The effect of ionizing radiation on tryptophan fluorescence of thymocyte and erythrocyte plasma membranes

The effect of ionizing radiation on tryptophan fluorescence of thymocyte and erythrocyte plasma membrane preparations was studied. The intensity of tryptophan fluorescence decreased after applying radiation doses up to 15 Gy. The radiosensitivity of thymocyte membranes appeared to be higher than that of the erythrocyte ghosts. Tryptophan radiolysis did not significantly contribute to the effects of radiation. The fraction of tryptophan residues accessible for quenching by I- decreased from 0.87 in the untreated membranes to 0.63 and 0.49 in membranes after doses of 10 and 250 Gy, respectively. The effective quenching constant and the tryptophan fluorescence polarization increased after irradiation. The mechanisms producing these radiation-induced changes are discussed.     

Physicochemical study of the state of protein and lipid phases in synaptosome membranes following irradiation with fast electrons

No activation of lipid peroxidation and structural rearrangements were noted in the lipid phase of synaptosome membranes after exposure of animals to electrons of 300 Gy/min at doses inducing cerebral lesions. At the same time, in isolated synaptosome membranes, radiation induced defined changes manifested by a decrease in tryptophan fluorescence.     

Fluorescence spectral resolution of myelin basic protein conformers in complexes with lysophosphatidylcholine

The structure of (Deibler) myelin basic protein in solution and in a lysolecithin lipid complex has been studied by using the emission properties of the single tryptophan residue of the protein (Trp-115). The studies have been carried out using both static and time-resolved fluorescence techniques. Relative to the free protein, the lipid bound myelin basic protein showed a, twofold increase in fluorescence intensity and a marked blue-shift in the emission maximum wavelength. The multiexponential fluorescence decays and the decay associated spectra indicated that the protein exists in at least three different conformations both in buffer and in lipids. Fluorescence polarization and acrylamide quenching experiments showed that the tryptophan containing region of the protein is embedded in the lipid matrix. The binding of the protein to the lipid appears to be comparable with that predicted for the interaction of amphipathic helices with nonpolar lipids.     

Comparative antioxidant activity of individual herbal components used in Ayurvedic medicine

Four aqueous extracts from different parts of medicinal plants used in Ayurveda (an ancient Indian Medicine) viz., Momardica charantia Linn (AP1), Glycyrrhiza glabra (AP2), Acacia catechu (AP3), and Terminalia chebula (AP4) were examined for their potential as antioxidants. The antioxidant activity of these extracts was tested by studying the inhibition of radiation induced lipid peroxidation in rat liver microsomes at different doses in the range of 100-600 Gy as estimated by thiobarbituric acid reactive substances (TBARS). Of all these extracts, AP4 showed maximum inhibition in the TBARS formation and hence is considered the best antioxidant among these four extracts. The extracts were found to restore antioxidant enzyme superoxide dismutase (SOD) from the radiation induced damage. The antioxidant capacities were also evaluated in terms of ascorbate equivalents by different methods such as cyclic voltammetry, decay of ABTS(.-) radical by pulse radiolysis and decrease in the absorbance of DPPH radicals. The results were found to be in agreement with the lipid peroxidation data and AP4 showed maximum value of ascorbate equivalents. Therefore AP4, with high antioxidant activity, is considered as the best among these four extracts.     

Fluorescence spectral resolution of myelin basic protein conformers in complexes with lysophosphatidylcholine

The structure of (Deibler) myelin basic protein in solution and in a lysolecithin++ lipid complex has been studied by using the emission properties of the single tryptophan residue of the protein (Trp-115). The studies have been carried out using both static and time-resolved fluorescence techniques. Relative to the free protein, the lipid bound myelin basic protein showed a twofold increase in fluorescence intensity and a marked blue-shift in the emission maximum wavelength. The multiexponential fluorescence decays and the decay associated spectra indicated that the protein exists in at least three different conformations both in buffer and in lipids. Fluorescence polarization and acrylamide quenching experiments showed that the tryptophan containing region of the protein is embedded in the lipid matrix. The binding of the protein to the lipid appears to be comparable with that predicted for the interaction of amphipathic helices with nonpolar lipids.     

