Role Of Band Research Articles

D-Galactose induced early aging in human erythrocytes: Role of band 3 protein.

Aging, a time‐dependent multifaceted process, affects both cell structure and function and involves oxidative stress as well as glycation. The present investigation focuses on the role of the band 3 protein (B3p), an anion exchanger essential to red cells homeostasis, in a d‐galactose ( d‐Gal)‐induced aging model. Anion exchange capability, measured by the rate constant of SO₄²− uptake through B3p, levels of lipid peroxidation, oxidation of membrane sulfhydryl groups, B3p expression, methemoglobin, glycated hemoglobin (Hb), and the reduced glutathione/oxidized glutathione ratio were determined after exposure of human erythrocytes to 25, 35, 50, and 100 mmol/L d‐Gal for 24 h. Our results show that: (i) in vitro application of d‐Gal is useful to model early aging in human erythrocytes; (ii) assessment of B3p ion transport function is a sensitive tool to monitor aging development; (iii) d‐Gal leads to Hb glycation and produces substantial changes on the endogenous antioxidant system; (iv) the impact of aging on B3p function proceeds through steps, first involving Hb glycation and then oxidative events at the membrane level. These findings offer a useful tool to understand the mechanisms of aging in human erythrocytes and propose B3p as a possible target for new therapeutic strategies to counteract age‐related disturbances.

Cell physiology and molecular mechanism of anion transport by erythrocyte band 3/AE1

The major transmembrane protein of the red blood cell, known as band 3, AE1, and SLC4A1, has two main functions: 1) catalysis of Cl−/ exchange, one of the steps in CO2 excretion, and 2) anchoring the membrane skeleton. This review summarizes the 150-year history of research on red cell anion transport and band 3 as an experimental system for studying membrane protein structure and ion transport mechanisms. Important early findings were that red cell Cl− transport is a tightly coupled 1:1 exchange and band 3 is labeled by stilbenesulfonate derivatives that inhibit anion transport. Biochemical studies showed that the protein is dimeric or tetrameric (paired dimers) and that there is one stilbenedisulfonate binding site per subunit of the dimer. Transport kinetics and inhibitor characteristics supported the idea that the transporter acts by an alternating access mechanism with intrinsic asymmetry. The sequence of band 3 cDNA provided a framework for detailed study of protein topology and amino acid residues important for transport. The identification of genetic variants produced insights into the roles of band 3 in red cell abnormalities and distal renal tubular acidosis. The publication of the membrane domain crystal structure made it possible to propose concrete molecular models of transport. Future research directions include improving our understanding of the transport mechanism at the molecular level and of the integrative relationships among band 3, hemoglobin, carbonic anhydrase, and gradients (both transmembrane and subcellular) of , Cl−, O2, CO2, pH, and nitric oxide (NO) metabolites during pulmonary and systemic capillary gas exchange.

D‐Galactose‐induced accelerated aging model on human erythrocytes and role of Band 3 protein

Aging is a physiological process that induces several changes in cells and tissues, resulting in a greater risk of disease and death. Though this process has been extensively studied both in vivo and in vitro, including human erythrocytes which are an appropriate model to study aging, the involved pathways have not been fully elucidated. D-Galactose (D-Gal) has been widely used in vitro to model the characteristics of premature aging. Nevertheless, there is few information regarding its effect on Band 3 protein (B3p) in human erythrocytes. Band 3 protein is an anion exchanger, essential to maintain homeostasis of erythrocytes and, in turn, of the whole organism. In the present study, human erythrocytes were treated with high D-Gal concentrations (25-35-50-100 mM) for 24 hours at 25°C, and the rate constant for SO42-uptake through B3p was determined to investigate anion exchange capability. As oxidative damage is associated to aging, lipid peroxidation, membrane sulfhydryl oxidation (-SH groups) and % MetHb (methemoglobin) levels after D-Gal treatment were determined. Moreover, % A1c levels (glycated hemoglobin) and expression levels of B3p have been also measured. Our results suggest that D-Gal (35-50-100 mM) reduces anion exchange capability via B3p, putatively mediated both by -SH groups oxidation and lipid peroxidation of cellular membranes; conversely, neither B3p expression levels nor % MetHb were modified. However, D-Gal increased % A1c, known to crosslink with B3p, and involved in the alteration of anion exchange capability. The present findings show that erythrocytes are a good model to study the impact of aging on cell homeostasis. The D-Gal-induced aging model provokes changes at the erythrocyte's membrane level, altering thus anion exchange capability through B3p. High D-Gal concentrations induce elevation of oxidative stress levels, and successively increase % A1c in human erythrocytes. Our findings contribute to the understanding of mechanisms underlying in vitro-induced aging and provide a useful basis for developing therapeutic strategies to counteract age-related diseases and their oxidative impact on erythrocytes. In this view, future studies are recommended to assay antioxidant molecules and their beneficial effect on B3p function during aging process.

