Oxygen Affinity Of Hb Research Articles

The biologic implications of a rare hemoglobin mutant that decreases oxygen affinity.

Blood from seven newborns, a 13-y-old, and seven adult family members with a suspected hemoglobinopathy because of unexplained cyanosis was obtained for analysis to determine Hb oxygen affinity and to characterize and quantify the Hb variants. Their oxygen saturation was 76 to 84%. The P(50) was 30.3 +/- 2.9 for the newborns and 32.5 +/- 2.6 mm Hg for their related adults. In the same order, the plasma erythropoietin was 7.4 +/- 2.9 and 15.9 +/- 3.7 mU/mL, whereas 2,3-diphosphoglycerate was 16.1. +/- 2.9 and 15.9 +/- 3.7 micromol/g Hb. In four of the newborns with increased P(50), the mother had a normal P(50) (27 mm Hg), which indicated a greater maternal oxygen affinity than the fetus with no adverse effects on the fetus. Genetic analysis of alpha-globin genes demonstrated a heterozygous mutation on the alpha2 gene [alpha94(G1)Asp-->His] for each of the newborns and their related adults. The same mutation was found on the alpha1 gene in an adolescent and her father. The mRNA measurements showed that the alpha2- to alpha1-globin mRNA mean ratio was 2.5, alpha2 mutant globin mRNA/total alpha2-globin mRNA was 45.0%, whereas the alpha1 mutant globin mRNA/total alpha1-globin mRNA was 37.8%. The level of alpha2 mutant globin/total alpha-globin was 27.3 +/- 1%, and alpha1 mutant globin/total alpha-globin was 23.8 +/- 1%. The percentage of synthesized alpha2 and alpha1 mutant globins was 27.5 +/- 2 and 26.1 +/- 1, respectively. The ratio of the alpha2/alpha1 mutant globins was 1.1, which corresponded to a ratio at the mRNA level of alpha2/alpha1 of 2.5 +/- 0.5, which suggested that there is a less efficient translation of the alpha2 mRNA than alpha1 mRNA. The reversal of the physiologic fetomaternal oxygen affinity had no effects on fetal development.

Sudden infant death syndrome: a preconditioning approach to acute arterial hypoxemia

The blood hemoglobin F (HbF) concentration increases in response to chronic arterial hypoxemia and is abnormally elevated in sudden infant death syndrome (SIDS) post-mortem indicating a need for greater oxygen affinity of hemoglobin (Hb) or diminished oxygen usage by tissues or both.Modifying Hb oxygen affinity in rats revealed that increased, rather than decreased, hemoglobin-oxygen affinity permitted survival at greatly reduced environmental oxygen pressures equivalent to high altitude. Decreased Hb-oxygen affinity resulted in bradycardia 5–10 minutes before death. Cardiorespiratory recordings from infants dying suddenly and unexpectedly at home demonstrated cardiovascular failure with hypotension and bradycardia, rather than a cessation of breathing.A fall in blood pressure and acidosis due to hypoxemia in combination with reduced arterial oxygen saturation leads to circulatory failure, heart failure and death.It is speculated that the final mechanism of SIDS mimics failure to survive at high altitudes and very low environmental oxygen pressures when low arterial oxygen pressures combine with decreased Hb-oxygen affinity lead to severe hypoxemia and death.

Increased Glutathionyl Hemoglobin in Diabetes Mellitus and Hyperlipidemia Demonstrated by Liquid Chromatography/Electrospray Ionization-Mass Spectrometry

Erythrocytes contain a large amount of glutathione (GSH), which protects cells from oxidative injury. The purpose of this study was to examine whether hemoglobin (Hb) is modified with glutathione by oxidation of the thiol groups in diabetes mellitus and hyperlipidemia, and to determine the oxygen affinity of glutathionyl Hb. Hb samples obtained from patients with type 2 diabetes, patients with hyperlipidemia, and healthy subjects were analyzed by liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). Glutathionyl Hb was synthesized in vitro by incubating Hb with GSH. The oxygen affinity of glutathionyl Hb was determined by measuring its oxygen dissociation curve. We first demonstrated that the concentration of glutathionyl Hbbeta chains is markedly increased in the diabetic patients and hyperlipidemic patients compared with healthy subjects. The in vitro synthesis of glutathionyl Hb by incubation of Hb with GSH was enhanced by adding H(2)O(2), a reactive oxygen species, into the incubation solution. The glutathionyl Hb prepared in vitro by incubating Hb with GSH showed a marked increase in oxygen affinity and a marked decrease in the Hill coefficient compared with Hb incubated without GSH. Glutathionyl Hb may be useful as a clinical marker of oxidative stress. The increased concentrations of glutathionyl Hb with high oxygen affinity and low cooperativity in diabetes and hyperlipidemia may lead to reduced tissue oxygen delivery.

