FORMATION AND STRUCTURE OF CROSS-LINKING AND MONOMERIC PYRROLE AUTOXIDATION PRODUCTS IN 2,5-HEXANEDIONE-TREATED AMINO-ACIDS, PEPTIDES, AND PROTEIN

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 E-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 crosslinking. 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 H-1-NMR spectroscopy. C-13-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) = 335 nm) consistent with extended conjugation. Polymerization of pyrrolylated acetyllysine was accelerated by persulfate, a free-radical initiator, and inhibited by ascorbate, an antioxidant. Oxidative cross-linking of pyrrolylated RNase, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was competitively inhibited in the presence of pyrrolylated acetyllysine, These results provide structural identification of the protein crosslink in gamma-diketone neuropathy and demonstrate the free-radical dependence of autoxidative dimerization. In addition, a potentially important alternative pathway of pyrrole autoxidation, leading to stable, non-cross-linking products, is revealed.