The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

Abstract

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (^*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H₂O₂; ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.     

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H(2)O(2); ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.     

Nuclear magnetic resonance investigation of human erythrocytes in the presence of manganese ions. Evidence for a thermal transition

Water proton transverse relaxation was investigated in whole blood and washed erythrocytes samples, respectively, at various temperatures and manganese concentrations. Water diffusional exchange controls proton relaxation in whole blood samples at higher Mn²⁺ concentrations (20–30 mM) or in washed erythrocyte samples at low Mn²⁺ content (1–5 mM). Mn²⁺ uptake is significant in washed normal erythrocyte samples when its concentrations is about 18 mM or higher in the medium, at temperatures below about 26°C. The thermal transition as revealed by the NMR doping method represents a switch from a water exchange process, mainly seen in the higher temperature range, to a paramagnetic ion controlled water proton relaxation in the lower temperature range.     

Water of hydration in the intra- and extra-cellular environment of human erythrocytes

The proton nuclear magnetic resonance (NMR) titration method (which requires measurement of the relaxation rate at multiple measured levels of dehydration) was applied to the analysis of human erythrocytes, a hemoglobin solution, plasma, and serum. The results allowed identification of bulk water and four motionally perturbed water of hydration subfractions. Based on previous NMR studies of homopolypeptides we designated these subfractions as superbound, irrotationally bound, rotationally bound, and structured. The total water of hydration (sum of both structured and bound water subfractions) in plasma, serum, and hemoglobin ranged from 2.78 to 3.77 g H2O/g dry mass and the sum of the three bound water subfractions ranged from 1.23 to 1.72 g H2O/g dry mass. The total water of hydration on hemoglobin, as determined by (i) spin-lattice (T1) and spin-spin (T2) NMR data, (ii) quench ice-crystal imprint size, (iii) calculations based on osmotic pressure data, and (iv) two other methods, ranged from 2.26 to 3.45 g H2O/g dry mass. In contrast, the estimates of total water of hydration in the intact erythrocytes ranged from 0.34 to 1.44 g H2O/g dry mass, as determined by osmotic activity and spin-lattice titration, respectively. Studies on the magnetic-field dependence of the spin-lattice relaxation rate (1/T1 rho) of solvent water nuclei in protein solutions and in intact and disrupted erythrocytes indicated that hemoglobin aggregation exists in the intact erythrocytes and that erythrocyte disruption decreases the extent of hemoglobin aggregation. Together, the present and past data indicate that the extent of water of hydration associated with hemoglobin depends on the amount of salt present and the degree of aggregation of the hemoglobin molecules.     

New Insight into Erythrocyte through In Vivo Surface-Enhanced Raman Spectroscopy

The article presents a noninvasive approach to the study of erythrocyte properties by means of a comparative analysis of signals obtained by surface-enhanced Raman spectroscopy (SERS) and resonance Raman spectroscopy (RS). We report step-by-step the procedure for preparing experimental samples containing erythrocytes in their normal physiological environment in a mixture of colloid solution with silver nanoparticles and the procedure for the optimization of SERS conditions to achieve high signal enhancement without affecting the properties of living erythrocytes. By means of three independent techniques, we demonstrate that under the proposed conditions a colloid solution of silver nanoparticles does not affect the properties of erythrocytes. For the first time to our knowledge, we describe how to use the SERS-RS approach to study two populations of hemoglobin molecules inside an intact living erythrocyte: submembrane and cytosolic hemoglobin (Hb_sm and Hb_c). We show that the conformation of Hb_sm differs from the conformation of Hb_c. This finding has an important application, as the comparative study of Hb_sm and Hb_c could be successfully used in biomedical research and diagnostic tests.     

Confocal Raman microscopy on single living young and old erythrocytes

Raman confocal microscopy, including the techniques of point Raman spectra, line mapping, 2D mapping, and time-dependent spectrum monitoring performed with 514.5 nm excitation light, was used in a comparative study on the distribution and oxidation states of hemoglobin (Hb) in young and old mature erythrocytes. It is demonstrated that in contrast to the homogeneous distribution of the Hb in young cells, there are more Hb distribution around the cell membrane in old erythrocyte. The proteins exhibit some extent of aggregation and conformational change, present less ability of oxidation, and lower oxygenation speed than the Hb in young erythrocytes. Our results also provide the first direct evidence of some intermediate oxygenated states of Hb between the two fully oxygenated (R) and deoxygenated (T) states in living erythrocyte, and give detail information about the conformational change of the intracellular Hb with time during the reoxygenation process. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 951-959, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Oxidation of hemoglobin to methemoglobin in intact erythrocyte by a hydroperoxide induces formation of glutathionyl hemoglobin and binding of alpha-hemoglobin to membrane

