Premium
Electrophoretically mediated reaction of glycosidases at a nanoliter scale
Author(s) -
Kanie Yoshimi,
Kanie Osamu
Publication year - 2003
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200390130
Subject(s) - substrate (aquarium) , microanalysis , michaelis–menten kinetics , enzyme , chemistry , chromatography , glycoside hydrolase , reaction rate , order of reaction , enzyme assay , reaction rate constant , kinetics , stereochemistry , biochemistry , organic chemistry , catalysis , biology , ecology , physics , quantum mechanics
We have investigated electrophoretically mediated microanalysis (EMMA) for the assay of a native glyco‐enzyme. As a representative of this class of enzyme, β‐glucosidase was selected, and the reaction was analyzed. Our EMMA was based on the plug‐plug interaction of enzyme and substrate plugs, which is essential to reduce quantities of materials. Furthermore, we have addressed the problem of incompatibility of the enzymatic reaction and separation of the reactants. As a result, EMMA of native glycosidase was achieved with a reaction volume of ∼ 20 nL and the Michaelis constant was estimated according to the Lineweaver‐Burk plot. The current method may have advantages over traditional assay methods, especially in terms of the amount of enzyme (ng order) and substrate (pmol order) required for a reaction*.