Radiation-induced lipid peroxidation and the fluidity of erythrocyte membrane lipids

The effect of radiation-induced peroxidation on the fluidity of the phospholipids of the erythrocyte membrane was studied using both erythrocyte ghosts and liposomes formed from the polar lipids of erythrocytes. In liposomes, the oxidation of the phospholipids increased with radiation dose, but there was no change in the fluidity of the lipids as measured by spin-label motion. Under the same conditions of irradiation, no oxidation of phospholipid was detected in erythrocyte ghosts, although changes occurred in the motion of spin labels intercalated with the membrane. These changes were attributed to radiation-induced alterations in the membrane proteins. It is concluded that alterations in motion of spin labels, observed with intact membranes after irradiation, are most likely the result of changes in the structure of membrane proteins rather than the lipids.     

Structural alterations of the human erythrocyte membrane upon influenza virus attachment

Molecular events on the human erythrocyte membrane subsequent to influence virus binding were investigated by electron spin resonance (ESR) measurements after spin labeling of the cell membrane at different positions. Virus binding affected the glycocalyx structure as well as the physical state of the cytoskeleton at the inner leaflet, but not the lipid phase. A lateral reorganization of spin-labeled glycophorin was not indicated after virus attachment.     

Comparison of the photodynamic action of Rose Bengal and tetrachlorosalicylanilide on isolated porcine erythrocyte membranes

The light-mediated effects of Rose Bengal and 3,3',4',5-tetrachlorosalicylanilide (T4CS) on porcine erythrocyte membranes have been investigated. Irradiation in the presence of Rose Bengal and oxygen causes extensive destruction of the unsaturated fatty acids of the erythrocyte membrane. This results in a decrease in the membrane flexibility as measured by a nitroxide spin probe. Irradiation in the presence of T4CS and oxygen had no measurable effect on the unsaturated fatty acids or on the membrane flexibility. Irradiation of erythrocyte membranes both in the presence of Rose Bengal and oxygen and of T4CS gave rise to polymerisation of the membrane proteins. This was evident by polyacrylamide gel electrophoresis and by freeze-fracture electron microscopy. Aggregation of membrane proteins could be observed with low levels of Rose Bengal and short irradiation times at which no loss of unsaturated fatty acids could be detected. Irradiation of the n-butanol-extracted apoprotein of porcine erythrocyte membranes both in the presence of Rose Bengal and of T4CS resulted in polymerisation of the protein as measured by gel electrophoresis and electron microscopy. The results obtained from the two photodynamic compounds are compared in terms of their different mechanisms of action on the membrane. The implications of the results in determining the molecular events leading to photohaemolysis are discussed.     

Effect of cholesterol on human erythrocyte membrane. A spin label study

The effect of cholesterol on the membrane fluidity of human erythrocytes has been studied by electron spin resonance (ESR) spectroscopy, sensing the motion of androstane and fatty acid spin labeles in the cell membrane and in vesicles made from extracted phospholipids. 1. Androstane spin label (ASL) was incorporated from ASL-containing phospholipid vesicles into the erythrocyte membrane, essentially by a partition mechanism in proportion to their phospholipid contents. 2. On increasing the cholesterol or ASl content in the cell membrane, the spin label was gradually immobilized. 3. ASL motion in the cell membrane seemed to be primarily determined by the cholesterol/phospholipid molar ratio, regardless of the membrane protein-lipid interaction, as judged from the temperature effects on the ESR spectra of both membranes. 4. However, glutaraldehyde pretreatment induced considerable changes of the cholesterol-lipid interaction in the cell membrane, i.e., strong immobilization and cluster formation of ASL were observed.     

Electron spin resonance analysis of irreversible changes induced by calcium perturbation of erythrocyte membranes.

