The use of HPLC-pulsed amperometry for the characterization and assay of glycosidases and glycosyltransferases.
A sensitive and reproducible high performance chromatographic procedure is described for the assay of jack bean beta-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.
Willenbrock
F W
FW
Celltech Inc., Slough Berks, UK.
Neville
D C
DC
Jacob
G S
GS
Scudder
P
P
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004563:Electrochemistry / Q000379:methods; D007887:Fabaceae / Q000201:enzymology; D005260:Female; D005690:Galactose / Q000032:analysis; D005700:Galactosyltransferases / Q000032:analysis* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000032:analysis*; D016695:Glycosyltransferases / Q000032:analysis*; D007700:Kinetics; D007785:Lactose / Q000096:biosynthesis; D008892:Milk / Q000201:enzymology; D010946:Plants, Medicinal; D001616:beta-Galactosidase / Q000032:analysis* / Q000378:metabolism
D016695:Glycosyltransferases; D005700:Galactosyltransferases; D006026:Glycoside Hydrolases; D001616:beta-Galactosidase; D007785:Lactose; D005690:Galactose