J. Funct. Biomater. 2012, 3(1), 61-78; doi:10.3390/jfb3010061
Article

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

1,2,†email, 1,2,‡email, 2, 2,§email, 2,||email, 1,2email and 2,¶,* email
Received: 3 November 2011; in revised form: 29 December 2011 / Accepted: 5 January 2012 / Published: 13 January 2012
(This article belongs to the Special Issue Blood Substitutes)
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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.
Keywords: polyhemoglobin; dihemoglobin; tetrahemoglobin; hemoglobin engineering; extravasation; pressor response
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MDPI and ACS Style

Marquardt, D.A.; Doyle, M.P.; Davidson, J.S.; Epp, J.K.; Aitken, J.F.; Lemon, D.D.; Anthony-Cahill, S.J. Monodisperse 130 kDa and 260 kDa Recombinant Human Hemoglobin Polymers as Scaffolds for Protein Engineering of Hemoglobin-Based Oxygen Carriers. J. Funct. Biomater. 2012, 3, 61-78.

AMA Style

Marquardt DA, Doyle MP, Davidson JS, Epp JK, Aitken JF, Lemon DD, Anthony-Cahill SJ. Monodisperse 130 kDa and 260 kDa Recombinant Human Hemoglobin Polymers as Scaffolds for Protein Engineering of Hemoglobin-Based Oxygen Carriers. Journal of Functional Biomaterials. 2012; 3(1):61-78.

Chicago/Turabian Style

Marquardt, David A.; Doyle, Michael P.; Davidson, Jeffrey S.; Epp, Janet K.; Aitken, Jacqueline F.; Lemon, Douglas D.; Anthony-Cahill, Spencer J. 2012. "Monodisperse 130 kDa and 260 kDa Recombinant Human Hemoglobin Polymers as Scaffolds for Protein Engineering of Hemoglobin-Based Oxygen Carriers." J. Funct. Biomater. 3, no. 1: 61-78.

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