To deal with this problem we evaluated calcium contamination in our BAXoligo preparation using the Ca2 selective electrode. These experiments unveiled that BAXoligo preparations used in our experiments did not contain appreciable amounts of Ca2. Nevertheless, we analyzed the cytochrome c release induced by BAXoligo in HSP90 inhibition the clear presence of 1 mM EGTA and didn’t find any difference with studies where we used 10 uM EGTA. Hence, all data obtained with recombinant BAXoligo might be caused by the action of this protein and to Ca2 contamination. Earlier in the day, it had been suggested that oxidative stress and lipid peroxidation could subscribe to BAXoligo induced cytochrome c release from isolated liver mitochondria. In the following experiments, natural compound library we addressed the question of whether the intensity of oxidative stress, evaluated because the price of ROS generation by mitochondria, correlated with the release of cytochrome c caused by BAXoligo or alamethicin. In mitochondria, superoxide radical O2?U, a main reactive oxygen species, is converted by Mn superoxide dismutase into H2O2 which is often easily followed with Amplex Red analysis. With succinate as a, mitochondrial generation of ROS is linked to the reverse electron flow from Complex II to Complex I of the respiratory cycle and may be efficiently inhibited by mild mitochondrial depolarization. Within our experiments, BAXoligo lowered the rate of ROS generation in a dependent manner, according to its ability to depolarize mitochondria. FCCP and alamethicin created even stronger reduction of ROS generation. CsA and ADP attenuated inhibition of ROS generation by BAXoligo, but not by FCCP or alamethicin. A mix of CsA and ADP attenuated the inhibition of ROS generation by BAXoligo possibly due to security of? and, for that reason, maintenance of the reverse electron Mitochondrion move in the respiratory cycle. In the clear presence of mPT inhibitors, ROS generation was large, but the release of cytochrome c was somewhat diminished. On another hand, mPT CDK2 inhibitor inhibitors did not influence the inhibition of ROS generation caused by alamethicin. Thus, within our experiments with isolated mind mitochondria the intensity of oxidative stress and the release of cytochrome c caused by BAXoligo or alamethicin had an inverse relationship. Therefore, it appears unlikely that lipid peroxidation linked to the oxidative stress brought to the release of cytochrome c from isolated brain mitochondria. 3. Discussion The release of mitochondrial intermembrane proteins plays a vital role in performance of the apoptotic program. The cell free experimental design of isolated mitochondria in combination with the use of recombinant pro apoptotic proteins became a very helpful tool in the elucidation of those systems.