Biochemical consequences of oxidation of hemoglobin (Hb) in intact human erythrocytes were studied. The incubation of washed erythrocyte with 1mM tert-butylhydroperoxide induced an increase in glutathionyl-Hb (G-Hb). The formation of G-Hb occurred linearly until 10 min in parallel with the formation of methemoglobin (metHb) after exhaustion of reduced glutathione. The results show that metHb, but not normal Hb, reacts with oxidized glutathione to form G-Hb. G-Hb was confirmed by immunoblotting with anti-glutathione antibody and the formation of G-Hb was accompanied by parallel decrease in beta-globin detected with a reversed phase HPLC. Electrophoretic studies showed time-dependent increase in membrane-associated alpha-Hb until 10 min, indicating that a part of unpaired alpha-Hb bound to the membrane. Pre-beta-globin increased despite the decrease in beta-globin and a part of the increase was independent of the decrease in beta-globin. Pre-beta-globin reacted with anti-glutathione antibody, but it differs from G-Hb in many features.     

The oxidation of glutathione by nitroprusside: Changes in glutathione in intact erythrocytes during incubation with sodium nitroprusside as detected by 1H spin echo NMR spectroscopy

Spin echo ¹H NMR spectroscopy showed that when the hypotensive agent sodium nitroprusside, Na₂[Fe(CN)₅NO]·2H₂O, was incubated with intact erythrocytes in ²H₂O saline, glutathione in the erythrocytes was oxidised to diglutathione. This was confirmed by ¹H FT NMR for the in vitro reaction. ¹³C FT NMR showed that the stoichiometry of the glutathione nitroprusside reaction was 1:1; the inorganic products were nitric oxide and hexacyanoferrate(II), [Fe(CN)₆]⁻. At no stage was free cyanide liberated. The reaction of nitroprusside with glutathione, which occurs after the nitroprusside has crossed the erythrocyte membrane, is compared with the reaction of nitroprusside with haemoglobin. In neither of these reactions with major erythrocyte components was any free cyanide liberated by sodium nitroprusside.     

Effect of pH on molecular constitution and distribution of hemoglobin in living erythrocyte

The molecular constitution of in situ hemoglobin (Hb) and their distribution in living erythrocyte were investigated versus pH using the technique of confocal Raman microscopy. Both Raman point spectra and line mapping measurements were performed on living erythrocytes in suspensions with pH values from 4.82 to 9.70. It was found that the Hb inside a living erythrocyte would dissociate into monomer/dimer when the cells are in low and high pH environments. In contrast to the homogeneous distribution of the Hbs in the cells in neutral suspension, there are more Hbs distributing around the cell membrane or binding to the membrane as pH increases. While in low pH, as the cell become spherical, most of the Hbs distribute to the central part of the cell. In summary, our investigation suggests that the variation of the external pH not only brings changes in the morphology and membrane structure of an erythrocyte, but also affects the constitution and distribution of its intracellular Hbs, thereby the flexibility of the cell membrane and the oxygenation ability of the Hb. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 348–354, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Erythrocyte signal transduction pathways, their oxygenation dependence and functional significance.

Erythrocytes play a key role in human and vertebrate metabolism. Tissue O2 supply is regulated by both hemoglobin (Hb)-O2 affinity and erythrocyte rheology, a key determinant of tissue perfusion. Oxygenation-deoxygenation transitions of Hb may lead to re-organization of the cytoskeleton and signalling pathways activation/deactivation in an O2-dependent manner. Deoxygenated Hb binds to the cytoplasmic domain of the anion exchanger band 3, which is anchored to the cytoskeleton, and is considered a major mechanism underlying the oxygenation-dependence of several erythrocyte functions. This work discusses the multiple modes of Hb-cytoskeleton interactions. In addition, it reviews the effects of Mg2+, 2,3-diphosphoglycerate, NO, shear stress and Ca2+, all factors accompanying the oxygenation-deoxygenation cycle in circulating red cells. Due to the extensive literature on the subject, the data discussed here, pertain mainly to human erythrocytes whose O2 affinity is modulated by 2,3-diphosphoglycerate, ectothermic vertebrate erythrocytes that use ATP, and to bird erythrocytes that use inositol pentaphosphate.     

