Abstract
A recombinant 130 kDa dihemoglobin which is made up of a single-chain tetra-α globin and four β globins has been expressed as a soluble protein in E. coli. The sequence of the single chain tetra-α is: αI-Gly-αII-(SerGlyGly)5Ser-αIII-Gly-αIV. This dihemoglobin has been purified and characterized in vitro by size exclusion chromatography, electrospray mass spectroscopy, equilibrium oxygen binding, and analytical ultracentrifugation. The observed values of P50 and nmax for the dihemoglobin are slightly lower than those observed for the recombinant hemoglobin rHb1.1 (a “monohemoglobin” comprised of two β globins and an αI-Gly-αII diα-globin chain). Titration of the deoxy form of dihemoglobin with CO shows that all eight heme centers bind ligand. In vivo, dihemoglobin showed increased circulating halflife and a reduced pressor response in conscious rats when compared to rHb1.1. These observations suggest that dihemoglobin is an oxygen carrying molecule with desirable in vivo properties and provides a platform for an isooncotic hemoglobin solution derived solely from a recombinant source. A 260 kDa tetrahemoglobin has also been produced by chemical crosslinking of a dihemoglobin that contains a Lys16Cys mutation in the C-terminal α-globin subunit. Tetrahemoglobin also shows reduced vasoactivity in conscious rats that is comparable to that observed for dihemoglobin.
Highlights
IntroductionHemoglobin-based oxygen carriers have been studied for many years as potential therapeutics [1,2,3,4,5]
Hemoglobin-based oxygen carriers have been studied for many years as potential therapeutics [1,2,3,4,5].The hemoglobins required to produce candidate therapeutics have been obtained from outdated human blood, transgenic and other animal sources, and bacterial or yeast fermentation
The globin chains of the purified material were separated by C4 reversed phase high performance liquid chromatography (RP-HPLC) on a Hewlett Packard model 1090 HPLC equipped with a Vydac
Summary
Hemoglobin-based oxygen carriers have been studied for many years as potential therapeutics [1,2,3,4,5]. Replacement of Asn108 with Lys in the β globins and covalent linkage of the two α globins in the human α2β2 tetramer by insertion of a single Gly codon between two α genes yields a hemoglobin, rHb1.1, which binds oxygen in a cooperative manner with a P50 close to that of human blood [8] This example demonstrates that a relatively simple structural alteration (i.e., linking the α globins) can yield a protein with improved in vivo function. To explore further the effects of increased molecular size on the vasoactivity of hemoglobin solutions, we produced a Cys-containing dihemoglobin that can be chemically crosslinked to produce a monodisperse “tetrahemoglobin” of ~260 kDa (Figure 1) Both dihemoglobin and tetrahemoglobin show reduced vasoactivity compared to rHb1.1 in conscious rats
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