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 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.     

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.     

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.     

Nitroxide radicals protect against DNA damage in rat epithelial cells induced by nitric oxide,nitroxyl anion and peroxynitrite

In order to gain more knowledge on the antioxidant role of nitroxide radicals, in this study we investigate their possible protective action against DNA damage induced by nitric oxide (NO) and reactive nitrogen oxide species deriving from it, namely nitroxyl anion (NO(-)) and peroxynitrite (ONOO(-)). Rat trachea epithelial cells were exposed under aerobic conditions to (1) NO generated by 150 microM S-nitrosoglutathione monoethyl ester (GSNO-MEE), (2) NO(-) generated by 200 microM Angeli's salt (Na(2)N(2)O(3)) (3) ONOO(-) generated by 1mM SIN-1 (3-morpholino-sydnonimine) and (4) 100 microM synthesized ONOO(-), in the absence and presence of 5 microM of two indolinonic nitroxides synthesized by us and the piperidine nitroxide TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl). DNA damage was assessed using the comet assay-a rapid and sensitive, single-cell gel electrophoresis technique used to detect primary DNA damage in individual cells. The parameter tail moment, used as an index of DNA damage, showed that in all cases the nitroxides remarkably inhibited DNA strand breaks induced by the different nitrogen oxide species. All three nitroxides protect to the same extent, except in the case of synthesized peroxynitrite where the aromatic nitroxides 1 and 2 are more efficient than TEMPO. These findings are consistent with the antioxidant character of nitroxide compounds and give additional information on the potential implications for their use as therapeutic agents.     

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.     

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.     

Changes in the lipid physical state in human erythrocyte membranes subjected lead exposure in vitro

The effect of lead acetate on the physical state of membrane lipids in human erythrocytes in vitro was studied using the lipophilic fluorescence probe 1,6-diphenyl-1,3,5-hexatriene and spin probes 16-doxyl-stearate and iminoxyl palmitic acid. It was shown that 2-10 microM lead acetate causes an increase in both intensity and polarization of fluorescence of 1,6-diphenyl-1,3,5-hexatriene, indicating changes in the microviscosity of the lipid bilayer of erythrocyte membranes. Judging from the parameters of EPR spectra of 16-doxyl stearate and iminoxyl palmitic acid incorporated into erythrocyte membranes, 2-10 microM lead acetate increases the heterogeneity of the lipid bilayer in surface and deep hydrophobic layers of the erythrocyte membrane.     

Nitroxide radicals protect DNA from damage when illuminated in vitro in the presence of dibenzoylmethane and a common sunscreen ingredient

Indolinonic nitroxide radicals efficiently scavenge oxygen- and carbon-centered radicals. They protect lipid and protein systems against oxidative stress, but little is known about their capacity to protect DNA against radical-mediated damage. We compare indolinonic nitroxides and the piperidines TEMPO and TEMPOL for their ability to inhibit strand breaks inflicted on DNA when it is illuminated in vitro in the presence of dibenzoylmethane (DBM) and a relative, Parsol 1789, used as a UVA-absorbing sunscreen. We used spin-trapping EPR to examine the formation of radicals and plasmid nicking assays to evaluate DNA strand breakage. The results have a two-fold interest. First, they show that all the nitroxides tested efficiently prevent DNA damage in a dose-dependent fashion. Vitamin E had no effect under the conditions used. Second, they show that carbon-centered radicals are produced on illumination of DBM and its relative and that their formation is probably responsible for the direct strand breaks found when naked DNA is illuminated in vitro in their presence. Additional work on the ability of sunscreens to enter human cells and their response to the light that penetrates sunscreen-protected skin would be necessary before any conclusion could be drawn as to whether the results reported here are relevant to human use of sunscreens.     