Introduction of calcium during hemolysis of erythrocytes causes irreversible membrane changes, including protein aggregation. These changes have been investigated by incorporation of one protein and three fatty acid spin label probes into washed membranes from erythrocytes hemolyzed with a range of Ca2+ concentrations. Electron spin resonance spectra of the lipid probes were analyzed for changes in the order parameters, isotropic coupling constants and mean angular deviations of the lipid hydrocarbon chains. The results generally indicated an increased freedom of mobility of the probes with increased Ca2+ concentration during hemolysis, but the response of each probe showed a different concentration dependence. The maximal response was obtained with the I(5, 10) probe. Variations in the responses were interpreted to reflect different modes of protein-lipid or protein-probe interactions arising from Ca2+ -induced membrane protein alterations. Spectra from membranes treated with the protein spin label showed an increased ratio of immobilized to mobile label with increased Ca2+ concentrations at hemolysis. This is consistent with the membrane protein aggregation phenomena previously observed. It is suggested that the increased protein-protein interactions formed as a result of calcium treatment permit an increased lipid mobility in the membrane regions monitored by the fatty acid probes.     

Electron paramagnetic resonance investigation of sucrose irradiated with heavy ions

We investigated the potential use of sucrose to estimate linear energy transfer (LET) for heavy-ion irradiation. We also made a quantitative comparison between heavy-ion and gamma irradiation in terms of spin concentration. Heavy-ion irradiation of sucrose produces stable free radicals. Based on the electron paramagnetic resonance (EPR) spectra obtained, the stable sucrose radicals are the same among helium ions, carbon ions and gamma rays. The EPR spectrum was approximately 70 G wide and was composed of several hyperfine structures. The total spin concentration obtained after the heavy-ion irradiation increased linearly as the absorbed dose increased and decreased logarithmically as LET increased. Production of the spin concentration of helium ions was two times more dependent on LET than that for carbon-ion irradiation. The empirical relationships obtained imply that LET at a certain dose can be determined by the spin concentration. Furthermore, the results of gamma irradiation of deuterated sucrose suggest that one of the persistent radicals is a carbon-centered radical.     

Effects of phytohaemagglutinin, wheat-germ agglutinin, and concanavalin-A on the physical state of sialic acid and membrane proteins in human erythrocyte ghosts: a spin label study

Parallel experiments employing sialic acid- and protein specific spin labels have been performed to monitor the effects on the physical state of this carbohydrate and membrane proteins of human erythrocytes induced by the binding of three lectins, $\text{Phaseolus vulgaris}$ phytohaemagglutinin (PHA), wheat germ agglutinin (WGA), and Concanavalin-A (Con-A). PHA and WGA, both of which are known to bind at different sites on the principal sialoglycoprotein of human erythrocytes, glycophorin, had markedly different effects: compared to control values, PHA decreased the apparent correlation time of the sialic acid specific spin probe by 10% while this parameter was decreased by 33% by WGA. The protein specific spin label also monitored differential effects of these lectins: the relevant electron spin resonance parameter (the W/S ratio) was reduced 33% by PHA and increased by WGA over 17% from that of control values. Con-A, which is known to bind to the principal transmembrane protein, Band 3, had no effect on sialic acid or membrane proteins as assessed by the two spin labels employed. These results suggest that (1) the effects of binding of these different lectins, two of which bind to the same cell surface receptor, can be discriminated by use of spin labeling methods; (2) binding events occuring at the cell surface have distinct and pronounced effects on the physical state of proteins within the membrane; (3) the different results with PHA and WGA both of which bind to glycophorin are indicative of multiple and complex interactions of this glycoprotein with the membrane proteins in the erythrocyte; and (4) that the spin labelling technique has the potential to investigate the relationships between cell-surface binding events to membrane structural-functional interactions.     

