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.     

Different types of glutathionylation of hemoglobin can exist in intact erythrocytes

Glutathionylation of hemoglobin (Hb) was studied by incubation of intact human erythrocytes with 1 mM tert-butylhydroperoxide (tBHP). Electrophoresis of the membranes showed a time dependent increase of membrane-bound Hb alpha chain until 10 min, and immunoblotting study showed that membrane-bound Hb alpha chain reacted with anti-glutathione antibody only after 10 min. Concomitant with the Hb alpha chain, membrane associated actin, spectrin, and glyceraldehyde 3-phosphate dehydrogenase reacted with the antibody. Cytosolic Hb of the control erythrocytes reacted with anti-glutathione antibody. Together with our previous paper, the present study indicates that at least three different types of glutathionylation of Hb can exist in erythrocytes. The first type is a mixed disulfide bond between reduced glutathione (GSH) and normal Hb. The second type is a disulfide bond between the cysteine 93 of metHb beta chain and oxidized glutathione (GSSG), and the third type is a disulfide bond between the other cysteine residues of metHb alpha chain and/or metHb beta chain and GSSG.     

Peroxiredoxin 2, glutathione peroxidase,and catalase in the cytosol and membrane of erythrocytes under H2O2-induced oxidative stress

Abstract

Erythrocytes are continuously exposed to risk of oxidative injury due to oxidant oxygen species. To prevent damage, they have antioxidant agents namely, catalase (Cat), glutathione peroxidase (GPx), and peroxiredoxin 2 (Prx2). Our aim was to contribute to a better understanding of the interplay between Prx2, Cat, and GPx under H₂O₂-induced oxidative stress, by studying their changes in the red blood cell cytosol and membrane, in different conditions. These three enzymes were quantified by immunoblotting. Malondialdehyde, that is, lipoperoxidation (LPO) in the erythrocyte membrane, and membrane-bound hemoglobin (MBH) were evaluated, as markers of oxidative stress. We also studied the erythrocyte membrane protein profile, to estimate how oxidative stress affects the membrane protein structure. We showed that under increasing H₂O₂ concentrations, inhibition of the three enzymes with or without metHb formation lead to the binding of Prx2 and GPx (but not Cat) to the erythrocyte membrane. Prx2 was detected mainly in its oxidized form and the linkage of metHb to the membrane seems to compete with the binding of Prx2. Catalase played a major role in protecting erythrocytes from high exogenous flux of H₂O₂, since whenever Cat was active there were no significant changes in any of the studied parameters. When only Cat was inhibited, Prx2 and GPx were unable to prevent H₂O₂-induced oxidative stress resulting in increasing MBH and membrane LPO. Additionally, the inhibition of one or more of these enzymes induced changes in the anchor/linker proteins of the junctional complexes of the membrane cytoskeleton–lipid bilayer, which might lead to membrane destabilization.     

The influence of metmyoglobin and ferrylmyoglobin on the human erythrocyte membrane

Preliminary experiments revealed that ferrylmyoglobin decayed more slowly in the absence than in the presence of intact erythrocytes and erythrocyte membranes. This suggested the existence of interactions between FerrylMb and the erythrocyte membrane. Subsequent studies examined the influence of FerrylMb on the membrane of intact erythrocytes and on isolated erythrocyte membranes. The incubation of intact erythrocytes with FerrylMb did not influence their osmotic fragility or the fluidity of their membranes; the level of peroxidation of the membrane lipids increased only slightly (there was only a slight increase in the level of membrane lipid peroxidation). The activity of acetylcholinesterase significantly increased after 15 minutes of incubation, whereas longer incubation did not lead to any changes in the activity of this enzyme. The incubation of isolated erythrocyte membranes with FerrylMb resulted in an increase in their fluidity and a significant rise in the level of lipid peroxidation.     

