Decrease in 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO) EPR signal in ozone-treated erythrocyte membranes

In ozone-treated erythrocyte membrane suspension a slow decrease occurs in the EPR signal of 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO). Because of the absence of such a phenomenon in control membranes and ozonized buffer, this effect must be caused by reaction of nitroxide radicals with products of ozone reactions with membrane components. To find out which components are responsible for the decrease in EPR signal we studied this effect in simple model systems. The same phenomenon was observed both in lipid and protein systems treated by ozone. For unsaturated fatty acids, the correlation between the rate of decrease in EPR signal and the number of double bonds in the lipid molecule was very strong. This suggests that the observed decrease in the nitroxide radical TEMPO EPR signal in ozone-treated erythrocyte membranes is a complex process, but probably the most important reaction is recombination of nitroxide radicals with organic free radicals produced both in the process of lipid peroxidation and ozonolysis of double bonds.     

Decrease in 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO) EPR signal in peroxynitrite-treated erythrocyte membranes.

The treatment of erythrocyte membranes with peroxynitrite (ONOO-), a cytotoxic species formed in vivo by the almost completely diffusion controlled reaction of nitric oxide (NO*) and the superoxide anion (O2*-), led to the loss of the EPR signal of the nitroxide radical 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO). The decrease in the TEMPO EPR signal was peroxynitrite concentration dependent in the studied peroxynitrite concentration range (100-1000 microM). The absence of such a phenomenon in the control membranes (not treated with peroxynitrite) and in a buffer treated with peroxynitrite indicates that the effect must be caused by nitroxide radicals reacting with the products of peroxynitrite reactions with membrane components. To find out which membrane components are responsible for the decrease in EPR signal, this effect was studied in simple model systems (protein and lipid suspensions). The same phenomenon was observed in both lipid and protein systems treated with peroxynitrite, but in protein solutions the effect was greater and occurred for lower peroxynitrite concentrations. A clear effect of the loss of the EPR signal was observed for both erythrocyte membranes and bovine serum albumin (BSA) solution for a peroxynitrite concentration of 100 microM, while in the case of linolenic acid suspension, a significant difference between control and peroxynitrite-treated samples was achieved for a peroxynitrite concentration of 1000 microM. A comparison of the results obtained for the lipid and protein systems suggests that the reaction of nitroxide radicals with protein derived species plays the main role in the observed decrease in the TEMPO EPR signal in peroxynitrite treated erythrocyte membranes.     

Electron paramagnetic resonance studies of membrane fluidity in ozone-treated erythrocytes and liposomes

Doxyl stearate spin probes which differed in the attachment of the nitroxide free radical to the fatty acid have been used to study membrane fluidity in ozone-treated bovine erythrocytes and liposomes. Analysis of EPR spectra of spin labels incorporated into lipid bilayer of the erythrocyte membranes indicates an increase in the mobility and decrease in the order of membrane lipids. In isolated erythrocyte membranes (ghosts) the most significant changes were observed for 16-doxylstearic acid. In intact erythrocytes statistically significant were differences for 5-doxylstearic acid. The effect of ozone on liposomes prepared from a lipid extract of erythrocyte lipids was marked in the membrane microenvironment sampled by all spin probes. Ozone apparently leads to alterations of membrane dynamics and structure but does not cause increased rigidity of the membrane.     

