Inactivation of glucose oxidase by diperoxovanadate-derived oxidants

Inactivation of glucose oxidase occurred in the presence of bromide, vanada te, H2O2, and phosphate (the bromide system), and this was prevented by NAD H or phenol red, a bromine acceptor. Glucose oxidase present during the rea ction between diperoxovanadate and a reduced form of vanadate, vanadyl (the vanadyl system), but not added after mixing the reactants, was inactivated , and this was accompanied by a loss of binding of the dye, Coomassie blue, to the protein. The transient intermediate of the type OVOOV(O-2), known t o form in these reactions and used in the oxidation of bromide ion and NADH , appears to be responsible for inactivating glucose oxidase. In both syste ms, the inactivation of the enzyme was prevented by histidine and DTT, know n to quench singlet-oxygen. By direct measurement of 1270-nm emission of si nglet-oxygen, its generation was demonstrated in the bromide system, and in the reaction of hypohalous acids with diperoxovanadate, but not in the van adyl system. By themselves both hypohalous acids, HOCl and HOBr inactivated glucose oxidase, and their prior reaction with H2O2 during which singlet-o xygen was released, protected the enzyme. The results provide support for p ossible oxidative inactivation of glucose oxidase by diperoxovanadate-deriv ed oxidants. (C) 1999 Academic Press.