Interactions Of Vitamin E With Free Radicals And Membranes

-Tocopherol performs an antioxidant role in biological membranes by acting as a one-electron reductant. In micellar solutions it has been observed by pulse radiolysis that the micellar charge has a pronounced effect on the rate constant for repair of organic free radicals by -tocopherol. The interactions between -tocopherol and model bilayer lipid membranes have been studied by fluorescence spectroscopy. Quencing of -tocopherol fluorescence by acrylamide and some n-doxyl stearates shows the transverse distribution of -tocopherol in membranes to be affected by the physical state of the membrane lipids and by the salt concentration in the aqueous phase. Time-resolved fluorescence depolarization measurements, with a diphenylhexatriene-phospholipid conjugate as probe. demonstrate an increase in the bilayer order parameter on incorporation of -tocopherol into a membrane     

Changes of structural and dynamic properties of model lipid membranes induced by α-tocopherol: implication to the membrane stabilization under external electric field

The effects of α-tocopherol on electric properties of bilayer lipid membranes were investigated. Planar bilayer membranes formed by the Mueller-Rudin method were used. Voltammetric and chronopotentiometric measurements were performed using a four-electrode potentiostat-galvanostat. It was demonstrated that registration of membrane capacitance, resistance, and voltammetric characteristics provided information about the change in the structure and permeability of bilayer lipid membranes. The results suggested that incorporation of α-tocopherol into lipid membrane destabilized its structure and facilitated the electrogeneration of pores. The possible role of observed changes in physiological functions of α-tocopherol was discussed.     

Molecular modeling of the reactive configuration of peroxidized lipid and α-tocopherol in the membrane

The mutual arrangement of a phospholipid molecule containing a peroxyl radical and a molecule of membrane-acting antioxidant α-tocopherol (vitamin E) in the lipid bilayer has been studied by molecular dynamics simulation. The geometry of molecules in the membrane is revealed at which the hydrogen atom can be transferred from the exocyclic hydroxyl of α-tocopherol to the peroxyl lipid radical. It is shown that, under equilibrium conditions, the peroxidized fatty acid segment rises nearer to the polar surface of the membrane, while α-tocopherol submerges into the hydrophobic part of the lipid bilayer.     

Effects of α-tocopherol on the lipid peroxidation and fluidity of porcine intestinal brush-border membranes

The effect of α-tocopherol on the lipid fluidity of porcine intestinal brush-border membranes was studied using pyrene as a fluorescent probe. Addition of α-tocopherol to the medium decreased fluorescence intensity and lifetime, but increased the fluorescence polarization of pyrene-labeled membranes. β-, γ-, and δ-Tocopherols gave no appreciable effect on the fluorescence intensity and polarization of the complex. The apparent dissociation constant (3.1 ± 0.12 μM) of the interaction of α-tocopherol with the membranes, estimated from the change in the fluorescence intensity with varying concentrations of α-tocopherol, was in good agreement with the concentration required to cause the half-maximal inhibition of lipid peroxidation of the membranes performed by incubation with 100 μM ascorbic acid and 10 μM Fe²⁺. Decrease of the slope in the thermal Perrin plot of the polarization of pyrene-labeled membranes by α-tocopherol suggests that the movement of pyrene molecules in the membranes is restricted by binding of the tocopherol. This interpretation was confirmed by an increased harmonic mean of the rotational relaxation time of the dye molecules in the membranes from 10.9 ± 0.16 to 18.5 ± 0.51 μs after addition of 25 μM α-tocopherol to the medium. The perturbation of lipid phase in the membranes induced by α-tocopherol was also suggested from a decreased quenching rate constant of pyrene fluorescence in the membranes for Tl⁺. Based on these results, the effect of α-tocopherol on the lipid fluidity of the membranes is discussed.     

