Characterization of Isothiocyanates, Thioureas, and Other Lysine Adduction Products in Carbon Disulfide-Treated Peptides and Protein

Anthony P. DeCaprio, David C. Spink, Chen Xi, Jay H. Fowke, Mingshe Zhu, Shelton Bank

Research output: Contribution to journalArticle

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Abstract

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 ε-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 14CS2, 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 13C-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 crosslinking was obtained. Identical results were obtained with cysteinyl-blocked BSA, indicating the lack of formation of N,S-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 proteinbound isothiocyanate adducts. A comprehensive direct reaction scheme for this neurotoxicant under physiological conditions is proposed.

Original languageEnglish (US)
Pages (from-to)496-504
Number of pages9
JournalChemical Research in Toxicology
Volume5
Issue number4
DOIs
StatePublished - Jul 1 1992
Externally publishedYes

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Isothiocyanates
Carbon Disulfide
Thiourea
Bovine Serum Albumin
Lysine
Dipeptides
Peptides
Intermediate Filaments
Proteins
Thiram
High Pressure Liquid Chromatography
Spectrophotometry
Radioactivity
Xenobiotics
Photodiodes
Sulfhydryl Compounds
Disulfides
Crosslinking
Nuclear magnetic resonance spectroscopy
Amines

All Science Journal Classification (ASJC) codes

  • Toxicology

Cite this

Characterization of Isothiocyanates, Thioureas, and Other Lysine Adduction Products in Carbon Disulfide-Treated Peptides and Protein. / DeCaprio, Anthony P.; Spink, David C.; Xi, Chen; Fowke, Jay H.; Zhu, Mingshe; Bank, Shelton.

In: Chemical Research in Toxicology, Vol. 5, No. 4, 01.07.1992, p. 496-504.

Research output: Contribution to journalArticle

DeCaprio, Anthony P. ; Spink, David C. ; Xi, Chen ; Fowke, Jay H. ; Zhu, Mingshe ; Bank, Shelton. / Characterization of Isothiocyanates, Thioureas, and Other Lysine Adduction Products in Carbon Disulfide-Treated Peptides and Protein. In: Chemical Research in Toxicology. 1992 ; Vol. 5, No. 4. pp. 496-504.
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N2 - 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 ε-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 14CS2, 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 13C-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 crosslinking was obtained. Identical results were obtained with cysteinyl-blocked BSA, indicating the lack of formation of N,S-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 proteinbound isothiocyanate adducts. A comprehensive direct reaction scheme for this neurotoxicant under physiological conditions is proposed.

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