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L. Earle Arnow OSINE MIXTURES3,4-DIHYDROXYPHENYLALANINETYR

OF THE COMPONENTS OFCOLORIMETRIC DETERMINATIONARTICLE:

1937, 118:531-537.J. Biol. Chem.

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COLORIMETRIC DETERMINATION OF THE COMPO-NENTS OF 3,4-DIHYDROXYPHENYLALANINE-

TYROSINE MIXTURES

BY L. EARLE ARNOW

(From the Laboratory of Physiological Chemistry, University of Minnesota,Minneapolis)

(Received for publication, January 14, 1937)

It has proved necessary in recent investigations concerned witha possible mechanism for melanin formation to determine quanti-tatively the components of 3,4-dihydroxyphenylalanine-tyrosinemixtures. The method suggested by Schmalfuss and Lindemann(9) is based upon the previous observation of Schmalfuss andWerner (10) that the tyrosinase from the hemolymph of Arctia

caja catalyzes the conversion of 3,4-dihydroxyphenylalanine andtyrosine to melanin, this conversion being much more rapid inthe case of 3,4-dihydroxyphenylalanine than in the case oftyrosine. The melanin thus produced is determined colori-metrically.

The scheme presented in this paper is based on simple chemicalprocedures, no enzyme being required.

Determination of . I,&Dihydroxyphenylalanin,e

Reagents-1. 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 distilledwater.

3. 1 N sodium hydroxide.4. Standard solution. Dissolve 50 mg. of pure 3 ,Cdihydroxy-

phenylalanine in 500 cc. of distilled water contained in a liter

volumetric flask. Add 2 cc. of 0.1 N hydrochloric acid andenough distilled water to make a volume of 1 liter. Preserveunder toluene.

531


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532 3 ,4-Dihydroxyphenylalanine-Tyrosine

5. Alternative standard. If 3,4-dihydroxyphenylalanine isnot available, catechol can be used as a standard. Dissolve192 mg. of catechol in enough distilled water to make a volumeof 1 liter. Preserve under toluene. Dilute 10 cc. of this stocksolution to a volume of 100 cc. to make a standard solution.A green Wratten No. 61 filter (supplied by the Eastman KodakCompany) must be used in making the reading if this standardis used. 1 cc. of the catechol standard is equivalent to 1 cc. ofthe 3,4-dihydroxyphenylalanine standard described above.

Procedure-Place .l cc. of unknown solution (containing 0.02to 1.0 mg. of 3,4-dihydroxyphenylalanine) in a test-tube gradu-ated at 5 cc. Place 1 cc. of standard solution in a similar test-tube. Add to each test-tube, in the order given, the followingreagents, mixing well after each addition: 1 cc. of 0.5N hydro-

TABLE I

Determination of S,&Dihydroxyphenylalanine

Theoretical Determined Theoretical Determinedconcentration ooncentrstion concentration concentration

ng. per 1. mg. per 1. nag. psr 1.

20.0 20.5 60.830.0 30.5 69.240.0 40.1 79.450.0 50.4 100.2

mg. per 1.

60.070.080.0

100.0

chloric acid, 1 cc. of nitrite-molybdate reagent (a yellow color

results at this point), 1 cc. of 1 N sodium hydroxide (a red colorresults), and enough distilled water to make a volume of 5 cc.Compare in a Duboscq calorimeter. The green Wratten No. 61filter must be used if the catechol standard is used; it may beused with the 3,4-dihydroxyphenylalanine standard if theanalyst has difficulty in matching red colors. Tyrosine does notinterfere with this determination.

