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BBA - Proteins and Proteomics (v.1784, #10)
Ligand reactivity and allosteric regulation of hemoglobin-based oxygen carriers by Luca Ronda; Stefano Bruno; Stefania Abbruzzetti; Cristiano Viappiani; Stefano Bettati (pp. 1365-1377).
Historically, exogenous administration of hemoglobin solutions to implement the oxygen transport capacity for clinical applications suffered from dramatic drawbacks, resulting in the failure of many attempts. In the last decades, the biochemical and physiological basis responsible for the therapeutic failures has been extensively investigated. It is now widely accepted that they mostly arise because, out of the confined and controlled environment of the red blood cell, hemoglobin exhibits tetramer instability, increased auto-oxidation rate, higher oxygen affinity, altered cooperativity and nitric oxide reactivity. Moreover, it became evident that the design of a hemoglobin-based oxygen carrier that exactly reproduces the “physiological” oxygen-binding curve is not only an overly ambitious task, but may also represent a wrong approach for many potential clinical applications. Under these premises, and given the complex chemical nature of blood, it is obvious that any strategy undertaken to modify the stability and function of the hemoglobin tetramer for clinical use should be driven by a detailed knowledge of its structure, dynamics and mechanism of allosteric regulation. We briefly review the most recent theories and experiments that increased our understanding of the mechanism of homo- and heterotropic effects in human hemoglobin, trying to interpret, on a biophysical basis, how diverse approaches like polymerization, cross-linking, site-directed mutagenesis, surface decoration and encapsulation may affect ligand affinity and allosteric regulation.
Keywords: Abbreviations; BZF; bezafibrate; DPG; 2,3 diphosphoglicerate; HbCO; carbonmonoxy-Hb; IHP; inositol hexaphosphate; MWC; Monod, Wyman and Changeux; NO; nitric oxide; PEG; polyethylene glycol; pO; 2; oxygen partial pressure; (propyl-PEG5k); 6; -Hb; hemoglobin PEGylated with 6 PEG 5000 chains by reductive alkylation chemistry; (propyl-PEG2k); 6; -Hb; hemoglobin PEGylated with 6 PEG 2000 chains by reductive alkylation chemistry; TTS model; Tertiary Two State modelHemoglobin; Allosteric properties; Tertiary conformation; Oxygen binding; Silica gel
All hemoglobin-based oxygen carriers are not created equally by Paul W. Buehler; Abdu I. Alayash (pp. 1378-1381).
Hemoglobin (Hb)-based oxygen carriers (HBOCs) also known as “blood substitutes” have been under active clinical development over the last two decades. Cell-free Hb outside its natural protective red blood cell environment, as is the case with all HBOCs, has been shown to be vasoactive in part due to the scavenging of vascular endothelial nitric oxide (NO) and may in some instances induce heme-mediated oxidative stress. Chemical modification intended to stabilize HBOCs in the tetrameric or polymeric forms introduces conformational constraints that result in proteins with diverse allosteric responses as well as oxidative and nitrosative redox side reactions. Intra and inter-molecular cross-linking may in some instances also determine the interactions between HBOCs and normal oxidative inactivation and clearance mechanisms. Oxygen and oxidative reactions of normal and several cross-linked Hbs as well as their interactions with endogenous plasma protein (haptoglobin) and cellular receptor pathways (macrophage CD163) differ significantly. Therefore, safety and efficacy may be addressed by designing HBOCs with modifications that limit hypertension, minimize heme destabilization and take into account endogenous Hb removal mechanisms to optimize exposure times for a given indication.
Keywords: Hemoglobin; Blood substitutes; Oxidation; Nitric oxide
Cell-free oxygen carriers: Scientific foundations, clinical development, and new directions by Robert M. Winslow (pp. 1382-1386).
