Human erythrocyte membrane band 3 protein influences hemoglobin cooperativity. Possible effect on oxygen transport

Hemoglobin function can be modulated by the red cell membrane but some mechanistic details are incomplete. For example, the 43-kDa chymotryptic fragment of the cytoplasmic portion of red cell membrane Band 3 protein and its corresponding N-terminal 11-residue synthetic peptide lower the oxygen affinity of hemoglobin but effects on cooperativity are unclear. Using highly purified preparations, we also find a lowered Hill coefficient (n values <2) at subequivalent ratios of Band 3 fragment or of synthetic peptide to Hb, resulting in an oxygen affinity that is moderately decreased and a partially hyperbolic shape for the O2 binding curve. Both normal HbA and sickle HbS display this property. Thus, the determinant responsible for the Hb cooperativity decreases by the 43-kDa fragment resides within its first 11 N-terminal residues. This effect is observed in the absence of chloride and is reversed by its addition. As effector to Hb ratios approach equivalence or with saturating chloride normal cooperativity is restored, and oxygen affinity is further lowered because the shape of the oxygen binding curve becomes completely sigmoidal. The relative efficiencies of 2,3-diphosphoglycerate (DPG), the 43-kDa Band 3 fragment, and the 11-residue synthetic peptide in lowering cooperativity are very similar. The findings are explained based on the stereochemical mechanism of cooperativity because of two populations of T-state hemoglobin tetramers, one with bound effector and the other with free (Perutz, M. F. (1989) Q. Rev. Biophys. 22, 139-237). As a result of this property, hemoglobin at the membrane inner surface in contact with the N-terminal region of Band 3 could preferentially bind O2 at low oxygen tension and then release it upon saturation with 2,3-diphosphoglycerate in the interior of the red cell. Membrane modulation of hemoglobin oxygen affinity has particularly interesting implications for the polymerization of hemoglobin S in the sickle red cell.     

Nitric oxide effect on the hemoglobin-oxygen affinity.

The biological roles of nitric oxide (NO)-hemoglobin (Hb) derivatives are obscure. It is proposed that NO can function as an allosteric regulator of hemoglobin oxygen-binding properties. We aimed to estimate the effects of NO donors and NO-synthase substrate (L-arginine) on hemoglobin-oxygen affinity (HOA) in experiments in vitro with the various ratios between NO formed and Hb and various oxygen pressures. HOA index (p50), blood pH, plasma and red blood cell (RBC) concentrations of nitrite/nitrate and methemoglobin amounts were measured after the experiments. In our experiments, blood incubation with NO donors (glyceryltrinitrate, molsidomine, sodium nitroprusside, S-nitrosocysteine) or NO-synthase substrate (L-arginine) did not change HOA even at NO:Hb ratio of 1:1. At the same time our results showed that oxygenated blood incubation with S-nitrosocysteine induced an oxyhemoglobin dissociation curve shift leftwards. This indicates a leading role of met-Hb in a modification of Hb oxygen-binding properties. However other NO-modified forms of hemoglobin (S-nitroso- and nitrosylhemoglobin) also may be involved in the regulation of HOA. The results obtained indicate that nitric oxide can be the allosteric effector of hemoglobin, increasing or decreasing its oxygen affinity - possibly, through the generation of different NO-Hb derivatives.     

Single amino acid substitution in important hemoglobinopathies does not disturb molecular function and biological process

Hemoglobin is an important protein found in the red cells of many animals. In humans, the hemoglobin is mainly distributed in the red blood cell. Single amino acid substitution is the main pathogenesis of most hemoglobin disorders. Here, the author used a new gene ontology technology to predict the molecular function and biological process of four important hemoglobin disorders with single substitution. The four studied important abnormal hemoglobins (Hb) with single substitution included Hb S, Hb E, Hb C, and Hb J-Baltimore. Using the GoFigure server, the molecular function and biological process in normal and abnormal hemoglobins was predicted. Compared with normal hemoglobin, all studied abnormal hemoglobins had the same function and biological process. This indicated that the overall function of oxygen transportation is not disturbed in the studied hemoglobin disorders. Clinical findings of oxygen depletion in abnormal hemoglobin should therefore be due to the other processes rather than genomics, proteomics, and expression levels.     

