Anesthetic-protein interaction Random versus helix polylysine monolayers and interaction with 1-alkanols

Penetration of 1-alkanols into monolayers of hydrophobic polypeptides, poly(ε-benzyloxycarbonyl-l-lysine) and poly(ε-benzyloxycarbonyl-dl-lysine), was compared with their adsorption on the air/water interface in the absence of monolayers. The polypeptide prepared from l-lysine is generally considered to be in the α-helical form whereas dl-copolymer polypeptide contains random-coiled portions due to the structural incompatibility between the two isomers. The free energy of adsorption of 1-alkanols on the air/water interface at dilute concentrations was ?0.68 kcal·mol^?1 per methylene group and 0.15 kcal·mol^?1 for the hydroxyl group at 25°C. In the close-packed state, the surface area occupied by each molecule of 1-alkanols of varying carbon chain-lengths showed nearly a constant value of about 27.2 Ų, indicating perpendicular orientation of the alkanol molecules at the interface. About 75% of the water surface was covered by 1-butanol in this close-packed state. The mode of adsorption of 1-alkanols on the vacant air/water interface followed the Gibbs surface excess while the mode on the polypeptide membranes followed the Langmuir adsorption isotherm, indicating that the latter is characterized by the presence of a finite number of binding sites. The free energies of adsorption of 1-alkanols on the l-polymer monolayers were more negative than those on the vacant air/water interface and less negative than those on the dl-copolymer monolayers. Thus, the affinity of 1-alkanols to the interface was in the order of vacant air/water interface <l-polymer <dl-copolymer. The difference between the air/water interface and l-polymer was about 0.54 kcal·mol^?1 and that between l-polymer and dl-copolymer was 0.17 kcal·mol^?1 at 25°C: the adsorption of 1-alkanols to the dl-copolymer was favored compared to the l-polymer. The polar moieties of the backbone of the dl-copolymer may be exposed to the aqueous phase at the disordered portion. Dipole interaction between this portion and 1-alkanol molecules may account for the enhanced adsorption of the alkanols to the dl-copolymer.     

Spread mixed monolayers of deoxycholic and dehydrocholic acids at the air-water interface, effect of subphase pH. Characterization by axisymmetric drop shape analysis

Bile acids (deoxycholic and dehydrocholic acids) spread mixed monolayers behavior at the air/water interface were studied as a function of subphase pH using a constant surface pressure penetration Langmuir balance based on the Axisymmetric Drop Shape Analysis (ADSA). We examined the influence of electrostatic, hydrophobic and hydration forces on the interaction between amphiphilic molecules at the interface by the collapse area values, the thermodynamic parameters and equation of state virial coefficients analysis. The obtained results showed that at neutral (pH=6.7) or basic (pH=10) subphase conditions the collapse areas values are similar to that of cholanoic acid and consistent with the cross-sectional area of the steroid nucleus (approximately 40 A(2)). The Gibbs energy of mixing values (DeltaG(mix)<0) and the first virial coefficients of the equation of state (b(0)<1) indicated that a miscible monolayer with laterally structured microdomains existed. The aggregation number (1/b(0)) was estimated within the order of 6 (pH=6.7) and 3 (pH=10). At pH=3.2, acidic subphase conditions, no phase separation occurs (DeltaG(mix)<0) but a high expanded effect of the monolayer could be noted. The mixed monolayer behavior was no ideal and no aggregates were formed (b(0)> or =1). Such behavior indicates that the polar groups of the molecules interacts each other more strongly by repulsive electrostatic forces than with the more hydrophobic part of the molecule.     