Direct observation of alpha-lactalbumin,adsorption and incorporation into lipid membrane and formation of lipid/protein hybrid structures

The interaction between proteins and membranes is of great interest in biomedical and biotechnological research for its implication in many functional and dysfunctional processes. We present an experimental study on the interaction between model membranes and alpha-lactalbumin (α-La). α-La is widely studied for both its biological function and its anti-tumoral properties. We use advanced fluorescence microscopy and spectroscopy techniques to characterize α-La-membrane mechanisms of interaction and α-La-induced modifications of membranes when insertion of partially disordered regions of protein chains in the lipid bilayer is favored. Moreover, using fluorescence lifetime imaging, we are able to distinguish between protein adsorption and insertion in the membranes. Our results indicate that, upon addition of α-La to giant vesicles samples, protein is inserted into the lipid bilayer with rates that are concentration-dependent. The formation of heterogeneous hybrid protein-lipid co-aggregates, paralleled with protein conformational and structural changes, alters the membrane structure and morphology, leading to an increase in membrane fluidity.     

Antioxidative effect of α-tocopherol incorporation into lecithin liposomes on ascorbic acid-Fe2+-induced lipid peroxidation

The antioxidative effect of α-tocopherol incorporated into lecithin liposomes was studied. Lipid peroxidation of liposome membranes, assayed as malondialdehyde production, was catalyzed by ascorbic acid and Fe²⁺. The peroxidation reaction, which did not involve the formation of singlet oxygen, superoxide, hydrogen peroxide, or a hydroxyl radical, was inhibited by α-tocopherol and a model compound of α-tocopherol, 2,2,5,7,8-pentamethyl-6-hydroxy-chroman (TMC), but not by phytol, α-tocopherylquinone, or α-tocopheryl acetate. One mole of α-tocopherol completely prevented peroxidation of about 100 moles of polyunsaturated fatty acid. Decrease in membrane fluidity by lipid peroxidation, estimated as increase of fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) embedded in the membrane, was also inhibited by α-tocopherol and TMC, reflecting their antioxidant functions. Cholesterol did not act as an antioxidant, even when incorporated in large amount into the liposome membranes, but it increased the antioxidative efficiency of α-tocopherol. When a mixture of liposomes with and without α-tocopherol was incubated with Fe²⁺ and ascorbic acid, α-tocopherol did not protect the liposomes not containing α-tocopherol from peroxidation. However, preincubation of the mixture, or addition of Triton X-100 allowed the α-tocopherol to prevent peroxidation of the liposomes not containing α-tocopherol. In contrast, in similar experiments, liposomes containing TMC prevented peroxidation of those without TMC without preincubation. Tocopherol in an amount so small as to exhibit only a slight antioxidative effect was oxidized when incorporated in egg lecithin liposomes, but it mostly remained unoxidized when incorporated in dipalmitoyllecithin liposomes, indicating that oxygen activated by ascorbic acid-Fe²⁺ does not oxidize α-tocopherol directly. Thus, decomposition of α-tocopherol may be caused by its interaction with peroxy and/or alkoxyl radicals generated in the process of lipid peroxidation catalyzed by Fe²⁺ and ascorbic acid.     

Formation of α-tocopherol radical and recycling of α-tocopherol by ascorbate during peroxidation of phosphatidylcholine liposomes: An electron paramagnetic resonance study

The events accompanying the inhibitory effect of α-tocopherol and/or ascorbate on the peroxidation of soybean L-α-phosphatidylcholine liposomes, which are an accepted model of biological membranes, were investigated by electron paramagnetic resonance, optical and polarograpic methods. The presence of α-tocopherol radical in the concentration range 10^?8–10^?7 M was detected from its EPR spectrum during the peroxidation of liposomes, catalysed by the Fe³⁺-triethylnetatramine complex. The α-tocopherol radical, generated in the phosphatidylcholine bilayer, is accessible to ascorbic acid, present in the aqueous phase at physiological concentrations. Ascorbic acid regenerates from it the α-tocopherol itself. A kinetic rate constant of about 2·10⁵ M^?·s^?1 was estimated from the reaction as it occurs under the adopted experimental conditions. The scavenging effect of α-tocopherol on lipid peroxidation is maintained as long a ascorbic acid is present.     

