CHARACTERIZATION OF THE PH-DEPENDENCE OF HEMOGLOBIN BINDING TO BAND-3- EVIDENCE FOR A PH-DEPENDENT CONFORMATIONAL CHANGE WITHIN THE HEMOGLOBIN-BAND-3 COMPLEX
Jm. Salhany et al., CHARACTERIZATION OF THE PH-DEPENDENCE OF HEMOGLOBIN BINDING TO BAND-3- EVIDENCE FOR A PH-DEPENDENT CONFORMATIONAL CHANGE WITHIN THE HEMOGLOBIN-BAND-3 COMPLEX, Biochimica et biophysica acta. Biomembranes, 1371(1), 1998, pp. 107-113
The pH dependence of hemoglobin binding to inside-out red cell membran
e 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 th
ese saturation curves yielded the apparent K-d and the maximum light s
cattering signal change (Delta LSmax). The apparent K-d for hemoglobin
binding did not change substantially between pH 5.5 and 7.0, while at
pK 8, there is no binding. In contrast, Delta LSmax decreased by abou
t 20-fold between pH 5.5 and 7.0, with an apparent pK of 6.5. These re
sults suggest that hemoglobin binds to CDB3 with high affinity at both
neutral and acid pH, a suggestion that was confirmed using a centrifu
gation method. Thus, the pK for the light scattering signal change is
significantly lower than the pK for the actual binding process. We sho
w that the change in Delta LSmax 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 Delta LSmax under our specific experimental conditions. On the
basis of these and previous fluorescence quenching results in the lit
erature, we propose a new model for hemoglobin binding to band 3, wher
e raising the pH from 6 and 7 causes the CDB3-hemoglobin complex to un
dergo a conformational change leading to the movement of the bound hem
oglobin away from the surface of the bilayer. The possible implication
of this new mechanistic interpretation is discussed briefly. (C) 1998
Elsevier Science B.V.