Modulation of Interfacial Hydration by Carbonyl Groups in Lipid Membranes.

The lack of carbonyl groups and the presence of ether bonds give the lipid interphase a different water organization around the phosphate groups that affects the compressibility and electrical properties of lipid membranes. Generalized polarization of 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (14:0 diether PC) in correlation with Fourier transform infrared (FTIR) analysis indicates a higher level of polarizability of water molecules in the membrane phase around the phosphate groups both below and above Tm. This reorganization of water promotes a different response in compressibility and dipole moment of the interphase, which is related to different H bonding of water molecules with phosphates (PO) and carbonyl (CO) groups.

Functional role of water in membranes updated: A tribute to Träuble.

The classical view of a cell membrane is as a hydrophobic slab in which only nonpolar solutes can dissolve and permeate. However, water-soluble non-electrolytes such as glycerol, erythritol, urea and others can permeate lipid membranes in the liquid crystalline state. Moreover, recently polar amino acid's penetration has been explained by means of molecular dynamics in which appearance of water pockets is postulated. According to Träuble (1971), water diffuses across the lipid membranes by occupying holes formed in the lipid matrix due to fluctuations of the acyl chain trans-gauche isomers. These holes, named "kinks" have the molecular dimension of CH2 vacancies. The condensation of kinks may form aqueous spaces into which molecular species of the size of low molecular weight can dissolve. This molecular view can explain permeability properties considering that water may be distributed along the hydrocarbon chains in the lipid matrix. The purpose of this review is to consolidate the mechanism anticipated by Träuble by discussing recent data in literature that directly correlates the molecular state of methylene groups of the lipids with the state of water in each of them. In addition, the structural properties of water near the lipid residues can be related with the water activity triggering kink formation by changes in the head group conformation that induces the propagation along the acyl chains and hence to the diffusion of water.

Antiradical activity of gallic acid included in lipid interphases.

Polyphenols are well known as antioxidant agents and by their effects on the hydration layers of lipid interphases. Among them, gallic acid and its derivatives are able to decrease the dipole potential and to act in water as a strong antioxidant. In this work we have studied both effects on lipid interphases in monolayers and bilayers of dimyristoylphosphatidylcholine. The results show that gallic acid (GA) increases the negative surface charges of large unilamellar vesicles (LUVs) and decreases the dipole potential of the lipid interphase. As a result, positively charged radical species such as ABTS(+) are able to penetrate the membrane forming an association with GA. These results allow discussing the antiradical activity (ARA) of GA at the membrane phase which may be taking place in water spaces between the lipids.

Connected and isolated CH2 populations in acyl chains and its relation to pockets of confined water in lipid membranes as observed by FTIR spectrometry.

Analysis of the band corresponding to the frequency of vibrational symmetric stretching mode of methylene groups in the lipid acyl chains and the bands of water below and above the phase transition of different lipids by Fourier transform infrared spectroscopy gives strong support to the formation of confined water pockets in between the lipid acyl chains. Our measures and analysis consolidate the mechanism early proposed by Traüble, in the sense that water is present in kinks formed by trans-gauche isomers along the hydrocarbon tails. The formation of these regions depends on the acyl lipid composition, which determines the presence of different populations of water species, characterized by its degree of H bond coordination in fluid saturated or unsaturated lipids. The free energy excess due to the reinforcement of the water structure along few water molecules in the adjacencies of exposed membrane residues near the phase transition is a reasonable base to explain the insertion and translocation of polar peptides and amino acid residues through the biomembrane on thermodynamic and structural grounds.

Water state and carbonyl distribution populations in confined regions of lipid bilayers observed by FTIR spectroscopy.

