Ligand Binding and Release of an Analogue of 2,3-Diphosphoglycerate from Human Hemoglobin

Abstract

Abstract The fluorescent substance 8-hydroxy-1,3,6-pyrenetrisulfonate (HPT) has been shown to combine with deoxyhemoglobin with a stoichiometry of 1 molecule of HPT per hemoglobin tetramer. It is displaced with the same stoichiometry by 2,3-diphosphoglycerate and by inositol hexaphosphate. When added to human hemoglobin solutions of low ionic strength it diminishes oxygen affinity. HPT has been used to study anion release associated with carbon monoxide binding. This occurs late in the reaction with hemoglobin A at pH 6, probably after 3 molecules of CO have been bound, and somewhat earlier at pH 7.5. Release is earlier with des-his(β146) hemoglobin, hemoglobins Hiroshima (β146 asp), Chesapeake (α92 leu), and hemoglobin A treated with p-mercuribenzoate. In static titrations with carbon monoxide there was good proportionality between ligand binding and anion release. With oxygen as ligand there was little or no lag between oxygen binding and anion release in kinetic experiments at low ligand concentrations. Detailed analysis suggests that more than two hemoglobin conformations are required to account for the results. The chief kinetic contribution to cooperativity in the CO binding reaction due to the combination velocity constants occurs after binding of 3 CO molecules.