Effects of Increased Anionic Charge in the β-Globin Chain on Assembly of Hemoglobin In Vitro

Abstract

AbstractStudies on assembly in vitro of α-globin chains with recombinant β16 Gly→Asp, β95 Lys→Glu, β120 Lys→Glu and β16 Gly→Asp, 120 Lys→Glu human β-globin chain variants in addition to human βA- and βS-globin chains were performed to evaluate effects of increased anionic charge in the β chain on hemoglobin assembly using soluble recombinant β-globin chains expressed in bacteria. A β112 Cys→Asp change was also engineered to monitor effects on assembly of increased negative charge at α1β1 interaction sites. Order of tetramer formation in vitro under limiting α-globin chain conditions showed Hb βG16D, K120E = Hb βK120E = Hb βK95E > Hb βG16D > Hb A > Hb S >>> Hb βC112D. In addition, β112 Cys→Asp chains exist as monomers rather than β4tetramers in the absence of α chains, and the β chain in Hb βC112D tetramers was readily exchanged by addition of βs. These results suggest that affinity between α and β chains is promoted by negatively-charged β chains up to a maximum of two additional net negative charges and is independent of location on the surface except at the α1β1 interaction site. In addition, our findings show that β112 Cys on the G helix is critical for facilitating formation of stable αβ dimers, which then form functional hemoglobin tetramers, and that β112 Cys→Asp inhibits formation of stable α1β1 and β1β2 interactions in α2β2 and β4 tetramers, respectively.