Reactivity differences between haemoglobins. Part VII. The effect of ionic strength on the affinity of methaemoglobins A and C for azide ion. A test of the dielectric cavity model for methaemoglobins

Abstract

The variation with ionic strength of the formation constants of the azide complexes of methaemoglobins A and C has been determined as a function of pH. An ionic strength extrapolation function is derived from a dielectric cavity model for a protein, and using this function the net charges on the methaemoglobins have been calculated as a function of pH. These charges are compared with those calculated from the titration curves of the two methaemoglobins which are also reported here. The charges obtained by the two methods are the same within experimental error except over a narrow pH range, if the radius of the cavity in the solvent representing the protein is assumed to be that obtained from crystallographic studies plus the diameter of two water molecules. The observed differences between the standard free energies of formation at zero ionic strength of the complex of the two methaemoglobins, and the variation of ΔG° with pH for each complex, are compared with the values calculated from the dielectric cavity model. Good agreement is obtained except over a small pH range in the region of the isoelectric point; the same pH region in which the values of the net charges calculated from the dielectric cavity model differ from those calculated from the titration curves. The disagreement is explained in terms of a postulated structural perturbation of the protein. The effect of temperature on the titration curves of the two proteins is discussed and, in agreement with earlier workers, it is found to be consistent with a dielectric cavity model for the protein. The differences between the net charges on methaemoglobins A and C calculated from the titration curves are discussed in relation to the known difference in amino-acid composition.