Antioxidant effects of water- and lipid-soluble nitroxide radicals in liposomes

Liposomes are today useful tools in different fields of science and technology. A lack of stability due to lipid peroxidation is the main problem in the extension of the use of these formulations. Recent investigative works have reported the protective effects of stable nitroxide radicals against oxidative processes in different media and under different stress conditions. Our group has focused its attention on the natural aging of liposomes and the protection provided by the water- and lipid-soluble nitroxide radicals 2,2,6,6-tetramethylpiperdine-1-oxyl (TEMPO) and doxylstearic acids (5-DSA, 12-DSA, and 16-DSA), respectively. Unilamellar liposomes were incubated under air atmosphere at 37°C, both in the absence and in the presence of these radicals. Conjugated dienes, lipid hydroperoxides, TBARS, membrane fluidity, and nitroxide ESR signal intensity were followed as a function of time. Our results demonstrated that doxylstearic acids were more efficient than TEMPO in retarding lipid peroxidation at all the concentrations tested. The inhibition percentages, depending on the total nitroxide concentration, were not proportional to the lipid–water partition coefficient. Furthermore, time-course ESR signals showed a slower decrease for doxylstearic acids than for TEMPO. No significant differences were found among 5-DSA, 12-DSA, and 16-DSA. We concluded that the nitroxide radical efficiency as antioxidant directly depends on both nitroxide concentration and lipophilicity.     

Disruption of erythrocyte membranal organization by superoxide

Human erythrocyte ghosts were covalently labeled with 4-maleimide-2,2,6,6-tetramethylpiperidinooxyl. Electron paramagnetic resonance (EPR) spectrometry revealed two major binding environments representing strongly (S) and weakly (W) immobilized species. The disorder parameter, W/S, determined from the respective peak amplitudes, was shown to be irreversibly elevated following treatment of the labeled ghosts with superoxide, indicating an increase in membrane fluidity. Labeled ghosts reduced with ascorbate showed no nitroxide EPR signals. However, following exposure of these membranes to superoxide, the nitroxide spectrum returned with a W/S ratio of 25. In contrast, the disorder parameter for spin-labeled ghosts decreased following exposure to hydroxyl radicals suggesting decreased fluidity, as a result of lipid peroxidation. This effect could be prevented by the inclusion of mannitol. These changes in membrane fluidity and/or protein mobility observed by EPR are compared with previous results obtained by other methods and provide additional evidence for physiologic alterations initiated by superoxide.     

Interaction of photosensitizers with liposomes containing unsaturated lipid

Small unilamellar liposomes were made of dipalmitoyl-phosphatidylcholine and dioleoyl-phosphatidylcholine, and photosensitized by a symmetrically or an asymmetrically substituted glycosilated tetraphenyl-porphyrin derivative. As differential scanning calorimetry and electron paramagnetic resonance spectroscopy (EPR) revealed these porphyrin derivatives were localized in different depth within the lipid bilayer. Both porphyrin derivatives were able to induce photoreaction and consequent structural changes in the membrane. 5-, 12-, or 16-doxyl stearic acid labeled lipid bilayers were applied and the efficiency of photoinduced reaction was followed by the decay of their EPR signal amplitude. Light dose-dependent destruction of nitroxide radical proved to be dependent on the position of spin label. In this process the porphyrin localized in closer connection with the double bond of unsaturated fatty acid was more effective. EPR signal decay was also dependent on the unsaturated fatty acid content of the liposome and the oxygen saturation of the solvent.     

Aging of the erythrocyte IV. Spin-label studies of membrane lipids,proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Effects of in vitro treatment with ozone on the physical and chemical properties of membranes

Treatment of microsomal membranes from cotyledons of Phaseolus vulgaris with ozone raises the liquid-crystalline to gel lipid phase transition temperature and results in the formation of distinct domains of gel phase lipid in the membranes. Liposomes prepared from the total lipid extracts of ozone-treated membranes undergo phase separations just a few degrees below the transition temperature for intact membranes, indicating that the formation of gel phase lipids is largely attributable to ozone-induced alterations in the membrane lipids. Levels of unsaturated fatty acids as well as the sterol to phospholipid ratio are markedly reduced in the ozone-treated membranes, and the neutral lipid fraction from treated membranes shows, an increased propensity to induce the formation of gel phase phospholipid when incorporated into liposomes of egg phosphatidylcholine. Since gel phase phospholipid also forms in naturally senescing plant membranes and appears to be attributable to changes in the neutral lipid fraction, the effects of natural senescence and ozone on membranes have been compared.     

Aging of the erythrocyte. IV. Spin-label studies of membrane lipids, proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Very high frequency electron paramagnetic resonance of 2,2,6,6-tetramethyl-1-piperidinyloxy in 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine liposomes: partitioning and molecular dynamics.

