Formation of disulfide bonds between glutathione and membrane sh groups in human erythrocytes

In erythrocytes treated with the SH-oxidizing agent, diamide, mixed disulfide bonds between membrane proteins and GSH are formed involving 20% of the membrane SH groups. To study the distribution of these mixed disulfides over the membrane protein fractions, intracellular GSH was labelled biosynthetically with [2-³H]glycine prior to diamide treatment of the cells and the radioactivity of defined membrane peptide fractions determined. Mixed disulfides preferentially occur in the extrinsic protein, spectrin (six SH groups), in addition to the formation of peptide disulfides. Intrinsic proteins are much less reactive: only one SH group of the major intrinsic protein (band 3) reacts with GSH, which accounts for previously observed impossibility to dimerize band 3 via disulfide bonds in intact cells. The labelling method described offers a promising strategy to label and map exposed endofacial SH groups of membrane proteins with a physiological, impermeable marker, GSH.In ghosts treated with diamide and GSH the number of mixed disulfides formed is greater than in erythrocytes. Polymerization of spectrin via intermolecular disulfide bridges is suppressed, while intramolecular disulfides are still formed, providing a means for the analysis of spectrin structure.The diamide-induced mixed membrane-GSH disulfides are readily reduced by GSH. This suggests, that GSH may also be able to reduce mixed disulfides formed in the erythrocyte membrane under oxidative stress in vivo. The reversible formation of mixed disulfides may serve to protect sensitive membrane structures against irreversible oxidative damage.     

Hemolysis of human erythrocytes under hydrostatic pressure is suppressed by cross-linking of membrane proteins

The effects of cross-linking of membrane proteins on hemolysis of human erythrocytes under high pressure (2.0 kbar) were examined. The membrane proteins were cross-linked by oxidation of their SH-groups with diamide (0.05-0.5 mM) under different pressures (1-1,000 bar) at which no hemolysis occurs. As the pressure during diamide treatment was raised, the degree of hemolysis under 2.0 kbar and the quantity of cytoskeletal proteins extracted in a low ionic strength medium were gradually decreased. However, both values were increased by reduction with dithiothreitol. From the determination of membrane SH-groups, it was found that cross-linking of membrane proteins by diamide was accelerated under pressure. Only in erythrocytes treated with diamide under pressure were parts of spectrin and ankyrin, in addition to band 3 and band 4.2 proteins, extracted by using Triton X-100. One- and two-dimensional SDS-PAGE of membrane proteins showed that cross-linking of the membrane with cytoskeletal meshwork through linking proteins, in addition to that of membrane proteins themselves, was formed only in the diamide treatment under pressure. These results indicate that pressure-induced hemolysis is greatly suppressed by the supramolecular-weight polymers formed among membrane proteins, and that the high pressure technique is useful for cross-linking membrane proteins with diamide.     

Spectrin as a stabilizer of the phospholipid asymmetry in the human erythrocyte membrane

After treatment of intact human erythrocytes with SH-oxidizing agents (e.g. tetrathionate and diamide) phospholipase A₂ cleaves approx. 30% of the phosphatidylserine and 50% of the phosphatidylethanolamine without causing hemolysis (Haest, C.W.M. and Deuticke, B. (1976) Biochim. Biophys. Acta 436, 353–365). These phospholipids are scarcely hydrolysed in fresh erythrocytes and are assumed to be located in the inner lipid layer of the membrane (Verkleij, A.J., Zwaal, R.F.A., Roelofsen, B., Comfurius, P., Kastelijn, D. and van Deenen, L.L.M. (1973) Biochim. Biophys. Acta 323, 178–193). The enhancement of the phospholipid cleavage is now shown to be accompanied by a 50% decrease of the membrane SH-groups and a cross-linking of spectrin, located at the inner surface of the membrane, to oligomers of < 10⁶ dalton.Blocking approx. 10% of the membrane SH groups with N-ethylmaleimide suppresses both the polymerization of spectrin and the enhancement of the phospholipid cleavage. N-Ethylmaleimide, under these conditions, reacts with three SH groups per molecule of spectrin, 0.7 SH groups per major intrinsic 100 000 dalton protein (band 3) and 1.1 SH groups per molecule of an extrinsic protein of 72 000 daltons (band 4.2). Blocking studies with iodoacetamide demonstrate that the SH groups of the 100 000-dalton protein are not involved in the effects of the SH-oxidizing agents.It is suggested that a release of constraints imposed by spectrin enables phosphatidylserine and phosphatidylethanolamine to move from the inner to the outer lipid layer of the erythrocyte membrane and that spectrin, in the native erythrocyte, stabilizes the orientation of these phospholipids to the inner surface of the membrane.     

