Recombinant hemoglobins as artificial oxygen carriers.

This paper describes the approaches we have taken to construct a) mutant hemoglobins with different oxygen affinities, and b) mutant hemoglobins and myoglobins that polymerize to high molecular weight aggregates in an effort to prevent extravasation and the associated vasoactivity. In vivo testing indicates that exchange transfusion of polymeric hemoglobins in mice does not result in vasoactivity and that polymeric hemoglobins are effective oxygen carriers to ischemic tissues irrespective of their oxygen affinity and cooperativity.

Salvage of focal cerebral ischemic damage by transfusion of high O2-affinity recombinant hemoglobin polymers in mouse.

Cell-free hemoglobin solutions with high oxygen affinity might be beneficial for selectively delivering oxygen to ischemic tissue. A recombinant hybrid hemoglobin molecule was designed using the human alpha-subunit and the bovine beta-subunit, with placement of surface cysteines to permit disulfide bond polymerization of the tetramers. The resulting protein generated from an Escherichia coli expression system had a molecular mass >1 MDa, a P50 of approximately 3 Torr, and a cooperativity of n = 1.

A recombinant polymeric hemoglobin with conformational, functional, and physiological characteristics of an in vivo O2 transporter.

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.

Introduction of a new regulatory mechanism into human hemoglobin.

Previous studies on bovine hemoglobin (HbBv) have suggested amino acid substitutions, which might introduce into human hemoglobin (HbA) functional characteristics of HbBv, namely a low intrinsic oxygen affinity regulated by Cl(-). Accordingly, we have constructed and characterized a multiple mutant, PB5, [beta(V1M + H2 Delta + T4I + P5A + A76K)] replacing four amino acid residues of HbA with those present at structurally analogous positions in HbBv, plus an additional substitution, beta T4I, which does not occur in either HbBv or HbA. This 'pseudobovine' hemoglobin has oxygen binding properties very similar to those of HbBv: the P(50) of HbA, PB5 and HbBv in the absence of Cl(-) are 1.