A Hydrogen-Donating Monohydroxamate Scavenges Ferryl Myoglobin Radicals

The addition of 25μM hydrogen peroxide to 20μM metmyoglobin produces ferryl (Fe^IV = O) myoglobin. Optical spectroscopy shows that the ferryl species reaches a maximum concentration (60-70% of total haem) after 10 minutes and decays slowly (hours). Low temperature EPR spectroscopy of the high spin metmyoglobin (g = 6) signal is consistent with these findings. At this low peroxide concentration there is no evidence for iron release from the haem. At least two free radicals are detectable by EPR immediately after H₂O₂ addition, but decay completely after ten minutes. However, a longer-lived radical is observed at lower concentrations that is still present after 90 minutes. The monohydroxamate N-methylbutyro-hydroxamic acid (NMBH) increases the rate of decay of the fenyl species. In the presence of NMBH, none of the protein-bound free radicals are detectable; instead nitroxide radicals produced by oxidation of the hydroxamate group are observed. Similar results are observed with the trihydroxamate, desferoxamine. “Ferryl myoglobin” is still able to initiate lipid peroxidation even after the short-lived protein free radicals are no longer detectable (E.S.R. Newman, C.A. Rice-Evans and M.J. Davies (1991) Biochemical and Biophysical Research Communications 179, 1414-1419). It is suggested that the longer-lived protein radicals described here may be partly responsible for this effect. The mechanism of inhibition of initiation of lipid peroxidation by hydroxamate drugs, such as NMBH, may therefore be due to reduction of the protein-derived radicals, rather than reduction of ferryl haem.     

A Hydrogen-Donating Monohydroxamate Scavenges Ferryl Myoglobin Radicals

The addition of 25μM hydrogen peroxide to 20μM metmyoglobin produces ferryl (Fe^IV = O) myoglobin. Optical spectroscopy shows that the ferryl species reaches a maximum concentration (60-70% of total haem) after 10 minutes and decays slowly (hours). Low temperature EPR spectroscopy of the high spin metmyoglobin (g = 6) signal is consistent with these findings. At this low peroxide concentration there is no evidence for iron release from the haem. At least two free radicals are detectable by EPR immediately after H₂O₂ addition, but decay completely after ten minutes. However, a longer-lived radical is observed at lower concentrations that is still present after 90 minutes. The monohydroxamate N-methylbutyro-hydroxamic acid (NMBH) increases the rate of decay of the fenyl species. In the presence of NMBH, none of the protein-bound free radicals are detectable; instead nitroxide radicals produced by oxidation of the hydroxamate group are observed. Similar results are observed with the trihydroxamate, desferoxamine. “Ferryl myoglobin” is still able to initiate lipid peroxidation even after the short-lived protein free radicals are no longer detectable (E.S.R. Newman, C.A. Rice-Evans and M.J. Davies (1991) Biochemical and Biophysical Research Communications 179, 1414-1419). It is suggested that the longer-lived protein radicals described here may be partly responsible for this effect. The mechanism of inhibition of initiation of lipid peroxidation by hydroxamate drugs, such as NMBH, may therefore be due to reduction of the protein-derived radicals, rather than reduction of ferryl haem.     

Interaction of dipyridamole with micelles of lysophosphatidylcholine and with bovine serum albumin: fluorescence studies.

