Enzymatic staining of sialidase and b-galactosidase in polyacrylamide gel using chromogenic and fluorigenic substrates

Abstract

Lysosomal sialidase is a membrane-bound enzyme, which found to be a complex with ?-galactosidase and carboxypeptidase A. Due to its low stability a simple detection in polyacrylamide gels using chromogenic substrate was developed to speed up exploring this enzyme. Vibrio cholera sialidase and Escherichia coli ?-galactosidase were used as amodel. In a polyacrylamide gels using SDS-PAGE under non-reducing condition, bands showing enzyme activities of sialidase and ?-galactosidase were detected following staining with the chromogenic substrates, 5-bromo-4-chloro-3-indolyl-?-D-N-acetylneuraminic acid (X-Neu5Ac) and 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside (X-Gal) respectively. With this staining method, protein band corresponds to the enzyme activities could be located and detected as a permanently sharp blue band, which could be very useful to detect the protein band correspond to the enzyme activities even from partially purified enzyme. Detection of the Vibrio cholera sialidase and Escherichia coli ?-galactosidase using fluorigenic substrates, sialyl-methylumbelliferyl ?-glycoside (MU-Neu5Ac) and galactosyl-methylumbelliferyl ?-glycoside, respectively, were used as comparison.