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Abstract
As part of an effort to understand the interactions in HbS polymerization, we have produced and studied a recombinant triple mutant, D6A(alpha)/D75Y(alpha)/E121R(beta), and a quadruple mutant comprising the preceding mutation plus the natural genetic mutation of sickle hemoglobin, E6V(beta). These recombinant hemoglobins expressed in yeast were extensively characterized, and their structure and oxygen binding cooperativity were found to be normal. Their tetramer-dimer dissociation constants were within a factor of 2 of HbA and HbS. Polymerization of these mutants mixed with HbS was investigated by a micromethod based on volume exclusion by dextran. The elevated solubility of mixtures of HbS with HbA and HbF in dextran could be accurately predicted without any variable parameters. Relative to HbS, the copolymerization probability of the quadruple mutant/HbS hybrid was found to be 6.2, and the copolymerization probability for the triple mutant/HbS hybrid was 0.52. The pure quadruple mutant had a solubility slightly above that of its hybrid with HbS. One way to explain these results is to require significant cis-trans differences in the polymer and that HbA assemble above 42.5 g/dl. A second way to explain these data is by the modification of motional freedom, thereby changing vibrational entropy in the polymer.
Highlights
MethodsSDS-PAGE of the recombinant mutants was performed on the Phast System from Amersham Biosciences
Glu is close to the positive charge of Lys-17 in both cis and trans position
The replacement Arg or Lys 121 makes an unfavorable contact with these Lys amino acids. 121 is close to 116 and 117 in the trans position from which repulsion would be expected between His (116 and 117) and Lys or Arg
Summary
SDS-PAGE of the recombinant mutants was performed on the Phast System from Amersham Biosciences. The protein bands were stained with Coomassie Brilliant Blue R-250. Isoelectric focusing was performed on the pH 7–10 Hb-Resolve system from Isolab. The ␣- and -globin chains from recombinant hemoglobins were separated by reverse phase HPLC on a Vydac C4 column with a gradient of 20 – 60% acetonitrile containing 0.1% trifluoroacetic acid. Amino acid analysis of globin chains isolated by this procedure was performed on a Beckman 6300 instrument with a System Gold data handling system. The spectrum of each mutant was recorded on a Shimadzu 1601 UV-visible spectrophotometer
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