Role of Band 3 Tyrosine Phosphorylation in Causing the Vaso-Occlusive Events in Sickle Cell Disease

Background: Sickle cell disease (SCD) derives from a mutation in sickle hemoglobin (HbS) that leads to vaso-occlusive events that eventually cause organ failure. Based on observations from multiple labs, we hypothesize that the events linking HbS denaturation to vaso-occlusion are: HbS denaturation→oxidative stress→inhibition of erythrocyte tyrosine phosphatases→enhanced tyrosine phosphorylation of band 3→dissociation of the spectrin/actin cytoskeleton from the membrane→release of membrane-derived microparticles (MPs) and discharge of cell-free HbS→induction of microthromboses and activation of the vascular endothelium→vascular occlusion. In the studies below, we document each step of this pathway and demonstrate that inhibitors of Band 3 tyrosine phosphorylation block all downstream events. Methods: Band 3 tyrosine phosphorylation, plasma MP counts, plasma cell-free Hb concentration, sickle cell deformability, sickle cell flow through micro-capillaries, and adhesion of sickle erythrocytes to activated endothelial cells are quantitated using fresh sickle blood both before and after incubation with inhibitors of Band 3 tyrosine phosphorylation. Findings: Imatinib and other Syk kinase inhibitors block Band 3 tyrosine phosphorylation, suppress release of membrane MPs, reduce discharge of cell-free HbS, improve sickle cell deformability, enhance flow of sickle cells through microcapillaries, and reduce adherence of sickle cells to heme-activated endothelium. Interpretation: Inhibition of Band 3 Tyrosine phosphorylation restores membrane integrity and thereby suppresses the downstream sequelae that lead to vaso-occlusive events in SCD. Inhibitors of Band 3 tyrosine phosphorylation may therefore constitute a novel effective therapy for SCD. Funding Statement: NIH grants R01GM24417-40 (P.S.L.), 1RF1 NS110049-01 (J.W.), R01HL133574 (U.A.G. and J.A.L.), T32HL134622 (R.A.), and NSF #1552782 (U.A.G.). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: All sickle cell blood samples were obtained following informed consent using procedures approved by the local IRBs.

Band 3 Erythrocyte Membrane Protein Acts as Redox Stress Sensor Leading to Its Phosphorylation by p72 Syk

In erythrocytes, the regulation of the redox sensitive Tyr phosphorylation of band 3 and its functions are still partially defined. A role of band 3 oxidation in regulating its own phosphorylation has been previously suggested. The current study provides evidences to support this hypothesis: (i) in intact erythrocytes, at 2 mM concentration of GSH, band 3 oxidation, and phosphorylation, Syk translocation to the membrane and Syk phosphorylation responded to the same micromolar concentrations of oxidants showing identical temporal variations; (ii) the Cys residues located in the band 3 cytoplasmic domain are 20-fold more reactive than GSH; (iii) disulfide linked band 3 cytoplasmic domain docks Syk kinase; (iv) protein Tyr phosphatases are poorly inhibited at oxidant concentrations leading to massive band 3 oxidation and phosphorylation. We also observed that hemichromes binding to band 3 determined its irreversible oxidation and phosphorylation, progressive hemolysis, and serine hyperphosphorylation of different cytoskeleton proteins. Syk inhibitor suppressed the phosphorylation of band 3 also preventing serine phosphorylation changes and hemolysis. Our data suggest that band 3 acts as redox sensor regulating its own phosphorylation and that hemichromes leading to the protracted phosphorylation of band 3 may trigger a cascade of events finally leading to hemolysis.

