Release of flavine adenine dinucleotide on adsorption of the enzyme glucose oxidase to clays

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Abstract
The adsorption–desorption characteristics of glucose oxidase holoenzyme (EC 1.1.3.4; β-D-glucose: oxygen-1-oxidoreductase), glucose oxidase apoenzyme, and the coenzyme flavine adenine dinucleotide (FAD) todays were studied. The fixation of apoenzyme, at a solution pH of 4.5, was rapid and almost complete within 1 h of incubation, since less than 5% of adsorbed apoenzyme was desorbed on changing the pH to 6.5. On the other hand, the fixation of the holoenzyme was much slower, since after I and 24 h of incubation at pH 4.5, raising the pH to 6.5 resulted in the desorption of 45 and 35% of initially adsorbed protein respectively. Flavine adenine dinucleotide was adsorbed to clay at pH 4.5, but only minimal adsorption occurred at pH 6.5.Experiments were performed to determine the release of flavenoid(s) into supernates when the holoenzyme was fixed to clay at pH 4.5. Determinations of the increases in fluorescence of supernates indicated that up to 80% of the original FAD in reacted enzyme was released into supernates. These supernates contained two compounds which possessed different Rf values than the reference compounds, flavine mononucleotide, riboflavine, lumichrome, and lumiflavine. The addition of apoenzyme to supernates obtained from holoenzyme–clay complex to reconstitute enzyme activity indicated that less than 3% of the original FAD could be detected in supernates.When the enzyme–clay complex structured at pH 4.5 was adjusted to pH 6.5, data on desorption of both protein and FAD allowed the conclusion that the coenzyme was desorbed as an integral part of the enzyme.
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