Role of monochloramine in the oxidation of erythrocyte hemoglobin by stimulated neutrophils

Stimulation of the oxygen (O2) metabolism of isolated human neutrophilic leukocytes resulted in oxidation of hemoglobin of autologous erythrocytes without erythrocyte lysis. Hb oxidation could be accounted for by reduction of O2 to superoxide (O-2) by the neutrophils, dismutation of O-2 to yield hydrogen peroxide (H2O2), myeloperoxidase-catalyzed oxidation of chloride (Cl-) by H2O2 to yield hypochlorous acid (HOCl), the reaction of HOCl with endogenous ammonia (NH+4) to yield monochloramine ( NH2Cl ), and the oxidative attack of NH2Cl on erythrocytes. NH2Cl was detected when HOCl reacted with the NH+4 and other substances released into the medium by neutrophils. The amount of NH+4 released was sufficient to form the amount of NH2Cl required for the observed Hb oxidation. Oxidation was increased by adding myeloperoxidase or NH+4 to increase NH2Cl formation. Due to the volatility of NH2Cl , Hb was oxidized when neutrophils and erythrocytes were incubated separately in a closed container. Oxidation was decreased by adding catalase to eliminate H2O2, dithiothreitol to reduce HOCl and NH2Cl , or taurine to react with HOCl or NH2Cl to yield taurine monochloramine . NH2Cl was up to 50 times more effective than H2O2, HOCl, or taurine monochloramine as an oxidant for erythrocyte Hb, whereas HOCl was up to 10 times more effective than NH2Cl as a lytic agent. NH2Cl contributes to oxidation of erythrocyte components by stimulated neutrophils and may contribute to other forms of neutrophil oxidative cytotoxicity.     

The interaction of hemoglobin with hexadecyltrimethylammonium bromide

The interaction of hemoglobin (Hb) with hexadecyltrimethylammonium bromide (CTAB) is investigated by UV–vis absorption spectra and fluorescence spectra method. CTAB monomer can convert methemoglobin (metHb) to hemichrome, and CTAB molecular assemblies, such as micelle, microemulsion and lamellar liquid crystal, can induce heme monomer to leave the hydrophobic cavity of Hb. TEM results show that Hb maintains the spherical structure in CTAB microemulsions while it is unfolded in CTAB lamellar liquid crystals. The existence of proton in the above systems can increase the stability of metHb.     

The alkylation of hemoglobin S by nitrogen mustard. High resolution proton nuclear magnetic resonance studies.

Sickle cell hemoglobin (Hb S) treated with nitrogen mustard (bis(beta-chloroethyl)methylamine hydrochloride) gives two reaction products, one labile and one stable. After dialysis against buffer solution, the remaining stable product is found to inhibit the polymerization of deoxyhemoglobin S. High resolution proton nuclear magnetic resonance has been used to study the structure and function of this stable product and to investigate the nature of the binding sites of nitrogen mustard to the hemoglobin molecule. The NMR results suggest that the nitrogen mustard treatment of Hb S does not alter the heme environment or the subunit interfaces of the hemoglobin molecule. Moreover, the NMR spectra have also shown that the nitrogen mustard reacts with the beta2 histidines of the hemoglobin molecule and have suggested that several other surface amino acid residues of the hemoglobin molecule are also affected by the nitrogen mustard alkylation. These NMR findings are in good agreement with the data obtained from biochemical studies of nitrogen mustard-treated Hb S. The NMR spectra also indicate that nornitrogen mustard (which is also effective in inhibiting sickling) binds with the hemoglobin molecule in a manner identical with nitrogen mustard. Sulfur mustard, on the other hand, produces no observable changes in the aromatic proton resonances, which is consistent with the fact that it does not inhibit the polymerization of deoxy-Hb S.     

