Kinetic Studies of Ligand Binding to Hemoglobin and Its Isolated Subunits by the Temperature Jump Relaxation Method

Abstract

Abstract The temperature jump pulse relaxation method has been used to study the kinetics of oxygen binding to sperm whale myoglobin, to the α and β chains of human hemoglobin, and to native human and sheep hemoglobins. In addition we also investigated the kinetics of binding of ethyl isocyanide to human hemoglobin by the same method. The kinetics of the reaction has been followed through changes in absorbance. The results show that binding of oxygen to the isolated α and β subunits, as well as to sperm whale myoglobin, is kinetically a simple process, in that the reaction follows bimolecular kinetics. The relaxation kinetics of ligand binding to hemoglobin is complex, and the analysis given in this paper is still preliminary. Under a variety of experimental conditions, the relaxation spectrum can be described by two relaxation times (τf and τs). The concentration dependence of the slower relaxation time (τs) follows the behavior expected for a bimolecular process; this suggests that the final process by which equilibrium is re-established is not followed by any first order process, such as a slow intramolecular change in structure. The similarity of the relaxation spectrum at different ionic strengths (from 0.2 to 2.2 m) suggests that the relaxation behavior of hemoglobin is consistent with a model in which the (αβ) dimer is functionally important.