Structural, functional, and genetic characterization of Gastrophilus hemoglobin.
Hemoglobin of Gastrophilus intestinalis (Insecta, Diptera), was purified and characterized. At least two isoforms have been identified by isoelectrofocusing, mass spectrometry, and genomic Southern blotting. Functional studies show a high oxygen affinity due to a low ligand dissociation rate (koff = 2.4 s-1) and a relatively high autoxidation rate (t1/2 = 1.6/h). The globins were separated under denaturing conditions, and the sequence of Hb1 (Mr = 17,965 +/- 2) was determined at the protein and DNA level. The open reading frame codes for a polypeptide of 150 amino acids. Although the globin is distantly related to globins from other species, it has a low penalty score against globin templates. Freshly isolated hemoglobin was crystallized from polyethylene glycol. Crystals contain two hemoglobin molecules per asymmetric unit. Solution of the three-dimensional structure by molecular replacement could not be achieved, possibly due to the presence of three protein isoforms in the crystals. In order to determine its three-dimensional structure, G. intestinalis Hb1 was overexpressed in Escherichia coli, resulting in a fully functional molecule as confirmed by ligand binding affinity. The globin gene contains two introns at positions D7.0 and G7.0. The D7.0 intron is unprecedented, suggesting that globin gene evolution is much more complex than originally thought.
Dewilde
S
S
Department of Biochemistry, University of Antwerp, B-2610 Antwerp, Belgium.
Blaxter
M
M
Van Hauwaert
M L
ML
Van Houte
K
K
Pesce
A
A
Griffon
N
N
Kiger
L
L
Marden
M C
MC
Vermeire
S
S
Vanfleteren
J
J
Esmans
E
E
Moens
L
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004175:Diptera / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis
D018076:DNA, Complementary; D006454:Hemoglobins