Effects of heme addition on formation of stable human globin chains and hemoglobin subunit assembly in a cell-free system

Our previous assembly studies to form hemoglobin hetero-dimers and -tetramers using a coupled transcription/translation cell-free system suggested that α-globin chains bind to nascent non-α chains during and/or soon after translation to promote hemoglobin formation [Adachi et al., J. Biol Chem. 2002 (277) 13415]. In this report effects of CN–hemin on subunit assembly were studied using this cell-free system. Addition of CN–hemin and excess unlabeled heme-containing partner chains during synthesis leads to formation of radiolabeled heme-containing αhβh hetero-dimers. In contrast, in the absence of added CN–hemin, unlabeled heme-containing α or β chains can assemble with newly synthesized radiolabeled β- and α-globin chains to form heme-containing αhβh and semi-α (αhβ0) or semi-β (α0βh) hetero-dimers, respectively. These results suggest the existence of semi-hemoglobins as intermediates prior to formation of heme-containing αhβh and indicate transfer of heme from α and/or β chains into semi-hemoglobin hetero-dimers to form heme-containing hetero-dimers.