Analytical Sciences

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

Hajime KATANO,* Kohei UEMATSU,* Takao HIBI,* Tokuji IKEDA,* and Toshihide TSUKATANI**

*Department of Bioscience, Fukui Prefectural University, Eiheiji, Fukui 910-1195, Japan
**Research and Development Division, Nicca Chemical, Co. Ltd., Bunkyo, Fukui 910-8670, Japan

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.

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