[Introduction] [Results and Discussion] [Title page]

Materials and Methods

Materials. SPQ was synthesized by heating equimolar amounts of 6-methoxyquinoline and 1,3-propane sultone (Aldrich, Milwaukee, WI) at 100 °C for 45 min (9) and purified by recrystallization from ethanol. Biogel A-50m was obtained from Biorad (Richmond, CA). DIDS was obtained from Pierce Chemical (Rockford, IL). DBDS was synthesized by the method of Kotaki et al. (10). All other chemicals were obtained from Sigma (St. Louis, MO). Red cells less than four days old were obtained from the Binghamton Chapter of the Red Cross Blood Bank.

Ghost preparation. Red cell ghosts were prepared by a hemolysis-resealing procedure performed on a gel filtration column (11). A 7 x 31 cm column containing 1250 ml of Biogel A-50m was pre-equilibrated with 0.1 mM EDTA and 20 mM Pipes, pH 6.0. The column was then loaded with 480 ml of 20 mM Pipes, pH 7.6, followed by 120 ml of 150 mM NaCl, 20 mM Pipes, pH 7.6 (isotonic buffer). Red cells were washed three times in isotonic buffer and suspended in isotonic buffer at 10% hematocrit. The washed red cell suspension (100 ml) was applied to the column and eluted with isotonic buffer. Red cells hemolyzed in the region of the column equilibrated with 20 mM Pipes. Unsealed ghost membranes were eluted in the void volume of the column while hemoglobin and other intracellular constituents were retained on the column. All operations were performed at 0.0 ± 0.1 C. This procedure typically removed >98% of the hemoglobin as determined by Drabkin's assay (Sigma). Ghost membranes were concentrated by centrifugation at 10,400 x g, 0 °C, for 10 min, and resealed by resuspending the pellet in 20 volumes of 150 mM NaCl, 20 mM HEPES, pH 7.5 (isotonic chloride buffer), at 37 °C for 45 min.

Resealed ghosts were loaded with SPQ by incubation of equal volumes of packed ghosts and 20 mM SPQ in isotonic buffer overnight at 4 °C, then washed at least 5 times with 20 volumes of either isotonic buffer or isotonic bicarbonate buffer (150 mM NaHCO₃, 20 mM HEPES, pH 7.5). Ghosts were kept at 4 °C prior to use. Samples for stopped-flow experiments were prepared by adding 1 volume of packed, SPQ-labeled ghosts to 14 volumes of buffer solution.

DIDS-labeled membranes were prepared by incubating washed intact red cells at 33% hematocrit with 66-100 M DIDS for 1 hour at 37 °C, then washing three times with isotonic chloride buffer. Anion transport inhibition was assessed by measuring the time course of pH equilibration following a 1 pH unit perturbation of a 1.4% hematocrit red cell suspension in isotonic chloride buffer. Inhibition was > 98%. Ghost membranes were prepared from DIDS-labeled cells as described above.

Fluorescence measurements. The time course of fluorescence was measured in a Dionex Model D-110 stopped-flow apparatus (Sunnyvale, CA) interfaced to a PDP 11/23 computer. Equal volumes of SPQ-labeled ghosts and buffer were mixed. In some experiments, experimental conditions were set to produce a varied chloride concentration gradient across the membrane at constant equilibrium chloride concentration. In other experiments, equilibrium chloride concentration was varied at constant chloride gradient. In all cases, the sum of chloride and bicarbonate concentrations was constant at 150 mM. Stock solutions of isotonic bicarbonate buffer were prepared immediately before use by adding sodium bicarbonate to HEPES acid. SPQ excitation wavelength was 360 nm (20 nm bandwidth); emission wavelengths were >400 nm as viewed with a Schott Glass GG-400 cut-on filter (Duryea, PA). Typically, ten time courses were averaged before data analysis.

Equilibrium SPQ fluorescence was measured as a function of chloride and bicarbonate concentration in a SLM 8000 fluorescence spectrometer. Solutions of 5 M SPQ were prepared in buffer consisting of 20 mM HEPES, x mM NaCl, and (150-x) mM NaHCO₃. Fluorescence was measured with 352 nm excitation and 450 nm emission (slit width 10 nm). Fluorescence lifetimes of SPQ were measured in an SLM 68000 phase-modulation spectrometer.

Data Analysis. As shown in Results and Discussion, both chloride and HEPES quench SPQ fluorescence by a collisional mechanism. In the presence of these two quenchers, the fluorescence, F, of SPQ is related to chloride and HEPES concentration by

[1]

[2]

[3]

The intracellular chloride concentration should depend exponentially on time if anion exchange occurs by a ping-pong mechanism (see below)

[4]

[5]

[Introduction] [Results and Discussion] [Title page]