Ionizing radiation-induced structural modification of human red blood cells

Summary The effect of gamma radiation on red blood cells have been examined using a spin labeling method. For this purpose two spin labels were used to monitor membrane fluidity: methyl 5-doxylpalmitate (Met 5-DP) and methyl 12-doxylstearate (Met 12-DS). The irradiation of red cells with the doses of 200 and 500 Gy caused decrease of microviscosity in certain regions of lipid bilayer (as indicated by Met 5-DP and Met 12-DS spectra) but did not affect lipid order parameter. The behavior of two other spin labels, maleimide(4-malei-mido-2,2,6,6-tetramethylpiperidine-1-oxyl) and TEMPONE (4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl) indicated:1) conformational changes of membrane proteins,2) modification of cell internal peptides and proteins,3) decreased internal viscosity of red blood cells.     

Interaction energies in lectin-induced erythrocyte aggregation

Two N-acetylgalactosamine-reactive lectins, Helix pomatia (HPA) and Dolichos biflorus (DBA), were used to study the energies involved in cell-cell interactions through the specific binding of these lectins to their membrane receptors on genotype AO human erythrocytes (red blood cells) (RBCs). The energy required to dissociate a unit of aggregated membrane area (gamma d) of two RBCs bridged by lectin molecules was determined from the shear force needed to dissociate two-cell aggregates in a flow channel. When HPA were used as bridging molecules, gamma d (0.4 X 10(-4) to 3.8 X 10(-4) dyn/cm) was proportional to the density (D = 175 to 1,060 molecules/micron 2) of HPA molecules bound on the RBC membrane. A similar gamma d/D ratio was also obtained for DBA. These results indicate that the number of lectin molecules bound on the interface plays an important role in determining the energy required for cell-cell dissociation. The aggregation energy per unit membrane area (gamma a) in lectin-induced aggregates was calculated from the degree of encapsulation of a lectin-bound, heat-sphered human RBC by a normal discoid RBC. A minimum of approximately 1,800 HPA molecules/micron 2 on the spheres was required to form stable aggregates with the RBC. By using spheres having a surface HPA density of 1,830 to 2,540 molecules/micron 2, or 1.1-1.5 X 10(12) combining sites/cm2, the gamma a value for HPA-induced aggregation was found to be 2.2 X 10(-3) dyn/cm. This higher value of gamma a than gamma d has been explained on the basis of several differences in aggregation and disaggregation processes. The gamma a value for DBA-induced aggregation was not obtainable by the sphere encapsulation method because of the relative low D values. A comparison of the present results with the published value of the free energy change of 5 kcal/mol for the interactions of HPA and DBA with their ligands suggests that only a small fraction of the lectin molecules bound to RBC surface participate in the bridging of adjacent cells.     

Detection of irradiated spices by different physical techniques

We used thermoluminescence, electron spin resonance, and viscosimetric measurements to establish whether or not a spice had been irradiated. Thermoluminescence, using the 1788 EN official protocol with an alternative method for the extraction of mineral impurities, led to proof of irradiation or proof of no treatment. Electron spin resonance led to different spectrum shapes depending on the chemical composition of the spices; ESR could only be used as proof of irradiation up to several weeks after irradiation, and only for some spices. Viscosimetric measurements carried out on spice suspensions led to a presumption of treatment (or of no treatment) and possibly to proof of irradiation.     

Ionophoric activity of truncated gramicidin A analogue in phospholipid bilayers and mitochondrial and erythrocyte membranes

Ionophoric activities of an N-terminus truncated gramicidin A (gA) analogue (mini-gramicidin) and its covalent dimer were studied in planar bilayer phospholipid membranes (BLM) using macroscopic current measurements (at high concentrations of the peptides) and single-channel recordings. As with gA-induced currents, mini-gramicidin-stimulated macroscopic currents through BLM underwent sensitized photoinactivation, i.e. were suppressed after irradiation with visible light in the presence of a photosensitizer generating singlet oxygen. The sensitivity of the tested compounds to photoinactivation descended in the following order: minigramicidin dimer > mini-gramicidin monomer > gA. The data from single-channel measurements and kinetic analysis of flash-induced photoinactivation obtained at different levels of macroscopic currents suggest that mini-gramicidin and its covalent dimer induce a variety of conducting states; their ratio depends on membrane thickness. Analysis of natural (mitochondrial and erythrocyte) membranes established that ionophoric activities of mini-gramicidin and its covalent dimer depend essentially on the membrane type.     

