 600 610 620
-0.01
Sourcee W W W M T W W S S
See footnote a, Table 11. Number of crystallizations in parentheses. c Average of triplicate determinations. d Average of duplicate determinations. e W, Worthington Biochemical Corp., Freehold, N. J.; M, L. R. McDonnell, see acknowledgment; T, Teikoku Chemical Industry Co., LTD, Osaka, Japan; S , Sigma Chemical Co., St. Louis, Mo. 5
b
Table IV. Tryptophan Content of Miscellaneous Proteins" Pronase hydrolysis proc. W, %" 0.872 f 0.001
Intact Protein Wave length max abs., Proc. N, mfi 0.872 570
Alkaline hydrolysis ~Protein Proc. V,* Proc. U,b Sourced Apoferritin 0.954 f 0.001 0.966 f 0.055 Bovine serum albumin (crystalline) 0.574 i 0.007 0.594 f 0.01 0.545 f 0,011 0.620 610 P Edestin 1.53 f 0.04 1.55 f 0.04 1.45 f 0.01 1.61 600 J Egg albumin D 1.33 f 0.05 1.42 f 0.02 1.26 f 0.05 (1) Conalbumin 2.61 f 0.04 2.82 It 0.02 2.87 f 0.10 3.35 610 N Human serum albumin 0.358 580 P 0.282 f 0.006 0.347 i 0.011 0.372 f 0.011 (crystalline) 600 J 1.44 f 0.02 1.54 1.50 f O . 0 4 1.44 f 0.02 Ox muscle 4.32 590 L 4.15 f 0.04 4.46 f 0.07 3.95 f 0.04 Subtilin 4 See footnote C , Table 11. b Average of triplicate determinations. c Average of duplicate determinations. P, Pentex Inc., Kankakee, Ill.; J, D. B. Jones ( I ) ; N, Nutritional Biochemical Corp., Cleveland, Ohio; L, J. C. Lewis, see acknowledgment.
1414
ANALYTICAL CHEMISTRY
z
RESULTS Table I shows analytical recoveries of free tryptophan after heating alone in oxygen-free 5N, sodium hydroxide under racemizing conditions (150’ C) and after similar heating with other substances : cystine and serine; histidine and basic lead acetate; cystine, serine, and histidine; cystine, serine, and basic lead acetate ; cystine, serine, histidine, basic lead acetate and 1 :100 ketone-free, 2-octanol in toluene. The tryptophan contents of some milk proteins, enzymes, and miscellaneous proteins, as determined by Procedures U, V, W, and N (1) are shown in Tables 11, 111, and IV, respectively. The number of tryptophan residues per mole in some of the proteins, calculated from values obtained by Procedure U, is shown in Table V.
DISCUSSION Results in Table I provide convincing evidence that protective agents can prevent destruction of tryptophan during hydrolysis of proteins with sodium hydroxide. These results show that 37% of a 50-pg sample of tryptophan was destroyed by 1 mg each of cystine and serine per test on heating at 150” C in oxygen-free, 5N sodium hydroxide in the absence of protective agents. But, recoveries were 98%, 93 %, and 101 % when 50 pg of free tryptophan was similarly heated with 5 mg of histidine per test, with saturated basic lead acetate, and with histidine and saturated basic lead acetate combined, respectively. Apparently histidine is an effective protective agent against both cystine and serine while basic lead acetate protects mainly against cystine. However, both histidine and basic lead acetate were used in Procedure U to enhance protection of tryptophan during hydrolysis inasmuch as using both required n o inconvenient modification of procedure. Other protein constituents d o not interfere in tryptophan analysis by Procedures U and W as shown by the essentially zero tryptophan values obtained on ribonucleases A and B by both methods. Although the wave length of maximum absorption obtained with various proteins analyzed by the intact protein method [Procedure N ( I ) ]ranged from 550 t o 620 mp (Tables 11, 111, and IV), maximum absorption of all proteins analyzed by Procedures U and W was the same as that of free tryptophan, 590-600 mp. The generally good agreement between the results obtained by Procedures U and W is substantiating evidence of the essential accuracy of the two methods. Thus, of 30 proteins analyzed by both methods, the differences between values were < 3 x with 13, < 6 % with 20, and
