Detection of Dopachrome Isomerase Activity on Gels

Abstract

Dopachrome isomerase is a recently discovered enzyme associated with the melanogenesis process occurring in most vertebrates and invertebrates. It catalyzes the conversion of dopachrome to 5,6-dihydroxyindole(s). Based on the fact that 5,6-dihydroxyindoles are rapidly oxidized to melanochrome pigment by tyrosinases, we have developed a rapid, sensitive, and specific staining procedure for detection of dopachrome isomerase activity after gel electrophoresis. The method employs the use of commercially available mushroom tyrosinase entrapped in polyacrylamide gels for electrophoretic separation of dopachrome isomerase. Staining is achieved by the use of dopa solution. The dopachrome formed by the action of mushroom tyrosinase entrapped in the gel is converted to 5,6-dihydroxyindole(s) by dopachrome isomerase initially. The latter compound is subsequently oxidized by tyrosinase to purple-colored melanochrome. Therefore, dopachrome isomerase appears as a bluish-purple band against a pale orange-red background within 10 min. With the use of the new detection technique, the presence of hitherto unknown isozymes of dopachrome isomerase could be readily detected in polyacrylamide gels. This procedure is more sensitive than silver staining for detection of dopachrome isomerase and as little as 15 ng of purified protein could be easily detected on gels.