THE CHEMISTRY OF MELANIN

V. OXIDATION OF DIHYDROXYPHENYLALANINE BY TYROSINASE*

BY HOWARD S. MASON AND CHARLES I. WRIGHT

(From Ihe Clinical Investigations Section, Division of Industrial Hygiene, and the Experimental Biology and Medicine Institute, National Institutes of Health,

United States Public Health Service, Bethesda, Maryland)

(Received for publication, April 13, 1949)

The sequence of reactions which occurs when 3,4-dihydroxyphenylalan- ine is oxidized in the presence of tyrosinase is now considered to be as in the accompanying diagram (1, 2). This process has been reported to require from 3.2 to 4.12 atoms of oxygen per molecule of dihydroxyphenyl- alanine (3-5). Detection of the carbon dioxide which should be evolved during this reaction sequence has not, to our knowledge, been reported. In the present study the oxygen consumed under varying conditions of pH, enzyme concentration, and buffer salts has been determined; carbon dioxide evolved under corresponding conditions but at a fixed pH, 5.1, was also measured. The results were found to support and extend the above concept of melanogenesis.

EXPERIMENTAL

The measurements of gaseous exchange were made with Barcroft type differential manometers fitted with side arm flasks of approximately 18 ml. capacity. The reagents in each flask included 0.2 ml. of 10 per cent KOH or buffer in the central well. The fluid volume was brought to 3.0 ml. by employing 2.3 ml. of buffer containing 0.65 mg. of dihydroxyphenyl- alanine (3.3 X lo-” mole) and 0.5 ml. of tyrosinase in distilled water (side arm). Further details of the manometric technique have been given in a previous paper (6). Carbon dioxide was determined by difference in the apparent oxygen consumption in the presence and absence of KOH, prop- erly corrected by the COZ factor. By experiment it was shown that no carbon dioxide was retained as bicarbonate at pH 5.1.

The tyrosinase employed in this study was purchased from the Tree- mond Company’ and was labeled to contain 3500 catecholase units per ml, (8). 3,4-Dihydroxyphenyl-L-alanine and 3,4-dihydroxyphenyl-m-al- anine were obtained from Hoffmann-La Roche, Inc. They melted at

* For the preceding paper in this series, see Mason, H. S., Kahler, H., MacCardle, R. C., and Dalton, A. J., Proc. Sot. Exp. Biol. and Med., 66,421 (1947).

1 The enzyme was prepared according to the procedure of Mallette et al. (7) from the common mushroom (personal communication).

235

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236 CHEMISTRY OF MELANIN. V

272-278’ and 270-274” (corrected) respectively. Unless otherwise noted, the raceme was employed as substrate in the experiments described below. The phosphate-citrate buffers referred to had the following compositions: at pH 6.6, 0.273 M disodium phosphate and 0.027 M citric acid; at pH 7.4, 0.305 M disodium phosphate and 0.012 M citric acid. Other buffers were prepared from 0.2 M phosphate, citrate, and acetate solutions of the de- sired pH by direct dilution. Measurement showed that dilution from 0.2 to 0.1 M caused a pH increase in the phosphate buffers of less than

10 0 HO HH

0 Enzyme 1

0 HO

0’ - H HO- H

0 1

Enzyme (?)

Melanin

+ co2

0 H

OH- t-

0 HH Hallachrome

H+

HO

COOH HO H

0.1 unit, and in the citrate buffer, 0.2 unit. Dilution of from 0.2 to 0.01 M caused a pH increase of 0.2 to 0.3 unit in the phosphate buffers, and 0.4 unit in the citrate buffer. The acetate buffer was not affected by dilution in the range of pH and concentration utilized. The effect of dilution upon hydrogen ion concentration must be taken into account in interpreting Figs. 2, 5, 7, and 8. Buffer concentrations are given below in terms of final values in the reaction vessels.

Results

Fig. 1 shows the results obtained when 0.65 mg. of dihydroxyphenyl- alanine was oxidized in the presence of increasing amounts of enzyme at pH 7.4 and 6.6. In 0.164 M phosphate buffer (pH 7.4) concentrations

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H. S. MASON AND C. I. WRIGHT 237

of enzyme between 22 and 175 units catalyzed the consumpt.ion of oxygen to 170 ~1. in 240 minutes; with 11 units the oxygen consumption dropped to 132 ~1. In phosphat,e-citrate buffer at the same pH, increasing the tyrosinase concentration from 11 to 44 units increased the oxygen con- sumed in 240 minutes from 130 to 170 ~1.; further increase of enzyme

ol’ 0 60 I60 240 0 00 160 240

MINUTES

FIG. 1. The oxygen consumed during the eneymic oxidation of 0.65 mg. of dihy- droxyphenyl-nn-alanine with increasing quantities of tyrosinase in (A) 0.164 M ghos- phate buffer, pH 7.4; (B) phosphate-citrate, pH 7.4; (C) 0.164 M phosphatje buffer, pH 6.6; and (D) phosphate-citrate buffer, pH 6.6. The catecholase units of enzyme employed are indicated at each curve. The dotted lines show the volumes equiva- lent to 2 and 4 atoms of oxygen per molecule of dihydroxyphenylalanine.

caused no further increase in oxygen consumption. At pH 6.6, in 0.164 M phosphate buffer, increasing the enzyme concentration from 11 to 22 units per 2.8 ml. of reaction volume caused an increase in the oxygen con- sumed at 240 minutes from 125 to 165 ~1.; further increase of enzyme con- centration had no effect. In phosphate-citrate buffer at the same pH, increasing enzyme concentration to 44 units caused an increase of oxygen

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238 CHEMISTRY OF MBL4NIN. V

consumed at 240 minutes to 155 ~1.; further addition of enzyme had no effect.

The influence of buffer concentration upon oxygen consumption at approximately neutral pH is shown in Fig. 2. In these experiments the initial tyrosinase concentration was 88 units per 2.8 ml. to insure that oxygen consumption would be independent of enzyme concentration (Fig. 1). Fig. 2, A shows that, as buffer concentration was changed from 0.008 M (pH 7.6) to 0.164 M (pH 7.4) phosphate, the oxygen consumed at 240 minutes increased from 145 to 170 ~1. As the buffer concentration was similarly varied at approximately pH 6.6 (Fig. 2, B), the oxygen con- sumed at 240 minutes increased from 112 to 165 ~1.

--I

- I

0 80 160 240 i, 80 160 240

MINUTES

FIG. 2. The oxygen consumed during the oxidation of 0.65 mg. of dihydroxy- phenyl-nbalanine in the presence of 88 units of tyrosinase with increasing concen- trations of phosphate (A) at pH 7.4 and (B) at pH 6.6. The dotted lines show the volumes equivalent to 2 and 4 atoms of oxygen per molecule of dihydroxyphenyl- alanine.

The effect of increasing enzyme concentration upon oxygen consump- tion and carbon dioxide evolution from 0.164 M phosphate buffer at pH 5.1 is shown in Fig. 3. The buffer capacity of phosphate at this pH is very low, but checks before and after the experiments described showed no variation. At concentrations above 11 units per 2.8 ml., approximately 74 ~1. of oxygen (2 atoms per molecule of dihydroxyphenylalanine) are consumed rapidly (Curves A) ; a break in the consumption rate then occurs and the oxygen uptake continues at a much lower rate, but without reach- ing a definite limiting value within the period of observation, which ex- tended to 18 hours. When the central well of the reaction vessels con- tained buffer instead of KOH, carbon dioxide accumulated in the gaseous phase. Curves B represent the course of volume changes in these vessels. In the presence of 44 or more units of enzyme a volume change equal to

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II. S. MASON AND C. I. WRIGHT 239

consumption of approximately 74 ~1. of oxygen took place rapidly. Little further change occurred in vessels containing 66 or more units of enzyme, indicating that the carbon dioxide produced equaled the oxygen con-