The accuracy of this method is indicated by Table I, which

lists results obtained by the analysis of pure 3,4-dihydroxy-phenylalanine solutions.Discussion-The determination of 3,4-dihydroxyphenylalanine

is based upon the fact that this substance gives a yellow colorwith nitrous acid, the yellow color changing to an intense orange-red in the presence of excess sodium hydroxide. Castiglioni (1)


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L. E. Arnow 533

has observed that compounds containing phenolic OH groupsyield colored compounds when heated with sodium nitrite. Hebelieves that hydrogen ions from the phenolic OH groups unitewith nitrite ions from the sodium nitrite, the molecular nitrousacid then forming NO compounds with the phenols. Various

10 BO-

I I I I I I-O@

450 500 550 600 650 700WAVE-LENGTH (MILLIMICRONS)

FIG. 1. Absorption spectra for colored solutions and transmission curvefor green Wratten No. 61 filter. Curve 1, 3,4-dihydroxyphenylalaninederivative; Curve 2, catechol derivative; Curve 3, tyrosine derivative;Curve 4, Wratten filter.

investigators (6, 8) have used nitrites to detect the presence ofepinephrine.

Purpose of the Sodium Molybdate-If sodium nitrite is added toan acid solution of 3,4-dihydroxyphenylalanine, the nitrous acidwhich is formed decomposes airly rapidly, and the final intensity


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of color which is produced depends to some extent on the timeduring which the reaction is allowed to proceed. The sodiummolybdate prevents the rapid decomposition of the nitrous acid.In addition, it causes an increase in color production of about50 per cent in the case of 3,4-dihydroxyphenylalanine, and ofabout 15 per cent in the case of catechol.

Purpose of Acidifying Standard Solution-Alkaline, neutral, oreven very slightly acid solutions of 3,4-dihydroxyphenylalanine

rapidly turn red, later depositing a precipitate of melanin. Theaddition of small amounts of hydrochloric acid prevents thischange.

TABLE IIColors Produced by Various Compounds

CompoundColor after add ition of

HCl and nitrite-molybdate

Ephedrine ... ... ... ... ... ... ... ... . Colorless

Phenol ............................. Light yellow(faint)Tyrosine ..... .... ..... ..... ..... ... ColorlessCatechol.. ......................... YellowEpinephrine .......................3,4-Dihydroxyphenylalanine .......Resorcinol.. ....................... Yellow-brownOrcinol.. ..... ..... ..... .... ..... .. Yellow (faint)

Pyrogallol.. ... ... ... ... ... ... ... ... Dark brown

Phloroglucinol.. ................... Orange ppt.

Color after add ition ofNaOH

Colorless

Yellow-brown(faint)

ColorlessRed

I

Dark brownYellow-brown

(faint)Dark red-brown

Yellow-brownsolution

Use of Wratten Filter-The color produced by 3,4-dihydroxy-phenylalanine is an orange-red; that produced by catechol is abright red. These colors can be matched by using the greenWratten No. 61 filter. The absorption spectra for the coloredcompounds and the transmission curve for the filter are shown in

Fig. 1. The extinction coefficient is defined by the equation

Extinction coefficient = f log 1n

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


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L. E. Arnow 535

droxyphenylalanine), d is the length in cm. of the column offluid through which the light is passing, and n is the fraction ofthe incident light transmitted by d cm. of colored fluid. It willbe observed that the wave-length of maximum light absorptionof the colored solutions (510 mp) is close to the wave-length ofmaximum light transmission of the filter (530 mp). The absorp-tion data were obtained with a Bausch and Lomb spectropho-tometer.

Xpecijkity of the Reaction-Table II. indicates that compoundscontaining only one phenolic OH group react weakly or not atall with the reagents. Compounds having two or three phenolicOH groups react strongly, orcinol being an exception to this rule.

Stability of the Color-The color is stable for at least 1 hour, butfades if left overnight.

Determination of Tyr osine

Reagents-

1. hilercuric sulfate reagent. Dissolve 15 gm. of mercuricsulfate in 100 cc. of 5N sulfuric acid.

2. Nitrite reagent. Dissolve 0.2 gm. of sodium nitrite in 100cc. of distilled water.

3. Standard solution. Dissolve 100 mg. of pure tyrosine inenough distilled water to make a volume of 1 liter. Preserveunder toluene.

