Studies on the dissociation of flavin adenine dinucleotide from metalloflavoproteins.

Treatment of milk xanthine oxidase and chicken liver xanthine dehydrogenase with high concentrations of salts for several hours resulted in the loss of reactivity toward O2 and NAD, respectively. Oxidation of xanthine by either enzyme in the presence of electron acceptors such as dichlorophenol indophenol was unimpaired under these conditions, in the presence of 3 M KI. Prolonged treatment of xanthine oxidase and xanthine dehydrogenase with 3 M KI followed by extensive dialysis in 0.5 M KC1 yielded flavin-free metalloproteins which had the absorption characteristics of their iron-sulfur chromophore. Both flavin-free enzymes showed markedly enhanced rates of electron transfer to cytochrome c and nitroblue tetrazolium compared to the native enzymes. The cytochrome c activity of flavin-free xanthine oxidase, in contrast to the same activity of native xanthine oxidase, was completely insensitive to superoxide dismutase. Prior reduction of the enzymes with their electron donor substrates resulted in a dramatic increase in the lability of FAD in the presence of 3 M KI. Thus, in the case of xanthine dehydrogenase reduced with NADH, the FAD was nearly completely dissociated in less than 5 min compared to the several hours required with the native enzyme. The flavin-free enzymes prepared by the modified procedure were much more stable, and showed superior ability to be reconstituted with FAD.