Anion binding characteristics of the band 3 / 4,4 '-dibenzamidostilbene-2,2 '-disulfonate binary complex: Evidence for both steric and allosteric interactions

A novel kinetic approach was used to measure monovalent anion binding to be tter define the mechanistic basis for competition between stilbenedisulfona tes and transportable anions on band 3. An anion-induced acceleration in th e release of 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) from its comp lex with band 3 was measured using monovalent anions of various size and re lative affinity for the transport site. The K-1/2 values for anion binding were determined and correlated with transport site affinity constants obtai ned from the literature and the dehydrated radius of each anion. The result s show that anions with ionic radii of 120-200 pm fall on a well-defined co rrelation line where the ranking of the K-1/2 values matched the ranking of the transport site affinity constants (thiocyanate < nitrate congruent to bromide < chloride < fluoride). The K-1/2 values for the anions on this lin e were about 4-fold larger than expected for anion binding to inhibitor-fre e band 3. Such a lowered affinity can be explained in terms of allosteric s ite-site interactions, since the K-1/2 values decreased with increasing ani onic size. In contrast, iodide, with an ionic radius of about 212 pm, had a 10-fold lower affinity than predicted by the correlation line established by the smaller monovalent anions. These results indicate that smaller monov alent anions have unobstructed access to the transport site within the band 3 / DBDS binary complex, while iodide experiences significant steric hindr ance when binding. The observation of steric hindrance in iodide binding to the band 3 / DBDS binary complex, but not in the binding of smaller monova lent anions, suggests that the stilbenedisulfonate binding site is located at the outer surface of an access channel leading to the transport site.