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

M. Naoi, M. Kondoh, T. Mutoh, T. Takahashi, T. Kojima, T. Hirooka, T. Nagatsu

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)75-82
Number of pages8
JournalJournal of Chromatography B: Biomedical Sciences and Applications
Volume426
Issue numberC
DOIs
Publication statusPublished - 1988
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Chemistry(all)

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