80

0

120 v) a

2 80 ? 0 a

‘1 40 I

80

2 UNITS

A

__________------------.

5

ii

I I I 1 I I

80 180 240

44 UNITS

5

A

---- -------------- c B

II UNITS

B !zs G

80 160 240

MINUTES

FIG. 3. The oxygen consumed and the carbon dioxide evolved during the oxida- tion of 0.65 mg. of dihydroxyphenyl-nr,-alanine in 0.164 M phosphate buffer, pH 5.1, in the presence of increasing amounts of tyrosinase. The catecholase units of en- zyme are indicated on each graph. Curves A represent oxygen consumption, Curves B the observed volume changes without absorption of carbon dioxide, and Curves C the carbon dioxide evolution calculated from the difference between Curves A and B. The dotted lines show the volume of oxygen or carbon dioxide equiva- lent to 1 molecule per molecule of dihydroxyphenylalanine.

sumed. The carbon dioxide evolution calculated from these sets of curves showed that, in the presence of 22 or more units, approximately 55 ~1. were evolved in 240 minutes (Curves C).

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240 CHEMISTRY OF MELANIN. V

The data in Figs. 1 and 3 referring to oxygen consumption at 240 min- utes in 0.164 M phosphate buffers as a function of enzyme concentration have been recalculated as atoms of oxygen per molecule of dihydroxy- phenylalanine in Fig. 4. This figure demonstrates that at 240 minutes 22 or more unit.s of enzyme catalyze the uptake of 4.6 atoms of oxygen at pH 7.4 under the conditions of the experiment; 44 or more units catalyze t,he uptake of 4.5 atoms of oxygen at pH 6.6 and 44 or more units catalyze the uptake of 3.4 atoms of oxygen at pH 5.1.

I I I L I I I

20 40 60 00 IO0 120 140 160

ENZYME CONCENTRATION (CATECHOLASE UNITS)

FIG. 4. The atoms of oxygen consumed per molecule of dihydroxyphenyl-DL- alanine with increasing tyrosinase concentration. The values were observed at 240 minutes of oxidation of 0.65 mg. of substrate dissolved in 2.8 ml. of 0.164 M phos- phate buffers of the indicated pH.

The effect of buffer salts upon oxygen consumption and carbon dioxide evolution during the oxidation of 0.65 mg. of dihydroxyphenylalanine is shown in Fig. 5. The enzyme concentration was fixed at 88 units and the pH, 5.1, was held constant within the limits mentioned above. The buffer concentrations were varied as indicated on each curve. In 0.016, 0.082, and 0.164 of citrate buffers the oxygen consumption at 240 minutes was 122, 144, and 150 PI., respectively; the corresponding carbon dioxide evolutions were 43, 64, and 78 ~1. Measurements of gaseous exchange in 0.016 M phosphat,e buffer at this pH were erratic, but at 0.082 and 0.164 M, 110 and 124 ~1. of oxygen were consumed and 49 and 55 ~1. of carbon dioxide were evolved in 240 minutes. In the corresponding concentra- tions of acetate buffer the values observed were 106, 110, and 132 ~1. of oxygen consumed, and 43,48, and 58 ~1. of carbon dioxide evolved.

The effect of neutral salt is also depicted in Fig. 5. When reaction

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H. S. MASON AND C. I. WRIGHT 241

mixtures either 0.016 or 0.164 M in citrate were made up to 0.164 M in sodium chloride (pH 5.2 and 5.0, respe&ively), the rate of oxygen con- sumption throughout the period of observation was depressed; at 240 minutes 93 and 110 ~1. were taken up.

The effect of increasing buffer concentration upon the rate of carbon dioxide evolution from hallachrome2 in the absence of enzyme was also observed. Solutions of hallachrome were prepared by oxidation of di-

-/Y dt.l64MNoCll // ill rm7 ______________ r __________________--------- r ------------------------ 1

CITRATE

I

PHOSPHATE

I

ACETATE

-i

MINUTES

FIG. 5. The oxygen consumption and carbon dioxide evolution with increasing buffer concentration during the oxidation of 0.65 mg. of dihydroxyphenyl-DL-alanine at approximately pH 5.1 in the presence of 88 enzyme units. Buffer molarities are indicated on each graph. The effect of 0.164 M NaCl upon oxygen consumption in citrate buffers is also shown. The dotted lines indicate t.he volume equivalent to 2 atoms of oxygen per molecule of dihydroxyphenylalanine.

hydroxyphenylala,nine with silver oxide (2, 3). Assuming quantitative conversion, concentrations were adjusted to 0.65 mg. of hallachrome per 2.8 ml. of 0.016 and 0.164 M citrate buffers adjusted to exactly pH 5.1. The carbon dioxide evolved was followed manometrically. Fig. 6 de- scribes the results: carbon dioxide evolution from 0.164 M citrate solution was the more rapid.

