398 P. A. Riley Two isomers are formed in vitro: 2-cysteinyl dopa and 5-cysteinyl dopa, the latter in proportionately much larger amounts. If the product is oxidized to cysteinyl quinone spontaneous cyclization takes place with reduction to dihydrobenzothiazine (Fig. 6). This reaction is linked to the oxidation of a diphenol such as cysteinyl-dopa so that the generation of dihydrobenzothiazine, once initiated, can proceed non-enzymatically. This may explain the apparently paradoxical findings of Prota's group (6) that 3-x•C - cysteine incorporation by pigment cells is related to tyrosinase activity whereas Gesch- wind, Huseby and Nishioka (7) showed that MSH administration to A ¾ mice switched melanocytes from pheomelanin to eumelanin production. o•CO OH Ho•CO OH -,. Figure 6. Reduction of 5-S-cysteinylquinone to form dihydrobenzothiazine. It has been shown that the pheomelanins are composed of more or less complex polymers of dihydrobenzothiazine. A number of trichochromes which are composed of dimers of dihydrobenzothiazine have been identified and degradation studies of gallo- pheomelanin obtained from New Hampshire hen feathers have shown it to be an irregular and complex polymer perhaps containing benzothiazole and tetrahydroisoquinoline ring systems. A similar lack of simplicity is evident in the composition of eumelanins. Polymeriza- tion of the oxidation products of tyrosine takes place by a series of reductive additions. A random assortment of oxidized intermediates is incorporated into the polymer which therefore lacks any specific structure. The most regular structure is one produced by polymerization between C4 and C7 atoms of adjacent indole quinone rings. X-ray diffraction studies indicate that stacking of the rings occurs forming a lattice arrangement and this constraint may be responsible for introducing some regularity into the polymer by restricting access to sterically affine structures. Two major differences between eumelanins and pheomelanins emerge from a knowledge of their biosynthesis. Pheo- melanins, because they are polymers of less conjugated precursor molecules, show a much more restricted spectral absorbance than eumelanins and thus give the structures which they pigment a reddish or yellowish appearance in contrast to the dark brown or black of the highly conjugated eumelanins. Secondly, because the cyclization of cysteinyl-quinone replaces one of the hydroxyl groups on the phenol ring, the pheomelanin polymer cannot contain reactive quinones and this prevents the formation of cross-linkages with protein and leads to the characteristic lack of organization within pheomelanin pigment granules. In the case of eumelanins the protein tanning effect of quinone constituents of the pigment is very marked and seems to be of importance in the hardening of insect cuticles and sclerotization reactions in other invertebrates (1). In mammalian melanosomes the reaction of eumelanin quinones with protein probably accounts both of the melanin deposition on the matrix and the inhibition of tyrosinase in the fully melanized granule. The highly conjugated structure of eumelanins permits electron movements and electron exchanges with neighbouring molecules take place readily, i.e. melanins are chemically highly reactive. Commoner, Townsend and Pake (8) first demonstrated the paramagnetic

The mechanism of skin pigment production 399 properties of melanins and subsequently Mason's group (9) showed that the free radical property of melanin is due to semiquinones, stabilized by resonance in the highly con- jugated polymer and steric restrictions on internal radical annihilation reactions. Mole- cular orbital calculations by Pullman and Pullman (10), based on an assumed structure for dopa-melanin, showed that several redox states are possible and predicted electron acceptor properties which have been confirmed by several studies (11, 12, 13, 14). This reactivity of melanins may be the reason which necessitates its compartmentation in membrane-bounded melanosomes in the melanocytes (15). When transferred to the cytoplasm of keratocytes, melanin granules probably act as initiators of cytoplasmic damage in cells exposed to radiation which is absorbed by the pigment and generates free radicals (9, 16). The evidence for this proposal is discussed elsewhere (1, 17, 18) but it is probable that the selective advantage to hairless mammals is the destruction of cells which have received a radiation dosage sufficient to cause deleterious mutations. Clearly, from what has been said about their structure, the pheomelanins are much less effective in this respect and this is borne out by the statistics on the susceptibility of various ethnic groups to skin cancer. TYROSINASE The enzyme responsible for the oxidations involved in melanogenesis is tyrosinase. At least two forms (a and [•) of the enzyme are recognized and minor differences in activity exist but the general properties are broadly identical. They are widespread in nature, occurring in both eukaryotic and prokaryotic organisms. In vertebrates the enzyme is synthesized only in specialized cells (melanocytes) and is active only in specialized cyto- plasmic organelles (melanosomes). In contrast to many other enzymes whose structures have been determined and for which the catalytic mechanisms are well understood, tyro- sinase is still very poorly comprehended and no clear reaction mechanism has emerged. It is known that tyrosinases contain copper and that they bind oxygen. There are two main classes of oxidation catalysed by tyrosinases: the dehydrogenation of diphenols (catecholase activity) and the ortho-hydroxylation of monophenols (cresolase activity). CATECHOLASE ACTIVITY The enzyme isolated from Neurospora crassa has been studied by a number of groups. The enzyme has a molecular weight of about 33 000 daltons and contains one atom of copper per mole of protein (19). The kinetic studies of Gutteridge and Robb (20) in- dicate a single binding site for diphenolic substrates. Electron spin resonance studies show that only 4•o of the copper is in the cupric form and spectral absorbance data indicate that 35•o is complexed with monomolecular oxygen leaving about 60•o of the enzyme free to combine with the substrate. Binding to substrate and oxygen is thought to take place in a random sequence (Fig. 7). The Neurospora enzyme appears to be incapable of oxidizing monophenols. CRESOLASE ACTIVITY Mushroom (Agoricus bisporus) tyrosinase is a tetramer of 130 000 daltons molecular weight and contains four atoms of copper. On the basis of the reaction with hydrogen peroxide, Jolley et al. (21) have proposed a dimeric active site containing two copper