The most significant hurdle to the development of a safe and effective hemoglobin-based oxygen carrier (“blood substitute”) is generally thought to be its propensity to cause vasoconstriction in the microcirculation and hypertension. Two theories for this effect are currently being studied: in one, scavenging NO by hemoglobin reduces vasorelaxation; in the other, cell-free hemoglobin oversupplies O2 (a known vasoconstrictor) to vascular walls by facilitated diffusion. While both mechanisms might lead to reduction of local NO concentration, the important distinction between the two is that if the NO scavenging theory is correct, it greatly diminishes the prospects to develop any solution based on free hemoglobin. However, if the O2-oversupply theory is correct, modifications to the hemoglobin molecule can be envisioned that can prevent oversupply and reduce toxicity. This review summarizes the development of Hemospan®, a novel modification of human hemoglobin whose design is based on the O2-oversupply theory. Because of its low P50 and increased molecular size, the release of O2 in resistance vessels (arterioles) by Hemospan is restricted, and vasoconstriction is greatly reduced.
Keywords: Blood substitutes; Oxygen; Hemoglobin; Transfusion; Oxygen carriers
Insensitivity of cerebral oxygen transport to oxygen affinity of hemoglobin-based oxygen carriers by Raymond C. Koehler; Clara Fronticelli; Enrico Bucci (pp. 1387-1394).
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-oxygen-carrying 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 P50 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 P50 of 3 and 18 Torr reduces infarct volume from experimental stroke. Cell-free hemoglobins do not require a P50 as high as red blood cell hemoglobin to facilitate oxygen delivery.
Keywords: Abbreviations; 20-HETE; 20-hydroxyeicosatetraenoic acid; CBF; cerebral blood flow; CMRO; 2; cerebral metabolic rate of oxygen; Hb; hemoglobin; HBOC; hemoglobin-based oxygen carriers; NO; nitric oxide; P; 50; partial pressure of oxygen at 50% oxyhemoglobin saturation; PVP; polyvinylpyrrolidoneAnemia; Blood substitute; Cerebral blood flow; Hemoglobin; Oxygen affinity; Oxygen transport
Non-conservative surface decoration of hemoglobin: Influence of neutralization of positive charges at PEGylation sites on molecular and functional properties of PEGylated hemoglobin by Dongxia Li; Tao Hu; Belur N. Manjula; Seetharama A. Acharya (pp. 1395-1401).
High hydrodynamic volume, high viscosity and high colloidal osmotic pressure (COP) of PEGylated hemoglobin (Hb) have been suggested to neutralize the vasoactivity of acellular Hb. Consequences of non-conservative PEGylation (positive charge of the amino groups at the PEGylation sites is neutralized) using succinimidyl-ester of propionic acid PEG5K on the properties of PEGylated Hb have now been investigated. Non-conservative PEGylation of Hb leads to a much higher increase in the COP and viscosity of Hb than conservative extension arm facilitated (EAF) PEGylation of Hb. Introduction of αα-fumaryl crosslinking decreased the COP of non-conservative PEGylated Hb by stabilization of interdimeric interactions. Compared to the EAF-PEGylated αα-fumaryl Hb, non-conservative PEGylated product shows a comparable COP and higher viscosity. Conservative PEGylation of αα-fumaryl Hb by reductive alkylation chemistry does not increase the COP to this level, but enhanced the molecular volume and viscosity comparable to EAF-PEGylated product. Thus, the molecular properties of PEGylated Hb can be fine tuned using different PEGylation platforms and provide a unique opportunity for the design of second generation PEGylated Hbs.
Keywords: Abbreviations; Hb; hemoglobin; PEG; polyethylene glycol; COP; colloidal osmotic pressure; EAF; extension arm facilitated; PEGylation; conjugation of PEG chains to proteins; HBOC; hemoglobin based oxygen carrier; CD; circular dichroism; SEC; size exclusion chromatography; P; 50; oxygen pressure at half saturation; 4-PDS; 4,4′-dithiodipyridine; DPG; 2,3-diphosphoglycerate; L35; 2-[4-(3,5-dichlorophenylureido)phenoxy]-2-methylpropionic acid; SPA-PEG5K; succinimidyl-ester of PEG5K propionic acidPEGylation; Hemoglobin; Blood substitute; Colloidal osmotic pressure; Acylation chemistry
Towards a novel haemoglobin-based oxygen carrier: Euro-PEG-Hb, physico-chemical properties, vasoactivity and renal filtration by Portoro I. Portörő; L. Kocsis; Herman P. Hermán; D. Caccia; M. Perrella; L. Ronda; S. Bruno; S. Bettati; C. Micalella; A. Mozzarelli; A. Varga; M. Vas; K.C. Lowe; A. Eke (pp. 1402-1409).
