QUANTITATIVE-ANALYSIS OF THE KINETICS OF STILBENEDISULFONATE BINDING TO BAND-3

Stilbenedisulfonates are potent inhibitors of erythrocyte band 3 chlor ide/bicarbonate exchange. Band 3 exists as dimers and tetramers in sit u, and each monomer binds one stilbenedisulfonate molecule. We determi ne: (a) whether stilbenedisulfonates exhibit cooperativity in reversib le binding to the Band 3 dimer, and (b) whether stilbenedisulfonates d irectly compete with chloride, Stopped-flow and static fluorescence sp ectroscopy were used to measure the kinetics and equilibrium of DBDS ( 4,4'-dibenzamido-2,2'-stilbenedisulfonate) binding to isolated and mem brane-bound Band 3, DBDS binding showed biphasic kinetic time courses which were consistent with a two step mechanism: [GRAPHICS] Static bin ding studies showed no evidence for cooperativity, in agreement with t he kinetic measurements. Chloride (150 mM) strongly affected the secon d step in the binding process by increasing k(-2) about 20-fold, witho ut 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 th e transport site, but rather interacts with the transport site alloste rically.