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

Kevin M. Bobofchak, Toshiaki Mito, Sarah J. Texel, Andrea Bellelli, Masaaki Nemoto, Richard J. Traystman, Raymond C Koehler, William S. Brinigar, Clara Fronticelli

Research output: Contribution to journalArticle

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 α-human and β-bovine chains. Polymerization of Hb Minotaur through S-S intermolecular cross-linking was obtained by introducing a Cys at position β9 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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume285
Issue number2 54-2
StatePublished - Aug 1 2003

Fingerprint

Hemoglobins
Oxygen
Polymerization
Reducing Agents
Brain Ischemia
Heme
Albumins
Arterial Pressure
Polymers
Escherichia coli
Pressure

Keywords

  • Artificial oxygen carriers
  • Blood substitutes
  • Exchange transfusion
  • Extravasation
  • Focal cerebral ischemia
  • Hemoglobin polymeric
  • Hemoglobin recombinant
  • Hemoglobin retention time
  • Hemoglobin synthetic
  • Transfusional fluids

ASJC Scopus subject areas

  • Physiology

Cite this

A recombinant polymeric hemoglobin with conformational, functional, and physiological characteristics of an in vivo O2 transporter. / Bobofchak, Kevin M.; Mito, Toshiaki; Texel, Sarah J.; Bellelli, Andrea; Nemoto, Masaaki; Traystman, Richard J.; Koehler, Raymond C; Brinigar, William S.; Fronticelli, Clara.

In: American Journal of Physiology - Heart and Circulatory Physiology, Vol. 285, No. 2 54-2, 01.08.2003.

Research output: Contribution to journalArticle

Bobofchak, Kevin M. ; Mito, Toshiaki ; Texel, Sarah J. ; Bellelli, Andrea ; Nemoto, Masaaki ; Traystman, Richard J. ; Koehler, Raymond C ; Brinigar, William S. ; Fronticelli, Clara. / A recombinant polymeric hemoglobin with conformational, functional, and physiological characteristics of an in vivo O2 transporter. In: American Journal of Physiology - Heart and Circulatory Physiology. 2003 ; Vol. 285, No. 2 54-2.
@article{5fcc4bf08bed4c5fafcdc0b274cd5dbd,
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 α-human and β-bovine chains. Polymerization of Hb Minotaur through S-S intermolecular cross-linking was obtained by introducing a Cys at position β9 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.",
keywords = "Artificial oxygen carriers, Blood substitutes, Exchange transfusion, Extravasation, Focal cerebral ischemia, Hemoglobin polymeric, Hemoglobin recombinant, Hemoglobin retention time, Hemoglobin synthetic, Transfusional fluids",
author = "Bobofchak, {Kevin M.} and Toshiaki Mito and Texel, {Sarah J.} and Andrea Bellelli and Masaaki Nemoto and Traystman, {Richard J.} and Koehler, {Raymond C} and Brinigar, {William S.} and Clara Fronticelli",
year = "2003",
month = "8",
day = "1",
language = "English (US)",
volume = "285",
journal = "American Journal of Physiology",
issn = "0363-6135",
publisher = "American Physiological Society",
number = "2 54-2",

}

TY - JOUR

T1 - A recombinant polymeric hemoglobin with conformational, functional, and physiological characteristics of an in vivo O2 transporter

AU - Bobofchak, Kevin M.

AU - Mito, Toshiaki

AU - Texel, Sarah J.

AU - Bellelli, Andrea

AU - Nemoto, Masaaki

AU - Traystman, Richard J.

AU - Koehler, Raymond C

AU - Brinigar, William S.

AU - Fronticelli, Clara

PY - 2003/8/1

Y1 - 2003/8/1

N2 - 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 α-human and β-bovine chains. Polymerization of Hb Minotaur through S-S intermolecular cross-linking was obtained by introducing a Cys at position β9 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.

AB - 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 α-human and β-bovine chains. Polymerization of Hb Minotaur through S-S intermolecular cross-linking was obtained by introducing a Cys at position β9 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.

KW - Artificial oxygen carriers

KW - Blood substitutes

KW - Exchange transfusion

KW - Extravasation

KW - Focal cerebral ischemia

KW - Hemoglobin polymeric

KW - Hemoglobin recombinant

KW - Hemoglobin retention time

KW - Hemoglobin synthetic

KW - Transfusional fluids

UR - http://www.scopus.com/inward/record.url?scp=0037623907&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037623907&partnerID=8YFLogxK

M3 - Article

VL - 285

JO - American Journal of Physiology

JF - American Journal of Physiology

SN - 0363-6135

IS - 2 54-2

ER -