Interaction of haemoglobin with ions. Interactions among magnesium, adenosine 5'-triphosphate, 2,3-bisphosphoglycerate, and oxygenated and deoxygenated human haemoglobin under simulated intracellular conditions.

Abstract

Under simulated intracellular conditions (pH 7.2, 37 °C, 130 mM KCl, 20 mM NaCl and haemoglobin concentrations > 1.5 mM) the conditional association constants for the binding of ATP and 2,3‐bisphosphoglycerate (P2‐glycerate) to deoxygenated and oxygenated haemoglobin (Hb and HbO2, respectively) and the influence of Mg2+ on the binding of phosphocompounds were determined by means of a cation‐sensitive electrode and ultrafiltration.The K′ass for the binding of ATP to Hb is 2.6 mM−1 and 0.36 mM−1 for HbO2. P2‐glycerate is bound to Hb with K′ass of 5 mM−1 and to HbO2 with a K′ass of 0.25 mM−1. The anions compete with each other in binding. Mg2+ is not bound to haemoglobin but it reduces the binding of the phosphocompounds by complexing with them. It decreases the shift of the oxygen binding curve produced by ATP. MgATP is weakly bound to Hb with a K′ass of 0.14 mM−′ and to HbO2 with a K′ass of 0.039 mM−1. Binding of the Mg ·P2‐glycerate complex to haemoglobin was not established.The calculated values for the dependence of p50 (the partial pressure of oxygen at half‐saturation of haemoglobin) on the free concentration of P2‐glycerate, based on the K′ass of Hb and HbO2 complexes, agree well with the experimental data on erythrolysates and intact human red blood cells. The K′ass are furthermore an appropriate basis for the estimation of the intraerythrocatic state of Mg2+ and the metabolites.