Quantitative Kinetic Characterization of Glycoside Hydrolases Using High-Performance Anion-Exchange Chromatography (HPAEC)
Autor: | Gregory Arnal, Harry Brumer, Nicholas McGregor |
---|---|
Rok vydání: | 2017 |
Předmět: |
0301 basic medicine
chemistry.chemical_classification Chromatography 030102 biochemistry & molecular biology Chemistry Kinetics Substrate (chemistry) Oligosaccharide High-performance liquid chromatography Amperometry 03 medical and health sciences Hydrolysis chemistry.chemical_compound 030104 developmental biology Glycoside hydrolase Glycosyl |
Zdroj: | Methods in Molecular Biology ISBN: 9781493968985 |
Popis: | 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. |
Databáze: | OpenAIRE |
Externí odkaz: |