Microviscosity Of Erythrocyte Membranes Research Articles

Effects of nitric oxide on microviscosity and polarity of erythrocyte membranes in experiment

Enhancement of methods for protecting target organs during coronary artery bypass surgery determined the development of a technology for delivering nitric oxide (NO) to the systemic blood flow using a cardiopulmonary bypass (CB) apparatus, which makes NO available to all organs and tissues.The aim of the study was to access the effect of perioperative NO conditioning on the coefficients of microviscosity and polarity of sheep etythrocyte membranes during experimental surgical intervention using CB.Material and methods. Study was carried out on 20 sheep weighing 30–34 kg. Two groups were formed. In the CB group, 10 sheep underwent the standard clinical protocol of artificial lung ventilation (ALV) and CB. In the CB + NO group, 10 sheep received NO at a dose of 80 ppm through the circuit of ALV apparatus immediately after tracheal intubation. At the start of CB, NO was delivered to the extracorporeal circulation circuit at a dose of 80 ppm for 90 min. After disconnection from CB, NO supply continued through the ALV apparatus at a dose of 80 ppm for 60 min. The coefficients of microviscosity and polarity of sheep erythrocyte membranes were determined by spectrofluorimetry using pyrene probe.Results and discussion. The implementation of CB was accompanied by a statistically significant decrease in the microviscosity coefficient in the zones of proteinlipid contact of sheep erythrocyte membranes. In the zone of total lipids, the microviscosity coefficient did not change after CB implementation. The membrane polarity coefficient at the final stage of the surgery increased significantly in the zone of annular lipids and did not change in the zone of total lipids. NO supply to the circuit of the extracorporeal circulation neutralizes the revealed increase in the microviscosity and polarity of the annular lipids.Conclusions. The introduction of NO into the extracorporeal circulation circuit at the concentration of 80 ppm prevents a decrease in the coefficients of microviscosity and polarity of annular lipids of erythrocyte membranes that occurs during cardiac surgery.

STUDIES OF ALTERATIONS OF THE CELLULAR MEMBRANE BARRIER FUNCTION AT LARYNGEAL CANCER

Introduction. Cellular membrane barrier alterations lead to metabolic and functional disorders. However, in the case of laryngeal cancer (LC) they are insufficiently studied.
 The aim of the study – to learn the nature of the interaction of erythrocyte membranes with introduced spin probes as an indicator of changes in the barrier function of membranes at LC.
 Research Methods. Samples of the erythrocyte membranes from 40 patients with LC stages II and III and 20 healthy volunteers were probed by EPR with AdTEMPO test. Microviscosity of erythrocyte membranes was determined by the τeff and the decreasing in RSSI. The content of MWM was identified in the blood plasma and in erythrocyte. The partition coefficient between blood plasma proteins and erythrocyte glycocalyx was calculated. SCEM was evaluated by amount of unabsorbed methylene blue.
 Results and Discussion. It was established that LC patient’s endogenous intoxication is characterized by excessive accumulation of the total pool of MWM both in blood plasma and glycocalyx of erythrocyte. SCEM was significantly decreased in samples of both LC stages in comparison to control. The most apparent decline in τeff was observed prior to washing of erythrocytes for 5 min after probe insertion. The deceleration after 60 min was observed only in LC stage II. The value of τeff was at control values levels after washing of erythrocytes of LC stage II 5 min after probe insertion and was significantly reduced in stage III LC in comparison to control. RSSI in samples both stage of patients prior to and after washing of erythrocytes was on average 1.5-fold higher than that of control.
 Conclusions. It was established that the LC patient’s endogenous intoxication is characterized by excessive accumulation of the total pool of MWM both in blood plasma and glycocalyx of erythrocytes, activation of catabolic processes in plasma, redistribution of MWM between the pool of erythrocyte proteins, which corresponds to the second stage of endotoxicosis. The reduction of the SCEM is shown, which is a manifestation of pathological changes in the surface functional activity of erythrocyte membranes. The effectiveness of AdTEMPO for the eva­luation of microviscosity of erythrocyte membranes in patients with LC was confirmed.

