[Quenching of SPQ] [Hydration and Dehydration of CO2] [Title page]

Results and Discussion: Time course of fluorescence quenching

The kinetics of chloride-bicarbonate exchange were examined by creating inwardly and outwardly-directed gradients of chloride across the membrane in the stopped-flow apparatus. The experimental conditions were set so that the total intracellular and extracellular anion concentration at the start of the experiment was 150 mM. Since anion exchange in the red cell is a 1-for-1 exchange, the total intracellular and the total extracellular anion concentrations remained constant throughout the exchange time course. Furthermore, at equilibrium, the intracellular and extracellular chloride concentrations must be equal because the Donnan ratio for red cell ghosts is equal to 1. The chloride concentration was manipulated to present an inwardly-directed or outwardly-directed chloride gradient across the membrane, with an equal but oppositely-directed bicarbonate gradient.

Figure 2 shows the time course of SPQ fluorescence at five different chloride gradients. In these experiments, initial intracellular chloride concentration ranged from 0 to 150 mM, while the final intracellular and extracellular chloride concentrations at equilibrium were always 75 mM. Because the extracellular volume was much greater than the intracellular volume, the extracellular chloride concentration was constant throughout the experiment. Inwardly-directed chloride gradients (top three traces in Figure 2) produce a time-dependent quenching of SPQ fluorescence, while outwardly-directed chloride gradients (bottom two traces) produce an enhancement time course. The kinetic experiments shown in Figure 2 measure chloride-bicarbonate exchange. Chloride-chloride or bicarbonate-bicarbonate exchange would not change the intracellular chloride concentration nor the fluorescence intensity of SPQ.

Figure 2.
Figure 2. Time course of SPQ fluorescence. SPQ-labeled, resealed ghost membranes (0.2 mg protein/ml) in buffer solution of chloride, bicarbonate and 20 mM HEPES, pH 7.5, were mixed with an equal volume of chloride, bicarbonate and 20 mM HEPES, pH 7.5. The composition of the two solutions was varied to produce varying chloride gradients across the membrane; the sum of chloride and bicarbonate concentrations was 150 mM. From top to bottom, the chloride gradient was -65, -45, -25, +25, +45 mM. (A negative chloride gradient corresponds to an inwardly-directed chloride gradient.) The final equilibrium intracellular and extracellular chloride and bicarbonate concentrations were 75 mM. The data represent the average of six to eight scans. For these and the following figures, SPQ fluorescence is in arbitrary units.

As shown in Figure 3, the time courses are well-described by an equation that combines first-order kinetics with Stern-Volmer quenching (equation 5).

Figure 3. Figure 3.
Figure 3. Fit of fluorescence time course. Experiments were performed as in figure 2. (a) Inwardly-directed chloride gradient is -65 mM; equilibrium chloride concentration is 75 mM. A fit of equation 5 to the data (solid line) gives a time constant of 0.74 ± 0.04 s. The graph below the data is the deviation between the experimental data and the fit, expressed as a fraction of the signal amplitude. (b) As in (a), but with outwardly-directed chloride gradient of +65 mM. The fit gives a time constant of 0.69 ± 0.04 s.

First-order kinetics are expected from the commonly accepted ping-pong mechanism of anion exchange (Figure 4).

Figure 4.
Figure 4. Ping-pong mechanism of anion exchange. The translocation steps are slow compared to the anion binding steps. Equilibrium constants are expressed as dissociation constants. The symbol E stands for band 3.

If anion binding and unbinding are rapid compared with translocation steps across the membrane (12), the exchange system behaves as an apparent first-order kinetic system characterized by an inverse exponential time constant, [tau]-1, given by3
equation 6            [6]
where the symbols are defined in Figure 4. The inverse exponential time constant, [tau]-1, is a function of the rate constants, equilibrium dissociation constants, and equilibrium concentrations of both anions on both sides of the membrane. The parameter can be thought of as a relaxation time constant. When the anion exchange system is perturbed by the imposition of an anion concentration gradient, the system will relax back to equilibrium with first order kinetics with inverse time constant given by equation 6.

[Quenching of SPQ] [Hydration and Dehydration of CO2] [Title page]