The various colorimetric methods now employed for the estimation of tryptophan are not specific for tryptophan but give colored products with many compounds containing the indole nucleus, including indole itself.  In order to facilitate studies of the enzymatic synthesis of tryptophan from indole and serine by extracts of Neurospora (1), a search for a rapid, quantitative method for the estimation of tryptophan in the presence of indole was undertaken.  Tauber (2) reported that tryptophan gives a green fluorescence when treated with 70 to 72 per cent perchloric acid at room temperature.  Modification of this procedure has led to a method for the rapid estimation of tryptophan, without preliminary extraction of indole, in hydrolyzed or unhydrolyzed tryptophan-containing materials

Gordon, Malcolm

Mitchell, Herschel K.

English

Caltech Authors - Main

A FLUOROMETRIC METHOD FOR THE ESTIMATION OF 
TRYPTOPHAN* 
BY MALCOLM GORDON? AND HERSCHEL K. MITCHELL 
(From the Kerckhoff Laboratories of Biology, California Institute of Technology, 
Pasadena) 
(Received for publication, Ma,y 7, 1949) 
The various calorimetric methods now employed for the estimation of 
tryptophan are not specific for tryptophan but give colored products 
with many compounds containing the indole nucleus, including indole 
itself. In order to facilitate studies of the enzymatic synthesis of tryp- 
tophan from indole and serine by extracts of Neurospora (l), a search for a 
rapid, quantitative method for the estimation of tryptophan in the pres- 
ence of indole was undertaken. Tauber (2) reported that tryptophan 
gives a green fluorescence when treated with 70 to 72 per cent perchloric 
acid at room temperature. Modification of this procedure has led to a 
method for the rapid estimation of tryptophan, without preliminary ex- 
traction of indole, in hydrolyzed or unhydrolyzed tryptophan-containing 
materials. 
EXPERIMENTAL 
Apparatus-Intensity of fluorescence is measured with a Coleman elec- 
tronic photofluorometer, model 12B. The light entering from the mercury 
vapor lamp is caused to pass through a PC-3 filter and the resulting fluores- 
cence through a PC-10 filter. A 2 in. round test-tube of the type ordinarily 
used with this instrument is fitted with a leucite ring bored to support a 
22 X 100 mm. test-tube inside the larger tube. Test solutions are placed 
in the inner tube. A volume of 2.5 ml. is sufficient to allow the entering 
light to focus in the test solution. Under these conditions readings are 
reproducible to within f2 scale divisions. 
Reagents- 
1. 70 to 72 per cent perchloric acid (Merck reagent). 
2. Aqueous indole. Dissolve 117 mg. of indole (Eastman) in 100 ml. 
of distilled water. 
3. Aqueous -r-(indole-3)-n-butyric acid. Dissolve 10 mg. of r-(indole3)- 
n-butyric acid (Eastman) in 100 ml. of distilled water. 
* This work was supported by funds from the Rockefeller Foundation and by funds 
from the Atomic Energy Commission, administered through contract with the Office 
of Naval Research, United States Navy (contract N-6-onr-244, Task Order 5). 
t Merck Fellow of the National Research Council. 
1065 
1066 DETERMINATION OF TRYPTOPHAN 
4. 10 per cent acid-hydrolyzed casein (Nutritional Biochemicals Corpo- 
ration). 
5. Phosphate buffer, pH 7.5 to 8.0. Dissolve 14.7 gm. of disodium 
hydrogen phosphate (Merck reagent) in distilled water and make up to a 
liter. Adjust pH with phosphoric acid. 
6. Supplement. Mix 1 ml. of indole solution, 1 ml. of r-(indole-3)-n- 
butyric acid solution, and 1 ml. of 10 per cent acid-hydrolyzed casein. 
Dilute to 10 ml. with phosphate buffer. The supplement should be made 
up fresh weekly and kept in a refrigerator when not in use. 
Procedure for Assay-In order to obtain results which have quantitative 
significance it is necessary to stabilize the fluorescence. Such stabili- 
zation can be effected by the use of the supplement described above. 
The sample to be tested is taken up in the phosphate buffer and 0.4 ml. 
TABLE I 
Fluorescence Obtained by Treating Certain Indcle Derivatives with 70 to 7.2 Per Cent 
Perchloric Acid 
Each tube contains 0.05 PM of the compound. 
Scale I I reading 
Indole* 2 j3- (Indole-3)-n-propionic acid 70 
Indole-2-carboxylic acid 1 y- (Indole-3) -n-butyric acid 35 
Indole-3-carboxylic acid 2 Tryptophan* 100 
3-Methylindole 38 
Indole-a-acetic acid 55 
- 
*The photofluorometer is adjusted to a zero scale reading with a mixture of 0.5 
