Direct evidence for co-binding of cisplatin and cadmium to a native zinc- and cadmium-containing metallothionein

Cisplatin is widely used to treat a number of cancers, and its covalent binding to DNA is believed to cause cell death; however, the roles of cisplatin-protein interactions in the mechanisms of action, toxicity, and resistance of the drug largely remain to be elucidated. Here, we investigate the interactions of cisplatin and a native rabbit metallothionein (MT), containing 1.4% zinc and 7.9% cadmium, using nanospray tandem quadrupole time-of-flight mass spectrometry (MS) and size-exclusion high-performance liquid chromatography with inductively coupled plasma MS. At near-neutral pH conditions, reactions between cisplatin and MT resulted in the formation of complexes that contained Cd-4-Pt-n-MT (n = 1-7). While zinc was displaced by cisplatin, both platinum and cadmium were bound to the same MT molecule. This is the first report to provide direct evidence for the co-binding of cadmium and platinum to MT, which suggests that the mechanism of the binding of cisplatin to the native MT may not be through the displacement of cadmium as previously proposed. A tandem MS investigation into the binding sites of the platinum and cadmium to MT showed platinum- and cadmium-related fragments, such as (PtS2C2H7N)(+) and (CdS3C5H17N2)(+), demonstrating the platinum- cysteine and cadmium-cysteine binding. In addition, detection of Cd-4-Pt-7-MT demonstrated more than ten metals bound to a single MT molecule. This finding was extended to the binding of MT with a five-fold excess of CdCl2. As many as 14 metal atoms (13 cadmium and one zinc) were detected bound to a single MT molecule, the complexes being Cd-x-Zn-MT (x = 5-13). The high binding capacity of MT for cadmium and platinum is consistent with the role of MT in reduction of metal toxicity and its involvement in drug resistance. Copyright (C) 2003 John Wiley Sons, Ltd.