STRUCTURAL AND BINDING-PROPERTIES OF THE STILBENEDISULFONATE SITES ONERYTHROCYTE BAND-3 - AN ELECTRON-PARAMAGNETIC-RESONANCE STUDY USING SPIN-LABELED STILBENEDISULFONATES

Two new spin-label derivatives of 4,4'-diaminodihydrostilbene-2,2'-dis ulfonate (H-2-DADS) have been chemically synthesized and employed in e lectron paramagnetic resonance (EPR) studies of binding to the anion e xchange protein (band 3) in intact human erythrocytes. Equilibrium bin ding studies with the 4-monoacyl-spin-label derivative (mono-SL-H-2-DA DS) indicated an effective dissociation constant of 11 muM and substan tial negative cooperativity in isotonic citrate buffer, pH 7.4, at 20- degrees-C. The 4,4'-diacyl-spin-label derivative (di-SL-H-2-DADS) boun d with an effective dissociation constant of 54 muM and no detectable cooperativity under the same binding conditions. The findings of subst antial negative cooperativity in binding of the less bulky mono-SL-H-2 -DADS and no cooperativity for di-SL-H-2-DADS suggest the presence of an allosteric coupling between the stilbenedisulfonate sites on adjace nt band 3 monomers rather than steric interactions. There were approxi mately 1 x 10(6) binding sites per erythrocyte for both the mono- and di-SL-H-2-DADS derivatives, and the binding of each was blocked by pre treatment of intact cells with 4,4'-diisothiocyanostilbene-2,2'-disulf onate (DIDS), a highly specific covalent inhibitor of anion exchange. EPR spectra collected over a wide range of concentrations of mono-SL-H -2-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. E PR spectra collected using di-SL-H-2-DADS revealed the presence of int ramolecular dipole-dipole interactions between spin-label moieties on opposite ends of this biradical probe, but no intermolecular dipole-di pole interactions between separate bound probes. These data indicate t hat di-SL-H-2-DADS binds to the stilbenedisulfonate binding site on ba nd 3 in a bent conformation and further suggest that the termini of th ese binding sites on adjacent monomers are greater than 20 angstrom ap art.