THE INFLUENCE OF 2 ANION-TRANSPORT INHIBITORS, '-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' and 4,3'-dibenzoylstilbene-2 ,2'-disulfonate potently inhibit the erythrocyte anion transporter. Th ese inhibitors act by binding, with a 1:1 stoichiometry, to the band 3 transport protein. We have studied, by sedimentation equilibrium anal ysis in an analytical ultracentrifuge, the effect of the two closely r elated stilbenedisulfonates on the state of association of band 3 in t he nonionic detergent nonaethyleneglycol lauryl ether. It was found th at covalent binding of 4,4'-diisothiocyanatodihydrostilbene-2,2 to ban d 3 did not significantly disturb the monomer/dimer/tetramer associati on equilibrium shown by the unliganded protein. An entirely different result was obtained after addition of 4,4'-dibenzoylstilbene-2,2'-disu lfonate to the protein, at both low and high chloride concentrations. The amount of band 3 dimer in the samples increased with increasing in hibitor concentration c(1), and for c(1) greater than or equal to 15 m u M virtually all of the protein was present as dimer. After removal o f the inhibitor (by gel filtration or dialysis), the original monomer/ dimer/tetramer distribution of the band 3 protein was restored. Our da ta 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.