Measurements of membrane potentials in Escherichia coli K-12 inner membrane vesicles with the safranine method.

The use of safranine, a positively-charged dye, as a probe for the determination of membrane potentials in Escherichia coli vesicles has been studied. 1. Shifts in the spectrum of safranine were observed during induction of potassium ion diffusion potentials with valinomycin or during oxidation of formate by vesicles prepared from cells of E. coli K-12 or ML 308-225 subjected to anaerobic growth with nitrate. The extent of the valinomycin-dependent spectral change correlated linearly with the magnitude of the K+ equilibrium potential, as calculated from the Nernst equation, from 50 to 160 mV (interior negative). The formate-induced changes could also be calibrated by increasing the concentration of potassium in the presence of valinomycin, after the formation of formate-dependent responses. In this case, results identical to those obtained with the first method were obtained. 2. O2 or nitrate-dependent oxidation of formate resulted in a membrane potential of the order of 170 mV. The oxidation of ascorbate-reduced N-methylphenazonium methosulphate resulted in a potential of similar magnitude, but anaerobically with nitrate only a small but definite potential was formed. 3. The water-soluble quinones, duroquinone and menadione, could produce membrane potentials when used in their oxidized or reduced forms in the presence of formate or nitrate (or oxygen). 2-Hydroxy-1,4-naphthoquinone was not only ineffective but was found to be inhibitory. 4. N,N'-dicyclohexylcarbodiimide at suitable concentrations increased the rate of formation and the extent of membrane potentials induced by respiration or by artificial means.