Oxidative damages in erythrocytes of patients with metabolic syndrome

The aim of the study was to estimate the changes caused by oxidative stress in structure and function of membrane of erythrocytes from patients with metabolic syndrome (MS). The study involved 85 patients with MS before pharmacological treatment and 75 healthy volunteers as a control group. Cholesterol level, lipid peroxidation, glutathione level (GSH), and antioxidant enzyme activities in erythrocytes were investigated. The damage to erythrocyte proteins was also indicated by means of activity of ATPase (total and Na⁺,K⁺ ATPase) and thiol group level. The membrane fluidity of erythrocytes was estimated by the fluorescent method. The cholesterol concentration and the level of lipid peroxidation were significantly higher, whereas the concentration of proteins thiol groups decreased in the patient group. ATPase and GSH peroxidase activities diminished compared to those in the control group. There were no differences in either catalase or superoxide dismutase activities. The membrane fluidity was lower in erythrocytes from patients with MS than in the ones from control group. These results show changes in red blood cells of patients with MS as a consequence of a higher concentration of cholesterol in the membrane and an increased oxidative stress.     

Vesiculation induced by hydrostatic pressure in human erythrocytes.

When human erythrocytes were subjected to hydrostatic pressure (1.1-2.0 kbar), it was found that membrane vesicles were released from the red cells above 1.4 kbar. As with hemolysis under high pressure, the amount of released vesicles was increased with increasing pressure but decreased by the cross-linking of membrane proteins with diamide. Vesicles obtained at 2.0 kbar were heterogeneous in size but similar to intact erythrocytes in phospholipid composition. Although it has been reported that spectrin-free vesicles are released by echinocytogenic agents, pressure-induced vesicles did contain considerable and similar amounts of spectrin irrespective of the difference in size. These results suggest that vesiculation by high pressure is associated with the disruption of the membrane skeleton, as previously seen in pressure-induced hemolysis [Yamaguchi et al. (1989) J. Biochem. 106, 1080-1085].     

Alteration of rheological properties of human erythrocytes by crosslinking of membrane proteins

The crosslinking of membrane proteins of human erythrocytes by diamide (diazene dicarboxylic acid bis(N,N-dimethylamide) ) was quantified by 4% polyacrylamide gel electrophoresis in 1% sodium dodecyl sulfate. The relation between the crosslinking of membrane proteins and erythrocyte functions (rheological and oxygen transporting) was quantitatively examined. (i) The crosslinking of membrane protein was induced by diamide, without changing the shape and the contents of intracellular organic phosphates (adenylates and 2,3-diphosphoglycerate). The intensity of spectrin 2 in SDS-polyacrylamide gel electrophoresis decreased proportionally to diamide concentration. The percentage decrease in spectrin 2 (using band 3 as an internal standard) was the most appropriate indicator for crosslinking ("% crosslinking'). (ii) The suspension viscosity of erythrocytes increased in proportion to the percentage of crosslinking, in the range of applied shear rates of 3.76-752 s-1. (iii) Erythrocyte deformability (measured by a high-shear rheoscope) was reduced by the crosslinking. The change was detectable even at 5% crosslinking. (iv) Rouleaux formation (measured by a television image analyzer combined with a low-shear rheoscope) was inhibited by the crosslinking. The inhibition was also sensitively detected at more than 5% crosslinking. (v) Hemoglobin in erythrocytes was chemically modified by higher dose of diamide (probably by the binding of diamide with sulfhydryl groups). Also the oxygen affinity of hemoglobin increased and the heme-heme interaction decreased. (vi) The reduction of the crosslinking of membrane proteins by dithiothreitol apparently reversed the intensity of spectrin bands in SDS-polyacrylamide gel electrophoresis and the erythrocyte functions (the suspension viscosity and the deformability), though not completely.     

