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J Biol Chem, Vol. 275, Issue 17, 12424-12429, April 28, 2000

ACCELERATED PUBLICATION
Assembly of - with -Globin Chains to Form Human Fetal Hemoglobin in Vitro and in Vivo^*

Kazuhiko Adachi^§, Yi Zhao, Takamasa Yamaguchi, and Saul Surrey^¶

From  The Children's Hospital of Philadelphia, Division of Hematology and University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 and ^¶ Department of Pediatrics, Jefferson Medical College, Philadelphia, Pennsylvania 19104 and A. I. duPont Hospital for Children, Wilmington, Delaware 19803

Soluble -globin chains were expressed in bacteria and purified to assess the mechanism of - and -chain assembly to form Hb F. Formation of Hb F in vitro following incubation of equimolar mixtures of  and  chains was about 4 × 10⁵-fold slower than assembly of  and  chains to form Hb A in vitro. Results of assembly for ^116IleHis and ^112ThrAsp chains with  chains were similar to that of  chains, whereas assembly of ^112ThrCys and  chains was similar to wild type  chains, indicating that amino acid differences at 11 and 11 interaction sites between 116 Ile and 116 His are responsible for the different assembly rates in vitro in the formation of Hb F and Hb A. Homoassembly in vitro of individual  chains as assessed by size-exclusion chromatography shows that  and ^112ThrCys chains form stable dimers like and that do not dissociate readily into monomers like  chains. In contrast, ^116IleHis chains form monomers and dimers upon dilution. These results are consistent with the slower assembly rate in vitro of  and ^112ThrCys with  chains, whereas the faster rate of assembly of ^116IleHis and ^112ThrAsp chains with  chains, like  chains, may be caused by dissociation to monomers. These results suggest that dissociation of ₂ dimers to monomers limits formation of Hb F in vitro. However, yields of soluble Hb F expressed in bacteria were similar to Hb A, and no unassembled  and  chains were detected. These results indicate that  chains assemble in vivo with  chains prior to forming stable ₂ dimers, possibly binding to  chains as partially folded nascent -globin chains prior to release from polyribosomes.

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