红细胞膜变形能力与胞内分子聚合状态和细胞形态的相关变化

以显微激光散射光谱和图像分析技术,对单个活态红细胞在无扰、在位、实时的情况下,测量了人血红细胞膜的变形能力,以及与之同时对应的胞内血红蛋白的聚合状态和细胞的形态结构。并据此研究了它们的相关关系及随各种生理条件而交互变化的情况。     

Active nitric oxide produced in the red cell under hypoxic conditions by deoxyhemoglobin-mediated nitrite reduction

Recent studies have generated a great deal of interest in a possible role for red blood cells in the transport of nitric oxide (NO) to the microcirculation and the vascular effect of this nitric oxide in facilitating the flow of blood through the microcirculation. Many questions have, however, been raised regarding such a mechanism. We have instead identified a completely new mechanism to explain the role of red cells in the delivery of NO to the microcirculation. This new mechanism results in the production of NO in the microcirculation where it is needed. Nitrite produced when NO reacts with oxygen in arterial blood is reutilized in the arterioles when the partial pressure of oxygen decreases and the deoxygenated hemoglobin formed reduces the nitrite regenerating NO. Nitrite reduction by hemoglobin results in a major fraction of the NO generated retained in the intermediate state where NO is bound to Hb(III) and in equilibrium with the nitrosonium cation bound to Hb(II). This pool of NO, unlike Hb(II)NO, is weakly bound and can be released from the heme. The instability of Hb(III)NO in oxygen and its displacement when flushed with argon requires that reliable determinations of red blood cell NO must be performed on freshly lysed samples without permitting the sample to be oxygenated. In fresh blood samples Hb(III)NO accounts for 75% of the red cell NO with appreciably higher values in venous blood than arterial blood. These findings confirm that nitrite reduction at reduced oxygen pressures is a major source for red cell NO. The formation and potential release from the red cell of this NO could have a major impact in regulating the flow of blood through the microcirculation.     

Purification of hemoglobin from red blood cells using tangential flow filtration and immobilized metal ion affinity chromatography

Two methods for purifying hemoglobin (Hb) from red blood cells (RBCs) are compared. In the first method, red blood cell lysate is clarified with a 50 nm tangential flow filter and hemoglobin is purified using immobilized metal ion affinity chromatography (IMAC). In the second method, RBC lysate is processed with 50 nm, 500 kDa, and 50-100 kDa tangential flow filters, then hemoglobin is purified with IMAC. Our results show that the hemoglobins from both processes produce identical Hb products that are ultrapure and retain their biophysical properties (except for chicken hemoglobin, which shows erratic oxygen binding behavior after purification). Therefore, the most efficient method for Hb purification appears to be clarification with a 50 nm tangential flow filter, followed by purification with IMAC, and sample concentration/polishing on a 10-50 kDa tangential flow filter.     

Steady-State, Hemoglobin-Facilitated O2 Transport in Human Erythrocytes

We measured the rate of oxygen transport through thin (165 µ) films of packed erythrocytes (Hb concentration = 30 g/100 ml). Under optimal conditions steady-state O₂ diffusion was nearly three times that found when the hemoglobin was prevented from acting as a carrier molecule by carbon monoxide binding or high oxygen back pressure. After each experiment we measured hemolysis and found that it averaged less than 1%. Hemolysis could not account for the facilitation, thus proving that facilitated transport of O₂ by hemoglobin can occur in red blood cells. The rate of facilitated transport was identical for Hb solutions of equal concentration to the cells. We interpret this to mean that under the conditions of our experiments the red cell membrane offers no detectable diffusion resistance to O₂ and that the mobility of Hb in intact red cells is the same as in concentrated Hb solution.     

Effects of superoxide anions on red cell deformability and membrane proteins.

The effect of superoxide anions (O2-) on red blood cells (RBC) deformability and membrane proteins was investigated using hypoxanthine-xanthine oxidase system. Exposure of RBC to O2- caused a marked decrease in RBC deformability with a concomitant increase in cell volume and shape changes. The RBC exposed to O2- also displayed pronounced degradation of membrane proteins such as band 3 protein and spectrin; new bands of low molecular weight products appeared as the original membrane proteins tended to diminish, without the appearance of high molecular weight products. Since the membrane proteins are involved in processes regulating membrane properties such as permeability and viscoelasticity, the decreased deformability induced by O2- may be attributable to changes in membrane proteins. Interestingly, resealed ghosts exposed to O2- did not show any significant change in membrane proteins, which suggests the existence of further generation of O2- and subsequent production of other active oxygen species mediated by O2(-)-initiated autoxidation of hemoglobin in intact RBC. Furthermore, electrophoretic analysis suggested that active oxygens increased the endogenous proteolytic susceptibility of RBC. In conclusion, a close linkage was suggested between RBC deformability and the membrane proteins.     

