Purification and properties of a catechol oxidase from blood cells of the ascidian Pyura stolonifera.

A catechol oxidase (EC 1.10.3.1) was purified to homogeneity from blood cells of the ascidian Pyura stolonifera using gel filtration on Sephadex G-50 and hydrophobic interaction chromatography on PhenylSuperose. Two peaks of activity were eluted from PhenylSuperose, one with a decreasing salt gradient and the other with nonionic detergent. The latter represents an aggregated form of the enzyme. The enzyme has a molecular weight of 56 kd and shows a preference for catechols with uncharged hydrophobic side chains (e.g., 4-t-butylcatechol) but does not hydroxylate free tyrosine. Inhibition of the enzyme by diethyldithiocarbamic acid and thiol reagents implicate copper at the active site. Sequence analysis of a peptide generated by incubation with Staphylococcus aureus V8 protease demonstrated considerable homology to one of the conserved copper binding regions of tyrosinases. This enzyme is found in the same cells as the dopa-containing protein ferreascidin. When ferreascidin is incubated with the enzyme, its spectrum changes rapidly, indicating that the catechol oxidase uses it as a substrate. The P. stolonifera enzyme differs from an enzyme involved in adhesion, isolated from the mussels, M. edulis and G. demissa: it is isolated as a soluble enzyme that does not appear to exist as a latent precursor.