INFLUENCE OF THE CHEMICAL NATURE OF SIDE-CHAIN AT BETA-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 beta 108 in HbA. Transgenic pigs expressed these variants at a level up to 11% and were healthy. Both Hb(Presbyterian) and Hb(Yoshizuka) exhibited low O-2 affinity (P-50 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 O-2 affinity of these variants appears to be distinct: the consequence of mutation at beta 108 is a function of the chemical nature of the side chain. This is further confirmed by the sensitivity of the O-2 affinity of the variants to the presence of Cl-. The O-2 affinity of Hb(Yoshizuka) is insensitive to changes in Cl- concentration, whereas the O-2 affinity of Hb(Presbyterian) exhibited a pronounced and dramatic chloride effect. In fact, P-50 of Hb(Presbyterian) was identical to that of HbA at very low Cl- concentrations, and the P-50 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 epsilon-amino group of Lys(beta 108) with either the guanidino group of Arg(beta 104) or the epsilon-amino group of Lys(alpha 99), resulting in the stabilization of the ''T'' structure. The utility of these low O-2 affinity hemoglobins as cell-free oxygen carriers is discussed.