Reference "Expression, purification, and properties of recombinant barley (Hordeum sp.) hemoglobin. Optical spectra and reactions with gaseous ligands"

Reference ID

8648

Title

Expression, purification, and properties of recombinant barley (Hordeum sp.) hemoglobin. Optical spectra and reactions with gaseous ligands

Source

The Journal of biological chemistry, 1997, vol. 272, pp. 16746-16752

Authors (3)

Duff-S-M	Wittenberg-J-B
Hill-R-D

Abstract

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.