Molecular characterization of the human erythrocyte anion transport protein in octyl glucoside

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Abstract
Band 3 protein, the anion transport protein of the human erythrocyte membrane, was purified in the presence of the nonionic detergent octyl glucoside. A molecular characterization was carried out to investigate whether the native structure of the protein was retained in the presence of this detergent. Band 3 bound octyl glucoside below the critical micelle concentration (cmc) of the detergent, approaching saturation above the cmc. At 40 mM octyl glucoside, close to saturating concentrations, 0.64 g of octyl glucoside is bound per gram of band 3 protein, corresponding to 208 molecules of detergent bound per monomer of band 3. Sedimentation velocity and gel filtration studies, performed at 40 mM octyl glucoside, indicated that the band 3-octyl glucoside complex had an average molecular weight of 1.98 .times. 106, which corresponds to a dodecamer. Sedimentation equilibrium experiments confirmed that band 3 in octyl glucoside exists in a heterogeneous and high oligomeric state. This high oligomeric state did not change dramatically over octyl glucoside concentrations ranging from 6 to 60 mM. The circular dichroism spectrum of band 3 changed only slightly over this range of octyl glucoside concentrations. The .alpha.-helical and .beta.-sheet contents of band 3 in 2 mM octyl glucoside were calculated to be 40% and 27%, respectively, indicating that no gross alteration in the secondary structure of the protein had occurred in octyl glucoside. The ability of band 3 to bind 4-benzamido-4''-aminostilbene-2,2''-aminostilbene-2,2''-disulfonate (BADS), a potent inhibitor (Ki = 1 .mu.M) of anion transport, was measured to assess the integrity of the inhibitor binding site of the protein in octyl glucoside. Band 3 in octyl glucoside bound BADS (Kd = 0.8-1.5 .mu.M), but at high octyl glucoside concentrations, an irreversible decrease in the binding affinity (to Kd .simeq. 10 .mu.M) was observed. The results indicate that while band 3 in octyl glucoside retained a high degree of secondary structure, the inhibitor binding site had been altered during purification in this detergent.