Blood transfusion is still a critical therapy in many diseases, traumatic events and war battlefields. However, blood cross-matching and storage may limit its applicability, especially in Third World countries. Moreover, haemoglobin, which in red blood cells is the key player in the oxygen transport from lung to tissues, when free in the plasma causes hypertension and renal failure. This investigation was aimed at the development of a novel haemoglobin-based oxygen carrier with low vasoactivity and renal filtration properties. Human haemoglobin was chemically conjugated with polyethylene glycol (PEG) under either aerobic or anaerobic conditions, following different chemical procedures. The resulting PEGylated haemoglobin products were characterized in terms of oxygen affinity, cooperativity, effects of protons and carbon dioxide concentration, and oxidation stability, and were transfused into rats to evaluate vasoactivity and renal filtration. A deoxyhaemoglobin, conjugated with seven PEG and seven propionyl groups, which we called Euro-PEG-Hb, did not produce profound hypertension, was 99% retained within 6 h, and exhibited oxygen binding properties and allosteric effects more similar to human haemoglobin A than the other tested PEGylated haemoglobin derivatives, thus appearing a very promising candidate as blood substitute.
Keywords: Abbreviations; EDTA; ethylenediamine tetraacetic acid; EU; endotoxin unit; HbA; purified human haemoglobin A; HBOC; haemoglobin-based oxygen carrier; Hct; arterial haematocrit; IHP; inositol hexaphosphate; IMT; 2-imino thiolane; LAL; limulus amebocyte lysate; LPS; lipopolysaccharide; MAL-PEG; maleimido polyethylene glycol; NEM; N; -ethyl maleimide; NPM; N; -propionyl maleimide; PBS; phosphate buffered saline; PEG; polyethylene glycol; SABP; systemic arterial blood pressure; SFH; stroma free haemoglobinBlood substitute; Haemoglobin; (Hb)-based oxygen carrier; Protein PEGylation; Urine Hb; Vasoactivity
Effect of cross-linker length on the stability of hemoglobin by Kevin M. Bobofchak; Eugene Tarasov; Kenneth W. Olsen (pp. 1410-1414).
A series of cross-linking reagents with 4 to 7 carbons have been synthesized and used to modify human hemoglobin. The product yields and biochemical properties of these cross-linked hemoglobins are compared to those made with both longer and shorter cross-linkers. Several trends become apparent. The yields decrease as the cross-linker becomes longer, which correlates well with molecular dynamics studies of reagent binding pathways presented here. The autooxidation rates increase while thermal stability decreases with longer reagents. Cross-linking under deoxy conditions also increases autooxidation rates, but the effect is less than that of increased cross-linker length. The results suggest that shorter reagents may provide better-stabilized tetramers for the construction of more complex hemoglobin-based oxygen carriers.
Keywords: Hemoglobin; Cross-link; Oxygen carrier; Stability; Autooxidation
Peroxidase activity of hemoglobin towards ascorbate and urate: A synergistic protective strategy against toxicity of Hemoglobin-Based Oxygen Carriers (HBOC) by Chris E. Cooper; Radu Silaghi-Dumitrescu; Martine Rukengwa; Abdu I. Alayash; Paul W. Buehler (pp. 1415-1420).
Acellular hemoglobins developed as oxygen bridging agents with volume expanding properties (“blood substitutes”) are prone to autoxidation and oxidant-mediated structural changes in circulation. In the presence of hydrogen peroxide and either ascorbate or urate we show that ferric hemoglobin functions as a true enzymatic peroxidase. The activity saturates with both substrates and is linearly dependent on protein concentration. The activity is enhanced at low pH with a p Ka of 4.7, consistent with protonation of the ferryl species (Fe(IV)−OH) as the active intermediate. To test whether these redox reactions define its behaviour in vivo we exchanged transfused guinea pigs with 50% polymerized bovine Hb (PolyHbBv) and monitored plasma levels of endogenous ascorbate and urate. Immediately after transfusion, met PolyHbBv levels increased up to 30% of total Hb and remained at this level during the first 24 h post transfusion. Plasma ascorbate decreased by 50% whereas urate levels remained unchanged after transfusion. A simple kinetic model, assuming that ascorbate was a more active ferric heme reductase and peroxidase substrate than urate, was consistent with the in vivo data. The present finding confirms the primary and secondary roles of ascorbate and urate respectively in maintaining the oxidative stability of infused Hb.