Strong interactions between a spin-labeled cholesterol analog and erythrocyte proteins in the human erythrocyte membrane

We have used a spin label analog of cholesterol bearing a nitroxide on the alkyl chain (26-nor-25-doxylcholestanol) to study cholesterol-protein interactions in the human erythrocyte membrane. As judged from the ESR spectrum, the spin label is readily incorporated into the membrane when added from a concentrated ethanolic solution to a cell or ghost suspension. With intact erythrocytes or white ghosts in isotonic buffer, the ESR spectrum is a superposition of a mobile component and a strongly immobilized component (outer hyperfine splitting 61–63 G). The latter corresponds to approx. 45% of the signal, a percentage which is barely affected by varying the temperature between 5 and 37°C. Removal of the cytoskeletal proteins spectrin and actin by low ionic strength treatment or of all extrinsic proteins by alkali treatment of ghosts reduces the immobilized fraction to approx. 25%. The effect of controlled proteolysis of intrinsic proteins was also tested. Pre-treatment of cells with chymotrypsin or pre-treatment of unsealed ghosts with trypsin has no effect on the ESR spectrum obtained with alkali-treated membranes. On the other hand, after chymotrypsin treatment of unsealed ghost, which reduces the band 3 protein to a 17.5 kDa membrane fragment, the strongly immobilized component is no longer observable. These data show that the cholesterol analog 26-nor-25-doxylcholestanol interacts strongly with one or several proteins of the erythrocyte membrane. That the intrinsic protein band 3 is involved is suggested by the disappearance of the immobilized fraction occurring upon chymotrypsin digestion of this protein. Our results are thus consistent with the proposal of a selective cholesterol-band 3 interaction in the erythrocyte membrane (Schubert, D. and Boss, K. (1982) FEBS Lett. 150, 4–8). Our data also suggest that this interaction is influenced by cytoskeletal proteins, an effect which can be explained considering the known linking of band 3 to the erythrocyte cytoskeleton via ankyrin. Experiments have also been carried out with 3-doxylandrostanol, a more commonly used cholesterol spin-label analog. With this spin label, at all temperatures investigated, we found it impossible to demonstrate unambiguously the existence of two separate spectral components. It is suggested that 26-nor-25-doxylcholestanol is a better reporter of cholesterol behavior in membranes.     

A spin label study of human erythrocyte ghost membranes damaged by methyl phenyldiazenecarboxylate (azoester)

The mechanism of methyl phenyldiazenecarboxylate (azoester) damage to human erythrocyte membranes has been investigated by means of spin labels. Azoester treatment exposed protein binding sites for non-covalently bound stearic acid and androstane spin labels that were occult in the untreated membrane. Experiments with iodoacetamide and N-alkylmaleimide spin labels suggested that azoester destroyed membrane sulfhydryl groups. No change in the structural integrity of membrane lipid components could be detected.     

Features of organic nonelectrolyte binding to the erythrocyte membrane

The capacity of erythrocyte membranes for organic nonelectrolytes from different chemical groups of chemical compounds was studied by the spin probe method and scanning electron microscopy. Hydrophobic spin-labeled derivatives of gamma-carbolin and stearic acid and the screened phenol antioxidant fenozan-1 were used as nonelectrolytes. Based on the analysis of electron spin resonance spectra of the hydrophobic spin-labeled nonelectrolytes and electron micrographs of erythrocytes, differences in the capacity of distribution regions in the intramembrane space of the derivative of gamma-carbolin and fenozan-1, on the one hand, and the spin-labeled derivative of fatty acid, on the other hand, were found. The first group has at least two membrane distribution regions, whereas in the second case only one type of distribution was found. The influence of limited membrane capacity on the realization of biological activity of organic nonelectrolytes is discussed.     

