title: Oxygen binding and other physical properties of human hemoglobin made in yeast.
abstract: Wagenbach et al. (1991, BioTechnology, 9, 57-61) have recently developed a system for producing soluble recombinant tetrameric hemoglobin in yeast: hemoglobin begins to appear 4-5 h after induction with galactose, alpha- and beta-globin chains fold in vivo and endogeneously produced heme is incorporated into hemoglobin tetramers. We have further characterized the oxygen-binding properties, as well as the tetramer stability, of recombinant human Hb A made in yeast. After purification by ion-exchange chromatography, a single band at the same position as normal human Hb A was obtained using cellulose acetate electrophoresis. Although the oxy and deoxy forms of purified recombinant Hb A made in yeast were spectrophotometrically identical to native human Hb A, the oxygen-binding curve was shifted slightly left of that for native human Hb A. Further purification of recombinant hemoglobin by FPLC revealed two fractions: one (fraction B) with low cooperativity and high oxygen affinity, and the other (fraction A) with almost identical cooperativity and oxygen affinity compared with native human Hb A. The Bohr effect of fraction A was also identical to native human Hb A. Hemoglobin in fraction B with lowered cooperativity precipitated approximately 1.5 times faster than normal human Hb A during mechanical agitation, while hemoglobin in fraction A with normal cooperativity precipitated with kinetics identical to native human Hb A. These results suggest that some of the recombinant molecules made in yeast fold improperly, and that these molecules may exhibit decreased cooperativity for oxygen binding and decreased stability.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Children's Hospital of Philadelphia, Division of Hematology, PA.
authors.forename: K
authors.initials: K
authors.lastname: Adachi
authors.forename: P
authors.initials: P
authors.lastname: Konitzer
authors.forename: C H
authors.initials: CH
authors.lastname: Lai
authors.forename: J
authors.initials: J
authors.lastname: Kim
authors.forename: S
authors.initials: S
authors.lastname: Surrey
mesh_terms: D000494:Allosteric Regulation; D014644:Genetic Variation; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010100:Oxygen / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014162:Transfection
chemical_list: D006454:Hemoglobins; D011994:Recombinant Proteins; D010100:Oxygen