Microassay for GM₁ ganglioside β-galactosidase activity using high-performance liquid chromatography

A simple and sensitive assay for GM₁ ganglioside (GM₁) β-galactosidase activity was devised by direct measurement of released d-galactose using high-performance liquid chromatography (HPLC). GM₁ β-galactosidase activity in crude samples such as brain homogenates could be measured by this method. After incubation of brain homogenate for 1 h with GM₁ at 37°C and pH 4.4 in the presence of sodium taurodeoxycholate, the reaction was terminated by heating at 100°C for 2 min and the supernatant from the centrifuged sample was analysed directly by HPLC. d-Galactose isolated by HPLC was converted into a fluorescent compound by a post-column reaction with arginine at 150°C and the fluorescence intensity at 430 nm was measured with excitation at 320 nm. By this method 10 pmol of d-galactose could be measured and the fluorescence intensity was linear up to 1 mmol of d-galactose. Using this method, the optimal conditions for the activity of this enzyme were re-examined. As an application, the enzyme activity in the brain of a patient with GM₁ gangliosidosis was examined. This method can be applied to any natural substrates, glycolipids or glycoproteins, the terminal galactose of which is hydrolysed by this enzyme.