Contribution of arginine (HC3) 141 alpha to the Bohr effect of the fourth binding step in the reaction of ligand with human hemoglobin

A few years ago we reported that histidine (HC3) 146 beta plays a major role in the pH-dependent properties of the R-state of human hemoglobin, accounting for close to 50% of the R-state Bohr effect. We have extended these studies by examining the role of arginine 141 alpha, another group known to affect the overall Bohr effect. We have compared the pH dependencies of the rate constants for the dissociation and combination of the fourth carbon monoxide molecule, l4 and l'4, respectively, for native hemoglobin A (HbA) and a control reconstituted HbA, and des-(Arg 141 alpha) HbA, the hemoglobin molecule resulting from the enzymatic removal of the C-terminal arginine of the alpha-chain of human Hb. From these kinetic constants the pH dependence of L4, the affinity constant for the fourth carbon monoxide molecule, has been estimated. We find that the removal of arginine 141 alpha reduces the pH dependence of log L4 by about 80% between pH 6 and 8, where the alkaline Bohr effect normally occurs. The sum of the effects of the removal of His 146 beta and of Arg 141 alpha is greater than 100%. This suggests that at least one of these modifications alters the contributions of other residues of this Bohr effect.