In Russian

Based on the pharmacokinetics of nitazole, proprietary oral administration of the rabbit analyzes various aspects of its use as auxiliary substances in the preparation of dosage forms with controlled absorption of starch, carboxymethyl starch, dialdehyde starch, sodium alginate and chitosan. Our analysis of pharmacokinetic curves revealed a correlation between the presence and magnitude of the negative charge in the polymer of starch derivatives and an increase in the bioavailability of nitazole, the absorption rate estimated by tmax and from the equations describing the curves in the framework of the single-particle absorption model. It can be assumed that the change in the bioavailability of nitazole in the presence of starch derivatives is associated with the ion-ion or ion-dipole interaction of the positively charged imine group of nitazole and negatively charged groups of starch derivatives. Obviously, in the mechanism of absorption of nitazole, the limiting stage is the stage of dissolution of nitazole in the stomach.Since the absorption of nitazole may also depend on the microviscosity of the membranes of the cells of the walls of the stomach, the effect of the above polysaccharides on the microviscosity of the membranes of red blood cells as model cells has been studied. However, the work revealed a noticeable effect of only chitosan on the microviscosity of erythrocyte membranes, in which there was some immobilization of the conformational mobility of the lipid bilayer of the cell membranes upon binding of the oppositely charged chitosan to the surface of the erythrocyte membranes. The remaining negatively charged polysaccharides did not significantly affect the microviscosity of the membranes, apparently due to the processes of electrostatic repulsion from red blood cell cells. The totality of the data obtained has expanded our knowledge about the mechanisms of the possible effect of high molecular weight polysaccharides on the bioavailability of various drugs when creating drugs with controlled absorption.

Study of cytotoxicity performance of carbon nanohorns by method of spin probes

The effects of as-produced and treated by HNO3(3M) carbon nanohorns on the microviscosity of rat erythrocyte membranes and the viscosity of the water-containing plasma protein matrix were investigated by the method of spin probes. Addition of nanohorns at the concentration of 100 μg/ml to a suspension of erythrocytes led to an increase in membrane microviscosity during 4 h (about 60% effect). In addition, it was shown that nanohorns also induced an increased polarity of the microenvironment for lipophilic probes in the outer layer of membrane phospholipids, as well as disorders in erythrocytes membranes. Addition of nanohorns to plasma led to a little decrease in the viscosity of water and protein matrix, apparently, due to its partial destruction, impacting especially albumin. Pristine and treated by HNO3(3M) acid nanohorns was found more cytotoxic than nanoparticles of oxidized graphene, and significantly less than carbon nanotubes, which are known to dramatically increase the microviscosity of the membranes of erythrocytes and disrupt their integrity.

In Russian

The microviscosity of erythrocyte membranes is directly related to their integrity, the violation of which in the presence of foreign objects manifests itself in a sharp increase in the microviscosity index, which certainly indicates the degree of cytotoxic effect of objects in contact with cells of a living organism. Using the spin probe method, a comparative study was made of the effect of a number of carbon nanoparticles on the microviscosity of rat erythrocyte membranes. It has been found that the presence of oxidized graphene (OG) in suspension of red blood cells practically does not affect the microviscosity index of membranes. In the presence of carbon nanotubes (CNTs) of various structures during prolonged (24 h) incubation with erythrocytes, an increase in the microviscosity of erythrocyte membranes is 1.5–2 times. Oxidized multi-walled nanotubes exert the greatest effect. Hydrophobic multi-walled CNTs have a smaller effect, and single-walled nanotubes are characterized by a slight effect on the membranes. Incubation of erythrocytes with carbon nanochorns (CNCh) leads to a significant increase in the microviscosity of erythrocyte membranes by 60 % and higher; after 1 h of incubation, the polarity of the surface of the lipid layer of the membranes increases stepwise, apparently due to the destruction of the membrane and the ingress of water molecules there, as well as the disorder of phospholipids. The introduction of detonation nanodiamond (DND) nanoparticles into a suspension of erythrocytes at a concentration of 25 μg/ml produced no noticeable change in the membrane microviscosity index. An increase in the concentration of DND in suspension of red blood cells to 50 and 75 μg/ml led to a decrease in the microviscosity of erythrocyte membranes (increase in fluidity of the membranes) by 20 and 28 %, respectively. Thus, among carbon nanoparticles, nanotubes and nanochorns can exhibit cytotoxicity, which is mainly due to their structure, size, and shape.

