Expression, purification, and properties of recombinant barley (Hordeum sp.) hemoglobin. Optical spectra and reactions with gaseous ligands.
A cDNA encoding barley hemoglobin (Hb) has been cloned into pUC 19 and expressed in Escherichia coli. The resulting fusion protein has five extra amino acids at the N terminus compared with the native protein, resulting in a protein of 168 amino acids (18.5 kDa). The recombinant Hb is expressed constitutively. Extracts made from the bacteria containing the recombinant fusion construct contain a protein with a subunit molecular mass of approximately 18.5 kDa comprising approximately 5% total soluble protein. Recombinant Hb was purified to homogeneity according to SDS-polyacrylamide gel electrophoresis by sequential polyethylene glycol precipitation and fast protein liquid chromatography. Its native molecular mass as assessed by fast protein liquid chromatography-size exclusion was 40 kDa suggesting that it is a dimer. Ligand binding experiments demonstrate that 1) barley Hb has a very slow oxygen dissociation rate constant (0.0272 s-1) relative to other Hbs, and 2) the heme of ferrous and ferric forms of the barley Hb is low spin six-coordinate. The subunit structure, optical spectrum, and oxygen dissociation rate of native barley hemoglobin are indistinguishable from those obtained for the recombinant protein. The implications of these kinetic data on the in vivo function of barley Hb are discussed.
Duff
S M
SM
Department of Plant Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
Wittenberg
J B
JB
Hill
R D
RD
D000595:Amino Acid Sequence; D002248:Carbon Monoxide / Q000378:metabolism*; D019281:Dimerization; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006420:Hemeproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism; D001467:Hordeum / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010100:Oxygen / Q000378:metabolism*; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012639:Seeds / Q000737:chemistry; D013053:Spectrophotometry
C106902:HB protein, Hordeum vulgare; D006420:Hemeproteins; D006454:Hemoglobins; D008024:Ligands; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D002248:Carbon Monoxide; D010100:Oxygen