TOXI 89 |
| Cytochrome c is a key respiratory protein that may be modified in oxidative stress. In particular, as the mitochondrial membrane is rich in esterified linoleic acid, cyt c may become adducted by its oxidation products, including 4-hydroxy-2-nonenal (HNE). Modification of cytochrome c by HNE was studied by both MALDI mass spectrometry and HPLC-ESI-MS/MS under different incubation and workup conditions (e.g., at different temperatures, with and without gel filtration, dialysis, and NaBH4 reduction). Stable HNE Michael adducts were found on His33 even without NaBH4 reduction, whereas HNE Michael adducts on Lys residues, formed reversibly, were found by ESI-MS only when the adducts were trapped by NaBH4 reduction prior to denaturation. Through a study involving the mixing of aliquots of HNE-d0-modified cyt c as a function of time (10 min to 12 h) with aliquots of 12 h HNE-d11-modified cyt c, we could obtain information on the relative rates of side-chain modification as a function of residue position. Rates varied by a factor of four and reflected a complex combination of factors including microenvironmental pKa and dielectric constant, and solvent accessibility. |
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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 |