Synthesis and characterization of melanins from dihydroxyindole-2-carboxylic acid and dihydroxyindole.

Several studies have confirmed that a melanocyte-specific enzyme, dopachrome tautomerase (EC 5.3.2.3), catalyzes the isomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) (Pawelek, 1991). Here we report that DHICA, produced either enzymatically with dopachrome tautomerase or through chemical synthesis, spontaneously polymerized to form brown melanin that was soluble in aqueous solutions above pH 5. Under the same reaction conditions, solutions of either DOPA, DOPAchrome, or 5,6-dihydroxyindole (DHI) formed black, insoluble melanin precipitates. When DHICA and DHI were mixed together, with DHICA in molar excess, little or no precipitation of DHI-melanin occurred and the rate and extent of soluble melanin formation was markedly enhanced over that achieved with DHICA alone, suggesting co-polymerization of DHICA and DHI. With or without DHI, DHICA-melanins absorbed throughout the ultraviolet and visible spectra (200-600 nm). The DHICA-melanins precipitated below pH 5, at least in part because of protonation of the carboxyl groups. DHICA-melanins could be passed through 0.22 micron filters but could not be dialyzed through semi-permeable membranes with exclusion limits of 12,000-14,000 daltons. HPLC/molecular sieve analyses revealed apparent molecular weights ranging from 20,000 to 200,000 daltons, corresponding to 100-1,000 DHICA monomers per molecule of melanin. DHICA-melanins were stable to boiling, lyophilization, freezing and thawing, and incubation at room temperature for more than 1 year. The natural occurrence of oligomers of DHICA was first reported by Ito and Nichol (1974) in their studies of the brown tapetal pigment in the eye of the sea catfish (Arius felis L.).(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the final phase of mammalian melanogenesis. The role of dopachrome tautomerase and the ratio between 5,6-dihydroxyindole-2-carboxylic acid and 5,6-dihydroxyindole.

The regulation of the final steps of the melanogenesis pathway, after L-2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome) formation, is studied. It is shown that both tyrosinase and dopachrome tautomerase are involved in the process. In vivo, it seems that tyrosinase is involved in the regulation of the amount of melanin formed, whereas dopachrome tautomerase is mainly involved in the size, structure and composition of melanin, by regulating to the incorporation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into the polymer. Moreover, using L-3,4-dihydroxyphenylalanine (dopa) and related compounds, it was shown that the presence of dopachrome tautomerase mediates an initial acceleration of melanogenesis since L-dopachrome is rapidly transformed to DHICA, but that melanin formation is inhibited because of the stability of this carboxylated indole compared to 5,6-dihydroxyindole (DHI), its decarboxylated counterpart obtained by spontaneous decarboxylation of L-dopachrome. Using L-dopa methyl ester as a precursor of melanogenesis, it is shown that this carboxylated indole does not polymerize in the absence of DHI, even in the presence of tyrosinase. However, it is incorporated into the polymer in the presence of both tyrosinase and DHI. Thus, this study suggests that DHI is essential for melanin formation, and the rate of polymerization depends on the ratio between DHICA and DHI in the medium. In the melanosome, this ratio should be regulated by the ratio between the activities of dopachrome tautomerase and tyrosinase.     

After dopachrome?

Dopachrome, an intermediate in melanin biosynthesis, exhibits some unusual properties. At physiologic pH (e.g., pH 6-8) it is unstable and spontaneously loses its carboxyl group to form 5,6-dihydroxyindole (DHI) and CO2. However, over this same pH range, if various metals or a melanocyte-specific enzyme are present, it rapidly rearranges to its isomer form--5,6-dihydroxyindole-2-carboxylic acid (DHICA)--which is far more stable than dopachrome in its ability to retain the carboxyl group. Whether or not the carboxyl group is retained could have important implications for the regulation of melanogenesis, since in the presence of oxygen DHI spontaneously forms a black precipitate, whereas DHICA forms a golden-brown solution. The solubility of "DHICA-melanin" is due to the presence of carboxyl groups, which provide negative charges and hydrophilicity. Thus, in vivo, the extent to which dopachrome is converted to DHI or DHICA may well influence the solubility and color of the melanin formed. The purpose of this article is to review recent findings in these areas and to discuss the possible significance of dopachrome conversion in the regulation of melanogenesis and color formation.     

