title: Effects of heterotropic allosteric effectors on the equilibrium and kinetics of the reaction of a single ligand molecule with an alpha or beta subunit of deoxygenated HbA.
abstract: Symmetrical FeZn hybrids of human HbA have been used to measure K(1)(alpha) and K(1)(beta), the dissociation constants for the binding of a single molecule of oxygen to unliganded HbA at an alpha subunit and at a beta subunit, respectively. The kinetic constants, l(1)'(alpha) and l(1)'(beta), for the combination of the first CO molecule to unliganded HbA at an alpha or a beta subunit, respectively, were also measured. Measurements were carried out between pH 6 and pH 8 in the presence and absence of inositol hexaphosphate (IHP). Both equilibrium constants exhibit a significant Bohr effect in the absence of IHP. The addition of IHP to a concentration of 0.1 mM increases both dissociation constants in a pH-dependent manner with the result that both Bohr effects are greatly reduced. These results require a negative thermodynamic linkage between the binding of a single oxygen at either an alpha or a beta subunit and the binding of IHP to the T quaternary structure of HbA. Although the beta hemes are relatively near the IHP binding site, a linkage between that site and the alpha hemes, such that the binding of a single oxygen molecule to the heme of one alpha subunit reduces the affinity of the T state for IHP, requires communication across the molecule. l(1)'(alpha) exhibits a very slight pH dependence, with a maximum variation of 20%, while l(1)'(beta) varies with pH three times as much. IHP has no effect on the pH dependence of either rate constant but reduces l(1)'(alpha) marginally, 20%, and l(1)'(beta) by 2-fold at all pH values.
authors.affiliation: Department of Medicine, University at Buffalo, VA Western New York Healthcare System, Building 20, 3495 Bailey Avenue, Buffalo, New York 14215, USA.
authors.forename: Ellen
authors.initials: E
authors.lastname: Karasik
authors.forename: Laura D
authors.initials: LD
authors.lastname: Kwiatkowski
authors.forename: Hilda L
authors.initials: HL
authors.lastname: Hui
authors.forename: Judith F
authors.initials: JF
authors.lastname: Colby
authors.forename: Robert W
authors.initials: RW
authors.lastname: Noble
mesh_terms: D000494:Allosteric Regulation; D006441:Hemoglobin A / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D010100:Oxygen / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D008024:Ligands; D006441:Hemoglobin A; D010100:Oxygen