Oxygen Binding Properties Of Hemoglobin Research Articles

Cryopreserved red blood cells maintain allosteric control of oxygen binding when utilizing trehalose as a cryoprotectant

One of the most common life-saving medical procedures is a red blood cell (RBC) transfusion. Unfortunately, RBCs for transfusion have a limited shelf life after donation due to detrimental storage effects on their morphological and biochemical properties. Inspired by nature, a biomimetics approach was developed to preserve RBCs for long-term storage using compounds found in animals with a natural propensity to survive in a frozen or desiccated state for decades. Trehalose was employed as a cryoprotective agent and added to the extracellular freezing solution of porcine RBCs. Slow cooling (−1 °C min−1) resulted in almost complete hemolysis (1 ± 1 % RBC recovery), and rapid cooling rates had to be used to achieve satisfactory cryopreservation outcomes. After rapid cooling, the highest percentage of RBC recovery was obtained by plunging in liquid nitrogen and thawing at 55 °C, using a cryopreservation solution containing 300 mM trehalose. Under these conditions, 88 ± 8 % of processed RBCs were recovered and retained hemoglobin (14 ± 2 % hemolysis). Hemoglobin's oxygen-binding properties of cryopreserved RBCs were not significantly different to unfrozen controls and was allosterically regulated by 2,3-bisphosphoglycerate. These data indicate the feasibility of using trehalose instead of glycerol as a cryoprotective compound for RBCs. In contrast to glycerol, trehalose-preserved RBCs can potentially be transfused without time-consuming washing steps, which significantly facilitates the usage of cryopreserved transfusible units in trauma situations when time is of the essence.

Hemoglobin: A General Review

Hemoglobin (Hb) is red blood corpuscle pigment causing red color in blood with centrally located iron atom, responsible for oxygen transport having molecular weight of a tetramer of 64,458 g/mol. The oxygen binding properties of hemoglobin are affected by pH and presence of carbon dioxide. It cause different clinical problems due to its high or low concentration in blood. Poisoning of Hb can also occur by combination of Hb with carbon monoxide resulting in formation of carboxyhemoglobin. Besides carboxyhemoglobin, sulfhemoglobin and methemoglobin are also derivatives of Hb.

Improving Oxygen Binding of Desiccated Human Red Blood Cells

Desiccating human red blood cells (RBCs) to increase their storage life has been the subject of intense research for a number of years. However, drying RBCs invariably compromises their integrity and has detrimental effects on hemoglobin function due to autoxidation. We have previously demonstrated an RBC desiccation and rehydration process that preserves RBC antigenic epitopes better than frozen RBCs. This study expands on those observations by examining what effects this desiccation process has on RBC hemoglobin function with respect to oxygen binding properties. In this paper, we examined RBCs from normal donors which were desiccated to 25% moisture content and stored dry for 2 weeks at room temperature prior to rehydration with plasma followed by structural and functional studies. Our data showed that approximately 98% of the RBCs were intact upon rehydration based on hemolysis assays. Oxygen dissociation curves for the desiccated/rehydrated RBCs showed a left shift compared to fresh RBCs (pO2 = 17 mmHg vs. 26 mmHg, respectively). The desiccated/rehydrated RBCs also showed an increase in methemoglobin compared to fresh RBCs (4.5% vs 0.9%, respectively). 2,3-Diphosphoglycerate concentration of the desiccated/rehydrated RBCs was reduced by 20%. In conclusion, although this RBC dehydration process preserves RBC integrity and hemoglobin oxygen binding properties better than most other dehydration techniques described so far, further optimization and long-term studies are needed to make this procedure acceptable for human transfusion.

Studies of the blood antioxidant system and oxygen-transporting properties of human erythrocytes during 105-day isolation

Effects of strict 105-day isolation on the blood antioxidant status, processes in erythrocyte membranes, and oxygen-binding properties of hemoglobin were studied in six male volunteers (25 to 40 years old) in ground-based simulation of a mission to Mars (Mars-105 experiment). The study was performed with venous blood samples and red blood cells isolated from them, which were collected during the baseline data collection period, on days 35, 70, and 105 of the experiment, and on days 7 and 14–15 after its completion. Biochemical (determination of enzyme activity and thin-layer chromatography) and biophysical (laser interference microscopy and Raman spectroscopy) methods showed changes in the relative content of lipid and phospholipid fractions, suggesting an increase in the membrane microviscosity and the content of TBA-RP (active lipid peroxidation products interacting with thiobarbituric acid). A significant increase in the activity of glucose-6-phosphate dehydrogenase and superoxide dismutase and a reduction in the catalase activity was found, which indicates both reparative processes in red blood cells and imbalance between the amount of generated reactive oxygen species and antioxidant protection mechanisms in cells. The hemoglobin affinity for oxygen and the blood level of oxyhemoglobin also increased. It is assumed that the adaptation of the body to stresses experienced during and after the experiment can disturb the balance between the antioxidant defense systems. The latter, in turn, leads to peroxidation of membrane phospholipids, alteration in their content, increase in membrane microviscosity, and eventual disturbance of the gas-exchange function of red blood cells.

