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When azide ion reacts with methemoglobin in unbuffered solution the pH of the solution increases. This phenomenon is associated with increases in the pK values of heme-linked ionizable groups on the protein which give rise to an uptake of protons from solution.We have determined as a function of pH the proton uptake, Ah', on azide binding to methemoglobin at 20°C. Data for methemoglobins A (human), guinea pig and pigeon are fitted to a theoretical expression based on the electrostatic effect of three sets of heme-linked ionizable groups on the binding of the ligand. From these fits the pK values of heme-linked ionizable groups are obtained for liganded and unliganded methemoglobins. In unliganded methemoglobin pK1, which is associated with carboxylic acid groups, ranges between 4.0 and 5.5 for the three methemoglobins; pK,, which is associated with histidines and terminal amino groups, ranges from 6.2 to 6.7. In liganded methemoglobin pK1 lies between 5.8 and 6.3 and pK, varies from 8.1 to 8.5.The pH dependences of the apparent equilibrium constants for azide binding to the three methemoglobins at 20°C are well accounted for with the pK values calculated from the variation of Ah' with pH.The reaction of ligands with heme proteins is influenced to an appreciable extent by the state of ionization of groups on the protein [l -71. This effect is reciprocal, and pKchanges of ionizable groups on the protein may occur when ligands bind to the iron atoms of heme proteins [7-91. The number and nature of such heme-linked ionizable groups have been determined in some ferric heme proteins from the pH dependence of the kinetics of ligand binding [l -71.By studying the pH dependence of the apparent secondorder complex-formation rate constant and of the first-order complex-dissociation rate constant of turnip peroxidases P1 and P7, Job and Ricard [7] demonstrated that the pK values of heme-linked ionizable groups on these proteins increase on ligand binding. A method such as this for determining the pK values of heme-linked ionizable groups of liganded and unliganded heme proteins can only be applied to methemoglobin with difficulty. We have shown that such pK values can be determined for unliganded methemoglobin with cyanide ion as ligand 161. Unfortunately, however, the apparent dissociation rate constant of cyanide-methemoglobin complex is so small [6] that its determination is subject to a great deal of uncertainty. Thus the pK values of ionizable groups of liganded methemoglobin cannot be determined by this method with cyanide as the ligand. In contrast to the simple kinetic time course for cyanide binding [6], the complex kinetic time course for the binding of other ligands to methemoglobin precludes application of the method of Job and Ricard [7] to these ligands.It is known that the binding of azide ion to methemoglobin in unbuffered solution gives rise to an uptake of protons from solution [lo]. We have therefore determined the pH dependence of the proton uptake on azide binding to methemoglobin, in an attempt to mea...

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