Effect of pH on elementary steps of dopachrome conversion from first‐principles calculation

Dopachrome conversion, in which dopachrome is converted into 5,6‐dihydroxyindole (DHI) or 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA) upstream of eumelanogenesis, is a key step in determining the DHI/DHICA monomer ratio in eumelanin, which affects the antioxidant activity. Although the ratio of DHI/DHICA formed and the conversion rate can be regulated depending on pH, the mechanism is still unclear. To clarify the mechanism, we carried out first‐principles calculations. The results showed the kinetic preference of proton rearrangement to form quinone methide intermediate via β‐deprotonation. We also identified possible pathways to DHI/DHICA from the quinone methide. The DHI formation can be achieved by spontaneous decarboxylation after proton rearrangement from carboxyl group to 6‐oxygen. α‐Deprotonation, which leads to DHICA formation, can also proceed with a significantly reduced activation barrier compared with that of the initial dopachrome. Considering the rate of the proton rearrangements in a given pH, we conclude that the conversion is suppressed at acidic pH.     

Neutral pH and copper ions promote eumelanogenesis after the dopachrome stage

The diversity of pigmentation in the skin, hair, and eyes of humans has been largely attributed to the diversity of pH in melanosomes with acidic pH being proposed to suppress melanin production. Tyrosinase has an optimum pH of 7.4 and its activity is suppressed greatly at lower pH values. The first step of eumelanogenesis is the oxidation of tyrosine to dopachrome (DC) via dopaquinone. However, how eumelanogenesis is controlled by pH beyond this stage is not known. In this study, we examined the effects of pH (5.3–7.3) on the conversion of DC to 5,6‐dihydroxyindole (DHI) and 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA) and the subsequent oxidation of DHI and DHICA to form eumelanin. The effects of Cu²⁺ ions on those reactions were also compared. The results indicate that an acidic pH greatly suppresses the late stages of eumelanogenesis and that Cu²⁺ ions accelerate the conversion of DC to DHICA and its subsequent oxidation.     

Inhibitory Effects of Melanin Monomers,Dihydroxyindole-2-Carboxylic Acid (DHICA) and Dihydroxyindole (DHI) on Mammalian Tyrosinase,With a Special Reference to the Role of DHICA/DHI Ratio in Melanogenesis

DOPAchrome tautomerase (DCT) is known to control the ratio of DHICA/DHI formed within the melanocyte, but physiologic significance of this activity is not yet fully elucidated. In this study the two melanin monomers are shown to inhibit with different efficacy the initial, tyrosinase-controlled, melanogenic reaction, namely conversion of L-tyrosine to DOPAchrome (2-carboxy-2,3-dihydroindole-5,6-quinone). This is demonstrated in the test tube assay system whereby formation of DOPAchrome is catalyzed by i) isolated premelanosomes (PMS), ii) tyrosinase-rich PMS glycoproteins, or iii) tyrosinase purified from fibroblasts transfected with human tyrosinase gene. Both DHI and DHICA suppress the conversion of L-tyrosine to DOPAchrome when added to reaction mixture but the inhibitory effect is far more strongly pronounced by DHI. DHI inhibits both activities of tyrosinase—tyrosine-hydroxylation and DOPA-oxidation—more strongly than DHICA. The different extent of inhibition is shown to reflect i) the ability of the two monomers to compete with tyrosinase substrates for the enzyme's active center and ii) the rate of interaction between melanin monomers and DOPAquinone. Consequently, we demonstrate that the tyrosinase-catalyzed DOPAchrome formation can be modulated by the ratio of DHICA/DHI among melanin monomers with the increased proportion of DHICA resulting in more efficient DOPAchrome formation. These results raise the possibility that DOPAchrome tautomerase plays a role in positive control of the tyrosinase-catalyzed early phase of melanogenesis.     