X-ray diffraction and foam film investigations of PC head group interaction in water/ethanol mixtures

The influence of ethanol on single phospholipid monolayers at the water/air interface and in foam films has been investigated. Grazing incidence X-ray diffraction investigations (GIXD) of Langmuir monolayers from 1,2-distearoyl-phosphatidylcholine (DSPC) spread on water subphases with different amounts of ethanol were performed. The thickness and free specific energy of formation of foam films stabilized by 1,2-dimyristoyl-phosphatidylcholine (DMPC) at different concentrations of ethanol in the film forming dispersions were measured. The GIXD investigations show that the tilt angle of the alkyl chains in the PC lipid monolayer decreases with increasing concentration of ethanol caused by a decrease of the diameter of the head groups. With increasing ethanol content of the solution also the thickness of the aqueous core of PC lipid foam films decreases. We assume that ethanol causes a decreasing probability for the formation of hydrogen bonds of water molecules to the PC head groups. The distinct difference between the effects of ethanol on lipid bilayers as described in the literature and on monolayers and foam films found in this study is discussed. Whereas PC monolayers at the water/air interface become unstable above 25 vol.% ethanol, the PC foam films are stable up to 50 vol.% ethanol. This is related to the decrease of the surface excess energy per lipid molecule by the interaction between the two film surfaces.     

Differences in surface behaviour of galactolipoids originating from different kind of wheat tissue cultivated in vitro

The aim of presented researches was to investigate the physicochemical properties of Langmuir monolayer of galactolipids extracted from two different kinds of plastids: immature embryos and inflorescences. Differences between the physicochemical properties of the plastid membranes may help to explain different physiological processes, such as plant regeneration. Surface pressure (pi) vs. molecular area (A) isotherms of the monogalactosyldiacylglycerol (MGDG)/digalactosyldiacylglycerol (DGDG) monolayers of various molar ratios were measured at 15 degrees C. Galactolipids were extracted from two different types of tissue: inflorescences and embryos. Based on the analysis of the pi-A isotherms, the properties of monolayers, such as collapse pressure (pi(coll)), limiting area (A(lim)), compressibility modulus (C(s)(-1)), excess free energy of mixing (DeltaG(EXC)) and free energy of mixing (DeltaG(MIX)), were calculated. The results show that pure MGDG and DGDG and their mixtures form liquid-expanded monolayers, independently on the kind of tissue. Galactolipids originating from inflorescences produce more compressible films at the air/water interface, with larger limiting area per molecule and lower stability against the collapse process. MGDG and DGDG are miscible and form non-ideal mixed monolayers at the air/water interface. Negative values of DeltaG(EXC) were calculated for the mixture of galactolipids originating from inflorescences, with the content of MGDG, x(MGDG)>0.6. In the case of embryos, the negative values of DeltaG(EXC) were found for x(MGDG) approximately 0.5. Therefore, the attractive interactions between MGDG and DGDG exist in the mixtures of these compositions. As it is shown by negative values of DeltaG(MIX), mixed monolayers are more stable compared with unmixed ones.     

The interaction of liposomes containing intrinsic erythrocyte membrane proteins with lipid monolayers at air/water and oil/water interfaces

The main intrinsic membrane proteins of the human erythrocyte membrane, glycophorin and the anion transporter, were isolated by extraction with Triton X-100 and ion-exchange chromatography. After removal of detergent the extract consisted of proteolipid vesicles with a lipid:protein molar ratio in the range 50-60 and a diameter of the order of 200 nm. The interaction between these vesicles and dipalmitoylphosphatidylcholine (DPPC), cholesterol and cholesterol:DPPC (2:1 molar ratio) monolayers at air/water and n-decane/water interfaces has been studied. The vesicles interact with the monolayers, rapidly causing large increases in surface pressure. Limiting values of surface pressure, 39.4-43 mN . m-1 at air/water and 31.5-33.4 mN . m-1 at the n-decane/water interface, were reached at protein levels above 1 microgram . ml-1. At the air/water interface, and probably at the n-decane/water, surface pressure increases were limited by monolayer collapse. Compression isotherms and surface potential measurements indicated that material from the proteolipid vesicles entered the monolayer phase. In contrast to proteolipid vesicles, injection of protein-free liposomes beneath the monolayer resulted in smaller, slower increases in surface pressure. Thus, the presence of intrinsic membrane proteins in vesicles greatly facilitated the transfer of material into the lipid monolayer.     

