ENZYMATIC AND NONENZYMATIC OXYGENATION OF TYROSINE

Tyrosinase isolated from cultured human melanoma cells was studied for tyrosine oxygenation activity. L-Tyrosine and D-tyrosine were used as substrates and dopa was measured with HPLC and electrochemical detect ion as the product of oxygenation. Incubations were performed in the p resence or absence of dopamine as co-substrate. Oxygenation of L-tyros ine occurred only in the presence of dopamine as co-substrate. No oxyg enation of D-tyrosine was found, and we conclude that human tyrosinase is characterised by exclusive specificity for the L-isomer of tyrosin e in its oxygenase function. It has recently been suggested that super oxide anion is a preferential oxygen substrate for human tyrosinase. I ncubations were therefore performed with L- and D-tyrosine, human tyro sinase, and xanthine/xanthine oxidase in the system, generating supero xide anion and hydrogen peroxide. Considerable formation of dopa was o bserved, but the quantity was the same irrespective of whether D-tyros ine or L-tyrosine was used as the substrate. Furthermore, formation of dopa occurred in a xanthine/xanthine oxidase system when bovine serum albumin (BSA) was substituted for tyrosinase. Our results provide no evidence that superoxide anion is an oxygen substrate for human tyrosi nase. In the incubate containing xanthine/xanthine oxidase, catalase c ompletely inhibited dopa formation, and superoxide dismutase and manni tol each strongly inhibited dopa formation. The results are compatible with hydroxyl radicals being responsible for the formation of dopa, s ince such radicals may be secondarily formed in the presence of supero xide anion and hydrogen peroxide.