Protocols for high-resolution respirometry experiments to test the activity of electron transfer system of pig brain mitochondria
Abstract
The aim of our study was to develop protocols of high-resolution respirometry optimized for pig brain mitochondria. Optimal concentrations of components controlling oxidative phosphorylation were determined by titration of isolated mitochondria with digitonin, adenosine diphosphate, respiratory substrates (malate, pyruvate, succinate, TMPD), inhibitors (rotenone, malonate, antimycin, cyanide, azide, oligomycin), and uncoupler.
Multiple substrate-uncoupler-inhibitor titration (SUIT) protocols were proposed for Complex I-, Complex II-, Complex I+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 biological model for research of mitochondrial respiration.