Purification, crystallization and preliminary X-ray analysis of haemoglobin from ostrich (Struthio camelus)

Haemoglobin is a tetrameric protein that carries oxygen from the lungs to tissues and carbon dioxide from tissues back to the lungs. The oxygen-binding properties of haemoglobin are regulated through the binding of allosteric effectors. The respiratory system of avian species is unique and complex in nature when compared with that of mammals. In avian species, inositol pentaphosphate (inositol-P(5)) is present in the erythrocytes of the adult and is thought to be the major factor responsible for the relatively high oxygen affinity of the whole blood. The ostrich (Struthio camelus) is a large flightless bird which contains inositol tetrakisphosphate (inositol-P(4)) in its erythrocytes and its whole blood oxygen affinity is higher. Efforts have been made to explore the structure-function relationship of ostrich haemoglobin. Ostrich haemoglobin was purified using ion-exchange chromatography. Haemoglobin crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant in 50 mM phosphate buffer pH 7.2. Data were collected using a MAR345 image-plate detector system. The crystals of ostrich haemoglobin diffracted to 2.2 A resolution. They belonged to the orthorhombic space group P2(1)2(1)2(1) with one whole biological molecule in the asymmetric unit; the unit-cell parameters were a = 80.93, b = 81.68, c = 102.05 A.

Root effect hemoglobins

The Root effect describes an extreme pH sensitivity expressed in the hemoglobins of certain fish, in which it plays a unique physiological role. This review describes our general understanding of the effect of protons on the oxygen binding properties of hemoglobin and the particular properties which characterize Root effect proteins. The development of our understanding of the molecular origins of this effect is outlined and the role played by our ever expanding knowledge of protein structure is highlighted. The present state of our knowledge is detailed.

Physiological and genetical adaptation to temperature in fish populations

The physiological and genetical adaptation of fishes to environmental temperatures has been studied by analyzing data concerning: (i) the oxygen binding properties of haemoglobin recorded during growth experiments on Atlantic cod, and (ii) the primary structure of haemoglobin (Hb) and lactate dehydrogenase (LDH) of several fish species living in polar and temperate areas. The results on the oxygen binding properties of cod's haemoglobin indicate that for this species a temperature of around 12°C is the most favourable one, irrespective of the haemoglobin genotype, and are in line with recent evidence challenging the existence of significant evolutionary differences between cod stocks in North Atlantic. The primary structures of both Hb and LDH from species living under temperate environments show a higher variability as compared to that from polar species, although the difference in the recurrent patterns of hydrophobicity between the two areas is much larger for Hb. These results highlight the dominant role of physiological and genetical factors in shaping the adaptation to temperature at the individual and at the species level, respectively.

Functional characterisation of Eskimo dog hemoglobin: I. Interaction of Cl- and 2,3-DPG and its importance to oxygen unloading at low temperature.

The oxygen binding properties of hemoglobin and some hematological parameters in Eskimo dogs (belonging to Canis lupus familiaris) in Ilulissat/Jacobshavn, Greenland were analysed. The average [2,3-DPG] and [Hb] (n = 16) were 3.14 +/- 0.34 mmol l-1 blood and 9.53 +/- 0.65 g dl-1 (1.49 mmol l-1), respectively, giving a stoichiometric ratio of 2.11 mol 2,3-DPG/mol Hb. Oxygen binding analysis carried out on hemolysate in HEPES buffer at 20 and 37 degrees C revealed a high oxygen affinity (1.2 mmHg at pH 7.4, 20 degrees C) in the desalted condition, which decreased markedly in the presence of chloride and 2,3-DPG. A low apparent equilibrium constant for the binding of 2,3-DPG (1.0 x 10(-5) mol l-1) was found at pH 7.2 and 20 degrees C in the absence of chloride. Moreover, we show that chloride ions have an additive effect on oxygen affinity in the concentration range 10-300 mmol l-1 in the presence of 3 mmol l-1 2,3-DPG at low pH and temperature (pH < 7.4 and 20 degrees C). This feature may be of physiological importance to oxygen unloading under acidotic conditions when tissue temperature is low. Thermodynamic analysis reveal that in the presence of 3 mmol l-1 2,3-DPG and 100 mmol l-1 chloride, the Eskimo dog hemoglobin exhibits a low heat of oxygenation, which places this animal close to arctic ruminants with respect to the influence of temperature on oxygen binding in vivo.