The surface properties of apolipoproteins A-I and A-II at the lipid/water interface

The monolayer system was employed to investigate the relative affinities of apolipoproteins A-I and A-II for the lipid/water interface. The adsorption of reductively 14C-methylated apolipoproteins to phospholipid monolayers spread at the air/water interface was determined by monitoring the surface pressure of the mixed monolayer and the surface concentration of the apoprotein. ApoA-II has a higher affinity than apoA-I for lipid monolayers; for a given initial surface pressure, apoA-II adsorbs more than apoA-I to monolayers of egg phosphatidylcholine (PC), distearoyl-PC and human high-density lipoprotein (HDL3) surface lipids. Comparison of the molecular packing of apolipoproteins A-I and A-II suggests that apoA-II adopts a more condensed conformation at the lipid/water interface compared to apoA-I. The ability of apoA-II to displace apoA-I from egg PC and HDL3 surface lipid monolayers was studied by following the adsorption and desorption of the reductively 14C-methylated apolipoproteins. At saturating subphase concentrations of the apoproteins (3.10(-5) g/100 ml), two molecules of apoA-II absorbed for each molecule of apoA-I displaced. This displacement was accompanied by an increase in surface pressure. An identical stoichiometry for the displacement of apoA-I from HDL particles by apoA-II has been reported by others. At low subphase concentrations of apoproteins (5.10(-6) g/100 ml), the apoA-I/lipid monolayer was not fully compressed and could accommodate the adsorbing apoA-II molecules without displacement of apoA-I molecules. ApoA-I molecules were unable to displace apoA-II from the lipid/water interface. The average residue hydrophobicity of apoA-II is higher than that of apoA-I; this may contribute to the higher affinity of apoA-II for lipids compared to apoA-I. The probable helical regions in apolipoproteins A-I and A-II were located using a secondary structure prediction algorithm. The analysis suggests that the amphiphilic properties of the alpha-helical regions of apoA-I and apoA-II are probably not significantly different. Further understanding of the differences in surface activity of these apolipoproteins will require more knowledge of their secondary and tertiary structures.     

Surface activity and interaction of StarD7 with phospholipid monolayers

StarD7 protein forms stable Gibbs and Langmuir monolayers at the air-buffer interface showing marked surface activity. The latter is enhanced by penetration into phospholipid films at an initial surface pressure above the protein’s own equilibrium adsorption surface pressure to a lipid-free interface. The protein-phospholipid stabilizing interactions at the interface depend on the lipid, with preference for phosphatidylserine, cholesterol, and phosphatidylglycerol, and the increases of lateral surface pressure generated are comparable to those of other membrane-active proteins. The surface activity of StarD7 is strong enough to thermodynamically drive and retain StarD7 at the lipid membrane interface where it may undergo lipid-dependent reorganization as indicated by changes of surface pressure and electrostatics.     

Association of protein kinase C with phospholipid monolayers: two-stage irreversible binding

