Surface Pressure Of Monolayers Research Articles

Lipolytic activity of ricin from Ricinus sanguineus and Ricinus communis on neutral lipids

The present study was carried out with a view of determining ricin lipolytic activity on neutral lipids in emulsion and in a membrane-like model. Using 2,3-dimercapto-1-propanol tributyrate (BAL-TC4) as substrate, the lipolytic activity of ricin was found to be proportional to ricin and substrate concentrations, with an apparent Km (Km,app) of 2.4mM, a kcat of 200min−1 and a specific activity of 1.0unit/mg of protein. This work was extended to p-nitrophenyl (pNP) fatty acid esters containing two to twelve carbon atoms. Maximum lipolytic activity was registered on pNP decanoate (pNPC10), with a Km,app of 3.5mM, a kcat of 173min−1 and a specific activity of 3.5units/mg of protein. Ricin lipolytic activity is pH and galactose dependent, with a maximum at pH7.0 in the presence of 0.2M galactose. Using the monolayer technique with dicaprin as substrate, ricin showed a lipolytic activity proportional to the ricin concentration at 20mN/m, which is dependent on the surface pressure of the lipid monolayer and is detectable up to 30mN/m, a surface pressure that is of the same order of magnitude as that of natural cell membranes. The methods based on pNPC10 and BAL-TC4 hydrolysis are simple and reproducible; thus they can be used for routine studies of ricin lipolytic activity. Ricin from Ricinus communis and R. sanguineus were treated with diethyl p-nitrophenylphosphate, an irreversible serine esterase inhibitor, and their lipolytic activities on BAL-TC4 and pNPC10, and cytotoxic activity, were concurrently recorded. A reduction in lipolytic activity was accompanied by a decrease in cytotoxicity on Caco2 cells. These data support the idea that the lipolytic activity associated with ricin is relevant to a lipase whose activity is pH and galactose dependent, sensitive to diethyl p-nitrophenylphosphate, and that a lipolytic step may be involved in the process of cell poisoning by ricin. Both colorimetric tests used in this study are sensitive enough to be helpful in the detection of possible lipolytic activities associated with other cytotoxins or lectins.

Molecular Dynamics Simulation of the Surface Pressure of Colloidal Monolayers

The compression of two-dimensional colloidal monolayers consisting of polystyrene particles at an oil−water interface is investigated theoretically. Expressions for the pair−particle interactions are derived both for point particles and for particles of radius R. The resulting expressions for the interaction energies are employed in a molecular dynamics simulation of the surface pressure of these monolayers. A comparison of the theoretical results with recent observations allows the determination of the dominant interaction mechanisms in the experimental system. Possible physical interpretations of the interaction mechanisms between the particles are discussed.

Halofantrine-phospholipid interactions: monolayer studies.

The antimalarial agent halofantrine penetrates dipalmitolylphosphatidylcholine (DPPC) monolayers resulting in an increase in surface pressure and an expansion in area occupied by the lipid components of the monolayer. This phenomenon is observed at concentrations (0.05-0.2 microm) of halofantrine that have no surface activity. Penetration increases with drug concentration and is greatest at low initial surface pressures of the monolayer. A critical surface pressure of the DPPC monolayer has been determined from constant area and constant pressure conditions. The magnitude of these values support the hypothesis that halofantrine readily penetrates the DPPC monolayers. The presence of cholesterol in the DPPC monolayer hampers penetration and a lower critical surface pressure is obtained under such conditions. Even then, a slower rate of penetration is observed only in monolayers maintained at high initial surface pressures (10, 15 mN/m), corresponding to the liquid condensed phase of the monolayer, and not at low surface pressures (2.5, 5.0 mN/m). These results help to give a better understanding of the dynamics of the halofantrine-phospholipid interaction as well as the pharmacodynamic character of the drug.

Surface-active properties of vasoactive intestinal peptide☆

The purpose of this study was to determine whether human vasoactive intestinal peptide (VIP) aggregates in aqueous solution and, if so, whether the peptide interacts with a biomimetic phospholipid monolayer and increases surface pressure. Using a custom-made Teflon trough containing HEPES buffer (pH 7.4) at room temperature and a surface tensiometer, we found that the critical micellar concentration (CMC) of VIP is 0.4 μM. Surface pressure of a dipalmitoylphosphatidylcholine (DPPC) monolayer spread over the HEPES buffer declined significantly over 120 min because of phospholipid decomposition. However, injection of VIP at concentrations above CMC into the subphase of the monolayer elicited a significant concentration-dependent increase in surface pressure that persisted for 120 min ( P < 0.05). Unlike VIP, injection of [ 8Arg]-vasopressin at an equimolar concentration only prevented the time-dependent decline in DPPC monolayer surface pressure. Taken together, these data indicate that human VIP aggregates in aqueous solution and expresses surface-active properties at physiological concentrations in vitro. We suggest that these attributes could have a role in modulating the bioactive effects of the peptide in vivo.