Change in the structure of rat liver mitochondrial membranes under the effect of ionizing radiation

The effect of gamma-irradiation (under 0-10(3) Gy) on the intensity of lipid peroxidation, the microviscosity of annular and free lipids and the polarization of membrane proteins tryptophan fluorescence was studied in the outer and inner mitochondrial membranes. Some specific individual peculiarities of the mitochondrial membranes post-radiation changes were established.     

Tryptophan fluorescence of erythrocyte ghosts following irradiation and Fe2+ initiation of lipid peroxidation

It was shown that under the effect of Fe2+-initiated lipid peroxidation and ionizing radiation tryptophan fluorescence parameters (i.e. intensity and polarization) were subjected to similar changes. Shortly (15 min) after irradiation no changes were observed in the level of products reacting with thiobarbituric acid. It is concluded that the process and products of lipid peroxidation do not markedly contribute to the postirradiation alteration of tryptophan fluorescence. At the same time additional postirradiation damages to proteins can be attributed to activation of lipid peroxidation.     

Study of the time-resolved tryptophan fluorescence of crystalline alpha-chymotrypsin

The tryptophan environments in crystalline alpha-chymotrypsin were investigated by fluorescence. The heterogeneous emission from this multitryptophan enzyme was resolved by time-correlated fluorescence spectroscopy. The fluorescence decays at 296-nm laser excitation and various emission wavelengths could be characterized by a triple-exponential function with decay times tau 1 = 150 +/- 50 ps, tau 2 = 1.45 +/- 0.25 ns, and tau 3 = 4.2 +/- 0.4 ns. The corresponding decay-associated emission spectra of the three components had maxima at about 325, 332, and 343 nm. The three decay components in this enzyme can be correlated with X-ray crystallographic data [Birktoft, J.J., & Blow, D.M. (1972) J. Mol. Biol. 68, 187-240]. Inter- and intramolecular tryptophan-tryptophan energy-transfer efficiencies in crystalline alpha-chymotrypsin were computed from the accurately known positions and orientations of all tryptophan residues. These calculations indicate that the three fluorescence decay components in crystalline alpha-chymotrypsin can be assigned to three distinct classes of tryptophyl residues. Because of the different proximity of tryptophan residues to neighboring internal quenching groups, the decay times of the three classes are different. Decay tau 1 can be assigned to Trp-172 and Trp-215 and tau 2 to Trp-51 and Trp-237, while the tryptophyl residues 27, 29, 141, and 207 all have decay time tau 3.     

Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase

A procedure is described for using nanosecond time resolved fluorescence decay data to obtain decay-associated fluorescence spectra. It is demonstrated that the individual fluorescence spectra of two or more components in a mixture can be extracted without prior knowledge of their spectral shapes or degree of overlap. The procedure is also of value for eliminating scattered light artifacts in the fluorescence spectra of turbid samples. The method was used to separate the overlapping emission spectra of the two tryptophan residues in horse liver alcohol dehydrogenase. Formation of a ternary complex between the enzyme, NAD+, and pyrazole leads to a decrease in the total tryptophan fluorescence. It is shown that the emission of both tryptophan residues decreases. The buried tryptophan (residue 314) undergoes dynamic quenching with no change in the spectral distribution. Under the same conditions, the fluorescence intensity of tryptophan (residue 15) decreases without a change in decay time but with a red shift of the emission spectrum. There is also a decrease in tryptophan fluorescence intensity when the free enzyme is acid denatured (succinate buffer, pH 4.1). The denatured enzyme retains sufficient structure to provide different microenvironments for different tryptophan residues as reflected by biexponential decay and spectrally shifted emission spectra (revealed by decay association). The value of this technique for studies of microheterogeneity in biological macromolecules is discussed.     

