Abstract

The relationship between the metal-binding properties of metallothionein (MT) and its ability to interact with peroxides and free radicals was explored in vitro. The binding of 109Cd to MT and the thiol density of the protein were determined after incubation of a purified Zn/Cd-metallothionein preparation with either hydrogen peroxide alone, or with a number of free radical generating systems. Exposure of MT to H 2O 2, whether in the presence or absence of Fe 2+, resulted in the progressive loss of the thiol residues of the protein and led to a parallel decrease of its 109Cd-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 H 2O 2, alone or plus Fe 2+, on MT were completely prevented by catalase, but totally unaffected by superoxide dismutase or desferrioxamine. Exposure of MT to xanthine/xanthine oxidase also led to thiol oxidation and to a concomitant loss of the Cd-binding properties. In this system, both changes correlated with an r of 0.993 (P = 0.001) and were completely inhibited by superoxide dismutase. Exposure of MT to the peroxyl radical generator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), resulted in the progressive loss of its the metal-binding properties and its thiol residues, both changes correlating with an r of 0.986 (P = 0.002). The ability of MT to bind 109Cd, lost as a result of its prior exposure to either H 2O 2 alone, H 2O 2 plus Fe 2+, xanthine/xanthine oxidase, or to AAPH was, in all cases, completely recovered after incubation of the modified protein with dithiothreitol. These results indicate that H 2O 2 alone, and/or the oxygen-derived species, superoxide anion and peroxyl radicals, can all directly interact in vitro with MT to modify the protein oxidatively, and suggest that, under in vivo conditions, these species may be implicated as modifying factors of the metal-binding capacity of metallothionein.