ml. of distilled water and 2 ml. of 70 to 72 per cent perchloric acid. The sensitivity 
is so adjusted that the tryptophan tube reads 100. 
is added to 0.1 ml. of the supplement solution. 2 ml. of perchloric acid 
are added rapidly at room temperature, the solution is well mixed, warmed 
for 1 hour at 40”, and allowed to cool to room temperature. A series of 
tubes prepared in the same manner containing 0, 2, 4, 6, and 8 y of tryp- 
tophan is used to adjust the photofluorometer. The instrument is adjusted 
to a zero scale reading with the blank tube and the sensitivity is adjusted 
with the 8 y tube to a scale reading of 100. The intermediate tubes will 
read approximately 25, 50, and 75, respectively. The unknown solutions 
are read with this setting of the instrument. Occasional readjustments 
of the zero and 100 readings are desirable during the course of a large num- 
ber of determinations. Since the intensity of fluorescence slowly dimin- 
ishes, it is necessary to adjust the instrument with a control series prepared 
at the same time as the unknown solutions. The linear relationship 
between the intensity of fluorescence and the concentration of tryptophan 
persists for about 24 hours. 
M. GORDON AND H. K. MITCHELL 1067 
In&ewe of Various Materials on Relative Stability of Fluorescence--The 
relative stability of fluorescence is a sensitive function of several materials 
which do not contain tryptophan. High concentrations of many inorganic 
TABLE II 
Tryptophan Found in Various Materials (in Per Cent 7 
Material 
Albumin egg stalest 
Unhydrolyzed* 
- 
. - 
- 
Hydrolyzed 
‘r!i - 
_ 
Crystalline bovine serum al- 
bumin$ 
Caseins 
Edestinll 
Gelatin7 
Gluten** 
Zeintt 
Hot water Neurospora extract 
(autolyzed) 
Acid-hydrolyzed casein + 2% 
added tryptophan 
2.04 
1.98 
2.04 
0.72 
0.76 
0.69 
1.40 
1.32 
1.38 
1.44 
1.46 
1.38 
0.016 
0.021 
0.017 
0.84 
0.86 
0.94 
0.22 
0.20 
0.34 
0.32 
2.00 
2.08 
2.04 
1.88 
1.96 
0.68 
0.70 
0.65 
1.33 
1.27 
1.24 
1.30 
1.29 
1.32 
0.010 
0.012 
0.75 
0.81 
0.21 
0.19 
0.30 
0.29 
1.89 
1.84 
1.90 
- 
rptophan) 
Literature values 
0.57 (4) 
0.65 (5) 
0.68 (5) 
1.32 (4) 
1.38 (5) 
1.54 (6) 
1.3 (7) 
1.3 (8) 
1.2 (4) 
1.4 (10) 
0.0 (11) 
0.0 (9) 
0.1 (12) 
0.91 (4) 
0.93 (6) 
1.2 (13) 
0.2 (14) 
0.2 (9) 
0.0 (12) 
* Separate determinations on the same sample. 
t Albumin egg scales, J. T. Baker Chemical Company. 
# Crystallined bovine serum albumin, the Armour Laboratories, G 4502 plasma. 
$ Casein, vitamin-free S. M. A. Corporation batch No. 12537. 
11 Edestin, laboratory sample of unknown origin. 
pI Gelatin, Difco batch No. 326614. 
** Gluten, Braun, Knecht, Heimann Company. 
tt Zein (corn), Pfanstiehl Chemical Company, batch No. 536. 
ions, concentrations of organic materials above 10 mg. per tube, prolonged 
heating or exposure to direct sunlight or ultraviolet light, and either too 
high or too low a concentration of hydrogen ion cause uneven fading of 
fluorescence. The NazHPOl adjusts for this last effect, except in strongly 
1068 DETERMINATION OF TRYPTOPHAN 
alkaline or acidic solutions. In such cases the sample should be neutralized 
before being taken up in the buffer solution. 
Occasional samples of perchloric acid, when mixed with tryptophan 
by the described procedure, will not produce the substances which fluoresce 
under ultraviolet light. It seems probable that these samples are con- 
taminated with one or more agents which inhibit fluorescence. 
TIME (in hours) 
FIG. 1. Per cent conversion of indole to tryptophan by extracts of Neurospora. 
0, calculated from tryptophan found by perchloric acid method; 0, calculated from 
indole found by xanthydrol method of Fearon. 
Speci$city of Test-Table I gives data comparing the fluorescence ob- 
tained when 0.05 PM of several compounds containing an indole nucleus is 
treated with 70 to 72 per cent perchloric acid. Tests for tryptophan 
based upon its reaction with aldehydes in the presence of mineral acids 
are thought to be characteristic of indole and indole derivatives containing 
a hydrogen-substituted 2-carbon atom. In contrast, the fluorescence ob- 
tained in this test appears to be a function of both a hydrogen-substituted 
2-carbon atom and a side chain in the 3 position of the indole nucleus. 
Estimation of Tryptophan in Various Organic Matetials-Table II gives 
M. GORDON AND H. K. MITCHELL 1069 