Role of band 3 in the erythrocyte membrane structural changes under thermal fluctuations -multi scale modeling considerations.

An attempt was made to discuss and connect various modeling approaches on various time and space scales which have been proposed in the literature in order to shed further light on the erythrocyte membrane rearrangement caused by the cortex-lipid bilayer coupling under thermal fluctuations. Roles of the main membrane constituents: (1) the actin-spectrin cortex, (2) the lipid bilayer, and (3) the trans membrane protein band 3 and their course-consequence relations were considered in the context of the cortex non linear stiffening and corresponding anomalous nature of energy dissipation. The fluctuations induce alternating expansion and compression of the membrane parts in order to ensure surface and volume conservation. The membrane structural changes were considered within two time regimes. The results indicate that the cortex non linear stiffening and corresponding anomalous nature of energy dissipation are related to the spectrin flexibility distribution and the rate of its changes. The spectrin flexibility varies from purely flexible to semi flexible. It is influenced by: (1) the number of band 3 molecules attached to single spectrin filaments, and (2) phosphorylation of the actin-junctions. The rate of spectrin flexibility changes depends on the band 3 molecules rearrangement.

External electric field induced band dispersion engineering in Si1−xGex nanowires

Abstract The effects of external electric field F on the structures and the electronic and optical properties of (1 1 1)-oriented Si 1− x Ge x alloy nanowires (NWs) are quantitatively investigated using density functional theory. We demonstrate that F has little effect on structural composition, but can induce robust modulation of energy band dispersions by redistributing charge density and orbital levels of the NWs. The significant variations of conduction band minimum (CBM) of the alloy NWs play a key role in band engineering at lower F , while changes of valence band maximum (VBM) also become important at higher F . The incorporation of Ge atoms in the alloy NWs enhances the sensitivity of CBM and VBM to the applied F . Finally, nonlinear variation of optical properties of the alloy NWs under F is predicted and understood based on F induced band engineering. Our results suggest an effective way to externally tune band dispersion features of Si 1− x Ge x alloy NWs and may be important for potential applications in SiGe based devices at nanoscale.

Endoscopic variceal ligation as a safe and effective alternative to endoscopic sclerotherapy in bleeding ectopic duodenal varix—report of a rare case