The interaction of the coronary vasodilator dipyridamole with biological systems, protein and membranes has been studied through optical absorption and fluorescence spectroscopies. Using the analysis of the spectra and fluorescence intensity of dipyridamole (DIP) in solution, the interaction of this compound with the transport protein albumin (BSA) and with a model of cell membranes, namely micelles of lysophosphatidylcholine (L-PC), was investigated. Measurements were performed at pH 5.0 and pH 7.0 where the molecule of DIP is fully protonated and partially protonated, respectively. The quenching of fluorescence with nitroxide-stable radicals 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) as well as with acrylamide and iodide allowed the localization of the drug in the polar interface of micelles. Quenching by acrylamide and iodide in L-PC micelles demonstrated the effect of micelle protonation which increased the accessibility of iodide to the chromophore. An effective association constant was obtained both at pH 7.0 (7.5 x 10(3) M-1) and pH 5.0 (2.5 x 10(3) M-1) and a very good agreement with the proposed binding model was observed. The quantum yields of fluorescence data agree very well with the fluorescence lifetimes. The measurement of lifetimes was important to understand the kinetic data obtained from Stern-Volmer plots both of radical, acrylamide and iodide quenching of fluorescence. It was observed that, in the presence of micelles, the kq value increased for TEMPO while decreased for TEMPOL. This result, together with the vanishing solubility of DIP in saturated hydrocarbons and the preferential partition of TEMPO in micelles, suggested the localization of DIP in the polar micellar interface. This is also supported by the enhanced iodide quenching at pH 5.0, constancy of acrylamide quenching in the range of pH 7.0-5.0 and the partition of TEMPO and TEMPOL in SDS micelles. The association constant of DIP to BSA was also estimated both at pH 7.0 (2 x 10(4) M-1) and pH 5.0 (4 x 10(3) M-1). Quenching studies with nitroxide radicals, acrylamide and iodide also suggested the binding of the drug to a hydrophobic region of the protein. At pH 5.0, the protein undergo a conformational change which leads to a loosening of the overall structure so that the accessibility of the nitroxide radicals for DIP is increased at this pH. The differences in kq values at pH 7.0 and pH 5.0 suggested that at pH 7.0 the chromophore is protected in the protein site.(ABSTRACT TRUNCATED AT 400 WORDS)     

EPR detection of glutathionyl and protein-tyrosyl radicals during the interaction of peroxynitrite with macrophages (J774)

Peroxynitrite is one of the biological oxidants whose addition to cells has been shown to either activate signaling pathways or lead to cell injury, depending on cell type and oxidant concentration. The intermediacy of free radicals in these processes has been directly demonstrated only during the interaction of peroxynitrite with erythrocytes, a particular cell type, due to its high hemoglobin content. Here, we demonstrate that the addition of peroxynitrite to a macrophage cell line (J774) led to the production of glutathionyl and protein-tyrosyl radicals. The glutathionyl radical was characterized by EPR spin-trapping experiments with 5,5-dimethyl-1-pyrroline-N-oxide. Protein-tyrosyl radical formation was suggested by direct EPR spectroscopy and confirmed by EPR spin-trapping experiments with 3,5-dibromo-4-nitrosobenzenesulfonic acid and Western blot analysis of nitrated proteins in treated macrophages. Time dependence studies of free radical formation indicate that intracellular glutathione and unidentified proteins are the initial peroxynitrite targets in macrophages and that their derived radicals trigger radical chain reactions. The results are likely to be relevant to the understanding of the bioregulatory and biodamaging effects of peroxynitrite.     

Oxygen concentration dependence of lipid peroxidation and lipid-derived radical generation: application of profluorescent nitroxide switch

Lipid-derived radicals and peroxides are involved in the pathogenesis of oxidative stress diseases and, although lipid peroxide production is a required reaction between a lipid radical and molecular oxygen, a useful lipid radical detection method has remained tentative. Also, the effect of oxygen concentration on lipid peroxide production must be considered because of the hypoxic conditions in cancer and ischemic regions. In this study, the focus was on nitroxide reactivity, which allows spin trapping with carbon-centred radicals via radical-radical reactions and fluorophore quenching through interactions with nitroxide's unpaired electron. Thus, the aim here was to demonstrate a useful detection method for lipid-derived radicals as well as to clarify the effects of oxygen concentration on lipid peroxide production using profluorescent nitroxide. This latter compound reacted with lipid-derived radicals in a manner inversely dependent on oxygen concentration, resulting in fluorescence due to alkoxyamine formation and, conversely, lipid peroxide concentrations decreased with lower oxygen in the reaction system. Furthermore, nitroxide inhibited lipid peroxide production and stopped oxygen consumption in the same solution. These results suggested that the novel application of profluorescent nitroxide could directly and sensitively detect lipid-derived radicals and that radical and peroxide production were dependent on oxygen concentration.     

