Mitochondrial Respiratory Chain Coupling and Electron Flux

Mitochondria were isolated using a standard differential centrifugation method. ⁵ Briefly, minced hearts were placed in mitochondrial isolation buffer 1 in 67 mM sucrose, 50 mM Tris/HCl, 50 mM KCl, 10 mM EDTA, and 0.2% BSA with a pH=7.2, homogenized with a glass Teflon pestle, and subsequently centrifuged at 700g for 10 minutes at 4 °C. The supernatant was collected and centrifuged at 8000g for 10 minutes at 4 °C. The pellet was then resuspended in ice‐cold buffer (isolation buffer 2) containing 250 mM sucrose, 3 mM EGTA/Tris, and 10 mM Tris/HCl with a pH=7.2, followed by centrifugation at 8000g for 10 minutes at 4 °C. Mitochondria were then resuspended and stored in isolation buffer 2. Protein concentration was determined using the Bradford Assay. Coupling and electron flux assays were performed as described. ³⁸