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 alpha-globin chains bind to nascent non-alpha 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 alpha(h)beta(h) hetero-dimers. In contrast, in the absence of added CN-hemin, unlabeled heme-containing alpha or beta chains can assemble with newly synthesized radiolabeled beta- and alpha-globin chains to form heme-containing alpha(h)beta(h) and semi-alpha (alpha(h)beta(0)) or semi-beta (alpha(0)beta(h)) hetero-dimers, respectively. These results suggest the existence of semi-hemoglobins as intermediates prior to formation of heme-containing alpha(h)beta(h) and indicate transfer of heme from alpha and/or beta chains into semi-hemoglobin hetero-dimers to form heme-containing hetero-dimers.