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Title
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Functional properties of human hemoglobins synthesized from recombinant mutant beta-globins.
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Authors
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M.L.Doyle,
G.Lew,
A.De Young,
L.Kwiatkowski,
A.Wierzba,
R.W.Noble,
G.K.Ackers.
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Ref.
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Biochemistry, 1992,
31,
8629-8639.
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PubMed id
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Abstract
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The previous and following articles in this issue describe the recombinant
synthesis of three mutant beta-globins (beta 1 Val----Ala, beta 1 Val----Met,
and the addition mutation beta 1 + Met), their assembly with heme and natural
alpha chains into alpha 2 beta 2 tetramers, and their X-ray crystallographic
structures. Here we have measured the equilibrium and kinetic allosteric
properties of these hemoglobins. Our objective has been to evaluate their
utility as surrogates of normal hemoglobin from which further mutants can be
made for structure-function studies. The thermodynamic linkages between
cooperative oxygenation and dimer-tetramer assembly were determined from global
regression analysis of multiple oxygenation isotherms measured over a range of
hemoglobin concentration. Oxygen binding to the tetramers was found to be highly
cooperative (maximum Hill slopes from 3.1 to 3.2), and similar patterns of
O2-linked subunit assembly free energies indicated a common mode of cooperative
switching at the alpha 1 beta 2 interface. The dimers were found to exhibit the
same noncooperative O2 equilibrium binding properties as normal hemoglobin. The
most obvious difference in oxygen equilibria between the mutant recombinant and
normal hemoglobins was a slightly lowered O2 affinity. The kinetics of CO
binding and O2 dissociation were measured by stopped-flow and flash photolysis
techniques. Parallel studies were carried out with the mutant and normal
hemoglobins in the presence and absence of organic phosphates to assess their
allosteric response to phosphates. In the absence of organic phosphates, the
CO-binding and O2 dissociation kinetic properties of the mutant dimers and
tetramers were found to be nearly identical to those of normal hemoglobin.
However, the effects of organic phosphates on CO-binding kinetic properties of
the mutants were not uniform: the beta 1 + Met mutant was found to deviate
somewhat from normalcy, while the beta 1 Val----Met mutant reproduced the native
allosteric response. Further characterization of the allosteric properties of
the beta 1 Val----Met mutant was made by measuring the pH dependence of its
overall oxygen affinity by tonometry. Regulation of oxygen affinity by protons
was found to be nearly identical to normal hemoglobin from pH 5.8 to 9.3 (0.52
+/- 0.07 protons released per oxygen bound at pH 7.4). The present study
demonstrates that the equilibrium and kinetic functional properties of the
recombinant beta 1 Val----Met mutant mimic reasonably well those of normal
hemoglobin. We conclude that this mutant is well-suited to serve as a surrogate
system of normal hemoglobin in the production of mutants for structure-function
studies.
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