It has been suggested that water in confined regions presents different properties than bulk water, mainly because of the changes in water population species that may be induced by the adjacent walls of different polarities in terms of hydrogen bond formation. In this context, it would be expected that lipids in the gel and the fluid states should offer different templates for water organization. The presence of water pockets or defects in lipid bilayers has been proposed to explain the insertion of charged and polar peptides and amino acids in membranes. In this work, we provide direct evidence by means of FTIR spectroscopy that water band profiles are changed whether lipids are in the solid state, in the gel state after heating and cooling across the phase transition, or in the fluid state. The different bands found in each case were assigned to different H-bonded water populations in agreement with the exposure of carbonyl groups.

Breakdown of classical paradigms in relation to membrane structure and functions.

Updates of the mosaic fluid membrane model implicitly sustain the paradigms that bilayers are closed systems conserving a state of fluidity and behaving as a dielectric slab. All of them are a consequence of disregarding water as part of the membrane structure and its essential role in the thermodynamics and kinetics of membrane response to bioeffectors. A correlation of the thermodynamic properties with the structural features of water makes possible to introduce the lipid membrane as a responsive structure due to the relaxation of water rearrangements in the kinetics of bioeffectors' interactions. This analysis concludes that the lipid membranes are open systems and, according to thermodynamic of irreversible formalism, bilayers and monolayers can be reasonable compared under controlled conditions. The inclusion of water in the complex structure makes feasible to reconsider the concept of dielectric slab and fluidity.

Interaction of chlorogenic acid with model lipid membranes and its influence on antiradical activity.

Chlorogenic acid (CGA) is a strong phenolic antioxidant with antibacterial properties composed by a caffeoyl ester of quinic acid. Although a number of benefits has been reported and related to interactions with the red blood cell membranes, details on its membrane action and how composition and membrane state may affect it, is not yet well defined. In this work, the interaction of CGA with lipid monolayers and bilayers composed by 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC); 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (14:0 diether PC); 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine (16:0 diether PC) were studied at different surface pressures (π). The kinetics of interaction was found to be more rapid in DMPC than in the absence of carbonyl groups. Measurements by FTIR-ATR at different water activities confirm specific interactions of CGA with carbonyl and phosphate groups affecting water level along hydrocarbon region. The antioxidant activity of CGA in the presence of DMPC unilamellar vesicles, evidenced by the absorbance reduction of the radical cation ABTS•+, is significantly different with respect to aqueous solution. The influence of CGA on antiradical activity (ARA) with lipid membranes depending on the hydration state of the lipid interface is discussed.

Effect of cholesterol on the hydration properties of ester and ether lipid membrane interphases.

Fluorescence spectroscopy and Molecular Dynamics results show that cholesterol reduces water along the chains in ether lipids by changing the water distribution pattern between tightly and loosely bound water molecules. Water distribution was followed by emission spectra and generalized polarization of 6-dodecanoyl-2-dimethyl aminonaphthalene (Laurdan) inserted in 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (14: 0 Diether PC) membranes. Molecular Dynamics simulations indicate that the action of cholesterol could be different in ether PC in comparison to ester PC. In addition, Cholesterol seems to act "per se" as an additional hydration center in ether lipids. Regardless of the phase state, cholesterol both in DMPC and 14:0 Diether PC vesicles, changed the distribution of water molecules decreasing the dipole relaxation of the lipid interphase generating an increase in the non-relaxable population. Above 10% Cholesterol/14:0 Diether PC ratio vesicles' interphase present an environment around Laurdan molecules similar to that corresponding to ester PC.

Structural and dynamical surface properties of phosphatidylethanolamine containing membranes.