Release of potassium,lipids, and proteins from nonionic detergent treated chicken red blood cells

The plasma membrane of erythrocytes, as of other cells, is thought to act as the barrier responsible for maintaining intracellular gradients of most ions and small molecular species between the cell and its environment. Controlled application of the nonionic detergent Brij 58 effectively opened the erythrocyte plasma membrane, as judged by electron microscopy and lipid mobilization, but the cytoplasm maintained much of its integrity for about 30 min. Release of K⁺ correlated well with release of protein into the surrounding medium. The results demonstrate that permeabilization of the erythrocyte plasma membrane does not result in an instantaneous equilibration of small ions, such as K⁺, between the cell and its environment. A comparison was made between erythrocytes treated with Brij 58 and Triton X-100. The lipid and protein solubilizing actions of Triton X-100 were not as easily separable in time as those of Brij 58. The results of treatment of the erythrocytes with different types of nonionic detergents suggest that the membranolytic and cytoplasmic protein destabilizing actions of nonionic detergents correspond with their hydrophilic-lipophilic balance numbers (HLB values). © 1994 wiley-Liss, Inc.     

Viscoelasticity of packed erythrocyte suspensions subjected to low amplitude oscillatory deformation.

Concentrated adult erythrocyte suspensions were subjected to low amplitude oscillatory shear in a Weissenberg rheogoniometer equipped with a cone-and-plate assembly. The dynamic viscoelastic properties of the suspension were measured over a broad range of frequency by a numerical solution that accounted for fluid inertia. Variation of shear amplitude and cell volume percent, and comparison of buffered saline, plasma, and dextran as suspending media showed that the cellular elements had undergone small bending and shearing deformations. Studies of normal adult erythrocytes, hypotonically swollen cells, temperature-altered cells, and erythrocyte ghosts suggested that the method was evaluating membrane material properties. The normal membrane was found to exhibit a shear rate dependent elastic modulus that increased by more than a factor of 20 over a frequency range from 0.0076 Hz to 60 Hz. The membrane viscosity showed a substantial drop with frequency indicative of a frequency thinning phenomenon. At high frequency of deformation the viscous response of normal erythrocytes was no longer indicative of a membrane property due to the dominant influence of the internal hemoglobin solution. The studies generally supported the ability of the method to quantify relative membrane material properties and detect changes in membrane structure.     

Aqueous Diaminsilver Hydroxide as a Precursor of Pure Silver Nanoparticles for SERS Probing of Living Erythrocytes

A new effective and simple preparation method of pure metallic hydrosols consisting of silver nanoparticles is proposed using aqueous diaminsilver hydroxide as a precursor freed of special reducing agents, surfactants, or anionic pollutants. The process is driven by NH₃ ligand loss and silver complex dissociation followed by silver ion reduction with hydroxyl ions or ammonia itself present in the solution. Self-reduction of aqueous diaminsilver hydroxide occurs for 20–60 min at 90–100 °C in water and results in a wide range of silver nanoparticles, with their sizes dependent on silver complex concentration and reaction time. The pure silver hydrosol is found to attach to a cell membrane without its damage thus allowing measurements of SERS spectra of submembrane hemoglobin inside living erythrocytes.     

Differential time‐dependent volumetric and surface area changes and delayed induction of new permeation pathways in P. falciparum‐infected hemoglobinopathic erythrocytes

During intraerythrocytic development, Plasmodium falciparum increases the ion permeability of the erythrocyte plasma membrane to an extent that jeopardizes the osmotic stability of the host cell. A previously formulated numeric model has suggested that the parasite prevents premature rupture of the host cell by consuming hemoglobin (Hb) in excess of its own anabolic needs. Here, we have tested the colloid‐osmotic model on the grounds of time‐resolved experimental measurements on cell surface area and volume. We have further verified whether the colloid‐osmotic model can predict time‐dependent volumetric changes when parasites are grown in erythrocytes containing the hemoglobin variants S or C. A good agreement between model‐predicted and empirical data on both infected erythrocyte and intracellular parasite volume was found for parasitized HbAA and HbAC erythrocytes. However, a delayed induction of the new permeation pathways needed to be taken into consideration for the latter case. For parasitized HbAS erythrocyte, volumes diverged from model predictions, and infected erythrocytes showed excessive vesiculation during the replication cycle. We conclude that the colloid‐osmotic model provides a plausible and experimentally supported explanation of the volume expansion and osmotic stability of P. falciparum‐infected erythrocytes. The contribution of vesiculation to the malaria‐protective function of hemoglobin S is discussed.     

Comparison between internal microviscosity of low-density erythrocytes and the microviscosity of hemoglobin solutions: an electron paramagnetic resonance study.

