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Quantitative Kinetic Characterization of Glycoside Hydrolases Using High-Performance Anion-Exchange Chromatography
Nicholas McGregor1,2, Gregory Arnal1, Harry Brumer3,4,5
1Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, Canada, V6T 1Z4.
Abstract:
High-performance anion-exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD) is a powerful analytical technique enabling the high-resolution separation and sensitive quantification of oligosaccharides. Here, we describe a general method for the determination of glycoside hydrolase kinetics that harnesses the intrinsic power of HPAEC-PAD to simultaneously monitor the release of multiple products under conditions of low substrate conversion. Thus, the ability to track product release under initial-rate conditions with substrate concentrations as low as 5 μM enables the determination of Michaelis-Menten kinetics for glycosidase activities, including hydrolysis and transglycosylation. This technique may also be readily extended to other carbohydrate-active enzymes (CAZymes), including polysaccharide lyases, and glycosyl transferases.