Effect of pH on low-density lipoprotein oxidation by O2-./HO2. free radicals produced by gamma radiolysis.

This study aimed to analyze quantitatively the initiation and the consequences of low-density lipoprotein (LDL) peroxidation by O2-./HO2. free radicals produced by gamma radiolysis. The action of increasing radiation doses on aqueous LDL solutions has been monitored simultaneously by several parameters: a decrease in endogenous vitamin E, the formation of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes, the appearance of a differential fluorescence (excitation wavelength = 360 nm), and an increase of the relative electrophoretic mobility. Initial radiation yields (decrease in vitamin E, formation of TBARS) have been determined at pH 7 and pH 5.7 as a function of LDL concentration (from 0.75 to 9 g liter-1). From the comparison of these yields with those of O2-. radicals produced by water radiolysis, we have deduced reaction mechanisms for LDL peroxidation initiated by O2-./HO2. free radicals.     

Calcium-induced conformational change in fragment 1-86 of factor X

Time-resolved fluorescence of the single tryptophan residue Trp41 in fragment 1-86 of factor X (FX F1-86) is studied using a time-correlated single photon counting technique with synchrotron radiation as the excitation source. Calcium ions are believed to induce a conformational change in the N-termini of the activated factor X and other vitamin K dependent proteins, which is accompanied by a decrease in fluorescence intensity. The titration with calcium yields a sigmoidal fluorescence titration curve with a transition midpoint concentration of 0.44 mM. The wavelength-dependent tryptophan fluorescence decays of the apo-FX F1-86 (in the absence of calcium) and Ca-FX F1-86 are characterized by conventional multiexponential analysis and fluorescence lifetime distribution analysis. In the absence of calcium there are three significant classes of fluorescence lifetimes (ns) that are nearly wavelength independent: 0.55 +/- 0.08 (component A), 2.6 +/- 0.1 (component B), and 5.3 +/- 0.3 (component C). However, their preexponential amplitudes vary with wavelength. The decay associated emission spectra of the individual components show that components B and C contribute over 85% to the total fluorescence for all examined wavelengths. However, in the presence of calcium, the analysis of the time-resolved fluorescence data of Ca-FX F1-86 yields four wavelength-independent lifetimes (ns) of 0.30 +/- 0.09 (component D), 0.65 +/- 0.10 (component A), 2.7 +/- 0.2 (component B), and 5.4 +/- 0.3 (component C). Calcium addition to the apo-FX F1-86 leads to a decrease in the fluorescence intensities of components B and C while their decay times remain unaffected. In Ca-FX F1-86 an additional component D arises that has a decay time of 0.30 ns and that contributes up to 35% to the total fluorescence intensity. A comparison with a previous investigation of prothrombin fragment 1 demonstrates the extensive structural and functional homology between the N termini of prothrombin and factor X(a).     

Study of the aggregation of membrane proteins of erythrocyte ghosts using SDS-PAAG electrophoresis

Using the method of electrophoresis in SDS-PAAG the authors showed a diminution of proteins of bands I + II (spectrins) and III (major integral protein) after irradiation of erythrocyte ghosts with doses of 50 to 1000 Gy. We failed to ascertain that radiation-induced lipid peroxidation is involved into membrane protein aggregation. Among the radiolysis products, OH-radicals were shown to contribute markedly to the radiation effect observed.     

New fluorescent octadecapentaenoic acids as probes of lipid membranes and protein-lipid interactions.

