Electrostatic attraction governs the dimer assembly of human hemoglobin
Journal of Biological Chemistry
We have investigated the effect of surface charge on the rate of assembly of alpha beta dimers of human hemoglobin A: alpha + beta k a----alpha beta. Heme intact beta A subunits were compared with four mutant subunits which differ by integral units of charge: beta N(Lys-95----Glu) (2-); beta J(Gly-16----Asp) (1-); beta S(Glu-6----Val) (1+); beta C(Glu-6----Lys) (2+). Subunit competition experiments were performed as follows. Varying amounts of 3H-labeled alpha A subunits were added to a mixture
... added to a mixture containing equal amounts of beta A and beta X subunits so that alpha/(beta A + beta X) ranged from 0.05-1.0. The reconstituted 3H-labeled Hbs A and X were analyzed by ion-exchange high pressure liquid chromatography as well as by gel electrofocusing and fluorography. Under the solvent conditions employed (10 mM PO4(Na), pH 7.0, 0 degrees C) a predominant proportion of the beta subunits was monomeric. Therefore, the ratio of Hb X to Hb A formed from subunit reconstitution when alpha/(beta X + beta A) approached zero provides a direct measure of the relative rates of monomer combination: kXa/kAa. The experimental values of this ratio decreased monotonically with the overall charge of the variant beta subunit: beta N = 2.6; beta J = 1.5; beta S = 0.41; beta C = 0.13. In contrast surface charge had no significant effect on the rate of dissociation of the alpha beta dimer: alpha beta kd----alpha + beta. At pH 8.0, where the alpha chains lack a net surface charge, they combined equally well to beta A and beta C chains. These experiments are consistent with a two-step mechanism, alpha + beta in equilibrium (alpha...beta) in equilibrium alpha beta, where the oppositely charged monomers diffuse together under the influence of their mutual electrostatic interaction to form a nonspecifically bound encounter complex [alpha...beta] that undergoes a surface charge-independent rearrangement to form the stable dimer.