Dimer-tetramer association equilibria of human adult hemoglobin and its mutants as observed by analytical ultracentrifugation

Dimer-tetramer equilibrium of human adult hemoglobin in CO form (COHb A) and its mutants were measured by sedimentation velocity and sedimentation equilibrium. In sedimentation velocity, the association constants were estimated by measuring the concentration dependence of the weight average sedimentation coefficients at pH 6 and 7 and fitting the data to the theoretical binding isotherms with association constants as a parameter. Association constants of wild type Hb A and three mutant Hbs, Hb Hirose(beta W37S), recombinant (r)Hb(beta W37H) and rHb(alpha Y42S), in which an amino acid was replaced at the alpha(1)beta(2) interface, were measured in the presence and absence of inositol hexaphosphate (IHP). All the three mutations lowered the value of association constants, but the presence of IHP shifted the equilibrium toward tetramer. Although the association constant between dimer and tetramer of rHb(beta W37H) and rHb(alpha Y42S) were similar, sedimentation coefficient distribution function, c(s), analysis indicated that the association and dissociation rate constants of the former is higher than the latter. (C) 2011 Elsevier Inc. All rights reserved.