Determination of equilibrium constants for a sequential model of dioxygen binding by hemoglobin-inositol hexaphosphate complexes: the structural pathway from deoxy- to oxy-hemoglobin

The affinity of human hemoglobin (Hb4) for dioxygen was determined in 0.050 M bistris, 0.005 M inositol hexaphosphate (IHP) at pH 7.0 and 20.0 degrees C. Binding of dioxygen by Hb4 was determined by detailed spectroscopic analysis of the absorption spectrum in the region from 460 to 620 nm. The absorption spectrum of samples at intermediate values of fractional saturation (F) could not be resolved into components of Hb4 and (HbO2)4 without generating a residual spectrum, the amplitude of which was greatest at F from 0.4 to 0.5 and least at values of F of 0 and 1. An equation of state for dioxygen binding by the Hb4-IHP complex was formulated and tested by its ability to predict (i) the equilibrium binding curve and (ii) the variation in amplitude of the residual spectrum with F. The equilibrium binding data was fitted to the following equation of state: (Formula: see text) where K1 is the equilibrium constant for binding of dioxygen to an alpha chain of the Hb4-IHP complex, K2 is the constant for the second alpha chain, K3 is the equilibrium constant for the large-scale conformational change, K4 is the equilibrium constant for binding of oxygen by both beta chains, and (L) is the ligand concentration. The best-fitting values were as follows: K1, 0.03497 mm Hg-1; K2, 0.01368 mm Hg-1; K3, 2.44; K4, 0.0008867 mm Hg-2. The residual spectra were attributed to differential loading of dioxygen by the alpha and beta chains. Equations of state for F of each chain are presented, and the amplitude of the residual spectra was shown to be accurately predicted by the differences in F of each chain when subjected to the constraint that the best-fitting values of K1-K4 be used in predicting saturation of each chain with dioxygen.