Turkey liver xanthine dehydrogenase: further observations on the reaction with arsenite.

The catalytically essential persulphide groups at the molybdenum centres of xanthine oxidase and xanthine dehydrogenase are essential to interaction with arsenite (Edmonson et al., 1972; Cleere et al., 1974) Inhibition of xanthine hydroxylation may result from arsenite forming a complex with the persulphides and vicinal thiol groups (Massey & Edmonson, 1970). The NADH-dichlorophenol-indophenol and NADH-trinitrobenzenesulphonate oxidoreductase activities of chicken liver xanthine are enhanced on incubation of the oxidized enzyme with arsenite (Rajagopalan & Handler, 1967). However, similar treatment of enzyme pre-reduced with NADH was found to inhibit the former activity. We wished to determine whether the effects of arsenite on NADH diphorase activity are restricted to the electron acceptors above and whether interaction with the inhibitor is confined to the persulphide groups. The experimental procedures used have been reported elsewhere (Cleere & Coughlan, 1975). Stopped-flow studies are described in the preceding paper (Ni Fhaolain et al., 1976). The inactivation of oxidized and of reduced enzyme by arsenite was carried out by the methods described by Rajagopalan & Handler (1967). Fig. l(a) shows the time-course of inhibition (pseudo-first-order with respect to enzyme) of xanthine-NAD+ oxidoreductase activity accompanying incubation of oxidized enzyme with arsenite. In agreement with the findings of Rajagopalan & Handler (1967), such treatment increased the oxidation of NADH by dichlorophenolindophenol. In complete contrast, however, with the report by these workers, we found that incubation of oxidized turkey enzyme with arsenite decreased its ability to catalyse the oxidation of NADH by trinitrobenzenesulphonate (Fig. la). The same trend was found in five separate experiments with different batches of the enzyme. These contrasting findings may reflect subtle differences in the environment of the persulphide groups in two otherwise-similar enzymes. The rate of increase in NADH-dichlorophenol-indophenol oxidoreductase activity on arsenite treatment paralleled that of the decrease in activity with trinitrobenzenesulphonate as acceptor when enzyme samples at the same concentration and content of functional active sites were used. However, unlike the time-course of inhibition of xanthineNAD+ oxidoreductase activity, neither followed first-order kinetics. This may reflect the fact that enzyme lacking completely the active-centre persulphide groups, and presumably incapable of reacting with arsenite, retains about 10% of the NADHdichlorophenol-indophenol and NADH-trinitrobenzenesulphonate oxidoreductase activities of the fully functional enzyme (Ni Fhaolain & Coughlan, 1976). Fig. l(b) confirms the inability of arsenite (incubated with oxidized enzyme) to alter either activity of the non-functional enzyme and shows that the extent of the increase in activity with dichlorophenol-indophenol, or of the decrease in activity with trinitrobenzenesulphonate as acceptor, is dependent on the content of functional active sites in the enzyme samples used. In contrast with the above findings, incubation of the oxidized enzyme with arsenite had no effect whatsoever on the oxidation of NADH by Methylene Blue, by ferricyanide or by 02. Moreover, stopped-flow studies at 450nm showed that the rate constants for the initial fast phase of reduction by NADH of native and arsenite-treated enzyme were not significantly different. These were 40.0s-' and 41 .Os-' respectively. Preliminary treatment of enzyme with xanthine or with NADH followed by exposure to arsenite did not affect the oxidation of NADH by Methylene Blue, ferricyanide or 02, nor did it affect subsequent reduction of the enzyme by NADH. The rate constant in