A fluorometric microassay procedure for monitoring the enzymatic activity of GM 1-ganglioside β-galactosidase by use of high-performance liquid chromatography

Abstract

For the measurement of the enzymatic activity of GM 1-ganglioside (II 3 NeuAcGgOse 4Cer, galactosyl- N-acetylgalactosaminyl-( N-acetylneuraminosyl)-galactosyl-glucosylceramide) β-galactosidase in crude enzyme samples, a microassay using nonradioisotopic GM 1-ganglioside was devised. To reduce the volume of the reaction mixture and eliminate the interferences due to the fluorescent contaminants in the reaction mixture, NADH, a product after the oxidation of the released galactose with NAD and β-galactose dehydrogenase, was fluorometrically estimated by use of high-performance liquid chromatography. By this method, as little as 10 pmol of galactose can be detected. Using rat brain homogenates as an enzyme sample, the several parameters were reexamined to define the optimal conditions for the assay. This assay method was also applied to human cultured skin fibroblast homogenates, and it was found that this method can be used for the diagnosis of GM 1-gangliosidosis, instead of the usual method using the radioisotopelabeled natural substrate.