Binding of chloride and a disulfonic stilbene transport inhibitor to red cell band 3.

The effect of chloride on 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS) binding to band 3 in unsealed red cell ghost membranes was studied in buffer [NaCl (0 to 500 mM) + Na citrate] at constant ionic strength (160 or 600 mM), pH 7.4, 25 degrees C. In the presence of chloride, DBDS binds to a single class of sites on band 3. At 160 mM ionic strength, the dissociation constant of DBDS increases linearly with chloride concentration in the range [Cl] = 10 to 120 mM; at 600 mM ionic strength, the DBDS dissociation constant saturates hyperbolically with half-saturating [Cl] = 450 mM. The observed rate of DBDS binding to ghost membranes, as measured by fluorescence stopped-flow kinetic experiments, increases with chloride concentration at both 160 and 600 mM ionic strength. The equilibrium and kinetic results have been incorporated into the following model of the DBDS-band 3 interaction: (formula; see text) The equilibrium and rate constants of the model at 600 mM ionic strength are K1 = 0.67 +/- 0.16 microM, k2 = 1.6 +/- 0.7 sec-1, k-2 = 0.17 +/- 0.09 sec-1, K'1 = 6.3 +/- 1.7 microM, k'2 = 9 +/- 4 sec-1 and k'-2 = 7 +/- 3 sec-1. The apparent dissociation constants of chloride from band 3, KCl, are 40 +/- 4 mM (160 mM ionic strength) and 11 +/- 3 mM (600 mM ionic strength). Our results indicate that chloride and DBDS have distinct, interacting binding sites on band 3.