Assembly of human hemoglobin (Hb) β- and γ-globin chains expressed in a cell-free system with α-globin chains to form Hb A and Hb F

Abstract

Rates of in vitro synthesis of radiolabeled γ and β chains made in a cell-free transcription/translation system were similar, but expressed globin chains were unstable. The addition of unlabeled β or γ chains at the start of chain synthesis generated radiolabeled β4 or γ2 and γ4 chains, respectively. If unlabeled α-globin chains were added at the start of chain synthesis, then approximately equal amounts of radiolabeled αβ or αγ bands were generated. If unlabeled Hb A or Hb F was added to reactions containing radiolabeled αβ or αγ prior to electrophoresis, then radiolabeled Hb A or Hb F tetramers, respectively, were generated. If α chains were added after synthesis of radiolabeled γ chains made in the presence of unlabeled γ chains, then little radiolabeled αγ formed. In contrast, if α chains were added after synthesis of radiolabeled β chains made in the presence of unlabeled β chains, then radiolabeled α2β2 formed. These findings suggest that β and γ chains associate with α chains during or soon after translation. This would prevent the formation of unstable monomers as well as stable γ2 dimers and suggests that α chains may bind to nascent non-α chains, acting as folding catalysts to promote functional tetrameric hemoglobin formation in vivo.