RSR13, a Synthetic Allosteric Modifier of Hemoglobin, Improves Myocardial Recovery Following Hypothermic Cardiopulmonary Bypass

Background —During hypothermic blood cardioplegia, oxygen delivery to myocytes is minimal with ineffective anaerobic metabolism predominating. RSR13, 2-[4-[[(3,5-dimethylanilino) carbonyl]methyl]phenoxy]-2-methylpropionic acid, a synthetic allosteric modifier of hemoglobin (Hb), increases release of oxygen from Hb, increasing oxygen availability to hypoxic tissues, and reverses the hypothermia-dependent increase in Hb oxygen affinity. We studied recovery of myocardial mechanical and metabolic function and examined myocardial morphology after cardioplegia, comparing RSR13 (1.75 mmol/L)-supplemented blood (RSR13-BC) to standard blood cardioplegia (BC). Methods and Results —Twelve dogs underwent 15 minutes of 37°C global ischemia on cardiopulmonary bypass, followed by 75 minutes of hypothermic cardioplegia (13°C) with either BC (n=6) or RSR13-BC (n=6). There were no differences in baseline function between groups. Cardiac function was assessed after 30 minutes of 37°C reperfusion (BC versus RSR13-BC, respectively) by measuring: % return to normal sinus rhythm (0/100%), % of baseline+dP/dt (33.7±1.7/76.3±1.9), % of baseline−dP/dt (26.6±2.0/81.1±1.6), stroke volume (3.5±0.5/7.1±0.9 mL), cardiac output (340±20/880±40.3 mL/min), and LVEDP (11.3±2.2/0.3±2.9 mm Hg). Postischemic oxidative and metabolic parameters including myocardial lactate, pyruvate, ATP content, and percent water content also were determined. Histological analysis demonstrated preservation of endothelial and myocyte morphology in hearts receiving RSR13-BC compared with BC. Conclusions —These results indicate that in the setting of hypothermic cardiopulmonary bypass, RSR13 improves recovery of myocardial mechanical and metabolic function compared with standard hypothermic BC. Findings from this study suggest that RSR13-BC, by decreasing hemoglobin oxygen affinity, improves oxidative metabolism and preserves cellular morphology, resulting in significantly improved contractile recovery on reperfusion.

RSR13, a synthetic allosteric modifier of hemoglobin, improves myocardial recovery following hypothermic cardiopulmonary bypass.

During hypothermic blood cardioplegia, oxygen delivery to myocytes is minimal with ineffective anaerobic metabolism predominating. RSR13, 2-[4-[[(3,5-dimethylanilino) carbonyl]methyl]phenoxy]-2-methylpropionic acid, a synthetic allosteric modifier of hemoglobin (Hb), increases release of oxygen from Hb, increasing oxygen availability to hypoxic tissues, and reverses the hypothermia-dependent increase in Hb oxygen affinity. We studied recovery of myocardial mechanical and metabolic function and examined myocardial morphology after cardioplegia, comparing RSR13 (1.75 mmol/L)-supplemented blood (RSR13-BC) to standard blood cardioplegia (BC). Twelve dogs underwent 15 minutes of 37 degrees C global ischemia on cardiopulmonary bypass, followed by 75 minutes of hypothermic cardioplegia (13 degrees C) with either BC (n=6) or RSR13-BC (n=6). There were no differences in baseline function between groups. Cardiac function was assessed after 30 minutes of 37 degrees C reperfusion (BC versus RSR13-BC, respectively) by measuring: % return to normal sinus rhythm (0/100%), % of baseline+dP/dt (33.7+/-1.7/76.3+/-1.9), % of baseline-dP/dt (26.6+/-2.0/81.1+/-1.6), stroke volume (3.5+/-0.5/7.1+/-0.9 mL), cardiac output (340+/-20/880+/-40.3 mL/min), and LVEDP (11.3+/-2.2/0. 3+/-2.9 mm Hg). Postischemic oxidative and metabolic parameters including myocardial lactate, pyruvate, ATP content, and percent water content also were determined. Histological analysis demonstrated preservation of endothelial and myocyte morphology in hearts receiving RSR13-BC compared with BC. These results indicate that in the setting of hypothermic cardiopulmonary bypass, RSR13 improves recovery of myocardial mechanical and metabolic function compared with standard hypothermic BC. Findings from this study suggest that RSR13-BC, by decreasing hemoglobin oxygen affinity, improves oxidative metabolism and preserves cellular morphology, resulting in significantly improved contractile recovery on reperfusion.