Keywords: Abbreviations; Hb; hemoglobin; OxyHb; oxyhemoglobin; MetHb; methemoglobin; deoxyHb; deoxyhemoglobin; Mb; myoglobin; HBOC; hemoglobin-based oxygen carrier; PolyHbBv; polymerized bovine Hb (commercial name Oxyglobin ®), LGO,; l; -gulonolactone oxidase; RBC; red blood cells; PEG; polyethylene glycolHemoglobin; Ascorbate; Urate; Peroxidase; Ferryl; Blood substitute
Microvascular experimental evidence on the relative significance of restoring oxygen carrying capacity vs. blood viscosity in shock resuscitation by Salazar Vazquez Beatriz Y. Salazar Vázquez; Reto Wettstein; Pedro Cabrales; Amy G. Tsai; Marcos Intaglietta (pp. 1421-1427).
The development of volume replacement fluids for resuscitation in hemorrhagic shock comprises oxygen carrying and non carrying fluids. Non oxygen carrying fluids or plasma expanders are used up to the transfusion trigger, and upon reaching this landmark either blood, and possibly in the near future oxygen carrying blood substitutes, are used. An experimental program in hemorrhagic shock using the hamster chamber window model allowed to compare the relative performance of most fluids proposed for shock resuscitation. This model allows investigating simultaneously the microcirculation and systemic reactions, in the awake condition, in a tissue isolated from the environment. Results from this program show that in general plasma expanders such as Ringer's lactate and dextran 70 kDa do not sufficiently restore blood viscosity upon reaching the transfusion trigger, causing microvascular collapse. This is in part restored by a blood transfusion, independently of the oxygen carrying capacity of red blood cells. These results lead to the proposal that effective blood substitutes must be designed to prevent microvascular collapse, manifested in the decrease of functional capillary density. Achievement of this goal, in combination with the increase of oxygen affinity, significantly postpones the need for a blood transfusion, and lowers the total requirement of restoration of intrinsic oxygen carrying capacity.
Keywords: Plasma expander; Blood viscosity; Oxygen carrying capacity; Blood substitute; Hemorrhagic shock; Microcirculation
Modulation of the NO/cGMP pathway reduces the vasoconstriction induced by acellular and PEGylated haemoglobin by Anna Caretti; Monica Fantacci; Dario Caccia; Michele Perrella; Kenneth C. Lowe; Michele Samaja (pp. 1428-1434).
Activation of the NO/cGMP pathway modulates smooth muscle cells relaxation and hence vasoconstriction, a major hindrance for the use of cell-free haemoglobin (Hb) as blood substitute, despite conjugation with 5-kDa maleimide poly(ethylene)-glycol (PEG) reduces vasoconstriction in vivo. We aimed at assessing how a recently developed PEGylated-Hb (Deoxy-PEGHb) and manipulation of the NO/cGMP pathway enable modulation of vasoconstriction in isolated rat hearts. Hearts were Langendorff-perfused with oxygenated Krebs–Henseleit (15 ml/min) while monitoring the coronary pressure (CPP) after injection (1 min) of 50 nM norepinephrine followed by a 1 μM Hb or Deoxy-PEGHb bolus, without altering the flow. Deoxy-PEGHb induced less vasoconstriction than Hb. Although the presence of PEG could contribute to vasoconstriction, Deoxy-PEGHb did not contain appreciable amounts of free PEG. Whereas reducing endothelial NO release by 0.2 mMl-NAME increased vasoconstriction, abolishing NO scavenging by Hb using its cyanomet derivative almost completely blunted it. Furthermore, maintaining intracellular cyclic GMP by inhibiting phosphodiesterase-5 with 0.02 mM sildenafil enabled control of Hb-induced vasoconstriction. We conclude that, although PEG-Hb represents a possible approach to limit Hb-induced vasoconstriction, manipulating the NO/cGMP pathway may provide a powerful way to circumvent this problem.