The chemical and spectroscopic properties of the new fluorescent acids all(E)-8, 10, 12, 14, 16-octadecapentaenoic acid (t-COPA) and its (8Z)-isomer (c-COPA) have been characterized in solvents of different polarity, synthetic lipid bilayers, and lipid/protein systems. These compounds are reasonably photostable in solution, present an intense UV absorption band (epsilon(350 nm) approximately 10(5) M(-1) cm(-1)) strongly overlapped by tryptophan fluorescence and their emission, centered at 470 nm, is strongly polarized (r(O) = 0.385 +/- 0.005) and decays with a major component (85%) of lifetime 23 ns and a faster minor one of lifetime 2 ns (D,L-alpha-dimyristoylphosphatidylcholine (DMPC), 15 degrees C). Both COPA isomers incorporate readily into vesicles and membranes (K(p) approximately 10(6)) and align parallel to the lipids. t-COPA distributes homogeneously between gel and fluid lipid domains and the changes in polarization accurately reflect the lipid T(m) values. From the decay of the fluorescence anisotropy in spherical bilayers of DMPC and POPC it is shown that t-COPA also correctly reflects the lipid order parameters, determined by 2H NMR techniques. Resonance energy transfer from tryptophan to the bound pentaenoic acid in serum albumin in solution, and from the tryptophan residues of gramicidin in lipid bilayers also containing the pentaenoic acid, show that this probe is a useful acceptor of protein tryptophan excitation, with R(O) values of 30-34 A.     

Antioxidant effect of ethanol toward in vitro peroxidation of human low-density lipoproteins initiated by oxygen free radicals

This study was designed to evaluate the effect of ethanol on the peroxidation of human low-density lipoprotein (LDL) initiated by oxygen free radicals (O(2)(.-) and (.)OH in the absence of ethanol; O(2)(.-) and ethanol-derived peroxyl radicals, RO(2)(.), in the presence of ethanol) generated by gamma radiolysis. Initial radiolytic yields as determined by several markers of lipid peroxidation [i.e. decrease in endogenous antioxidants alpha-tocopherol and beta-carotene, formation of conjugated dienes and of thiobarbituric acid-reactive substances (TBARS)] were determined in 3 g liter(-1) LDLs (expressed as total LDL concentration) in the absence of ethanol or its presence at six different concentrations (0.42-17 x 10(-2) mol liter(-1)). Ethanol acted as an antioxidant by decreasing the rate of consumption of LDL endogenous antioxidants and the yields of formation of lipid peroxidation products, and by delaying the onset of the propagation phase for conjugated dienes and TBARS. With regard to the different markers studied, except for alpha-tocopherol and beta-carotene consumption, the effect of ethanol did not appear to be dependent on its concentration. Indeed, (.)OH were scavenged by ethanol at the lowest ethanol concentration (0.42 x 10(-2) mol liter(-1)), leading to RO(2)(.). These RO(2)(.) resulted in lower radiation-induced yields related to endogenous antioxidant consumption or to formation of lipid peroxidation products (for example, approximately 10% of RO(2)(.) oxidized LDLs from TBARS). Thus, under our in vitro conditions, ethanol behaved as an antioxidant when added to the LDL solutions. This should be taken into account in the reported antioxidant activity of wine. This is also of interest when lipophilic compounds have to be added as ethanolic solutions to LDLs to evaluate in vitro their antioxidant activity toward LDL peroxidation.     

Action of some hydroxyl radical scavengers on radiation-induced haemolysis

Human and bovine erythrocytes (RBCs) from peripheral blood were gamma-irradiated in vitro to a dose of 500 Gy in the presence of three efficient hydroxyl radical (OH) scavengers: ethanol, ethylene glycol and dimethyl sulphoxide (DMSO). Bovine erythrocytes were strongly protected from radiation induced haemolysis by each of the three scavengers over a concentration range from 10(-4) to 10(-2) molar, presumably as a result of OH scavenging. Human cells were protected as efficiently as bovine RBCs by ethanol and ethylene glycol over the same concentration range, however DMSO failed to protect human cells from haemolysis over a six-decade concentration range up to one molar. Exogenously supplied vitamin E (alpha-tocopherol) protected human RBCs from haemolytic effects of 500 Gy radiation in a dose-dependent fashion; however, bovine cells were not protected over the same concentration range. These preliminary results support evidence from model membrane systems suggesting that secondary radicals of DMSO generated during radiation may be of sufficient reactivity to initiate lipid peroxidation and are suggestive of species differences in the protection of biological membranes from oxidative stress.     

