Melanin Accelerates the Tyrosinase‐Catalyzed Oxygenation of p‐Hydroxyanisole (MMEH)

Abstract

Although pigment melanin has long been though of as "inert," recent work has attested to its chemical reactivity. In this communication, we report that either commercial synthetic melanin prepared by persulfate oxidation of tyrosine ("Sigma melanin") or sepia melanin extracted from cuttlefish markedly accelerates the in vitro oxygenation of p-hydroxyanisole (MMEH), catalyzed by mushroom or B-16 melanoma tyrosinase. Kinetics of 4-methoxy-1,2-benzoquinone formation (lambda max = 413 nm) or of molecular O2 uptake were biphasic, with an initial slow rate ("lag time") followed by a fast linear increase. The biphasic response reflects an initial slow hydroxylation followed by a fast dehydrogenation. Added melanin markedly decreased the lag time but had little effect on subsequent dehydrogenation. Similar effects were observed for tyrosine itself. A complex between MMEH and melanin appears to be the "active" species in these reactions. The results indicate that melanin acts as an electron conduit, which accepts electrons from the substrate and transfers them to tyrosinase. The magnitude of the effect depends on the type of melanin as well as on its oxidation state. Kinetic analysis indicates that both melanins are very efficient at transferring electron to tyrosinase, and that Sigma melanin is roughly threefold more efficient than sepia melanin. The qualitative similarity of reaction between the synthetic and "natural" melanins suggests that the former may serve as a first approximation to the in vivo situation. On the other hand, the observed quantitative differences and the sensitivity of these results to the chemical state of melanin suggests that this methodology might eventually be adapted as a non-destructive probe of melanin in situ.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosinase Kinetics: A Semi-quantitative Model of the Mechanism of Oxidation of Monohydric and Dihydric Phenolic Substrates

“…The action of this co-factor has variously been ascribed to an allosteric e!ect when bound to a secondary site (Hearing et al, 1981) or to a modifying action on the active site. In view of the similar e!ect of alternative reductants (Menter et al, 1990;Palumbo et al, 1990;Pomerantz, 1966), it now seems most probable that dopa is responsible for activating tyrosinase in the so-called met-state. In the met-enzyme the copper atoms of the active site are in the oxidized, i.e.…”

Tyrosinase Kinetics: Failure of Acceleration in Oxidation of Ring‐Blocked Monohydric Phenol Substrate

“…Monophenolase action exhibits unusual lag phase kinetics (Lerner et al, 1949), which has received considerable attention. It is not clear whether the lag period is a significant controlling feature of tyrosinase activity in vivo where microenvironmental factors including the effects of melanosomal pH (Ramaiah, 1996) and availability of alternative electron donors (Pomerantz, 1966;Menter et al, 1990;Palumbo et al, 1990;Naish-Byfield et al, 1994) may play a role. It is, however, a characteristic feature of in vitro tyrosinase activity.…”

Electron Transfer and Photoprotective Properties of Melanins in Solution

“…The initial velocity of ferricyanide reduction was monitored at 420 nm. %osinase-catalysed oxygenation of MMEH was monitored at 413 nm (4-methoxy-1,2-benzoquinine); that of tBC at 400 nm (4-t-butyl-1,2-benzoquinone), and that of DOPA at 475 nm (dopachrome), as described in Menter et al (1990).…”

“…A dimensionless enhuncementfactor, E F = k*K&* reflects the ability of melanin to enhance ( E F > 1) or retard ( E F < 1) the rate of ferricyanide reduction by DF! Melanin-mediated acceleration of tyrosinase reaction (Menter et al, 1990). Stock enzyme solution (0.3 ml) was rapidly pipetted into a stirred solution containing 300 pM MMEH, tBE or tyrosine plus G166 pg melanin in 3.0 ml.…”

“…Although eumelanin has been considered a relatively inert biopolymer that protects the organism against solar radiation by absorption and scattering of harmful W (Pathak et al, 1987), there has recently been a new appreciation of melanin's ability to bind a wide variety of compounds (Larssen, 1993), to act as a radical scavenger in photoprotection (Musk and Parsons, 1987;, to act as an electron transport conduit (Gan et al, 1976;Menter and Willis, 1986;Menter et al, 19901, and to photochemically generate reactive radicals (Sarna at al., 1985;Korytowski et al, 1987;Schmitz et al, 1995, Chedekel, 1995. There has been a growing awareness that these very same chemical processes could also result in "antiprotective" behavior Cesarini, 1988;Menter and Willis, 1986;Menter et al, 1990) which could be deleterious to the cell or its surroundings under appropriate conditions.…”