The hypothesis that the internal viscosity of erythrocytes is governed by the intracellular hemoglobin (Hb) concentration is examined. Here viscosity is determined by labeling of the cytoplasmic reduced glutathione with the spin label maleimido-Tempo. Erythrocyte populations with different Hb concentrations in isosmotic conditions were obtained through incomplete lysis, followed by cell resealing, and discontinuous density gradient separation. This procedure maintains normal cell shape and volume. Microviscosity of membrane-free Hb solutions was measured by addition of spin labeled glutathione. It was found that microviscosity values are similar for the erythrocyte cytoplasm and for Hb solutions of equivalent concentrations, showing that the erythrocyte membrane does not have any influence on internal microviscosity. The dependence of the microviscosity on the concentration of Hb solutions was compared with results of macroscopic viscosity obtained by other authors. It is concluded that microviscosity is sensitive to individual properties of the Hb molecule (intrinsic viscosity), but that it is not sensitive to intermolecular interactions. As the microviscosity behavior as a function of Hb concentration is the same in Hb solutions as in the erythrocyte cytoplasm, the inferences regarding macroscopic viscosity in Hb solutions could be translated to the rheological properties of the erythrocyte cytoplasm. Thus, these properties could be predicted from the values of the mean corpuscular Hb concentration.     

Roles of hemoglobin Allostery in hypoxia-induced metabolic alterations in erythrocytes: simulation and its verification by metabolome analysis

When erythrocytes are exposed to hypoxia, hemoglobin (Hb) stabilizes in the T-state by capturing 2,3-bisphosphoglycerate. This process could reduce the intracellular pool of glycolytic substrates, jeopardizing cellular energetics. Recent observations suggest that hypoxia-induced activation of glycolytic enzymes is correlated with their release from Band III (BIII) on the cell membrane. Based on these data, we developed a mathematical model of erythrocyte metabolism and compared hypoxia-induced differences in predicted activities of the enzymes, their products, and cellular energetics between models with and without the interaction of Hb with BIII. The models predicted that the allostery-dependent Hb interaction with BIII accelerates consumption of upstream glycolytic substrates such as glucose 6-phosphate and increases downstream products such as phosphoenolpyruvate. This prediction was consistent with metabolomic data from capillary electrophoresis mass spectrometry. The hypoxia-induced alterations in the metabolites resulted from acceleration of glycolysis, as judged by increased conversion of [(13)C]glucose to [(13)C]lactate. The allostery-dependent interaction of Hb with BIII appeared to contribute not only to maintenance of energy charge but also to further synthesis of 2,3-bisphosphoglycerate, which could help sustain stabilization of T-state Hb during hypoxia. Furthermore, such an activation of glycolysis was not observed when Hb was stabilized in R-state by treating the cells with CO. These results suggest that Hb allostery in erythrocytes serves as an O(2)-sensing trigger that drives glycolytic acceleration to stabilize intracellular energetics and promote the ability to release O(2) from the cells.     

Testosterone action on erythropoiesis does not require its aromatization to estrogen: Insights from the testosterone and estrogen treatment of two aromatase-deficient men

Androgens act on erythropoiesis, but the relative role of testosterone (T) and estradiol (E₂) on erythropoietic parameters in men is a poorly investigated issue. In order to evaluate separately the effects on erythropoiesis of high-dose T administration alone and of physiological dose of E₂ administration alone two adult men with aromatase deficiency were assessed before and during each treatment. Blood cell count, hemoglobin (Hb), hematocrit (Hct), erythrocyte mean cell volume (MCV), erythrocyte mean corpuscular hemoglobin (MCH), erythrocyte mean corpuscular hemoglobin concentration (MCHC), serum ferritin, iron and total iron-binding capacity (TIBC), serum erythropoietin, serum total testosterone and estradiol were evaluated. Hb, Hct and red cell count rose during testosterone treatment, consistently with the increase in circulating testosterone, but failed to increase during estradiol treatment. A decrease in Hb, Hct and red cell count was recorded in one of the two subjects during estradiol treatment, with a concomitant decrease in serum testosterone. Circulating T alone is capable of and sufficient to influence erythropoiesis, especially at supraphysiological dosage, while circulating E₂ have not the same effect on erythropoietic parameters, suggesting the hypothesis that the erythropoietic changes induced by androgens are not mediated via its aromatization to estrogens.