The Physicochemical Characteristics of Serum Albumin and Erythrocyte Cell Membranes under Normal and Heart Failure Symptom Conditions

Abstract—Spin labeling EPR spectroscopy was used to study the structural and conformational characteristics of human serum albumin and human erythrocyte membranes under normal conditions and with symptoms of heart failure. 5-Doxyl stearic acid and 16-doxyl stearic acid were used as spin labels, whose paramagnetic NO fragments are bound to different sites of the hydrocarbon chain. The EPR spectra of 16-doxyl stearic acid indicate that in the physiological temperature range serum albumin molecules are characterized by several types of fatty acid binding sites, which differ in parameters of spin-label rotational diffusion. This distribution of fatty-acid binding sites was typical for the blood serum of all patients who participated in our study, regardless of deviations from the normal blood parameters. The microviscosity of erythrocyte membranes from patient blood was measured using both 5-doxyl stearic and 16-doxyl stearic spin labels, whose paramagnetic fragments are located at different depths inside the lipid bilayer. It was found that in patients with an increased erythrocyte distribution width, the membrane lipid microviscosity is statistically significantly higher than under normal conditions.

In Russian

The method of spin probes investigated the effect of various concentrations of detonation nanodiamonds (DND) 25, 50 and 75 µg/ml on the microviscosity of rat erythrocyte membranes. For this, a lipophilic spin probe based on palmetic acid was introduced into the erythrocyte membrane. The introduction of DND into a suspension of erythrocytes at a concentration of 25 μg/ml did not lead to any changes in the microviscosity of erythrocyte membranes within the experimental error. At the same time, an increase in the concentration of DND in suspension of erythrocytes to 50 and 75 µg/ml resulted in to a marked decrease in the microviscosity of erythrocyte membranes (increase in membrane fluidity) by 20 and 28 %, respectively. A noticeable decrease in the intensity of the EPR spectrum after 4 h of incubation of erythrocytes with DNA (50 µg/ml) indicates the antioxidant activity of nanodiamond — the ability to be an electron donor and restore the nitroxyl stable radical (spin probe) to hydroxylamine. Physiologically, an increase in erythrocyte membrane turnover is of great importance for the organism, since allows red blood cells to better penetrate thin or narrowed capillaries and more efficiently nourish the tissues and organs of the body with oxygen. In addition, an increase in cell membrane fluidity increases the activity of membrane enzymes, which should lead to activation of metabolic and regulatory processes in cells and in the body as a whole. Perhaps this may explain the successful use of DND in oncology, treatment of the gastrointestinal tract, etc. At the same time, an excessive increase in the fluidity of cell membranes at high concentrations of DND can lead to irreversible changes in the native structure of cell membranes.

Change in the Microviscosity of Erythrocyte Membranes and Proteins in Blood Plasma after Graphene Oxide Addition: The ESR Spectroscopy Study

The change in the microviscosity of erythrocyte membranes and the proteins in blood plasma after graphene oxide addition is studied by the ESR spectroscopy exploiting two spin probes with different lipophilic components in the structures. Experiments with charged spin probe 2 embedded into the erythrocyte membrane showed that the introduction of graphene oxide in small concentrations (∼70 μg/ml) into a suspension of erythrocytes did not lead to significant changes in the microviscosity of their membranes. Correlation times of hydrophobic spin probe 1 adsorbed to hydrophobic pockets of plasma proteins demonstrate a gradual slowdown at the graphene oxide injection into blood plasma that indicates a small deformation of the hydrophobic cavity of protein at the adsorption. However, this protein binding with graphene oxide does not cause the displacement of the spin probe from their hydrophobic cavities, which is evidence about small changes in the protein secondary structure. The obtained results indicate the insignificant cytotoxicity effect of small concentrations of graphene oxide for erythrocytes and blood plasma.

EPR SPECTROSCOPY STUDIES OF CHANGES IN ERYTHROCYTE MEMBRANES IN PATIENTS WITH LARYNGEAL CANCER

To evaluate microviscosity and sorption capacity of erythrocyte membranes (SCEM) from patients with laryngeal cancer (LC). Samples from 35 patients with LC of stages II and III and 20 healthy volunteers were investigated by electron paramagnetic resonance with Bis(1-oxyl-2,2,6,6-tetramethylpiperidinyl-4)-ester of 5,7-dimethyladamantane-1,3-dicarbonic acid (AdTEMPO) probe. SCEM was evaluated by amount of unabsorbed methylene blue. Microviscosity of erythrocyte membranes was determined by the effective rotational diffusion correlation times (τeff) and a decrease in radical spectrum signal intensity per hour. The most apparent decrease in mobility of the AdTEMPO in erythrocytes was observed prior to washing of erythrocytes with 0.9% NaCl for 5 min after probe insertion. The deceleration after 60 min was observed only in stage II LC. τeff was at control values after washing of erythrocytes of stage II LC 5 min after probe insertion and was significantly reduced in stage III LC in comparison to control. Radical spectrum signal intensity per hour in samples of stage II and III patients prior to and after washing of erythrocytes was on average 1.5-fold higher than that of control. SCEM in samples of stage II and III LC was found in 40 and 33% cases, respectively and was on average significantly reduced in comparison to control. The initial interaction of AdTEMPO with erythrocyte membranes of stage II and III LC patients is accompanied by an increase in τeff, indicating deceleration of probe rotation. τeff of the probe in membranes remains unchanged in 60 min, indicating changes in the structural organization of lipid bilayer and its associated proteins in particular. The similarity of SCEM for both studied groups reflects the pathological changes in function of erythrocyte membranes.

