Engineering of low oxygen affinity hemoglobins

Our objective is to prepare variant human Hb's whose oxygen affinity is regulated by normal components of the blood plasma rather than 2,3-DPG. The design of some of these mutants is based on the functional characteristics of bovine Hb whose oxygen affinity is modulated by Cl^- opposed to 2,3-DPG. A model has been proposed which correlates the regulation of oxygen affinity by Cl^- with the presence of particular amino acid residues at a particular position in the B-chains of bovine Hb. Creation of a Cl^- binding site in T state Hb is postulated to result in part from the N-terminal end of the β-subunit being positioned closer to the center of the molecule compared to normal human Hb. We have expressed and purified the mutant βNA1Val→met, βNA2His→deleted, which has an N-terminal end identical to that of bovine Hb. Oxygen equilibrium measurements performed in the absence and in the presence of a varying amount of Cl^- showed a constant decrease in oxygen affinity with respect to human Hb. However, in no case was the oxygen affinity as low as that of bovine Hb under the same conditions. The results demonstrate that these substitutions alone are insufficient to increase the sensitivity of Hb to Cl^-. However, the decreased oxygen affinity suggests an increased stabilization of the T state, probably resulting from a displacement of the N-terminal end toward the interior of the molecule.