Analytical Sciences

Abstract − Analytical Sciences, 24(11), 1421 (2008).

Application of Polyammonium Cations to Enzyme-immobilized Electrode: Application as Enzyme Stabilizer for Bilirubin Oxidase
Hajime KATANO,* Hirosuke TATSUMI,** Takao HIBI,* Tokuji IKEDA,* and Toshihide TSUKATANI***
* Department of Bioscience, Fukui Prefectural University, Eiheiji-cho, Fukui 910-1195, Japan
** International Young Researchers Empowerment Center, Shinshu University, Matsumoto 390-8621, Japan
*** Research and Development Division, Nicca Chemical, Co. Ltd., Bunkyo, Fukui 910-8670, Japan
Bilirubin oxidase was stored in the solutions containing polyammonium salts for a given time at 30°C, and the activity was assayed. The enzyme catalyzes the reaction: 4[Fe(CN)6]4- + 4H+ + O2 → 4[Fe(CN)6]3- + 2H2O, and the activity can be measured by the absorbance at the wavelength for the absorption maxima of [Fe(CN)6]3-. The results show that polyammonium cations comprising quaternary ammonium in the main chain and hydrophilic groups, such as hydroxyl and amide groups, stabilize the enzyme in solution. These polyammonium cations may act like a protective colloid. The membrane-covered electrode containing the polyammonium cations, the enzyme, and [Fe(CN)6]4-/3- in the internal solution phase was constructed. The electrode gave a well-defined current-potential curve with a steady state limiting current due to the polycation-[Fe(CN)6]4-/3- complex-mediated bioelectrocatalytic current for the reduction of O2. The time-dependent decrease of the limiting current indicates again the stabilizing effect of the polyammonium cations on the enzyme.