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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.)
(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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