Oxygen binding properties of human mutant hemoglobins synthesized in Escherichia coli.
Human beta-globin was synthesized in Escherichia coli as a cleavable fusion protein, using the expression vector pLcIIFX beta-globin [Nagai, K. & Thøgersen, H. C. (1984) Nature (London) 309, 810-812]. The fusion protein cIIFX beta-globin was purified to homogeneity and cleaved at the junction by blood coagulation factor Xa; the authentic beta-globin was liberated. Beta-globin was folded in vitro and reconstituted with heme and alpha subunits to form alpha 2 beta 2 tetramers. The oxygen binding properties of reconstituted Hb are essentially the same as those of human native Hb. Two mutant Hbs (Hb Nymphéas [Cys-93 beta----Ser] and Hb Daphne [Cys-93 beta----Ser, His-143 beta----Arg]) were constructed by site-directed mutagenesis using synthetic oligonucleotides. Hb Nymphéas showed a slightly increased oxygen affinity and diminished cooperativity with normal 2,3-diphosphoglyceric acid and slightly reduced alkaline Bohr effects. Hb Daphne showed low cooperativity with high oxygen affinity. The alkaline Bohr effect was slightly reduced but the diphosphoglycerate effect was enhanced by 50% by the His-143 beta----Arg mutation. As arginine is fully charged at physiological pH and has a long flexible side chain, diphosphoglycerate binds more strongly to Hb Daphne.
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D005170:Factor X / Q000378:metabolism; D015951:Factor Xa; D005822:Genetic Vectors; D005914:Globins / Q000235:genetics / Q000378:metabolism; D006441:Hemoglobin A / Q000378:metabolism; D006454:Hemoglobins / Q000235:genetics / Q000378:metabolism*; D006455:Hemoglobins, Abnormal / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D010100:Oxygen / Q000378:metabolism*; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D006454:Hemoglobins; D006455:Hemoglobins, Abnormal; D011994:Recombinant Proteins; D005170:Factor X; D005914:Globins; D006441:Hemoglobin A; C046954:hemoglobin Daphne; C046955:hemoglobin Nympheas; D015951:Factor Xa; D010100:Oxygen