Lipoxygenase/H2O2-catalyzed oxidation of dihydroxyindoles: synthesis of melanin pigments and study of their antioxidant properties

5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), which are important intermediates in melanogenesis, can be converted into the corresponding melanin pigments by the action of the lipoxygenase/H₂O₂ system. Kinetic and HPLC analyses indicate that both DHI and DHICA are good substrates for this enzymatic system. Enzyme activity on both substrates was measured in comparison with peroxidase and tyrosinase; the oxidizing behaviour of lipoxygenase is more similar to that of peroxidase rather than that of tyrosinase. The antioxidant properties of DHI- and DHICA-melanins have been investigated in comparison with other kinds of melanins. DHICA-melanin shows a more pronounced antioxidant effect than that of DHI-melanin and this behaviour can be ascribed to the different structure and solubility of the two pigments. The mixed polymer synthesized from DHI and DHICA is the most effective one. Some implications about the possible explanation of the above mentioned behaviour are discussed.     

Synthesis, discovery and mechanism of 2,6-dimethoxy-N-(4-methoxyphenyl)benzamide as potent depigmenting agent in the skin

In this study, a new skin-depigmenting agent, 2,6-dimethoxy-N-(4-methoxyphenyl)benzamide (DMPB), was synthesized using a combination of benzoic acid and aniline. DMPB exhibited significant depigmentation ability on the UV B-induced hyperpigmentation of the brown guinea pig skin. In addition, the 100ppm treatment with this compound had a 30% inhibitory effect on melanin pigment generation in the melan-a cell line without significant cell toxicity. To search for relationship with the depigmentation, the effects of DMPB on the tyrosinase and dopachrome tautomerase were evaluated. DMPB had no effect on tyrosinase. However, it accelerated dopachrome transformation into 5,6-dihydroxyindole-2-carboxylic acid (DHICA) in the presence of dopachrome tautormerase. In addition, intracellular level of dopachrome tautomerase in melan-a cells was increased by treatment of DMPB. These results suggest that the pigment-lightening effects of DMPB might be due to biased production of DHICA-eumelanin induced by dopachrome tautormerase activation.     

Mechanism of dopachrome tautomerization into 5,6-dihydroxyindole-2-carboxylic acid catalyzed by Cu(II) based on quantum chemical calculations

Background

Tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) is a biologically crucial reaction relevant to melanin synthesis, cellular antioxidation, and cross-talk among epidermal cells. Since dopachrome spontaneously converts into 5,6-dihydroxyindole (DHI) via decarboxylation without any enzymes at physiologically usual pH, the mechanism of how tautomerization to DHICA occurs in physiological system is a subject of intense debate. A previous work has found that Cu(II) is an important factor to catalyze the tautomerization of dopachrome to DHICA. However, the effect of Cu(II) on the tautomerization has not been clarified at the atomic level.

Methods

We propose the reaction mechanism of the tautomerization to DHICA by Cu(II) from density functional theory-based calculation.

Results

We clarified that the activation barriers of α-deprotonation, β-deprotonation, and decarboxylation from dopachrome are significantly reduced by coordination of Cu(II) to quinonoid oxygens (5,6-oxygens) of dopachrome, with the lowest activation barrier of β-deprotonation among them. In contrast to our previous work, in which β-deprotonation and quinonoid protonation (O5/O6-protonation) were shown to be important to form DHI, our results show that the Cu(II) coordination to quinonoid oxygens inhibits the quinonoid protonation, leading to the preference of proton rearrangement from β-carbon to carboxylate group but not to the quinonoid oxygens.

Conclusion

Integrating these results, we conclude that dopachrome tautomerization first proceeds via proton rearrangement from β-carbon to carboxylate group and subsequently undergoes α-deprotonation to form DHICA.

General significance

This study would provide the biochemical basis of DHICA metabolism and the generalized view of dopachrome conversion which is important to understand melanogenesis.     

Maintenance of immune hyporesponsiveness to melanosomal proteins by DHICA-mediated antioxidation: Possible implications for autoimmune vitiligo

