Kinetics of the alkaline tetramer leads to dimer dissociation in liganded human hemoglobin: a laser light-scattering stopped-flow study.

Abstract

The 1st-order dissociation of tetrameric HbCO to the dimer was studied over the pH range 10.30-11.57 in a light-scattering stopped-flow apparatus using argon-ion laser excitation. The 1st-order dissocation rate constant varies from 0.25 s-1 to 24.0 s-1 over this pH interval. A semilogarithmic plot of k vs. pH has a slope of 2.56 at pH 11.07, the midpoint. The pH-dependence of the dissociation of the tetramer is consistent with progressive titration of .alpha.1-.alpha.2 and .beta.1-.beta.2 salt bridges. At pH 10.66, the dissociation rates of HbO2, HbCO, methemoglobin and HbCN vary less than 20% from their mean value. A study of the dissociation kinetics as a function of protein concentration allows one to obtain both association and dissocation rate constants, and hence equibilibrium constants, for the tetramer .dblarw. dimer reaction. In this manner, equilibrium constants were obtained on protein solutions with less than 15 s of exposure to dissociating conditions.