Molecular engineering of a polymer of tetrameric hemoglobins

Fronticelli, Clara; Arosio, Daniele; Bobofchak, Kevin M.; Vasquez, Gregory B.

doi:10.1002/prot.1086

Cited by 24 publications

(21 citation statements)

References 28 publications

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“…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. Hb (Polytaur)n is a large, heterogeneous polymer with average MW of ∼1.0 MDa, a P 50 near 3 Torr, and no oxygen-binding cooperativity.…”

Section: Recombinant Hemoglobinmentioning

confidence: 99%

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

Koehler

Fronticelli

Bucci

2008

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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The cerebrovascular effects of exchange transfusion of various cell-free hemoglobins that possess different oxygen affinities are reviewed. Reducing hematocrit by transfusion of a non-oxygencarrying solution dilates pial arterioles on the brain surface and increases cerebral blood flow to maintain a constant bulk oxygen transport to the brain. In contrast, transfusion of hemoglobins with P 50 of 4-34 Torr causes constriction of pial arterioles that offsets the decrease in blood viscosity to maintain cerebral blood flow and oxygen transport. The autoregulatory constriction is dependent on synthesis of 20-HETE from arachidonic acid. This oxygen-dependent reaction is apparently enhanced by facilitated oxygen diffusion from the red cell to the endothelium arising from increased plasma oxygen solubility in the presence of low or high-affinity hemoglobin. Exchange transfusion of recombinant hemoglobin polymers with P 50 of 3 and 18 Torr reduces infarct volume from experimental stroke. Cell-free hemoglobins do not require a P 50 as high as red blood cell hemoglobin to facilitate oxygen delivery.

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Section: Recombinant Hemoglobinmentioning

confidence: 99%

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

Koehler

Fronticelli

Bucci

2008

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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“…Polymerization has been obtained by introducing, on the protein surface, a cysteine at position β9 to promote the formation of intermolecular S-S bonds. Polymers of human Hb [9] and of a human/bovine hybrid, Hb Minotaur, have been obtained [2,3]. The latter has the functional characteristics of the HbA polymer and is endowed with a high expression level that has allowed preparation of the necessary amounts for in vivo experimentation.…”

Section: Polymeric Hemoglobinsmentioning

confidence: 99%

“…Polymerization of tetrameric Hb prevents the vasoconstriction associated with the infusion of Hb solutions [7,8]. Polymers have been obtained through the formation of intermolecular S-S bonds between cysteine residues introduced on the Hb surface [2,3,9]. S-S bonds are approximately 2.5 Å long, and their accessibility to the reducing agents present in blood is hindered by the numerous intermolecular interactions that result from the crosslinks.…”

mentioning

confidence: 99%

Recombinant Hemoglobins as Artificial Oxygen Carriers

Fronticelli

Koehler

Brinigar

2007

Artificial Cells, Blood Substitutes, and Biotechnology

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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. KeywordsBlood substitute; Heme pocket mutants; Molecular engineering; Polymeric hemoglobin; Surface mutant HEME POCKET MODIFICATIONSThe functional properties of the heme pocket are sensitive to the polar character of the amino acid side chains that line the pocket [1]. Accordingly, we modified the polarity of the heme pocket and obtained three mutant hemoglobins with a range of oxygen affinities (Fig. 1) [2].In the B-helix of the β-chains, we replaced leucine 28, a highly conserved residue in the hydrophobic cluster on the heme distal side, with the isosteric, but polar, asparagine. This residue, by interacting with bound O 2 , stabilizes the oxygenated R-state, resulting in a ten-fold increase in oxygen affinity (P 50 = 0.65 torr). HbA under the same conditions has a P 50 of 6.5 torr.In the E-helix of the α or β chains, we replaced valine at position E11 with the isosteric, but polar, threonine. When this mutation was introduced in the β-chains, a two-fold decrease in oxygen affinity (P 50 = 12 torr) with respect to HbA was observed. However, when this mutation was introduced at position E11 of the α-chains, the functional effects were strikingly different. The oxygen affinity of αV62T was decreased four-fold (P 50 = 25 torr). The significantly decreased oxygen affinity measured in αV62T was attributed to stabilization of the water molecule present in the distal heme pocket of deoxy, T-state, which must dissociate prior to O 2 bonding to the heme-Fe. Cooperativity was present in the mutants βL28N (n = 1.8) and βV67T (n = 2.2), but was essentially absent in αV62T (n = 1.1). A troublesome aspect of this approach was the increased autoxidation rate observed in all of the heme-pocket mutants, Address correspondence to Clara Fronticelli, Professor, Department of Anesthesiology/Critical Care Medicine, 600 N. Wolfe Street, Blalock 1404, Baltimore, MD 21287, USA. E-mail: cfrontic@jhmi.edu. NIH Public Access Author ManuscriptArtif Cells Blood Substit Immobil Biotechnol. Author manuscript; available in PMC 2008 July 24. Published in final edited form as:Artif SURFACE MUTATIONSWe also used surface mutations to modify the oxygen affinity of human Hb. By increasing the hydrophobic interactions between the A-helix and the hydrophobic core of the β-subunits, we constructed an Hb molecule with a three-fold decrease in oxygen affinity with respect to HbA. The amino acid substitutions introduced in the β-chain of HbA are Val1 → Met, His2 → deleted, Thr4 → Ile, Pro5 → Ala, and Ala 76 → Lys. In...

