Interspecies hybrids of HbA and Hb from mouse C57BL/10 [alpha 2M beta 2H and alpha 2H beta 2M (H = human, M = mouse)], representing 19 and 27 sequence differences per alpha beta dimers (as compared with human alpha beta dimer) have been generated in vitro. The efficiency of the assembly of the interspecies hybrids by the alloplex intermediate pathway is about twofold higher than the low-pH-mediated subunit approach. The interspecies hybrids exhibit a cooperative O2 binding. The intrinsic O2 affinity of mouse Hb is slightly lower than HbA, while the 2,3-diphosphoglycerate (DPG) effect is comparable. Interestingly, the interspecies hybrid alpha 2M beta 2H has high O2 affinity (compared to either human or mouse Hb), while the interspecies hybrid alpha 2H beta 2M exhibits a very low O2 affinity. These results suggest that the mouse beta chain generates a tetramer with very low oxygen affinity. However, the complementarity of the mouse alpha and beta chains generates a set of unique interactions that compensate for the low-oxygen-affinity propensity of the mouse beta chain. DPG binds the tetramer in the central cavity formed by the two beta subunits, hence the DPG effects on the interspecies hybrids should be as in the parent molecule. However, the results of the present study demonstrate that the DPG binding pocket is influenced by the nature of the alpha chain present in the tetramer. The mouse alpha chain reduces considerably the DPG right shift of the O2 affinity of the human beta-chain containing hybrid.(ABSTRACT TRUNCATED AT 250 WORDS)