Abstract
Recombinant alpha 2 beta 2 tetrameric Hb expressed and assembled in Escherichia coli has been characterized extensively. Electrospray mass spectrometry and optical and electron paramagnetic resonance spectroscopy suggest that the overexpressed protein is identical to native human Hb. Although the functional properties of this recombinant Hb are nearly identical to native Hb, crucial differences exist between the two molecules. The recombinant Hb expressed in E. coli has a lower Hill coefficient even though oxygen equilibrium binding studies indicate cooperative binding. The most significant difference observed between the recombinant and native Hb is the loss of oxygen affinity regulation by 2,3-diphosphoglyerate and protons. CO binding to the deoxy tetramer was found to be biphasic with both phases sensitive to the presence of allosteric effectors. The recombinant chains were isolated, and the ligand binding properties demonstrated that the recombinant chains behave in a similar fashion to native alpha-sh and beta-sh. To investigate whether the chains were capable of forming a well behaved tetramer, the isolated chains were reassembled into a tetramer and purified to homogeneity. Oxygen binding properties of the reassembled recombinant Hb now show an increased Hill coefficient of 2.5, close to, but still slightly lower than, that observed for native Hb. Additionally, reassembly of recombinant Hb produces a protein that is subject to regulation by allosteric effectors. Furthermore, CO binding to the reassembled recombinant deoxy tetramer was found to be monophasic under all conditions.
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