J. Biol. Chem.

COLORIMETRIC DETERMINATION OF THE NENTS OF 3,4-DIHYDROXYPHENYLALANINETYROSINE MIXTURES

(From

the

Laboratory

BY L. EARLE ARNOW of Physiological Chemistry, Minneapolis)

(Received for publication,

January

COMPO-

University

of Minnesota,

14, 1937)

Determination of .!I,&Dihydroxyphenylalanin,e Reagents1. 0.5 N hydrochloric acid. 2. Nitrite-molybdate reagent. Dissolve 10 gm. of sodium nitrite and 10 gm. of sodium molybdate in 100 cc. of distilled water. 3. 1 N sodium hydroxide. 4. Standard solution. Dissolve 50 mg. of pure 3 ,Cdihydroxyphenylalanine in 500 cc. of distilled water contained in a liter volumetric flask. Add 2 cc. of 0.1 N hydrochloric acid and enough distilled water to make a volume of 1 liter. Preserve under toluene. 531

Downloaded from http://www.jbc.org/ by guest on May 4, 2015

It has proved necessary in recent investigations concerned with a possible mechanism for melanin formation to determine quantitatively the components of 3,4-dihydroxyphenylalanine-tyrosine mixtures. The method suggested by Schmalfuss and Lindemann (9) is based upon the previous observation of Schmalfuss and Werner (10) that the tyrosinase from the hemolymph of Arctia caja catalyzes the conversion of 3,4-dihydroxyphenylalanine and tyrosine to melanin, this conversion being much more rapid in the case of 3,4-dihydroxyphenylalanine than in the case of tyrosine. The melanin thus produced is determined colorimetrically. The schemepresented in this paper is based on simple chemical procedures, no enzyme being required.

532

3 ,4-Dihydroxyphenylalanine-Tyrosine

TABLE Determination Theoretical concentration ng. per 1.

20.0 30.0 40.0 50.0

I

of S,&Dihydroxyphenylalanine Determined ooncentrstion mg. per 1.

20.5 30.5 40.1 50.4

Theoretical concentration mg. per 1.

60.0 70.0 80.0 100.0

Determined concentration nag. psr 1.

60.8 69.2 79.4 100.2

chloric acid, 1 cc. of nitrite-molybdate reagent (a yellow color results at this point), 1 cc. of 1 N sodium hydroxide (a red color results), and enough distilled water to make a volume of 5 cc. Compare in a Duboscq calorimeter. The green Wratten No. 61 filter must be used if the catechol standard is used; it may be used with the 3,4-dihydroxyphenylalanine standard if the analyst has difficulty in matching red colors. Tyrosine does not interfere with this determination. The accuracy of this method is indicated by Table I, which lists results obtained by the analysis of pure 3,4-dihydroxyphenylalanine solutions. Discussion-The determination of 3,4-dihydroxyphenylalanine is based upon the fact that this substance gives a yellow color with nitrous acid, the yellow color changing to an intense orangered in the presence of excess sodium hydroxide. Castiglioni (1)


5. Alternative standard. If 3,4-dihydroxyphenylalanine is not available, catechol can be used as a standard. Dissolve 192 mg. of catechol in enough distilled water to make a volume of 1 liter. Preserve under toluene. Dilute 10 cc. of this stock solution to a volume of 100 cc. to make a standard solution. A green Wratten No. 61 filter (supplied by the Eastman Kodak Company) must be used in making the reading if this standard is used. 1 cc. of the catechol standard is equivalent to 1 cc. of the 3,4-dihydroxyphenylalanine standard described above. Procedure-Place .l cc. of unknown solution (containing 0.02 to 1.0 mg. of 3,4-dihydroxyphenylalanine) in a test-tube graduated at 5 cc. Place 1 cc. of standard solution in a similar testtube. Add to each test-tube, in the order given, the following reagents, mixing well after each addition: 1 cc. of 0.5 N hydro-

L. E. Arnow

533

has observed that compounds containing phenolic OH groups yield colored compounds when heated with sodium nitrite. He believes that hydrogen ions from the phenolic OH groups unite with nitrite ions from the sodium nitrite, the molecular nitrous acid then forming NO compounds with the phenols. Various

