The use of HPLC-pulsed amperometry for the characterization and assay of glycosidases and glycosyltransferases
- 1 March 1991
- journal article
- research article
- Published by Oxford University Press (OUP) in Glycobiology
- Vol. 1 (2) , 223-227
- https://doi.org/10.1093/glycob/1.2.223
Abstract
A sensitive and reproducible high performance chromatographic procedure is described for the assay of jack bean β-galactosidase in which the reaction products are separated on a Dionex AS6 ion exchange column under alkaline conditions and detected by triple-pulsed amperometry. Quantition of the enzyme-released galactose is accomplished by using either fucose or lactose, the substrate, as an internal standard. The validity of the procedure as a general method for the assay and kinetic characterization of exoglycosidases was demonstrated by performing parallel measurements of galactose using an established coupled-enzyme assay, and using these values to calculate Km and Vmax values against lactose. Additional data are presented which establish the applicability of using a similar HPLC approach for the assay of glycosyltransferases.Keywords
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