Heterogeneity in the fluidity of intact erythrocyte membrane and its homogenization upon hemolysis.

Intact erythrocytes were spin-labeled with various classes of phospholipid label. The ESR spectrum for phosphatidylcholine spin label was distinctly different from those for phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidic acid spin labels. The overall splitting for the former (52.5 G) was markedly larger than those for the others (approx. 47 G), suggesting a more rigid phosphatidylcholine bilayer phase and more fluid phosphatidylethanolamine and phosphatidylserine phases in the erythrocyte membrane. Evidence for asymmetric distribution of phospholipids in the membrane was obtained. Spin-labeled phosphatidylcholine incorporated into erythrocytes was reduced immediately by cystein and Fe3+, while the reduction of spin-labeled phosphatidylserine was very slow. The present results therefore suggest asymmetric fluidity in erythrocyte membrane; a more rigid outer layer and a more fluid inner layer. The heterogeneity in the lipid structure was also manifested in the temperature dependence of the fluidity. The overall splitting for phosphatidylcholine spin label showed two inflection points at 18 and 33 degrees C, while that for phosphatidylserine spin label had only one transition at 30 degrees C. When the spin-labeled erythrocytes were hemolyzed, the marked difference in the ESR spectra disappeared, indicating homogenization of the heterogenous fluidity. Mg2+ or Mg2+ + ATP prevented the hemolysis-induced spectral changed. Ca2+ did not prevent the homogenization and acted antagonistically to Mg2+. The heterogeneity preservation by Mg2+ was nullified by trypsin, pronase or N-ethylmaleimide added inside the cell. Some inner proteins may therefore be involved in maintaining the heterogeneous structure. The protecting action of Mg2+ was dependent on hemolysis temperature, starting to decrease at 18 degrees C and vanishing at 40 degrees C. The present study suggests that the heterogeneity in the fluidity of intact erythrocyte membranes arises from interactions between lipids and proteins in the membrane and also from interactions between the membrane constituents and the inner proteins. Concentration of cholesterol in the outer layer may also partly contribute to the heterogeneity.     

Spin label study of erythrocyte deformability I. Electron spin resonance spectral change under shear flow

A spin labeling method in electron spin resonance spectroscopy (ESR) is applied for the first time to study the deformability of human red blood cells (RBC). ESR measurements of a RBC suspension incubated with a fatty acid spin label were performed, using a narrow-gap flat ESR sample cell under various flow shear stresses (tau). Remarkable changes were observed in ESR spectra with tau, indicating that RBC are oriented in such a way that the greater part of the membrane surface is aligned parallel to the ESR cell walls. The diamide-treated, hardened RBC, in which the biconcave discoid shape remains intact under no shear stress, exhibit a smaller ESR spectral change with tau than the intact, demonstrating that the present method can be used to assess the deformation of RBC occurring with flow orientation. In particular, the relative amplitude of an ESR difference spectrum may be used as a measure of the elongation of RBC. The conclusion is further supported by experiments using glutaraldehyde-treated or heat-denatured RBC. All these ESR results are in good agreement with the corresponding results obtained by several different methods. The present spin labeling technique is thus proven to be applicable for evaluating RBC deformability.     