Risk of cerebrovascular disease incidence in the cohort of Mayak production association workers first employed during 1948-1958

Incidence of cerebrovascular diseases (CVD) has been studied in a cohort of 12210 workers first employed at one of the main plants (reactors, radiochemical or plutonium) of the Mayak nuclear facility during 1948-1958 and followed up to the end of 2000. Information on external gamma doses is available for virtually all (99.9%) of these workers; the mean (+/- one standard deviation) total gamma dose was 0.91 +/- 0.95 Gy (99% percentile 3.9 Gy) for men and 0.65 +/- 0.75 Gy (99% percentile 2.99 Gy) for women. Plutonium body burden was measured only for 30.0% of workers. Amongst those monitored, the mean (+/- standard deviation) cumulative liver dose from plutonium alpha exposure was 0.40 +/- 1.15 Gy (99% percentile 5.88 Gy) for men and 0.81 +/- 4.60 Gy (99% percentile 15.95 Gy) for women 4418 cases (first diagnosis) of CVD were identified in the studied cohort. A statistically significant increasing trend in CVD incidence with total external gamma dose was revealed after adjustment for non-radiation factors and internal exposure from incorporated plutonium-239. Excess relative risk per Gy was 0.464 (95% confidence interval 0.360-0.567). Incidence of CVD was statistically significantly higher for the workers chronically exposed to external gamma rays at a dose above 1.0 Gy A statistically significant increasing trend in CVD incidence with internal liver dose from plutonium alpha exposure was observed after adjustment for non-radiation factors and external exposure. ERR per Gy was 0.155 (95% confidence interval 0.075-0.235). CVD incidence was statistically significantly higher among workers with a plutonium liver dose above 0.1 Gy, although the trend estimates differed between workers at different plants. The incidence risk estimates for external radiation are generally compatible with estimates from the study of Chernobyl clean-up workers, although the incidence data point to higher risk estimates compared to those from the Japanese A-bomb survivors.     

Surfactant sealing of membranes permeabilized by ionizing radiation

Acute tissue injury and subsequent inflammation, including tissue edema and erythema, can be caused by sufficiently high levels of exposure to gamma radiation. The mechanism of this tissue injury is related to the generation of reactive oxygen intermediates (ROI) which chemically alter biological molecules and cell physiology. Cell membrane lipids are vulnerable to ROI-mediated lipid peroxidation that then leads to many of the acute tissue effects. We hypothesize that increased cell membrane permeability leading to osmotic swelling and vascular transudation is one of these effects. Thus we used adult postmitotic rhabdomyocytes in culture and microscopic fluorescence techniques to quantify radiation-induced changes in cell membrane permeability. Based on time-resolved dye flux measurements, a characteristic lag time of 34 +/- 3 min was determined between exposure to 160 Gy of gamma radiation and the decrease in membrane permeability. Administration of 0.1 mM nonionic surfactant Poloxamer 188 added to the cell medium after irradiation completely inhibited the dye loss over the time course of 2 h. Thus a reproducible model was developed for studying the mechanism of acute radiation injury and the efficacy of membrane-sealing agents. As only supportive measures now exist for treating the acute, nonlethal injuries from high-dose radiation exposure, agents that can restore cell membrane function after radiation damage may offer an important tool for therapy.     

