Location of the stilbenedisulfonate binding site of the human erythrocyte anion-exchange system by resonance energy transfer

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Abstract
The stilbenedisulfonate inhibitory site of the human erythrocyte anion-exchange system was characterized by using several fluorescent stilbenedisulfonates. The covalent inhibitor 4-benzamido-4''-isothiocyanostilbene-2,2''-disulfonate (BIDS) reacted specifically with the band 3 protein of the plasma membrane when added to intact erythrocytes, and the reversible inhibitors 4,4''-dibenzamidostilbene-2,2''-disulfonate (DBDS) and 4-benzamido-4''-aminostilbene-2,2''-disulfonate (BADS) showed a fluorescence enhancement upon binding to the inhibitory site on erythrocyte ghosts. The fluorescence properties of all 3 bound probes indicated a rigid, hydrophobic site with nearby tryptophan residues. The Triton X-100 solubilized and purified band 3 protein had similar affinities for DBDS, BADS and 4,4''-dinitrostilbene-2,2''-disulfonate (DNDS) to those observed on intact erythrocytes and erythrocyte ghosts, showing that the anion binding site was not perturbed by the solubilization procedure. The distance between the stilbenedisulfonate binding site and a group of cysteine residues on the 40,000 dalton amino-terminal cytoplasmic domain of band 3 was measured by the fluorescence resonance energy transfer technique. Four different fluorescent sulfhydryl reagents were used as energy transfer donors or energy transfer acceptors in combination with the stilbenedisulfonates (BIDS, DBDS, BADS and DNDS). Efficiencies of transfer were measured by sensitized emission, donor quenching and donor lifetime changes. Although these sites were approchable from opposite sides of the membrane by impermeant reagents, they were separated by only 34-42 .ANG., indicating that the anion binding site was located in a protein cleft which extends some distance into the membrane.