TARGETING GLUCOSE-OXIDASE AT ASPARTATE AND GLUTAMATE RESIDUES WITH ORGANIC 2-ELECTRON REDOX MEDIATORS

The bimolecular rate constants for the reactions of five organic two-e lectron redox mediators with reduced glucose oxidase (GOx) were determ ined by measuring voltammetric electrocatalytic currents at glassy car bon electrodes in the presence of excess glucose under anaerobic condi tions. The mediators studied were thionine, brilliant cresyl blue, azu re A, daunomycin, and dopamine, and the bimolecular rate constants for electron transfer between GOx and the oxidized mediator (M(-1) s(-1)) are 1.6 x 10(4), 4.0 x 10(2), 9.8 x 10(2), 9.0 x 10(3), and 1.2 x 10( 6), respectively. GOx was covalently derivatized using 1-ethyl-3-[3-(d imethylamino)propyl]carbodiimide and N-hydroxysulfosuccinimide to form amide bonds between the aliphatic primary amine groups on daunomycin and dopamine and carboxylate side chains of aspartate and glutamate re sidues. Derivatives with 2.5 +/- 0.1 daunomycin groups and 4 +/- 1 dop amine groups were obtained, with activities of 50% and 75%, respective ly, relative to native GOx in a dye-peroxidase assay. Although the dau nomycin derivative did not show measurable intramolecular electron-tra nsfer rates, the dopamine derivative rapidly transfers electrons from active-site FADH(2) groups to the oxidized (quinone) form of dopamine. Because the heterogeneous oxidation of dopamine is relatively slow, t he currents measured at +0.75 V vs Ag/AgCl were not at their limiting (plateau) values, and only a minimum value of the intramolecular rate constant (4.5 s(-1)) could be determined. This value is >20 times larg er than values obtained for GOx-ferrocene derivatives in which surface lysine residues were covalently modified using identical coupling rea gents and similar reaction conditions. This work shows that targeting GOx carboxylate groups with electron-transfer mediators may represent a promising approach to the design of reagentless glucose biosensors.