Lipase Catalysis on Monolayers at the Air/Water Interface. 1. Kinetic Rate Constants on Quasi-Two-Dimension

Chemical reactions on a cell membrane surface are of pivotal importance for cellular functions such as intracellular signal transduction. Of the variety of membrane proteins, those imbedded in hemi-leaflets of bilayer membranes can be mimicked structurally and dynamically on monomolecular layers of phospholipids at the air/water interface. This is to report the kinetics of lipase (Pseudomonas cepacia) catalyzed hydrolysis of a substrate (umbelliferone stearate) on uniphasic l-α-dilauroylphosphatidylcholine monolayers at the air/water interface. A novel experimental protocol of the interfacial reaction is employed to probe the enzymatic kinetics. The kinetic rate constants on the monolayers are extracted from the substrate and the enzyme concentration dependences of the initial hydrolysis rate. The lipase on monolayers at the air/water interface is highly activated over that in bulk solution, and the turnover number of the lipase catalysis is strongly dependent on the surface pressure of monolayers.

Adsorption of Glucose Oxidase into Lipid Monolayers. Effect of Lipid Chain Lengths on the Stability and Structure of Mixed Enzyme/Phospholipid Films

The effect of lipid hydrophobic chain lengths on adsorption of glucose oxidase (GOx) into phospholipid monolayers has been investigated. The results show that at low initial surface pressures, penetration of GOx depended both on hydrophobic interactions and on the orientation of lipid chains with respect to the interface plane. Compared with dimyristoyl-phosphatidylcholine (DMPC) and dipalmitoyl-phosphatidylcholine (DPPC), dibehenoyl-phosphatidylcholine (DBPC) favored enzyme adsorption at low surface pressures. This was attributed to its long hydrophobic chains and to their vertical orientation. At higher initial surface pressures, the fluidity and packing of these monolayers appeared to play a key role in the penetration process. The presence of a double bond in the hydrocarbon chains enhanced enzyme adsorption, and this effect was independent of the initial surface pressure of lipid monolayers.

Single Molecules as Probes of Lipid Membrane Microenvironments

The single-molecule dynamics of the membrane probe diIC18 dispersed in well-controlled lipid environments were studied. Lipid monolayers and bilayers of DPPC, transferred onto a solid substrate using the Langmuir−Blodgett technique, were utilized to systematically control the environment around single diIC18 molecules. The single-molecule emission trajectories revealed large intensity fluctuations that were strongly coupled to the lipid environment surrounding the probe molecule. For example, as the surface pressure of a DPPC monolayer was increased from the liquid-expanded/liquid-condensed (π = 5 mN/m) region to the solid condensed (π = 30 mN/m) region, the characteristic fluorescence fluctuation times increased from approximately 440 ms to over 1 s. For bilayer films, we found characteristic fluctuation times on the order of 2 s, regardless of which side of the bilayer the probe molecule resided. The monolayer and bilayer results are most consistent with a mechanism for intensity fluctuations driven by ...

Effect of Hydrophobic Surfactant Peptides SP-B and SP-C on Binary Phospholipid Monolayers. I. Fluorescence and Dark-Field Microscopy

The influence of the hydrophobic proteins SP-B and SP-C, isolated from pulmonary surfactant, on the morphology of binary monomolecular lipid films containing phosphocholine and phosphoglycerol (DPPC and DPPG) at the air-water interface has been studied using epifluorescence and dark-field microscopy. In contrast to previously published studies, the monolayer experiments used the entire hydrophobic surfactant protein fraction (containing both the SP-B and SP-C peptides) at physiologically relevant concentrations (∼1 wt %). Even at such low levels, the SP-B/C peptides induce the formation of a new phase in the surface monolayer that is of lower intrinsic order than the liquid condensed (LC) phase that forms in the pure lipid mixture. This presumably leads to a higher structural flexibility of the surface monolayer at high lateral pressure. Variation of the subphase pH indicates that electrostatic interaction dominates the association of the SP-B/C peptides with the lipid monolayer. As evidenced from dark-field microscopy, monolayer material is excluded from the DPPC/DPPG surface film on compression and forms three-dimensional, surface-associated structures of micron dimensions. Such exclusion bodies formed only with SP-B/C peptides. This observation provides the first direct optical evidence for the squeeze-out of pulmonary surfactant material in situ at the air-water interface upon increasing monolayer surface pressures.

