DOPAchrome

Melanin biosynthesis in animals is a complex process starting with the L-tyrosine amino acid. In the first step, L-tyrosine is converted first into DOPA and then into dopaquinone, a process catalyzed by tyrosinase. In the biosynthesis of eumelanins, dopaquinone undergoes a cyclization to form dopachrome and subsequently a tau-tomerization into 5,6-dihydroxyindole-2-carboxylic acid (DHICA). DHICA is further oxidized to indole-5,6-quinone2-carboxylic acid, the precnrsor of DHICA eumelanins. Tyrosinase-related proteins TRP-2 and TRP-1, respectively, are responsible for the last two steps, and they are under the control of the tyrosinase promoter. 

Dopachrome also undergoes a nonenzymatic reaction to form dihidroxyindole (DHI), the precursor of DHI-eumelanins. For the formation of phaeomelanins, dopaquinone is first transformed in cysteinil-DOPA and then in cysteinyl-dopaquinone which suffers a nonenzymatic polymerization. The polymerization of monomers and the association of melanins with proteins is not yet completely elucidated and may involve other intermediates. ... 

Figure 1. The biosynthetic pathway from tyrosine to melanin (according to Hearing and Tsukamoto, 1991 Tsukamoto et al., 1992). Tyrosinase catalyzes three different reactions in this pathway (1, 2, 3). The reaction catalyzed by the product of TRP-2, DOPAchrome tautomerase, is indicated by 4. DOPA = 3,4-dihydroxyphenylalanine DHICA = 5,6-dihydroxyin-dole-2-carboxylic acid DHI = 5,6-dihydroxyindole.

Tsukamoto, K., Jackson, I. J., Urabe, K., Montague, P. M., and Hearing, V. J. (1992). A second tyrosinase-related protein, TRP-2, is a melanogenic enzyme termed DOPAchrome tautomerase. EMBO J. 11 519-526. 

Oxidative polymerization of phenol derivatives is also important pathway in vivo, and one example is the formation of melanin from tyrosine catalyzed by the Cu enzyme, tyrosinase. The pathway from tyrosine to melanin is described by Raper (7) and Mason (8) as Scheme 8 the oxygenation of tyrosine to 4-(3,4-dihydro-xyphenyl)-L-alanin (dopa), its subsequent oxidation to dopaqui-none, its oxidative cyclization to dopachrome and succeeding decarboxylation to 5,6-dihydroxyindole, and the oxidative coupling of the products leads to the melanin polymer. The oxidation of dopa to melanin was attempted here by using Cu as the catalyst. 

Figure 5. The pH dependence of rate constant of dopachrome and melanin formation (Q) imidazole-Cu (O) PVIm-Cu (A) PIPo—Cu (9) tyrosinase 30°C, air, phosphate buffer... 

From Lys hydrochloride pyrolyzed in vacuo at 600°C for 8-10 min, a tricyclic compound 134 is formed in low yield (80TL2679). Cystine reacts with dopachrome to give an unstable product, but the methyl ester of dopachrome gave a stable pyrrolo[2,3-/i][l,4]benzothiazine 135 (87T5357). 

The oxidation of DOPA and adrenaline to dopachrome and adrenochrome, respectively, by a horse radish peroxidase-H202 system has been reported by Herzmann.29,30 The oxidation process was activated by trace quantities of caffeic acid, its esters, and related compounds.30 Ascorbic acid inhibited the oxidation of adrenaline by this enzyme in the initial stages of the reaction, but later had a stimulatory effect.30... 

However, Van der Wender and Spoerlein have recently described the presence of an enzyme system in rat brain that is capable of oxidizing DOPA to melanitic pigments43 (an aminochrome, i.e. dopachrome, must be formed as an essential intermediate in this process) the same enzyme system apparently oxidizes adrenaline to adrenochrome.43 Kaliman has demonstrated the presence of an enzyme system in rabbit heart tissue which oxidizes adrenaline via the quinonoid pathway (presumably to adrenochrome).44 Heart, kidney, and brain tissues of white rats were also shown by Kaliman and Koshlyak to possess similar activity.45... 

In 1937 Arnow showed that tyrosine could be converted into DOPA by ultraviolet radiation51 and that the DOPA produced in this manner was subsequently destroyed by further irradiation, the solutions becoming red-brown in color (presumably due to the formation of dopachrome).51 In 1939 Konzett and Weis reported that the blood pressure-raising effect of adrenaline solutions was lost on ultraviolet irradiation and that the solutions became colored and fluorescent the initial red color fades to reddish yellow.62 This phenomenon suggests the initial formation of adrenochrome, followed by its isomerization to adrenolutin, both of these compounds being virtually void of pressor activity. Similarly to the radiation-induced hydroxylation of tyrosine mentioned above, synephrine was first... 

In his classical studies on melanin formation from DOPA (3), Raper proposed the following scheme for the formation of the red pigment now known to be the aminochrome dopachrome (4). The first stage involved the oxidation of the catechol nucleus of-3 to give the quinone dopa-quinone (16). The second stage was the non-oxidative intramolecular cyclization of 16 to leuco-dopachrome (17), which was in turn oxidized to dopachrome (4).72,73 Since... 

