title: Inhibition of 2,5-hexanedione-induced protein cross-linking by biological thiols: chemical mechanisms and toxicological implications.
abstract: n-Hexane is metabolized to the gamma-diketone 2,5-hexanedione (2,5-HD), a derivative that covalently binds to lysine residues in neurofilament (NF) protein to yield 2,5-dimethylpyrrole adducts. Studies comparing the pyrrole-forming potential and neurotoxic potency of gamma-diketones have demonstrated that pyrrolylation is an absolute requirement in the neuropathogenesis. Autoxidative cross-linking of pyrrolylated NF proteins occurs and is proposed as a second required event. In the present study, the role of nucleophilic thiols and amines in the pyrrole-mediated cross-linking reaction was investigated. When pyrrolylated ribonuclease was incubated with N-acetyllysine, N-acetylcysteine, or glutathione in physiologic buffer (pH 7.4) under air, pyrrole-to-pyrrole cross-linking was inhibited only by the thiol-containing compounds. Stable thiol--pyrrole conjugates containing a bridge from the pyrrole ring at C-3 to the sulfur atom of the thiol were characterized by thermospray LC/MS and 1H-NMR spectroscopy. In contrast to low-molecular-mass thiols, SDS--PAGE studies indicated that, under the same incubation conditions, free thiols present in proteins did not undergo reaction with pyrrole adducts to form cross-links. Further experiments using a low-molecular-mass pyrrole derivative indicated that glutathione may also able to suppress pyrrole dimerization without conjugate formation, possibly via inhibition of a free radical-dependent mechanism. The results suggest the following: (1) 2,5-HD-induced protein cross-linking is mediated primarily by pyrrole-to-pyrrole bridging under physiologic conditions, and (2) glutathione and other low-molecular-mass thiols may inhibit the pyrrole dimerization reaction by two distinct pathways. These findings have significant implications for the mechanism of gamma-diketone neuropathy.
authors.affiliation: Department of Environmental Health and Toxicology, University at Albany, State University of New York, USA.
authors.forename: M
authors.initials: M
authors.lastname: Zhu
authors.forename: D C
authors.initials: DC
authors.lastname: Spink
authors.forename: B
authors.initials: B
authors.lastname: Yan
authors.forename: S
authors.initials: S
authors.lastname: Bank
authors.forename: A P
authors.initials: AP
authors.lastname: DeCaprio
mesh_terms: D000588:Amines / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000378:metabolism; D006588:Hexanones / Q000737:chemistry* / Q000633:toxicity*; D011506:Proteins / Q000378:metabolism*; D011758:Pyrroles / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D000588:Amines; D003432:Cross-Linking Reagents; D006588:Hexanones; D011506:Proteins; D011758:Pyrroles; D013438:Sulfhydryl Compounds; C011269:2,5-hexanedione; D005978:Glutathione