The hydration of solid dimyristoylphosphatidylethanolamine (DMPE) produces a negligible shift in the asymmetric stretching frequency of the phosphate groups in contrast to dimyristoylphosphatidylcholine (DMPC). This suggests that the hydration of DMPE is not a consequence of the disruption of the solid lattice of the phosphate groups as occurs in DMPC. The strong lateral interactions between NH(3) and PO(2)(-) groups present in the solid PEs remain when the lipids are fully hydrated and seem to be a limiting factor for the hydration of the phosphate group hindering the reorientation of the polar heads. The lower mobility is reflected in a higher energy to translocate the phosphoethanolamine (P-N) dipoles in an electrical field. This energy is decreased in the presence of increasing ratios of PCs of saturated chains in phosphoethanolamine monolayer. The association of PC and PE in the membrane affecting the reorientation of the P-N groups is dependent of the chain-chain interaction. The dipole potentials of PCs and PEs mixtures show different behaviors according to the saturation of the acyl chain. This was correlated with the area in monolayers and the hydration of the P-N groups. In spite of the low hydration, DMPE is still able to adsorb fully hydrated proteins, although in a lower rate than DMPC at the same surface pressure. This indicates that PE interfaces possess an excess of surface free energy to drive protein interaction. The relation of this free energy with the low water content is discussed.

Water Behavior at the Phase Transition of Phospholipid Matrixes Assessed by FTIR Spectroscopy.

Lipid membranes are one of the most important biological matrixes in which biochemical processes take place. This particular lipid arrangement is driven by different water disposition interacting with it, which is related to different water states with different energy levels at the interphase. In our work, we report changes in water content and distinctive water states by Fourier transform infrared (FTIR) spectroscopy of this self-assembled matrix at different water contents and temperatures. To determine whether water properties at lipid interphases depend on the group of the lipid molecule at which it is bound the phase-transition temperature of 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (14:0 diether PC) was followed by the changes in frequency of the different groups of the lipids by attenuated total reflection (ATR)-FTIR spectroscopy at different humidities. A comparison of these two lipids enables us to put into relevance the contribution of the CO groups as a hydration site. These changes were compared with those occurring at the water band, and a value of the enthalpic change was evaluated from them. The -OH stretching in the liquid water IR spectrum is the principal region used to understand its molecular organization (4000-3000 cm-1). The strength of hydrogen bonding depends on the cooperative/anticooperative nature of the surrounding hydrogen bonds, with the strongest hydrogen bonds giving the lowest vibrational frequencies. Thus, we can use water as a mirror of the membrane state in this kind of biological systems. Different phospholipids associate water at particular modes according to their structures. This may produce modulation of packing and hydration suitable for the incorporation of amino acids, peptides, and enzymes.

Structural and functional properties of hydration and confined water in membrane interfaces.

The scope of the present review focuses on the interfacial properties of cell membranes that may establish a link between the membrane and the cytosolic components. We present evidences that the current view of the membrane as a barrier of permeability that contains an aqueous solution of macromolecules may be replaced by one in which the membrane plays a structural and functional role. Although this idea has been previously suggested, the present is the first systematic work that puts into relevance the relation water-membrane in terms of thermodynamic and structural properties of the interphases that cannot be ignored in the understanding of cell function. To pursue this aim, we introduce a new definition of interphase, in which the water is organized in different levels on the surface with different binding energies. Altogether determines the surface free energy necessary for the structural response to changes in the surrounding media. The physical chemical properties of this region are interpreted in terms of hydration water and confined water, which explain the interaction with proteins and could affect the modulation of enzyme activity. Information provided by several methodologies indicates that the organization of the hydration states is not restricted to the membrane plane albeit to a region extending into the cytoplasm, in which polar head groups play a relevant role. In addition, dynamic properties studied by cyclic voltammetry allow one to deduce the energetics of the conformational changes of the lipid head group in relation to the head-head interactions due to the presence of carbonyls and phosphates at the interphase. These groups are, apparently, surrounded by more than one layer of water molecules: a tightly bound shell, that mostly contributes to the dipole potential, and a second one that may be displaced by proteins and osmotic stress. Hydration water around carbonyl and phosphate groups may change by the presence of polyhydroxylated compounds or by changing the chemical groups esterified to the phosphates, mainly choline, ethanolamine or glycerol. Thus, surface membrane properties, such as the dipole potential and the surface pressure, are modulated by the water at the interphase region by changing the structure of the membrane components. An understanding of the properties of the structural water located at the hydration sites and the functional water confined around the polar head groups modulated by the hydrocarbon chains is helpful to interpret and analyze the consequences of water loss at the membranes of dehydrated cells. In this regard, a correlation between the effects of water activity on cell growth and the lipid composition is discussed in terms of the recovery of the cell volume and their viability. Critical analyses of the properties of water at the interface of lipid membranes merging from these results and others from the literature suggest that the interface links the membrane with the aqueous soluble proteins in a functional unit in which the cell may be considered as a complex structure stabilized by water rather than a water solution of macromolecules surrounded by a semi permeable barrier.

