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(Pre sbyterian) and Hb(Yoshizuka), were produced in transgenic pigs and pur ified to homogeneity by ion-exchange chromatography. These two variant s contain either lysine (Hb(Presbyterian)) or aspartic acid (Hb(Yoshiz uka)) instead of the normal asparagine residue at position beta 108 in HbA. Transgenic pigs expressed these variants at a level up to 11% an d were healthy. Both Hb(Presbyterian) and Hb(Yoshizuka) exhibited low O-2 affinity (P-50 of 21.2 and 18.9, respectively, compared with contr ol HbA value of 11.8 in 0.1 M NaCl, pH 7.5) and retained normal cooper ativity with Hill coefficients of 2.9 and 2.5, respectively. Hb(Presby terian) exhibited Bohr effect comparable with HbA. In contrast, Hb(Yos hizuka) had a diminished response to changes in pH. Thus the structura l basis of reduced O-2 affinity of these variants appears to be distin ct: the consequence of mutation at beta 108 is a function of the chemi cal nature of the side chain. This is further confirmed by the sensiti vity 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- concentr ation, whereas the O-2 affinity of Hb(Presbyterian) exhibited a pronou nced and dramatic chloride effect. In fact, P-50 of Hb(Presbyterian) w as 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 a bolished when Hb(Presbyterian) was stabilized at the 2,3-diphosphoglyc erate pocket by interdimeric cross-linking. Molecular modeling studies demonstrate that in Hb(Presbyterian), Cl- can bridge the epsilon-amin o group of Lys(beta 108) with either the guanidino group of Arg(beta 1 04) or the epsilon-amino group of Lys(alpha 99), resulting in the stab ilization of the ''T'' structure. The utility of these low O-2 affinit y hemoglobins as cell-free oxygen carriers is discussed.