The association of protein kinase C (PKC) with phospholipid (PL) monolayers spread at the air-water interface was examined. PKC-PL binding induced surface pressure changes that were dependent on the amount of PKC, the phospholipid composition of the monolayers, the presence of Ca2+, and the initial surface pressure of the monolayer (pi 0). Examination of surface pressure increases induced by PKC as a function of phospholipid surface pressure, pi 0, revealed that PKC-phosphatidylserine (PS) association had a critical pressure of 43 dyn/cm. Above this surface pressure, PKC cannot cause further surface pressure changes. This high critical pressure indicated that PKC should be able to penetrate many biological membranes which appear to have surface pressures of about 30 dyn/cm. PKC-induced surface pressure changes were Ca2+ dependent only for PL monolayers spread at a pi 0 greater than 26 dyn/cm. PKC alone (in the absence of PL) formed a film at the air-water interface with a surface pressure of about 26 dyn/cm. Calcium-dependent binding was studied at the higher surface pressures which effectively excluded PKC from the air-water interface. Subphase depletion measurements suggested that association of PKC with PS monolayers consisted of two stages: a rapid Ca2+-dependent interaction followed by a slower process that resulted in irreversible binding of PKC to the monolayer. The second stage appeared to involve penetration of PKC into the hydrocarbon region of the phospholipid. The commonly used in vitro substrates for PKC, histone and protamine sulfate, also associated with and penetrated PS monolayers with critical pressures of 50 and 60 dyn/cm, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Surface active properties of amphiphilic sequential isopeptides: Comparison between alpha-helical and beta-sheet conformations

Poly(Leu-Lys-Lys-Leu) and poly(Leu-Lys) are sequential amphiphilic peptide isomers that adopt respectively an alpha-helical conformation and a beta-sheet structure in saline solutions and at the air/water interface. The surface active properties of LKKL and LK sequential isopeptides containing 16, 20, and n residues have been compared in order to evaluate the contributions of the alpha-helical and beta-sheet conformations. Both have a natural tendency to spread at the surface of a saline solution and the values of the equilibrium spreading pressure pi(e) lie in the same range. When dissolved in a saline solution, alpha-helical peptides diffuse faster and adsorb faster at the interface than the beta-sheet isomers. From the compression isotherms of LKKL and LK peptide monolayers it is possible to extract parameters that characterize the behavior of alpha-helical and beta-sheet conformations: beta-sheet peptide monolayers are more stable and less compressible than the monolayers formed with the alpha-helical isomers. The LK peptides differ also by their high degree of self-association at the air/water interface. Copyright 1999 John Wiley & Sons, Inc.     

The Equilibria Between Monovalent Ions and Phosphatidylcholine Monolayer at the Air/Water Interface

The effect of monovalent ion (Li⁺, Na⁺, Cs⁺) interaction with monolayers of phosphatidylcholine (lecithin, PC) was investigated at the air/water interface. We present surface tension measurements of lipid monolayers obtained using a Langmuir method as a function of monovalent ion concentration. Measurements were carried out at 22 °C using a Teflon trough and a Nima 9000 tensiometer. Interactions between lecithin and monovalent ions result in significant deviations from the additivity rule. An equilibrium theory to describe the behavior of monolayer components at the air/water interface was developed in order to obtain the stability constants and area occupied by one molecule of PC–monovalent ion complexes (PC^?Me⁺).     

The interaction of synthetic analogs of the N-terminal fusion sequence of influenza virus with a lipid monolayer. Comparison of fusion-active and fusion-defective analogs

The amino terminus of subunit-2 of influenza virus hemagglutinin (NHA2) plays a crucial role in the induction of fusion between viral and endosomal membranes leading to the infection of a cell. Three synthetic analogs with an amino acid sequence corresponding to NHA2 of variant hemagglutinins were studied in a monolayer set up. Comparison of the interaction of a fusion-active and two fusion-defective analogs with a lipid monolayer revealed a greater surface activity of the fusion-active analog. Pronounced differences were found if the pure peptides were spread at the air/water interface; the fusion-active analog showed a higher collapse pressure and a greater limiting molecular area. Circular dichroism measurements on collected lipid monolayers indicated a high content of alpha-helical structure for the fusion-active and one of the fusion-defective analogs. A simple relation between alpha-helical content and fusogenicity does not seem to exist. Instead, the extent of penetration, a defined tertiary structure or orientation of the alpha-helical peptide may be essential for its membrane perturbing activity.     

