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 bacteriumLeuconostoc mesenteroideswith 4-hydroxy-2-nonenal (HNE), a major product of lipid peroxidation, results in the formation of cross-linked protein. HNE-modified 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 formation of cross-linked Glu-6-PDH is associated with the nearly exclusive loss of lysine residues. For this reason the reaction ofN-acetyllysine with HNE has been investigated. The ϵ-amino group of lysine reacts with the double bond (C3) and the carbonyl (C1) functions of HNE via Michael addition and Schiff base formation resulting in the production of a 2:1 amino acid-HNE cross-link. Chromatographic detection of this adduct in the acid hydrolysate of HNE-treated Glu-6-PDH reveals that this 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 intermolecular protein–HNE cross-links. The results of the present study indicate that modifications to protein by lipid peroxidation products may be physiologically relevant and could contribute to the disease- and age-related buildup of damaged protein.