Chemical characterization of a protein-4-hydroxy-2-nonenal cross-link: Immunochemical detection in mitochondria exposed to oxidative stress

Jeffrey A. Cohn, Lin Tsai, Bertrand Friguet, Luke I. Szweda

Research output: Contribution to journalArticle

121 Scopus citations

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 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.

Original languageEnglish (US)
Pages (from-to)158-164
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume328
Issue number1
DOIs
StatePublished - Apr 1 1996

Keywords

  • 4-hydroxy-2-nonenal
  • Mitochondria
  • free radicals
  • lipid peroxidation
  • protein cross- linking

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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