B

O-

-O@ I

450

I

500

I

550

I

I

I

600

650

700

WAVE-LENGTH (MILLIMICRONS) FIG. 1. Absorption spectra for colored solutions and transmission curve for green Wratten No. 61 filter. Curve 1, 3,4-dihydroxyphenylalanine derivative; Curve 2, catechol derivative; Curve 3, tyrosine derivative; Curve 4, Wratten filter.

investigators (6, 8) have used nitrites to detect the presence of epinephrine. sodium nitrite is added to Purpose of the Sodium Molybdate-If an acid solution of 3,4-dihydroxyphenylalanine, the nitrous acid which is formed decomposesfairly rapidly, and the final intensity


10-

534


TABLE

Colors Produced

by

II Various

Compounds

Color after addition HCl and nitritemolybdate

Compound

Ephedrine ......................... Phenol ............................. Tyrosine ........................... Catechol.. ......................... Epinephrine ........................ 3,4-Dihydroxyphenylalanine Resorcinol.. ....................... Orcinol.. .......................... Pyrogallol.. ........................ ................... Phloroglucinol..

........

of Color after addition of NaOH

Colorless yellow Light (faint) Colorless Yellow “ ” Yellow-brown Yellow (faint) Dark brown Orange ppt.

Colorless Yellow-brown (faint) Colorless Red ‘I Dark brown Yellow-brown (faint) Dark red-brown Yellow-brown solution

Use of Wratten Filter-The color produced by 3,4-dihydroxyphenylalanine is an orange-red; that produced by catechol is a bright red. These colors can be matched by using the green Wratten No. 61 filter. The absorption spectra for the colored compounds and the transmission curve for the filter are shown in Fig. 1. The extinction coefficient is defined by the equation Extinction

coefficient

= f

log 1 n

where c is the concentration in moles per liter (assuming 1 molecule of colored compound per molecule of catechol or 3,4-dihy-


of color which is produced depends to some extent on the time during which the reaction is allowed to proceed. The sodium molybdate prevents the rapid decomposition of the nitrous acid. In addition, it causes an increase in color production of about 50 per cent in the case of 3,4-dihydroxyphenylalanine, and of about 15 per cent in the case of catechol. Purpose of Acidifying Standard Solution-Alkaline, neutral, or even very slightly acid solutions of 3,4-dihydroxyphenylalanine rapidly turn red, later depositing a precipitate of melanin. The addition of small amounts of hydrochloric acid prevents this change.

L. E. Arnow

535

Determination

of Tyr osine

Reagents1. hilercuric sulfate reagent. Dissolve 15 gm. of mercuric sulfate in 100 cc. of 5 N sulfuric acid. 2. Nitrite reagent. Dissolve 0.2 gm. of sodium nitrite in 100 cc. of distilled water. 3. Standard solution. Dissolve 100 mg. of pure tyrosine in enough distilled water to make a volume of 1 liter. Preserve under toluene. Procedure-Place 1 cc. of unknown tyrosine solution (containing 0.05 to 0.15 mg. of tyrosine) in a test-tube graduated at 5 cc. Place 1 cc. of standard solution in a similar test-tube. Add to each tube 1 cc. of mercuric sulfate reagent. After mixing well, immerse both tubes in a boiling water bath for 10 minutes. Cool and add 1 cc. of nitrite reagent to each tube. Add enough distilled water to make a volume of 5 cc. If 3,4-dihydroxyphenylalanine is present, the solution will be turbid. Centrifuge until clear; pipette off 3 to 4 cc. of the clear, red supernatant liquid, and compare with the standard in a Duboscq calorimeter. If centrifugation is not done, wait 5 to 10 minutes after the addition of the nitrite reagent before comparing in the calorimeter. The author has less difficulty in matching greens than reds and has used the Wratten filter in obtaining his readings. The absorption spectrum of the tyrosine derivative is given in Fig. 1.


droxyphenylalanine), d is the length in cm. of the column of fluid through which the light is passing, and n is the fraction of the incident light transmitted by d cm. of colored fluid. It will be observed that the wave-length of maximum light absorption of the colored solutions (510 mp) is close to the wave-length of maximum light transmission of the filter (530 mp). The absorption data were obtained with a Bausch and Lomb spectrophotometer. Xpecijkity of the Reaction-Table II. indicates that compounds containing only one phenolic OH group react weakly or not at all with the reagents. Compounds having two or three phenolic OH groups react strongly, orcinol being an exception to this rule. Stability of the Color-The color is stable for at least 1 hour, but fades if left overnight.

