Effects of Increased Anionic Charge in the 
-Globin Chain on
Assembly of Hemoglobin In Vitro
Kazuhiko Adachi,
Takamasa Yamaguchi,
Jian Pang, and
Saul Surrey
From the Division of Hematology, The Children's Hospital of
Philadelphia, and Department of Pediatrics, University of Pennsylvania
School of Medicine, Philadelphia, PA; the Departments of Research and
Pediatrics, duPont Hospital for Children, Wilmington, DE; and the
Department of Pediatrics, Jefferson Medical College, Philadelphia, PA.
Studies on assembly in vitro of 
-globin chains with recombinant
16 Gly
Asp, 95 LysGlu, 120 LysGlu
and 16 GlyAsp, 120 LysGlu human -globin chain
variants in addition to human A- and
S-globin chains were performed to evaluate effects of
increased anionic charge in the chain on hemoglobin assembly using
soluble recombinant -globin chains expressed in bacteria. A 112
CysAsp change was also engineered to monitor effects on
assembly of increased negative charge at 11 interaction sites.
Order of tetramer formation in vitro under limiting -globin chain
conditions showed Hb G16D, K120E = Hb K120E = Hb K95E > Hb G16D > Hb A > Hb S >>> Hb C112D. In addition, 112
CysAsp chains exist as monomers rather than 4
tetramers in the absence of chains, and the chain in Hb C112D tetramers was readily exchanged by addition of
s. These results suggest that affinity between and
chains is promoted by negatively-charged chains up to a maximum
of two additional net negative charges and is independent of location on the surface except at the 11 interaction site. In addition, our findings show that 112 Cys on the G helix is critical for facilitating formation of stable dimers, which then form
functional hemoglobin tetramers, and that 112 CysAsp
inhibits formation of stable 11 and 12 interactions in
22 and 4 tetramers, respectively.
Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1438-1445
© 1998 by The American Society of Hematology.