Crystallization and X-ray diffraction data analysis of human deoxyhaemoglobin A0 fully stripped of any anions

In this work, initial crystallographic studies of human haemoglobin (Hb) crystallized in isoionic and oxygen-free PEG solution are presented. Under these conditions, functional measurements of the O(2)-linked binding of water molecules and release of protons have evidenced that Hb assumes an unforeseen new allosteric conformation. The determination of the high-resolution structure of the crystal of human deoxy-Hb fully stripped of anions may provide a structural explanation for the role of anions in the allosteric properties of Hb and, particularly, for the influence of chloride on the Bohr effect, the mechanism by which Hb oxygen affinity is regulated by pH. X-ray diffraction data were collected to 1.87 A resolution using a synchrotron-radiation source. Crystals belong to the space group P2(1)2(1)2 and preliminary analysis revealed the presence of one tetramer in the asymmetric unit. The structure is currently being refined using maximum-likelihood protocols.

Effect of high altitude and in vivo adenosine/(&bgr;)-adrenergic receptor blockade on ATP and 2,3BPG concentrations in red blood cells of avian embryos

In chick embryos, developmental changes of the blood oxygen tension control hemoglobin (Hb) oxygen affinity via modulation of ATP and 2, 3BPG concentrations in red blood cells. Hypoxia, which is a normal developmental condition for late chick embryos, causes a decrease of the red cell ATP concentration (and increase of red cell oxygen affinity) as well as activation of 2,3BPG synthesis via cyclic AMP-dependent signaling. Adenosine and catecholamines have been implicated as signaling substances in these red cell responses. To assess the extent to which adenosine and catecholamines are involved in vivo in the control of red cell ATP/2,3BPG concentrations, day 13 chick embryos were treated for 24 h with adenosine A(2) and/or (&bgr;)-adrenergic receptor blockers and red cell ATP and 2,3BPG levels were determined. The data suggest that adaptive effects later in development in chick embryos induced by adenosine and catecholamines are vital. We have also tested whether avian embryos of the free-living, high-altitude, native white-tailed ptarmigan (Lagopus leucurus) alter their organic phosphate pattern in red cells in response to incubation at different altitudes. Embryos incubated at 3600-4100 m decrease their red cell ATP concentration much more rapidly than embryos of the same clutch incubated at 1600 m. From these data it can be inferred that the oxygen affinity of high altitude embryos will be adjusted to the altitude at which the eggs are incubated.

Storage parameters affecting red blood cell survival and function after transfusion

