Analytical Sciences

Abstract − Analytical Sciences, 25(9), 1077 (2009).

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.