O2 release from Hb vesicles evaluated using an artificial,narrow O2-permeable tube: comparison with RBCs and acellular Hbs

A phospholipid vesicle that encapsulates a concentrated hemoglobin (Hb) solution and pyridoxal 5'-phosphate as an allosteric effector [Hb vesicle (HbV) diameter, 250 nm] has been developed to provide an O2 carrying ability to plasma expanders. The O2 release from flowing HbVs was examined using an O2-permeable, fluorinated ethylenepropylene copolymer tube (inner diameter, 28 microm) exposed to a deoxygenated environment. Measurement of O2 release was performed using an apparatus that consisted of an inverted microscope and a scanning-grating spectrophotometer with a photon-count detector, and the rate of O2 release was determined based on the visible absorption spectrum in the Q band of Hb. HbVs and fresh human red blood cells (RBCs) were mixed in various volume ratios at a Hb concentration of 10 g/dl in isotonic saline that contained 5 g/dl albumin, and the suspension was perfused at the centerline flow velocity of 1 mm/s through the narrow tube. The mixtures of acellular Hb solution and RBCs were also tested. Because HbVs were homogeneously dispersed in the albumin solution, increasing the volume of the HbV suspension resulted in a thicker marginal RBC-free layer. Irrespective of the mixing ratio, the rate of O2 release from the HbV/RBC mixtures was similar to that of RBCs alone. On the other hand, the addition of 50 vol% of acellular Hb solution to RBCs significantly enhanced the rate of deoxygenation. This outstanding difference in the rate of O2 release between the HbV suspension and the acellular Hb solution should mainly be due to the difference in the particle size (250 vs. 7 nm) that affects their diffusion for the facilitated O2 transport.     

The effects of antibiotics on hemolytic behavior of red cells

Human blood was sheared between rotating polyethylene disks and plasma hemoglobin measured at intervals to produce kinetic hemolysis curves (KHC), plotted as free hemoglobin concentration vs time. The KHC produced by blood samples incubated in the presence of penicillin, streptomycin, gentamicin, and amikacin lie always below those for control samples, indicating a reduction in hemolysis; this reduction was greater as the drug concentration was increased. Explanations in terms of alterations in red cell structure were sought by several characterization tests of amikacin-loaded blood samples. Drug-localization studies demonstrated that significant fractions of the total dosage were associated with the red-cell membrane. Resistive pulse spectroscopy was used to show how amikacin affected cell size, deformability, and osmotic fragility; results were sensitive to storage age of the blood. In all cases, the effect of shearing was to reduce cell size, deformability, and osmotic fragility. Mechanisms for hemolytic protection by drugs are proposed.     

Temperature acclimation and oxygen binding properties of blood of the European eel, Anguilla anguilla

Temperature acclimation of the European eel, Anguilla anguilla, resulted in red cell GTP/Hb molar ratios of 1.20, 1.77 and 0.80 at 2, 17 and 29 degrees C, respectively. A small increase in blood oxygen capacity was present in 29 degrees C acclimated eels. The CO2 Bohr effect and the shape of the oxygen binding curve (n-Hill) were invariant with both temperature and GTP/Hb. The significant differences in the GTP/Hb ratio corresponded with a strong enhancement of the temperature effect on blood oxygen affinity between 2 and 17 degrees C and a similarly strong compensation between 17 and 29 degrees C. Predicted in vivo P50 values were 3.0, 13.8 and 17.6 mmHg at 2 degrees C, 17 and 29 degrees C, respectively. The adaptational value of these findings are discussed in relation to standard metabolic rates at the various temperatures. A tentative hypothesis is proposed that the present study confirms and expands earlier work and supports the contention that adjustments in blood oxygen affinity of thermally acclimated teleosts serve to provide them with an unloading O2 tension for diffusion closely matching the standard oxygen requirements at the various temperatures.     

The effects of antibiotics on hemolytic behavior of red cells

Human blood was sheared between rotating polyethylene disks and plasma hemoglobin measured at intervals to produce kinetic hemolysis curves (KHC), plotted as free hemoglobin concentration vs time. The KHC produced by blood samples incubated in the presence of penicillin, streptomycin, gentamicin, and amikacin lie always below those for control samples, indicating a reduction in hemolysis; this reduction was greater as the drug concentration was increased. Explanations in terms of alterations in red cell structure were sought by several characterization tests of amikacin-loaded blood samples. Drug-localization studies demonstrated that significant fractions of the total dosage were associated with the red-cell membrane. Resistive pulse spectroscopy was used to show how amikacin affected cell size, deformability, and osmotic fragility; results were sensitive to storage age of the blood. In all cases, the effect of shearing was to reduce cell size, deformability, and osmotic fragility. Mechanisms for hemolytic protection by drugs are proposed.     

