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

Abstract

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.