Mechanism Of Cytomegalovirus Penetration Through Erythrocyte Membrane

Objective: To study the effect of cytomegalovirus infection on histidine concentration in erythrocytes of peripheral blood in pregnant women at 27-28th week of gestation and virus penetration through the erythrocyte membrane and to demonstrate the morphofunctional alterations of the latter. The objective of this study is to demonstrate the mechanism of cytomegalovirus penetration through the erythrocyte membrane as well as to describe the process of its structure damage which leads to the accumulation in the peripheral blood of degenerative forms of erythrocytes that lose their ability to deformability. This creates the conditions for the formation of hemic anaemia in a pregnant woman. The research has been conducted on the basis of current data and our own studies. Materials and methods: 75 pregnant women at 27-28th weeks of gestation were examined, including 55 CMV-seropositive ones with CMV-infection exacerbation at 27th week of gestation. All the studies were carried out at the Federal State Budget Scientific Institution “Far Eastern Scientific Centre of Physiology and Pathology of Respiration” during one year time. Histidine activity in peripheral blood erythrocytes was found according to the method suggested by Lucenko Michael T and Andrievskaya Irina A.; fatty acid peroxides in erythrocyte membrane-by Winkler-Schultze method; erythrocyte membrane microviscosity was found by spectrofluorimetric method of lateral diffusion of pyrene. Erythrocyte deformability was also found in accordance with the method of Lucenko Michael T and Andrievskaya Irina A. The calculation of deformed erythrocytes was done using automated cytophotometric device “Mekos” (Russia). Erythrocyte membrane condensation was measured in pixels by means of cytophotometric camera “Pixera” (USA). Results: During the exacerbation of CMV infection at 27-28th weeks of gestation the amount of distal histidine was lower 2.13 times per one erythrocyte. The level of unsaturated fatty acids increased 2.9 times, of fatty acid peroxides in erythrocyte membranes increased 3.3 times, which resulted in 1.7 times increase in erythrocyte membrane microviscosity in the lipid bilayer and in 6.8 times increase in erythrocyte deformability. Conclusion: The exacerbation of chronic CMV infection at 27-28th weeks of gestation causes the virus intrusion into erythrocyte membrane damaging the distal histidine content, which promotes further penetration of proteins of viral envelope and tegument into the erythrocyte. That results in the disturbance of phospholipids bond with erythrocyte membrane proteins, increase in fatty acid peroxides in erythrocytes and decrease in the membrane microviscosity. This promotes the accumulation of deformable erythrocytes in peripheral blood thus causing the threat of anemia formation in pregnant woman at late gestation.

Влияние пропиленгликоля и полиэтиленгликоля с молекулярной массой 1500 на микровязкость мембран эритроцитов

Electron paramagnetic resonance of spin probes was used to study the effect of low and high molecular weight CPAs on microviscosity of erythrocyte membranes. Research data on rotational mobility of the fatty acid spin probes in erythrocyte membranes after 5 min incubation in 15 % propylene glycol solution indicate the absence of strong changes in membrane microviscosity. Introduced polyethylene glycol with m. w. 1500 (PEG-1500) at a final concentration of 15% has been shown to increase the membrane microviscosity at 60 min exposure. This is explained by the fact that the molecules of PEG-1500 do not pass inside the cells and are localized on the membrane surface to cause immobilization and inhibiting the rotational mobility of the spin probe. In the initial period of time under these concentrations of PEG-1500 the injury of liquid crystal membrane structure and disorder of the protein-lipid interactions in the membrane prevail over the dehydration of cells. Probl Cryobiol Cryomed 2016; 26(1):35-44.