Conformation of poly(l-glutamic acid) at the air/water interface

The conformation of the monolayer of poly(l-glutamic acid) on subsolutions of different pH values was studied by the film-balance technique, obtaining surface pressure measurements, together with polarized infrared spectroscopy and Raman spectroscopy. The monolayers of poly(l-glutamic acid) gave different surface pressure-area curves on subsolutions of various pH values. It was found that the conformation of poly(l-glutamic acid) monolayer spread at the air/water interface differs from that in solution. It can be presumed that poly(l-glutamic acid) in a monolayer is in the form of an α-helix at pH 2.0, in the β-form at pH 3.5 and in an ‘intramolecular’ heterogeneous conformation (consisting of a random coil and an α-helix) at pH 4.0.     

Specific anchoring modes of two distinct dystrophin rod sub-domains interacting in phospholipid Langmuir films studied by atomic force microscopy and PM-IRRAS

Dystrophin rod repeats 1-3 sub-domain binds to acidic phosphatidylserine in a small vesicle binding assay, while the repeats 20-24 sub-domain does not. In the present work, we studied the adsorption behaviour of both sub-domains at the air/liquid interface and at the air/lipid interface in a Langmuir trough in order to highlight differences in interfacial properties. The adsorption behaviour of the two proteins at the air/liquid interface shows that they display surface activity while maintaining their alpha-helical secondary structure as shown by PM-IRRAS. Strikingly, R20-24 needs to be highly hydrated even at the interface, while this is not the case for R1-3, indicating that the surface activity is dramatically higher for R1-3 than R20-24. Surface-pressure measurements, atomic force microscopy and PM-IRRAS are used in a Langmuir experiment with DOPC-DOPS monolayers at two different surface pressures, 20 mN/m and 30 mN/m. At the lower surface pressure, the proteins are adsorbed at the lipid film interface while maintaining its alpha-helical structure. After an increase of the surface pressure, R1-3 subsequently produces a stable film, while R20-24 induces a reorganization of the lipid film with a subsequent decrease of the surface pressure close to the initial value. AFM and PM-IRRAS show that R1-3 is present in high amounts at the interface, being arranged in clusters representing 3.3% of the surface at low pressure. By contrast, R20-24 is present at the interface in small amounts bound only by a few electrostatic residues to the lipid film while the major part of the molecule remains floating in the sub-phase. Then for R1-3, the electrostatic interaction between the proteins and the film is enhanced by hydrophobic interactions. At higher surface pressure, the number of protein clusters increases and becomes closer in both cases implying the electrostatic character of the binding. These results indicate that even if the repeats exhibit large structural similarities, their interfacial properties are highly contrasted by their differential anchor mode in the membrane. Our work provides strong support for distinct physiological roles for the spectrin-like repeats and may partly explain the effects of therapeutic replacement of dystrophin deficiency by minidystrophins.     

Large-scale recrystallization of the S-layer of Bacillus coagulans E38-66 at the air/water interface and on lipid films.

S-layer protein isolated from Bacillus coagulans E38-66 could be recrystallized into large-scale coherent monolayers at an air/water interface and on phospholipid films spread on a Langmuir-Blodgett trough. Because of the asymmetry in the physiochemical surface properties of the S-layer protein, the subunits were associated with their more hydrophobic outer face with the air/water interface and oriented with their negatively charged inner face to the zwitterionic head groups of the dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine (DPPE) monolayer films. The dynamic crystal growth at both types of interfaces was first initiated at several distant nucleation points. The individual monocrystalline areas grew isotropically in all directions until the front edge of neighboring crystals was met. The recrystallized S-layer protein and the S-layer-DPPE layer could be chemically cross-linked from the subphase with glutaraldehyde.     