Sir, Upper gastrointestinal bleeding (UGIB) following devascularization procedures for portal hypertension is rare. Ectopic varices (EVs), outside the esophagogastric region, pose significant diagnostic and therapeutic challenges and bleeding can bemassive and life-threatening. Endoscopic sclerotherapy (EST) has been used to eradicate EV. Endoscopic variceal ligation (EVL) is often not practised because of higher chances of recurrence and increased chances of rebleeding as reported in the literature. A 25-year-old lady presented with massive UGIB. She was known to be cirrhotic with portal hypertension (PHT) who underwent emergency devascularization with splenectomy, after failed endoscopic sclerotherapy 2 years earlier. She was on regular beta-blocker prophylaxis but had episodes of upper gastrointestinal bleeding after devascularization. The residual esophageal varices were obliterated with endoscopic sclerotherapy. She was conscious, oriented and hemodynamically stable. Esophagogastroduodenoscopy revealed varices in the first part of the duodenum. There were no residual esophageal or gastric varices or portal hypertensive gastropathy. A single band was applied over the duodenal varix (Fig. 1). She had no further episodes of UGIB and was discharged after a week of observation. She is on regular follow up for the past 18 months with no recurrence of varices. We report this case to highlight the successful use of EVL for duodenal EV. EVs account for 1 % to 5 % of all variceal bleeding [1–3] and need a high index of suspicion for diagnosis. They occur predominantly in the duodenum, jejunum, ileum, colon, rectum and enterostomy sites. Bleeding from these EVs is rare in patients with portal hypertension. The duodenum is a common site of EV hemorrhage in patients with PHT. Duodenal variceal bleeding is generally massive and life-threatening with mortality as high as 40 % from the initial bleeding [3–5]. The most common cause of duodenal variceal bleeding is extrahepatic PHT, less commonly intrahepatic PHT [6, 7]. Duodenal varices develop from collateral veins originating from the portal vein trunk or superior mesenteric vein, which empty into the inferior vena cava [1]. Diagnosis of ruptured duodenal varices and control of bleeding are difficult [1]. EST has been reported to be successful in the control of duodenal variceal bleeding [8, 9], but there are reports of rebleeding after EST [5]. Literature discussing the role of banding in patients with ectopic varices is limited. EVL for bleeding duodenal varices is challenging because of the difficulty in maintaining the field of vision. EVL has been tried for EV, but those patients received EST also. Hence, it was not clear as to which modality controlled the bleeding. EVL can be risky if the band applied does not completely encircle a characteristically large ectopic varix which can slough off causing a S. C. Sistla :D. Ram : S. M. Ali :N. Rajkumar Department of Surgery, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry 605 006, India

The Role of Banding in Raptor Conservation: Proceedings of a Special Session at the 2010 RRF Annual Meeting, Fort Collins, Colorado

The Role of Banding in Raptor Conservation: Proceedings of a Special Session at the 2010 RRF Annual Meeting, Fort Collins, Colorado

New antimalarial indolone- N-oxides, generating radical species, destabilize the host cell membrane at early stages of Plasmodium falciparum growth: role of band 3 tyrosine phosphorylation

Although indolone- N-oxide (INODs) genereting long-lived radicals possess antiplasmodial activity in the low-nanomolar range, little is known about their mechanism of action. To explore the molecular basis of INOD activity, we screened for changes in INOD-treated malaria-infected erythrocytes ( Pf-RBCs) using a proteomics approach. At early parasite maturation stages, treatment with INODs at their IC 50 concentrations induced a marked tyrosine phosphorylation of the erythrocyte membrane protein band 3, whereas no effect was observed in control RBCs. After INOD treatment of Pf-RBCs we also observed: (i) accelerated formation of membrane aggregates containing hyperphosphorylated band 3, Syk kinase, and denatured hemoglobin; (ii) dose-dependent release of microvesicles containing the membrane aggregates; (iii) reduction in band 3 phosphorylation, Pf-RBC vesiculation, and antimalarial effect of INODs upon addition of Syk kinase inhibitors; and (iv) correlation between the IC 50 and the INOD concentrations required to induce band 3 phosphorylation and vesiculation. Together with previous data demonstrating that tyrosine phosphorylation of oxidized band 3 promotes its dissociation from the cytoskeleton, these results suggest that INODs cause a profound destabilization of the Pf-RBC membrane through a mechanism apparently triggered by the activation of a redox signaling pathway rather than direct oxidative damage.

