title: 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.
authors: Research and Development, Baxter Hemoglobin Therapeutics, 2545 Central Avenue, Boulder, CO 80301, USA. marquardt_david_a@lilly.com.
authors: David A
authors: DA
authors: Marquardt
authors: Research and Development, Baxter Hemoglobin Therapeutics, 2545 Central Avenue, Boulder, CO 80301, USA. mdoyle@clovisoncology.com.
authors: Michael P
authors: MP
authors: Doyle
authors: Somatogen Inc., 2545 Central Avenue, Boulder, CO 80301, USA.
authors: Jeffrey S
authors: JS
authors: Davidson
authors: Somatogen Inc., 2545 Central Avenue, Boulder, CO 80301, USA. info@pisces-molecular.com.
authors: Janet K
authors: JK
authors: Epp
authors: Somatogen Inc., 2545 Central Avenue, Boulder, CO 80301, USA. j.aitken@auckland.ac.nz.
authors: Jacqueline F
authors: JF
authors: Aitken
authors: Research and Development, Baxter Hemoglobin Therapeutics, 2545 Central Avenue, Boulder, CO 80301, USA. douglemon@yahoo.com.
authors: Douglas D
authors: DD
authors: Lemon
authors: Somatogen Inc., 2545 Central Avenue, Boulder, CO 80301, USA. sacahill@chem.wwu.edu.
authors: Spencer J
authors: SJ
authors: Anthony-Cahill