A LTHOUGH OUR understanding of the various parameters influencing the collection, separation, and storage of blood cells has progressively improved during past decades, with the exception of apheresis technologies, there have been surprisingly few advances in the practice of routine blood collection and component separation. This review attempts to summarize recent developments and to suggest steps that might be taken to improve the quality of stored red blood cells (RBCs) to maximize their safety and their benefits for the transfused recipient. The RBC is a highly specialized cell .without a nucleus and devoid of protein synthesis. This means that it has to rely on its original contents of enzymes and other proteins for the whole of its limited lifetime. In addition, because it lacks mitochondria, it cannot use oxidative phosphorylation for adenosine triphosphate (ATP) regeneration but has to rely on metabolizing glucose to lactate. This results in a net gain of 2 moles of ATP, 2 H +, and 2 moles of lactate per 1 mole of glucose metabol ized--an ineffective mechanism for energy supply compared with mitochondrial oxidation via the citric acid cycle. Approximately 10% of the metabolic flux passes by way of the pentose phosphate pathway in which oxygen is consumed and carbon dioxide formed. A simplified metabolic outline is presented in Figure 1, which also illustrates some substances that can be used to influence the metabolism under in vitro storage conditions. The most important function of RBCs, oxygen transport, exposes the cell to toxic oxygen species, free oxygen radicals such as O 2 and OH', and to hydrogen peroxide. For protection against denaturation by peroxidation of the lipids in the membrane bilayer, its proteins and enzymes, a number of antioxidants such as vitamin E are available in both the liquid and cellular components of the blood, and redox machinery is provided within the red cell. A highly flexible membrane is essential for rapid uptake and release of oxygen and carbon dioxide. In this way, the RBC will have a great contact surface area between its own membrane and that of the capillaries as it rolls through these vessels like a small wheel with a big flat tire. This rolling also helps mix the viscous intracellular content. The degree of saturation of the RBC hemoglobin (Hb) and its dependence on the surrounding partial pressure of oxygen (pO2), can be graphically represented by a sigmoidal curve, the oxygen dissociation curve (ODC). The inflection point of this curve is at 50% Hb saturation. This point, the p50, normally has a value of approximately 3.6 kPa (27 mm Hg). The factors that most potently influence p50 are pH (H + and CO2) and 2,3-diphosphoglycerate (2,3-DPG). As pH is lowered, the ODC is shifted to the right, which means that the affinity of Hb for oxygen is reduced, oxygen is less readily bound and more readily released. In the normally functioning lung, pO2,is sufficiently high to result in nearly complete saturation, at both normal and moderately rightshifted ODC. In the peripheral circulation, the pH is lower than in the lungs, which promotes oxygen release. If the ODC is left-shifted, less oxygen will be delivered at a certain pO2. Because of competition between 2,3-DPG and oxygen at the same site on the Hb molecule, an increased concentration of 2,3-DPG will cause reduced oxygen affinity and a right shift of the ODC. Patients with chronic anemia usually have an increased erythrocyte 2,3-DPG concentration and a right-shifted ODC, which seems to give some compensation for the lower concentration of circulating cellular Hb. All currently used methods for the liquid storage of RBCs result in depletion of 2,3-DPG, generally after I to 2 weeks of storage. These RBCs will have a left-shifted ODC, greater oxygen affinity, and may supply less oxygen to the tissues. Cells with depleted 2,3-DPG will normalize within 48 to 72 hours after transfusion. 1-3

Additive Effects of β Chain Mutations in Low Oxygen Affinity Hemoglobin βF41Y,K66T

In order to decrease significantly the oxygen affinity of human hemoglobin, we have associated the mutation betaF41Y with another point mutation also known to decrease the oxygen affinity of Hb. We have synthesized a recombinant Hb (rHb) with two mutations in the beta chains: rHb betaF41Y,K66T. In the absence of 2, 3-diphosphoglycerate, additive effects of the mutations are evident, since the doubly mutated Hb exhibits a larger decrease in oxygen affinity than for the individual single mutations. In the presence of 2,3-diphosphoglycerate, the second mutation did not significantly increase the P(50) value relative to the single mutations. However, the kinetics of CO binding still indicate combined effects on the allosteric equilibrium, as evidenced by more of the slow bimolecular phase characteristic of binding to the deoxy conformation. Dimer-tetramer equilibrium studies indicate an increase in stability of the mutants relative to rHb A; the double mutant rHb betaF41Y, K66T at pH 7.5 showed a K(4,2) value of 0.26 microM. Despite the lower oxygen affinity, the single mutant betaF41Y and double mutant betaF41Y,K66T show only a moderate increase of 20% in the autoxidation rate. These mutations are thus of interest in developing a Hb-based blood substitute.

