Protocols for high-resolution respirometry experiments to test the activity of electron transfer system of pig brain mitochondria

258-272Isolated mitochondria serve as a biological model for analysis of oxidative phosphorylation and understanding of mitochondrial respiration control, effects of various biologically active substances, and pathophysiology of mitochondrial diseases. The aim of our study was to develop protocols of high-resolution respirometry optimized for pig brain mitochondria. Optimal and/or minimal concentrations of components controlling oxidative phosphorylation were determined by titration of isolated mitochondria with digitonin, adenosine diphosphate, respiratory substrates (malate, pyruvate, succinate, TMPD), inhibitors (antimycin, azide, cyanide, malonate, oligomycin, rotenone), and uncoupler. Multiple substrate-uncoupler-inhibitor titration (SUIT) protocols were proposed for Complex I-, Complex II-, Complex I AND II-, and Complex IV linked respiration. Crude mitochondrial fraction and mitochondria purified on sucrose gradient were compared. We confirmed that pig brain mitochondria can serve as a biological model for research of mitochondrial respiration using SUIT protocols analogous to those, which were previously published for rodent mitochondria and permeabilized cells. We suppose that difference between a substance effect on respiration of crude and purified mitochondria is due to higher content of mitochondrial enzymes and lower content of the non-mitochondrial contaminants in purified mitochondria. High-resolution respirometry of pig brain mitochondria can be recommended as a suitable assay in research of neuroprotective effects and/or mitochondrial toxicity of novel medical drugs