FIGURE 5.

Quantification of the extent of oxidation of complex I 75-kDa subunit Cys residues during diamide treatment of mitochondria. Heart mitochondria were incubated ± diamide; some diamide-treated samples were further incubated with DTT, and then all samples were treated with NEM. The peptides containing NEM-alkylated Cys residues were then analyzed by MALDI-TOF, and their peak intensities are expressed as a percentage of an appropriate non-Cys containing reference peptide peak from the 75-kDa subunit digest. A, representative MALDI-TOF spectra of the NEM-alkylated peptides containing Cys-531 (1591.8 Da) or Cys-704 (1637.7 Da) from control, diamide-treated, and diamide- and DTT-treated bovine heart mitochondria. B, effect of treatment of bovine heart mitochondria with diamide or with diamide and DTT on peak intensities of NEM-alkylated peptides. Peak intensities are plotted as a percentage of the control incubation (-diamide) for that experiment. Data are the means ± S.E. of three to four independent experiments, except for the +diamide/DTT experiments, which were done once. ^*, p ≤ 0.05 relative to control samples by a paired Student's t test.