A recombinant polymeric hemoglobin with conformational, functional, and physiological characteristics of an in vivo O₂transporter

Abstract

With the objective of developing a recombinant oxygen carrier suitable for therapeutic applications, we have employed an Escherichia coli expression system to synthesize in high-yield hemoglobin (Hb) Minotaur, containing alpha-human and beta-bovine chains. Polymerization of Hb Minotaur through S-S intermolecular cross-linking was obtained by introducing a Cys at position beta9 and substituting the naturally occurring Cys. This homogeneous polymer, Hb Polytaur, has a molecular mass of approximately 500 kDa and was resistant toward reducing agents present in blood. In mice, the circulating half-time (3 h) was fivefold greater than adult human Hb (HbA). The half-time of autooxidation measured in blood (46 h) exceeded the circulating retention time. Hypervolemic exchange transfusion resulted in increased arterial blood pressure similar to that with albumin. The increase in pressure was less than that obtained by transfusion of cross-linked tetrameric Hb known to undergo renovascular extravasation. The nitric oxide reactivity of Hb Polytaur was similar to HbA, suggesting that the diminished pressor response to Hb Polytaur was probably related to diminished extravasation. Transfusion of 3% Hb Polytaur during focal cerebral ischemia reduced infarct volume by 22%. Therefore, site-specific Cys insertion on the Hb surface results in uniform size polymers that do not produce the large pressor response seen with tetrameric Hb. Polymerization maintains physiologically relevant oxygen and heme affinity, stability toward denaturation and oxidation, and effective oxygen delivery as indicated by reduced cerebral ischemic damage.

Insensitivity of cerebral oxygen transport to oxygen affinity of hemoglobin-based oxygen carriers

“…A hypervolemic exchange transfusion was performed in mice over a 20-min period starting 10 min after occlusion of the middle cerebral artery. Infarct volume was reduced by transfusion of Hb Polytaur, a homogeneous polymer of seven tetrameric Hb molecules (MW∼500 MDa) with an intermediate P 50 (18 Torr) and cooperativity (n = 1.8) [8]. Unexpectedly, Hb (Polytaur)n, a heterogeneous polymer with average MW of ∼1 MDa, a low P 50 (3 Torr), and no cooperativity (n = 1), also markedly reduced infarct volume (Fig.…”

“…Optimal expression of recombinant Hb was obtained using a hybrid protein, Hb Minotaur, which contained α-human and β-bovine chains [8]. Polymerization of Hb Minotaur spontaneously occurred through the oxidation of thiol groups that were introduced on the surface of the β-chains.…”

“…Polymerization of Hb Minotaur spontaneously occurred through the oxidation of thiol groups that were introduced on the surface of the β-chains. Two polymers have been prepared: Hb Polytaur and Hb (Polytaur)n [8,9]. Like Hb Prisca [10], Hb Polytaur is a homogeneous globular polymer with a molecular weight (MW) of ∼500 kDa, P 50 of 18 Torr, and oxygen-binding cooperativity of n=1.8.…”

“…Pial arteriolar diameter was measured by intravital microscopy through closed cranial windows in anesthetized cats and rats [14]. Focal cerebral ischemia was produced in mice by occlusion of the middle cerebral artery, as described [8].…”

“…These products are endowed with high oncotic pressure and viscosity, which may limit the concentration of Hb that can be administered. Alternatively, recombinant molecular engineering is used to obtain Hb polymers of different sizes in the absence of chemical modification [8,9]. Although the amount of recombinant HBOCs that can be prepared is currently limited, the continuous development of molecular engineering could result in largescale production capacity of recombinant oxygen carriers that (a) are free from infectious agents that could be present in mammalian blood and (b) have functional and conformational characteristics that are geared to various clinical applications.…”