Hemoglobin calais [β 76 (E20) Ala → pro]: a hemoglobin variant with decreased intrinsic oxygen affinity

Hb Calais [β 76 (E20) Ala → Pro] is a new human hemoglobin variant displaying a decreased oxygen affinity. The only electrophoretical difference with Hb A was a slight more acidic isoelectric point. A 2-fold decrease in the oxygen affinity was found by equilibrium measurements performed in a suspension of intact red blood cells and in the lysate. It was confirmed by kinetic studies of the purified abnormal hemoglobin. The rte of methamoglobin formation at 37°C of Hb Calais was also increased realtive to Hb A. The mechanism by which the Pro for Ala substitution of an external residue in the β-chains results in these profound functional abnormalities is nuclear. Subtle changes at the heme pocket, at a distance from teh mutation, may be a plausible explanation for the effects observed.     

Measurement of biophysical properties of red blood cells by resistive pulse spectroscopy: volume, shape, surface area, and deformability

This paper presents a simple, new approach to the determination of size, shape, surface area, and deformability information for cells, notably red blood cells. The results are obtained by combining experimental measurements from resistive pulse spectroscopy (an extension of electronic cell-sizing methodology) with theoretical calculations for model cell systems. Assuming constancy of surface area and approximating red cell shapes by both prolate and oblate ellipsoids of revolution, values are determined for cell shape factor and volume under a variety of conditions. For red blood cells under low-stress conditions, shape factor, volume, and surface area results are found to be consistent with those available from the literature, when the oblate model is used. The applicability of this approach for determination of red cell properties under altered conditions is demonstrated by results for cell volume, at varying osmotic pressure and mechanical shear (tensile) stress. By quantitating the change in cell shape with stress, a new numerical scale for measuring cell deformability is also obtained, and data are presented on its variation for red cells at different osmolalities, over the range of 140 to 500 mOsm.     

Effects of 4-nonylphenol on blood cells of the African catfish Clarias gariepinus (Burchell, 1822)

In the present work, the destructive effects of the 4-nonylphenol on one of the most economically important Nile fishes, namely African catfish (Clarias gariepinus) were studied. Apoptosis, erythrocytes alterations, micronucleus test and blood parameters count were used as biological indicators to detect those effects. After exposure to sublethal concentrations of 4-nonylphenol (0, 0.05, 0.08 and 0.1 mg/l), apoptotic red blood cells with many malformations and micronucleated erythrocytes were recorded. Decrease in the blood parameters such as red blood cells (RBCs), hemoglobin (Hb), package cell volume (PCV), mean corpuscular hemoglobin concentration (MCHC), platelets, white blood cells (WBCs), lymphocytes, basophils, monocytes and increase in mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), neutrophils, eosinophils indicated the negative effects of 4-nonylphenol. It was concluded that, the 4-nonylphenol caused genotoxicity in erythrocytes with many malformations in shape and number indicated with other blood parameters.     

Human fetal hemoglobin synthesis and its NH2-terminal acetylation in a rabbit reticulocyte lysate translational system

The biosynthesis of the acetylated (Hb FIc) and the non-acetylated (Hb F0) human fetal hemoglobin components has been examined in a cell-free translational system. The poly(A)-RNA was isolated from umbilical cord blood samples and translated in the heterologous translational system derived from rabbit reticulocyte lysates in the presence of labeled amino acid(s) or acetyl-CoA. The amount of each hemoglobin or globin chain made in the system was determined by separating the synthesis products by cation-exchange chromatographic methods. The in vitro synthesis ratios were close to the FIc/Ftotal values of the respective hemolysates. The same conclusion could be reached by determining the specific activity ratios of Hb FIc/Hb F0. Co-migration of radioactivity peaks with absorbance peaks indicated the synthesis of that hemoglobin or globin chain. Confirmation of the synthesis of true gamma 0 and gamma Ic was accomplished by high-pressure liquid chromatographic separation of 3H-labeled tryptic peptides. Each peptide corresponded well with the radioactivity peak. Labeled acetyl-group incorporation into Hb FIc and gamma IcT-1 provided direct evidence for acetylation of gamma chains in Hb FIc. The data indicate that the mRNA itself dictates whether a protein is acetylated and, if so, to what extent. The control appears to be not unique to the human red cell system.     

