SYMMETRICAL INTERSPECIES HYBRIDS OF MOUSE AND HUMAN HEMOGLOBIN - MOLECULAR-BASIS OF THEIR ABNORMAL OXYGEN-AFFINITY

Interspecies hybrids of HbA and Hb from mouse C57BL/10 [alpha(2)(M) be ta(2)(H) and alpha(2)(H) beta(2)(M) (H = human, M=mouse)], representin g 19 and 27 sequence differences per alpha beta dimers (as compared wi th human alpha beta dimer) have been generated in vitro. The efficienc y of the assembly of the interspecies hybrids by the alloplex intermed iate pathway is about twofold higher than the low-pH-mediated subunit approach. The interspecies hybrids exhibit a cooperative O-2 binding. The intrinsic O-2 affinity of mouse Hb is slightly lower than HbA, whi le the 2,3-diphosphoglycerate (DPG) effect is comparable. Interestingl y, the interspecies hybrid alpha(2)(M) beta(2)(H) has high O-2 affinit y (compared to either human or mouse Hb), while a the interspecies hyb rid alpha(2)(H) beta(2)(M) exhibits a very low O-2 affinity. These res ults suggest that the mouse beta chain generates a tetramer with very low oxygen affinity. However, the complementarity of the mouse alpha a nd beta chains generates a set of unique interactions that compensate for the low-oxygen-affinity propensity of the mouse beta chain. DPG bi nds the tetramer in the central cavity formed by the two beta subunits , hence the DPG effects on the interspecies hybrids should be as in th e parent molecule. However, the results of the present study demonstra te that the DPG binding pocket is influenced by the nature of the cu c hain present in the tetramer, The mouse alpha chain reduces considerab ly the DPG right shift of the O-2 affinity of the human beta-chain con taining hybrid. Sequence analysis suggest that perturbations of the al pha(1) beta(1) (not the alpha(1) beta(2)) are communicated to the DPG binding pocket in the presence of the alien subunit, and are the prima ry determinant of the ligand binding properties. The results have impl ications for the design of Hb-based blood substitutes and understandin g of the inhibitory potential of mouse alpha chains in transgenic mous e expressing human beta(S) chains.