The presence of 0.001 M concentrations of cupric, barium, or calcium ions did not affect the rate of carbon dioxide evolution during the enzymic

2 When the term hallachrome is used in this paper, 2,3-dihydro-2-carboxyindole- 5,6-quinone is indicated.

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242 CHEMISTRY OF MELANIN. V

oxidation of dihydroxyphenylalanine in 0.016 M acetate buffer at pH 5.1. Mercuric ion at this concentration inhibited the consumption of oxygen. Zinc ion slightly catalyzed the evolution of carbon dioxide at a concentra- tion of 0.005 M under these conditions.

-5 -

0 40 80 120 160 200 240 MINUTES

FIG. 6. The non-enzymic carbon dioxide evolution from solutions of 0.65 mg. of hallachrome in 2.8 ml. of citrate buffers, Curve A, 0.164 M and, Curve B, 0.016 M,

both adjusted to pH 5.1.

No difference in the rates of oxygen consumption or carbon dioxide evolution was observed during the oxidation of dihydroxyphenykal- anine and dihydroxyphenyl-DL-alanine in 0.164 M acetate solutions at pH 5.1 in the presence of 88 units of enzyme.

DISCUSSION

During the oxidation of a fixed weight of dihydroxyphenylalanine an increase in the concentration of tyrosinase, within limits, causes an in- crease in the amount of oxygen consumed within a given time (Figs. 1, 3, 4). Increase in enzyme concentration beyond these limits results in no further increase in oxygen consumption. The rate of consumption of the first 2 atoms of oxygen per molecule of dihydroxyphenylalanine is generally too high to permit accurate measurements of the effect of enzyme con- centration upon it (Figs. 1, 3), but the rate and amount of oxygen con- sumed beyond this point may be satisfactorily determined. Since there is a range in which the amount and rate of oxygen consumption beyond the second atom is not affected by changes in enzyme concentration, it is probable that the velocity of a non-enzymic step is the rate-controlling factor. This step will presently be shown to be the decarboxylative rear- rangement of hallachrome.

In the range of concentration in which the amount of enzyme limits the oxygen consumption, the behavior observed parallels that of catechol

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H. S. MASON AND C. I. WRIQHT 243

(6) and is caused by inactivation of tyrosinase during the reaction. In Figs. 1, 3, and 4, it is shown that the amount of enzyme necessary to catalyze limiting oxygen consumption is influenced by the electrolytes in solution.

In the presence of amounts of enzyme optimal for all conditions (88 units) a variation of the ionic composition of the solution will also affect the rate and amount of oxygen consumed beyond the first 2 atoms (Figs. 2, 5). At low buffer concentrations either at pH 7.6 or 6.9 a break in oxygen consumption rate is observed at 2 atoms of oxygen per molecule of dihydroxyphenylalanine. This is the amount of oxygen required to convert dihydroxyphenylalanine into hallachrome, and the speed with which it is consumed, in the presence of excess enzyme, together with the marked change of rate at the end of this phase, indicates that halla- chrome is formed quantitatively under the conditions utilized.

The change in rate at 2 atoms of oxygen is more clearly demonstrable ‘at pH 5.1 and can then be correlated with the initiation of carbon dioxide evolution (Figs. 3, 5). Accordingly, carbon dioxide must evolve after hallachrome is formed, in agreement with the hypothesis (1, 2). Fur- thermore in the presence of 66 or more units of enzyme Curves B of Fig. 3 remain approximately level after the initial consumption of 2 atoms of oxygen, indicating that roughly 1 molecule of oxygen is being utilized for each molecule of carbon dioxide formed. This relationship shows that, in so far as the manometric method reveals, decarboxylation and rear- rangement of hallachrome take place simultaneously.