The contribution of surface residues to membrane binding and ligand transfer by the α-tocopherol transfer protein (α-TTP)

Previous work has shown that the α-tocopherol transfer protein (α-TTP) can bind to vesicular or immobilized phospholipid membranes. Revealing the molecular mechanisms by which α-TTP associates with membranes is thought to be critical to understanding its function and role in the secretion of tocopherol from hepatocytes into the circulation. Calculations presented in the Orientations of Proteins in Membranes database have provided a testable model for the spatial arrangement of α-TTP and other CRAL-TRIO family proteins with respect to the lipid bilayer. These calculations predicted that a hydrophobic surface mediates the interaction of α-TTP with lipid membranes. To test the validity of these predictions, we used site-directed mutagenesis and examined the substituted mutants with regard to intermembrane ligand transfer, association with lipid layers and biological activity in cultured hepatocytes. Substitution of residues in helices A8 (F165A and F169A) and A10 (I202A, V206A and M209A) decreased the rate of intermembrane ligand transfer as well as protein adsorption to phospholipid bilayers. The largest impairment was observed upon mutation of residues that are predicted to be fully immersed in the lipid bilayer in both apo (open) and holo (closed) conformations such as Phe165 and Phe169. Mutation F169A, and especially F169D, significantly impaired α-TTP-assisted secretion of α-tocopherol outside cultured hepatocytes. Mutation of selected basic residues (R192H, K211A, and K217A) had little effect on transfer rates, indicating no significant involvement of nonspecific electrostatic interactions with membranes.     

α-Tocopherol incorporation in mitochondria and microsomes upon supranutritional vitamin E supplementation

Vitamin E (α-tocopherol) is a major lipid-soluble chain-breaking antioxidant in humans and mammals and plays an important role in normal development and physiology. The localization of α-tocopherol within the highly unsaturated phospholipid bilayer of cell membranes provides a means of controlling lipid oxidation at the initiation site. Mitochondria are the site for major oxidative processes and are important in fat oxidation and energy production, but a side effect is leakage of reactive oxygen species. Thus, incorporation of α-tocopherol and other antioxidants into mitochondria and other cellular compartments is important in order to maintain oxidative stability of the membrane-bound lipids and prevent damage from the reactive oxygen species. Many studies regarding mitochondrial disease and dysfunction have been performed in relation to deficiency of vitamin E and other antioxidants, whereas relatively sparse information is available regarding the eventual beneficial effects of antioxidant-enriched mitochondria in terms of health and function. This may be due to the fact that only little scientific information is available concerning the effect of supranutritional supplementation with antioxidants on their incorporation into mitochondria and other cellular membranes. The purpose of this review is therefore to briefly summarize experimental data performed with dietary vitamin E treatments in relation to the deposition of α-tocopherol in mitochondria and microsomes.     

Vibrational circular dichroism study of polypeptide model-membrane systems

In this article, we describe the mutual structural effect of the interaction between the model membranes and polylysine and poly-l-arginine. Vibrational circular dichroism (VCD), a method exceptionally sensitive to the polypeptide structure that has not been established in such studies before, was the primary method of this study. A complementary technique, electronic circular dichroism, was applied to verify the newly obtained results and as a bridge to the previous studies. We used micelles composed of sodium dodecyl sulfate (SDS) as a monolayer membrane model and large unilamellar vesicles composed of phospholipids as a bilayer membrane model. We describe the conformational changes of the polypeptides caused by the interaction with the model membranes. Among others, the presence of the liposomes in the solution generated special conditions for the formation of the α-helical structure of poly-l-arginine; the presence of SDS induced the formation of the β-structure of polylysine. From a methodological point of view, we emphasize the advantages of infrared spectroscopic techniques for the liposomic membrane studies as well as the preference of ultraviolet techniques for smaller micellar systems.     