A study of mass transfer kinetics of alanyl-alanine on a chiral crown ether stationary phase

The mass transfer kinetics of alanyl-alanine enantiomers in a column packed with a chiral stationary phase (CSP) ChiroSil RCA(+) was studied by means of the moment method. Methanol–water solutions acidified with sulphuric acid were used as the mobile phase. It was shown that the spreading of peaks in the column was strongly affected by abnormal eddy diffusion. This effect was well described within the framework of the Giddings coupling theory. The comprehensive four-term Giddings equation for eddy diffusion was applied, considering simultaneous contribution of the trans-column, trans-channel, short-range inter-channel, and long-range inter-channel dispersion factors. Through these calculations, a predominant importance of the trans-column flow velocity bias was revealed. Besides eddy diffusion, the adsorption kinetic resistance to mass transfer plays a noticeable role in band broadening, all the other contributions (from longitudinal molecular diffusion, external and intraparticle mass transfer) being of minor significance. A relative importance of the mass transfer kinetics increases correlatively with a growth of the retention factor. Both the retention and kinetics of the adsorption of alanyl-alanine on the CSP in study are enantioselective. The influence of the column pressure on retention as well as corrections required because of this influence are also discussed.

Failures Induced by Abnormal Banding in Steels

In industrial applications failures of mechanical parts made of carbon and alloyed steels may develop either during heat treatment steps or final finishing operations. Such failures have high impact costs for manufacturers, since heat treated steel products, in general, are high value products which increase in value with each step in the production process until the final life-cycle manufacturing steps are completed. This work highlights the selection of steels to avoid premature ruptures developing during either the heat treatment steps or finishing operations with emphasis on the role of banding in the failure process. Failure does not have to involve fracture but may simply imply a decrease in performance of surface treated components as consequence of surface properties, even in the presence of correct heat treatment parameters. The root causes for banding in steels, are described in literature, and banding has major effects on final product properties (and causal relationships). Therefore, the causes of banding are studied and classified. Conclusions suggest that microstructural defects such as (micro)segregation bands and other defects such as slag and oxides inclusions are developed in the early fabricating cycle steps and can cause premature failure of either semi-finished or finished products.

Changes in band 3 structure as determinants of erythrocyte integrity during storage and survival after transfusion.

Storage of erythrocytes for transfusion purposes is accompanied by a number of morphological and biochemical changes, the storage lesions. Some of these lesions, such as the almost complete, fast disappearance of 2,3-diphoshosphoglycerate and the relatively small, slow decrease in intracellular ATP, may be reversible. Other changes, such as the loss of haemoglobin, and of phospholipid and protein by vesiculation, are not1. Especially the irreversible lesions are likely to affect erythrocyte survival and function after transfusion. Elucidation of the underlying mechanisms may not only result in prolonged storage times, but also - and probably more importantly - in a higher quality of the erythrocyte transfusion product. One of the first requirements for a higher quality is prolonged survival time in vivo or, more specifically, a higher fraction of erythrocytes that survive the first 24 hours after transfusion. The data of recent studies, that used minor blood group antigens and compared the survival characteristics of various products in one and the same recipient, indicate that the percentage erythrocytes that is removed within a few hours after transfusion may be as high as 30%2. This fraction does not only represent nonfunctional erythrocytes, but also may be a major factor in the interaction between the transfused erythrocyte and the patient's immune system. The resulting pathological reactions may cause long-term transfusion side effects such as the development of anti-erythrocyte antibodies, especially in transfusion-dependent patients, and in patients with a chronic inflammation3–6. Since the pivotal studies showing that physiological removal of old erythrocytes is initiated by their specific recognition by the immune system7, it has become obvious to apply the knowledge of causes and effects of the aging process in vivo to the study of the changes that erythrocytes undergo during storage, an aging process in vitro. In fact, in one of the first attempts to mimick the physiological aging process, storage in blood bank-like conditions was the treatment that yielded the most complete set of biologically relevant aging parameters then available8. These parameters were concentrated on structural and functional changes in band 3, the anion exchanger and major protein of the erythrocyte membrane: increased breakdown as deduced from immunoblot patterns and decreased anion transport capacity as deduced from sulfate exchange characteristics8. Since then, our knowledge on composition and insight in the organisation of the erythrocyte membrane and the role of band 3 therein has vastly expanded. However, our knowledge on the molecular changes that band 3 undergoes during aging in vivo, the effects of these changes on binding of physiological, naturally occurring autoantibodies, and on the induction of pathological autoantibodies, has lagged behind. This hampers a critical evaluation of claims on the occurrence of erythrocyte aging during storage8–10. Here we will review the data that are presently available on changes in band 3 structure and function during storage in blood bank conditions, focusing on their relevance for the generation of immunological removal signals as biomarkers of old and/or damaged erythrocytes.

