The use of HPLC-pulsed amperometry for the characterization and assay of glycosidases and glycosyltransferases
1991; Oxford University Press; Volume: 1; Issue: 2 Linguagem: Inglês
10.1093/glycob/1.2.223
ISSN1460-2423
AutoresFrances Willenbrock, David C. A. Neville, Gary S. Jacob, Peter Scudder,
Tópico(s)Biochemical Analysis and Sensing Techniques
ResumoA 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.
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