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Abstract
Abstract 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 KCl 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.
Highlights
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
Summary
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. 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. Electron paramagnetic resonance studies on milk xanthine oxidase (4, 5) and hepatic aldehyde oxidasc (6, 7), both of which utilize molecular 02 as an electron acceptor, led to the tentative conclusion that an electron transfer sequence of molybdenum-FAD-iron was present in both enzymes, with t,he molybdenum component being closest to the substrate-oxidizing site and the iron complex closest to the 02 reducing site. Massey, and Palmer (8) have recently succeeded in preparing reconstitutable deflavoxanthine oxidase of milk through the use of CaCIZ
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