The rates and amounts of oxygen consumed and carbon dioxide evolved are dependent not only upon enzyme concentration (up to optimal levels) but also upon the concentration and kind of buffer employed (Fig. 5). The slight increases of pH due to dilution of buffers discussed under “Ex- perimental” should increase oxygen consumption according to the results shown in Fig. 4, but the influence of electrolyte dilution in the opposite direction is apparently greater, because dilution of buffer solutions under these conditions caused a decrease in rate of both oxygen consumption and carbon dioxide evolution.

The rate of carbon dioxide evolution observed in the presence of a series of citrate, phosphate, and acetate buffers has been plotted as a function of the concentration of unchanged hallachrome (Fig. 7). Linear relation- ships are found to hold, indicating that decarboxylation is a reaction of the first order with respect to hallachrome concentration. By plotting the specific first order rate constants so derived against anion concentration an approximately linear relationship is again found (Fig. 8). No regu- larity is observable if the specific rate constants are plotted against cation concentrations. The decarboxylative rearrangement of hallachrome is

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244 CHEMISTRY OF MELANIN. V

accordingly subject to general base catalysis, but the analysis cannot be carried farther in terms of the Bronsted law because of the depressivt action of neutral salt upon the rate of formation of hallachrome (Fig. 5) In the latter connection, Lea (9) has reported an inhibiting effect of NaCl on melanin formation. The present results indicate that ihe effect is probably due to the influence of chloride ion upon enzymic steps in the melanogenic sequence.

-5.6

b&J 3

-5.5

a zi a 3

-5.7

2

cn -5.9 w

$2

4

-6. I

-6.3

-6.5 1 0 40 60 120 160 200 240

MINUTES

FIG. 7. The first order character of the decarboxylation of hallachrome, from the data of Fig. 5. hallachrome.

Carbon dioxide evolution is expressed as moles of unchanged The molarity of the buffers is indicated on each curve.

The effect of anions upon the decarboxylative rearrangement of halla- chrome does not involve the presence of a decarboxylase in mushroom tyrosinase, because the same effect occurs in the absence of enzyme (Fig. 6). In the absence of a catechol oxidase, however, hallachrome may be reduced by the products of its own rearrangement; this loss of hallachrome is reflected in a low rate of carbon dioxide evolution. In addition to the anion effect upon non-enzymic decarboxylation, increase of enzyme con-

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II. S. MASON AND C. I. WRIGHT 245

centration beyond 22 units per reaction volume at pH 5.1 failed to in- crease the rate or amount of carbon dioxide evolved (Fig. 3).

The oxygen consumed when dihydroxyphenylalanine is oxidized in the presence of tyrosinase has been measured by Enselme and Vigneau (5) and Raper and coworkers (3, 4). The highest value reported was 4.1 atoms per molecule, observed at pH 8 as an unchanging quantity. In this study higher values were observed. The atoms of oxygen consumed and the molecules of carbon dioxide evolved per molecule of dihydroxy- phenylalanine in the presence of excess enzyme have been calculated from

IO -

8-

s

x 6- x

IO. .02 .04 .06 .00 .I0 .I2 .I4 .I6 ANION MOLARITY

FIGI. 8. The relationship between specific rate constants of hallachrome decar- boxylation in citrate, phosphate, and acetate buffers and anion concentration, calculated from the data of Fig. 7.

the data given above and are listed in Table I as functions of pH and buffer concentration. At 240 minutes from 2.9 to 4.6 atoms of oxygen were required; at pH 5.1 the carbon dioxide evolution amounted to 0.6 to 1.0 molecule. Gaseous exchange continued beyond this time and at 18 hours much higher values were observed, but these reflected respiration of microorganisms to an undeterminable extent. In general, increasing pH led to higher oxygen consumption. Increasing buffer concentration led to both higher oxygen consumption and carbon dioxide evolution.

