Radioassay for Oxidation of Tyrosine by Tyrosinase using L-[Ring-3H] tyrosine and L-[Carboxyl-14C] tyrosine

Two intermediates involved in the synthesis of melanin from L-tyrosine by mushroom tyrosinase (EC 1.14.18.1) were assayed simultaneously by using L-[ring-3H] tyrosine and L-[carboxyl-14C] tyrosine. Tritium ions released from the 3-position of L-[3, 5-3H] tyrosine or the 6-position of L-[2, 6-3H] tyrosine were isolated as 3H2O and employed to determine the quantity of L-dopa or leucodopachrome, respectively (3H2O method). 5, 6-Dihydroxyindole formed at the decarboxylation step was estimated from the radioactivity remaining after the evolution of 14CO2 from L-[carboxyl-14C] tyrosine (14CO2-release method). The lower limits of determination in the 3H2O and the 14CO2-release methods were 0.1 pmol and nmol of the intermediates, respectively. The formations of L-dopa, leucodopachrome, and 0.1 5, 6-dihydroxyindole were measured at various concentrations of L-tyrosine by both methods, and dopachrome was determined colorimetrically by measuring the absorbance changes at 475nm. The maximum velocities were estimated from Lineweaver-Burk plots.