Effect of shear stress and free radicals induced by ultrasound on erythrocytes

The present study was undertaken to elucidate the mechanism of hemolysis induced by ultrasound. Ar or N2O gas was used to distinguish between cavitation with or without free radical formation (hydroxyl radicals and hydrogen atoms). Free radical formation was examined by the method of spin trapping combined with ESR. After sonication of erythrocyte suspensions, several structural and functional parameters of the erythrocyte membrane--hemolysis, membrane fluidity, membrane permeability, and membrane deformability--were examined. Although free radical formation was observed in the erythrocyte suspensions sonicated in the presence of Ar, no free radical formation was observed in the presence of N2O. However, the hemolysis behavior induced by ultrasound was similar in the presence of Ar or N2O. The membrane fluidity, permeability, and deformability of the remaining unlysed erythrocytes after sonication in the presence of Ar or N2O were unchanged and identical to those of the control cells. On the other hand, after gamma irradiation (700 Gy), the hemolysis behavior was quite different from that after sonication, and the membrane properties were significantly changed. These results suggest that hemolysis induced by sonication was due to mechanical shearing stress arising from cavitation, and that the membrane integrity of the remaining erythrocytes after sonication was the same as that of control cells without sonication. The triatomic gas, N2O, may be useful for ultrasonically disrupting cells without accompanying free radical formation.     

ESR spectral changes induced by chlorpromazine in spin-labeled erythrocyte ghost membranes

chlorpromazine interacted preferentially with membrane proteins rather than membrane lipids in the initial incorporation into human erythrocyte ghosts, as demonstrated by means of the fluorescence quenching and a maleimide spin label. In this state the membrane fluidity increased. At higher concentrations of chlorpromazine, the membrane fluidity decreased and a motionally restricted signal from fatty acid spin labels appeared predominantly. However, no such signal appeared in protein-free vesicles. The temperature and pH dependences of the outer hyperfine splitting of this restricted signal were very similar to those of bovine serum albumin. On the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chlorpromazine-treated and -untreated ghosts, it was found that there was no significant difference in membrane proteins between both samples except for the changes of a few bands which were not directly concerned with the occurrence of this restricted signal. These results suggest that the fatty acid spin labels bind preferably to membrane proteins as the lipid domain becomes packed with chlorpromazine.     

ESR spectral changes induced by chlorpromazine in spin-labeled erythrocyte ghost membranes

Chlorpromazine interacted preferentially with membrane proteins rather than membrane lipids in the initial incorporation into human erythrocyte ghosts, as demonstrated by means of the fluorescence quenching and a maleimide spin label. In this state the membrane fluidity increased. At higher concentrations of chlorpromazine, the membrane fluidity decreased and a motionally restricted signal from fatty acid spin labels appeared predominantly. However, no such signal appeared in protein-free vesicles. The temperature and pH dependences of the outer hyperfine splitting of this restricted signal were very similar to those of bovine serum albumin. On the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chlorpromazine-treated and -untreated ghosts, it was found that there was no significant difference in membrane proteins between both samples except for the changes of a few bands which were not directly concerned with the occurrence of this restricted signal. These results suggest that the fatty acid spin labels bind preferably to membrane proteins as the lipid domain becomes packed with chlorpromazine.     

Conformational alterations within the glycocalyx of erythrocyte membranes studied by spin labelling

The structure of the glycocalyx of the membrane of human erythrocytes and spectrin-depleted vesicles was studied under various conditions by two spin-labelling approaches: covalently labelling sialic acid residues of the glycocalyx and incorporation of a charged hydrophobic spin probe, CAT 16, being sensitive to alterations on the membrane surface into the lipid phase. Although cell electrophoretic measurements which were performed, additionally, indicated an erection of the glycocalyx upon decreasing the ionic strength of the suspension medium a more restricted mobility of spin-labelled sialic acid residues was found, in this case probably due to electrostatic interactions. The enhanced mobility of the spin probe CAT 16 at low ionic strength as well as in the case of neuraminidase-treated cells could be caused by reduced steric and electrostatic interaction with glycoproteins and glycolipids. La3+ adsorption and virus attachment on the human erythrocyte membrane were accompanied with a reduced mobility of sugar headgroups of the surface coat. No indication of cluster formation or lateral segregation of glycophorin molecules was found upon virus binding. After denaturation of the spectrin cytoskeleton of intact erythrocytes, increased mobility of spin-labelled sialic acid residues was observed.