Determination of Tryptophane m vVhale :Yieat
Shichiro Akiya, Keiichi Takahashi·Since the discovery in 1906 by Rose, Willock, and Abderhalden,' thattryptophalie was an indispensable amino acid in human body, many reports
have been published regarding its change and mechanism in the livingbody. The authors, in order to determine the content of this amino acidin whale meat and to determine whether whale meat could be utilized ail
resource for peptone production, carried out comparative tests on quantitative detennination of tryptophane and thereby found a novel revised
method.
Theoretical
There are already numerous methods of determining tryptophane,
which, unlike other amino acids, possesses indole nucleus in its structureso that there are qualitative, quantitative and colorimetric methods for its
determination. Homer1> led tryptophane to its bromo compound and thentitrated excess amount of bromine by iodometry. Homer maintains thattryptophane gives octabromo-tryptophane by bromination. However, Levnersays that it forms tribromo compound and this point has not been settled
as yet. If this point can be settled, bromination methhod is excellent andsimple because the :i::eaction by halogen is limited to free tryptophane
except ablin (N-methyltryptophane). All other tryptophane derivatives andaddition products do not ree:tct to this reagent.
Quantitative determination by Hopkins-Cole reag~nt of mercury-tryptophane method and its modification by Onslon~> by the determination of Nvalue in mercury-tryptophane are some of other methods but the mostordinary method is to use colorimetric measurement. Colorimetric reagent
employs, chiefly, glyoxylic acid, formaldehyde and p-dimethylaminobenzaldehyde, which possess carboxyl radical. Others employ xanthoprotein3> andPauly's diazo reactions but these are also positive to amino acids having
benzene ring, such as tyrosine and phenylalanine, so that a more complicated processes are required and cannot, moreover, be taken as a specificreaction for tryptophane. It follows, therefore, that colorimetric determi-

52Shichiro Akiya, Kciichi Takahashi

nation of tryptophane should employ reagents possessing C£!rboxyl radical,
especially the Ehrich reagent recommend by Neuber and Rohde, Hohm4 \May and Rose5).
Ehrlich reagent employs p-dimethylaminobenzaldehyde which is condensed to tryptophane in H1S04-acidity and the dyestuff is thus formed.
Chemical mechanism of the dyestuff formation can be explained as follows :/""-_I

II

NH..

I -.

II CH:!-CH-COOH

\/V
N

NII

I ~

11;
I+ O=C-/-\,""-=/

-N

/CH.

"-cH,,

H""""· .

-;. III

CH.-CH-COOH\/"'/'N-/-,/CH3 Hr

C-"- /-N,=~~-..

Colouring

The above reaction necessitates the presence of H+ so that, if OH is present, the reagent aldehyde does not condense to the nucleus but with theamino radical in the side chain. Ehrlich reagent, therefore, colors only
under strong acidity.
If tryptopnane itself undergo=s change only under this condition, thisreaction would become unsuitable for the determination of tryptophane.
The authors, therefore, observed the change in tryptophane, with considerations of the effects of reaction time and temperature, and found, as is
generally known, that tryptophane resisted acids. In other words, nochange could be observed when tryptophane was boiled in aqueous solutionof mineral acids. However, when carbohydrates, beside tryptophane werepresent, these reacted under strong acidity and formed the so-called Fumine
substance of unknown structure. In these cases, there is no sense incolorimetric determination of tryptophane. However, if this reaction iscarried out under 40°C, Fumirte substance is not formed, even under strong
acidity. This leaves the question of reaction time. As a result of determining minimum time required for total tryptophane in protein to be isolted by hydrolys it was found that, although it differed slightly according to
the proteins used, the hydrolysis was completed in 5-10 days with proteinsfrom whale meat and casein. Side reactions o~curred when the reaction
time was prolonged over two weeks and the amount of tryptophane found
was decreased considerably. These experiments were conducted under theDetermination of Tryptophane in Whale meat

53

presence of p-dimethylaminobenzaldehyde. The reason for this is that, evenif carbohydrates are isolated from proteins by hydrolysis, tryptophane will
have condensed with p-dimethylaminobenzaldehyde before it had time tobond with carbohydrates to form _Fumine substances. Rose determined theamount of tryptophane in protein by using Ehrljch reagent and 20% HCl,with the reaction at 35-37° for 24 hours and at room temperature for 48
hours, determining the coloration here formed. The authors learned that,uder thes~ conditions, complete hydrolyses of protein cannot be expected
and therfore, determineed the time where coloration by Ehrlich reagent
was at its maximum by determing amino form nitrogen by Van Slyke
method by which the degree of protein hydrolysis would be measured.
As a result, the afore-mentioned reaction time of 5-10 days, temperatureof 35"C, and degree of protein hydrolysis of 85-90% were obtained.
Reaction above these figures would give discoloration whkh eventually
gave decreased amount of tryptophane.
Results obtained by Boyd6, Hohm and Komn7, on pure tryptophane
coincided with the results obtained by the authors.
Experimental
Colorimetric Standard and Test Solution:
Preparation of Standard Solution : For the preparation of this solution,
Protiens not containing tryptophane, such as gelatine or zein, are used. 3a(a=50 mg) of this protein is added to 1 mg of pure tryptophane.
Test Solution: (1-2) a of sample protein is added to (1- 2) a of gelatine
bringing the amount of total protein to 3a. The object of the addition ofgelatine is to shorten reaction time somewhat and to control the coloration.
Method:100 g of the sample protein and .50 mg of gelatine are placed in lOOc.c.
measuring flask. On the other hand, standard substance is prep<:J.red byplacing 150 mg of gelatine and a determined quantity of pure trytophanein 100 c.c. measuring flask. 1 c.c. of Ehrlich reagent (5% p-dimethylaminobenzaldehyde in 10% HcSO. solution) is added to each of the above flasks,
shaken well, and bring the amount of solution to 100 c.c. by adding 20%
HCl. This is left in a thermostat at 35°C and the coloration observed everyday. Colorimetric determination with Dubosqui's colorimeter is made when
the coloration seems to be at a maximum. In the present experiment, theShichiro Akiya, Keiichi Takahshi

54

reaction required 5-10 days. Following are the results obtained :Content of tryptophane in whale meat.
After dehydration of whale meat by acetone, degveasing and drying, thesample is taken of this dried whale meat.. Crude protein contained in thesample was 66.3% as determined by Kjeldahl method. Content of tryptophane in whale meat.
---------------------------------

SampleTryptophane (%)

Average(%)

A

0.97
0.91
0.96
0.94

0.95

BcD

Content of Tryptophane in Casein not purified.
SampleTryptophan (%)

AB. 1.66
1.83
1.89c

References1) Homer: J. Biol. Chem. (1915) XXII 369.
2)

Onslow : Biochem. J. (1924) XVIII 63.

3) Tillmann: Biochcm. Z. (1928) 198 3719.
4) Hohm: J. Am. Chem. Soc. (1923)XIV 1788.
5) May and Rose : J. Biol. Chem. (1922) 54 213.
6) Boyd Biochem : J. (1929) 23 78.
7) Komm:

J. Physiol. Chem. (1922-~3) CXXIV 287.

Average(%)

1.79