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Measurements of Membrane Potentials in Plant Mitochondria with the Safranine Method ¹

Department of Biochemistry, University of Sussex, Falmer, Brighton BN1 9QG United Kingdom, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19174

The positively charged dye, safranine, has been used as an indicator of membrane potentials in mung bean (Phaseolus aureus) and Voodoo lily (Sauromatum guttatum) mitochondria under a variety of metabolic conditions. The spectral response of safranine has been calibrated with respect to a K⁺ diffusion potential and was found to be linearly related to the developed potential within the range of 50 to 160 millivolts. Both respiration and ATP hydrolysis gave rise to a membrane potential of approximately 135 millivolts. Respiratory inhibitors such as cyanide and antimycin depolarized the potential, whereas rotenone has little effect. No potentials were developed during NADH supported cyanide insensitive respiration. It is concluded that safranine may be a useful spectrophotometric probe of the mitochondrial membrane potential.

¹ Supported by grants from the National Science Foundation (W. D. B.) and S. E. R. C. (A. L. M.).

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