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Issue 3, 2020
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Haemoglobin(βK120C)–albumin trimer as an artificial O2 carrier with sufficient haemoglobin allostery

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Abstract

The allosteric O2 release of haemoglobin (Hb) allows for efficient O2 delivery from the lungs to the tissues. However, allostery is weakened in Hb-based O2 carriers because the chemical modifications of the Lys- and Cys-β93 residues prevent the quaternary transition of Hb. In this paper, we describe the synthesis and O2 binding properties of a recombinant Hb [rHb(βK120C)]–albumin heterotrimer that maintains sufficient Hb allostery. The rHb(βK120C) core, with two additional cysteine residues at the symmetrical positions on its protein surface, was expressed using yeast cells. The mutations did not influence either the O2 binding characteristics or the quaternary transition of Hb. Maleimide-activated human serum albumins (HSAs) were coupled with rHb(βK120C) at the two Cys-β120 positions, yielding the rHb(βK120C)–HSA2 trimer, in which the Cys-β93 residues were unreacted. Molecular dynamics simulation demonstrated that the HSA moiety does not interact with the amino acid residues around the haem pockets and the α1β2 surfaces of the rHb(βK120C) core, the alteration of which retards Hb allostery. Circular dichroism spectroscopy demonstrated that the quaternary transition between the relaxed (R) state and the tense (T) state of the Hb core occurred upon both the association and dissociation of O2. In phosphate-buffered saline solution (pH 7.4) at 37 °C, the rHb(βK120C)–HSA2 trimer exhibited a sigmoidal O2 equilibrium curve with the O2 affinity and cooperativity identical to those of native Hb (p50 = 12 Torr, n = 2.4). Moreover, we observed an equal Bohr effect and 2,3-diphosphoglycerate response in the rHb(βK120C)–HSA2 trimer compared with naked Hb.

Graphical abstract: Haemoglobin(βK120C)–albumin trimer as an artificial O2 carrier with sufficient haemoglobin allostery

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Supplementary files

Article information


Submitted
29 Apr 2020
Accepted
25 Jun 2020
First published
13 Jul 2020

This article is Open Access

RSC Chem. Biol., 2020,1, 128-136
Article type
Paper

Haemoglobin(βK120C)–albumin trimer as an artificial O2 carrier with sufficient haemoglobin allostery

Y. Morita, A. Saito, J. Yamaguchi and T. Komatsu, RSC Chem. Biol., 2020, 1, 128
DOI: 10.1039/D0CB00056F

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