Purification of human skin tyrosinase and its protein inhibitor: properties of the enzyme and the mechanism of inhibition by protein

Tyrosinase from normal human skin was purified to high specific activity; 228 nmol of dopa formed/min/mg protein. The properties of the purified enzyme differ from those of the same enzyme in crude homogenates. The activity of the purified enzyme is not affected by dopa. It is not inhibited by excess tyrosine and exhibits no lag in its rate at 4 mm concentration of ascorbic acid. This preparation is free of peroxidase and yet will catalyze both hydroxylation of tyrosine to dopa and its further oxidation to dopa quinone with fourfold more activity with dopa as substrate suggesting that mammalian tyrosinase catalyzes both reactions rather than dopa oxidation alone as suggested by M. Okun, L. Edelstein, R. Patel, and B. Donnellan (1973, Yale J. Biol. Med.46, 535–540). A protein present in the cytosol and melanosomes that constitutes 30% of soluble epidermal proteins was purified and found to inhibit tyrosinase competitively with tyrosine. Its molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 66,000.