Title: Shifts in affinity and enthalpy of oxygen binding to human hemoglobin A induced by pyridoxal and pyridoxal 5′-phosphate
Abstract: A therapeutic agent capable of inhibiting the polymerization of hemoglobin S would relieve the symptoms of sickle cell disease. The P50 of oxygen binding to hemoglobin A (HbA) in the presence of an effector has been used for the initial screening of potential antisickling agents. We evaluated the thermodynamic parameters, P50, ΔG, ΔH, and ΔS and Hill coefficient for oxygen binding to human HbAo (HbA stripped of all endogenous effectors) in the presence of two antisickling agents, pyridoxal (PL) and pyridoxal 5′-phosphate (PLP), and two sickling-enhancing agents, 2,3-diphosphoglycerate (DPG) and inositol hexaphosphate (IHP). The P50 of oxygen binding to HbAo in phosphate/EDTA buffer, pH 7.6, was found to be 560 Pa. The P50 was increased to 653 Pa and 1746 Pa by DPG and IHP, respectively. The antisickling agents, PLP, and PL, had opposite effects on the P50, shifting it to 840 Pa and 187 Pa, respectively, indicating that a decrease in P50 may not be the best predictor of an antisickling agent. The Hill coefficient was 2.9 for both HbAo and HbAo + DPG and 2.6 for HbAo + IHP. The Hill coefficient was reduced by both PLP to 2.3 and by PL to 2.1. The enthalpies for oxygen binding to HbAo, HbAo + DPG, and HbAo + IHP were −48.1 kcal/mol Hb, −48.0 kcal/mol Hb, and −42.2 kcal/mol Hb, respectively. In contrast, the enthalpies for HbAo + PLP and HbAo + PL were −57.0 kcal/mol Hb and −51.1 kcal/mol Hb. Our study suggests that decreases in cooperativity and enthalpy, alone or in combination, may be useful indices of potential antisickling agents.