Identification of the binding sites of chromium in cytochrome c

Studies have shown that chromium (VI) is a potent carcinogen. Chromate, which is highly soluble at physiological pH, is easily transported into the cell where its reduction to Cr(III) allows the formation of DNA-Cr(III)-protein crosslinks. These crosslinks are believed to be involved in the initiation of carcinogenesis. The purpose of this project was to synthesize a Cr(III)-cytochrome c complex and identify the chromium-containing peptides. The complex was obtained by reduction of Cr(VI), at pH 7.4 and 25$\sp\circ$C, by the dropwise addition of L-cysteine ethyl ester in presence of the protein. The sample was then purified and the metal concentration determined, after oxidation using both a colorimetric and a spectrophotometric method; the complex was found to contain 1 to 6 chromiums per cytochrome c. Following protein digestion and HPLC separation of the digests, the chromium-containing peptide was isolated. Amino acid sequencing of this peptide indicated the presence of 1 aspartic acid, 1 glutamic acid, 2 glycine, and 5 lysine residues.