Determination of Tryptophane, Phenylalanine, and Methionine A Rapid Procedure W. C. HESS
AND
M. X. SULLIVAN,
Cherno-Medical Research Institute, Georgetown University, Washington,
P
ROTEISS may be split into their constituent amino acids by enzymes or by hydrolysis with acids or alkalies. Because of the absence of humin in alkaline hydrolysis, such hydrolysis has been found to have some advantage over acid hydrolysis. For some amino acids, such as cystine which can be estimated readily in acid hydrolyzates, alkaline hydrolysis cannot be employed, since the cystine is more or less destroyed by alkali. On the other hand, for certain amino acids-for example, tryptophane-an acid hydrolysis is as a rule destructive, whereas as noted by many workers an alkaline hydrolysis is highly satisfactory. I n fact, practically the same value for tryptophane is obtained for a number of proteins by hydrolyzing for 18 to 24 hours with 5 N sodium hydroxide as was obtained with intact proteins (IS). In more recent work ( 7 ) , it has been reported that phenylalanine can be estimated colorimetrically following alkaline hydrolysis for 4 to 18 hours. The authors have estimated methionine (8, 11) satisfactorily in acid hydrolysis. Since Mueller (18) isolated methionine from hasein that had been hydrolyzed with 18% sodium hydroxide, it was probable that this amino acid also could be quantitatively determined by alkaline hydrolysis. In the determination of phenylalanine ( 7 ) it was found that digesting the protein in a sealed tube at 110" C. for 2 hours gave as good a value as 18 hours in an open tube. Accordingly similar hydrolyzates in a sealed tube were prepared and tryptophane, phenylalanine, and methionine were determined therein with satisfactory results.
D. C,
1,2-naphthoquinone-4sodium sulfonate (7) ; and methionine by the sodium nitroprusside method of McCarthy and Sullivan (11) modified slightly by Hess and Sullivan (8). The proteins used were all highly purified samples, for the most part prepared in this laboratory. The alpha- and beta-globulins of the mung and adzuki beans were preparations furnished some years ago by D. B. Jones of the U. S. Department of Agriculture. Simultaneously determinations of tryptophane were made upon the intact proteins by the modified Bates procedure as described by Sullivan and Hess (13). As detailed in previous publications (8, 11, 13) the procedures for estimating phenylalanine, methionine, and tryptophane are highly specific, sensitive, and readily reproducible with an error of 2 to 3%. For the present it can be said that the tryptophane and methionine methods will detect 0.02 mg. in 1 cc., while the phenylalanine procedure is sensitive to 6 micrograms per cc. The results corrected for moisture and ash, given in Table I, are average values for closely agreeing findings from two to four determinations. They may be compared with findings in the literature made on various types of hydrolysis, 6 to 18 hours' digestion with 55 to 57% hydriodic acid, 2 to 20 hours' hydrolysis with 25% sulfuric acid or 20y0 hydrochloric acid with and without urea, or 20% hydrochloric acid and concentrated formic acid, and 6 hours' digestion with 5 N sodium hydroxide. SUMMARY
The results for tryptophane are substantially the same whether the alkaline hydrolyzate or intact protein is analyzed. The phenylalanine contents of squash-seed globulin and casein are likewise the same as those previously obtained by the same method. The methionine content of casein is the same as that determined following hydrolysis with 20% hydrochloric acid, while the value for squash-seed globulin is but slightly higher. This method of hydrolysis permits a rapid assay of these three dietary essential amino acids in the same hydrolyzate, and is being applied to such determinations on a number of other purified proteins in conjunction with separate determinations of cystine and cysteine.
EXPERIMENTAL. The protein (150 mg.) was placed in a piece of glass tubing, about 12.5 cm. (5 inches) in length, previously sealed a t one end, 1cc. of 5 N sodium hydroxide was added, and the tube was sealed by pulling out the open end in a Bunsen flame and then placed in an oven a t 110" for 2 hours. The tube was occasionally inverted t o be certain of complete solution of the protein. When the tube was cool it was opened, and the contents with washings were poured into a 25-cc. graduate. The hydrolyzate was then acidified by the addition of 1 cc. of 14 -Y sulfuric acid and diluted, with water, to 20 cc. The resulting solution, approximately 0.5 N sulfuric acid, while usually clear, at times had some suspended silica, and therefore was always filtered. Determinations for the particular amino acids were made upon 5-cc. aliquots of the filtrate. Tryptophane waB determined by the modified Bates method (131 ; phenylalanine by nitration, reduction, and coupling w t h
LITERATURE CITED
(1) Baernstein, H. D., J. Biol. Chem., 97, 669 (1932); 115, 26 (1936). (2) Beach, E. P., and Teague, D. >I., Ibid., 142, 277 (1942). (3) Block, R. J., and Bolling, D., "hmino Acid Composition of Proteins and Foods", Springfield, Ill., Charles C Thomas, 1945. (4) Block, R. J.. Jervis. G. A.. Bollinn. D. and Webb, M., J. BioZ: Chem.:'134, 567 (1940). ' 5 ) Brand; E., Block, R. J., Kassell, B. Table 1. Tiyptophane, Methionine, and Phenylalanine Content of Protein and Cahill, G. F., J. Biol. Chem., Authors' Values Literature Values 119, 669 (1937). Phen, 1TryptoPhenir (6) Brown, 13'. L., Ibid., 142, 299 (1942). Proteir Tryptophane Methionine alanine phane Methionine alanine (7) Hess, W. C., and Sullivan, M. X., Arch. Biochem., 5, 166 (1944). 1 . 7 1 ( I S ) 2 . 3 9 (3) 2.78 5.85 1.60 Squash-seed globulin 1.690 (8) Hess, W. C., and Sullivan, M. X., J . 2 . 2 ( 6 ) 6 . 4 2 2 . 1 4 1 . 0 2 (9) 0.99 0.90 Conarachin Biol. Chem., 151, 635 (1943). 0 . 7 0 (9) 0 . 5 4 (I), 0 . 5 7 5.99 0.45 0.88 Arachin 0.87 ( 2 ), 0 . 6 7 (6') (9) Horn, M. H., and Jones, D. B., Ibid., ,... 2.41 1.39 0.81 0.78 a-Globulin mung 157, 153 (1946). .... 2.16'(f')'hv 5.1s 1.06 0.53 0.57 0-Globulin mung (10) Kapeller-Adler, R., Bioehem. Z., 252, ... 1.22 ( I ) 4.27 2.32 0.88 0.87 a-Globulin adruki ..... .... .... .... 185 (1938). . . . 1 :04 0-Globulin adsuki 2.31 (1) 3.99 1.81 1.02 a-Globulin lima (11) McCarthy, T. E., and Sullivan, M. X., 0.73 ( I ) 3.49 1. o i '(9) 1.55 0.96 0.98 @-Globulinlima J . Biol. Chem., 141, 871 (1941). 1 , 2 4 ( I 3) 3 . 1 0 to 3 . 5 2 5.79 3.52 1.28 1.17 Casein (f), 3.20 ( 5 ) (12) Mueller, J. H., Ibid., 56, 157 (1923). (13) Sullivan, M.X., and Hess, W. C.,Ibid., 155, 441 (1944). Phaseolin 0.51 0.49 1.59 4.53 0.70 (9) .... (14) Virtanen, H . I., Laine, T., and * All the values in thin vertical column are upon the intact proteins, the otheri upon the sealed-tube Toivonen, T . , Z. physiol. Chem., hydrolycates. 266, 173 (1940).
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