CHEMICAL CHARACTERIZATION OF A PROTEIN-4-HYDROXY-2-NONENAL CROSS-LINK- IMMUNOCHEMICAL DETECTION IN MITOCHONDRIA EXPOSED TO OXIDATIVE STRESS

We have previously shown that incubation of the model protein glucose- 6-phosphate dehydrogenase (Glu-6-PDH) from the bacterium Leuconostoc m esenteroides with 4-hydroxy-2-nonenal (HNE), a major product of lipid peroxidation, results in the formation of cross-linked protein, HNE-mo dified protein is resistant to proteolytic degradation and acts as an inhibitor of the multicatalytic proteinase, It was therefore important to establish the chemistry of the cross-linking reaction. The formati on of cross-linked Glu-6-PDH is associated with the nearly exclusive l oss of lysine residues, For this reason the reaction of N-acetyllysine with HNE has been investigated. The E-amino group of lysine reacts wi th the double bond (C3) and the carbonyl (C1) functions of HNE via Mic hael addition and Schiff base formation resulting in the production of a 2:1 amino acid-HNE cross-link. Chromatographic detection of this ad duct in the acid hydrolysate of HNE-treated Glu-6-PDH reveals that thi s chemistry is responsible for the formation of cross-linked protein. Antibody to the reduced form of the 2:1 lysine-HNE adduct was prepared , The antibody was used to demonstrate that exposure of isolated liver mitochondria to oxidative stress led to the formation of intra- and i ntermolecular protein-HNE cross-links. The results of the present stud y indicate that modifications to protein by lipid peroxidation product s may be physiologically relevant and could contribute to the disease- and age-related buildup of damaged protein. (C) 1996 Academic Press, Inc.