Assembly of γ- with α-globin chains to form human fetal hemoglobin in vitro and in vivo

Abstract

Soluble γ-globin chains were expressed in bacteria and purified to assess the mechanism of γ- and α-chain assembly to form Hb F. Formation of Hb F in vitro following incubation of equimolar mixtures of γ and chains was about 4 x 105-fold slower than assembly of and β chains to form Hb A in vitro. Results of assembly for γ(116Ile→His) and γ(112Thr→Asp) chains with α chains were similar to that of β chains, whereas assembly of γ(112Thr→Cys) and α chains was similar to wild type γ chains, indicating that amino acid differences at α1β1 and α1γ1 interaction sites between γ116 Ile and β116 His are responsible for the different assembly rates in vitro in the formation of Hb F and Hb A. Homoassembly in vitro of individual γ chains as assessed by size-exclusion chromatography shows that γ and γ(112Thr→Cys) chains form stable dimers like αβ and αγ that do not dissociate readily into monomers like β chains. In contrast, γ(116Ile→His chains form monomers and dimers upon dilution. These results are consistent with the slower assembly rate in vitro of γ and γ(112Thr→Cys) with α chains, whereas the faster rate of assembly of γ(116Ile→His) and γ(112Thr→Asp) chains with α chains, like β chains, may be caused by dissociation to monomers. These results suggest that dissociation of γ2 dimers to monomers limits formation of lib F in vitro. However, yields of soluble Hb F expressed in bacteria were similar to Hb A, and no unassembled α and γ chains were detected. These results indicate that γ chains assemble in vivo with chains prior to forming stable γ2 dimers, possibly binding to α chains as partially folded nascent γ-globin chains prior to release from polyribosomes.