HYDRODYNAMIC EXAMINATION OF THE DIMERIC CYTOPLASMIC DOMAIN OF THE HUMAN ERYTHROCYTE ANION TRANSPORTER, BAND-3

Solution studies of the cytoplasmic domain (molecular mass approximate to 40kDa) of band 3, the anion exchanger from human erythrocyte membr anes, previously suggested a dimeric molecule on the basis of the rela tive techniques of calibrated gel filtration and calibrated preparativ e ultracentrifugation, This dimeric behavior is firmly established on an absolute basis by a combination of calibrated gel chromatography an d absolute ultracentrifugation techniques. Sedimentation velocity in t he analytical ultracentrifuge combined with calibrated gel chromatogra phy give a molecular mass M of (77 +/- 4) kDa, a value confirmed by lo w-speed sedimentation equilibrium. Velocity sedimentation in the analy tical ultracentrifuge gave a single sedimenting species with an s(20,w )(0) of (3.74 +/- 0.07)S. Sedimentation equilibrium analysis was also used to establish the strength of the binding via the dissociation con stant K-d, with a value from direct fitting of the concentration distr ibution curves of (2.8 +/- 0.5) mu M, confirmed by a value of similar to 3 mu M obtained from fitting a plot of molecular weight M(w,app) ve rsus cell loading concentration, Hydrodynamic calculations based on th e classical translational frictional ratio showed that the protein was highly asymmetric, with an axial ratio of similar to 10:1, consistent with observations from electron microscopy.