Ascorbate 6-palmitate protects human erythrocytes from oxidative damage

Lipid-soluble antioxidants, such as α-tocopherol, protect cell membranes from oxidant damage. In this work we sought to determine whether the amphipathic derivative of ascorbate, ascorbate 6-palmitate, is retained in the cell membrane of intact erythrocytes, and whether it helps to protect the cells against peroxidative damage. We found that ascorbate 6-palmitate binding to erythrocytes was dose-dependent, and that the derivative was retained during the multiple wash steps required for preparation of ghost membranes. Ascorbate 6-palmitate remained on the extracellular surface of the cells, because it was susceptible to oxidation or removal by several cell-impermeant agents. When bound to the surface of erythrocytes, ascorbate 6-palmitate reduced ferricyanide, an effect that was associated with generation of an ascorbyl free radical signal on EPR spectroscopy. Erythrocyte-bound ascorbate 6-palmitate protected membrane α-tocopherol from oxidation by both ferricyanide and a water-soluble free radical initiator, suggesting that the derivative either reacted directly with the exogenously added oxidant, or that it was able to recycle the α-tocopheroxyl radical to α-tocopherol in the cell membrane. Ascorbate 6-palmitate also partially protected cis-parinaric acid from oxidation when this fluorescent fatty acid was intercalated into the membrane of intact cells. These results show that an amphipathic ascorbate derivative is retained on the exterior cell surface of human erythrocytes, where it helps to protect the membrane from oxidant damage originating outside the cells.     

Binding of a cationic phenazinium dye in anionic liposomal membrane: a spectacular modification in the photophysics

Interaction of a cationic phenazinium dye, phenosafranin (PSF), with the anionic liposomal vesicle/bilayer of dimyristoyl-l-α-phosphatidylglycerol (DMPG) has been demonstrated using steady state and time resolved fluorescence and fluorescence anisotropy techniques. The charge transfer emission spectrum of PSF shows a dramatic modification in terms of fluorescence yield together with an appreciable hypsochromic shift in the lipid environment. The blue shift indicates a lowering in polarity inside the vesicle as compared to that in bulk water. The fluorescence and fluorescence quenching studies and micropolarity determination reveal that the cationic fluorophore has a profound binding interaction with the anionic DMPG membrane. Anisotropy study indicates the imposition of a motional restriction on the probe inside the bilayer. The electrostatic interaction between the cationic dye and the anionic lipid membrane has been argued to be the reason behind all these observations. The results could be useful in analyzing membrane organization and heterogeneity in natural membranes exploiting PSF or alike compounds as fluorescent probes.     

Transverse distribution of alpha-tocopherol in bilayer membranes studied by fluorescence quenching

The fluorescence properties of alpha-tocopherol in a range of solvents and in micelles and membrane vesicles have been measured. In solvents the fluorescence decay was fitted by a single exponential. In bilayer membranes of dipalmitoylphosphatidylcholine or egg phosphatidylcholine the fluorescence decay was more accurately fitted as a double exponential. This may indicate that alpha-tocopherol occupies two or more sites in such membranes. Depth-dependent quenching of alpha-tocopherol fluorescence by acrylamide and some doxyl stearates has also been studied. The results confirm that in gel-phase lipid the chromanol group has a transverse distribution close to the head-group region of the lipid. In fluid phase lipid in the presence of buffer the results indicate there is more penetration of the chromanol group into the bilayer.     

Role of membrane lipids in the mechanism of bacterial species selective toxicity by two α/β-antimicrobial peptides

We have previously shown that two synthetic antimicrobial peptides with alternating α- and β-amino acid residues, designated simply as α/β-peptide I and α/β-peptide II, had toxicity toward bacteria and affected the morphology of bacterial membranes in a manner that correlated with their effects on liposomes with lipid composition similar to those of the bacteria. In the present study we account for the weak effects of α/β-peptide I on liposomes or bacteria whose membranes are enriched in phosphatidylethanolamine (PE) and why such membranes are particularly susceptible to damage by α/β-peptide II. The α/β-peptide II has marked effects on unilamellar vesicles enriched in PE causing vesicle aggregation and loss of their internal aqueous contents. The molecular basis of these effects is the ability of α/β-peptide II to induce phase segregation of anionic and zwitterionic lipids as shown by fluorescence and differential scanning calorimetry. This phase separation could result in the formation of defects through which polar materials could pass across the membrane as well as form a PE-rich membrane domain that would not be a stable bilayer. α/β-Peptide II is more effective in this regard because, unlike α/β-peptide I, it has a string of two or three adjacent cationic residues that can interact with anionic lipids. Although α/β-peptide I can destroy membrane barriers by converting lamellar to non-lamellar structures, it does so only weakly with unilamellar vesicles or with bacteria because it is not as efficient in the aggregation of these membranes leading to the bilayer-bilayer contacts required for this phase conversion. This study provides further understanding of why α/β-peptide II is more toxic to micro-organisms with a high PE content in their membrane as well as for the lack of toxicity of α/β-peptide I with these cells, emphasizing the potential importance of the lipid composition of the cell surface in determining selective toxicity of anti-microbial agents.     

