LOSS OF THE METAL-BINDING PROPERTIES OF METALLOTHIONEIN INDUCED BY HYDROGEN-PEROXIDE AND FREE-RADICALS

The relationship between the metal-binding properties of metallothione in (MT) and its ability to interact with peroxides and free radicals w as explored in vitro. The binding of Cd-109 to MT and the thiol densit y of the protein were determined after incubation of a purified Zn/Cd- metallothionein preparation with either hydrogen peroxide alone, or wi th a number of free radical generating systems. Exposure of MT to H2O2 , whether in the presence or absence of Fe2+, resulted in the progress ive loss of the thiol residues of the protein and led to a parallel de crease of its Cd-109-binding capacity. These changes correlated with r values of 0.999 (P = 0.001) and 0.998 (P = 0.001), in the absence and presence of iron, respectively. The effects of H2O2, alone or plus Fe 2+, on MT were completely prevented by catalase, but totally unaffecte d by superoxide dismutase or desferrioxamine. Exposure of MT to xanthi ne/xanthine oxidase also led to thiol oxidation and to a concomitant l oss of the Cd-binding properties. In this system, both changes correla ted with an r of 0.993 (P = 0.001) and were completely inhibited by su peroxide dismutase. Exposure of MT to the peroxyl radical generator, 2 ,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), resulted in the p rogressive loss of its the metal-binding properties and its thiol resi dues, both changes correlating with an r of 0.986 (P = 0.002). The abi lity of MT to bind Cd-109, lost as a result of its prior exposure to e ither H2O2 alone; H2O2 plus Fe2+, xanthine/xanthine oxidase, or to AAP H was, in all cases, completely recovered after incubation of the modi fied protein with dithiothreitol. These results indicate that H2O2 alo ne, and/or the oxygen-derived species, superoxide anion and peroxyl ra dicals, can all directly interact in vitro with MT to modify the prote in oxidatively, and suggest that, under in vivo conditions, these spec ies may be implicated as modifying factors of the metal-binding capaci ty of metallothionein. (C) 1997 Elsevier Science Ireland Ltd.