Membrane skeletal protein structure and interactions in human erythrocytes after their treatment with diamide and calcium.

To analyse the role of native structures of membrane proteins in their structural modifications induced by the elevated intracellular free Ca2+ levels, we have studied the Ca(2+)-mediated effects on membrane skeletal proteins in human erythrocytes that were loaded with Ca2+ using the ionophore A23187 after their pretreatment with the sulphydryl oxidizing agent, diamide. The diamide treatment not only induced polymerization of the major membrane skeletal protein, spectrin, in the erythrocytes, but it also promoted intersubunit crosslinking within the tetramers and dimers of this protein. Loading of these diamide-treated cells with Ca2+ failed to induce significant structural modifications of spectrin as well as polypeptide 4.1, another major membrane skeletal protein, as compared to the erythrocytes that were loaded with Ca2+ without the diamide pretreatment. These results have been interpreted to suggest that the Ca(2+)-induced membrane skeletal protein changes in erythrocytes depend on both the shape and relative orientation of these proteins within the membrane skeleton.     

Peroxyl Radical-Mediated Hemolysis: Role of Lipid, Protein and Sulfhydryl Oxidation

The objective of this study was to define the relationship between peroxyl radical-mediated cytotoxicity and lipid, protein and sulfhydryl oxidation using human erythrocytes as the target mammalian cell. We found that incubation of human erythrocytes with the peroxyl radical generator 2,2' azobis (2-amidinopropane) hydrochloride (AAPH) resulted in a time and dose-dependent increase in hemolysis such that at 50 mM AAPH maximum hemolysis was achieved at 120min. Hemolysis was inhibited by hypoxia and by the addition of certain water soluble free radical scavengers such as 5-aminosalicylic acid (5-ASA), 4-ASA, N-acetyl-5-ASA and dimethyl thiourea. Peroxyl radical-mediated hemolysis did not appear to involve significant peroxidation of erythrocyte lipids nor did they enhance protein oxidation at times preceding hemolysis. Peroxyl radicals did however, significantly reduce by approximately 80% the intracellular levels of GSH and inhibit by approximately 90% erythrocyte Ca²⁺ -Mg²⁺ ATPase activity at times preceding the hemolytic event. Our data as well as others suggest that extracellular oxidants promote the oxidation of intracellular compounds by interacting with certain redox active membrane components. Depletion of intracellular GSH stores using diamide did not result in hemolysis suggesting that oxidation of GSH alone does not promote hemolysis. Taken together, our data suggest that neither GSH oxidation, lipid peroxidation nor protein oxidation alone can account for peroxyl radical-mediated hemolysis. It remains to be determined whether free radical-mediated inactivation of Ca²⁺-Mg²⁺ ATPase is an important mechanism in this process.     

Changes in membrane constituents and chemiluminescence in vitamin E-deficient red blood cells induced by the xanthine oxidase reaction

The oxidation of vitamin E-deficient rat red blood cells (RBCs) induced by the hypoxanthine-xanthine oxidase (HX-XOD) system has been performed in an aqueous suspension. The generation of chemiluminescence and the accumulation of thiobarbituric acid-reactive substances (TBARS) were observed initially and were followed by hemolysis. Interestingly, the total counts of chemiluminescence were closely related to the amount of TBARS. The predominant change of membrane proteins induced by the reaction was the depletion of spectrin bands in gel electrophoresis. When RBC ghosts were oxidized with HX-XOD, the sulfhydryl (SH) groups of membrane proteins decreased at an early stage of the incubation, which was coincident with the above protein alteration. Membrane alpha-tocopherol suppressed not only the formation of TBARS but also chemiluminescence and hemolysis; nevertheless, it did not inhibit the protein damage and the loss of SH groups. Moreover, it was concluded that the chemiluminescence observed during the oxidation of RBC membranes was associated mainly with the peroxidation of lipids and only to a minor extent with the oxidation of proteins.     

