An improved method for arylsulfatase a detection on polyacrylamide slab gels

Abstract

An improved method for the detection of arylsulfatase A on polyacrylamide slab gels has been developed using a four-step reaction sequence which leads to the formation of a stable, localized, visible product. The reaction sequence involves the formation of nitrocatechol which, in turn, reduces cupric ferricyanide to form Hatchett's brown, an insoluble brown compound. The next steps involve polymerization of 3,3′-diaminobenzidine and osmication of the polymer. Since the initial reaction product, nitrocatechol, reacts quickly with the cupric ferricyanide to form the Hatchett's brown precipitate, little diffusion occurs and the observed bands of Hatchett's brown are highly localized. The formation of the Hatchett's brown can be observed as the arylsulfatase A reaction proceeds. The final osmium-stained product is very stable and has been stored for weeks without appreciable loss of light absorption or increase in band width. Analysis of arylsulfatase A activity in human leukocyte lysates after discontinuous electrophoresis in polyacrylamide slab gels demonstrated the presence of four bands of activity.