Thermodynamic aspects of the linkage between binding of chloride and oxygen to human hemoglobin

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Abstract
O2 isotherms of human Hb measured in distilled water and in solutions of NaCl in the concentration range from 0.02-3.0 M indicate that the O2 affinity decreases up to about 1 M salt and then begins to increase. The isotherms obtained in the range from 0.02-0.6 M NaCl, at 37.degree. and pH 7.4, were analyzed in terms of changes in Gibbs free energy of heme ligation, resulting from the differential interaction between the chloride ion and the 2 forms of Hb. The maximal theoretical change in Gibbs free energy that Cl- can exert on the O2 binding of Hb amounts to 4.9 .+-. 0.2 kcal/mol (21 .+-. 0.8 kJ/mol) of Hb tetramer. A plot of the logarithm of O2 concentration at half saturation vs. the logarithm of the Cl- concentration has a slope of 0.40, suggesting 1.6 apparent chloride sites per Hb tetramer. Because the interaction between Cl- and Hb is dependent on pH, the apparent thermodynamic linkage between Cl- and O2 binding will also include the salt dependence of the Bohr effect at pH 7.4. The fractional change in Gibbs free energy, measured as a function of the Cl- concentration, can be approximated by the binding isotherm between a protein and a ligand, using an association constant of 11 M-1. If the number of O2-linked chloride sites is more than 1/Hb tetramer, these sites must be considered independent.