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.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - Jan 1 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology