CHARACTERIZATION OF ISOTHIOCYANATES, THIOUREAS, AND OTHER LYSINE ADDUCTION PRODUCTS IN CARBON DISULFIDE-TREATED PEPTIDES AND PROTEIN

Carbon disulfide (CS2) is an industrial solvent used in rayon production and as an organic synthetic precursor. It is also a member of the class of neuropathy-inducing xenobiotics known as the "neurofilament (NF) neurotoxicants". Current hypotheses propose direct reaction of CS2 with NF lysine epsilon-amine moieties as a step in the mechanism of this neuropathy. In this study, covalent CS2 binding in a lysine-containing dipeptide and in bovine serum albumin (BSA) in vitro was characterized. Dipeptide and BSA, incubated with (CS2)-C-14, exhibited stable incorporation of radioactivity after removal of unbound CS2 and reincubation in physiological buffer for up to 10 days. In contrast, free thiol levels decreased from a maximum immediately following CS2 exposure to near-base-line levels after 10 days, consistent with time-dependent conversion of initially formed N-substituted dithiocarbamate adducts into secondary products. HPLC/thermospray-MS and HPLC/UV photodiode-array analysis of CS2-dipeptide adducts confirmed dithiocarbamate formation and demonstrated their conversion into N-alkylisothiocyanates and, ultimately, N,N'-disubstituted thioureas and ureas. The results of UV spectrophotometry of CS2-treated BSA were also consistent with loss of dithiocarbamate and appearance of thioureas. Similar time-dependent formation of these products, in addition to N,N-disubstituted thiuram disulfides, was demonstrated in CS2-treated BSA by means of C-13-NMR spectroscopy. SDS-PAGE analysis of adducted protein revealed a discrete, higher mobility band, likely representing a specific intramolecular cross-link. In contrast, no evidence for intermolecular protein cross-linking was obtained. Identical results were obtained with cysteinyl-blocked BSA, indicating the lack of formation of NS-dialkyldithiocarbamate (dithiourethane) cross-links in these preparations. These findings clarify many chemical aspects of covalent CS2/polypeptide interaction and provide unequivocal evidence for the formation of protein-bound isothiocyanate adducts. A comprehensive direct reaction scheme for this neurotoxicant under physiological conditions is proposed.