title: Quaternary interactions in hemoglobin beta subunit tetramers. Kinetics of ligand binding and self-assembly.
abstract: We have investigated the rates of monomer in equilibrium with tetramer self-association of oxygenated beta SH subunits of human hemoglobin A as well as the influence of self-association on the binding kinetics for O2 and CO. A 4 beta in equilibrium with 2 beta 2 in equilibrium with beta 4 assembly pathway can be used to describe the association equilibria and kinetics. We have determined all four elementary rate constants for this assembly pathway at 15 degrees C in 0.1 M Tris-HCl, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4. These data imply that a significant amount (approximately 17%) of beta 2 can be present. Laser photolysis kinetic studies of O2 binding indicate that the O2 association rate constant is unaffected by the degree of self-association. In contrast, photolysis of beta CO solutions shows an overall rate of CO binding that increases at higher protein concentrations. These data are consistent with a concentration-dependent equilibrium between two protein species with CO association rates differing by a factor of 2.5, but they do not appear to be compatible with a direct assignment of different CO binding rates to the different assembly states. Rather, we believe the data imply that CO binding to beta oligomers is heterogeneous, with both a fast binding and a slow binding form being present in single association states. The fast binding form predominates (approximately equal to 87%) in beta 4, while the beta monomer has very little or none of the fast binding form. We propose that the slow binding component within beta 4 may be those subunits with rotationally disordered hemes (La Mar, G. N., Yamamoto, Y., Jue, T., Smith, K. M., and Pandey, R. K. (1985) Biochemistry 24, 3826-3831). The implications of these findings for the use of isolated subunits as models for the subunits within "R state" hemoglobin tetramers are discussed.
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs 06268.
authors.forename: J S
authors.initials: JS
authors.lastname: Philo
authors.forename: J W
authors.initials: JW
authors.lastname: Lary
authors.forename: T M
authors.initials: TM
authors.lastname: Schuster
mesh_terms: D000465:Algorithms; D002248:Carbon Monoxide / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D007700:Kinetics; D007834:Lasers; D046911:Macromolecular Substances; D010100:Oxygen / Q000378:metabolism; D010782:Photolysis
chemical_list: D006454:Hemoglobins; D046911:Macromolecular Substances; D002248:Carbon Monoxide; D010100:Oxygen