tryptophan values obtained by the above procedure for various substances 
both before and after hydrolysis for 1 hour with 2 N NaOH. All samples 
were dried in vucuo for 4 hours at 65” over PZ06. Literature values 
for tryptophan in similar materials are listed for comparison. 
Fig. 1 is a plot of the increase of tryptophan concentration found by 
this method when a crude dialyzed Neurospora extract is treated with 
pyridoxal phosphate, indole, and serine, and incubated at 35” at pH 
7.8. On the same graph is plotted the percentage conversion of indole to 
tryptophan obtained by measuring the decrease in indole concentration 
by the method of Fearon (3). 
DISCUSSION 
The nature of the reactions involved in the method herein described 
is unknown. Therefore, justification of the use of the recommended 
mixtures of amino acids, indole, and r-(indole-3)+butyric acid to obtain 
relative stability of fluorescence must be a strictly empirical one. How- 
ever, other workers have recommended the use of amino acid mixtures 
to stabilize tryptophan in highly acidic solutions (4, 15, 16). Graham 
et al. (4) suggest the use of 35 mg. of gelatin in order to stabilize the color 
obtained when tryptophan is estimated by the Bates (17) procedure. 
Since the samples of gelatin tested during this investigation contained 
detectable traces of tryptophan, attempts were made to obtain relative 
stability of fluorescence by use of acid-hydrolyzed casein. All experi- 
ments with this in view were negative and the addition of an indole-con- 
taining compound with a side chain in the 3 position was required before 
adequate stability was obtained. The test was standardized with 
r-(indole-3)-n-butyric acid because of the low fluorescence produced by 
it in this procedure. Although the test could no doubt be modified by 
using small amounts of tryptophan itself as the stabilizing indole compo- 
nent, it seems less ambiguous to use a material which is unknown in bio- 
logical systems. 
Readings on solutions containing 1 to 8 y of tryptophan can be re- 
produced to within f2 scale divisions. However, with scale readings 
below 50, this represents an error of at least 4 per cent, depending upon 
where the reading falls on the scale. If the samples to be tested are chosen 
so that they contain 6 to 8 y of tryptophan, determinations can be repeated 
with an error of not more than f5 per cent. 
Tauber (18) has reported that small quantities of dichromate enhance 
the fluorescence obtained in his qualitative test. With the quantities 
of tryptophan used in this work, dichromate was found to have a deleterious 
effect on the stability of the fluorescence. 
1070 DETERMINATION OF TRYPTOPHAN 
STJMMARY 
A new method for the estimation of tryptophan has been described. 
Tryptophan can be quantitatively determined in unhydrolyaed proteins. 
Determinations are reproducible approximately to within 5 per cent. 
BIBLIOGRAPHY 
1. Umbreit, W. W., Wood, W. A., and Gunsalus, I. C., J. Biol. Chem., 166,731 (1946). 
2. Tauber, H., J. Am. Chem. Sot., 70, 2615 (1948). 
3. Fear-on, W. R., Analyst, 69, 122 (1944). 
4. Graham, C. E., Smith, E. P., Hier, S. W., and Klein, D., J. Riol. Chem., 168, 
711 (1947). 
5. Hauschildt, J. D., Isaacs, T. L., and Wallace, W. B., J. BioZ. Chem., 167, 331 
(1947) . 
6. Greenhut, I. T., Schweigert, B. S., and Elvehjem, C. A., J. Biol. Chem., 166, 325 
(1946). 
7. Shsw, J. L. D., and McFarlane, W. D., J. BioE. Chem., 132,387 (1940). 
8. Folin, O., and Marenzi, A. D., J. Biol. Chem., 83, 103 (1929). 
9. Folin, O., and Looney, J. M., J. BioZ. Chem., 61,421 (1922). 
10. Kiesel, A., and Kusmin, S., Z. physiol. Chem., 238,145 (1936). 
11. Albanese, A. A., and Frankston, J. E., J. BioZ. Chem., 144,563 (1942). 
12. May, C. E., and Rose, E. R., J. BioZ. Chem., 64,213 (1922). 
13. Padoa, M. L., Ann. chim. appl., 21,544 (1938); Chem. Abstr., 32.6297 (1938). 
14. Folin, O., and Ciocalteu, V., J. BioZ. Chem., 73, 627 (1927). 
15. Sullivan, M. X., Milone, H. S., and Everitt, E. L., J. BioZ. Chem., 126,471 (1938). 
16. Doty, D. M., Id and Eng. Chem., Anal. Ed., 13,169 (1941). 
17. Bates, R. W., Proc. Am. Sot. BioZ. Chem., J. BioZ. Chem., 119, p. vii (1937). 
18. Tauber, H., J. BioZ. Chem., 1’77, 337 (1949). 


A fluorometric method for the estimation of tryptophan

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A fluorometric method for the estimation of tryptophan

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