Coexpression of human alpha- and circularly permuted beta-globins yields a hemoglobin with normal R state but modified T state properties.
For the first time, a circularly permuted human beta-globin (cpbeta) has been coexpressed with human alpha-globin in bacterial cells and shown to associate to form alpha-cpbeta hemoglobin in solution. Flash photolysis studies of alpha-cpbeta show markedly biphasic CO and O(2) kinetics with the amplitudes for the fast association phases being dominant due the presence of large amounts of high-affinity liganded hemoglobin dimers. Extensive dimerization of liganded but not deoxygenated alpha-cpbeta was observed by gel chromatography. The rate constants for O(2) and CO binding to the R state forms of alpha-cpbeta are almost identical to those of native HbA (k'(R(CO)) approximately 5.0 microM(-1) s(-1); k'(R(O(2))) approximately 50 microM(-1) s(-1)), and the rate of O(2) dissociation from fully oxygenated alpha-cpbeta is also very similar to that observed for HbA (k(R(O(2))) approximately 21-28 s(-1)). When the equilibrium deoxyHb form of alpha-cpbeta is reacted with CO in rapid mixing experiments, the observed time courses are monophasic and the observed bimolecular association rate constant is approximately 1.0 microM(-1) s(-1), which is intermediate between the R state rate measured in partial photolysis experiments (approximately 5 microM(-1) s(-1)) and that observed for T state deoxyHbA (k'(T(CO)) approximately 0.1 to 0.2 microM(-1) s(-1)). Thus the deoxygenated permutated beta subunits generate an intermediate, higher affinity, deoxyHb quaternary state. This conclusion is supported by equilibrium oxygen binding measurements in which alpha-cpbeta exhibits a P(50) of approximately 1.5 mmHg and a low n-value (approximately 1.3) at pH 7, 20 degrees C, compared to 8.5 mmHg and n approximately 2.8 for native HbA under identical, dilute conditions.
Asmundson
Anna L
AL
Department of Chemistry, Western Washington University, Bellingham, Washington 98225-9150, USA.
Taber
Alexandria M
AM
van der Walde
Adella
A
Lin
Danielle H
DH
Olson
John S
JS
Anthony-Cahill
Spencer J
SJ
D001665:Binding Sites; D002248:Carbon Monoxide / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D010100:Oxygen / Q000378:metabolism; D010782:Photolysis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D055542:alpha-Globins / Q000737:chemistry / Q000378:metabolism*; D055544:beta-Globins / Q000737:chemistry / Q000378:metabolism*
D006454:Hemoglobins; D008024:Ligands; D011994:Recombinant Proteins; D055542:alpha-Globins; D055544:beta-Globins; D002248:Carbon Monoxide; D010100:Oxygen