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ON THE QUANTITATIVE ESTIMATION OF TRYPTOPHAN
IN PROTEIN CLEAVAGE PRODUCTS.
BY P. A. LEVENE AND C. A. ROUJLLER.
(From the Rockefeller Ins&&e for Medical Research, New York.)
ON THE QUANTITATIVE ESTIMATION OF TRYPTOPHAN
IN PROTEIN CLEAVAGE PRODUCTS.
BY P. A. LEVENE AND C. A. ROUJLLER.
(From the Rockefeller Ins&&e for Medical Research, New York.)
ON THE QUANTITATIVE ESTIMATION OF TRYPTOPHAN
IN PROTEIN CLEAVAGE PRODUCTS.
BY P. A. LEVENE AND C. A. ROUJLLER.
(From the Rockefeller Ins&&e for Medical Research, New York.)
BY P. A. LEVENE AND C. A. ROUJLLER. (From the Rockefeller Ins&&e for Medical Research, New York.) (Received for publication, November 29, 1906.) The method of Hopkins and Cole i made possible the isolation of tryptophan from the other protein cleavage products. The methods of preparation of the pure substance always lead to
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appreciable loss and the yield of the pure substance does not furnish exact information regarding the real quantity of this constituent in the protein molecule. It seemed, therefore, de- sirable to devise a method by which tryptophan could be esti- mated quantitatively. A method which naturally suggested itself is the calorimeter, which, as is the case with all similar methods, is very imperfect. We preferred to resort to a method based on the following observation. When bromine water is added to a tryptophan solution a purple coloration develops. The intensity of the color increases with the continued addition of bromine water until a maximum is reached. At this phase the solution becomes very sensitive to further addition of the reagent. As little as one additional drop of bromine water causes the disappearance of the purple color. The nature of the chemical changes, which bring about the described reactions, will be studied in the future. In applying this property of bromine water in the quantitative estimation of tryptophan, one must bear .in mind that there are several other products of protein cleavage which combine with bromine, and some of them form colored substances, the presence of which renders it difficult to distinguish the end point of the tryptophan reaction. In order to obviate this difficulty it was found necessary to titrate, not the solution containing all the -products of protein cleavage, but only the fraction precipitated by the reagent of Hopkins and Cole. 1Journ. of Physiol., xxvii, p. 418, 1901. 461 482 Quantitative Estimation of Tqyptophan The process of tryptophan estimation in detail is then as follows: The solution of the products of protein digestion or hydrolysis is made to contain 5 per cent. of sulphuric acid and is then treated with the mercuric sulphate reagent i of Hopkins and Cole, which yields a precipitate. The reagent is added gradually, the supernatant liquid being tested with bromine water from time to time. Tt is preferable to end the addition of the bromine just at the point at which the supernatant liquid ceases to form the pur@e coloration. The mixture is then allowed to stand for twenty-four hours. The precipitate is filtered off, suspended in water containing not more than I to 2 per cent. of sulphuric acid,
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and decomposed by sulphuretted hydrogen. The filtrate from mercuric sulphide is heated on the water bath until all hydrogen sulphide is removed, brought to a definite volume. and is then ready for titration. Fifteen cubic centimeters of the solution are taken in a test tube for analysis. To this are added 2 cubic centimeters of amyl alcohol which serve for the extraction of the coloring matter. The bromine water is added gradually and the tube is shaken vigorously. The addition of bromine water is discontinued as soon as the purple color of the amyl alcoholic layer disappears. Duplicates of the same solution on titration consumed quantities of bromine water differing by from 0.05 to 0.10 cubic centimeter of the reagent. The concentration of the tryptophan, and the degree of acidity of th’e solution employed for analysis remain without influence on the end reaction. This can be seen from the following experiments. I. INPLIJENCEOPCONCENTRATJON. Tryptophan solution 10 cc. 5 C.C. Water 5 “ 10 “ Bromine water 1.85 “ 0.9 “ (2 cc. of amyl alcohol and G drops of a 15 per cent. solution of sulphuric acid were added to each tube.) II. INFLUENCE 0~ ACIDITY. Tryptophan solution 10 C.C. 10 c;p. Water 5 I‘ 5 Amy1 alcohol 2 ‘< 2 “ Sulphuric acid, 15 per cent. 6 drops - Sulphuric acid cont. - 6 drops
Bromine water 1.85 cc. 1.75 cc.