Structural modification of erythrocyte membranes during oxidative stress and activity of membrane bound NADH-methemoglobin reductase

The activity of NADH-methemoglobin reductase (metHb-reductase) in membranes isolated from human erythrocytes treated with phenylhydrazine at its sublytic concentration was studied. A decrease in the activity of membrane-bound metHb-reductase was shown to depend on the concentration of phenylhydrazine. Simultaneously, an increase in the level of membrane-bound methemoglobin and a change in the fluorescence parameters of membrane-bound 4,4'-diisothiocy-anatostilbene-2,2'-disulfonic acid were registered. In the case when Hb-free erythrocyte ghosts were treated with 0.2-2.0 mM phenylhydrazine, the activity of metHb-reductase did not change. The obtained results indicate that the inhibition of the activity of membrane-bound metHb-reductase by phenylhydrazine-induced oxidative stress in human erythrocytes is not caused by the direct action of the oxidant on the enzyme. The reason for this is the interaction of the products of hemoglobin oxidation with erythrocyte membrane (protein band 3) and structural changes in membrane proteins.     

Reaction of hydrogen peroxide with ferrylhemoglobin: superoxide production and heme degradation

The reaction of Fe(II) hemoglobin (Hb) but not Fe(III) hemoglobin (metHb) with hydrogen peroxide results in degradation of the heme moiety. The observation that heme degradation was inhibited by compounds, which react with ferrylHb such as sodium sulfide, and peroxidase substrates (ABTS and o-dianisidine), demonstrates that ferrylHb formation is required for heme degradation. A reaction involving hydrogen peroxide and ferrylHb was demonstrated by the finding that heme degradation was inihibited by the addition of catalase which removed hydrogen peroxide even after the maximal level of ferrylHb was reached. The reaction of hydrogen peroxide with ferrylHb to produce heme degradation products was shown by electron paramagnetic resonance to involve the one-electron oxidation of hydrogen peroxide to the oxygen free radical, superoxide. The inhibition by sodium sulfide of both superoxide production and the formation of fluorescent heme degradation products links superoxide production with heme degradation. The inability to produce heme degradation products by the reaction of metHb with hydrogen peroxide was explained by the fact that hydrogen peroxide reacting with oxoferrylHb undergoes a two-electron oxidation, producing oxygen instead of superoxide. This reaction does not produce heme degradation, but is responsible for the catalytic removal of hydrogen peroxide. The rapid consumption of hydrogen peroxide as a result of the metHb formed as an intermediate during the reaction of reduced hemoglobin with hydrogen peroxide was shown to limit the extent of heme degradation.     

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.     

Altered Membrane Structure and Surface Potential in Homozygous Hemoglobin C Erythrocytes

Background

Hemoglobin C differs from normal hemoglobin A by a glutamate-to-lysine substitution at position 6 of beta globin and is oxidatively unstable. Compared to homozygous AA erythrocytes, homozygous CC erythrocytes contain higher levels of membrane-associated hemichromes and more extensively clustered band 3 proteins. These findings suggest that CC erythrocytes have a different membrane matrix than AA erythrocytes.

Methodology and Findings

We found that AA and CC erythrocytes differ in their membrane lipid composition, and that a subset of CC erythrocytes expresses increased levels of externalized phosphatidylserine. Detergent membrane analyses for raft marker proteins indicated that CC erythrocyte membranes are more resistant to detergent solubilization. These data suggest that membrane raft organization is modified in CC erythrocytes. In addition, the average zeta potential (a measure of surface electrochemical potential) of CC erythrocytes was ≈2 mV lower than that of AA erythrocytes, indicating that substantial rearrangements occur in the membrane matrix of CC erythrocytes. We were able to recapitulate this low zeta potential phenotype in AA erythrocytes by treating them with NaNO₂ to oxidize hemoglobin A molecules and increase levels of membrane-associated hemichromes.

Conclusion

Our data support the possibility that increased hemichrome deposition and altered lipid composition induce molecular rearrangements in CC erythrocyte membranes, resulting in a unique membrane structure.     

Maintenance and mobility of hemoglobin and water within the human erythrocyte after detergent disruption of the plasma membrane.