Partitioning and molecular dynamics of 2,2,6,6,-tetramethylpiperedine-1-oxyl (TEMPO) nitroxide radicals in large unilamellar liposomes (LUV) composed from 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine were investigated by using very high frequency electron paramagnetic resonance (EPR) spectroscopy. Experiments carried out at a microwave frequency of 94.3 GHz completely resolved the TEMPO EPR spectrum in the aqueous and hydrocarbon phases. An accurate computer simulation method combined with Levenberg-Marquardt optimization was used to analyze the TEMPO EPR spectra in both phases. Spectral parameters extracted from the simulations gave the actual partitioning of the TEMPO probe between the LUV hydrocarbon and aqueous phases and allowed analysis of picosecond rotational dynamics of the probe in the LUV hydrocarbon phase. In very high frequency EPR experiments, phase transitions in the LUV-TEMPO system were observed as sharp changes in both partitioning and rotational correlation times of the TEMPO probe. The phase transition temperatures (40.5 +/- 0.2 and 32.7 +/- 0.5 degrees C) are in agreement with previously reported differential scanning microcalorimetry data. Spectral line widths were analyzed by using existing theoretical expressions for motionally narrowed nitroxide spectra. It was found that the motion of the small, nearly spherical, TEMPO probe can be well described by anisotropic Brownian diffusion in isotropic media and is not restricted by the much larger hydrocarbon chains existing in ripple structure (P beta') or fluid bilayer structure (L alpha) phases.     

Electron Spin Resonance of 2,2,6,6-Tetramethylpiperidine-1-Oxyl (TEMPO)-labeled Plant Leaves

Temperature dependence for partitioning of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) between aqueous and lipid components of whole leaf tissue was measured. TEMPO is an electron spin resonance nitroxide label that has been used in model systems to detect membrane phase separations. Measurements were made on chilling-sensitive tomato leaves, frost-sensitive potato leaves, and frost-hardy and supercooled wheat leaves. The results suggest a membrane phase separation at 11 C in tomato, 3 C in potato, and −11 C in wheat.     

Local anesthetics can interact electrostatically with membrane proteins

The fluorescent probe 1-anilinonaphthalene 8-sulfonate was used to examine the binding of spin-labeled local anesthetics to lipid model systems, to the membranes of human red blood cells, and rabbit sarcoplasmic reticulum. 1-Anilinonaphthalene 8-sulfonate exhibits two distinct fluorescent lifetimes when bound to these biological membranes. The shorter lifetime represents the probe associated with the purely lipid region while the longer lifetime is associated with the protein region. The spin-labeled local anesthetic quenches the fluorescence of both of these components as indicated by the decrease in the lifetimes. Since nitroxide free radicals are known to quench fluorophores upon 'contract', the results reflect the relative interaction of local anesthetics with membrane lipids and proteins. The evidence is consistent with the concept of multiple binding sites for local anesthetics in membranes. Local anesthetics, once intercalated into the bilayer, may diffuse laterally and interact with membrane components, lipid as well as proteins. In biological membranes, however, positively charged local anesthetics are better able to quench 1-anilinonaphthalene 8-sulfonate in protein regions, suggesting that the interaction between local anesthetics and membrane proteins can be electrostatic in nature.     

Effect of fatty acyl chain length of phosphatidylcholine on their transfer from liposomes to erythrocytes and transverse diffusion in the membranes inferred by TEMPO-phosphatidylcholine spin probes

