A colorimetric procedure for measuring the enzymatic hydrolysis of terminal galactose from GM ganglioside

The enzymatic hydrolysis of the terminal galactose from GM₁-ganglioside is monitored by a colorimetric procedure. The NADH generated from the oxidation of released galactose with NAD and galactose dehydrogenase is employed to reduce p-iodonitrotetrazolium and the absorbance of the product, p-iodonitrotetrazolium formazan, is measured. The method can detect as little as 0.5 nmol of galactose. Hydrolysis of GM₁-ganglioside is accomplished using β-galactosidase from the marine gastropod Turbo cornutus. The enzymatic release of galactose is maximal at pH 3.5, and the reaction rate is linearly proportional to incubation time for 30 min, under the conditions employed. The presence of GM₂-ganglioside in the reaction mixture, after hydrolysis has occurred, is demonstrated by thin-layer chromatography.