Is an intact plasma membrane responsible for keeping hemoglobin and water within the human erythrocyte? If not, what is responsible? How free is Hb to move about within the erythrocyte? To answer these questions erythrocytes were taken for phase contrast microscopy, transmission electron microscopy (TEM), determination of water-holding capacity, and proton NMR studies both before and after membrane disruption with a nonionic detergent (Brij 58). Addition of 0.2% Brij to a D₂O saline solution of hemoglobin (Hb) caused particles of Hb to appear and to aggregate. This aggregation of Hb caused the amplitude of the Hb proton NMR spectra to decrease. Thus, the less mobile the Hb the lower the Hb proton spectra amplitude. Erythrocytes washed in D₂O saline showed proton NMR spectra of relatively low amplitude. Addition of Brij (0.2%) to these erythrocytes caused increased Hb mobility within these erythrocytes. The TEM of fixed and thin-sectioned erythrocytes treated with Brij showed disruption of the plasma membrane of all erythrocytes regardless of whether or not they had lost Hb. Brij-permeabilized erythrocytes washed in D₂O saline or in a D₂O K buffer maintained a higher heavy water-holding capacity upon centrifugation as compared to nonpermeabilized erythrocytes. The TEM of Brij-treated and washed erythrocyte “shells” revealed a continuous submembrane lamina but no other evidence of cytoskeletal elements. The water-holding capacity of the erythrocyte can be accounted for by the water-holding capacity of hemoglobin. The evidence favors a relatively immobile state of Hb and of water in the erythrocyte that is not immediately dependent on an intact plasma membrane but is attributed to interactions between Hb molecules and the submembrane lamina.     

Cooperation between soluble and membrane-bound proteinases in the degradation of β-hemoglobin chains in intact human erythrocytes

Human erythrocytes are shown to contain soluble proteinase(s) that convert excess β-hemoglobin introduced by in vitro entrapment to modified forms that are bound to the erythrocyte membrane. The bound modified hemoglobin chains are degraded in the membrane to yield acid soluble products. Native hemoglobin chains are not bound to the membrane and are not degraded. The cooperative degradation of excess β-hemoglobin chains by soluble and membrane-bound enzymes occurs at neutral pH and does not require energy. The results provide a role for the membrane-bound acid proteinases.     

Hypochlorous acid-induced lysis of human erythrocytes. Inhibition of cellular damage by the isoflavonoid genistein-8-C-glucoside

Erythrocyte damage induced by hypochlorous acid (HOCl) results in cell lysis developing with time after the oxidant is removed (post-hemolysis). The apparent rate constant of post-hemolysis depends on time of incubation in the presence of HOCl and concentration of this oxidant. HOCl-dependent damage of erythrocyte membranes is associated with uncompetitive inhibition of the membrane-bound acetylcholinesterase. Genistein-8-C-glucoside is an isoflavonoid isolated from the flowers of Lupinus luteus L.; in aqueous solution, genistein-8-C-glucoside (0.5-2 mM) efficiently inhibited HOCl-induced damage to erythrocytes similar to the known HOCl scavengers taurine and reduced glutathione. This bioflavonoid can protect the erythrocyte membrane (and to a lesser extent, intraerythrocytic components) by interacting with the reactive chlorine species including hypochlorous acid and membrane-bound chloroamines formed in the reaction of HOCl with erythrocyte membrane proteins.     

Mechanistic studies of the oxidation of oxyhemoglobin by peroxynitrite

The strong oxidizing and nitrating agent peroxynitrite has been shown to diffuse into erythrocytes and oxidize oxyhemoglobin (oxyHb) to metHb. Because the value of the second-order rate constant for this reaction is on the order of 10(4) M(-)(1) s(-)(1) and the oxyHb concentration is about 20 mM (expressed per heme), this process is rather fast and oxyHb is considered a sink for peroxynitrite. In this work, we showed that the reaction of oxyHb with peroxynitrite, both in the presence and absence of CO(2), proceeds via the formation of oxoiron(iv)hemoglobin (ferrylHb), which in a second step is reduced to metHb and nitrate by its reaction with NO(2)(*). In the presence of physiological relevant amounts of CO(2), ferrylHb is generated by the reaction of NO(2)(*) with the coordinated superoxide of oxyHb (HbFe(III)O(2)(*)(-)). This reaction proceeds via formation of a peroxynitrato-metHb complex (HbFe(III)OONO(2)), which decomposes to generate the one-electron oxidized form of ferrylHb, the oxoiron(iv) form of hemoglobin with a radical localized on the globin. CO(3)(*)(-), the second radical formed from the reaction of peroxynitrite with CO(2), is also scavenged efficiently by oxyHb, in a reaction that finally leads to metHb production. Taken together, our results indicate that oxyHb not only scavenges peroxynitrite but also the radicals produced by its decomposition.     

