The pH Dependence of the Binding of d ‐Glycerate 2,3‐Bisphosphate to Deoxyhemoglobin and Oxyhemoglobin

The number of Bohr protons released upon oxygenation has been measured over a large range of human hemoglobin concentrations (0.02 to 4.5 mM) in the presence of equimolar amounts of d ‐glycerate 2,3‐bisphosphate. From these data the association constants for the binding of this organic phosphate to deoxyhemoglobin and oxyhemoglobin were calculated at different pH values. The maximum number of protons absorbed upon binding to oxyhemoglobin was determined as well. The maximum number of protons bound to deoxyhemoglobin upon binding of d ‐glycerate 2,3‐bisphosphate was measured independently. From the pH dependence of the association constants and the maximum number of protons absorbed it could be concluded that only one d ‐glycerate 2,3‐bisphosphate can be bound to both deoxyhemoglobin and oxyhemoglobin.