A Binding Site for Chlorambucil on Metallothionein

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Abstract
It is of interest to test the hypothesis that induced metallothionein (MT) acts in acquired drug resistance by covalent sequestration. In this study MT was incubated in vitro with chlorambucil (CHB) under conditions where only 1:1 covalent adducts were formed. The proteolytic products of these adducts were analyzed by HPLC and mass spectrometry to reveal two major sites of modification. These were the sulfur atoms of cysteines 33 and 48, which cochelate the same metal atom in native MT. The time course of the reaction was followed using on-line electrospray ionization with a double-focusing mass spectrometer. These experiments showed that drug-modified MT binds seven metal ions, as does the unmodified protein. Molecular docking experiments showed that the selectively of drug binding is influenced by the presence of the aziridinium ion in the drug structure and complementary charge densities in the protein structure.