title: A recombinant polymeric hemoglobin with conformational, functional, and physiological characteristics of an in vivo O2 transporter.
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.
authors.affiliation: Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, 600 N. Wolfe St., Baltimore, MD 21287, USA.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Bobofchak
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Mito
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Texel
authors.forename: Andrea
authors.initials: A
authors.lastname: Bellelli
authors.forename: Masaaki
authors.initials: M
authors.lastname: Nemoto
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Traystman
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Koehler
authors.forename: William S
authors.initials: WS
authors.lastname: Brinigar
authors.forename: Clara
authors.initials: C
authors.lastname: Fronticelli
mesh_terms: D000818:Animals; D001483:Base Sequence; D001802:Blood Substitutes / Q000737:chemistry; D001803:Blood Transfusion; D002417:Cattle; D006418:Heme / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D002546:Ischemic Attack, Transient / Q000188:drug therapy* / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D010108:Oxyhemoglobins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011108:Polymers; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001802:Blood Substitutes; D006454:Hemoglobins; D010108:Oxyhemoglobins; D011108:Polymers; D011994:Recombinant Proteins; D009569:Nitric Oxide; D006418:Heme; C020622:hemoglobin B; D010100:Oxygen