title: Two mutations in recombinant Hb beta F41(C7)Y, K82 (EF6)D show additive effects in decreasing oxygen affinity.
abstract: Based on the properties of two low oxygen affinity mutated hemoglobins (Hb), we have engineered a double mutant Hb (rHb beta YD) in which the beta F41Y substitution is associated with K82D. Functional studies have shown that the Hb alpha 2 beta 2(C7)F41Y exhibits a decreased oxygen affinity relative to Hb A, without a significantly increased autooxidation rate. The oxygen affinity of the natural mutant beta K82D (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 degrees 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 A) 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 degrees C. Nevertheless, the rate of autooxidation, 3.5-fold larger than that of Hb A, remains a problem.
authors.affiliation: INSERM U 299, Hôpital de Bicêtre, France.
authors.forename: A
authors.initials: A
authors.lastname: Dumoulin
authors.forename: L
authors.initials: L
authors.lastname: Kiger
authors.forename: N
authors.initials: N
authors.lastname: Griffon
authors.forename: C
authors.initials: C
authors.lastname: Vasseur
authors.forename: I
authors.initials: I
authors.lastname: Kister
authors.forename: P
authors.initials: P
authors.lastname: Génin
authors.forename: M C
authors.initials: MC
authors.lastname: Marden
authors.forename: J
authors.initials: J
authors.lastname: Pagnier
authors.forename: C
authors.initials: C
authors.lastname: Poyart
mesh_terms: D006454:Hemoglobins / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D006454:Hemoglobins; D011994:Recombinant Proteins; D010100:Oxygen