[Temperature Dependence] [Acknowledgements] [Title page]

Results and Discussion: Dependence of time course on anion concentration and gradient

To investigate the relative magnitudes of the binding and translocation rates of the transport mechanism of Figure 4, the time course of SPQ fluorescence was measured as a function of equilibrium anion concentration and anion concentration gradient. Stopped-flow experiments at varying chloride gradient (at constant equilibrium chloride concentration) showed that the inverse exponential time constant (1/[tau] in equation 5) does not depend on anion concentration (data not shown). This finding is consistent with an apparent first-order relaxation system (17).

Figure 7.
Figure 7. Concentration dependence of chloride-bicarbonate exchange rate. The exchange rate was determined at each equilibrium chloride concentration with one to three concentration gradients.

Because of the presence of rapid bimolecular steps preceding and following the slow unimolecular steps in the reaction mechanism of Figure 4, the exchange rate should depend on the equilibrium chloride concentration, as given in equation 6. Figure 7 shows that the exchange rate increases with increasing chloride concentration and that the dependence is concave upward. Similar data have been obtained by measuring chloride self-exchange in the presence of bicarbonate in red cells (6, 18, , 19) and in resealed ghosts (4).

The data in Figure 7 can be used to examine the affinities and translocation rates of chloride and bicarbonate. In principle, equation 6 can be fit to the data, but in practice, the eight parameters in equation 6 are not independent and the data have too much variation to extract parameter values with good precision. A qualitative comparison can be made, however. In the limit of zero equilibrium chloride concentration, the reaction kinetics of Figure 4 are governed by bicarbonate translocation. As chloride concentration increases, the chloride translocation steps begin to contribute to the observed exchange time until at zero bicarbonate concentration, the reaction kinetics are governed by chloride translocation. Since the exchange rate increases with chloride concentration, the sum of the rate constants for the EoCl to Ei Cl transition in Figure 4 (k1+k-1) is greater than the sum of rate constants for the Eo HCO3 to Eo HCO3 transition (k2+k-2). Furthermore, because the curve is concave upward, the dissociation constants of chloride from Eo and Ei (KoCl and KiCl) are greater than those of bicarbonate (KoHCO3 and KiHCO3 ) (18).

We have shown that the fluorescence of SPQ, trapped in red cell ghosts, can be used to monitor chloride-bicarbonate exchange through band 3, the anion transport protein. We have also shown how the observed time course of fluorescence can be analyzed to give a single exponential time constant that characterizes the exchange. The method should be applicable to any exchange system in which SPQ can be trapped intracellularly and in which intracelluar halide concentration changes during the exchange process. For example, chloride-bicarbonate exchange could be studied in cardiomyocytes (20), hepatocytes (21), or other systems that have transport proteins similar to erythrocyte band 3.

[Temperature Dependence] [Acknowledgements] [Title page]