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: [GRAPHICS] 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 k(1), k(-1), or k(2). 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.