Surface Electrostatics of Lipid Bilayers by EPR of a pH-Sensitive Spin-Labeled Lipid

Many biophysical processes such as insertion of proteins into membranes and membrane fusion are governed by bilayer electrostatic potential. At the time of this writing, the arsenal of biophysical methods for such measurements is limited to a few techniques. Here we describe a, to our knowledge, new spin-probe electron paramagnetic resonance (EPR) approach for assessing the electrostatic surface potential of lipid bilayers that is based on a recently synthesized EPR probe (IMTSL-PTE) containing a reversibly ionizable nitroxide tag attached to the lipids’ polar headgroup. EPR spectra of the probe directly report on its ionization state and, therefore, on electrostatic potential through changes in nitroxide magnetic parameters and the degree of rotational averaging. Further, the lipid nature of the probe provides its full integration into lipid bilayers. Tethering the nitroxide moiety directly to the lipid polar headgroup defines the location of the measured potential with respect to the lipid bilayer interface. Electrostatic surface potentials measured by EPR of IMTSL-PTE show a remarkable (within ±2%) agreement with the Gouy-Chapman theory for anionic DMPG bilayers in fluid (48°C) phase at low electrolyte concentration (50 mM) and in gel (17°C) phase at 150-mM electrolyte concentration. This agreement begins to diminish for DMPG vesicles in gel phase (17°C) upon varying electrolyte concentration and fluid phase bilayers formed from DMPG/DMPC and POPG/POPC mixtures. Possible reasons for such deviations, as well as the proper choice of an electrostatically neutral reference interface, have been discussed. Described EPR method is expected to be fully applicable to more-complex models of cellular membranes.     

Oxygen concentration dependence of lipid peroxidation and lipid-derived radical generation: Application of profluorescent nitroxide switch

Abstract

Lipid-derived radicals and peroxides are involved in the pathogenesis of oxidative stress diseases and, although lipid peroxide production is a required reaction between a lipid radical and molecular oxygen, a useful lipid radical detection method has remained tentative. Also, the effect of oxygen concentration on lipid peroxide production must be considered because of the hypoxic conditions in cancer and ischemic regions. In this study, the focus was on nitroxide reactivity, which allows spin trapping with carbon-centred radicals via radical–radical reactions and fluorophore quenching through interactions with nitroxide's unpaired electron. Thus, the aim here was to demonstrate a useful detection method for lipid-derived radicals as well as to clarify the effects of oxygen concentration on lipid peroxide production using profluorescent nitroxide. This latter compound reacted with lipid-derived radicals in a manner inversely dependent on oxygen concentration, resulting in fluorescence due to alkoxyamine formation and, conversely, lipid peroxide concentrations decreased with lower oxygen in the reaction system. Furthermore, nitroxide inhibited lipid peroxide production and stopped oxygen consumption in the same solution. These results suggested that the novel application of profluorescent nitroxide could directly and sensitively detect lipid-derived radicals and that radical and peroxide production were dependent on oxygen concentration.     

Singlet oxygen production in thylakoid membranes during photoinhibition as detected by EPR spectroscopy

Exposure of isolated spinach thylakoids to high intensity illumination (photoinhibition) results in the well-characterized impairment of Photosystem II electron transport, followed by degradation of the D1 reaction centre protein. In the present study we demonstrate that this process is accompanied by singlet oxygen production. Singlet oxygen was detected by EPR spectroscopy, following the formation of stable nitroxide radicals from the trapping of singlet oxygen with a sterically hindered amine TEMP (2,2,6,6-tetramethylpiperidine). There was no detectable singlet oxygen production during anaerob photoinhibition or in the presence of sodium-azide. Comparing the kinetics of the loss of PS II function and D1 protein with that of singlet oxygen trapping suggests that singlet oxygen itself or its radical product initiates the degradation of D1.Abbreviations HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonle acid - PS Photosystem - TEMP 2,2,6,6-tetramethylpiperidine - TEMPO 2,2,6,6-tetramethylpiperidine-1-oxyl     

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.     

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.