The in vitro influence of external electrostatic field on the lipid-protein interactions in membranes of rat erythrocytes

Purpose – The purpose of this paper is to investigate the in vitro influence of 200 kV/m electrostatic fields (ESF) on the microviscosity, viscosity and polarity of rat erythrocyte membranes for revealing the possible changes in lipid-protein interactions due to the field influence. Design/methodology/approach – The investigation of the parameters of erythrocyte membranes and their ghosts, particularly, their microviscosity, the amount and immersion degree of membrane proteins in lipid bilayer, polarity in depth of membrane and its viscosity carried out by the spectrofluorimetric method using the hydrophobic fluorescent probe pyrene. Findings – The carried out investigations shown that the in vitro influence of ESF changes membrane microviscosity, the quantity of membrane peripheral proteins and their immersion degree in the lipid bilayer, if the ghosts have prepared from erythrocytes previously exposed in the field. The analysis of the same parameters for previously prepared erythrocyte ghosts exposed to the 200 kV/m ESF during an hour did not reveal any changes. Originality/value – Data obtained and their comparison with the results of the previous works allow authors to conclude that the in vitro influence of ESF leads to the changes in the lipid-protein interactions in erythrocyte membranes.

Microviscosity of erythrocyte membranes during chronic heart failure in patients of middle and senior age groups

Microviscosity coefficients were studied in the area of integrated and annular lipids of erythrocyte membranes in the peripheral blood of patients aged 35–50 and 60–75 with chronic coronary heart disease (CHD), as well as healthy donors of corresponding age. It was found in healthy donors that the microviscosity coefficients in the area of integrated and of annular lipids are significantly higher in the senior age group (60–75 years). In CHD, both an increase and a decrease in the microviscosity parameters in the area of lipid–lipid interactions can be characteristic for erythrocyte membranes, regardless of the patient age. In the protein–lipid contacts, an increase in microviscosity parameters is characteristic for patients aged 35–50 and, in contrast, a decrease in the microviscosity coefficient was observed in 60- to 75-year-old patients. It was concluded that the indicators of erythrocyte membrane microviscosity in patients of different age groups reflect both the implementation of general mechanisms of adaptive changes in cell membranes and the individual characteristics determined by their lipid and protein composition during the development of chronic CHD.

Оценка влияния углеродных нанотрубок на микровязкость мембран эритроцитов методом спиновых зондов

Оценка влияния углеродных нанотрубок на микровязкость мембран эритроцитов методом спиновых зондов

Microviscosity of erythrocyte membranes in chronic coronary insufficiency in patients of middle and older age groups

We examined the ratios microviscosity in the area of integral and annularly lipid membranes of erythrocytes of peripheral blood in patients of 35-50 and 60-75 years with chronic ischemic heart disease, as well as age-matched healthy controls. In healthy donors coefficients microviscosity in the area of integral and annularly lipids proved to be significantly higher in the older age group (60-75 years). In CHD, regardless of the age of the patients, erythrocyte membranes can be characterized as raising and lowering the parameters microviscosity of lipid-lipid interactions. In the protein-lipid contacts for patients aged 35-50 years an increase is typical, while for patients of 60-75 years, on the contrary, a decrease of the coefficient of microviscosity. It is concluded that the values of the microviscosity of erythrocyte membranes in patients of different age groups in the development of chronic ischemic heart disease, reflect both the implementation of the general mechanisms of adaptive changes of cell membranes, and the specifics of an individual, determined by their lipid and protein composition.

Age-related characteristics of erythrocyte membrane microviscosity in experimental cardiosclerosis

Age-related changes in the microviscosity coefficient and polarity in the zones of lipid-lipid and lipid-protein contacts have been studied in erythrocyte membranes of 4- and 12-month-old rats (group 1, n = 20, and group 2, n = 20, respectively) using the pyrene lateral diffusion method. Each group included ten control animals and ten animals in which post-infarction cardiac remodeling had been induced. Increased microviscosity and polarity of annular and total membrane lipids were detected in erythrocyte membranes of 12-month-old animals. A pronounced increase in the microviscosity and polarity of erythrocyte membranes in the zone of annular and total lipids occurred in 4-month-old animals in which PICS was induced. Decreased microviscosity in the lipid bilayer and increased microviscosity in the annular lipid zone was characteristic of 12-month-old animals suffering from PICS; lipid polarity in this group was high and did not change.

