title: Chemical characterization of a protein-4-hydroxy-2-nonenal cross-link: immunochemical detection in mitochondria exposed to oxidative stress.
abstract: We have previously shown that incubation of the model protein glucose-6-phosphate dehydrogenase (Glu-6-PDH) from the bacterium Leuconostoc mesenteroides with 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 of N-acetyllysine with HNE has been investigated. The epsilon-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.
authors.affiliation: Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Cohn
authors.forename: L
authors.initials: L
authors.lastname: Tsai
authors.forename: B
authors.initials: B
authors.lastname: Friguet
authors.forename: L I
authors.initials: LI
authors.lastname: Szweda
mesh_terms: D000447:Aldehydes / Q000737:chemistry*; D000818:Animals; D003432:Cross-Linking Reagents / Q000737:chemistry*; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry; D006639:Histidine / Q000032:analysis; D007120:Immunochemistry / Q000379:methods; D015227:Lipid Peroxidation; D008239:Lysine / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry; D008297:Male; D008930:Mitochondria, Liver / Q000737:chemistry; D008956:Models, Chemical; D018384:Oxidative Stress; D011506:Proteins / Q000737:chemistry* / Q000276:immunology; D051381:Rats; D011916:Rats, Inbred F344; D012545:Schiff Bases / Q000737:chemistry
chemical_list: D000447:Aldehydes; D003432:Cross-Linking Reagents; D011506:Proteins; D012545:Schiff Bases; D006639:Histidine; C016949:N-epsilon-acetyllysine; D005954:Glucosephosphate Dehydrogenase; C027576:4-hydroxy-2-nonenal; D008239:Lysine