Haemoglobin(βK120C)–albumin trimer as an artificial O(2) carrier with sufficient haemoglobin allostery

The allosteric O(2) release of haemoglobin (Hb) allows for efficient O(2) delivery from the lungs to the tissues. However, allostery is weakened in Hb-based O(2) 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 O(2) 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 O(2) 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)–HSA(2) 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 α(1)β(2) 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 O(2). In phosphate-buffered saline solution (pH 7.4) at 37 °C, the rHb(βK120C)–HSA(2) trimer exhibited a sigmoidal O(2) equilibrium curve with the O(2) 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)–HSA(2) trimer compared with naked Hb.