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.

Comparison of the Pharmacokinetic Properties of Hemoglobin-Based Oxygen Carriers

“…A second generation product, rHb2.0, has been developed and its efficacy is being evaluated in preclinical trials [12,13]. Furthermore, Marquardt and colleagues prepared di-Hb in which two rHb1.1 units are covalently cross-linked by gene fusion using a peptide linker and they also prepared tetra-Hb that is chemically cross-linked with bismaleimidohexane between a cysteine residue into di-Hb [14]. A pharmacokinetic study in rats demonstrated that the half-life of di-Hb and tetra-Hb are approximately 1.4 and 1.6 times longer than that of rHb1.1 [14,15].…”