In vivo and in vitro binding of platinum to metallothionein

Abstract

The in vivo binding of platinum to metallothionein (MT) has been observed in rat tissues following injections of the cis and trans isomers of DDP (dichlorodiammineplatinum(II]. Platinum in either cis-DDP or trans-DDP does not directly induce MT; platinum-MT is produced by the replacement of previously bound zinc in the protein. The binding of Pt(II) to MT depends on the availability of SH groups in MT. Preinjection with CdCl2 significantly enhances the association of Pt(II) with MT fractions compared to the degree of association resulting from injections with either cis-DDP or trans-DDP without CdCl2 pretreatment. In vitro experiments in which tissue extracts including a known (Cd,Zn)-MT were incubated with either cis-DDP or trans-DDP show that these isomers differ with respect to the transfer of Pt to MT; the equilibrium in both cases was reached when approximately 40% of the available Pt is bound to MT but with this equilibrium value attained in 2 h in the case of trans-DDP and only after 72 h in the case of cis-DDP. Pt-MTs were also formed by a series of incubation steps in which a native MT was used to prepare the apoprotein which was subsequently incubated with either cis-DDP or trans-DDP. Spectrophotometry established that a shoulder occurs at 285 nm for the Pt-MTs resulting from the incubation with either isomer. A competitive double-antibody radioimmunoassay for MT demonstrated that these Pt-MTs had complete cross-reactivity with a native (Cd,Zn)-MT. Gel filtration of tissue extracts after either in vivo or in vitro treatment with DDP showed that Pt was bound to a molecular species with properties characteristic of MT. These results were verified by atomic absorption spectrophotometry and polyacrylamide gel electrophoresis assays.