INTRAMOLECULAR ELECTRON-TRANSFER RATES IN FERROCENE-DERIVATIZED GLUCOSE-OXIDASE

Glucose oxidase (GOx) was covalently modified at pH 7.2 with ferrocenecarboxylic acid (FCA), ferrocenedicarboxylic acid (FDA), and ferroceneacetic acid (FAA) using 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide to promote selective coupling to surface lysines. Reagent ratios were varied to obtain derivatives with 5-12 ferrocene groups per GOx dimer and greater-than-or-equal-to 60% activity. For comparison, GOx was derivatized with FCA in the presence of 3 M urea using only EDC as a promoter. Varying reagent ratios yielded derivatives with 4-39 FCA groups per GOx and less-than-or-equal-to 30% activity; linear sweep voltammetry results showed a slow but readily detectable release of FCA upon storage of these derivatives. Tryptophan fluorescence quenching in two media, 0.1 M phosphate buffer (pH 7.0) and 8 M urea, confirmed that GOx was covalently modified and not merely associated with ferrocene. In all cases, the GOx derivatives exhibited significantly greater quenching than controls containing native GOx with free ferrocenes. The results of voltammetric dilution experiments (performed in oxygen-free solutions in the presence of excess glucose) were consistent with rate-limiting intramolecular electron transfer from the reduced flavin centers to bound ferricinium. Using an expression derived here, values between 0.16 and 0.90 s-1 were obtained for intramolecular electron transfer in the FCA derivatives, suggesting that the location (rather than the number) of bound ferrocene groups is rate-determining. Approximately 10(3)-fold slower intramolecular electron transfer was measured in an FDA derivative, consistent with fluorescence quenching data which indicated that bound FDA is more solvent-exposed than bound FCA. The results of lysine-targeted modification of GOx are interpreted in light of the recently published 3-D structure of GOx; since the critical flavin-lysine separations are all > 23 angstrom, an alternative approach is necessary to obtain GOx derivatives for use in a practical, reagentless glucose sensor.