Procedure-Place 1 cc. of unknown tyrosine solution (contain-

ing 0.05 to 0.15 mg. of tyrosine) in a test-tube graduated at 5cc. Place 1 cc. of standard solution in a similar test-tube. Addto each tube 1 cc. of mercuric sulfate reagent. After mixingwell, immerse both tubes in a boiling water bath for 10 minutes.Cool and add 1 cc. of nitrite reagent to each tube. Add enoughdistilled water to make a volume of 5 cc. If 3,4-dihydroxy-phenylalanine is present, the solution will be turbid. Centrifugeuntil clear; pipette off 3 to 4 cc. of the clear, red supernatantliquid, and compare with the standard in a Duboscq calorimeter.If centrifugation is not done, wait 5 to 10 minutes after the addi-tion of the nitrite reagent before comparing in the calorimeter.The author has less difficulty in matching greens than reds andhas used the Wratten filter in obtaining his readings. The ab-sorption spectrum of the tyrosine derivative is given in Fig. 1.


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Results obtained by analyzing both pure tyrosine solutions andtyrosine-3,4-dihydroxyphenylalanine mixtures are recorded inTables III and IV.

Discussion-The determination of tyrosine is based on theMillon reaction. The procedure described is a modification ofthe methods reported by Folin and Ciocalteu (2) and by Folin andMarenzi (5). The phenol reagent (2-4, 7) cannot be used in theanalysis of mixtures, since both tyrosine and 3,4-dihydroxy-

phenylalanine give the reaction.TABLE III

Determination of Tyrosine in Pure Solution s

Theoretical Determined Theoretical Determinedconcentration concentration concentration concentration

ng. per 1. mg. per 1.

50.0 51.8

70.0 70.9

90.0 90.1100.0 100.9

mg. per 1. 8. per 2.

110.0 109.9

130.0 129.8

150.0 147.1

TABLE IV

Determination of Mixtures

I Tyrosine T 3,4-DihydroxyphenyManineSolution No. Theoretical Determined

ooncentrrttion concentration

mg. per 2. mg. per 1.

1 60.0 61.02 100.0 100.0

3 140.0 142.04 60.0 60.1

5 80.0 79.3

Theoretical Determinedconcentration concentration

ng. per 1.

105.075.0

45.030.075.0

n g. per 2.

104.075.0

44.029.575.5

E ect of 3 ,4-Dihydroxyphenylalanine on Tyrosine Analysis-If 3,4-dihydroxyphenylalanine solution and the mercuric sulfate

reagent are mixed and immersed for 10 minutes in a boiling waterbath, 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; cen-trifugation is sufficient to remove it quantitatively.

Changes in Intensity of Color with Time-For about 4 or 5


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L. E. Arnow 537

minutes after the addition of the nitrite reagent the color steadilyincreases. After this it remains constant for at least 1 hour, butfades overnight.

SUMMARY

1. A calorimetric method for the quantitative determinationof 3,4-dihydroxyphenylalanine in the presence of tyrosine isdescribed. The method is based on the observation that an acidsolution of 3,4-dihydroxyphenylalanine reacts with a nitrite-molybdate 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 oftyrosine in the presence of 3,4-dihydroxyphenylalanine is de-scribed. The method is a modification of the Millon reaction.

3. Absorption spectra of the various colored solutions and thetransmission curve of the green Wratten No. 61 filter are included.

BIBLIOGRAPHY

1. Castiglioni, A., Gazz. chim. ita l., 62, 1065 (1932).2. Folin, O., and Ciocalteu, V., J. Bio l. Chem., 73,627 (1927).3. Folin, O., and Den is, W., J. Bio l. Chem., 12,239 (1912).

4. Folin, O., and Looney, J. M., .I. Bio l. Chem., 61,421 (1922).5. Folin, O., and Marenzi, A. D., J. Bio l. Chem., 83 ,89 (1929).6. Kisch , B., Biochem . Z., 220,358 (1930).

7. Looney, J. M., J. BioZ. Chem., 69,519 (1926).8. Riegel, E. R., and William s, J. F., J. Am. Chem. Sot., 48, 2871 (1926).9. Schm alfuss, H., and Lindemann, H., Biochem . Z., 184,lO (1927).

10. Sch ma lfuss , H., and Werner, H., Fermentforschung , 8,423 (1925).


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