Kinetics and mechanism of the reaction of glucose oxidase with a surface tethered N-hydroxysuccinimide ester using SPR

CHED 1419

Amanda R. Bolger, bolgera@allegheny.edu and Alice A. Deckert, adeckert@allegheny.edu. Department of Chemistry, Allegheny College, Meadville, PA 16335
The kinetics for the reaction between glucose oxidase (GOX) and self-assembled monolayers fabricated from the N-hydroxysuccinimide (NHS) ester of 4, mercaptobenzoic acid were studied using surface plasmon resonance (spr). Glucose oxidase solutions of different pH's were used to provide insight for the mechanism of enzyme immobilization on the surface. The data were pH-independent and supported the mechanism of an equilibrium non-specific binding of GOX with binding rate constant, kon and desorption rate constant, koff, followed by a one-step substitution of the GOX for NHS affording the surface tethered GOX with rate constant, k. These data contradicted the previously proposed two-step mechanism. The rate constants for the GOX immobilization were calculated to be kon = 760 M-1s-1, koff = 0.0092 M-1s-1 and k = 540 M-1s-1.