Expression of soluble human beta-globin chains in bacteria and assembly in vitro with alpha-globin chains.
Authentic soluble human beta-globin chains were produced in Escherichia coli using an expression plasmid (pHE2beta) containing full-length cDNAs coding for human beta-globin chain and methionine aminopeptidase. Spectral properties of the purified beta-globin were identical to those of authentic beta-globin. Soluble beta-globin showed low (16 kDa) and high molecular mass (32 kDa) forms that could be separated by gel filtration chromatography. SDS-polyacrylamide gel electrophoresis and electrospray mass spectrometry revealed the 32-kDa species was dimeric beta-globin formed by an intermolecular disulfide bond, while the 16-kDa species was authentic monomeric beta-globin. Monomeric forms of beta-globin, like authentic native beta-globin, formed tetrameric hemoglobin (Hb) A (alpha2beta2) in vitro upon incubation with alpha-globin, while dimeric forms did not. When beta-globin dimers, however, were converted to monomers by incubation with dithiothreitol, the beta-globin chain monomers assembled with alpha-globin and formed hemoglobin tetramers. alpha-Globin was more thermally unstable than beta-globin, while assembled tetramers promoted higher stability. Disulfide-bonded beta-globin dimers showed a slight increase in thermal stability compared with beta-globin; however, dimers were still more unstable than tetrameric Hb A. These results indicate that presence of alpha chains favors assembly with beta-globin, beta-beta dimers cannot bind alpha chains, and that Hb A tetramer formation results in the most thermally stable species.
The Children's Hospital of Philadelphia, Division of Hematology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
T
T
Yamaguchi
J
J
Pang
K S
KS
Reddy
H E
HE
Witkowska
S
S
Surrey
K
K
Adachi
D001704:Biopolymers; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005914:Globins / Q000737:chemistry / Q000235:genetics*; D006358:Hot Temperature; D006801:Humans; D007536:Isomerism; D010449:Peptide Mapping; D014357:Trypsin
D001704:Biopolymers; D005914:Globins; D014357:Trypsin