In 1927 Raper showed that the red pigment obtained on oxidation of DOPA [i.e. 2,3-dihydroindole-5,6-quinone-2-carboxylic acid, dopachrome (4)] rearranged spontaneously by an autoreduction process in vacuo to 5,6-dihydroxyindole (29).72 The rearrangement process could be accelerated by the action of alkali or sulfur dioxide.72 In the latter case, decarboxylation did not accompany the rearrangement and the colorless derivative was 5,6-dihydroxyindole-2-carboxylic acid (17).72 Compounds 17 and 29 were isolated as their dimethyl ethers, (30A) and (30B).72 Immediate decolorization of epinochrome (27) solutions on addition of alkali was reported a few years later.134... 

Subsequent investigations have shown that Raper s suggestion that dopachrome (4) and related aminochromes decompose by an internal oxidation-reduction process forming 5,6-dihydroxyindoles was essentially correct.73,118,120,184-137 The 5,6-dihydroxyindoles obtained from aminochromes such as dopachrome (4) and epino-chrome (27) (i.e. with no substitution in the 3-position) show only a relatively weak blue to blue-mauve fluorescence.118,120 The intense yellow-green fluorescence shown by the rearrangement products of aminochromes with a 3-hydroxyl group is due to the formation of... 

Since certain aminochromes (e.g. dopachrome) are essential intermediates in the process of melanogenesis (see preceding section) and in view of the widespread distribution of melanitic pigments in both the plant and animal kingdoms, it would appear, that in this respect at least, aminochromes must presumably be formed in vivo from certain of the catecholamines. 

The aglycone of betaniu, the red-violet pigment of the beet (Beta vulgaris var. rubra), known as betanidin, has recently been shown to have the structure depicted in structural formula 113.170,282 Betanidin had previously been reported by Piattelli and Minale to contain a dopachrome unit in its structure and consequently would have been formulated as 114.109 The Italian workers obtained pyrrole-2,3,5-tricarboxylic acid and pyridine-2,4,6-tricarboxylic acid on permanganate oxidation of betanidin and they also prepared a semi-carbazone of the pigment, the absorption spectrum of which showed the pH dependency expected for an aminochrome semicarbazone.169... 

Wyler et al., however, believe that the aminochrome monosemicar-bazone obtained by Piattelli and Minale was in fact dopachrome monosemicar bazone, formed after decomposition of the betanidin molecule, which occurs readily in air.170,282... 

Figure 25-6 Postulated pathways for synthesis of the black pigment melanin and pigments (phaeomelanins) of reddish hair and feathers. Dopachrome reacts in two ways, with and without decarboxylation. The pathway without decarboxylation is indicated by green arrows. To the extent that this pathway is followed the green carboxylate groups will remain in the polymer. The black eumelanin is formed by reactions at the left and center while the reddish phaeomelanin is derived from polymers with cysteine incorporated by reactions at the right.

Tyrosinase, a copper-containing oxidoreductase, catalyzes the orthohydroxy-lation of monophenols and the aerobic oxidation of catechols. The enzyme activity will be assayed by monitoring the oxidation of 3,4-dihydroxyphenyl-alanine (dopa) to the red-colored dopachrome. The kinetic parameters Ku and Vmax will be evaluated using Lineweaver-Burk or direct linear plots. Inhibition of tyrosinase by thiourea and cinnamate will also be studied. Two stereoisomers, L-dopa and D-dopa, will be tested and compared as substrates. 

Many assays for tyrosinase activity have been developed. Procedures in the literature include use of the oxygen electrode, oxidation of tyrosine followed at 280 nm, and oxidation of dopa followed at 475 nm. The most convenient assay involves following the tyrosinase-catalyzed oxidation of dopa by monitoring the initial rate of formation of dopachrome at 475 nm (Figure E5.8). 

Two substrates are required in the tyrosinase-catalyzed reaction, phenolic substrate (dopa) and dioxygen. The conditions described in the experiment are such that the reaction mixtures are saturated with dissolved dioxygen. Therefore, when measurements are made for Ku, only the concentration of dopa is limiting, so the rate of the reaction depends on dopa concentration. The dopachrome assay is extremely flexible, as it can be applied to a variety of studies of tyrosinase. 

Before kinetic constants can be evaluated, it is critical to find the correct concentration of enzyme to use for the assays. If too little enzyme is used, the overall absorbance change for a reaction time period will be so small that it is difficult to detect differences due to substrate concentration changes or inhibitor action. On the other hand, too much enzyme will allow the reaction to proceed too rapidly, and the leveling off of the time course curve as shown in Figure E5.7 will occur very early because of the rapid disappearance of substrate. A rate that is intermediate between these two extremes is best. For the dopachrome assay, it is desirable to use the level of tyrosinase that gives a linear absorbance change at 475 nm for 2 minutes. 

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