Characterization of the pH dependence of hemoglobin binding to band 3. Evidence for a pH-dependent conformational change within the hemoglobin-band 3 complex.
The pH dependence of hemoglobin binding to inside-out red cell membrane vesicles was studied using 90 degrees light scattering (Salhany, J.M. et al., Biochemistry 19 (1980) 1447-1454). Hyperbolic binding curves were observed for high-affinity hemoglobin binding to the cytoplasmic domain of band 3 (CDB3) within the intact transporter. Analysis of these saturation curves yielded the apparent Kd and the maximum light scattering signal change (DeltaLSmax ). The apparent Kd for hemoglobin binding did not change substantially between pH 5.5 and 7.0, while at pH 8, there is no binding. In contrast, DeltaLSmax decreased by about 20-fold between pH 5.5 and 7.0, with an apparent pK of 6.5. These results suggest that hemoglobin binds to CDB3 with high affinity at both neutral and acid pH, a suggestion that was confirmed using a centrifugation method. Thus, the pK for the light scattering signal change is significantly lower than the pK for the actual binding process. We show that the change in DeltaLSmax is not related to a change in band 3 binding capacity, which remains constant as a function of pH. We also show that hemoglobin binding to non-band 3 sites contributes less than 10% to DeltaLSmax under our specific experimental conditions. On the basis of these and previous fluorescence quenching results in the literature, we propose a new model for hemoglobin binding to band 3, where raising the pH from 6 and 7 causes the CDB3-hemoglobin complex to undergo a conformational change leading to the movement of the bound hemoglobin away from the surface of the bilayer. The possible implication of this new mechanistic interpretation is discussed briefly.
Salhany
J M
JM
Veterans Administration Medical Center, Omaha, NE 68198-5290, USA.
Cordes
K A
KA
Sloan
R L
RL
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry*; D002918:Chymotrypsin; D003593:Cytoplasm / Q000737:chemistry; D004910:Erythrocyte Membrane / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D011485:Protein Binding; D011487:Protein Conformation; D012542:Scattering, Radiation
D001457:Anion Exchange Protein 1, Erythrocyte; D006454:Hemoglobins; D002918:Chymotrypsin