Studies on assembly in vitro of a-globin chains with recombinant b16 Gly=Asp, b95 Lys=Glu, b120 Lys=Glu and b16 Gly=Asp, 120 Lys=Glu human b-globin chain variants in addition to human bAand bS-globin chains were performed to evaluate effects of increased anionic charge in the b chain on hemoglobin assembly using soluble recombinant bglobin chains expressed in bacteria. A b112 Cys=Asp change was also engineered to monitor effects on assembly of increased negative charge at a1b1 interaction sites. Order of tetramer formation in vitro under limiting a-globin chain conditions showed Hb bG16D, K120E 5 Hb bK120E 5 Hb bK95E G Hb bG16D G Hb A G Hb S GGG Hb bC112D. In addition, b112 Cys=Asp chains exist as monomers rather than b4 tetramers in the absence of a chains, and the b chain in Hb bC112D tetramers was readily exchanged by addition of bs. These results suggest that affinity between a and b chains is promoted by negatively-charged b chains up to a maximum of two additional net negative charges and is independent of location on the surface except at the a1b1 interaction site. In addition, our findings show that b112 Cys on the G helix is critical for facilitating formation of stable ab dimers, which then form functional hemoglobin tetramers, and that b112 Cys=Asp inhibits formation of stable a1b1 and b1b2 interactions in a2b2 and b4 tetramers, respectively. r 1998 by The American Society of Hematology.