The influence of two anion-transport inhibitors, 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate and 4,4'-dibenzoylstilbene-2,2'-disulfonate, on the self-association of erythrocyte band 3 protein.

4,4'-Diisothiocyanatodihydrostilbene-2,2'-disulfonate and 4,4'-dibenzoylstilbene-2,2'-disulfonate potently inhibit the erythrocyte anion transporter. These inhibitors act by binding, with a 1:1 stoichiometry, to the band 3 transport protein. We have studied, by sedimentation equilibrium analysis in an analytical ultracentrifuge, the effect of the two closely related stilbenedisulfonates on the state of association of band 3 in the nonionic detergent nonaethyleneglycol lauryl ether. It was found that covalent binding of 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate to band 3 did not significantly disturb the monomer/dimer/tetramer association equilibrium shown by the unliganded protein. An entirely different result was obtained after addition of 4,4'-dibenzoylstilbene-2,2'-disulfonate to the protein, at both low and high chloride concentrations. The amount of band 3 dimer in the samples increased with increasing inhibitor concentration c1, and for c1 > or = 15 microM virtually all of the protein was present as dimer. After removal of the inhibitor (by gel filtration or dialysis), the original monomer/dimer/tetramer distribution of the band 3 protein was restored. Our data show that the (noncovalent) binding of 4,4'-dibenzoylstilbene-2,2'-disulfonate drastically changes the coupling between band 3 protomers. In addition, a reversible change in the state of association of band 3 induced by ligand binding is demonstrated.