Influence of the chemical nature of side chain at β 108 of hemoglobin a on the modulation of the oxygen affinity by chloride ions: Low oxygen affinity variants of human hemoglobin expressed in transgenic pigs: Hemoglobins presbyterian and yoshizuka

Hemoglobin A (HbA) and two low oxygen affinity variants of HbA, Hb^Presbyterian and Hb^Yoshizuka, were produced in transgenic pigs and purified to homogeneity by ion-exchange chromatography. These two variants contain either lysine (Hb^Presbyterian) or aspartic acid (Hb^Yoshizuka) instead of the normal asparagine residue at position β108 in HbA. Transgenic pigs expressed these variants at a level up to 11% and were healthy. Both Hb^Presbyterian and Hb^Yoshizuka exhibited low O₂ affinity (P⁵⁰ of 21.2 and 18.9, respectively, compared with control HbA value of 11.8 in 0.1 M NaCl, pH 7.5) and retained normal cooperativity with Hill coefficients of 2.9 and 2.5, respectively. Hb^Presbyterian exhibited Bohr effect comparable with HbA. In contrast, Hb^Yoshizuka had a diminished response to changes in pH. Thus the structural basis of reduced O₂ affinity of these variants appears to be distinct: the consequence of mutation at β108 is a function of the chemical nature of the side chain. This is further confirmed by the sensitivity of the O₂ affinity of the variants to the presence of Cl^-. The O₂ affinity of Hb^Yoshizuka is insensitive to changes in Cl^- concentration, whereas the O₂ affinity of Hb^Presbyterian exhibited a pronounced and dramatic chloride effect. In fact, P₅₀ of Hb^Presbyterian was identical to that of HbA at very low Cl^- concentrations, and the P₅₀ increased to >40 at 0.5 M Cl^-. The chloride effect was completely abolished when Hb^Presbyterian was stabilized at the 2,3-diphosphoglycerate pocket by interdimeric cross-linking. Molecular modeling studies demonstrate that in Hb^Presbyterian, Cl^- can bridge the ε-amino group of Lys^β108 with either the guanidino group of Arg^β104 or the ε-amino group of Lys^α99, resulting in the stabilization of the "T" structure. The utility of these low O₂ affinity hemoglobins as cell-free oxygen carriers is discussed.