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

Marquardt, David A.; Doyle, Michael P.; Davidson, Jeffrey S.; Epp, Janet K.; Aitken, Jacqueline F.; Lemon, Douglas D.; Anthony-Cahill, Spencer J.

doi:10.3390/jfb3010061

Open AccessArticle

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

by

David A. Marquardt

^1,2,†,

Michael P. Doyle

^1,2,‡,

Jeffrey S. Davidson

²,

Janet K. Epp

^2,§,

Jacqueline F. Aitken

^2,||,

Douglas D. Lemon

^1,2 and

Spencer J. Anthony-Cahill

^2,*,¶

¹

Research and Development, Baxter Hemoglobin Therapeutics, 2545 Central Avenue, Boulder, CO 80301, USA

²

Somatogen Inc., 2545 Central Avenue, Boulder, CO 80301, USA

^*

Author to whom correspondence should be addressed.

^†

Biopharmaceutical Research and Development, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA

^‡

Clovis Oncology, Inc., 2525 28th St. Suite 100, Boulder, CO 80301, USA

^§

Pisces Molecular LLC, 5311 Western Avenue, Suite E, Boulder, CO 80301, USA

^||

School of Biological Sciences, Faculty of Science, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand

^¶

Department of Chemistry, Western Washington University, Bellingham, WA 98225-9150, USA

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

Submission received: 3 November 2011 / Revised: 29 December 2011 / Accepted: 5 January 2012 / Published: 13 January 2012

(This article belongs to the Special Issue Blood Substitutes)

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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. https://doi.org/10.3390/jfb3010061

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. https://doi.org/10.3390/jfb3010061

Chicago/Turabian Style

Marquardt, David A., Michael P. Doyle, Jeffrey S. Davidson, Janet K. Epp, Jacqueline F. Aitken, Douglas D. Lemon, and Spencer J. Anthony-Cahill. 2012. "Monodisperse 130 kDa and 260 kDa Recombinant Human Hemoglobin Polymers as Scaffolds for Protein Engineering of Hemoglobin-Based Oxygen Carriers" Journal of Functional Biomaterials 3, no. 1: 61-78. https://doi.org/10.3390/jfb3010061

APA Style

Marquardt, D. A., Doyle, M. P., Davidson, J. S., Epp, J. K., Aitken, J. F., Lemon, D. D., & Anthony-Cahill, S. J. (2012). Monodisperse 130 kDa and 260 kDa Recombinant Human Hemoglobin Polymers as Scaffolds for Protein Engineering of Hemoglobin-Based Oxygen Carriers. Journal of Functional Biomaterials, 3(1), 61-78. https://doi.org/10.3390/jfb3010061

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

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