The subfractionation of mitochondria into the four basic compartments involves a combination of osmotic shock and differential centrifugation. Isolated mitochondria that have not previously been frozen are suspended in a low osmotic strength solution (0-50 mM sucrose, 2 mM EDTA, 10 mM MOPS, pH 7.2). The solution is gently stirred on ice for 15 min to osmotically shock mitochondria. The molarity of the solution then needs to be increased to 0.3 M sucrose by the slow addition with stirring of 2 M sucrose, 2 mM EDTA, 10 mM, pH 7.2 stock solutions. The solution is then centrifuged at 15,000g for 15 min at 4°C to pellet the mitoplasts comprising the inner membrane and matrix (IM and MA). The mitoplast may be stored frozen for fraction-ation at a later stage. The supernatant containing the outer membrane (OM) and the intermembrane space (IMS) is transferred to clean tubes and centrifuged at 200,000g for 90 min. The resulting supernatant contains the IMS and is removed and stored frozen. The crude OM pellet requires further purification to remove contaminating IM. To do this, the pellet is resuspended in a small volume of 100 mM Tris-HCl pH 7.4 and layered over a 4 mL 0.6 M/0.9 M step sucrose gradient. The gradient is then centrifuged in a swing-out rotor at 50,000g for 60 min. The OM band is collected from the 0.6 M/0.9 M interface and is only visible when performing fractionations using more than 100 mg of mitochondrial protein. Other visible bands are likely to be contaminants that should be discarded.
To separate the IM from the MA, the mitoplast pellet from the first centrifugation is diluted to 15 mg/mL low osmotic strength buffer and sonicated for 3 x 5 bursts with 20-s rest intervals on ice. The IM fraction is pelleted from the MA fraction by centrifugation at 80,000g for 60 min. Subcompartment sample purity can be assayed using cytochrome c oxidase for IM, fumarase for MA and antimycin A-insensitive NADH: cytochrome c oxidoreductase for OM. On average, enzymatic determination of cross-contamination from the compartments is less than 15%.
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