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

Abstract

Deuterium-exchange reaction were performed on human Hb in its carbon monoxy and deoxy forms at various pH values and 36.5.degree. C. Peptides containing only 1 histidine residue were separated from tryptic and chymotryptic digests of the deuterated Hb, except for 2 peptides which contained the .alpha.-87 and .alpha.-89 and the .beta.-116 and .beta.-117 histidine residues, respectively. The pseudo-1st-order rate constant for the exchange reaction of each histidine residue was measured using the mass spectrometric method. 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-1st-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 Hb molecule. No change was detected in the pKa values on ligation for the other histidine residues in deoxyhemoglobin.