Inhibition of erythrocyte Ca2+-ATPase by activated oxygen through thiol- and lipid-dependent mechanisms

We have studied erythrocyte Ca2+-ATPase as a model target for elucidating effects of activated oxygen on the erythrocyte membrane. Either intracellular or extracellular generation of activated oxygen causes parallel decrements in Ca2+-ATPase activity and cytoplasmic GSH, oxidation of membrane protein thiols, and lipid peroxidation. Subsequent incubation with either dithiothreitol or glucose allows only partial recovery of Ca2+-ATPase, indicating both reversible and irreversible components which are modeled herein using diamide and t-butyl hydroperoxide. The reversible component reflects thiol oxidation, and its recovery depends upon GSH restoration. The irreversible component is largely due to lipid peroxidation, which appears to act through mechanisms involving neither malondialdehyde nor secondary thiol oxidation. However, some portion of the irreversible component could also reflect oxidation of thiols which are inaccessible for reduction by GSH, since we demonstrate existence of different classes of thiols relevant to Ca2+-ATPase activity. Activated oxygen has an exaggerated effect on Ca2+-ATPase of GSH-depleted cells. Sickle erythrocytes treated with dithiothreitol show a heterogeneous response of Ca2+-ATPase activity. These findings are potentially relevant to oxidant-induced hemolysis. They also may be pertinent to oxidative alteration of functional or structural membrane components in general, since many components share with Ca2+-ATPase both free thiols and close proximity to unsaturated lipid.     

Direct involvement of spectrin thiols in maintaining erythrocyte membrane thermal stability and spectrin dimer self-association

Human erythrocytes vesiculate upon exposure to temperatures of 49 degrees C and above. Pretreatment of the cells with the thiol-alkylating agent N-ethylmaleimide (NEM) lowers the temperature needed to produce the same effect. Concomitant with the cells' heat susceptibility, skeletal mechanical instability and an increase in spectrin dissociation have been reported (Smith and Palek (1983) Blood 62, 1190). In the present study, similar results were achieved by preincubation of the cells with diamide, which could be reversed by reduction with dithiothreitol. Another oxidative agent, sodium tetrathionate, could only induce the temperature susceptibility, with little effect on spectrin dissociation. Incubation of spectrin solutions with NEM or diamide caused decreased association of spectrin dimers and increased dissociation of spectrin tetramers. Estimation of membrane and spectrin thiols in the treated cells showed that NEM was effective while blocking less than 20% of the thiols. Diamide and tetrathionate blocked more than 50% of the thiols, but were less effective than NEM. It is suggested that some very defined population of thiols is essential for spectrin self-association and for membrane thermal stability. They are more available to NEM than to diamide and less so to tetrathionate. Other thiols participate in maintaining the membrane thermal stability only.     

The effect of mild diamide oxidation on the structure and function of human erythrocyte spectrin

Oxidants can alter erythrocyte membrane properties and cause ultimate hemolysis, but the mechanisms responsible for these changes are not understood. A protein skeleton preserves the normal integrity of the erythrocyte membrane. In this study, we investigated the effects of limited chemical oxidation on the structure and function of the major skeletal protein, spectrin. After mild treatment of spectrin with 2.5 microM diamide, with formation of an average of only one disulfide bond, we observed a 50% reduction in the ability of protein 4.1 to amplify spectrin-actin binding. The oxidized spectrin specifically lacked the ability to bind protein 4.1, whereas all other spectrin functions remained intact. However, oxidation also produced a structural change in spectrin. A rapidly migrating species appeared on non-denaturing gels in a dose-dependent manner with increasing diamide concentrations. By electron microscopy, the oxidized spectrin appeared as single-stranded signet rings with irregular knob-like protrusions. Fifty per cent of spectrin was converted to the ring form after the formation of an average of two disulfide bonds. Both the structural and functional defects were reversed by chemical reduction. The loss of spectrin function or the structural transformation in spectrin may contribute to erythrocyte membrane failure in the oxidative environment.     

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.     