536


Results obtained by analyzing both pure tyrosine solutions and tyrosine-3,4-dihydroxyphenylalanine mixtures are recorded in Tables III and IV. Discussion-The determination of tyrosine is based on the Millon reaction. The procedure described is a modification of the methods reported by Folin and Ciocalteu (2) and by Folin and Marenzi (5). The phenol reagent (2-4, 7) cannot be used in the analysis of mixtures, since both tyrosine and 3,4-dihydroxyphenylalanine give the reaction. TABLE

Theoretical concentration

mg. per 1.

51.8 70.9 90.1 100.9

110.0 130.0 150.0

TABLE Determination

I mg. per 2.

1 2 3 4 5

60.0 100.0 140.0 60.0 80.0

Determined concentration “8.

per 2.

109.9 129.8 147.1

IV of Mixtures

T

Tyrosine Theoretical ooncentrrttion

Solutions

Theoretical concentration

mg. per 1.

50.0 70.0 90.0 100.0

No.

in Pure

Determined concentration

ng. per 1.

Solution

III

of Tyrosine

Determined concentration mg. per 1. 61.0 100.0 142.0 60.1 79.3

3,4-DihydroxyphenyManine Theoretical concentration

Determined concentration

ng. per 1.

n&g. per 2.

105.0 75.0 45.0 30.0 75.0

104.0 75.0 44.0 29.5 75.5

E$ect of 3 ,4-Dihydroxyphenylalanine on Tyrosine AnalysisIf 3,4-dihydroxyphenylalanine solution and the mercuric sulfate reagent are mixed and immersed for 10 minutes in a boiling water bath, the solution assumes a faintly yellow color. After cooling, this yellow compound precipitates, forming a cloudy solution. The addition of the nitrite has no effect on this precipitate; centrifugation is sufficient to remove it quantitatively. Changes in Intensity of Color with Time-For about 4 or 5


Determination

L. E. Arnow

537

minutes after the addition of the nitrite reagent the color steadily increases. After this it remains constant for at least 1 hour, but fades overnight. SUMMARY

BIBLIOGRAPHY

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Castiglioni, A., Gazz. chim. ital., 62, 1065 (1932). Folin, O., and Ciocalteu, V., J. Biol. Chem., 73,627 (1927). Folin, O., and Denis, W., J. Biol. Chem., 12,239 (1912). Folin, O., and Looney, J. M., .I. Biol. Chem., 61,421 (1922). Folin, O., and Marenzi, A. D., J. Biol. Chem., 83,89 (1929). Kisch, B., Biochem. Z., 220,358 (1930). Looney, J. M., J. BioZ. Chem., 69,519 (1926). Riegel, E. R., and Williams, J. F., J. Am. Chem. Sot., 48, 2871 (1926). Schmalfuss, H., and Lindemann, H., Biochem. Z., 184,lO (1927). Schmalfuss, H., and Werner, H., Fermentforschung, 8,423 (1925).


1. A calorimetric method for the quantitative determination of 3,4-dihydroxyphenylalanine in the presence of tyrosine is described. The method is based on the observation that an acid solution of 3,4-dihydroxyphenylalanine reacts with a nitritemolybdate reagent to give a compound having a yellow color, this color changing to red on addition of excess alkali. 2. A calorimetric method for the quantitative estimation of tyrosine in the presence of 3,4-dihydroxyphenylalanine is described. The method is a modification of the Millon reaction. 3. Absorption spectra of the various colored solutions and the transmission curve of the green Wratten No. 61 filter are included.

ARTICLE: COLORIMETRIC DETERMINATION OF THE COMPONENTS OF 3,4-DIHYDROXYPHENYLALANINETYR OSINE MIXTURES

Access the most updated version of this article at http://www.jbc.org/content/118/2/531.citation Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites . Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/118/2/531.citation.full.h tml#ref-list-1


L. Earle Arnow J. Biol. Chem. 1937, 118:531-537.