Oxidative phosphorylation by suspensions of mitochondria following freezing and drying by sublimation in vacuo

Abstract

The cryotolerances of suspensions of mitochondria prepared in solutions of sucrose, glucose, dextran, polyvinylpyrrolidone or dimethylsulphoxide, alone or in combination, and frozen slowly at —76° were determined. The levels of oxidative phosphorylation by suspensions prepared and frozen as the above, dried by sublimation in vacuo at bath temperatures of 0°, and rehydrated with water, physiologic saline, different tonicities of sucrose, dextran in physiologic saline, polyvinylpyrrolidone in glucose (fresh, autoclaved or aged) or sucrose, and dimethylsulphoxide in sucrose were measured. Three steps appear to be necessary for the successful freezing and freeze-drying of suspensions of mitochondria: (a) the addition of protective substances (b) the use of hypertonic sucrose in the final suspension, both to wash away excessive amounts of protective compounds and to produce a preliminary dehydration (c) rehydration with solutions of compounds of high molecular weight. Under these conditions the highest balanced rate of oxidative phosphorylation was by suspensions of mitochondria prepared initially in 10% dimethylsulphoxide in 0.25 M sucrose, suspended finally in 0.50 M sucrose, frozen slowly at —76°, dried by sublimation in vacuo at a bath temperature of 0°, and rehydrated with 5% dextran in physiologic saline.