Red Blood Cells: Centerpiece in the Evolution of the Vertebrate Circulatory System

All vertebrates except cold-water ice fish transport oxygenvia hemoglobin packaged in red blood cells (RBCs). VertebrateRBCs vary in size by thirtyfold. Differences in RBC size havebeen known for over a century, but the functional significanceof RBC size remains unknown. One hypothesis is that large RBCsare a primitive character. Agnathans have larger RBCs than domammals. However, the largest RBCs are found in urodele amphibianswhich is inconsistent with the hypothesis that large RBCs areprimitive. Another possibility is that small RBCs increase bloodoxygen transport capacity. Blood hemoglobin concentration ([Hb])and mean RBC hemoglobin concentration (MCHC) increase from Agnathato birds and mammals. However, the changes in [Hb] and MCHCdo not parallel changes in RBC size. In addition, RBC size doesnot affect blood viscosity. Thus, there is no clear link betweenRBC size and oxygen transport capacity. We hypothesize thatRBC size attends changes in capillary diameter. This hypothesisis based on the following observations. First, RBC width averages25% larger than capillary diameter which insures cell deformationduring capillary flow. Functionally, RBC deformation minimizesdiffusion limitations to gas exchange. Second, smaller capillariesare associated with increased potential for diffusive gas exchange.However, smaller capillaries result in higher resistances toblood flow which requires higher blood pressures. We proposethat the large capillary diameters and large RBCs in urodelesreflect the evolutionary development of a pulmonary vascularsupply. The large capillaries reduced systemic vascular resistancesenabling a single ventricular heart to supply blood to two vascularcircuits, systemic and pulmonary, without developing high pressureson the pulmonary side. The large RBCs preserved diffusive gasexchange efficiency in the large capillaries.     

Microspectrophotometric and scanning microphotometric studies of carp (Cyprinus carpio L.) erythrocytes

Carp (Cyprinus carpio) hemoglobin readily autoxidizes in blood smears. Quantification of Soret-band absorbance in individual erythrocytes by means of scanning cytophotometry therefore requires more elaborate methods of preparation of blood samples. Of the fixatives that have been tested, suspension of whole blood in isotonic salt solutions containing glutaraldehyde was most suitable. Glutaraldehyde-fixed red blood cells are totally resistant to hemolysis. In the course of fixation, hemoglobin is transformed to methemoglobin. Spectrophotometry indicated extensive similarities between glutaraldehyde-fixed carp methemoglobin and human methemoglobin. In aqueous solutions, the intensity of the Soret-peak was pH-dependent. The allosteric modifier organic polyphosphate caused an R----T transition, resulting in increased molar extinctions. Dried preparations showed Soret-spectra that were not influenced from either pH or organic polyphosphate concentration of the aqueous suspensions in which the erythrocytes had been stored. The same was true for slide preparations of cyanomethemoglobin, easily derived from methemoglobin on addition of potassium cyanide. In the absence of oxygen fresh blood cells from carp slowly transform their hemoglobin into deoxyhemoglobin. Spectra of the intermediate stages of deoxygenation, Hb4(O2)3, Hb4(O2)2 and Hb4(O2), as well as mixtures of these intermediates, could be monitored.     

Reversible cross-linking and CO treatment as an approach in red cell stabilization

We explored the use of the reversible cross-linking reagent dimethyl 3,3-dithiobispropionimidate (DTBP) in combination with CO treatment as an approach to stabilizing erythrocyte structure and function. Erythrocytes were cross-linked with different concentrations of DTBP for different times. DTBP increased erythrocyte osmotic stability, blocked lysolecithin-induced echinocytosis, and decreased erythrocyte deformability in a concentration- and time-dependent manner. Reversal of the cross-linking with the reducing agent dithioerythritol (DTE) restored osmotic fragility and response to lysolecithin as well as deformability. Complete reversal, however, is a function of the DTBP concentration and the time of cross-linking. The effects of cross-linking with 5 mM DTBP for 1 h were completely reversible after treatment with 10 mM DTE for 20 min. Longer incubation times or higher concentrations of DTBP resulted in partial reversal by DTE of the effects produced by DTBP. Cross-linking and reversal only slightly reduced the ATP content. The hemoglobin contained in the cross-linked and reversed cells could still undergo reversible oxygenation and deoxygenation. Erythrocytes were pretreated with CO, cross-linked with 5 mM DTBP for 1 or 3 h, loaded with a solution containing 500 mM glucose for 24 h, and freeze-dried in a medium containing 15% (w/v) albumin. Rehydration followed in distilled water. Complete recovery, measured as the percentage of free hemoglobin, was achieved for cells cross-linked with 5 mM DTBP for 3 h and freeze-dried to a final water content of 10-15%. Non-cross-linked cells lysed 100% on rehydration in distilled water. No methemoglobin (MetHb) formation as a result of freeze-drying was detected in CO-treated cells. In non-CO-treated cells 20% of the Hb was converted to MetHb.