Damage to hemoglobin by radiation-generated serum albumin radicals

We have studied the effects of the interaction of radiation generated human serum albumin radicals (HSA ) with human hemoglobin molecules (Hb). Diluted Hb aqueous solutions were irradiated under N 2O or argon without HSA and in the presence of HSA. Analysis of Hb absorbance spectra in the visible range, cross-linking of HSA radicals with Hb molecules and functional properties of Hb were investigated. The degree of Hb destruction estimated on the basis of changes in the absorption spectra indicated that the effectiveness of HSA radicals generated under N 2O for Hb destruction was approximately equal to that of OH radicals. In this case mainly OH radicals formed the secondary HSA radicals. However, during the irradiation Hb + HSA under argon the presence of equivalent amounts of oxidizing and reducing products of water radiolysis lowers the degree of Hb destruction. Some reactions of HSA radicals with Hb molecules lead to the formation of covalent bonds between the molecules of both proteins. The following types of hybrids could be distinguished: Hb monomer-HSA, Hb dimer-HSA and higher aggregates. Structural changes of Hb by HSA radicals were reflected by alterations in the oxygen affinity (increase) and cooperativity (decrease) of Hb. The results obtained indicate that in the experimental systems studied, the HSA radical reactions with Hb molecules are favoured over recombination reactions of HSA radicals. On this basis one can suggest that in the studied systems Hb plays the role of an acceptor of radical energy located on HSA.

Changes in the functional properties of bovine hemoglobin induced by covalent modification with polyethylene glycol.

Polyethylene glycol conjugation to proteins and peptides (PEGylation) has been shown to promote increased retention time in the circulation as well as to blunt immune or allergic reactions. PEGylated bovine hemoglobin (PEG-Hb) is being explored in human clinical trials as an oxygen delivering agent for the sensitization of solid tumors to radiation therapy. In this study the functional properties of PEG-Hb were compared to those of bovine hemoglobin (Hb), the mutant human hemoglobin Rothchild and bovine hemoglobin crosslinked between the beta chains. The rate of heme transfer from Hb to serum albumin at pH 9.0 was greatly increased by PEGylation, suggesting destabilization of the heme-globin linkage and of the bonds between alpha beta dimers. Measurement of oxygen binding equilibrium showed that the oxygen affinity of Hb became unusually dependent on temperature and Hb concentration after PEGylation. Evidence is presented to suggest that PEGylation of lysine beta-81 at the entrance to the central cavity of the Hb tetramer might be responsible for these observations. The alterations of the functional properties of Hb induced by PEGylation are consistent with the beneficial effects of PEG-Hb in exchange transfusion and radiation sensitization models of human conditions.

The bidirectional effect of vanadyl ion on the oxygen affinity of human hemoglobin

The bidirectional effects of vanadyl on the oxygen affinity of hemoglobin depend on the mole ratio of vanadyl to Hb( R). In low R, the vanadyl ion increases Hb's oxygen affinity due to DPG hydrolysis. The lowered oxygen affinity in higher R is mainly due to the reactive-oxygen-species, probably superoxide, induced oxidation of Fe(II)–Hb to Fe(III)–Hb. The conformation change due to vanadyl binding contribute also to the lowered oxygen binding, but is monotonously decreasing with increasing of R values.

Tetramer-dimer equilibrium of oxyhemoglobin mutants determined from auto-oxidation rates.

One of the main difficulties with blood substitutes based on hemoglobin (Hb) solutions is the auto-oxidation of the hemes, a problem aggravated by the dimerization of Hb tetramers. We have employed a method to study the oxyHb tetramer-dimer equilibrium based on the rate of auto-oxidation as a function of protein concentration. The 16-fold difference in dimer and tetramer auto-oxidation rates (in 20 mM phosphate buffer at pH 7.0, 37 degrees C) was exploited to determine the fraction dimer. The results show a transition of the auto-oxidation rate from low to high protein concentrations, allowing the determination of the tetramer-dimer dissociation coefficient K4,2 = [Dimer] 2/[Tetramer]. A 14-fold increase in K4,2 was observed for addition of 10 mM of the allosteric effector inositol hexaphosphate (IHP). Recombinant hemoglobins (rHb) were genetically engineered to obtain Hb with a lower oxygen affinity than native Hb (Hb A). The rHb alpha2beta2 [(C7) F41Y/(G4) N102Y] shows a fivefold increase in K4,2 at pH 7.0, 37 degrees C. An atmosphere of pure oxygen is necessary in this case to insure fully oxygenated Hb. When this condition is satisfied, this method provides an efficient technique to characterize both the tetramer-dimer equilibrium and the auto-oxidation rates of various oxyHb. For low oxygen affinity Hb equilibrated under air, the presence of deoxy subunits accelerates the auto-oxidation. Although a full analysis is complicated, the auto-oxidation studies for air equilibrated samples are more relevant to the development of a blood substitute based on Hb solutions. The double mutants, rHb alpha2beta2 [(C7) F41Y/(G4) N102A] and rHb alpha2beta2 [(C7) F41Y/(E10) K66T], show a lower oxygen affinity and a higher rate of oxidation than Hb A. Simulations of the auto-oxidation rate versus Hb concentration indicate that very high protein concentrations are required to observe the tetramer auto-oxidation rate. Because the dimers oxidize much more rapidly, even a small fraction dimer will influence the observed oxidation rate.

