Structural, functional, and genetic characterization of Gastrophilus hemoglobin

Hemoglobin of Gastrophilus intestinalis (Insecta, Diptera), was purified an d characterized. At least two isoforms have been identified by isoelectrofo cusing, mass spectrometry, and genomic Southern blotting. Functional studie s show a high oxygen affinity due to a low ligand dissociation rate (k(off) = 2.4 s(-1)) and a relatively high autoxidation rate (t(1/2) = 1.6/h), The globins were separated under denaturing conditions, and the sequence of Hb 1 (M-r = 17,965 +/- 2) was determined at the protein and DNA level, The ope n reading frame codes for a polypeptide of 150 amino acids. Although the gl obin is distantly related to globins from other species, it has a low penal ty score against globin templates. Freshly isolated hemoglobin was crystallized from polyethylene glycol. Crys tals 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 wa s overexpressed in Escherichia coli, resulting in a fully functional molecu le as confirmed by Ligand binding affinity. The globin gene contains two introns at positions D7.0 and G7.0. The D7.0 i ntron is unprecedented, suggesting that globin gene evolution is much more complex than originally thought.