Melittin-induced alterations in dynamic properties of human red blood cell membranes.

The interaction of bee venom melittin with erythrocyte membrane ghosts has been investigated by means of fluorescence quenching of membrane tryptophan residues, fluorescence polarization and ESR spectroscopy. It has been revealed that melittin induces the disorders in lipid-protein matrix both in the hydrophobic core of bilayer and at the polar/non-polar interface of melittin complexed with erythrocyte membranes. The peptide has been found to act most efficiently at the concentration of the order of 10(-10) mol/mg membrane protein. The apparent distance separating the membrane tryptophan and bound 1-anilino-8-naphthalenesulphonate (ANS) molecules is decreased upon melittin binding, which results in a significant increase of the maximum energy transfer efficiency. Significant changes in the fluorescence anisotropy of both 1,6-diphenyl-1,3,5-hexatriene and 1-anilino-8-naphthalenesulphonate bound to erythrocyte ghosts, which have been observed in the presence of melittin and crude venom, indicate membrane lipid bilayer rigidization. The effect of crude honey bee venom has been found to be of similar magnitude as the effect of pure melittin at the concentration of 10(-10) mol/mg membrane protein. Using two lipophilic spin labels, methyl 5-doxylpalmitate and 16-doxylstearic acid, we found that melittin at its increasing concentrations induces a well marked rigidization in the deeper regions of lipid bilayer, whereas the effect of rigidization near the membrane surface maximizes at the melittin concentration of 10(-10) mol/mg (10(-4) mol melittin per mole of membrane phospholipid). The decrease in the ratio hw/hs of maleimide and the rise in relative rotational correlation time (tau c) of iodacetamid spin label, indicate that melittin effectively immobilizes membrane proteins in the plane of the lipid bilayer. We conclude that melittin-induced rigidization of the lipid bilayer may induce a reorganization of lipid assemblies as well as the rearrangements in membrane protein pattern and consequently the alterations in lipid-protein interactions. Thus, the interaction of melittin with erythrocyte membranes is supposed to produce local conformational changes in membranes, which are discussed in the connection with their significance during the synergistic action of melittin and phospholipase of bee venom on red blood cells.     

Membrane binding of oligomeric alpha-synuclein depends on bilayer charge and packing

Membrane disruption by oligomeric α-synuclein (αS) is considered a likely mechanism of cytotoxicity in Parkinson’s disease (PD). However, the mechanism of oligomer binding and the relation between binding and membrane disruption is not known. We have visualized αS oligomer-lipid binding by fluorescence microscopy and have measured membrane disruption using a dye release assay. The data reveal that oligomeric αS selectively binds to membranes containing anionic lipids and preferentially accumulates into liquid disordered (Ld) domains. Furthermore, we show that binding of oligomers to the membrane and disruption of the membrane require different lipid properties. Thus membrane-bound oligomeric αS does not always cause bilayer disruption.     

α-Lactalbumin binding and membrane integrity—effect of charge and degree of unsaturation of glycerophospholipids

Several studies have shown that the physical state of the phospholipid membrane has an important role in protein-membrane interactions, involving both electrostatic and hydrophobic forces. We have investigated the influence of the interaction of the calcium-depleted, (apo)-conformation of bovine α-lactalbumin (BLA) on the integrity of anionic glycerophospholipid vesicles by leakage experiments using fluorescence spectroscopy. The stability of the membranes was also studied by measuring surface tension/molecular area relationships with phospholipid monolayers. We show that the degree of unsaturation of the acyl chains and the proportion of charged phospholipid species in the membranes made of neutral and acidic glycerophospholipids are determinants for the association of BLA with liposomes and for the impermeability of the bilayer. Particularly, tighter packing counteracted interaction with BLA, while unsaturation—leading to looser packing—promoted interaction and leakage of contents. Equimolar mixtures of neutral and acidic glycerophospholipids were more permeable upon protein binding than pure acidic lipids. The effect of lipid structure on BLA-membrane interaction and bilayer integrity may throw new light on the membrane disrupting mechanism of a conformer of human α-lactalbumin (HAMLET) that induces death of tumour cells but not of normal cells.     

