TOXI 59 |
| The lipid oxidation product 4-oxo-2-nonenal (ONE) has been shown to be a highly reactive protein cross-linking agent. The major family of cross-links reflects conjugate addition of side-chain nucleophiles to the C=C to give a 2- or 3-substituted 4-ketoaldehyde, which then can undergo Paal-Knorr condensation with the primary amino group of lysine side-chains. If ONE is intercepted in biological fluids by antielectrophiles such as glutathione (GSH) or ß-alanylhistidine (carnosine), this will lead to circulating 4-ketoaldehydes that could then bind covalently to the lysine side-chains of proteins. We have used mass spectrometry (MALDI and ESI-MS/MS) with both tryptic and chymotryptic digestion to investigate this type of modification using model lysine-rich proteins. Under the reaction conditions of 2 mM ONE, 1 mM GSH or carnosine, 0.25 mM ß-lactoglobulin, pH 7.4 phosphate buffer (10% ethanol), 24 h, 37oC, virtually every lysine was found to be cross-linked to GSH. A somewhat lower level of modification was seen for carnosine. These stable antielectrophile-protein ONE cross-links may serve as biomarkers of oxidative stress. |
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Poster Session and Awards
6:00 PM-10:00 PM, Tuesday, August 21, 2007 BCEC -- 204 A/B, Poster
Division of Chemical Toxicology |