Microassay for GM1 ganglioside beta-galactosidase activity using high-performance liquid chromatography.
A simple and sensitive assay for GM1 ganglioside (GM1) beta-galactosidase activity was devised by direct measurement of released D-galactose using high-performance liquid chromatography (HPLC). GM1 beta-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 degrees C and pH 4.4 in the presence of sodium taurodeoxycholate, the reaction was terminated by heating at 100 degrees 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 degrees 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.
D001921:Brain / Q000201:enzymology*; D002793:Cholic Acids / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D005696:Galactosidases / Q000032:analysis*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013050:Spectrometry, Fluorescence; D013657:Taurodeoxycholic Acid / Q000494:pharmacology; D001616:beta-Galactosidase / Q000032:analysis* / Q000493:pharmacokinetics
D002793:Cholic Acids; D013657:Taurodeoxycholic Acid; D005696:Galactosidases; C021502:acid beta-galactosidase; D001616:beta-Galactosidase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate