The effect of bovine serum albumin on the membrane potential and reactive oxygen species generation in succinate-supported isolated brain mitochondria

Characteristics of the succinate-supported H2O2 formation were compared in mitochondria prepared from guinea-pig brain either by Percoll gradient centrifugation or using digitonin. The high rate of H2O2 generation measured in mitochondria prepared with digitonin (600.6 ± 26.8 pmol/min/mg protein) was inhibited by rotenone, consistently with a reverse flow of electrons via complex I. The rate of H2O2 formation was significantly smaller in Percoll-purified mitochondria (252.6 ± 17.3 pmol/min/mg protein) and this was stimulated by rotenone. Since bovine serum albumin (BSA) is usually present in the isolation medium used in the digitonin method, systematic study was performed addressing the effect of BSA on H2O2 formation. Mitochondria prepared by the digitonin method (BSA present in the isolation medium) were highly polarized (185 ± 3.2 mV) and addition of BSA (0.025%) to the assay medium increased H2O2 generation by only 50%. In Percoll-purified mitochondria ΔΨm was more depolarized (171 ± 2 mV) and BSA caused hyperpolarization by 10.7 ± 1.9 mV. H2O2 formation, which was largely independent of ΔΨm, was stimulated by 400%, became highly dependent on ΔΨm and could be inhibited by rotenone in the presence of BSA. This shows that in Percoll-purified mitochondria ROS formation via reverse electron flow is preferred only when BSA is present in the assay medium. It is demonstrated that (i) the presence or absence of BSA could determine the mechanism by which ROS is generated in succinate-supported mitochondria and (ii) depolarization by about 10 mV eliminates reverse electron flow and the remaining ROS formation, which is smaller but still significant, is no longer dependent on ΔΨm.