Lysophosphatidylcholine-arbutin complexes form bilayer-like structures.

Arbutin is known to suppress melanin production in murine B16 melanoma cells and inhibit phospholipase action. This encourages the possibility to stabilize it in lipid aggregates for its administration in medical applications. Thus, it was of interest to demonstrate that monomyristoylphosphatidylcholine (14:0 lysoPC) and arbutin may form association complexes. This was studied by Electron Microscopy (EM), 31P Nuclear Magnetic Resonance (31P NMR), Electronic Paramagnetic Resonance (EPR) and Fourier Transform Infrared Spectroscopy (FTIR). EM images show the formation of particles of c.a. 6 nm in diameter. For a 1:1 lysoPC-arbutin molar ratio 31P NMR shows a spectrum with a shoulder that resembles the axially symmetric spectrum characteristic of vesicles. The addition of La3+ ions to the arbutin-lysoPC complex allows one to distinguish two phosphorous populations. These results suggest that arbutin-lysoPC forms vesicles with bilayers stabilized in an interdigitated array. FTIR spectroscopy shows that arbutin interacts with the hydrated population of the carbonyl groups and with the phosphates through the formation of hydrogen bonds. It is interpreted that hydrophobic interactions among the phenol group of arbutin and the acyl chain of lysoPC are responsible for the decrease in acyl chain mobility observed at the 5th C level by EPR. A model proposing the formation of interdigitated bilayers of arbutin-lysoPC could explain the experimental results.

Effect of dipole potential variations on the surface charge potential of lipid membranes.

When the dipole potential of dimyristoylphosphatidylcholine (DMPC) monolayers was decreased, either by the insertion of phloretin or by the elimination of carbonyl groups at the interphase, the surface charge potential was displaced to lower negative values. At low ionic strength, the decrease of the negative charge density can be ascribed to a different exposure of the phosphate to water, as there is a good correlation to an increase in the area per lipid. At high ionic strength, the magnitude of the changes in the zeta potential produced by the effects on the dipole potential was found to be dependent on the type of anions present in the subphase. Differences between Cl- and ClO4- were ascribed to the adsorption of anions according to their different hydrations and polarizabilities. The influence of a low dipole potential on the anion adsorption can be ascribed to a less positive image charge at the membrane interior, resulting from an increase in the hydrocarbon core permittivity. This is congruent with the neutralization of interfacial dipoles and the area increase, as well as with the decrease in packing of the hydrocarbon groups. Phloretin did not cause changes in the dipole potential of dimyristoylphosphatidylethanolamine (DMPE), and in consequence, no effects on the zeta potential were measured. It is concluded that changes in the inner water/hydrocarbon plane affect the electrostatic potential measured in the outer plane of the polar headgroup region.

A new model for lipid monolayer and bilayers based on thermodynamics of irreversible processes.