Interaction of plasma lipoproteins with erythrocytes. II. Modulation of membrane-associated enzymes

Intact erythrocytes incubated in the presence of low density lipoproteins (LDL) undergo a time-dependent morphologic transformation from biconcave discs to spherocytes within 4 h. No shape change is observed when erythrocytes are incubated with high density lipoproteins (HDL). The LDL-induced change in erythrocyte morphology occurs without concomitant leakage of hemoglobin from the cell or depletion of intracellular ATP; no change in the distribution of the major lipids of the erythrocyte membranes was detected. The alteration of morphology does require attachment of LDL to the erythrocyte surface. The LDL-induced morphologic alteration is inhibited by HDL, but not by serum albumin. HDL prevent the attachment of LDL to the cell membrane; however, the HDL subfractions, HDL₂ and HDL₃, are only partially effective. These data suggest that normal erythrocyte morphology and cell function may depend on the concentration and composition of the circulating lipoproteins.     

Interaction of plasma lipoproteins with erythrocytes. I. Alteration of erythrocyte morphology

Intact erythrocytes incubated in the presence of low density lipoproteins (LDL) undergo a time-dependent morphologic transformation from biconcave discs to spherocytes within 4 h. No shape change is observed when erythrocytes are incubated with high density lipoproteins (HDL). The LDL-induced change in erythrocyte morphology occurs without concomitant leakage of hemoglobin from the cell or depletion of intracellular ATP; no change in the distribution of the major lipids of the erythrocyte membranes was detected. The alteration of morphology does require attachment of LDL to the erythrocyte surface. The LDL-induced morphologic alteration is inhibited by HDL, but not by serum albumin. HDL prevent the attachment of LDL to the cell membrane; however, the HDL subfractions, HDL₂ and HDL₃, are only partially effective. These data suggest that normal erythrocyte morphology and cell function may depend on the concentration and composition of the circulating lipoproteins.     

Effects of surface proteins of human erythrocyte membrane on the interaction with lipopolysaccharides from <Emphasis Type="Italic">Escherichia coli</Emphasis> O55:B5

The role of erythrocytic surface membrane proteins and membrane charge in the interactions of the erythrocytes with lipopolysaccharides (LPS) isolated from Escherichia coli O55:B5 (LPS _{E. coli} , S-form) has been examined by two independent methods, flow cytometry and cell electrophoresis. Treatment of erythrocytes with trypsin that modifies stereochemical properties of cell surface resulted in a 16% increase in the level of the erythrocyte fluorescence measured after their incubation with fluorescently labeled LPS _{E. coli} . Electrophoretic mobility (EM) of the trypsin-treated erythrocytes was reduced by 16%. The removal of sialic acids from the erythrocyte surface with neuraminidase had no considerable effect either on the relative EM values or fluorescence intensity after the incubation of cells with LPS. The results suggest that the major role in the incorporation of the S-form LPS into the membrane of human erythrocytes is played by stereochemical factors, whereas the cell surface charge is less significant.     

Membrane effects of nitrite-induced oxidation of human red blood cells.

