Kinetics and thermodynamics of free flavins and the flavin-based redox active site within glucose oxidase dissolved in solution or sequestered withina sol-gel-derived glass

We report on the steady-state and time-resolved fluorescence from the redox active site flavine adenine dinucleotides (FADs) that are bound to glucose oxidase (GOx) when this enzyme is dissolved in aqueous solution or sequest ered within a sol-gel-derived glass. To the best of our knowledge, this rep resents the first report on the actual dynamics of an enzyme active site wh en the enzyme is part of a sol-gel-derived glass. The results from these ex periments show that the "free" FAD intramolecular folding/unfolding kinetic s are slowed 3-10-fold within the glass vs solution. The intramolecular exc iplex formation event (i.e., excited-state FAD residue folding/unfolding) i s completely arrested for the GOx-bound FAD if the enzyme is sequestered wi thin a glass in the absence of glucose. This is significantly different fro m the behavior of GOx dissolved in solution. However, despite this differen ce in behavior, the GOx molecules that are sequestered within the glasses c ontinue to function somewhat like GOx dissolved in aqueous solution if they are challenged with glucose. We also found that the GOx molecules do not l each from the glass and they exhibit rotational mobility that is only 2-fol d less than GOx dissolved in aqueous solution at 20 degrees C. In aqueous s olution or within these glasses, the enzyme pocket that hosts the FAD redox sites opens up by 25-30% when GOx is challenged with glucose. Finally, we present preliminary analytical results for film-based sol-gel-derived biose nsors that contain GOx, L-amino acid oxidase or cholesterol oxidase wherein the intrinsic FAD fluorescence produces the analytical signal.