Erythrocytes anion transport and oxidative change in β‐thalassaemias

beta-Thalassaemia is characterized by a decrease in globin beta-chain synthesis and an excess in free alpha-globin chains. This induces alterations in membrane lipids and proteins resulting from a reduction in spectrin/band 3 ratio, partial oxidation of band 4.1 and clustering of band 3. The membrane injury provokes hyperhaemolysis and bone marrow hyperplasia. The pathophysiology of thalassaemia is associated with iron overload that generates oxygen free radicals and oxidative tissue injury with ocular vessel alterations. The aim of this research is to investigate the influence of oxidative stress on band 3 efficiency, which is an integral membrane protein of RBCs (red blood cells). Band 3 protein, of which there are more than 1 million copies per cell, is the most abundant membrane protein in human RBCs. It mediates the anion exchange and acid-base equilibrium through the RBC membrane. Some experiments were performed on thalassaemic cells and beta-thalassaemia-like cells and tested for sulfate uptake. To test the antioxidant effect of Mg(2+), other experiments were performed using normal and pathological cells in the presence of Mg(2+). The oxidant status in thalassaemic cells was verified by increased K(+) efflux, by lower GSH levels and by increased G6PDH (glucose-6-phosphate dehydrogenase) activity. The rate constant of SO(4)(2-) uptake decreases in thalassaemic cells as well as in beta-thalassaemia-like cells when compared with normal cells. It increases when both cells are incubated with Mg(2+). Our data show that oxidative stress plays a relevant role in band 3 function of thalassaemic cells and that antioxidant treatment with Mg(2+) could reduce oxidative damage to the RBC membrane and improve the anion transport efficiency regulated by band 3 protein.

Role of band 3 in regulating metabolic flux of red blood cells

Deoxygenation elevates glycolytic flux and lowers pentose phosphate pathway (PPP) activity in mammalian erythrocytes. The membrane anion transport protein (band 3 or AE1) is thought to facilitate this process by binding glycolytic enzymes (GEs) and inhibiting their activity in an oxygen-dependent manner. However, this regulatory mechanism has not been demonstrated under physiological conditions. In this study, we introduce a (1)H-(13)C NMR technique for measuring metabolic fluxes in intact cells. The role of band 3 in mediating the oxygenated/deoxygenated metabolic transition was examined by treating cells with pervanadate, a reagent that prevents the GE-band 3 complex from forming. We report that pervanadate suppresses oxygen-dependent changes in glycolytic and PPP fluxes. Moreover, these metabolic alterations were not attributable to modulation of bisphosphoglycerate mutase, direct inhibition of GEs by pervanadate, or oxidation, which are the major side effects of pervanadate treatment. These data provide direct evidence supporting the role of band 3 in mediating oxygen-regulated metabolic transitions.

’Pity the Bandless Towns’: Brass Banding in Australian Rural Communities Before World War Two

Wind band music has been part of Australian life from the beginning of colonisation. Reed and brass and allbrass bands became central to the fabric of the social and cultural life of a vast number of small and large communities beyond the chief cities. Proud and progressive rural communities produced some of the best contesting bands of the pre-World War One era but urbanisation, metropolitan dominance of banding and other factors gradually reversed the fortunes of rural banding. This article surveys, quantifies, and describes the role of banding in rural communities and its substantial decline by 1940. The study is made possible though the author’s ownership of a unique private Australian band history archive, The Arthur Stirling Collection, which includes the only known extant runs of early (pre-1925) Australian band journals (Whiteoak 1995, pp. 4–10).

