Volume 76, Issue 4, Pages 2043-2055 (April 1999) Hydrodynamic Properties of Human Erythrocyte Band 3 Solubilized in Reduced Triton X-100  Andrew M. Taylor, Jonathan Boulter, Stephen E. Harding, Helmut Cölfen, Anthony Watts  Biophysical Journal  Volume 76, Issue 4, Pages 2043-2055 (April 1999) DOI: 10.1016/S0006-3495(99)77361-3 Copyright © 1999 The Biophysical Society Terms and Conditions

Figure 1 Size exclusion HPLC (SE-HPLC) analysis of purified band 3 and its oligomers applied to a 7.5× 300-mm TSK 4000 SW column, monitored at 225nm. (a) Band 3 was purified from membrane that had been stripped of its cytoskeleton by 2mM EDTA (pH 12) before purification (purification method 1; – – –), and band 3 was purified from membrane that had been depleted of band 6 but had not been stripped of cytoskeleton (purification method 2; ——). (b) SE-HPLC analysis of band 3 dimer prepared according to method 2, isolated by SE-HPLC chromatography, held at 4°C for 24h, and rechromatographed (——), and dimer prepared according to method 2, isolated by SE-HPLC chromatography, held at 20°C for 24h, and rechromatographed (– – –). (c) As in b, except that the higher oligomer prepared according to method 2 was isolated and rechromatographed. (d) As in b, except that band 3 was prepared according to method 1. The column was calibrated with standard proteins: thyroglobulin (RS=86Å), ferritin (RS=63Å), catalase (RS=52Å), and aldolase (RS=46Å). The void (V0) and total volumes (Vt) were determined by the elution positions of blue dextran and β-mercaptoethanol, respectively. The elution buffer was 100mM NaCl, 5mM phosphate (pH 7.0), containing 0.1% (w/v) rTX-100. The peak eluting at 12.5ml is β-mercaptoethanol, which is present in the protein sample. Biophysical Journal 1999 76, 2043-2055DOI: (10.1016/S0006-3495(99)77361-3) Copyright © 1999 The Biophysical Society Terms and Conditions

Figure 2 Concentration of BADS binding to isolated oligomers of band 3. Dimeric band 3 was prepared according to method 2 (RS=76Å) (□). Band 3 was prepared according to method 1 (RS=100Å) (▴). Band 3 was prepared according to either method 1 or method 2 after 24h of incubation at 20°C (RS=114Å) (♦). Oligomers were separated before fluorescence analysis by SE-HPLC. Biophysical Journal 1999 76, 2043-2055DOI: (10.1016/S0006-3495(99)77361-3) Copyright © 1999 The Biophysical Society Terms and Conditions

Figure 3 Sedimentation velocity analysis of band 3 prepared according to method 1 (a) and band 3 prepared according to method 2, where □ represents the measured s20,w value for dimeric band 3 (RS=76Å) and ■ represents the measured s20,w value for the oligomer with RS=114Å (b). Absorbances were measured at 280nm, except for 0.025 and 0.05mg/ml solutions, which were measured at 225nm. The rotor speed was 35,000rpm, and the temperature was 20°C. Band 3 was purified in 0.1% (w/v) rTX-100, 5mM phosphate (pH 8), containing 25mM β-mercaptoethanol. Biophysical Journal 1999 76, 2043-2055DOI: (10.1016/S0006-3495(99)77361-3) Copyright © 1999 The Biophysical Society Terms and Conditions

Figure 4 Sedimentation equilibrium analysis of band 3 form previously characterized by RS=114Å. (a) M* is plotted versus ξ, the radial displacement squared parameter, at 5000rpm. (b) The same sample analyzed by conventional point-averaged molecular weight determination. The initial loading concentration was 0.5mg/ml, the absorbance was measured at 280nm, and the temperature was 20°C. Band 3 was purified in 0.1% (w/v) rTX-100, 5mM phosphate, pH 8, containing 25mM β-mercaptoethanol. Biophysical Journal 1999 76, 2043-2055DOI: (10.1016/S0006-3495(99)77361-3) Copyright © 1999 The Biophysical Society Terms and Conditions

Figure 5 Omega analysis as a diagnostic test of self-association. Omega analysis of the protein with a rotor speed of 5000rpm shows an overlap of samples with different loading concentrations of protein. The initial loading concentrations were 0.2 (+), 0.6 (○), and 0.9 (×) mg/ml. cF=0.3mg/ml. Biophysical Journal 1999 76, 2043-2055DOI: (10.1016/S0006-3495(99)77361-3) Copyright © 1999 The Biophysical Society Terms and Conditions

Figure 6 Fitting of data obtained from the Omega function analysis. The best fit for the sample at 5000rpm was obtained from a dimer/tetramer/hexamer model of the pooled data from Fig. 5, with equilibrium constants K12=(1.6±0.5)×105 M−1 and K14=(7.3±0.8)×1016 M−3, and the second virial coefficient, B, calculated as (1.6±0.3)×10−7 L mol g−2. Biophysical Journal 1999 76, 2043-2055DOI: (10.1016/S0006-3495(99)77361-3) Copyright © 1999 The Biophysical Society Terms and Conditions

Biophysical Journal 1999 76, 2043-2055DOI: (10 Biophysical Journal 1999 76, 2043-2055DOI: (10.1016/S0006-3495(99)77361-3) Copyright © 1999 The Biophysical Society Terms and Conditions