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Analytical ultracentrifugation; sedimentation equilibrium; protein-protein association; band 3 protein; ankyrin; erythrocyte membrane
Abstract:
Associations between different water-soluble proteins can be studied by sedimentation equilibrium experiments in the analytical ultracentrifuge and subsequent mathematical analysis of thec(r)-distributions obtained. The analysis can be simplified by labelling one of the proteins with a dye absorbing at wavelengths >300 nm. The method can also be applied to intrinsic membrane proteins in solutions of a nonionic detergent. The present paper both reviews the method and reports application to the associations between two proteins of the human erythrocyte membrane: 1) band 3, the membrane's main intrinsic protein which, in detergent solutions and presumably also in the erythrocyte membrane, is in a monomer/dimer/tetramer association equilibrium, and 2) the cytoskeletal protein ankyrin which links the membrane skeleton to the lipid bilayer by binding to band 3. Ankyrin was labelled with fluorescein isothiocyanate and the detergent used was C12E9. It was found that the only aggregate of ankyrin and band 3 occurring in significant amounts was a complex of one ankyrin molecule and four band 3 molecules. This strongly suggests that, in the erythrocyte membrane, the band 3 tetramer represents the high affinity ankyrin binding site.
