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 o f 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 mec hanism of toxicity. Alkylpyrroles are susceptible to autoxidative dime rization, 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 mod el, lysine-containing dipeptide and examined mechanistic aspects of py rrole-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 tw o major pyrrole autoxidation products, as demonstrated by HPLC, on-lin e thermospray MS, and UV photodiode array detection. An autoxidative p yrrole 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 i dentical pyrrole-to-pyrrole bridge in autoxidized, pyrrolylated ribonu clease (RNase). MS analysis also revealed a second major product-a sta ble, oxygen-containing monomeric pyrrole derivative. This product exhi bited a UV absorbance maximum (lambda(max) = 335 nm) consistent with e xtended conjugation. Polymerization of pyrrolylated acetyllysine was a ccelerated by persulfate, a free-radical initiator, and inhibited by a scorbate, an antioxidant. Oxidative cross-linking of pyrrolylated RNas e, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophore sis, was competitively inhibited in the presence of pyrrolylated acety llysine, These results provide structural identification of the protei n crosslink in gamma-diketone neuropathy and demonstrate the free-radi cal dependence of autoxidative dimerization. In addition, a potentiall y important alternative pathway of pyrrole autoxidation, leading to st able, non-cross-linking products, is revealed.