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“…Hb-Polytaur has an MM of nearly 500 kDa and is also likely globular (7, 15) with a radius of 62.1 Å . We have estimated its D, based on MM and radius, to be ϳ0.05 ϫ 10 Ϫ5 cm 2 /s (14). Little is known regarding the overall structure of Hb-(Polytaur) n , although it likely includes globular domains; based on its size, we estimated its D to be ϳ0.02 ϫ 10 Ϫ5 cm 2 /s.…”

Section: Hb-induced P Dhmentioning

confidence: 99%

Quantitative assessment of hemoglobin-induced endothelial barrier dysfunction

Dull

DeWitt

Dinavahi

et al. 2004

Journal of Applied Physiology

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ticelli. Quantitative assessment of hemoglobin-induced endothelial barrier dysfunction. J Appl Physiol 97: 1930 -1937, 2004. First published July 23, 2004 doi:10.1152/japplphysiol.00102.2004.-Hemoglobin (Hb)-based O 2 carriers (HBOC) are undergoing extensive development as potential "blood substitutes." A major problem associated with these molecules is an increase in microvascular permeability and peripheral vascular resistance. In this paper, we utilized bovine lung microvascular endothelial cell monolayers and simultaneously measured Hb-induced changes in transendothelial electrical resistance, diffusive albumin permeability, and diffusive Hb permeability (P DH) for three forms of Hb: natural tetrameric human Hb-A and two polymerized recombinant HBOCs containing ␣-human and ␤-bovine chains designated Hb-Polytaur (molecular mass: 500 kDa) and Hb-(Polytaur) n (molecular mass: ϳ1,000,000 Da), respectively. Hb-Polytaur and Hb-(Polytaur)n are being evaluated for clinical use as HBOCs. All three Hb molecules induce a rapid decline of transendothelial electrical resistance to 30% of baseline. Diffusive albumin permeabiltiy increases, on average, approximately ninefold (2.78 ϫ 10 Ϫ7 vs. 2.47 ϫ 10 Ϫ6 cm/s) in response to Hb exposure. All three Hb molecules induce an increase in their own permeability, a process that we have called Hb-induced Hb permeability. The magnitude of change of P DH is also related to Hb size. When PDH is corrected for the diffusive coefficient for each Hb species, no evidence of restricted diffusion is found. Immunofluorescent images demonstrate Hb-induced actin stress fiber formation and large intercellular gaps. These data provide the first quantitative assessment of the effect of polymerized HBOC on their own diffusion rates over time. We discuss the importance of these findings in terms of Hb extravasation rates, molecular sieving, and clinical consequences of HBOC use.albumin; blood substitutes; restricted diffusion HEMOGLOBIN (HB)-BASED O 2 carriers (HBOC) have received considerable attention as blood substitutes, and interest in developing these therapeutic macromolecules remains high, given the shortage and limited shelf-life of banked red blood cells, infection concerns, and, at times, unacceptable delays in crossmatching banked blood (1,24,26,33). Unfortunately, a number of unexpected problems have been associated with the use of HBOC, including extravasation, vasospasm, renal toxicity, and patient death (1,24,33). The ultimate utility of HBOC is to provide adequate oxygen delivery to patients requiring immediate resuscitation, as may occur following major trauma or surgical procedures associated with large-volume blood losses. Two untoward consequences of HBOC are extravasation of the HBOC itself, which is no longer able to participate in oxygen delivery, and the associated vasoconstriction due, at least in part, to local nitric oxide (NO) scavenging. The decrease in plasma Hb and localized vasospasms in the setting of anemia may create regional hypoxia. Overcoming these issues will...

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Molecular engineering of a polymer of tetrameric hemoglobins

Cited by 24 publications

References 28 publications

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

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

Recombinant Hemoglobins as Artificial Oxygen Carriers

Quantitative assessment of hemoglobin-induced endothelial barrier dysfunction

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