Keywords: Haemoglobin-based oxygen carrier; Haemoglobin PEGylation; Nitric oxide; Phosphodiesterase inhibitor; Vasoconstriction; Langendorff-perfused heart
Nanobiotechnological modification of hemoglobin and enzymes from this laboratory by Thomas Ming Swi Chang (pp. 1435-1440).
Polyhemoglobin is formed by the nanobiotechnological assembling of hemoglobin molecules into soluble nanodimension complex. A further step involves the nanobiotechnological assembly of hemoglobin, catalase and superoxide dismutase into a soluble nanodimension complex. This acts both as oxygen carrier and antioxidant to prevent the oxidative effects of hemoglobin. A further step is the preparation of nanodimension artificial red blood cells that contain hemoglobin and all the enzymes present in red blood cells. Other approaches include a polyhemoglobin–fibrinogen that acts as an oxygen carrier with platelet-like activity, and a polyhemoglobin–tyrosinase to retard the growth of a fatal skin cancer, melanoma.
Keywords: Abbreviations; Hb; Hemoglobin; MetHb; metHb; PEG; polyethylene glycol; PLA; polylactide; PolyHb; polyHb; RBC; red blood cells; CAT; catalase; SOD; superoxide dismutase; RES; Reticuloendothelial systemNanobiotechnology; Hemoglobin; Polyhemoglobin; Catalase; Superoxide dismutase; Nanomedicine; Blood substitutes; Fibrinogen; Tyrosinase; Melanoma
NO and CO binding profiles of hemoglobin vesicles as artificial oxygen carriers by Hiromi Sakai; Atsushi Sato; Peter Sobolewski; Shinji Takeoka; John A. Frangos; Koichi Kobayashi; Marcos Intaglietta; Eishun Tsuchida (pp. 1441-1447).
Hemoglobin vesicles (HbVs) are artificial oxygen carriers encapsulating purified and concentrated Hb solution in phospholipid vesicles (liposomes). We examined in-vitro reaction profiles of a formulation of HbV with NO and CO in anaerobic and aerobic conditions using stopped-flow spectrophotometry and a NO electrode. Reaction rate constants of NO to deoxygenated and oxygenated HbV were considerably smaller than those of cell-free Hb because of the intracellular NO-diffusion barrier. The reaction of CO with deoxygenated HbV was slightly slower than that of cell-free Hb solely because of the co-encapsulated allosteric effector, pyridoxal 5'-phosphate. The NO depletion in an aerobic condition in the presence of empty vesicles was monitored using a NO electrode, showing that the hydrophobic bilayer membrane of HbV, which might have higher gas solubility, does not markedly facilitate the O2 and NO reaction, and that the intracellular Hb is the major component of NO depletion. In conclusion, HbV shows retarded gas reactions, providing some useful information to explain the absence of vasoconstriction and hypertension when they are intravenously injected.
Keywords: Abbreviations; HbV; hemoglobin vesicles; RBC; red blood cells; k; '; on; (; NO; ); apparent NO binding rate constant; k; '; on; (; CO; ); apparent CO binding rate constant; k; '; OX; (; NO; ); apparent oxidation rate by NO; PLP; pyridoxal 5'-phosphate; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphatidylcholineBlood substitutes; Artificial red cells; Liposome; NO; CO; Vasoconstriction
Octamers and nanoparticles as hemoglobin based blood substitutes by Véronique Baudin-Creuza; Cédric Chauvierre; Elisa Domingues; Laurent Kiger; Liliane Leclerc; Corinne Vasseur; Chantal Célier; Michael C. Marden (pp. 1448-1453).
Progress in developing a blood substitute is aided by new biotechnologies and a better understanding of the circulatory system. For Hb based solutions, there is still a debate over the best set of fundamental parameters concerning the oxygen affinity which is correlated with the oxidation rate, the cooperativity, the transporter size, and of course the final source of material. Genetic engineering methods have helped discover novel globins, but not yet the quantity necessary for the high demand of blood transfusions. The expanding database of globin properties has indicated that certain individual parameters are coupled, such as the oxygen affinity and the oxidation rate, indicating that one must accept a compromise of the best parameters. After a general introduction of these basic criteria, we will focus on two strategies concerning the size of the oxygen transporter: Hb octamers, and Hb integrated within a nanoparticle.
