Titration behavior of histidines in human, horse, and bovine hemoglobins

The titration curves of the human hemoglobins A 2 and F, the slow and fast components of horse hemoglobin, and bovine hemoglobin B have been analyzed, mainly to obtain information about the titration behavior of the histidines in these hemoglobins. The results were also compared with earlier data concerning human hemoglobin A and bovine hemoglobin A. In both hemoglobin A 2 and F, 18 titratable histidines were found, two less than in hemoglobin A. Assuming that those histidyl residues which in the three-dimensional model occupy the same sites will show similar titration behavior, we reached the conclusion that in human hemoglobin histidine G19 and histidine H21 are titratable and that histidine G18 is not titratable. Both the slow and fast components of horse hemoglobin were found to contain 22 titratable histidines and 70 titratable carboxyl groups. Combining these results with the known number of amides in the α chain we calculated that the β chain should contain 11 amides. It appeared that in bovine hemoglobin B the histidines at B1(18)β, which are absent in bovine hemoglobin A, are titratable. The pK of these histidines, as estimated from the difference titration curve, is about 7.8, both in oxy- and deoxyhemoglobin. This high pK is probably caused by the formation of a saltbridge with the carboxyl group of aspartic acid B3(20)β in the same chain. Both hemoglobins contain 72 titratable carboxyl groups. Comparing the human, horse, and bovine hemoglobins, assuming again similar titration behavior for structurally identical histidines, we were able to correlate the titration results of human and horse hemoglobin. The human and bovine hemoglobins failed to show an equally good correlation, although the discrepancy was small.