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Two mutations in recombinant Hb β F41(C7)Y, K82 (EF6)D show additive effects in decreasing oxygen affinityxs
Author(s) -
Dumoulin A.,
Kiger L.,
Griffon N.,
Vasseur C.,
Kister J.,
Génin P.,
Marden M.C.,
Pagnier J.,
Poyart C.
Publication year - 1996
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560050114
Subject(s) - autoxidation , oxygen , mutant , chemistry , hemoglobin , p50 , biophysics , stereochemistry , allosteric regulation , recombinant dna , binding site , biochemistry , biology , receptor , organic chemistry , gene , transcription factor
Based on the properties of two low oxygen affinity mutated hemoglobins (Hb), we have engineered a double mutant Hb (rHb βYD) in which the βF41Y substitution is associated with K82D. Functional studies have shown that the Hb α 2 /β 2 (C7)F41Y exhibits a decreased oxygen affinity relative to Hb A, without a significantly increased auto‐oxidation rate. The oxygen affinity of the natural mutant βK82D (Hb Providence‐Asp) is decreased due to the replacement of two positive charges by two negative ones at the main DPG‐binding site. The functional properties of both single mutants are interesting in the view of obtaining an Hb‐based blood substitute, which requires: (1) cooperative oxygen binding with an overall affinity near 30 mm Hg at half saturation, at 37 °C, and in the absence of 2,3 diphosphoglycerate (DPG), and (2) a slow rate of autooxidation in order to limit metHb formation. It was expected that the two mutations were at a sufficient distance (20 Å) that their respective effects could combine to form low oxygen affinity tetramers. The double mutant does display additive effects resulting in a fourfold decrease in oxygen affinity; it can insure, in the absence of DPG, an oxygen delivery to the tissues similar to that of a red cell suspension in vivo at 37 °C. Nevertheless, the rate of autooxidation, 3.5‐fold larger than that of Hb A, remains a problem.