Structural and binding properties of the stilbenedisulfonate sites on erythrocyte band 3: an electron paramagnetic...

Two new spin-label derivatives of 4,4'-diaminodihydrostilbene-2,2'-disulfonate (H2-DADS) have been chemically synthesized and employed in electron paramagnetic resonance (EPR) studies of binding to the anion exchange protein (band 3) in intact human erythrocytes. Equilibrium binding studies with the 4-monoacyl-spin-label derivative (mono-SL-H2-DADS) indicated an effective dissociation constant of 11 microM and substantial negative cooperativity in isotonic citrate buffer, pH 7.4, at 20 degrees C. The 4,4'-diacyl-spin-label derivative (di-SL-H2-DADS) bound with an effective dissociation constant of 54 microM and no detectable cooperativity under the same binding conditions. The findings of substantial negative cooperativity in binding of the less bulky mono-SL-H2-DADS and no cooperativity for di-SL-H2-DADS suggest the presence of an allosteric coupling between the stilbenedisulfonate sites on adjacent band 3 monomers rather than steric interactions. There were approximately 1 x 10(6) binding sites per erythrocyte for both the mono- and di-SL-H2-DADS derivatives, and the binding of each was blocked by pretreatment of intact cells with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), a highly specific covalent inhibitor of anion exchange. EPR spectra collected over a wide range of concentrations of mono-SL-H2-DADS indicated that binding resulted in immobilization of the probe and that, even upon near saturation of available binding sites, there were no detectable dipole-dipole interactions between bound probes. EPR spectra collected using di-SL-H2-DADS revealed the presence of intramolecular dipole-dipole interactions between spin-label moieties on opposite ends of this biradical probe, but no intermolecular dipole-dipole interactions between separate bound probes. These data indicate that di-SL-H2-DADS binds to the stilbenedisulfonate binding site on band 3 in a bent conformation and further suggest that the termini of these binding sites on adjacent monomers are greater than 20 A apart.