The multiple forms of mushroom tyrosinase. Purification and molecular properties of the enzymes.

We have undertaken the purification of mushroom tyrosinase to obtain homogeneous enzyme for a study of its mechanism, which is unknown. Purified mushroom tyrosinases have already been reported (l-9). Some of these preparations were homogeneous, or almost homogeneous, by either electrophoresis or ultracentrifugation; one was homogeneous by both criteria (3, 9). However, there are discrepancies among them. According to one report, the molecular weight is 34,000 (3) ; according to others, it is close to 100,000 (2,4-6,9). The copper which the enzyme contains is either partially (6) or entirely cuprous (10). Moreover, if tyrosinase is a single protein able to catalyze a-hydroxylation of monophenols and dehydrogenation of o-diphenols, the ratio of these two kinds of activities, often referred to as cresolase and catecholase activities, depends on the method of purification (2). In 1949, Mallette and Dawson (2) obtained mushroom tyrosinases with different physical and enzymic properties which they ascribed to chemical or physicochemical changes in an original single enzyme during the purification process. Their enzymes contained different amounts of copper and different ratios of cresolase to catecholase activity, but they could not be separated from one another by ultracentrifugation or electrophoresis. Recently, Smith and Krueger (8) showed that five tyrosinase activities can be separated from mushroom extracts by chromatography on hydroxylapatite columns. One of their active fractions moved as a single substance during starch gel electrophoresis, but the other fractions were heterogeneous. In this study, we have confirmed the multiplicity of mushroom tyrosinase. Using classical procedures and an efficient preparative electrophoresis followed by chromatography on hydroxylapatite, we obtained four proteins: the (Y-, fl-, y-, and d-tyrosinases, three in homogeneous form. In this article, we describe the preparation of the enzymes and some of their enzymic, chemical, and physical properties.