TEMPO-phosphatidylcholine (PC) spin probes which have homologous saturated acyl chains of 10, 12, 14 and 16 carbon atoms, were synthesized as analogues of PC. Transfer of TEMPO-PCs from liposomal membrane to the ghost membrane of human erythrocyte and transverse diffusion of TEMPO-PCs within the membrane of intact erythrocytes were determined by measurement of spontaneous increase and decrease in signal amplitude of an anisotropic triplet spectrum, due to dilution of the label by natural phospholipid of the membrane and reduction of the label by the cytoplasmic content of the erythrocyte, respectively. TEMPO-PC molecules in TEMPO-PC liposomes, except dipalmitoyl TEMPO-PC, were rapidly incorporated into the ghost membrane by incubation at 37 degrees C; the PC having shorter acyl chains was transferred faster. The cytoplasmic content of the erythrocyte rapidly reduced the nitroxide radical of the spin probe. The central peak height of ESR signal was once increased by incorporation of TEMPO-PC into the erythrocyte membrane and then was spontaneously decreased during further incubation at 37 degrees C. This decrease indicates that PC molecules traverse from the outer to the inner layer of the membrane lipid bilayer. The decrease of signal amplitude was faster with PC of shorter acyl chain. These findings suggest that both transfer between membranes and transverse diffusion in the membrane may be favored to the PC species with shorter acyl chains.     

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.     

山莨菪碱对人红细胞膜蛋白及膜脂运动的影响

本文采用自旋标记顺磁共振波谱技术,研究了山茛菪碱对人红细胞膜蛋白和膜脂运动的影响.结果表明:用马来酰亚胺标记的人红细胞膜,加入山茛菪碱后,其顺磁共振波谱中强、弱固定化作用谱的峰值比增大,膜蛋白的运动受到限制.山茛菪碱对红细胞膜脂的作用部位主要在极性头部,并影响膜脂的流动性.本文还对山茛菪碱与红细胞膜作用的可能机制进行了讨论.     

Photo-induced electron transfer between hypocrellins and nano-sized semiconductor CdS-- EPR study on the kinetics of photosensitized reduction in HA-CdS and HB-CdS systems

Both HA-CdS and HB-CdS (Hys-CdS, Hys represents HA, HB) complex systems were established according to the dynamics of heterogeneous electron-transfer process ＜0. In these systems, the electron transferring from 1Hys* to conduction band of CdS is feasible. Determined from the fluorescence quenching, the apparent association constants (Kapp) between Hypocrellin A (HA), Hypocrellin B (HB) and CdS sol. were about 940 (mol/L)-1, 934 (mol/L)-1, respectively. Fluorescence lifetime measurements gave the rate constant for the electron transfer process from 1HA*, 1HB* into conduction band of CdS semiconductor as 5.16×109 s-1, 5.10×109 s-1, respectively. TEMPO (2,2,6,6-tetramethy-1-piperdinyloxy), a stable nitroxide radical, was used in the kinetic study of the reduction reaction taking place on the surface of a CdS colloidal semiconductor, kinetics equation of the reaction was determined with the electron paramagnetic resonance (EPR) method, and the reaction order of TEMPO is zero. When Hys were added, the rate of EPR increased greatly. By comparing rate constants, the Hys-CdS systems were revealed to be about 350 times more efficient than CdS sol. alone in the photoreduction of TEMPO under visi-ble light. It suggests that Hys can be used as efficient sensitizers of a colloidal semiconductor in the application of solar energy.     

Aldehydes, hydrogen peroxide, and organic radicals as mediators of ozone toxicity

It is generally agreed that unsaturated fatty acids (UFA) are an important class of target molecule for reaction with ozone when polluted air is inhaled. Most discussions have implicated the UFA in cell membranes, but lung lining fluids also contain fatty acids that are from 20 to 40% unsaturated. Since UFA in lung lining fluids exist in a highly aquated environment, ozonation would be expected to produce aldehydes and hydrogen peroxide, rather than the Criegee ozonide. In agreement with this expectation, we find that ozonations of emulsions of fatty acids containing from one to four double bonds give one mole of H2O2 for each mole of ozone reacted. Ozonation of oleic acid emulsions and dioleoyl phosphatidyl choline gives similar results. with two moles of aldehydes and one mole of H2O2 formed per mole of ozone reacted. The net reaction that occurs when ozone reacts with pulmonary lipids is suggested to be given by equation 1. [formula: see text]. From 5 to 10% yields of Criegee ozonides also appear to be formed. In addition, a direct reaction of unknown mechanism occurs between ozone and UFA in homogeneous organic solution, in homogeneous solutions in water, in aqueous emulsions, and in lipid bilayers to give organic radicals that can be spin trapped. These radicals are suggested to be responsible for initiating lipid peroxidation of polyunsaturated fatty acids. Thus, aldehydes, hydrogen peroxide, and directly produced organic radicals are suggested to be mediators of ozone-induced pathology.     