Keywords: Hemoglobin octamer; Nanoparticle; Cooperativity; Oxidation
Polymeric nanoparticles for hemoglobin-based oxygen carriers by Anna Maria Piras; Alberto Dessy; Federica Chiellini; Emo Chiellini; Claudio Farina; Massimiliano Ramelli; Elena Della Valle (pp. 1454-1461).
This article reports on the current status of the research on blood substitutes with particular attention on hemoglobin-based oxygen carriers (HBOCs). Insights on the physiological role of hemoglobin are reported in the view of the development of both acellular and cellular hemoglobin-based oxygen carriers. Attention is then focused on biocompatible polymeric materials that find application as matrices for cellular based HBOCs and on the strategies employed to avoid methemoglobin formation. Results are reported regarding the use of bioerodible polymeric matrices based on hemiesters of alternating copolymer (maleic anhydride- co-butyl vinyl ether) for the preparation of hemoglobin loaded nanoparticles.
Keywords: Blood substitute; Hemoglobin-based oxygen carrier; Bioerodible polymeric nanoparticle
Hb(αα,ββ): A novel fusion construct for a dimeric, four-domain hemoglobin by Gianna Panetta; Alessandro Arcovito; Veronica Morea; Andrea Bellelli; Adriana Erica Miele (pp. 1462-1470).
Hemoglobin-based blood substitutes are one of the options available to derive a resuscitating fluid taking into account clinical and physiological demands. In this paper we investigated a novel protein, Hb(αα,ββ) obtained as a combination of two homodimers α2 and β2 both derived from a fusion gene containing two alfa chains or two beta chains, each respectively coupled via a specific linker. The construct here described is thus a novel heterodimeric hemoglobin carrying four heme groups. The protein cannot dissociate into dimers, as demonstrated by its absence of reactivity versus haptoglobin, and is expected to have a relatively long circulating half-life. The modification does not increase the autoxidation rate, but increases the oxygen affinity, due to a destabilization of the T quaternary state. Characterization of the biochemical properties of this protein in comparison with HbA is reported.
Keywords: Protein engineering; Gene fusion; Blood substitute; Oxygen carrier
Enhancing stability and expression of recombinant human hemoglobin in E. coli: Progress in the development of a recombinant HBOC source by Philip E. Graves; Douglas P. Henderson; Molly J. Horstman; Brian J. Solomon; John S. Olson (pp. 1471-1479).
The commercial feasibility of recombinant human Hb (rHb) as an O2 delivery pharmaceutical is limited by the production yield of holoprotein in E. coli. Currently the production of rHb is not cost effective for use as a source in the development of third and fourth generation Hb-based oxygen carriers (HBOCs). The major problems appear to be aggregation and degradation of apoglobin at the nominal expression temperatures, 28–37 °C, and the limited amount of free heme that is available for holohemoglobin assembly. One approach to solve the first problem is to inhibit apoglobin precipitation by a comparative mutagenesis strategy to improve apoglobin stability. α Gly15 to Ala and β Gly16 to Ala mutations have been constructed to increase the stability of the α helices of both subunits of HbA, based on comparison with the sequences of the more stable sperm whale hemoglobin subunits. Fetal hemoglobin is also known to be more stable than human HbA, and sequence comparisons between human β and γ (fetal Hb) chains indicate several substitutions that stabilize the α1β1 interface, one of which, β His116 to Ile, increases resistance to denaturation and enhances expression in E. coli. These favorable effects of enhanced globin stability can be augmented by co-expression of bacterial membrane heme transport systems to increase the rate and extent of heme uptake through the bacterial cell membranes. The combination of increased apoglobin stability and active heme transport appear to enhance holohemoglobin production to levels that may make rHb a plausible starting material for all extracellular Hb-based oxygen carriers.
Keywords: Hemoglobin-based oxygen carrier; HBOC; Globin folding; Globin expression; Protein engineering; Heme transport