Monodisperse 130 kDa and 260 kDa Recombinant Human Hemoglobin Polymers as Scaffolds for Protein Engineering of Hemoglobin-Based Oxygen Carriers

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.

Figure 1

Covalent crosslinking of diα-globins (red) by gene fusion using a peptide linker (green) yields dihemoglobin (“di-Hb”). Introduction of a Cys residue into di-Hb, followed by chemical crosslinking with bismaleimidohexane (BMH), yields “tetra-Hb”. The subunit composition of rHb1.1 is shown within the dashed-line rectangle.

Figure 2

(a) Map of pSGE1000. The vector was designed to have unique SpeI and PstI sites flanking the fusion region. (b) X-ray crystal structure of deoxy-rHb1.1 (Reference [24]; PDB ID 1c7b), rendered using PyMol with the diα-globin in blue and K16 highlighted in red.

Figure 3

Tris-glycine gels (4%–20%) stained with coomassie brilliant blue. (a) SDS-PAGE of SGE 953 and rHb1.1. Lane 1, MW markers. MWs in kDa are indicated. Lane 2, partially purified dihemoglobin from SGE 953. This material is the collected fraction from the first chromatography step. Lane 3, fully purified dihemoglobin from SGE 953. Lane 4, purified rHb1.1; (b) SDS-PAGE of rHb1.1, rHb1.1 K158C, SGE 953 and SGE 2812 before and after chemical crosslinking; (c) Native PAGE of rHb1.1, rHb1.1 K158C, SGE 953, SGE 2812 before and after chemical crosslinking, and a glutaraldehyde-crosslinked “penta-Hb”.

Figure 4

Analytical size exclusion chromatography of dihemoglobin SGE 953. (a) Partially purified SGE 953. This material is the collected fraction from the first chromatography step (see lane 2 of Figure 3A); (b) Purified SGE 953; (c) Coinjection of purified SGE 953 and purified rHb1.1.

Figure 5

Ligand binding to SGE 953. (a) The stoichiometry of ligand binding was determined by titrating deoxy dihemoglobin with CO-saturated buffer at 20 °C. An overlay of the spectra recorded after each CO addition during the titration is shown; (b) The data from panel (a) are plotted at 418 nm; (c) The (oxy di-Hb)-(deoxy di-Hb) difference spectra for SGE 953 (magenta, dashed line) and rHb1.1 (blue, solid line).

Figure 6

Determination of circulating halflife and hemodynamic response of di-Hb in vivo (a) Circulating halflife was determined in male Sprague-Dawley rats. Top-load doses of protein were administered via intravenous infusion to six rats each in experimental (SGE 953, filled circles) and control (rHb1.1, open circles) groups. Time-dependent curves of plasma hemoglobin concentrations are shown; (b) Hemoglobin concentrations in peritoneal lavage fluid at two time points following administration of rHb1.1 (open bars) or dihemoglobin from SGE 953 (filled bars) to separate groups of Balb/C mice; (c) Changes in mean arterial pressure following administration of hemoglobins to conscious, unrestrained rats. Top-load doses of rHb1.1 (filled triangles, orange), SGE 953 di-Hb (filled circles, blue), crosslinked SGE 2812 tetra-Hb (filled crosses, green), or HSA (50 mg/mL, filled squares, purple) were administered to separate groups of rats (n = 6 in all groups).

Figure 7

Analytical size exclusion chromatography of dihemoglobin SGE 2812. (A) Purified SGE 2812 prior to crosslinking; (B) Purified SGE 2812 after crosslinking with bismaleimidohexane; (C) Coinjection of rHb1.1 (t_r = 58.8 min) and purified SGE 2812 before (t_r = 53.9 min) and after (t_r = 49.0 min) BMH crosslinking.