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

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 (p ₅₀ = 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.