Synergistic modulation by chloride and organic phosphates of hemoglobin from bear ( Ursus arctos)

The oxygen binding properties of hemoglobin (Hb) from brown bear ( Ursus arctos) have been studied focussing on the effect of heterotropic ligands, and the behaviour has been compared with that of human HbA, taken as a prototype of mammalian Hbs. It has been observed that in bear Hb chloride ions and 2,3-diphosphoglyceric acid (Gri(2,3)P 2) can modulate the oxygen affinity in a synergistic way such that their individual effect is enhanced whenever they are both present in saturating amounts. The thermodynamic analysis of such a feature indicates that in bear Hb there are two classes of chloride binding sites, one acting synergistically with Gri(2,3)P 2 and another one, which likely overlaps with the organic phosphate interaction cleft, and is therefore fully operative only in the absence of Gri(2,3)P 2. The behaviour of the last site is similar to that observed in human HbA, where the effect of Cl − and Gri(2,3)P 2 is mutually exclusive. The interaction energy between chloride and Gri(2,3)P 2 synergistic binding sites appears to be O 2-linked so that the interplay may have a relevant physiological role in modulating the oxygen transport in brown bear. This behaviour is associated with a marked pH-dependence of the oxygenation enthalpy in bear Hb, such that under acidotic and hypercloruremic conditions, oxygen supply to peripheral tissues could be maintained essentially unaltered even under low temperature conditions.

A comparative study of the temperature dependence of the oxygen‐binding properties of mammalian hemoglobins

The effect of temperature on the oxygen-binding properties of hemoglobin (Hb) from ruminants, such as ox, reindeer, musk ox, mouflon and egyptian water buffalo is compared to that of human adult Hb (HbA). A striking difference emerges where in the presence of chloride ions and in the absence of 2,3-diphosphoglycerate [Gri(2,3)P2] a strongly reduced exothermic oxygenation process is observed for all ruminant Hb investigated with respect to HbA. Next, in the presence of physiological concentrations of Gri(2,3)P2, HbA displays a less exothermic oxygenation process, with values tending toward those observed in ruminant Hb [where Gri(2,3)P2 is not a physiological effector and for which the addition of Gri(2,3)P2 has essentially no effect on the oxygenation enthalpy]. Different from HbA, the intrinsically less exothermic oxygen binding seems to be independent of the experimental conditions for ruminant Hb, underlying specific structural characteristics which might be responsible for this feature.

Materials Biotechnology and Blood Substitutes

Blood is a dispersion of formed elements in an aqueous colloid. The combined mass of the formed elements of blood measure on average 30 ml per kg body weight, or about the same weight as the liver. The colloidal phase of blood contains numerous organic factors that play important primary and supporting roles in homeostasis, including immune surveillance, coagulation, and nutrient transport.Erythrocytes (red blood cells) are the principle formed elements and provide the life-sustaining function, in conjunction with the heart, lungs, blood vessels and kidneys, of transporting and protecting the oxygen-carrying pigment, hemoglobin, to the tissues. The oxygen-binding properties of hemoglobin are sensitive to factors such as the cooperative effects of O2binding, pH and CO2levels, and the presence of other metabolic intermediates such as 2,3-diphosphoglycerate. The synergistic effects of these factors produce a well-known sigmoidal curve plot of the relationship between oxygen affinity and the partial pressure of oxygen (pO2): there is high oxygen affinity in the lung where the pO2is high, and a low oxygen affinity in the tissues, where the pO2is low. Uptake and delivery of oxygen by hemoglobin is associated with considerable spatial rearrangement of the hemoglobin molecule.Blood is a non-Newtonian suspension. Its viscosity is a function of both the vascular diameter and the concentration of erythrocytes. At a normal hematocrit of 40%, the viscosity of blood ranges between 2 and 4 Pa s as measured in tubes ranging 10–1,500 μm diameter. The osmolality of blood serum is 275–295 mOsm/1.