Radioprotective effect of 2-mercaptopropionyl glycine on radiation-induced lipid peroxidation and enzyme release in erythrocytes

gamma-Irradiation of erythrocyte suspensions resulted in lipid peroxidation and enzyme (acetylcholinesterase, AChE) release. The presence of 2-mercaptopropionyl glycine (MPG) during irradiation decreased lipid peroxidation and enzyme (acetylcholinesterase, AChE) release. The presence of 2-mercaptopropionyl glycine (MPG) during irradiation decreased lipid peroxidation and from erythrocytes of high and low concentrations was observed at 480 and 320 Gy, respectively. This implied that the extent of enzyme release is likely to be masked when only a single dose of radiation is used, unless it happens to be an optimum dose. MPG mediated inhibition of lipid peroxidation and enzyme release indicated that lipid peroxidation may induce the breakdown of the phosphatidylinositol/enzyme interaction. Further, the enzyme damage was assigned to the direct and indirect effects of radiation on the enzyme in situ.     

Electrostatic interactions, but not the YGNGV consensus motif, govern the binding of pediocin PA-1 and its fragments to phospholipid vesicles.

The purpose of this study was to characterize in detail the binding of pediocin PA-1 and its fragments to target membranes by using tryptophan fluorescence as a probe. Based on a three-dimensional model (Y. Chen, R. Shapira, M. Eisenstein, and T. J. Montville, Appl. Environ. Microbiol. 63:524-531, 1997), four synthetic N-terminal pediocin fragments were selected to study the mechanism of the initial step by which the bacteriocin associates with membranes. Binding of pediocin PA-1 to vesicles of phosphatidylglycerol, the major component of Listeria membranes, caused an increase in the intrinsic tryptophan fluorescence intensity with a blue shift of the emission maximum. The Stern-Volmer constants for acrylamide quenching of the fluorescence of pediocin PA-1 in buffer and in the lipid vesicles were 8.83 +/- 0.42 and 3.53 +/- 0.67 M-1, respectively, suggesting that the tryptophan residues inserted into the hydrophobic core of the lipid bilayer. The synthetic pediocin fragments bound strongly to the lipid vesicles when a patch of positively charged amino acid residues (K-11 and H-12) was present but bound weakly when this patch was mutated out. Quantitative comparison of changes in tryptophan fluorescence parameters, as well as the dissociation constants for pediocin PA-1 and its fragments, revealed that the relative affinity to the lipid vesicles paralleled the net positive charge in the peptide. The relative affinity for the fragment containing the YGNGV consensus motif was 10-fold lower than that for the fragment containing the positive patch. Furthermore, changing the pH from 6.0 to 8.0 decreased binding of the fragments containing the positive patch, probably due to deprotonation of His residues. These results demonstrate that electrostatic interactions, but not the YGNGV motif, govern pediocin binding to the target membrane.     

Iron-induced lipid peroxidation and protein modification in endoplasmic reticulum membranes. Protection by stobadine

Treatment with FeSO(4)/EDTA (0.2 micromol Fe(II) per mg of protein) was used to study the effect of oxidative stress on lipid peroxidation and structural properties of endoplasmic reticulum (ER) membranes isolated from rabbit brain. Oxidative stress resulted in conjugated diene formation and a decrease of 1-anilino-8-naphthalenesulfonate (ANS) fluorescence in a time-dependent manner. In contrast, fluorescence anisotropy of 1, 6-diphenyl-1,3,5-hexatriene was increased early after the initiation of lipid peroxidation and no further increase was observed after 1, 2 and 3 h of peroxidation. FeSO(4)/EDTA treatment was accompanied by formation of conjugates of lipid peroxidation products with membrane proteins, as detected by the increase in fluorescence excitation (350-360 nm) and emission (440-450 nm) maximum. Oxidative stress also induced a marked decrease of the intrinsic fluorescence of aromatic amino acids, suggesting modification or changes in the environment of these amino acid residue(s). The lipid antioxidant, stobadine, completely prevented the changes of ANS fluorescence and production of peroxidized lipid-protein conjugates whereas tryptophan fluorescence was only partially protected. These results suggest that Fe(II) induces both lipid-mediated- and lipid peroxidation independent-modification of ER membrane proteins. The study also demonstrates that stobadine is a potent inhibitor of Fe(II)-induced protein modification.