Amino Acids Responsible for Decreased 2, 3-Biphosphosphoglycerate Binding to Fetal Hemoglobin

AbstractTo clarify the role of γN-terminal Gly, γ5 Glu, and γ143 Ser in 2, 3-biphosphosphoglycerate (BPG) binding to fetal hemoglobin (Hb F ), we engineered and produced normal human Hb F and two Hb F variants (Hb F γG1V, γS143H, and Hb F γG1V, γE5P, γS143H) using a yeast expression system and then compared their oxygen-binding properties with those of native human Hb F and adult Hb (Hb A). Oxygen affinity of Hb F γG1V, γS143H in the absence of 2, 3-BPG was slightly higher than that of normal Hb F. The decrease in oxygen affinities for Hb F γG1V, γS143H with increasing 2, 3-BPG concentrations was larger than that of normal Hb F, but significantly less than that of Hb A. In contrast, oxygen affinities of Hb F γG1V, γE5P, γS143H in the absence and presence of 2, 3-BPG were much lower than those of Hb F γG1V, γS143H and were similar to those of Hb A. These results indicate that differences between Pro and Glu at the A2 position in the A helix in Hb A and Hb F, respectively, are critical for reduced binding of 2, 3-BPG to Hb F, even though β5 Pro does not interact directly with 2, 3-BPG in Hb A. Hb F variants such as Hb F γG1V, γE5P, γS143H, which exhibit reduced oxygen affinity, should facilitate design of efficient antisickling fetal Hb variants for potential use in gene therapy for sickle cell disease.

Changes in Whole Blood Oxygen Affinity and Eggshell Permeability in High Altitude Chickens Translocated to Sea Level

High altitude (HA; n = 5) chickens (Gallus gallus) with a high oxygen hemoglobin (Hb) affinity were transported from their birthplace (Puno, Perú 4,000 m) down to sea level (Lima, Perú). The in vivo whole blood oxygen affinity (P50) and the eggshell permeability (P) were studied after several months living at sea level and in the first (F1) and second (F2) generations born at sea level. Our approach was to analyze changes in Hb affinity and eggshell permeability, considered as indicators of HA adaptation in birds. Our results show an increase of the P50 values (a decrease in Hb affinity) towards sea-level values. The results in P indicate that this variable increases towards sea level values in the F2 generation. We conclude that in the Andean chicken, a relative “newcomer” to high altitude (no more than 500 years), neither the Hb affinity for oxygen nor the eggshell permeability are invariable indicators of HA adaptation, in contrast with other native high altitude mammals and birds.

A Transgenic Mouse Model of Hemoglobin S Antilles Disease

AbstractHemoglobin (Hb) S Antilles is a naturally occurring form of sickling human Hb but causes a more severe phenotype than Hb S. Two homozygous viable Hb S Antilles transgene insertions from Tg58Ru and Tg98Ru mice were bred into MHOAH mice that express high oxygen affinity (P50 ∼24.5 mm Hg) rather than normal (P50 ∼40 mm Hg) mouse Hbs. The rationale was that the high oxygen affinity MHOAH Hb, the lower oxygen affinity of Hb S Antilles than Hb S (P50 ∼40 v 26.5 mm Hg), and the lower solubility of deoxygenated Hb S Antilles than Hb S (∼11 v 18 g/dL) would favor deoxygenation and polymerization of human Hb S Antilles in MHOAH mouse red blood cells (RBCs). The Tg58 × Tg98 mice produced have a high and balanced expression (∼50% each) of hα and hβS Antilles globins, 25% to 35% of their RBCs are misshapen in vivo, and in vitro deoxygenation of their blood induces 30% to 50% of the RBCs to form classical looking, elongated sickle cells with pointed ends. Tg58 × Tg98 mice exhibit reticulocytosis, an elevated white blood cell count and lung and kidney pathology commonly found in sickle cell patients, which should make these mice useful for experimental studies on possible therapeutic intervention of sickle cell disease.

