Kinetic studies on the binding affinity of human hemoglobin for the 4th carbon monoxide molecule, L₄

L₄, the affinity of hemoglobin for the 4th CO molecule, has been determined for human adult hemoglobin (HbA) as a function of pH and the presence of organic phosphates by measuring the kinetic parameters for the reaction. l'₄, the rate of combination of CO with the triliganded molecule, was measured by flash photolysis while l₄, the rate of CO dissociation for the ligand saturated molecule, was measured by ligand replacement. L₄ is pH dependent and affected by 2,3 diphosphoglycerate. Additionally, this pH dependence of the high affinity state is largely eliminated by carboxypeptidase A digestion. L₄ for human fetal hemoglobin (HbF) in phosphate buffers was also determined and found to be pH dependent. These results cannot be reconciled within the framework of the two state allosteric model. Additional structures in the conformational equilibrium due to either intermediates in the T to R transition or two or more R states must exist.