Regiospecific transfer of tritium into 3H2O from labeled estrogens by mushroom tyrosinase

The specificity of mushroom tyrosinase in displacing 3H from estradiol and catechol estrogens labeled at C-1, C-2, C-4 or C-6,7 was investigated under various conditions. [2-3H]E2 Yielded significant amounts of 3H2O, in the presence of NADH, and the rate of 3H loss from the steroid paralleled that of the radioactivity remaining in the aqueous fraction after extraction with organic solvents. NADH had little effect on the release of 3H from [1-3H]E2 or [4-3H]E2 but glutathione was highly active in this respect, with considerable differences being observed between lyophilizable 3H2O and yields of water-soluble products. It is proposed that 3H losses from C-2 of estradiol reflects oxidative displacement of this isotope by tyrosinase while the loss observed from C-1 and C-4 is the result of non-enzymatic conjugation with glutathione after the formation of the catechol estrogen. The difference between lyophilizable 3H2O and the yield of water-soluble products obtained with [1-3H]E2 and [4-3H]E2 provided a measure of the relative amount of conjugation occurring at C-1 and C-4. These findings were confirmed by double label experiments with 3H- and 14C-labeled estrogens and the isolation of the glutathionyl derivatives. The catechol estrogens did not serve as substrates for further hydroxylation by the enzyme even when C-2 was available for this reaction. These experiments give further information about the specificity of tyrosinase in its reaction with aromatic steroids and provide a simple and rapid method for confirming the distribution of 3H at C-2 or C-4 of estradiol.