Separation of Mitochondria from Contaminating Subcellular Structures Utilizing Silica Sol Gradient Centrifugation

Discontinuous Percoll density gradients have been developed for the purification of mitochondria, permitting rapid separation under isosmotic and low viscosity conditions. Mitochondria from several etiolated tissues have been successfully separated from contaminating subcellular structures by this method. For potato tuber the ratio of washed to purified mitochondrial protein was 2.6, similar to the increase in specific activity of cytochrome c oxidase following separation. The purification of mitochondria from green leaf tissues on Percoll gradients has reduced chlorophyll contamination of spinach mitochondria from about 70 micrograms chlorophyll per milligram protein to approximately 8 micrograms chlorophyll per milligram protein.The ratio of protein content of the washed mitochondria compared to that in the purified preparation was 7 for spinach and respiratory activity was retained. The physiological integrity and oxidative properties of washed and gradient mitochondria are compared.