Melanocyte destruction in the skin of vitiligo patients has been considered to be a consequence of an autoimmune response against melanosomal proteins. However, little is known about the molecular mechanisms by which the immune system recognizes these sequestered intracellular self-proteins, which are confined in specialized organelles termed melanosomes, and is provoked into an autoimmune response to melanocytes. Here, we utilize a sucrose density-gradient ultracentrifugation protocol to enrich melanosomal components from dopachrome tautomerase (Dct)-mutant or wild-type melanocytes exposed to a pulse of hydrogen peroxide at a noncytotoxic concentration to evaluate their immunogenicity in mice challenged with the corresponding melanosomal proteins. The results demonstrate that enhanced humoral and cellular immune responses to a challenge with late-stage melanosomal proteins, especially with those derived from Dct-mutant melanocytes, are found in the immunized mice. To elucidate whether a reduced 5,6-dihydroxyindole-2-carboxylic acid (DHICA) content in melanin might cause a loss in antioxidative protection to the proteins, we incubated these melanosomal proteins in vitro with synthetic 5,6-dihydroindole (DHI)-melanin or DHI/DHICA (1:1)-melanin and then used them to immunize mice. T cell proliferation and IgG antibody responsiveness to the challenges were significantly induced by melanosomal proteins treated with DHI-melanin, but not by those treated with DHI/DHICA (1:1)-melanin. Moreover, we observed that melanosomal proteins derived from Dct-mutant melanocytes are subject to oxidative modifications that alter their antigenic configurations to attain an enhanced immunogenicity compared with those derived from wild-type melanocytes. From these results, we conclude that DHICA-mediated antioxidation plays a critical role in the maintenance of immune hyporesponsiveness to melanosomal proteins.     

Function of dopachrome oxidoreductase and metal ions in dopachrome conversion in the eumelanin pathway

The conversion of dopachrome (DC) in the eumelanin pathway has been analyzed to determine the specific product and the role of enzyme control. 5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) were quantitated by HPLC with fluorescent detection, after DC incubation with heated and unheated preparations of B-16 melanoma derived dopachrome oxidoreductase (DCOR). The enzyme-catalyzed reaction produced DHICA as the major product, while DHI formed with the spontaneous reaction. It had originally been suggested that the major product of DC conversion was DHI, with DHICA being formed as a minor product of this conversion [Raper, H.S. (1927) Biochem. J. 21, 89-96]. Copper, nickel, and cobalt ions promoted conversion of DC, with nickel simulating DCOR activity. Removal of free ions from unheated DCOR did not alter DC conversion. We conclude that the major product of DC conversion is DHICA and that DCOR is responsible for this conversion.     

The Expression of Tyrosinase,Tyrosinase-Related Proteins 1 and 2 (TRP1 and TRP2), the Silver Protein,and a Melanogenic Inhibitor in Human Melanoma Cells of Differing Melanogenic Activities

The expression of various melanogenic proteins, including tyrosinase, the tyrosinase-related proteins 1 (TRP1) and 2 (TRP2/DOPAchrome tautomerase), and the silver protein in human melanocytes was studied in six different human melanoma cell lines and compared to a mouse derived melanoma cell line. Analysis of the expression of tyrosinase, TRP1, TRP2, and the silver protein using flow cytometry revealed that in general there was a positive correlation between melanin formation and the expression of those melanogenic enzymes. Although several of the melanoma cell lines possessed significant activities of TRP2, the levels of DOPAchrome tautomerase in extracts of human cells were relatively low compared to those in murine melanocytes. Melanins derived from melanotic murine JB/MS cells, from melanotic human Ihara cells and HM-IY cells, from sepia melanin, and from C57BL/6 mouse hair were chemically analyzed. JB/MS cells, as well as Ihara cells and HM-TY cells, possessed significant amounts of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) derived melanins, this being dependent on the activity of TRP2. Kinetic HPLC assays showed that 5,6-dihydroxyindole (DHI) produced during melanogenesis was metabolized quickly to melanin in pigmented KHm-1/4 cells, whereas DHI was stable in amelanotic human SK-MEL-24 cells. A melanogenic inhibitor that has been purified from SK-MEL-24 cells that suppressed oxidation of DHI in the presence or absence of tyrosinase, but had no effect on DHICA oxidation. The sum of these results suggest that the expression of melanogenic enzymes as well as the activity of a melanogenic inhibitor are critical to the production of melanin synthesis in humans.     

An oxygen transporter hemocyanin can act on the late pathway of melanin synthesis

5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) are precursors of eumelanin. The effects of crustacean hemolymph proteins on these eumelanin-related metabolites were investigated. Zymogram analysis indicated that polymers of hemocyanin (Hc) subunits converted DHI into black pigment while no effects were observed using DHICA as a substrate. Spectrum changes for mixtures of purified Hc and DHI showed a profile similar to oxidized DHI by mushroom tyrosinase while Hc had only slight effects on DHICA. Typical inhibitors of tyrosinase and phenoloxidase severely hampered the production of oxidized DHI. Taken together with previous results, these data indicate that Hc plays a crucial role in the conversion of DHI in the hemolymph of crustaceans, which promotes late reactions in the melanin synthetic pathway as well as early reactions (oxidation of tyrosine and DOPA to dopaquinone).     