Increase in vulnerability to oxidative damage in cholesterol-modified erythrocytes exposed to t-BuOOH

During the course of radical oxidation, cholesterol may exert seemingly contradictory effects. In order to gain a better understanding of the relationship between cholesterol levels and membrane susceptibility to oxidative damage induced by reactive oxygen species (ROS), here we analyze the integrity and structural stability of cholesterol-modified (enriched or depleted) and unmodified (control) erythrocytes exposed to tert-butyl hydroperoxide. The oxidant significantly increased ROS production, with almost complete oxidation of hemoglobin and a reduction in GSH content in the different erythrocyte groups at 2 mM concentration. These changes were accompanied by losses of cholesterol and total phospholipids, the main decreases being in phosphatidylethanolamine and phosphatidylcholine. The highest lipid loss was found in the cholesterol-depleted group. Fatty acid analyses revealed changes only in peroxidized cholesterol-modified erythrocytes, with decreases in linoleic and arachidonic acids. Fluorescence anisotropy studies showed an increase in the fluidity of the negatively charged surface of peroxidized control erythrocytes. Increased hemolysis and a positive correlation between cellular osmotic fragility and malondialdehyde contents were found in all peroxidized groups. These findings provide evidence that the modification of cholesterol levels in the erythrocyte membrane has provoking effects on peroxidation, with corresponding increases in oxidative damage in the treated cell, possibly as a consequence of lipid bilayer destabilization.     

Differential in vitro and in vivo behavior of mouse ascorbate/Fe3+ and diamide oxidized erythrocytes

Chemical oxidation of mouse erythrocytes has been carried out using two different oxidizing systems namely: Diamide and Ascorbate/Fe³⁺ together with different concentrations of the oxidant. These oxidation treatments produced different extents of modification in membrane proteins as was observed by electrophoretic analyses that showed a possible formation of high molecular weight aggregates. Lipid peroxidation was also observed as the result of these chemical treatments. The action of these two oxidation treatments produced different extents of lipid peroxidation in which the effect Ascorbate/Fe³⁺ reached higher values than that shown by diamide treatments. To study the resulting in vitro behavior of such oxidized erythrocytes, we have evaluated the recognition of oxidized erythrocytes by peritoneal macrophages. In the conditions used, diamide oxidized erythrocytes were more highly recognized by macrophages than Ascorbate/Fe³⁺ treated erythrocytes. However, in both cases an influence of serum factors in the recognition process can be inferred. Additionally, we have correlated on one side the action of different oxidation systems on mouse erythrocytes with different in vivo behavior and organ uptake of the oxidized erythrocytes. On the other side, differential targeting of oxidized erythrocytes to a liver or spleen was observed on dependence of the oxidant used.     

An immunochemical approach for the analysis of membrane protein alterations in Ca2+-loaded human erythrocytes

An increase in the intracellular concentration of Ca²⁺ in human erythrocytes results in the formation of γ-glutamyl-?-lysine cross-linked membrane protein polymers. Following solubilization of the membranes with SDS, these polymers can be isolated on a Lubrol-containing sucrose gradient. Immunoelectrophoresis of the polymeric material with a polyspecific rabbit antibody against human ghosts gave rise to a single, but heterogeneous, precipitate. The polymer was amphiphilic and, on addition to Triton-solubilized erythrocyte membrane proteins, it coprecipitated with spectrin. When the antighost antibody was absorbed with the polymer prior to cross immunoelectrophoresis of normal erythrocyte membrane proteins, the precipitates of glycophorin, acetylcholinesterase, and hemoglobin were normal, whereas the anti-body liters against band 3 protein, spectrin, and ankyrin became reduced. Furthermore, a rabbit antibody raised against the isolated human polymer reacted selectively with the same three membrane proteins. No reactions occurred with lysate proteins.     

A methemoglobin-dependent and plasma-stimulated experimental model of oxidative hemolysis

Incubation of human red blood cells with ter-butyl hydroperoxide (tBHP) causes depletion of GSH and the production of highly reactive oxygen derivatives, notably hydroxyl (OH^?) radicals, followed by lysis of the cells. These effects are related to the formation of methemoglobin (MetHb), which catalyzes the homolytic cleavage of tBHP to form OH^? radicals. Lysis of red blood cells is the result of lipid peroxidation of membrane components and formation of protein aggregates and is enhanced if the tBHP-treated cells are resuspended in autologous plasma or serum. The tBHP-treated cells provide a useful model for analysis of the sequence of events in oxidative hemolysis.     

