Changes in pKa values of individual histidine residues of human hemoglobin upon reaction with carbon monoxide.

We performed the deuterium-exchange reaction on human hemoglobin in its carbon monoixy and deoxy forms at various pH values and 36.5 degrees C. Peptides containing only one histidine residue were separated from tryptic and chymotryptic digests of the deuterated hemoglobin, except for two peptides which contained the alpha-87 and alpha-89 and the beta-116 and beta-117 histidine residues, respectively. The pseudo-first-order rate constant for the exchange reaction of each histidin residue was measured by using the mass spectrometric method. We obtained the following results. The pKa values for the alpha-20, alpha-89, and beta-146 histidine residues in deoxyhemoglobin decreased significantly, while that for the beta-143 histidine residue increased significantly on ligation. The pseudo-first-order rate constants were virtually zero for the alpha-45, alpha-58, alpha-87, beta-63, and beta-92 histidine residues which are linked with a heme group, and also for the alpha-122 histidine residue which is buried at the alpha 1 beta 1 contact in the hemoglobin molecule. No change was detected in the pKa values on ligation for the other histidine residues in deoxyhemoglobin.