Preparation of eumelanin-related metabolites 5,6-dihydroxyindole, 5,6-dihydroxyindole-2-carboxylic acid, and their O-methyl derivatives

5,6-Dihydroxyindole (5,6DHI) and 5,6-dihydroxyindole-2-carboxylic acid (5,6DHI2C) are ultimate precursors of the black melanin, eumelanin. These indolic metabolites and their O-methyl derivatives are excreted in urine of melanoma patients at high levels and of healthy persons at low levels. We describe here a simplified procedure for preparing milligram to subgram quantities of 5,6DHI and 5,6DHI2C and their O-methyl derivatives. Dopachrome generated in situ by ferricyanide oxidation of dopa at pH 6.5 underwent spontaneous decarboxylation to give 5,6DHI in 40% isolation yield, while treatment of dopachrome with alkali at pH 13 afforded 5,6DHI2C in 38% isolation yield. Two isomeric O-methyl derivatives of 5,6DHI were prepared by treatment with diazomethane, while those of 5,6DHI2C were prepared by treatment with diazomethane followed by alkaline hydrolysis of the methyl esters. 5,6DHI and 6-hydroxy-5-methoxyindole were also obtained by heating the corresponding carboxylic acids in decalin. 5-Hydroxy-6-methoxyindole and 6-hydroxy-5-methoxyindole-2-carboxylic acid could also be prepared by debenzylation of the commercially available O-benzyl derivatives.     

The Mouse Brown (b) Locus Protein Functions as a Dopachrome Tautomerase

The mouse b locus controls black/brown coat coloration. Its product, the b-protein or TRP-1, has significant homology to tyrosinase, and this has led to suggestions that the b-protein is itself a melanogenic enzyme. In order to investigate its function, we have used lines of mouse fibroblasts stably expressing the b-protein. We were unable to con-firm previous reports that the b-protein has tyrosinase or catalase activity, but detected stereospecific dopachrome tautomerase activity in b-protein-expressing fibroblasts. This dopachrome tautomerase binds to Concanavalin A-Sepharose, and the major product of its action on L-dopachrome is 5,6-dihydroxyindole-2-carboxylic acid, as expected for the mammalian enzyme. Since this activity is not present in untransfected fibroblasts we conclude that the b-protein has dopachrome tautomerase activity. Further supporting evidence comes from the analysis of melanin metabolites produced by fibroblasts expressing tyrosinase alone, or in combination with the b-protein. Culture medium from the line expressing both proteins contains significant amounts of methylated carboxylated indoles, such as 6-hydroxy-5-methoxyindole-2-carboxylic acid, which would be expected in cells with an active dopachrome tautomerase. The levels of these compounds in medium from cells expressing tyrosinase alone are approximately 20-fold lower, and not significantly above background. Hence, it appears that the b-protein acts as a dopachrome tautomerase in vivo as well as in vitro.     

Degree of polymerization of 5,6‐dihydroxyindole‐derived eumelanin from chemical degradation study

Eumelanin is a brown‐black pigment comprising 5,6‐dihydroxyindole (DHI) and its 2‐carboxy derivative (DHICA), but the detailed structure of eumelanin is unclear. Chemical degradation is a powerful tool for analyzing melanin. H₂O₂ oxidation degradation of eumelanin affords pyrrole‐2,3,5‐tricarboxylic acid (PTCA) and pyrrole‐2,3‐dicarboxylic acid (PDCA). The ratio of PDCA to PTCA provides information about the eumelanin structure. In this article, we propose simple equations on the basis of previous experimental results on dimer yields for evaluating the yields of PTCA and PDCA from any DHI oligomers. Assuming the chemical disorder model of DHI‐melanin, we solve an equation where a theoretical expression for the ratio of PDCA to PTCA is set to the corresponding experimental value to obtain a plausible Poisson distribution of DHI oligomers. The results demonstrate that the main contributors to DHI‐melanin are tetramers and pentamers as shown by the mass spectrometry.     

