Protein Adducts Generated from Products of Lipid Oxidation: Focus on HNE and ONE

Abstract

Modification of proteins in conditions of oxidative stress can contribute to protein dysfunction or tissue damage and disease progression. Bifunctional, most often α,β-unsaturated carbonyl compounds such as 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal (ONE), and acrolein, generated from oxidation of polyunsaturated fatty acids (PUFAs), readily bind to protein nucleophiles. Modification by bifunctional aldehydes can also lead to intramolecular or intermolecular protein crosslinking. Model studies are revealing the structure of adducts that can then be more readily identified in mass spectrometric studies on proteins exposed to the various pure aldehydes or to peroxidized PUFAs. Although simple Michael and Schiff base adducts are often formed initially, only some of these adducts, such as the HNE- and ONE-derived Michael adducts on Cys and His residues, are found to survive the conditions of proteolysis and HPLC-MS analysis. Reversibly formed adducts, such as the HNE-Lys Michael adduct, can be found on proteolytic peptides only if a NaBH₄-reduction step is used prior to proteolysis. Initial adducts can evolve by tautomerization, oxidation, cyclization, dehydration, and sometimes condensation with a second aldehyde molecule (the same or different), to give stable advanced lipoxidation end products (ALEs) that can be found by mass spectrometry. These include the HNE-Lys-derived 2-pentylpyrrole, the ONE-Lys-derived 4-ketoamide, the ONE-derived His-Lys pyrrole crosslink, and a Lys-derived 3-formyl-4-pentylpyrrole that results from combined action of ONE and acrolein. Michael adducts of α,β-unsaturated aldehydes such as HNE and ONE can be derivatized by 2,4-dinitrophenylhydrazine (DNPH) and can thus constitute significant DNPH-detectable protein-bound carbonyl activity that serves as a key indicator of oxidative stress in tissues. It appears that lipid oxidation is a more important contributor to such activity than metal-catalyzed oxidation of protein side-chains.

Key Words :

• Lipid oxidation
• 4-Hydroxy-2-nonenal
• 4-Oxo-2-nonenal
• Acrolein
• Oxidative stress
• Mass spectrometry

Notes

*Presented at the Seventh International Symposium on Biological Reactive Intermediates, Tucson, Arizona, January 4–7, 2006.