O2 Binding and Dissociation and Ligand Exchange Reaction of O2 with CO in Polymer Composite Films of Hemoglobin

A polymer composite film of hemoglobin (Hb–polymer film) was prepared by the casting of an Hb–polymer mixed solution (weight ratio of Hb to polymer is 1 to 1). The percentages of O2 and CO saturation of the Hb–dextran film were 46% and 70%, respectively. In the Hb solution, 100% saturation was observed for both ligands, and a humidified Hb–dextran film also showed 100% saturation. Water molecules would provide flexibility to the matrix polymer and promote a structural change in the Hb from a tense state (T) to a relaxed state (R). Thus the ligand binding to the Hb in the polymer films was strongly affected by the degree of interaction of Hb with the matrix polymers and the physical properties of the polymers. Inositol hexaphosphate (IHP) worked as an allosteric effector even in the solid polymer film and lowered the oxygen affinity of Hb. The O2 transport through an Hb–polyethyleneimine (PEI) film with IHP showed the facilitated O2 transport in comparison with the film without IHP because of the high dissociation rate of O2 from Hb with IHP. © 1997 John Wiley & Sons, Ltd.

O2 Binding and Dissociation and Ligand Exchange Reaction of O2 with CO in Polymer Composite Films of Hemoglobin

A polymer composite film of hemoglobin (Hb–polymer film) was prepared by the casting of an Hb–polymer mixed solution (weight ratio of Hb to polymer is 1 to 1). The percentages of O2 and CO saturation of the Hb–dextran film were 46% and 70%, respectively. In the Hb solution, 100% saturation was observed for both ligands, and a humidified Hb–dextran film also showed 100% saturation. Water molecules would provide flexibility to the matrix polymer and promote a structural change in the Hb from a tense state (T) to a relaxed state (R). Thus the ligand binding to the Hb in the polymer films was strongly affected by the degree of interaction of Hb with the matrix polymers and the physical properties of the polymers. Inositol hexaphosphate (IHP) worked as an allosteric effector even in the solid polymer film and lowered the oxygen affinity of Hb. The O2 transport through an Hb–polyethyleneimine (PEI) film with IHP showed the facilitated O2 transport in comparison with the film without IHP because of the high dissociation rate of O2 from Hb with IHP. © 1997 John Wiley & Sons, Ltd.

2179 A phase Ib study to evaluate repeated daily intravenous doses of RSR 13 administered to cancer patients receiving concurrent radiation therapy

Purpose: In animal models RSR13, a synthetic allosteric modifier of hemoglobin (I-Ib), oxygenates and thus radiosensitizes hypoxic solid tumors by decreasing the oxygen (Or) affinity of Hb. A phase Ib open label, multi-center dose-frequency escalation study was conducted to evaluate the safety, tolerance, pharmacokinetics, and pharmacodynamics of repeated daily intravenous (IV) infusions of RSRl3 administered to cancer patients receiving palliative radiation therapy (RT).

Oxygen Transport by Fetal Bovine Hemoglobin

The functional properties of fetal bovine hemoglobin have been studied as a function of temperature, chloride and 2,3-diphosphoglycerate (DPG) concentration. The fetal bovine erythrocyte has six times the concentration of the allosteric modulator DPG compared with the adult cell, and yet the oxygen affinity of the fetal hemoglobin still exceeds that of the adult molecule at the respective physiological concentration of DPG and at physiological temperature. We find that the allosteric modulator strongly affects the enthalpy of oxygen for the fetal hemoglobin but not for the adult protein. We propose that this may be an important mechanism for the exchange of heat from mother to fetus. In particular, under stripped conditions the oxygen affinity of fetal bovine Hb is considerably higher than that of the adult hemoglobin. Due to the higher DPG concentration that characterizes fetal bovine erythrocytes this difference is almost abolished in the presence of the respective physiological concentration of DPG and at 20°C. However, on going from 20°C to 37°C, the difference in O 2affinity between the two hemoglobins is restored, as it should if oxygen has to be transferred from maternal to fetal blood, by virtue of the lower overall heat of oxygenation (Δ H) displayed by fetal Hb when in the presence of DPG at physiological concentration. This behavior is reminiscent of that of human fetal Hb and outlines the role of temperature and of its interplay with heterotropic ligands in the modulation of hemoglobin function to fully meet the physiological needs of the organism.