Unexpected impact of esterification on the antioxidant activity and (photo)stability of a eumelanin from 5,6‐dihydroxyindole‐2‐carboxylic acid

To inquire into the role of the carboxyl group as determinant of the properties of 5,6‐dihydroxyindole melanins, melanins from aerial oxidation of 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA) and its DHICA methyl ester (MeDHICA) were comparatively tested for their antioxidant activity. MALDI MS spectrometry analysis of MeDHICA melanin provided evidence for a collection of intact oligomers. EPR analysis showed g‐values almost identical and signal amplitudes (ΔB) comparable to those of DHICA melanin, but spin density was one order of magnitude higher, with a different response to pH changes. Antioxidant assays were performed, and a model of lipid peroxidation was used to compare the protective effects of the melanins. In all cases, MeDHICA melanin performed better than DHICA melanin. This capacity was substantially maintained following exposure to air in aqueous buffer over 1 week or to solar simulator over 3 hr. Different from DHICA melanin, MeDHICA melanin was proved to be fairly soluble in different water‐miscible organic solvents, suggesting its use in dermocosmetic applications.     

Relationship of melanin degradation products to actual melanin content: application to human hair

Methods not only for characterizing but also for quantitating melanin subtypes from the two types of melanin found in hair--eumelanin and pheomelanin--have been established. In relation to testing for drugs of abuse in hair, these methods will allow for correction of drug binding to specific melanin subtypes and will serve to improve drug measurement in hair. 5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) make up the majority of the eumelanin polymer while benzothiazene units derived from 2-cysteinyl-S-Dopa (2-CysDopa) and 5-cysteinyl-S-Dopa (5-CysDopa) compose the majority of the pheomelanin polymer. Our results show that: (1) pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA), markers for DHI and DHICA units, respectively, are produced in 0.37 and 4.8% yields, respectively, when melanins are subjected to alkaline hydrogen peroxide degradation, (2) 3-aminotyrosine (3AT) and 4-amino-3-hydroxyphenylalanine (AHP), markers for 2-CysDopa and 5-CysDopa, respectively, are produced in 16 and 23% yield, respectively, when subjected to hydriodic acid hydrolysis, and (3) that black human hair contains approximately 99% eumelanin and 1% pheomelanin, brown and blond hair contain 95% eumelanin and 5% pheomelanin; and red hair contains 67% eumelanin and 33% pheomelanin. These data will allow deeper investigation into the relationship between melanin composition and drug incorporation into hair.     

Electrochemical identification of dopachrome isomerase in Drosophila melanogaster

A principal reaction in the eumelanin biosynthetic pathway is the conversion of dopachrome (DC) to dihydroxyindole(s). Dopachrome isomerase (DI), the enzyme that catalyzes this reaction, was detected for the first time in larvae of D. melanogaster. Unlike the enzyme from B16 mouse melanoma cells which converts dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA), the insect enzyme forms 5,6-dihydroxyindole (DHI). The activity of the insect DI was linear through 15 min incubation, and the amount of DHI produced was proportional to the amount of enzyme that was incorporated into the reaction mixtures.     

A second tyrosinase-related protein, TRP-2, is a melanogenic enzyme termed DOPAchrome tautomerase.

The production of melanin pigment in mammals requires tyrosinase, an enzyme which hydroxylates the amino acid tyrosine to DOPA (3,4-dihydroxyphenylalanine), thus allowing the cascade of reactions necessary to synthesize that biopolymer. However, there are other regulatory steps that follow the action of tyrosinase and modulate the quantity and quality of the melanin produced. DOPAchrome tautomerase is one such melanogenic enzyme that isomerizes the pigmented intermediate DOPAchrome to DHICA (5,6-dihydroxyindole-2-carboxylic acid) rather than to DHI (5,6-dihydroxyindole), which would be generated spontaneously. This enzyme thus regulates a switch that controls the proportion of carboxylated subunits in the melanin biopolymer. Efforts to clone the gene for tyrosinase have resulted in the isolation of a family of tyrosinase related genes which have significant homology and encode proteins with similar predicted structural characteristics. Using specific antibodies generated against synthetic peptides encoded by unique areas of several of those proteins, we have immuno-affinity purified them and studied their melanogenic catalytic functions. We now report that TRP-2 (tyrosinase related protein-2), which maps to and is mutated at the slaty locus in mice, encodes a protein with DOPAchrome tautomerase activity.