Introduction of a new regulatory mechanism into human hemoglobin

Previous studies on bovine hemoglobin (HbBv) have suggested amino acid substitutions, which might introduce into human hemoglobin (HbA) functional characteristics of HbBv, namely a low intrinsic oxygen affinity regulated by Cl^-. Accordingly, we have constructed and characterized a multiple mutant, PB5, [β(V1M+H2Δ+T4I+P5A+A76K)] replacing four amino acid residues of HbA with those present at structurally analogous positions in HbBv, plus an additional substitution, βT4I, which does not occur in either HbBv or HbA. This 'pseudobovine' hemoglobin has oxygen binding properties very similar to those of HbBv: the P₅₀ of HbA, PB5 and HbBv in the absence of Cl^- are 1.6, 4.6 and 4.8 torr, respectively, and in 100 mM Cl^- are 3.7, 10.5 and 12 torr, respectively. Moreover, PB5 has 3-fold slower autoxidation rate compared to HbA and HbBv. These are desirable characteristics for a human hemoglobin to be considered for use as a clinical artificial oxygen carrier. Although the functional properties of PB5 and HbBv are similar, van't Hoff plots indicate that the two hemoglobins interact differently with water, suggesting that factors regulating the R to T equilibrium are not the same in the two proteins. A further indication that PB5 is not a functional mimic of HbBv derives from PB5_control, a human hemoglobin with the same substitutions as PB5, except the βT4I replacement. PB5_control has a high oxygen affinity (P₅₀=2.3 torr) in the absence of Cl^-, but retains the Cl^- effect of PB5. The Cl^- regulation of oxygen affinity in PB5 involves lysine residues at β8 and β76. PB4, which has the same substitutions as PB5 except βA76K, and PB6, which has all the substitutions of PB5 plus βK8Q, both have a low intrinsic oxygen affinity, like HbBv and PB5, but exhibit a decreased sensitivity to Cl^-. Since HbBv has lysine residues at both β8 and β76, these results imply that Cl^- regulation in HbBv likewise involves these two residues. The mechanism responsible for the low intrinsic oxygen affinity of HbBv remains unclear. It is suggested that residues peculiar to HbBv at the α₁β₁ interface may play a role.