Journal
CHEMICAL RESEARCH IN TOXICOLOGY
Volume 22, Issue 6, Pages 1050-1059Publisher
AMER CHEMICAL SOC
DOI: 10.1021/tx9000144
Keywords
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Funding
- NHLBI NIH HHS [R01 HL053315, R01 HL053315-04, HL 53315] Funding Source: Medline
- NIA NIH HHS [AG 15885, P01 AG015885] Funding Source: Medline
- NIGMS NIH HHS [T32 GM007250] Funding Source: Medline
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [R01HL053315] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [T32GM007250] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE ON AGING [P01AG015885] Funding Source: NIH RePORTER
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The lipid oxidation product 4-oxo-2-nonenal (ONE) derived from peroxidation of polyunsaturated fatty acids is a highly reactive protein cross-linking reagent. The major family of cross-links reflects conjugate addition of side chain nucleophiles such as sulfhydryl or imidazole groups to the C=C of ONE to give either a 2- or 3-substituted 4-ketoaldehyde, which then undergoes Paal-Knorr condensation with the primary amine of protein lysine side chains. If ONE is intercepted in biological fluids by antielectrophiles such as glutathione (GSH) or beta-alanylhistidine (carnosine), this would lead to circulating 4-ketoaldehydes that could then bind covalently to the protein Lys residues. This phenomenon was investigated by SDS-PAGE and mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight and LC-ESI-MS/MS with both tryptic and chymotryptic digestion). Under the reaction conditions of 0.25-2 mM ONE, I mM GSH or carnosine, 0.25 mM bovine beta-lactoglobulin (beta-LG), and 100 mM phosphate buffer (pH 7.4, 10% ethanol) for 24 h at 37 degrees C, virtually every Lys of beta-LG was found to be fractionally cross-linked to GSH. Cross-linking of Lys to carnosine was less efficient. Using cytochrome c and RNase A, we showed that ONE becomes more protein-reactive in the presence of GSH, whereas protein modification by 4-hydroxy-2-nonenal is inhibited by GSH. Stable antielectrophile-ONE-protein cross-links may serve as biomarkers of oxidative stress and may represent a novel mechanism of irreversible protein glutathionylation.
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