Explanation of anomalous binding kinetics with a high yield immobilized enzyme system.
The activities of glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) and catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) from commercial preparations do not give typical adsorption curves upon immobilization on non-porous polyethylenimine-coated glass microbeads. The cause of this effect with glucose oxidase was investigated. Protein binding exhibited a rectangular hyperbolic adsorption isotherm, approaching saturation at high concentrations, however, enzyme activities did not. The isotherm for activities exhibited a maxima which corresponded to less than 50% saturation with regard to total protein adsorption. The enzyme preparation was found to contain small quantities of several low molecular weight impurities as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These impurities apparently compete with glucose oxidase for binding. When large excesses of protein are added to beads, the binding of impurities becomes significant and the amount of enzyme activity per unit of bead is reduced.
D002374:Catalase / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005898:Glass; D005949:Glucose Oxidase / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D011094:Polyethyleneimine; D011485:Protein Binding
D004800:Enzymes, Immobilized; D011094:Polyethyleneimine; D005949:Glucose Oxidase; D002374:Catalase