Human red blood cells have been incubated in the presence of nystatin, which allows Na and K, as well as CI and pH to equilibrate rapidly when cell volume is set with external impermeant sucrose. The intracellular mean ionic activity coefficients, relative to values in the extraceUular solution, for KCI and NaCl are 1.01 4" 0.02 and 0.99 + 0.02 (SD, n = 10), respectively, and are independent of external pH, pH o, and of [sucrose]o. With nystatin the dependence of red cell volume on [sucrose]~ deviates from ideal osmotic behavior by as much as a factor of three. A virial equation for the osmotic coefficient, ~k, of human hemoglobin, Hb, accounts for the cell volumes, and is the same as that which describes Adair's measurements of ~#nb for Hb isolated from sheep and ox bloods. In the presence of nystatin the slope of the acid-base titration curve of the cells is independent of cell volume, implying that the charge on impermeant cellular solutes is independent of Hb concentration at constant pH.By modifying the Jacobs-Stewart equations (1947. J. Cell Comp. PhysioL 30:. 79-103) with the osmotic coefficients of Hb and of salts, a nonideal thermodynamic model has been devised which predicts equilibrium Donnan ratios and red cell volume from the composition of the extracellular solution and from certain parameters of the cells. In addition to accounting for the dependence of cell volume on osmotic pressure, the model also describes accurately the dependence of Donnan ratios and cell volumes on pHo either in the presence or absence of nystatin.

The fluorescence, F, of two dicarbocyanine dyes, diS-Ca(5) and diI-Ca(5), depends both on the membrane potential, E, and on the intracellular pH, pile, of 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 %6~F/mV for diS-Ca(5) and + 0.6 %~tF/ mV for diI-Ca (5). While pHo does not affect F of either dye, changes in pHc of 0. I unit at constant E cause changes of F equivalent to those induced by 2-3 mV. Based on these results, a method is given for estimating changes in E from dye fluorescence in experiments in which E and pile 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 /tM valinomycin, yields a value for the permeability ratio, PK.VAL/Po, 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 diS-Ca(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 non fluorescent dimers contributes to fluorescence quenching of diS-Ca(5). In contrast, only a hydrophobic interaction of dye monomers need be considered for diI-Ca(5), indicating the occurrence of a simpler mechanism of fluorescence quenching.

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Net K and CI effluxes induced by valinomycin or by gramicidin have been determined directly at varied external K, denoted by [K]o, in the presence and absence of the anion transport inhibitors DIDS (4,4'-diiso-thiocyano-2,2'-disulfonic acid stilbene), and its less potent analogue SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). The results confirm that pretreatment with 10 I~M DIDS, or 100 I~M SITS, for 30 rain at 23~ inhibits conductive CI efflux, measured in the continued presence of the inhibitors at 1 mM [K] strictly be ruled out, the results are consistent with the hypothesis that DIDSinsensitive C1 conductance turns on at an Em of ~ -40 mV.

This series of papers addresses the mechanism by which certain impermeant oxonol dyes respond to membrane-potential changes, denoted delta Em. Hemispherical oxidized cholesterol bilayer membranes provided a controlled model membrane system for determining the dependence of the light absorption signal from the dye on parameters such as the wavelength and polarization of the light illuminating the membrane, the structure of the dye, and delta Em. This paper is concerned with the determination and analysis of absorption spectral changes of the dye RGA461 during trains of step changes of Em. The wavelength dependence of the absorption signal is consistent with an "on-off" mechanism in which dye molecules are driven by potential changes between an aqueous region just off the membrane and a relatively nonpolar binding site on the membrane. Polarization data indicate that dye molecules in the membrane site tend to orient with the long axis of the chromophore perpendicular to the surface of the membrane. Experiments with hyperpolarized human red blood cells confirmed that the impermeant oxonols undergo a potential-dependent partition between the membrane and the bathing medium.