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.

A systematic investigation was carried out concerning relationships between visible and Soret spectra of heme complexes and the nature of the axial ligands. Dioxygen (02), carbon monoxide (CO), and five-coordinate complexes were prepared from proto-, meso-, and 2,4-dimethyldeuteroheme dimethyl esters in N,N-dimethylformamide (DMF) solution. A variety of axial ligands was employed, including imidazoles, pyridines, aliphatic amines, and very weak bases such as DMF and acetone. Variations in the ligands included differences in basicity and differences in substituents which sterically hindered coordination to heme iron. In the five-coordinate complexes, a shift to shorter wavelength in both the visible and Soret peaks accompanied a change to a more hindered axial ligand. Difference spectra obtained by taking the spectrum of a heme complex with a more hindered ligand minus that of a less hindered ligand approximated the T minus R state difference spectrum reported by Perutz et al. [Perutz, M. F., Ladner, J. E., Simon, S. R., & Ho, C. (1974) Biochemistry 13, 21631 for deoxyhemoglobin (deoxy-Hb) Kempsey f inositol hexaphosphate (IHP). In the CO complexes, a decrease in the basicity of the ligand or an increase in the steric hindrance of the axial ligand also resulted in a blue-shifted A, , ,accompanied by an increase in the ratio of intensities of the long-wavelength visible peak ( a ) to that of the shorter wavelength visible peak (P). The latter parameter is termed the a/P ratio. Spectra of the O2 complexes were observed only at temperatures below -40 OC because of limited stability at T h e visible spectra of oxyhemoglobins and oxymyoglobins are characterized by two peaks, a (-575 nm) and / 3 (-540 nm), of which the a peak usually has slightly the greater intensity. The spectra of the CO complexes are similar, but higher temperatures. In O2 complexes, shifts in A, , , and changes in the a/P ratio with unhindered ligands showed much the same pattern as with the CO complexes, but hindered ligands such as 1,2-dimethylirnidazole and 1,2,4,5-tetramethylimidazole gave red-shifted visible A, , , and low a/P ratios compared to the unhindered 02-heme-l-n-butylimidazole complex. This observation is interpreted as being due to the greater ease with which the Fe is distorted from the porphyrin plane in O2 complexes than in CO complexes. Comparison of a / @ ratios of model 02and CO-protoheme complexes with those of hemoglobins and myoglobins formed the basis for the suggestion that the proximal histidine is restrained by the protein, producing a relatively weak axial Fe-N interaction, both in liganded R-state hemoglobins and in common myoglobins. T minus R state difference spectra reported by Perutz et al. [Perutz, M. F., Kilmartin, J. V., Nagai, K., Szabo, A., & Simon, S. R. (1976) Biochemistry 15, 3781 for 02and CO-Hb Kansas f IHP were approximated by model protoheme complexes of a stronger minus a weaker axial ligand, These results are interpreted in terms of T-state steric conflict between coordinated O2 or CO and amino acid...

Three variants of tetrameric human hemoglobin, with changes at the alpha1beta2/alpha2beta1-interface, at the alpha1beta1/alpha2beta2-interface, and at both interfaces, have been constructed. At alpha1beta2/alpha2beta1-interface the beta93 cysteine was replaced by alanine (betaC93A), and at the alpha1beta1/alpha2beta2-interface the beta112 cysteine was replaced by glycine (betaC112G). The alpha1beta2 interface variant, betaC93A, and the alpha1beta1/alpha1beta2 double mutant, beta(C93A+C112G), were crystallized in the T-state, and the structures determined at 2. 0 and 1.8 A resolution, respectively. A comparison of the structures with that of natural hemoglobin A shows the absence of detectable changes in the tertiary folding of the protein or in the T-state quaternary assembly. At the beta112 site, the void left by the removal of the cysteine side chain is filled by a water molecule, and the functional characteristics of betaC112G are essentially those of human hemoglobin A. At the beta93 site, water molecules do not replace the cysteine side chain, and the alanine substitution increases the conformational freedom of beta146His, weakening the important interaction of this residue with beta94Asp. As a result, when Cl- is present in the solution, at a concentration 100 mM, the Bohr effect of the two mutants carrying the beta93Cys-->Ala substitution, betaC93A and beta(C93A+C112G), is significantly modified being practically absent below pH 7.4. Based on the crystallographic data, we attribute these effects to the competition between beta94Asp and Cl- in the salt link with beta146His in T-state hemoglobin. These results point to an interplay between the betaHis146-betaAsp94 salt bridge and the Cl- in solution regulated by the Cys present at position beta93, indicating yet another role of beta93 Cys in the regulation of hemoglobin function.

A plasmid analogous to the one described by Nagai and Thogersen (Nature, 309, 810-812, 1984) has been constructed for the expression of globins in E. coli. Induction with nalidixic acid produces high yields of a fusion protein, NS1-FX-beta-globin, where NS1 represents 81 residues of a flu virus protein and FX represents a blood-clotting Factor Xa recognition sequence, Ile-Glu-Gly-Arg. This fusion protein is readily solubilized in 50 mM NaOH and remains in solution when the pH is adjusted to 8.6. Under these conditions, the fusion protein is hydrolyzed by activated Factor X, giving authentic beta-globin which can be folded in the presence of cyanohemin and native alpha-chains to produce a tetrameric hemoglobin with the functional properties of natural human hemoglobin.