Comparative least-squares analysis of hemoglobin oxygen equilibrium curves

The oxygen-binding properties of hemoglobin have been studied at 600 microM protein concentration with organic phosphate, and analyzed by a series of different nonlinear least-squares analysis methods to determine whether reports of negligibly small values of the third overall Adair parameter, A3, are consequences of the data or a product of the data analysis. Data from other laboratories were analyzed as well. The single most important factor in creating a measurement that yields a small A3 is the use of equally weighted fitting in the Adair equation, while end-weighted fitting generally yields a larger A3. Endpoint extrapolation is ruled out as a major cause of abnormal A3 values. Monte Carlo simulations of the 600 microM results suggest that, if a small A3 were present, end weighting is at least as sensitive to a small A3 as equal weighting. We conclude that equally weighted fitting of the tetrameric Adair equation is unable to resolve the upper asymptote of the oxygen-binding data, resulting in an unusually small value for A3.

Blood Gas Transport Properties in Endurance-Trained Athletes Living at Different Altitudes

Hemoglobin oxygen binding properties and acid-base status were investigated in Colombian athletes (A) and controls (C) from Cali (C-1000 m) and Bogotá (B-2600 m). [Hb] and Hct values were not influenced by altitude, but Hct was lower in the blood of athletes (in Cali 2.6%, in Bogotá 1.4%). Both training and altitude produced a right-shift of the standard oxygen dissociation curve (P50 in CC 28.5 +/- 0.9 mmHg, AC 31.0 +/- 1.4 mmHg, CB 29.6 +/- 1.5 mmHg) leading to highest P50 in blood of altitude athletes (32.3 +/- 1.1 mmHg). Opposite to the position of the ODC the slope "n" was only increased by altitude influence (delta "n" in controls 0.07, in athletes 0.28). The BCCO2 was increased in AC over the whole saturation range, whereas BCLac was neither significantly influenced by training nor by altitude. All altitude effects can be explained by higher [DPG] (delta[DPG] in controls 5.0 mumol/gHb, in athletes 3.9 mumol/gHb), but the cause for the training effects still remains unclear. The acid base status in altitude residents was characterized by low BE and pCO2, which was most pronounced in altitude athletes, the latter correcting the actual venous pH to normal values. No significant variations of the Hb-O2-binding properties could be detected in athletes one day after leaving high altitude when compared with blood samples of athletes taken at high altitude, whereas BE and venous pCO2 were already increased. It is concluded that high altitude athletes are favoured during aerobic and handicapped during anaerobic exercise after the rapid descent to low altitude.

Oxygen binding properties of hemoglobin from the white rhinoceros (| β2-GLU|) and the tapir

The β-chain of rhinoceros hemoglobin contains glutamic acid at position β 2, an important site for the binding of organic phosphates. We have investigated the oxygen binding properties of this hemoglobin and its interaction with ATP, 2,3-diphosphoglycerate, CO 2 and chloride. The results show that the presence of GLU at position β 2 nearly abolishes the effect of organic phosphates and CO 2, whereas the oxygen-linked binding of chloride is not affected. Thus rhinoceros hemoglobin has only protons and chloride anions as major allosteric effectors for the control of its oxygen affinity. From the results obtained with hemoglobin solutions it can be calculated that the blood oxygen affinity of the rhinoceros must be rather high with a P 50 of about 20 torr at pH 7.4 and 37°C, which conforms with observations obtained for other large mammals.

Hemoglobin function in the water-ethylene glycol cosolvent system: linkage between oxygen binding and hydration.

The effects of ethylene glycol (EG) on the oxygen binding properties of human hemoglobin are described in this report. Under the conditions used, the hemoglobin molecule remains in the intact tetrameric form in up to 70% (w/w) EG, corresponding to a mole fraction of EG of 0.4. Interaction between the cosolvent and the hemoglobin is quite weak. Only at high concentrations of EG are the effects on the oxygen binding curve detectable. In the range of mole fraction of EG up to 0.2, oxygen affinity is decreased. In the range of mole fraction of EG between 0.2 and 0.4 (corresponding to molar concentrations of 8-12 M EG), hemoglobin oxygen affinity increases, eventually becoming higher than the value obtained in the absence of EG. Experiments were carried out in the presence of 0.013, 0.10, and 1.0 M NaCl to evaluate the linkage between EG and chloride as allosteric effectors and the possible general effect of ionic strength on oxygen binding properties of hemoglobin in the presence of cosolvent. The effects of EG on hemoglobin ligation are discussed in terms of a model in which EG interacts with hemoglobin in a weak allosteric fashion at the lower concentration range (less than mole fraction of 0.2) while at the higher range (mole fraction of 0.2-0.4) perturbations of protein hydration lead to stabilization of the high-affinity form of hemoglobin.