Здатність до деформації мембран еритроцитів у щурів різного віку при гіпоксії

Using modern ("laser tweezers", low ionic strength Liss, definition of microviscosity of erythrocyte membranes by fluorescence probe pyrene) and classical methods of investigation the osmotic resistance of red blood cells and protein membrane of red blood cells, the authors have esteblisheshed that deformation of the red blood cells in hemic hypoxia varies depending on the animals' age. The maximum capacity of red blood cells to deform was detected in rats of prepubertal period (40.2%). In the group of old rats this parameter reached the minimum level (19.7%). Age changes in erythrocyte membranes during hypoxia are manifested in an increase in the viscosity of the lipid bilayer (microviscosity index reduced in old rats compared with young adult animals by 69.5%) and an increase in the percentage of skeletal protein spectrin (by 53.0%). These changes determine the reduction in deformation capacity of red blood cells. The relationship between the age-related changes of erythrocyte membrane deformation and the state of regional microcirculation has been determined. In old rats a perfusion volume is reduced by 43.8%, the fractional volume of red blood cells passing per unit time through microvessels reduced by 59.3%, the proportion of oxygen consumption in tissues decreased by 6.6% and the perfusion of oxygen saturation decreased by 50.0%.

Influence of hydrophobic properties of IKhFAN antioxidants on their membranotropic activity

The echinocytogenic and stomatocytogenic action on erythrocytes, the structure-modifying effect on erythrocyte membranes, and the hemolytic activity of IKhFAN antioxidants were established as functions of their hydrophobicity. The functions are nonlinear. The echinocytogenic, stomatocytogenic, and hemolytic activities first increase, pass through a maximum, and then decrease as the hydrophobicity parameter increases whereas the microviscosity of erythrocyte membranes varies in the opposite manner. These results may be useful in developing approaches to the synthesis of hybrid biologically active compounds including drugs with controlled and targeted delivery to the corresponding cell membrane structures.

Interaction Mechanism of Anabolic Steroid Hormones with Structural Components of Erythrocyte Membranes

The interaction of testosterone, androsterone, dehydroepiandrosterone (DHEA), and dehydroepiandrosterone sulfate (DHEAS) with erythrocyte membranes was studied. It was shown that testosterone and androsterone have a high constant of binding to the membranes (K(b) ≈ 10(6) M(-1)), whereas K(b)'s for DHEA and DHEAS are 2 orders of magnitude lower. Hydrogen bonds and hydrophobic interactions play an important role in binding of anabolic steroids. Hydrogen bonds form with CO and NH groups both of membrane proteins and phospholipids. This results in the formation of complex domains rising above the surface of membranes. Strengthening of hydrophobic interactions in the domains promotes the displacement of water dipoles to adjacent regions, thus loosening the phospholipid bilayer. Overall, microviscosity of erythrocyte membranes strongly increases, which decreases the plasticity of erythrocytes and hampers their motion in blood capillaries. This mechanism may underlie the development of diffusion myocardial hypoxia and hypoxic cardiac arrest.

Role of the interaction between puerarin and the erythrocyte membrane in puerarin-induced hemolysis

Adverse drug reactions (ADR), especially intravenous hemolysis, have largely limited the application of puerarin injections in clinics. This study investigated the underlying mechanisms of puerarin-induced hemolysis. Our results show that puerarin induced concentration-dependent and time-dependent hemolysis when human erythrocytes were incubated in saline solution with more than 2 mM puerarin for over 2 h. However, incubation in PBS or addition of 1 mM of lidocaine to the saline solution completely abolished the hemolysis. Providing materials that could start ATP synthesis did not reverse the hemolysis, and puerarin did not affect Na +–K +–ATPase activity. In addition, puerarin (0.1–2 mM) did not cause calcium influx or exhibited pro-oxidant activity in erythrocytes. Puerarin exhibited different influences on the membrane microviscosity of erythrocytes in saline and PBS. Moreover, 1 mM lidocaine inhibited 8 mM puerarin-induced reduction of membrane microviscosity in saline solution. SDS–PAGE analysis of membrane proteins revealed that 2 mM puerarin treatment induced the appearance of several new protein bands but attenuated the expression of protein bands 2.1, 3, 4.1, 4.2 and 5. These results suggest that high concentrations of puerarin-induced hemolysis were associated with the changes of membrane lipids and of the composition of erythrocytes membrane proteins but not with ATP depletion, pro-oxidation and calcium influx. These changes could be related to the intercalation of amphiphilic puerarin at high concentration into the erythrocyte membrane in certain media, resulting in membrane disorganization and, eventually, cytolysis. Hence, in clinics, determining the optimal dose of puerarin is critical to avoid overdosing and ADR.