Effect of hypervitaminosis A on hemolysis and lipid peroxidation in the rat

Erythrocytes from rats fed large doses of Vitamin A alone, or large doses of vitamin A and vitamin E or diphenyl-p-phenylene diamine (DPPD) were studied for H2O2-induced hemolysis. The vitamin A-dosed rats were more susceptible than normal rats to H2O2-induced hemolysis. Hemolysis was not accompanied by lipid peroxidation. Nevertheless, the antioxidants vitamin E and DPPD inhibited hemolysis in erythrocytes from vitamin A-dosed rats. These antioxidants had the same inhibitory effect when they were included in the diet or added to erythrocyte suspensions in vitro. Erythrocytes from vitamin A-dosed rats with or without added vitamin E or DPPD were less susceptible than the erythrocytes from normal rats to osmotic challenge, showing that vitamin A was present in levels sufficient to alter the structure of the erythrocyte membrane. These studies show that oxidative hemolysis occurs when the erythrocyte membrane is modified. Furthermore, this oxidative hemolysis is unrelated to lipid peroxidation.     

Damage to human erythrocytes by radiation-generated HO* radicals: molecular changes in erythrocyte membranes

The effectiveness of radiation-generated HO

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radicals in initiating erythrocyte hemolysis in the presence of oxygen and under anaerobic conditions and prehemolytic structural changes in the plasma-erythrocyte membrane were studied. Under anaerobic conditions the efficacy of HO

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radicals in induction of hemolysis was 16-fold lower than under air. In both conditions, hemolysis was the final consequence of changes of the erythrocyte membrane. Preceding hemolysis, the dominating process under anaerobic conditions was the aggregation of membrane proteins. The aggregates were principally formed by -S-S- bridges. A decrease in spectrin and protein of band 3 content suggests their participation in the formation of the aggregates. These processes were accompanied by changes in protein conformation determined by means of 4-maleimido-2,2,6,6-tetramethylpiperidine-N-oxyl (MSL) spin label attached to membrane proteins. Under anaerobic conditions, in the range of prehemolytical doses, the reaction of HO

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with lipids caused a slight (10-16%) increase in fluidity of the lipid bilayer in its hydrophobic region with a lack of lipid peroxidation. However, in the presence of oxygen, hemolysis was preceded by intense lipid peroxidation and by profound changes in the conformation of membrane proteins. At the radiation dose that normally initiates hemolysis a slight aggregation of proteins was observed. Changes were not observed in particular protein fractions. It can be suggested the cross-linking induced by HO

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radicals under anaerobic conditions and a lack of lipid peroxidation are the cause of a decrease in erythrocyte sensitivity to hemolysis. Contrary, under aerobic conditions, molecular oxygen suppresses cross-linking, catalysing further steps of protein and lipid oxidation, which accelerate hemolysis.     

Effect of diamide on protein oxidation and physico-chemical properties of lipids in erythrocyte membranes

The effect of diamide on the physicochemical state of proteins and lipids of human erythrocyte membrane was studied. It was found that diamide at a concentration of 1 mM decreases the content of the SH-groups of membrane proteins by approximately 50%, resulting in enhanced vesiculation of erythrocytes upon metabolic exhaustion of cells. It was shown using fluorescein isothiocyanate-labeled concanavalin A and 4,4'-diisothiocyano-2,2'-stilbene disulfonate that diamide changes the structural state of the main integral protein of erythrocyte membranes, the band 3 protein. Changes in the microviscosity of the membrane lipid bilayer depending on diamide concentration were determined from the changes in the fluorescence parameters of the lipophilic probes (pyrene and 1,6-diphenyl-3,5-hexatriene). The level of lipid peroxidation products in membranes remained unchanged. It follows from these data that the SH-oxidizing agent diamide does not directly interact with the lipid bilayer of membrane and produces changes in the physicochemical state of lipids presumably by disrupting protein-lipid interactions that take place upon oxidation of the SH-groups and cross-linking of membrane proteins.