Oxygen binding properties of hemoglobins from Daphnia pulex (De Geer)

1. 1. Hemoglobin extracts of Daphnia pulex (De Geer) electrophoreses in two bands (Hb A and Hb B) with PAGE at pH8.1. 2. 2. With preparative electrofocusing on granulated gel (PEGG) the purified Hb extracts are separated in at least 12 bands. 3. 3. Elution of the gel after PEGG shows five mean Hb fractions as indicated by spectrophotometry at 412 nm. They were, for practical reasons, reduced to four fractions (Hb 1, 2, 3 and 4). When submitted again to PAGE they apparantly reassociate to Hb A, Hb B or a mixture of both. 4. 4. A study of the oxygen binding properties of the four Hb fractions shows a distinct positive Bohr effect for Hb 3 between pH 6.5 and 7.5 and for Hb 4 between pH 7.5 and 8.0. The latter shows also the highest oxygen affinity (0.93 mm Hg at pH 8.1 and 17°C). 5. 5. The four Hb fractions show Hill coefficients ranging between 1.4 and 2.3.

Oxygen binding properties of hemoglobins from Antarctic fishes

1. 1. The half-saturation value, P 50, for ‘stripped’ hemoglobins from Trematomus spp. (fam. Nototheniidae) at pH 8.27 and −1.8°C varied from 12.1 mmHg in the pelagic species T. borchgrevinki to 1.3 mmHg in the sedentary benthic species T. centronotus. 2. 2. The nototheniid hemoglobins showed appreciable Bohr effects (Φ = Δ log P 50/ΔpH = −0.87 to −0.48) while the hemoglobin from a bathydraconid species, Gymnodraco acuticeps, was insensitive to pH; Φ = −0.02. No Root shifts were detected. 3. 3. Cooperative oxygen binding was present in all hemoglobins with Hill's coefficient, n, ranging from 1.21 ± 0.14 in Dissostichus mawsoni to 2.28 ± 0.88 in T. borchgrevinki among the Nototheniidae, and n = 1.50 ± 0.14 in G. acuticeps. 4. 4. A small increase in P 50 was promoted by ATP for hemoglobins from T. borchgrevinki, T. bernacchii and D. mawsoni but significant temperature effects on oxygen binding were manifested, with apparent heats of oxygenation, ΔH, = −56.13, −62.16, and −23.98 kJ mol −1, respectively.

Oxygen-binding properties of hemoglobins from estivating and active African lungfish.

The oxygen-binding characteristics and the multiplicity of the stripped hemoglobiin from active lungfish Protopterus amphibius, are the same as in specimens that have been estivating for about 30 months, showing that alteration in the hemoglobin molecules is not involved in the earlier reported increase in oxygen affinity of whole blood during estivation (Johansen et al., '76). At pH 7.0 and 26 degrees C the hemolysates show a high oxygen affinity (P50 = 3.1 Torr), a Bohr factor (delta log P50/delta pH) of - 0.33, and a cooperativity coefficient (n) of 1.7. Between 15 and 26 degrees C, the apparent heat of oxygenation (delta H) is - 8.6 Kcal-mole-1 at pH 7.0, corresponding with data for other fish. A low sensitivity of oxygen affinity to urea appears to be adaptive to the high urea concentrations in estivating lungfish. The salt sensitivity is, however, similar to human hemoglobin. The hemoglobin consists of two major (electrophoretically anodal) components, which differ slightly in oxygen affinity but are both sensitive to pH and nucleoside triphosphates (NTP). Guanosine triphosphate (GTP), the major erythrocytic organic phosphate, however, depresses the oxygen affinity of the composite and separated hemoglobins more effectively than ATP suggesting that GTP is the primary modulator of oxygen affinity. Comparative measurements reveal only one major hemoglobin component in P. annectens which has a markedly lower oxygen affinity and phosphate sensitivity than P. amphibius hemoglobins and thus seems less pliable to phosphate-mediated variation in oxygen affinity. The data are discussed in relation to the hemoglobin systems of other fish.

Structure, function and evolution of primate hemoglobins—II. A survey of the oxygen-binding properties

1.1. The oxygen-binding properties of hemoglobins from more than twenty species and subspecies of nonhuman primates were compared at 15 and 20°C in the pH range 5·0–8·5.2.2. Most species of primates have hemoglobins with distinct oxygen-binding properties3.3. Prosimian hemoglobins have a lower affinity for oxygen near physiological pH values than do monkey and ape hemoglobins.4.4. Human hemoglobin has oxygen-binding properties intermediate to those of other primate hemoglobins.5.5. Functional variation apparently parallels structural variation.6.6. As predicted from considerations of allosterism, variation in one oxygen-binding parameter is often associated with variation in additional parameters.7.7. This prohibits rapid evolutionary changes, but does allow for multiple and precise control of function.