Structural stability of glucose oxidase encapsulated in liposomes to inhibition by hydrogen peroxide produced during glucose oxidation

The glucose oxidase (GO) consists of two identical subunits each of which contains noncovalently bound flavin adenine dinucleotide (FAD) cofactor. GO is known to be inactivated due to hydrogen peroxide (H2O2) produced in the oxidation of glucose. In our previous paper, the liposomal GO showed a much higher stability to H2O2 than the free enzyme. In this work, to deduce the structure and state of the liposomal GO, the fluorescence properties of the tryptophan residue and FAD cofactor in free GO during the glucose oxidation were measured for its tertiary structure and redox state, respectively. The tryptophan fluorescence data revealed that the initial glucose concentration lower than 0.6 mM resulted in almost no alteration in the tertiary structure, while the higher concentration did in a remarkable change in the structure due to the increase in catalytic turnover. On the other hand, the FAD fluorescence data showed that the reduced FAD was accumulated in the initial stage of the reaction. When glucose was completely consumed, the FAD restored the initial oxidized form for the initial glucose concentrations lower than 0.6 mM, whereas for the higher concentrations the reduced FAD tended to form an inactive complex with H2O2 leading to the deactivated enzyme. In the case of the liposomal GO at even such a high initial glucose concentration as 10 mM, the glucose concentration inside liposome was previously estimated to be lower than 0.2 mM due to its low permeability to glucose. Consequently, the formation of the inactive complex was proved to be effectively depressed in the liposomal GO reaction.