EFFECT OF CHLORIDE ON THE BINDING-KINETICS OF VARIOUS STILBENEDISULFONATES TO BAND-3

To determine the mechanism of apparent competitive binding of chloride and stilbenedisulfonates ( S ) to band 3 ( B ), we have compared the binding kinetics of three stilbenedisulfonates [ DIDS, 4,4'-diisothioc yanato-2,2'-stilbenedisulfonate; H2DIDS 4,4'-diisothiocyanodihydro-2,2 '-stilbenefonate and DBDS, 4,4'-dibenzamido-2,2'-stilbenedisulfonate ] in the absence and presence of 150 mM sodium chloride at constant ion ic strength. Biphasic time courses were observed with the fast phase r ate constants following second-order kinetics, and the slow phase rate constants following saturation kinetics according to the mechanism: [ GRAPHICS] The results can be understood in terms of the effect of chlo ride on each of these reaction steps. Chloride increased k(1) by about 2-fold, but decreased k(-1) 8-fold for H2DIDS. Thus, 150 nM chloride increased the initial affinity of H2DIDS by about 19-fold. There was a 3-fold increase in the initial affinity for DIDS, but little or no ef fect of chloride on the initial affinity of DBDS. There was no effect of chloride on k(2), but, previous ''off'' rate measurements showed th at 150 mM chloride increases k(-2) about 16-fold for DBDS and about 12 -fold for H2DIDS. Taken together, these results indicate that chloride allosterically competes with stilbenedisulfonates for binding to band 3, predominantly by substantially shifting the second isomerization e quilibrium to the left.