INHIBITION OF 2,5-HEXANEDIONE-INDUCED PROTEIN CROSS-LINKING BY BIOLOGICAL THIOLS - CHEMICAL MECHANISMS AND TOXICOLOGICAL IMPLICATIONS

n-Hexane is metabolized to the gamma-diketone 2,5-hexanedione (2,5-HD) , a derivative that covalently binds to lysine residues in neurofilame nt (NF) protein to yield 2,5-dimethylpyrrole adducts. Studies comparin g the pyrrole-forming potential and neurotoxic potency of gamma-diketo nes 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 pre sent study, the role of nucleophilic thiols and amines in the pyrrole- mediated cross-linking reaction was investigated. When pyrrolylated ri bonuclease was incubated with N-acetyllysine, N-acetylcysteine, or glu tathione in physiologic buffer (pH 7.4) under air, pyrrole-to-pyrrole cross-linking was inhibited only by the thiol-containing compounds. St able thiol-pyrrole conjugates containing a bridge from the pyrrole rin g at C-3 to the sulfur atom of the thiol were characterized by thermos pray LC/MS and H-1-NMR spectroscopy. In contrast to low-molecular-mass thiols, SDS-PAGE studies indicated that, under the same incubation co nditions, free thiols present in proteins did not undergo reaction wit h 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, po ssibly via inhibition of a free radical-dependent mechanism. The resul ts suggest the following: (1) 2,5-HD-induced protein cross-linking is mediated primarily by pyrrole-to-pyrrole bridging under physiologic co nditions, and (2) glutathione and other low-molecular-mass thiols may inhibit the pyrrole dimerization reaction by two distinct pathways. Th ese findings have significant implications for the mechanism of gamma- diketone neuropathy.