Lipid monolayers are used as experimental model systems to study the physical chemical properties of biomembranes. With this purpose, surface pressure/area per molecule isotherms provide a way to obtain information on packing and compressibility properties of the lipids. These isotherms have been interpreted considering the monolayer as a two dimensional ideal or van der Waals gas without contact with the water phase. These modelistic approaches do not fit the experimental results. Based on Thermodynamics of Irreversible Processes (TIP), the expansion/compression process is interpreted in terms of coupled phenomena between area changes and water fluxes between a bidimensional solution of hydrated head groups in the monolayer and the bulk solution. The formalism obtained can reproduce satisfactorily the surface pressure/area per lipid isotherms of monolayer in different states and also can explain the area expansion and compression produced in particles enclosed by bilayers during osmotic fluxes. This novel approach gives relevance to the lipid-water interaction in restricted media near the membrane and provides a formalism to understand the thermodynamic and kinetic response of biointerphases to biological effectors.

Superficially active water in lipid membranes and its influence on the interaction of an aqueous soluble protease.

The purpose of this paper is to demonstrate that the interaction of an aqueous soluble enzyme with lipid membranes is influenced by the lipid composition of the interphase. The results show that the interaction of an aqueous soluble protease, Rennet from Mucor miehei, depends on the exposure of the carbonyl and phosphate groups at the membrane interphase. The changes produced by the protease on the surface pressure of monolayers of dimyristoylphosphatidylcholine (DMPC); dioleoylphosphatidylcholine (DOPC); diphytanoylphosphatidylcholine (DPhPC); dipalmitoylphosphatidylcholine (DPPC); di-O-tetradecylphosphatidyl-choline [D(ether)PC]; dimyristoylphosphatidylethanolamine (DMPE); di-O-tetradecyl-phosphatidylethanolamine [D(ether)PE] were measured at different initial surface pressures. The meaning of the DeltaPi vs. Pi curves was interpreted in the light of the concept of interphase given by Defay and Prigogine [R. Defay, I. Prigogine, Surface Tension and Adsorption, John Wiley & Sons, New York, 1966, pp. 273-277] considering the interphase as a bidimensional solution of polar head groups. With this approach, and based on reported evidences that carbonyls and phosphates are the main hydration sites of the lipid membranes, it is suggested that the mechanism of interaction of aqueous soluble protein involves water beyond the hydration shell. At high surface pressure, only water strongly bound to carbonyl and phosphate groups is present and the interaction is not occurring. In contrast, at low surface pressures, the protease-membrane interaction is a function of acyl chain for different polar groups. This is interpreted, as a consequence of the changes in the interfacial tension produced by the displacement of water confined between the hydrated head groups.

Characterization of liposomes coated with S-layer proteins from lactobacilli.

The stability of liposomes coated with S-layer proteins from Lactobacillus brevis and Lactobacillus kefir was analyzed as a previous stage to the development of a vaccine vehicle for oral administration. The interactions of the different S-layer proteins with positively charged liposomes prepared with soybean lecithin or dipalmitoylphosphatidylcholine were studied by means of the variation of the Z potential at different protein-lipid ratios, showing that both proteins were able to attach in a greater extent to the surface of soybean lecithin liposomes. The capacity of these particles to retain carboxyfluorescein or calcein by exposure to bile salts, pancreatic extract, pH change and after a thermal shock showed that both S-layer proteins increased the stability of the liposomes in the same magnitude. The non-glycosylated protein from L. brevis protects more efficiently the liposomes at pH 7 than those from L. kefir even without treatment with glutaraldehyde.

FTIR analysis of the interaction of arbutin with dimyristoyl phosphatidylcholine in anhydrous and hydrated states.

In this paper, the interaction of arbutin with dimyristoylphosphatidylcholine bilayers was studied by FTIR spectrometry. The results show that arbutin interacts in different extents with the phosphate and carbonyl groups of membranes in the gel state, the liquid crystalline state or subjected to osmotic stress. The effect, in the presence of water, on the antisymmetric stretching of the phosphate groups is qualitatively similar to that found with other molecules composed by a glucose moiety such as trehalose and sucrose. However, significant differences were found between these compounds and arbutin in the carbonyl region. Arbutin displaces the PO2- antisymmetric stretching to lower frequencies in lipids dispersed in water. This indicates strong hydrogen bonding. In contrast, in the solid state, this frequency increases. The effect on the carbonyl groups varies depending on the hydration state of the bilayer, which is achieved by changing the phase state of the bilayer or by osmotic stress. The hydrocarbon region is not affected by arbutin in the excess of water. However, symmetric and antisymmetric stretching of CH2 and CH3 are strongly affected in the dry state.

