Isolation of estradiol-2,3-quinone and its intermediary role in melanin formation

Abstract

We have reported previously that 2-hydroxyestradiol can be oxidized in the presence of catechol by mushroom We have reported previously that 2-hydroxyestradiol can be oxidized in the presence of catechol by mushroom tyrosinase, with a stoichiometric requirement of molecular oxygen (Jacobsohn, G.M. and Jacobsohn, M.K. (1984) Arch. tyrosinase, with a stoichiometric requirement of molecular oxygen (Jacobsohn, G.M. and Jacobsohn, M.K. (1984) Arch. Biochem. Biophys. 232, 189–196). It is then incorporated into melanin (Jacobsohn et al. (1988) J. Steroid Biochem. 31, Biochem. Biophys. 232, 189–196). It is then incorporated into melanin (Jacobsohn et al. (1988) J. Steroid Biochem. 31, 377–385). We now report on the isolation and characterization of the o-quinone as a product of the enzyme reaction 377–385). We now report on the isolation and characterization of the o-quinone as a product of the enzyme reaction from 2-hydroxyestradiol. The o-quinone was isolated from incubates and identified by its FTIR spectrum, in particular, from 2-hydroxyestradiol. The o-quinone was isolated from incubates and identified by its FTIR spectrum, in particular, by the appearance of a new band at 1652 cm−1, its migration in HPLC systems, its ultraviolet spectrum, its by the appearance of a new band at 1652 cm−1, its migration in HPLC systems, its ultraviolet spectrum, its derivatization with phenylenediamine and comparison of these properties with the periodate oxidation product of the derivatization with phenylenediamine and comparison of these properties with the periodate oxidation product of the same substrate. The enzyme oxidation of the catechol estrogen was performed at 37°C and did not require an activator; same substrate. The enzyme oxidation of the catechol estrogen was performed at 37°C and did not require an activator; dopa at concentrations higher than 5 μM was inhibitory. At concentrations lower than 5 μM, dopa acted catalytically dopa at concentrations higher than 5 μM was inhibitory. At concentrations lower than 5 μM, dopa acted catalytically and was not consumed during the course of reaction. Ascorbic acid inhibited the reaction. The quinone exhibited both and was not consumed during the course of reaction. Ascorbic acid inhibited the reaction. The quinone exhibited both reversible and irreversible binding to preformed melanin and to melanin actively synthesized by the enzyme. Incubation reversible and irreversible binding to preformed melanin and to melanin actively synthesized by the enzyme. Incubation of 18 μM newly synthesized [4-14C]estradiol-2,3- quinone with mushroom tyrosinase for 45 min at 37°C in presence of of 18 μM newly synthesized [4-14Clestradiol-2,3- quinone with mushroom tyrosinase for 45 min at 37°C in presence of 400 μM dopa showed incorporation (irreversible binding) of 6.3 ± 0.3% of label into melanin produced during the 400 μM dopa showed incorporation (irreversible binding) of 6.3 ± 0.3% of label into melanin produced during the course of reaction. Similar incubations for 45 min of pre-formed melanin prepared from 400 μM dopa showed course of reaction. Similar incubations for 45 min of pre-formed melanin prepared from 400 μM dopa showed incorporation of 4.4 ± 0.2% of the label. Reversible binding was 10-times greater than incorporation for both actively incorporation of 4.4 ± 0.2% of the label. Reversible binding was 10-times greater than incorporation for both actively synthesized and preformed melanins. In the absence of dopa or catechol, enzyme incubations of either 2-hydroxyestradiol synthesized and preformed melanins. In the absence of dopa or catechol, enzyme incubations of either 2-hydroxyestradiol or its quinone did not yield melanin. Data suggest that estradiol-2,3-quinone is an intermediate in the or its quinone did not yield melanin. Data suggest that estradiol-2,3-quinone incorporation of the catechol estrogen into melanin by tyrosinase.