4.6. Mitochondrial Respiration Measurements

Oxygen consumption of mitochondria was registered polarographically with a Clark oxygen electrode connected to a suitable recorder in a thermostated water-jacketed closed chamber with magnetic stirring [93]. The reactions were carried out at 30 °C in 1 mL of the standard medium (100 mM sucrose, 100 mM KCl, 2 mM KH₂PO₄, 5 mM Hepes and 10 µM EGTA, pH 7.4) with 0.8 mg of protein. States 4 and 3 respiration were initiated with 5 mM succinate in the presence of 2 μM rotenone (mitochondrial complex II energization). Exogenous ADP (155 nmol/mg protein) was added to initiate state 3. In some experiments, oligomycin (2 μg/mL) and FCCP (1 μM) were also added to inhibit passive flux through the ATP synthase and to uncouple respiration, respectively.