Arbutin blocks defects in the ripple phase of DMPC bilayers by changing carbonyl organization.

The effect of arbutin, a 4-hydroxyphenyl-beta-glucopyranoside, on dimyristoylphosphatidylcholine (DMPC) bilayers was studied by turbidimetry, EPR and FTIR spectroscopies. The disruption of DMPC multilamellar vesicles (MLV's) with monomyristoylphosphatidylcholine (lysoPC), a product of hydrolysis of phospholipase A(2) (PLA(2)), is more efficient at 18 degrees C, where DMPC MLV's are known to be in the ripple P(beta') phase, than at 10 degrees C (L(beta') flat gel phase). Disruption at 18 degrees C was inhibited by increasing concentrations of arbutin in the solution. This inhibition was correlated with the disappearance of the ripple phase in MLV's when arbutin is present. Shifts in FTIR carbonyl bands caused by arbutin or by temperature changes allow us to propose a model. It is interpreted that the changes in the water-hydrocarbon interface caused by arbutin, forcing a reaccommodation of the carbonyl groups, eliminate the topological defects in the lattice due to mismatches among regions with different area per lipid where lysoPC can insert.

Experimental and theoretical study of the hydration of phosphate groups in esters of biological interest.

We have studied the influence of different groups esterified to phosphates on the strength of the interaction of the PO bond with one water molecule. Experimental vibrational spectra of PO(4)3-, HPO4(2-), H2PO4-, phosphoenolpiruvate (PEP) and ortho-phosphocholamine (o-PC) were obtained by means of FTIR spectroscopy. Geometry calculations were performed using standard gradient techniques and the default convergence criteria as implemented in GAUSSIAN 98 Program. In order to assess the behaviour of such DFT theoretical calculations using B3LYP with 6-31G* and 6-311++G** basis sets, we carried out a comparative work for those compounds. The results were then used to predict the principal bands of the vibrational spectra and molecular parameters (geometrical parameters, stabilisation energies, electronic density). In this work, the relative stability and the nature of the PO bond in those compounds were systematically and quantitatively investigated by means of Natural Bond Order (NBO) analysis. The topological properties of electronic charge density are analysed employing Bader's Atoms in Molecules theory (AIM). The hydrogen bonding of phosphate groups with water is highly stable and the PO bond wavenumbers are shifted to lower experimental and calculated values (with the DFT/6-311++G** basis set). Accordingly, the predicted order of the relative stability of the hydrogen bonding of the water molecule to the PO bond of the investigated compounds is: PO(4)3->HPO4(2-)>H2PO4->phosphoenolpiruvate>phosphocholamine for the two basis sets used.

Hydration of inorganic phosphates in crystal lattices and in aqueous solution. An experimental and theoretical study.

B3LYP/6-31G* and 6-311++G** calculations have been carried out in order to study the hydration of phosphates in aqueous media. Optimized geometries and relative stabilities for PO4(-3), HPO(4)-2, H2PO4(-1) have been calculated considering the interaction with one, two, three, four and five discrete water molecules and taking into account the solvent effect by using the self-consistent reaction field theory (Onsager and PCM methods). The role of specific and bulk contributions of solvent effect on the observable properties of phosphate compounds is analysed. Good agreement between theoretical and available experimental results of harmonic vibration frequencies is found. Significant effects on the geometrical and vibrational frequencies are found for those studied phosphate anions. The results presented here provide a first step toward the understanding of the phosphate group as a hydration sensor in lipid bilayers.