The amounts of oxygen required and carbon dioxide set free limit struc- tures assignable to melanins formed by the enzymic oxidation of dihydroxy- phenylalanine. In the present investigation the extent of gaseous ex- change was dependent upon time and the electrolytic nature of the solution

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246 CHEMISTRY OF MELANIN. V

even in the presence of excess enzyme. Utilization of more than 4 atoms of oxygen per molecule was readily realized (Fig. 4), but the point at which enzymic oxidation stopped and autoxidation commenced is not determin- able from the data, nor is the point, if any, at which the polymeric prod- uct reaches its greatest complexity and commences to break down into simpler substances. Dopa melanin cannot therefore be regarded as a homogeneous reaction product, the composition of which is independent

TABLE I Atoms of Oxygen Consumed and Molecules of Carbon Dioxide Evolved per Molecule

of Dihydroxyphenylalanine Varying pH and buffers but enzyme concentration fixed at 88 unite per reaction

volume of 2.8 ml.

BUi3Z.r

0.164 M phosphate 0.082 “ u 0.164 M acetate 0.082 “ “ 0.016 “ “ 0 164 “ 0:082

citrate “ cc

0.016 “ cc Citrate-phosphate 0.164 Y phosphate 0.082 “ “ 0.016 “ u 0.008 “ “ Citrate-phosphate 0.164 M phosphate 0.082 “ “ 0.016 “ “ 0.008 “ “ Citrate-phosphate

PH

5.1 5.1 5.1 5.1 5.1 5.1 5.3 5.5 5.1 6.6 6.6 6.8 6.8 6.6 7.4 7.4 7.6 7.6 7.4

*40°min. 3.4 3.0 3.6 3.0 2.9 4.1 3.9 3.4 4.1 4.5 4.3 3.8 3.0 4.3 4.6 4.5 4.3 3.9 4.6

coa 24omin.

0.7 0.7 0.8 0.7 0.6 1.0 0.9 0.6 1.0

of conditions of preparation. These conditions should therefore be spe- cified precisely in any study of the structure of melanin to permit repro- ducibility of the substance under investigation.

SUMMARY

1. Increase in tyrosinase concentration causes increase in over-all oxy- gen consumption during the enzymic oxidation of dihydroxyphenylala- nine only within limits. Further increase leads to no further increase in oxygen consumption at a given time.

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H. 8. MASON AND C. I. WRIGHT 247

2. The electrolytic composition of the solution influences the amount of ensyme necessary to catalyse limiting consumptions of oxygen, as well as the total consumption of oxygen and evolution of carbon dioxide.

3. The decarboxylative rearrangement of hallachrome is base-catalyzed and controls the rate of oxygen consumption beyond the 2nd atom.

4. At 240 minutes under the conditions employed 2.9 to 4.6 atoms of oxygen were consumed and 0.6 to 1.0 molecule of carbon dioxide was evolved per molecule of dihydroxyphenylalanine.

The technical assistance of Anne H. Wright is gratefully acknowledged.

BIBLIOGRAPHY

1. Raper, H. S., J. Chem. Sot., 125 (1938). 2. Mason, H. S., J. Biol. Chem., 173,83 (1948). 3. Duliere, W. L., and Raper, H. S., Biochem. J., 26,239 (1939). 4. Heard, R. D. H., and Raper, H. S., Biochem. J., 27.36 (1933).

’ 5. Enselme, J., and Vigneau, J. L., Bull. Sot. chim. biol., 27,387 (1945). 6. Wright, C. I., and M;ison, H. S., J. Biol. Chem., 165,45 (1946). 7. Mallette, M. F., Lewis, S., Ames, S. R., Nelson, J. M., and Dawson, C. R., Arch.

Biochem., 16,283 (1948). 8. Miller, W. H., Mallette, M. F., Roth, L. J., and Dawson, C. R., J. Am. Chem. Sot.,

66,514 (1944). 9. Lea,A. J., Nature, 166,428 (1945).

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Howard S. Mason and Charles I. WrightTYROSINASE

DIHYDROXYPHENYLALANINE BYOXIDATION OF

THE CHEMISTRY OF MELANIN: V.

1949, 180:235-247.J. Biol. Chem. 

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