1 Ten per cent. of mercuric sulphate, dissolved in a 5 per cent, solution
of sulphuric acid. P. A. Levene and C. A. Rouiller
Since, however, the precipitate obtained by Hopkins’ reagent
contains cystin and tyrosin in addition to tryptophan, it was necessary to inquire into the influence of these substances on the titration with bromine. It was found that each of the two substances combines with bromine. T rosin solution 5 c;p. - d ater 10 15 C.C. Amy1 alcohol 2 “ 15 “ Sulphuric acid, 15 per cent. 6 drops 6 drops Bromine water 0.5 C.C. 0.15 C.C.
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Titrated until the amyl alcohol layer acquired a pale yellow coloration. Thus 5 cubic centimeters of the tyrosin solution cons&med 0.35 cubic centimeter of the bromine water. The bromine water consumed by a solution containing both tryptophan and tyrosin is equal to the sum of the quantities required for titration of each of the substances separately. This is seen from the following experiment. ‘J$yg;;Fhan solution 10 C.C. 10 C.C. 10 CF. 2.5 “ 5 d ater 2 I‘ 2.5 “ Amy1 Alcohol 2 “ 2 “ 5 “ Sulphuric acid, 15 per cent. 6 drops 6 drops 6 drops --- ---- --- Bromine water 1.85 C.C. 2.00 C.C. 2.2 C.C.
Deducting the bromine water required for saturation of the
tyrosin one finds that in every one of the three experiments the tryptophan required 1.8 cubic centimeters for its saturation. It should be remarked, however, that if precautions are taken the quantity of tyrosin present in the tryptophan fraction can be reduced to a mere trace. This is accomplished first by avoiding the addition of an excess of the mercuric sulphate solution. As already mentioned, the addition of the reagent is discontinued as soon as the remaining solution ceases to form the typical coloration with bromine water. The precipitate is to be washed with a 5 per cent. solution of sulphuric acid until the wash water contains no tyrosin. One can calculate the quantity of tryptophan in the presence of tyrosin by making a nitrogen estimation of the solution. Of greater importance is the presence of cystin as seen from the following experiment. 484 Quantitative Estimation of Tryptophan
Tryptophan solution 5 C.C. 5 C.C. -
Cystg$olutlon of sodium “ 5 C.C. Water lo “ ii 1: Amy1 alcohol 2 “ 2 l!j :: Sulphuric acid, 15 per cent. 6 drops 6 drops 6 drops --- - Bromine water 0.95 C.C. 1.75 C.C. 0.8 C.C. (The tryptophan solution contained 0.0926 gm. in 100 C.C. of water; of tyrosin 0.069 gm. in 50 c.c.; of cystin also 0.0708 gm. in 100 c.c.)
Since the quantity of cystin can be easily estimated by a
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sulphur determination there is no difficulty in calculating the quantity of tryptophan present in the solution. As already mentioned the quantity of tyrosin in the tryptophan fraction can be reduced to a mere trace, and since the solution binds but little bromine by its presence, can be disregarded. In esti- mating the tryptophan one should proceed in the following manner: (I) Titrate the solution of cystin and tryptophan; (2) In an aliquot part of the solution make a sulphur estimation and calculate the quantity of bromine required to saturate it; (3) Deduct the last figure from that obtained on titration of tthe solution containing both substances and the resulting figure represents the number of cubic centimeters of bromine water required to saturate the tryptophan. It is an advantage to standardize the bromine water with solutions of tryptophan and cystin before each analysis. A study of the conditions giving the best yield of tryptophan on protein cleavage, and of the quantity of the substance p&sent in various proteids is now in progress. ON THE QUANTITATIVE ESTIMATION OF TRYPTOPHAN IN PROTEIN CLEAVAGE PRODUCTS P. A. Levene and C. A. Rouiller J. Biol. Chem. 1907, 2:481-484.
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