Formation and structure of cross-linking and monomeric pyrrole autoxidation products in 2,5-hexanedione-treated amino acids, peptides, and protein

• Published in: Chemical research in toxicology 1994-07, Vol.7 (4), p.551-558
• Main Authors: MINGSHE ZHU, SPINK, D. C, BIN YAN, BANK, S, DECAPRIO, A. P
• Format: Article
• Language: English
• Subjects: Amino Acids - chemistry; Biological and medical sciences; Chemical and industrial products toxicology. Toxic occupational diseases; Chromatography, High Pressure Liquid; Cross-Linking Reagents - chemistry; Hexanones - chemistry; Magnetic Resonance Spectroscopy; Medical sciences; Oxidation-Reduction; Peptides - chemistry; Proteins - chemistry; Pyrroles - chemistry; Solvents; Toxicology
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/tx00040a011

2,5-Hexanedione (2,5-HD) is the neurotoxic gamma-diketone metabolite of the industrial solvent n-hexane. Substantial evidence indicates that 2,5-HD reacts with neurofilament protein lysine epsilon-amines to yield 2,5-dimethylpyrrole adducts and that this reaction is critical to the mechanism of toxicity. Alkylpyrroles are susceptible to autoxidative dimerization, a process that has also been suggested as an obligatory step in 2,5-HD neuropathy. In the present study, we characterized pyrrole autoxidation products of a 2,5-HD-treated lysine analogue and of a model, lysine-containing dipeptide and examined mechanistic aspects of pyrrole-mediated protein cross-linking. Incubation of 2,5-HD with N alpha-acetyllysine or the dipeptide N alpha-acetylglycyllysine methyl ester in physiological buffer (pH 7.4) under oxidative conditions resulted in time-dependent formation of the N epsilon-pyrrole derivative and two major pyrrole autoxidation products, as demonstrated by HPLC, on-line thermospray MS, and UV photodiode array detection. An autoxidative pyrrole dimer containing a methylene bridge between C-2 of one pyrrole ring and C-3 of a second ring was characterized by thermospray MS and 1H-NMR spectroscopy. 13C-NMR spectroscopy provided evidence for an identical pyrrole-to-pyrrole bridge in autoxidized, pyrrolylated ribonuclease (RNase). MS analysis also revealed a second major product--a stable, oxygen-containing monomeric pyrrole derivative. This product exhibited a UV absorbance maximum (lambda max = 355 nm) consistent with extended conjugation. Polymerization of pyrrolylated acetyllysine was accelerated by persulfate, a free-radical initiator, and inhibited by ascorbate, an antioxidant.