The Equilibria of Diosgenin–Phosphatidylcholine and Diosgenin–Cholesterol in Monolayers at the Air/Water Interface

Diosgenin (Dio) has shown many treatment properties, but the most important property is cytotoxic activity in cancer cells. In this study, we investigated monolayers of Dio, cholesterol (Ch), and phosphatidylcholine (PC) at the air/water interface. The measurements were carried with a Langmuir Teflon trough and a Nima 9000 tensiometer program. The surface tension values of pure and mixed monolayers were used to calculate π–A isotherms and determine molecular surface areas. We were able to demonstrate the formation of complexes between Dio and PC and Dio and Ch molecules also. We considered the equilibrium between individual components and the formed complexes. In addition, we established that diosgenin and the lipids formed highly stable 1:1 complexes.     

Interaction of GM(1) glycolipid in phospholipid monolayers with wheat germ agglutinin: effect of phospholipidic environment and subphase.

Mixed monolayers of GM(1) glycolipid and stearoyl-oleoyl-phosphatidylcholine (SOPC) or dipalmitoyl-phosphatidycholine (DPPC) phospholipids were studied by surface pressure measurements. The effects induced by GM(1) on the mean molecular areas of mixtures and DPPC phase transition were followed for GM(1) concentrations ranging from 1 to 20 mol.%. Under our experimental conditions, one main parameter influencing the behavior of phospholipid-GM(1) monolayers is the ionic strength of the subphase. Mixed monolayers are in a more expanded state on buffer than on pure water. This could be due to a change of GM(1) orientation at the interface. The interaction of wheat germ agglutinin (WGA), a lectin recognizing specifically GM(1), with these monolayers was quantified in terms of the Gibbs equation. Specific WGA-GM(1) interactions are clearly reduced in the presence of DPPC as compared with SOPC, probably because of the higher packing density of these monolayers. Phospholipid-GM(1) monolayers could also undergo some rearrangements induced by WGA binding.     

Influence of stearic acid monolayers upon the procaine adsorption from underlying alkaline aqueous solutions

Adsorption of procaine at the air/water interface and its penetration into stearic acid monolayers from aqueous subphase of pH 8 are studied by measuring surface tension of aqueous procaine solutions and by recording surface pressure vs. mean molecular area curves for stearic acid monolayers spread onto procaine solutions of different concentrations. The amount of procaine in the interface is derived by means of Gibbs' equation. Results are compared to those obtained earlier at pH 2 and on unbuffered subphases. With increasing pH an increasing procaine adsorption and procaine penetration is observed. This phenomenon is interpreted in terms of protolytic equilibria in which participate both surfactants procaine and stearic acid.     

Interaction of mitochondrial creatine kinase with model membranes. A monolayer study

The interaction of mitochondrial creatine kinase (Mi-CK; EC 2.7.3.2) with phospholipid monolayers and spread mitochondrial membranes at the air/water interface has been investigated. It appeared that Mi-CK penetrated into these monolayers as evidenced by an increase in surface pressure upon incorporation of Mi-CK. The increase in surface pressure was dependent on (1) the amount and (2) the oligomeric form of Mi-CK in the subphase, as well as on (3) the initial surface pressure and (4) the phospholipid composition of the monolayer. In this experimental system Mi-CK was able to interact equally well with both inner and outer mitochondrial membranes.     

Effects of α-tocopherol analogs on lysosome membranes and fatty acid monolayers

The surface pressures of α-tocopherol analogs, fatty acids, and their mixtures were measured in their spread monolayers at an air—water interface. The surface pressure—area isotherms for the mixed monolayers of α-tocopherol and either stearic acid, oleic acid or linoleic acid deviated positively from those calculated on the basis of the additivity rule, and the magnitude depended on the length of the phytyl side chain in α-tocopherol and on the degree of unsaturation of the fatty acid chains. Lysosome membranes of mouse liver were stabilized by addition of α-tocopherol. A decrease in the length of the phytyl side chain in α-tocopherol reduced its ability to stabilize lysosome membranes. A good correlation was obtained between the extent of stabilizing activity of α-tocopherol analogs on lysosome membranes and the degree of positive deviation of the surface pressure for their mixtures with fatty acids.