Application of Poly[oxyethylene(dimethylimino)propyl-(dimethylimino)ethylene] as Enzyme Stabilizer for Bilirubin Oxidase Immobilized Electrode

Abstract

It has been shown that polyammonium cations comprising quaternary ammonium and hydrophilic groups such as amide and hydroxyl groups stabilize a redox enzyme bilirubin oxidase (BOD). The BOD catalyzes the reaction: 4[Fe(CN)₆]^4– + 4H⁺ + O₂ → 4[Fe(CN)₆]^3– + 2H₂O, and has been a promising enzyme for use as a cathode catalyst in biofuel cells. In this study, the stabilizing effect of poly[oxyethylene(dimethylimino)propyl(dimethylimino)ethylene] (PA1) on BOD has been investigated. The sample solution containing BOD and the PA1 salt was kept at a given temperature, and the loss of the enzymatic activity was detected after given stored times. The activity decreased exponentially with stored time so that the first-order rate-constant of inactivation was determined. The inactivation rate-constant lowered with increasing the concentration of the PA1 salt, suggesting that BOD was stabilized by the association with the PA1 cation. The PA1 cation may act like a protective colloid or decrease the local disorder of BOD by its wrapping. A membrane-covered electrode containing BOD, PA1, and [Fe(CN)₆]^4–/3– in the internal solution phase was examined in air-saturated aqueous solution. The electrode gave a well-defined current–potential curve with a steady state limiting current due to the PA1-[Fe(CN)₆]^4–/3– polyion complex-mediated bioelectrocatalytic current for the reduction of O₂. The decreasing of the steady state limiting current became slower in the presence of the PA1 salt, indicating again the stabilizing effect of PA1 cation on BOD.