Radioassay for oxidation of tyrosine by tyrosinase using L-(ring-3H)tyrosine and L-(carboxyl-14C)tyrosine.

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Abstract
Two intermediates involved in the synthesis of melanin from L-tyrosine by mushroom tyrosinase were assayed simultaneously by using L-[ring-3H]tyrosine and L-[carboxyl-14C]tyrosine. 3H 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 (3H2O method) respectively. 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 0.1 nmol of the intermediates, respectively. The formation of L-dopa, leucodopachrome and 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 475 nm. The maximum velocities were estimated from Lineweaver-Burk plots.

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