Windsor W T, Philo J S, Potschka M, Schuster T M
Department of Molecular and Cell Biology, University of Connecticut, Storrs 06268.
Biophys Chem. 1992 May;43(1):61-71. doi: 10.1016/0301-4622(92)80042-4.
A thorough kinetic characterization of the O2-binding and self-association reactions of alpha-subunits of human hemoglobin A has been performed. All of the rate constants for a five step reaction model linking the monomer-dimer reaction to the O2-binding steps have been determined for the first time. Our analysis of the ligand binding reaction shows that both monomer and dimer have nearly identical intrinsic O2-association and dissociation rate constants and therefore identical affinities for oxygen. During this investigation we discovered a small absorbance difference between the oxy-monomer and oxy-dimer alpha-subunits. This difference spectrum enabled direct measurements of the alpha O2 self-association reaction. We find an association rate constant of, 2.0 10(5) M(-1)s-1, similar to that for other subunit assembly processes in the hemoglobin system. Our results also suggest that the deoxy-subunit assembly kinetics must be similar to that for the oxy-subunit. These kinetic results together with the equilibrium constants obtained for these solution conditions by Ackers and coworkers provides, for the first time, a complete kinetic and thermodynamic description of all the intrinsic ligand binding and association reactions for alpha-subunits.
对人血红蛋白Aα亚基的O₂结合和自缔合反应进行了全面的动力学表征。首次确定了将单体 - 二聚体反应与O₂结合步骤联系起来的五步反应模型的所有速率常数。我们对配体结合反应的分析表明，单体和二聚体具有几乎相同的固有O₂缔合和解离速率常数，因此对氧具有相同的亲和力。在这项研究中，我们发现氧合单体和氧合二聚体α亚基之间存在微小的吸光度差异。这种差异光谱使得能够直接测量αO₂自缔合反应。我们发现缔合速率常数为2.0×10⁵ M⁻¹s⁻¹，与血红蛋白系统中其他亚基组装过程的速率常数相似。我们的结果还表明，脱氧亚基组装动力学必须与氧合亚基的相似。这些动力学结果与Ackers及其同事在这些溶液条件下获得的平衡常数一起，首次提供了α亚基所有固有配体结合和缔合反应的完整动力学和热力学描述。
