4-Hydroxy-2-nonenal (HNE), a cytotoxic aldehyde produced from lipid peroxidation, is known for its ability to modify proteins through its reactions with nucleophilic functions on the side-chain of some amino acid residues, e.g. cysteine, histidine, and lysine. We have observed that upon prolonged reaction of HNE with N-acetyllysine, a fluorescent compound could be isolated in very low yield from the mixture of reaction products. We have now characterized this fluorophore as 1-alkyl-2-hydroxy-3- alkylimino-1,2-dihydropyrrol: the two alkyl groups are derived from two molecules of the amino compounds which react with one molecule of HNE. Thus, the lysine-HNE fluorophore was most likely formed by an oxidative cyclization process on the non-fluorescent 2:1 lysine-HNE Michael adduct-Schiff base which we had identified previously as being responsible for the cross-linking in the HNE-modified proteins. The structural assignment of this fluorophore is consistent with extensive mass spectral data as well as proton and carbon nuclear magnetic resonance spectra of a purified sample of the fluorophore. Furthermore, analogous fluorophores were obtained from the reaction of a number of primary amines with HNE. These results firmly established the generality of this mode of action of HNE. A polyclonal antibody was raised to the N-acetyllysine fluorophore. This antibody was shown to be highly specific to the chromophoric structure of the compound. It was used to demonstrate that the lysine-HNE fluorophore was indeed formed on the enzyme, glucose-6-phosphate dehydrogenase, after it was treated with HNE. Work is in progress to delineate the chromophoric structure on the protein.