Band Filling and Thermal Escape in CdTe/ZnTe Quantum Dots Grown by Molecular Beam Epitaxy

We have investigated the effects of band filling and thermal escape on the temperature dependence and excitation power dependence of photoluminescence of CdTe QDs grown by molecular beam epitaxy on ZnTe(100) substrate. The absorption spectrum indicates that the PL spectrum consists of two peaks. The experimental results of excitation power dependence were well simulated by the rate equation model taking into account the effect of band filling. The temperature dependence of PL showed significant difference for different excitation powers. It is suggested that either the wetting layer or excited states play an important role in band filling and thermal escape.

Stilbenedisulfonate Binding Kinetics to Band 3 (AE 1): Relationship between Transport and Stilbenedisulfonate Binding Sites and Role of Subunit Interactions in Transport

ABSTRACTStilbenedisulfonates are competitive inhibitors of band 3 (AE1) anion exchange. It has been assumed that competitive binding implies that the stilbenedisulfonates bind to the transport site. In this paper, I summarize briefly an extensive series of stopped-flow fluorescence kinetic studies which indicate that stilbenedisulfonates do not compete with substrate anions directly, but rather that they behave as allosteric competitive inhibitors of monovalent anion binding to band 3. Monovalent anions lower stilbenedisulfonate affinity by accelerating the rate of their release from band 3, without changing the value of the initial second-order “on” kinetic constant. In addition, partial covalent labeling of the band 3 population with stilbenedisulfonates revealed subunit interaction effects: (a) in steady-state and pre-steady-state transport kinetic studies, (b) in studies on the kinetics of reversible stilbenedisulfonate binding to the unlabeled portion of the band 3 population, and (c) in microcalorimetric studies of the thermal unfolding of the membrane domain of band 3. Studies on the kinetics of reversible stilbenedisulfonate binding to erythrocyte membranes from an individual with Southeast Asian ovalocytosis also revealed subunit interaction effects. The demonstration of allosteric competition between stilbenedisulfonates and substrate anions and the observation of numerous examples of subunit interaction effects suggest that allosteric effects may play a significant role in band 3 function.

Haemolysis of human and sheep red blood cells in glycerol media: the effect of pH and the role of band 3

Haemolysis of red blood cells (RBC) in glycerol media may be measured spectrophotometrically. The haemolytic process in a rapid phase obeys a first order rate law. The rate constant expresses the rate of haemolysis. To gain a better understanding of the mechanism of haemolysis in glycerol media, the effects of pH and band 3 inhibitors on the rate of haemolysis in human and sheep RBC were observed. Over the pH range used (pH 5.8–10.0), the rate of haemolysis decreased with increase in pH in sheep RBC. By contrast, the rate of haemolysis increased from pH 5.8 to 6.4 and decreased above pH 6.4 in human RBC. The different effects of pH on the rate of haemolysis are due to inhibition of glycerol permeability by H+ in human RBC but not in sheep RBC. This is supported by the different effects of temperature and Cu2+ on the rate of haemolysis in human and sheep RBC. We did not observe complete inhibition of haemolysis by the classical band 3 inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). Another band 3 inhibitor 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS) showed only weak inhibition. Phenylgloxal (PG), another band 3 inhibitor, had no effect whatsoever on the rate of haemolysis. These results indicate that the anion pathway of band 3 is not the preferred route of transport of glycerol in mammalian RBC.

The role of band 3 protein in oxygen delivery by red blood cells

The synergistic effects of hemoglobin, carbonic anhydrase and the band 3 protein make red blood cells the ideal vehicle for oxygen delivering to the tissues. As long as oxygen is supplied by these ideal vehicles, oxygen intoxication of the tissues is precluded. Band 3 protein mediates the "Chloride-Shift", i.e., the anion exchange of Cl(-)/HCO(3) (-). Because of the Chloride-Shift, red blood cells are able to recognize metabolically active tissues and to supply the minimum amount of oxygen to the tissues. Investigation into the molecular mechanisms of the anion exchange mediated by the band 3 protein was introduced.