Stilbenedisulfonates are potent inhibitors of Band 3 mediated anion exchange. They bind tightly to the protein and form a 1-to-l reversible complex. Those stilbenedisulfonates which contain isothocyanato groups such as DIDS (4,4'-diisothiocyanato-2,2'-stilbenedisulfonate) and H2DIDS (4,4'- diisothiocyanatodihydrostilbene-2,2'-disulfonate) can also react rapidly with lysine residues within the binding pocket to yield an irreversible covalent adduct. The reactive lysine residue is known as lysine-A, and is thought to have an unusually low pka. In this report, we characterize the kinetics of DIDS adduct formation with respect to the effect of substrate anions, competitive inhibitory anions, and pH on the rate of covalent adduct formation. We investigate the following: (a) whether stilbenedisulfonates bind to or block access of substrate anions to the transport site; (b) whether the rapidity of the covalent reaction of DIDS at neutral pH is due to a low pka for lysine-A within the binding pocket; and (c) whether once bound, DIDS and H2DIDS isothiocyanato groups are accessible to reagents. For this latter experiment, we have utilized a newly discovered reaction of the DIDS isothiocyanato groups with azide to test for accessibility. Our results show that substrate anions, DIDS, and Band 3 form a ternary complex. Significantly, the binding of large substrate anions, such as iodide, is not weakened by DIDS to any greater extent than is the binding of smaller substrates such as chloride or fluoride. These results are not consistent with a “partial blockade” hypothesis for the relationship between the stilbenedisulfonate and transport sites. Rather, they support an allosteric site-site interaction hypothesis. Our pH dependence results show that the apparent pka for the DIDS/lysine-A reaction is greater than 9.26. This is consistent with typical lysine pKa values, and indicates that lysine-A does not have an unusually low pKa. Finally, we show that azide can react with the isothiocyanato groups of DIDS and H2DIDS within their Band 3 complexes, indicating that the stilbenedisulfonate binding site is accessible to solute. These results support a view which suggests that the stilbenedisulfonate site is a superficial inhibitory site on Band 3 which inhibits transport by allosteric interactions within the protein, rather than by either direct or partial blockade of the transport site.
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