Measurements of reversible and irreversible inactivation processes of a redox enzyme, bilirubin oxidase, by electrochemical methods based on bioelectrocatalysis.
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: DeltaG(not equal), DeltaH(not equal), and DeltaS(not equal).
Ikeda
Tokuji
T
Department of Bioscience, Fukui Prefectural University, Fukui 910-1195, Japan. tikeda@fpu.ac.jp
Uematsu
Kohei
K
Ma
Haku
H
Katano
Hajime
H
Hibi
Takao
T
D055162:Biocatalysis; D004563:Electrochemistry; D004789:Enzyme Activation / Q000187:drug effects; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D006999:Hypocreales / Q000201:enzymology; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation / Q000187:drug effects; D013696:Temperature
D004800:Enzymes, Immobilized; D044925:Oxidoreductases Acting on CH-CH Group Donors; C042401:bilirubin oxidase; D019791:Guanidine