Evidence that eosin-5-maleimide binds close to the anion transport site of human erythrocyte band 3: a fluorescence quenching study.
The interaction between eosin-5-maleimide (EMA), an inhibitor of the anion transport protein, band 3, and I-, a transportable substrate, was investigated by fluorescence quenching. The Stern-Volmer plot for the quenching reaction between EMA-labeled band 3 and I- exhibits downward curvature both in human erythrocyte ghosts and in purified band 3. The quenching reaction is insensitive to the viscosity of the bulk phase. The shape of the Stern-Volmer plot becomes more linear with increasing temperature. Following the approach of Blatt et al. [(1986) Biophys. J. 50, 349-356], we have developed a binding-diffusion model which is in good agreement with the quenching data. The model supposes that EMA is located in a compartment or "pocket" in band 3 which is separate from the bulk phase and contains a binding site or sites for the quencher. Quenching of band 3-bound EMA fluorescence by I- is inhibited by DIDS and by the transportable anions Cl-, HCO3-, and Br-. Analysis of these experiments yields dissociation constants for the anions which are in reasonable agreement with those determined from transport kinetics and by NMR. We thus deduce that the quencher binding site is the anion binding/transport site on band 3. We propose that EMA is located in the wall of the anion access channel such that it does not inhibit anion binding. The methods described in this report should facilitate detailed studies of anion binding to the transport site on band 3 under a variety of experimental conditions.
Pan
R J
RJ
Department of Chemistry and Biological Chemistry, University of Essex, Colchester, U.K.
Cherry
R J
RJ
D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D000838:Anions; D001692:Biological Transport; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; C033531:eosin maleimide; D004801:Eosine Yellowish-(YS)