Quantitative analysis of the kinetics of stilbenedisulfonate binding to band 3

Stilbenedisulfonates are potent inhibitors of erythrocyte band 3 chloride/bicarbonate exchange. Band 3 exists as dimers and tetramers in situ, and each monomer binds one stilbenedisulfonate molecule. We determine: (a) whether stilbenedisulfonates exhibit cooperativity in reversible binding to the Band 3 dimer, and (b) whether stilbenedisulfonates directly compete with chloride. Stopped-flow and static fluorescence spectroscopy were used to measure the kinetics and equilibrium of DBDS (4,4′-dibenzamido-2,2′-stilbenedisulfonate) binding to isolated and membrane-bound Band 3. DBDS binding showed biphasic kinetic time courses which were consistent with a two step mechanism: {A figure is presented} Static binding studies showed no evidence for cooperativity, in agreement with the kinetic measurements. Chloride (150 mM) strongly affected the second step in the binding process by increasing k_-2 about 20-fold, without significantly affecting k₁, k_-1 or k₂. Our results indicate: (a) that DBDS binds independently to each monomer of the band 3 dimer, and (b) that DBDS is not competitive with chloride for binding to the transport site, but rather interacts with the transport site allosterically.