Chapter title |
Quantitative Kinetic Characterization of Glycoside Hydrolases Using High-Performance Anion-Exchange Chromatography (HPAEC)
|
---|---|
Chapter number | 2 |
Book title |
Protein-Carbohydrate Interactions
|
Published in |
Methods in molecular biology, April 2017
|
DOI | 10.1007/978-1-4939-6899-2_2 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6898-5, 978-1-4939-6899-2
|
Authors |
Nicholas McGregor, Gregory Arnal, Harry Brumer |
Editors |
D. Wade Abbott, Alicia Lammerts van Bueren |
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. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 6 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Ph. D. Student | 2 | 33% |
Student > Master | 2 | 33% |
Student > Postgraduate | 1 | 17% |
Researcher | 1 | 17% |
Readers by discipline | Count | As % |
---|---|---|
Biochemistry, Genetics and Molecular Biology | 2 | 33% |
Agricultural and Biological Sciences | 2 | 33% |
Medicine and Dentistry | 1 | 17% |
Engineering | 1 | 17% |