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

• Published in: American journal of physiology. Heart and circulatory physiology Vol. 285; no. 2; pp. H549 - H561
• Main Authors: Bobofchak, Kevin M, Mito, Toshiaki, Texel, Sarah J, Bellelli, Andrea, Nemoto, Masaaki, Traystman, Richard J, Koehler, Raymond C, Brinigar, William S, Fronticelli, Clara
• Format: Journal Article
• Language: English
• Published: United States 01.08.2003
• Subjects: Animals; Base Sequence; Blood Substitutes - chemistry; Blood Transfusion; Cattle; Heme - metabolism; Hemoglobins - chemistry; Hemoglobins - genetics; Hemoglobins - metabolism; Humans; Ischemic Attack, Transient - drug therapy; Ischemic Attack, Transient - pathology; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Nitric Oxide - metabolism; Oxidation-Reduction; Oxygen - metabolism; Oxyhemoglobins - chemistry; Oxyhemoglobins - genetics; Oxyhemoglobins - metabolism; Polymers; Protein Conformation; Protein Denaturation; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism
• ISSN: 0363-6135; 1522-1539
• DOI: 10.1152/ajpheart.00037.2003

1 Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287; 2 Consiglio Nazionale della Ricerca, Institute of Molecular Biology and Pathology and Department Biochemical Sciences University La Sapienza, Rome, Italy; and 3 Department of Chemistry, Temple University, Philadelphia, Pennsylvania 19122 Submitted 15 January 2003 ; accepted in final form 4 April 2003 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 -human and -bovine chains. Polymerization of Hb Minotaur through S-S intermolecular cross-linking was obtained by introducing a Cys at position 9 and substituting the naturally occurring Cys. This homogeneous polymer, Hb Polytaur, has a molecular mass of 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. hemoglobin recombinant; hemoglobin synthetic; hemoglobin polymeric; blood substitutes; transfusional fluids; artificial oxygen carriers; hemoglobin retention time; exchange transfusion; extravasation; focal cerebral ischemia Address for reprint requests and other correspondence: C. Fronticelli, Dept. Anesthesiology and Critical Care Medicine, Johns Hopkins Univ. School of Medicine, 600 N. Wolfe St., Baltimore, MD 21287 (E-mail: cfrontic{at}jhmi.edu ).