The pyrrolopyrimidine U101033E is a potent free radical scavenger and prevents Fe(II)-induced lipid peroxidation in synaptosomal membranes

The pyrrolopyrimidine U101033E is a therapeutic compound potentially useful in stroke, head injury and other oxidative stress conditions. Electron paramagnetic resonance (EPR) techniques of spin labeling and spin trapping in conjunction with measures of lipid and protein oxidation have been used to investigate the proposed antioxidant capacity of U101033E. We report potent antioxidant activity of this agent in aqueous cell-free solution as measured by spin trapping. U101033E significantly (P<0.005) reduces the formation of the EPR active spin trap N-t-butyl-alpha-phenylnitrone (PBN)-radical adduct by 17.1% at a concentration of 1 microM, four orders of magnitude less than the concentration of PBN. As measured by the decrease in signal intensity of lipid-resident nitroxide stearate spin probes, an EPR assay for lipid peroxidation, this pyrrolopyrimidine compound efficiently protected against hydroxyl radical-induced lipid peroxidation in cortical synaptosomal membranes deep within the membrane bilayer, but not closer to the membrane surface. In addition, U101033E partially prevents synaptosomal protein oxidation in the presence of Fe(II); however, U101033E demonstrates some protein oxidative effects itself. These results are supportive of the proposed role of U101033E as a lipid-specific antioxidant, especially for protection against lipid peroxidation that occurs deep within the membrane bilayer, but raise some potential concerns about the oxidative nature of this agent toward proteins.     

In vitro photodynamic effects of phthalocyaninatosilicon covalently linked to 2,2,6,6-tetramethyl-1-piperidinyloxy radicals on cancer cells

In this paper, we have investigated the ability to sensitize the phototoxicity toward HeLa cells in vitro, of tetra-tert-butylphthalocyaninatosilicon (SiPc) covalently linked to one or two 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) radicals (R1c or R2c), which are shown as photosensitizers efficiently producing singlet oxygen (1Delta(g)). Addition of R1c or R2c encapsulated in liposomes to cultures, followed by irradiation with a 680-nm dye laser, resulted in a highly significant phototoxicity toward HeLa cells, in contrast to negligible phototoxicity observed with (dihydroxy)SiPc (R0). EPR measurements indicate that R1c and R2c exist in some degree as nitroxide radicals even in HeLa cells. Electronic absorption spectra indicate that the degree of aggregation increases in the order R2c相似文献   

In vitro effects of ozone on human erythrocyte membranes: an EPR study

The effects of ozone at different concentrations (10, 30, 45 g/m3) on fluidity and thermotropic properties of erythrocyte membranes were investigated by EPR using two spin probes: 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA). The effect of ozone on the erythrocyte membrane fluidity was a dose-dependent process. The ozone at concentration of 10 g/m3 caused rigidization of the membrane while at concentration of 45 g/m3 increased fluidity both on the surface and in the deeper hydrocarbon region of the membrane. Temperature transitions close to the polar heads region (monitored by 5-DSA) were not sensitive to an increase in ozone concentration. In the case of 16-DSA, low temperature thermotropic transition (around 20 degrees C) gradually decreased with the increase of ozone concentration. High temperature transition (around 40 degrees C) significantly differed at the ozone concentration of 10 g/m3 and 45 g/m3, being higher and lower, respectively, as compared to untreated cells. For the ozone concentration of 45 g/m3 the disappearance of the low temperature break and the appearance of two breaks at 37 degrees C and 16 degrees C were observed.