Analytical Sciences

Measurements of Reversible and Irreversible Inactivation Processes of a Redox Enzyme, Bilirubin Oxidase, by Electrochemical Methods Based on Bioelectrocatalysis

Tokuji IKEDA, Kohei UEMATSU, Haku MA, Hajime KATANO, and Takao HIBI

Department of Bioscience, Fukui Prefectural University, Fukui 910-1195, Japan

The inactivation of a redox enzyme, bilirubin oxidase (BOD), by heat and guanidine hydrochloride (GuHCl) was studied by two bioelectrochemical methods. One is a conventional method, which measures the inactivation of BOD in solution, and the other is a method using a BOD-immobilized electrode (a membrane/BOD/GC electrode), which measures the inactivation of BOD in the immobilized state. The results for thermal inactivation revealed that BOD both in solution and in the immobilized state obeyed the same irreversible inactivation kinetics. The CD and absorption spectra of BOD confirmed that the irreversible thermal inactivation was accompanied by a change in the secondary structure and the dissociation of type-1 copper from BOD. The measurements in the presence of GuHCl demonstrated that the BOD activity was significantly decreased at 1 M GuHCl in both states, and that the decrease proceeded reversibly. The CD spectrum of BOD indicated that the secondary structure of BOD was little affected by GuHCl at this concentration. The effect of GuHCl on the thermal inactivation was studied and evaluated as the resulting values of the Arrhenius activation energy: ΔG^≠, ΔH^≠, and ΔS^≠.

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