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Potentiometric Determination of Interrelationships of Energy Conservation and Ion Gradients in Mitochondria

From the ¹ From the Department of Biophysics and Physical Biochemistry, Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 19104

The reduction potential of cytochrome c is monitored potentiometrically with N,N,N',N'-tetramethyl-p-phenylenediamine as a mediator of electrons from cytochrome c to a platinum electrode.

Rotenone and cyanide are used as respiratory inhibitors in simultaneous observation of reversal of electron transport between cytochrome b measured spectroscopically and cytochrome c measured potentiometrically. Rotenone, malonate, antimycin A, and cyanide are employed to inhibit respiration for observation of reversal of electron transfer between cytochrome a measured spectroscopically and cytochrome c measured potentiometrically. The midpoint potential of cytochrome a at pH 7.4 is estimated to be more than 330 mv.

Rotenone alone is used as an inhibitor of electron transfer in a study of the interrelation between the phosphate potential and the reduction potential of cytochrome c in nonrespiring mitochondria; reduction potential changes in cytochrome c are shown to give an accurate estimate of phosphate potential changes in mitochondria. The standard free energy of hydrolysis of bound ATP to bound ADP at pH 7.4 is estimated to be -9.4 kcal.

By means of the potentiometric technique with rotenone-inhibited mitochondria to follow phosphate potential changes, the interaction of ion gradient and ion transport with the energy conservation system is investigated by adding calcium, valinomycin, nigericin, or uncoupling agents to alter ion permeability or ion gradients.

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