Abstract

The interaction between glutathione (GSH) and copper ions was investigated in vitro to determine whether such interaction could affect the free-radical scavenging properties of the tripeptide. To this end, the bleaching (decrease in OD734 nm) of a coloured solution containing the stable free-radical cation ABTS(.+), (which results from the addition of thiols to such a solution) was employed as an in vitro indication of the ability of the tripeptide to scavenge free radicals. While GSH bleached concentration-dependently (1.0-7.5 muM) the ABTS(.+)-containing solution, its prior incubation (5 muM) in the presence of Cu+1 or Cu+2 ions (1-7.5 M) led to a metal concentration-dependent decrease of the bleaching capacity. At a ratio equal to one (5 muM each), the bleaching capacity of the copper plus GSH mixture was 50% of that seen for GSH alone. Further additions of copper (reaching ratios up to 2) did not result in greater decreases in the GSH-bleaching capacity. Noteworthy at the ratio of one was that the copper plus GSH solutions maintained their bleaching capacity despite the lack of any DTNB-reactivity, i.e., the complete absence of thiols in the mixture. Mixtures of increasing concentrations of a fixed ratio (equal to 2) of copper plus GSH, which were found not to exhibit any DTNB-reactivity, showed a linear and concentration-dependent increase in bleaching capacity. The bleaching capacity remained unaltered when TRIEN, EDTA or histidine were added to pre-incubated (1:1) mixtures of copper plus GSH. However, the incubation of copper with TRIEN or EDTA (but not histidine) prior to GSH addition, totally prevented the loss of the original GSH-bleaching capacity. The present data supports the formation of a copper-glutathione complex which is stable to the presence of some copper-chelators, lacks all thiol reactivity, but fully conserves the free-radical scavenging properties of GSH.