The fluorescence, F, of two dicarbocyanine dyes, diS-C3(5) and diI-C3(5), depends both on the membrane potential, E, and on the intracellular pH, pHc, or human red blood cells. Compositions of isotonic media have been devised in which the equilibrium Donnan potential, E, varies at constant pHc and in which pHc varies at constant E. Dye fluorescence measurements in these suspensions yield calibrations of +1.7 % delta F/mV for diS-C3(5) and +0.6 % delta F/mV for diI-C3 (5). While pHo does not affect F of either dye, changes in pHc of 0.1 unit at constant E cause changes of F equivalent to those induced by 2--3mV. Based on these results, a method is given for estimating changes in E from dye fluorescence in experiments in which E and pHc co-vary. The relation of F to E also depends in a complex way on the type and concentration of cells and dye, and the wavelengths employed. The equilibrium calibration of dye fluorescence, when applied to diffusion potentials induced by 1 microM valinomycin, yields a value for the permeability ratio, PK.VAL/PCl, of 20 +/- 5, in agreement with previous estimates by other methods. The calibration of F is identical both for diffusion potentials and for equilibrium potentials, implying that diC-C3(5) responds to changes in voltage independently of ionic fluxes across the red cell membrane. Changes in the absorption spectra of dye in the presence of red cells in response to changes in E show that formation of nonfluorescent dimers contributes to fluorescence quenching of diS-C3(5). In contrast, only a hydrophobic interaction of dye monomers need be considered for diI-C3(5), indicating the occurrence of a simpler mechanism of fluorescence quenching.

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