Ferricenium salts as true substrates of glucose oxidase

Abstract

The steady-state kinetics of D-glucose oxidation by ferricenium dyes RFc⁺PF₆ ^- (R = H, Me, Et, n-Bu, MeCH₂CMe₂, and Cl) and 1,1′-Et₂Fc+PF₆-catalyzed by glucose oxidase from Aspergillus niger was investigated as a function of RFc⁺ and D-glucose concentrations at pH 6.7, 25°C in the presence of 2% (v/v) Triton X-100. The enzymatic bleaching is characterized by large steady-state portions on the kinetic curves for all ferricenium ions studied. The reaction follows the MichaelisMenten kinetics demonstrating a high affinity of RFc⁺ toward the active site of reduced glucose oxidase (GO). The reaction rate is weakly sensitive to the nature of RFc⁺, and the apparent V_m(app) values decrease only twofold on going from the most to the least reactive salt in the series (l,l′-Et₂Fc⁺ and CIFc⁺, respectively), although their observed redox potentials differ by 160 mV. Remarkably, the reactivity of RFc⁺ does not increase with increasing their oxidative power. The apparent Michaelis constants K_m(app) are also weakly sensitive to the nature of RFc⁺. The profiles for the steady-state rate vs [HFc⁺] and [D-glucose] were rationalized in terms of the “ping-pong” mechanism typical of the catalysis by GO. Ferrocenecarboxylic acid (FcCOOH) appeared to be a competitive inhibitor of GO with the inhibition constant of (3 ±1) x 10-³M. The pH profile for the ferricenium fading is bell-shaped with the optimum around 7.5. A simple routine for a rapid in situ preparation of the ferricenium dye, which is ready for spectrophotometric assaying of the GO activity, is presented. The apparent V_m(app) and K_m(app) values for this substrate are similar to those for HFc⁺PF₆.