Aggregation equilibria of tyrosinase of Harding-Passey mouse melanoma

Abstract

The purification of 2 isoenzymes of tyrosinase was carried out in Harding-Passey mouse melanoma. One is found in the cytosol and the other one bound to melanosomes. Both migrate as single bands on sodium dodecyl sulphate[SDS]/polyacrylamide gels, having an apparent MW of 58,000. Solubilized particulate tyrosinase showed an aggregation equilibrium involving a monomer, tetramer, octamer and a high-MW micellar form with Brij 35, the solubilizing agent. HPLC [high performance liquid chromatography] studies indicated a interconversion between those species, the monomer contribution increasing with the sample dilution. The tetramer and the octamer probably represent the predominant forms in vivo. Soluble tyrosinase showed a simpler aggregation equilibrium, involving 2 forms, monomer and tetramer, with the same interconversion pattern. Fluorescence studies suggested that tryptophan residues were exposed to the aqueous environment when tyrosinase was dissociated by dilution. Tyrosinase shows a tendency to aggregate, at low protein concentration and a resistance to dissociation by urea or SDS so remarkable that gel-permeation chromatography in 4 M-urea does not affect the equilibrium, and the band obtained on SDS/polyacrylamide-gel electrophoresis is a dimer.