Interactions between biliary lipid micelles and intestinal brush border membranes investigated by 1H and 31P nuclear magnetic resonance

The effect of taurocholate and lecithincholesterol-taurocholate mixed micelles on the structure of isolated intestinal brush border membranes was investigated by nuclear magnetic resonance (NMR). Rabbit brush border membranes isolated by a Mg²⁺ precipitation step were chosen for this study because of their stability and integrity as revealed by ³¹P NMR. Incubation of taurocholate with the brush border membranes does not induce significant solubilization of these membranes even when the taurocholate/phospholipid ratio reaches 3.0 ¹H NMR studies indicate that taurocholate is included in the membrane bilayer at low concentration (3 mM). However this biliary salt produces a size diminution of the vesicles when its concentration increases. Incorporation of lecithin or lecithin-cholesterol in micelles of taurocholate and subsequent incubation with brush border membranes lead simultaneously to a decrease in the ³¹P NMR isotropic/bilayer line ratio, and to an increase in . These results indicate a protective effect of these compounds against lytic damage of taurocholate. Futhermore the equilibrium distribution of lecithin between mixed micelles and the membrane bilayer is strongly in favour of complete integration of micellar components in the bilayer. These data suggest that uptake of lipids from the micellar phase by isolated brush border membranes involves an interaction of the micelles with membranes followed by a fusion process.     

Stimulation of α-synuclein amyloid formation by phosphatidylglycerol micellar tubules

α-Synuclein (α-Syn) is a presynaptic protein that is accumulated in its amyloid form in the brains of Parkinson's patients. Although its biological function remains unclear, α-syn has been suggested to bind to synaptic vesicles and facilitate neurotransmitter release. Recently, studies have found that α-syn induces membrane tubulation, highlighting a potential mechanism for α-syn to stabilize highly curved membrane structures which could have both functional and dysfunctional consequences. To understand how membrane remodeling by α-syn affects amyloid formation, we have studied the α-syn aggregation process in the presence of phosphatidylglycerol (PG) micellar tubules, which were the first reported example of membrane tubulation by α-syn. Aggregation kinetics, β-sheet content, and macroscopic protein-lipid structures were observed by Thioflavin T fluorescence, circular dichroism spectroscopy and transmission electron microscopy, respectively. Collectively, the presence of PG micellar tubules formed at a stochiometric (L/P = 1) ratio was found to stimulate α-syn fibril formation. Moreover, transmission electron microscopy and solid-state nuclear magnetic resonance spectroscopy revealed the co-assembly of PG and α-syn into fibril structures. However, isolated micellar tubules do not form fibrils by themselves, suggesting an important role of free α-syn monomers during amyloid formation. In contrast, fibrils did not form in the presence of excess PG lipids (≥L/P = 50), where most of the α-syn molecules are in a membrane-bound α-helical form. Our results provide new mechanistic insights into how membrane tubules modulate α-syn amyloid formation and support a pivotal role of protein–lipid interaction in the dysfunction of α-syn.     

Tocopherol and plastoquinone synthesis in spinach chloroplasts subfractions

Subfractions isolated from intact purified spinach chloroplasts are able to prenylate the aromatic moiety of α-tocopherol and plastoquinone-9 precursors. The biosynthesis of α-tocopherol and plastoquinone-9 is a compartmentalized process. The chloroplast envelope membranes are the only site of the enzymatic prenylation in α-tocopherol synthesis whereas the thylakoid membrane is also involved in the prenylation and methylation sequence of plastoquinone-9 biosynthesis. A very active kinase which forms phytyl-PP is localized in the stroma. Phytol but not geranylgeraniol is the polyprenol precursor of the side chain of α-tocopherol in spinach chloroplasts.