Comparison of protein oxidation and aldehyde formation during oxidative stress in isolated mitochondria

Oxidative stress is known to cause oxidative protein modification and the g eneration of reactive aldehydes derived from lipid peroxidation. Extent and kinetics of both processes were investigated during oxidative damage of is olated rat liver mitochondria treated with iron/ascorbate. The monofunction al aldehydes 4-hydroxynonenal (4-HNE), n-hexanal, n-pentanal, n-nonanal, n- heptanal, 2-octenal, 4-hydroxydecenal as well as thiobarbituric acid reacti ve substances (TBARS) were detected. The kinetics of aldehyde generation sh owed a lag-phase preceding an exponential increase. In contrast, oxidative protein modification, assessed as 54-dinitrophenylhydrazine (DNPH) reactive protein-bound carbonyls, continuously increased without detectable lag-pha se. Western blot analysis confirmed these findings but did not allow the id entification of individual proteins preferentially oxidized. Protein modifi cation by 4-HNE, determined by immunoblotting, was in parallel to the forma tion of this aldehyde determined by HPLC. These results suggest that protei n oxidation occurs during the time of functional decline of mitochondria, i .e. in the lag-phase of Lipid peroxidation. This protein modification seems not to be caused by 4-HNE.