Kinetics of the alkaline tetramer dimer dissociation in liganded human hemoglobin: A laser light-scattering stopped-flow study

The first-order dissociation of tetrameric HbCO to the dimer has been studied over the pH range 10.30-11.57 in a light-scattering stopped-flow apparatus using argon-ion laser excitation. The first-order dissociation rate constant varies from 0.25 sec-1 to 24.0 sec-l over this pH interval. A semilogarithmic plot of k versus 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 ai-a2 and #I-,62 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 dissociation rate constants, and hence equilibrium constants, for the tetramer >± dimer reaction. In this manner, equilibrium constants were obtained on protein solutions withless than 15 sec of exposure to dissociating conditions. The tetramer-dimer equilibrium for human carbon monoxide-hemoglobin (HbCO) has been studied in the ultracentrifuge from pH 7.0 to 10.75 by Edelstein et al. (1) and Andersen et al. (2). Alkaline denaturation at high pH values during the long times required for the equilibrium studies (12 hr) did not allow the collection of sufficiently extensive data for fitting to proton equilibrium models because a plot of the equilibrium constant as a function of pH did not approach a plateau at high pH values. The rate of the spectroscopic change at 245 nm following a rapid pH jump in the stopped-flow apparatus was assigned to the tetramer -dimer process. These rate studies were at pH 10.6. A flow-flash study by Gibson and Antonini (3) at pH 7.0 and 0.25 ,gM initial concentration of tetramer gave a relaxation rate constant of 1.24 sec-1. Observations of slowly varying spectroscopic changes at 429 nm of deoxygenated hemoglobin at pH 7.0 following rapid deoxygenation were interpreted by Kellett and Gutfreund (4) to give a dissociation constant for the tetramer of 1.35 sec-1. In this paper we report the direct study of the dissociation process by laser light-scattering stopped-flow techniques following rapid changes in pH from 7.0 to values between 10.30 and 11.57.