cytochrome-c-t and 2-4-decadienal

2(E),4(E)-Decadienal (DDE), a lipid peroxidation product, was found to covalently modify Lys residues of different proteins by different reactions using mass spectrometry (MALDI-TOF-MS and LC-ESI-MS). DDE mainly formed Lys Schiff base adducts with cytochrome c and ribonuclease A at 10 min, but these reversibly formed adducts almost disappeared after 24 h. In contrast, beta-lactoglobulin (beta-LG) was highly modified by DDE after 24 h. In addition to the Lys Schiff base adducts, DDE formed novel Lys pyridinium adducts as well as Cys Michael adducts with beta-LG.

Topics: Aldehydes; Amino Acid Sequence; Cytochromes c; Lactoglobulins; Mass Spectrometry; Molecular Sequence Data; Proteins; Ribonuclease, Pancreatic; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Modification of biomolecules by reactive aldehydes is believed to play a role in biological processes, including aging, atherosclerosis, and Alzheimer's disease. Here, the modification of cytochrome c promoted by trans, trans-2,4-decadienal (DDE) was investigated. Matrix-assisted laser desorption/ionization time-of-flight experiments indicated increases in the molecular weight of cytochrome c, consistent with the formation of DDE adducts. Our data show that the protein modification was time-, pH-, and DDE concentration-dependent, leading to the formation of at least six adducts after 2 h of incubation at pH 7.4. Electrospray ionization quantitative TOF mass spectrometry analysis of tryptic digests indicated that His-33, Lys-39, Lys-72, and Lys-100 were modified by DDE. These adducts could have significant effects considering that His-33, Lys-72, and Lys-100 are present in clusters of basic amino acid residues, which are believed to participate in the interaction of cytochrome c with cardiolipin in the inner mitochondrial membrane and cytochrome c oxidase. A blue shift in the cytochrome c Soret band from 409 to 406 nm was also observed after DDE reaction, indicating heme crevice opening and displacement of heme sixth ligand (Met-80) coordination in modified protein. The covalent modifications in cytochrome c could play a role in mitochondrial dysfunction associated with oxidative stress.

Topics: Aldehydes; Amino Acid Sequence; Cardiolipins; Cytochromes c; Electron Transport Complex IV; Histidine; Hydrogen-Ion Concentration; Lysine; Mitochondrial Membranes; Molecular Sequence Data; Molecular Weight; Oxidative Stress; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Time Factors