The aim of our investigation was to study the red blood cell (RBC) membrane effects of NaNO(2)-induced oxidative stress. Hyperpolarization of erythrocyte membranes and an increase in membrane rigidity have been shown as a result of RBC oxidation by sodium nitrite. These membrane changes preceded reduced glutathione depletion and were observed simultaneously with methemoglobin (metHb) formation. Changes of the glutathione pool (total and reduced glutathione, and mixed protein-glutathione disulfides) during nitrite-induced erythrocyte oxidation have been demonstrated. The rates of intracellular oxyhemoglobin and GSH oxidation highly increased as pH decreased in the range of 7.5-6.5. The activation energy of intracellular metHb formation obtained from the temperature dependence of the rate of HbO(2) oxidation in RBC was equal to 16.7+/-1.6 kJ/mol in comparison with 12.8+/-1.5 kJ/mol calculated for metHb formation in hemolysates. It was found that anion exchange protein (band 3 protein) of the erythrocyte membrane does not participate significantly in the transport of nitrite ions into the erythrocytes as band 3 inhibitors (DIDS, SITS) did not decrease the intracellular HbO(2) oxidation by extracellular nitrite.     

Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins

Interactions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of “leaky” erythrocytes with a water-soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate-labeled integral membrane proteins (> 70% of label in band 3 and PAS-1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus-fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside-out erythrocyte vesicles and has been purified (V). Bennett, J Biol Chem 253:2292 (1978). This 72,000-dalton fragment binds to spectrin in solution, competitively inhibits association of ³²P-spectrin with inside-out vesicles with a K_i of 10^?7M, and causes rapid dissociation of ³²P-spectrin from vesicles. Both acid-treated 72,000-dalton fragment and the 45,000 dalton-cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from these vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte.     

Membrane-bound enzymes: III. Protease activity in leucocytes in relation to erythrocyte membranes

Protease activity was detected in membranes of human and bovine erythrocytes prepared by the conventional procedures which include washing and removal of the “buffy layer”. The enzyme was extracted by 0.75 M KCNS or (NH₄)₂SO₄ and was activated by 0.4 to 0.5 M of the same salts. Colored, particulate hide powder-azure, membrane fractions and soluble proteins such as hemoglobin, casein or albumin were susceptible to hydrolysis by the membraneous protease.Partial purification of the enzyme was accomplished through disc-gel electrophoresis on polyacrylamide in the presence of 0.25% positively charged detergents like cetyltrimethylammonium bromide. An alkaline protease (pH 7.4) with properties similar to those of the erythrocyte enzyme was found in leucocytes. The similarity between the properties of the leucocytic and erythrocytic proteases and the correlation of the activity in erythrocyte membranes with the content of white cells in these preparations, suggest that enzymatic activities in the contaminating leucocytes are responsible for the activity of membraneous proteases in erythrocytes.     

Effect of reduced and oxidized glutathione on physico-chemical properties of erythrocyte membranes]

The parameters describing the structural and functional state of membranes depending on the level of reduced glutathione in erythrocytes were studied. It was shown, that the decrease in the concentration of reduced intracellular glutathione in erythrocytes upon metabolic depletion (prolonged incubation of cells at 37 degrees C in the absence of glucose) or a rapid irreversible depletion of glutathione with 1-chloro-2,4-dinitrobenzene enhances lipid peroxidation processes in membranes, inhibits the membrane-bound NAD.H methemoglobin reductase activity and decreases the intensity of 1,6-diphenyl-1,3,5-hexatrien fluorescence. The data obtained suggest that the depletion of reduced intracellular glutathione causes changes in the physicochemical state of the erythrocyte membrane: the accumulation of lipid peroxidation products, changes in the physical state of lipid bilayer and the inhibition of membrane-bound NAD.H-methemoglobin reductase activity.     

Changes in erythrocyte membranes after fractioned hyperthermia

The structural changes in erythrocytes membranes were examined before and after the second heat shock of erythrocytes. Electrophoretic separation of protein erythrocyte membranes for cells incubated at 48.5°C was different from control i.e. from erythrocytes incubated at 37°C. No quantitative or qualitative changes were spotted in comparison with protein membranes isolated from the erythrocytes following single or double heat shock. Fluidity of erythrocytes membranes was determined by using spin labels, 5-doxylstearic acid and 16-doxylstearic acid. The membranes were more rigid in their hydrophobic regions after incubation of cells at 44°C. It can be suggested that erythrocyte membranes play some role in thermotolerance and heat damage of enuclate cells.