Anion transport inhibitor binding to band 3 in red blood cell membranes.
The inhibitor of anion exchange 4,4'-dibenzoamido-2,2'-disulfonic stilbene (DBDS) binds to band 3, the anion transport protein in human red cell ghost membranes, and undergoes a large increase in fluorescence intensity when bound to band 3. Equilibrium binding studies performed in the absence of transportable anions show that DBDS binds to both a class of high-affinity (65 nM) and low-affinity (820 nM) sites with stoichiometry equivalent to 1.6 nmol/mg ghost protein for each site, which is consistent with one DBDS site on each band 3 monomer. The kinetics of DBDS binding were studied both by stopped-flow and temperature-jump experiments. The stopped-flow data indicate that DBDS binding to the apparent high-affinity site involves association with a low-affinity site (3 microM) followed by a slow (4 s-1) conformational change that locks the DBDS molecule in place. A detailed, quantitative fit of the temperature-jump data to several binding mechanisms supports a sequential-binding model, in which a first DBDS molecule binds to one monomer and induces a conformational change. A second DBDS molecule then binds to the second monomer. If the two monomers are assumed to be initially identical, thermodynamic characterization of the binding sites shows that the conformational change induces an interaction between the two monomers that modifies the characteristics of the second DBDS binding site.
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D002498:Centrifugation; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008954:Models, Biological; D013267:Stilbenes / Q000378:metabolism*; D013696:Temperature
D008565:Membrane Proteins; D013267:Stilbenes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C036010:4,4'-dibenzamido-2,2'-stilbenedisulfonic acid