Effect of Gramicidin A on the Dipole Potential of Phospholipid Membranes

The effect of channel-forming peptide gramicidin A on the dipole potential of phospholipid monolayers and bilayers has been studied. Surface pressure and surface potential isotherms of monolayers have been measured with a Langmuir trough equipped with a Wilhelmy balance and a surface potential meter (Kelvin probe). Gramicidin has been shown to shift pressure-area isotherms of phospholipids and to reduce their monolayer surface potentials. Both effects increase with the increase in gramicidin concentration and depend on the kind of phosphatidylcholine used. Application of the dual-wavelength ratiometric fluorescence method using the potential-sensitive dye RH421 has revealed that the addition of gramicidin A to dipalmitoylphosphatidylcholine liposomes leads to a decrease in the fluorescence ratio of RH421. This is similar to the effect of phloretin, which is known to decrease the dipole potential. The comparison of the concentration dependences of the fluorescence ratio for gramicidin and phloretin shows that gramicidin is as potent as phloretin in modifying the membrane dipole potential.

Monolayers of Some Poly(oxyethylene)-Based Surfactants at the Air–Water Interface: The Effect of Structural Variations and Salt Concentration

The Langmuir surface balance technique has been used to study the interfacial behavior of six structurally different poly(oxyethylene) (POE)-based polymer surfactants at the air–water interface. On a pure water subphase the surfactants have collapse surface pressures dependent on the POE chain length. The surfactant monolayers collapse at well-determined surface pressures, and the lower POE chain-length surfactants collapse at higher pressures than those with high POE content. This difference vanishes as increasing amounts of salt are added to the subphase. The ΠA-isotherms are smooth, which is normal for polymeric surfactants. A closer analysis of the isotherms reveals characteristic behavior that can be attributed to structural differences. Similarities in thermodynamic behavior suggest that the molecular orientation is the same despite the structural differences. A new expression for the compressibility factor is developed to explain the relationship between this parameter and surface pressure for polymeric monolayers.

Adaptation of bulk constitutive equations to insoluble monolayer collapse at the air-water interface.

A constitutive equation based on stress-strain models of bulk solids was adapted to relate the surface pressure, compression rate, and temperature of an insoluble monolayer of monodendrons during collapse at the air-water interface. A power law relation between compression rate and surface pressure and an Arrhenius temperature dependence of the steady-state creep rate were observed in data from compression rate and creep experiments in the collapse region. These relations were combined into a single constitutive equation to calculate the temperature dependence of the collapse pressure with a maximum error of 5 percent for temperatures ranging from 10 degrees to 25 degrees C.

Brain spectrin (fodrin) interacts with phospholipids as revealed by intrinsic fluorescence quenching and monolayer experiments

We demonstrate that phospholipid vesicles affect the intrinsic fluorescence of isolated brain spectrin. In the present studies we tested the effects of vesicles prepared from phosphatidylcholine (PtdCho) alone, in addition to vesicles containing PtdCho mixed with other phospholipids [phosphatidylethanolamine (PtdEtn) and phosphatidylserine] as well as from total lipid mixture extracted from brain membrane. The largest effect was observed with PtdEtn/PtdCho (3:2 molar ratio) vesicles; the effect was markedly smaller when vesicles were prepared from egg yolk PtdCho alone. Brain spectrin injected into a subphase induced a substantial increase in the surface pressure of monolayers prepared from phospholipids. Results obtained with this technique indicated that the largest effect is again observed with monolayers prepared from a PtdEtn/PtdCho mixture. The greatest effect was observed when the monolayer contained 50-60% PtdEtn in a PtdEtn/PtdCho mixture. This interaction occurred at salt and pH optima close to physiological conditions (0.15 M NaCl, pH7.5). Experiments with isolated spectrin subunits indicated that the effect of the beta subunit on the monolayer surface pressure resembled that measured with the whole molecule. Similarly to erythrocyte spectrin-membrane interactions, brain spectrin interactions with PtdEtn/PtdCho monolayer were competitively inhibited by isolated erythrocyte ankyrin. This also suggests that the major phospholipid-binding site is located in the beta subunit and indicates the possible physiological significance of this interaction.

Surface Pressure of Adsorbed Polymer Layers. Effect of Sticking Chain Ends

Using a mean-field theory with two order parameters and scaling laws, we study the properties of adsorbed polymer layers in contact with a pure solvent, at a fixed amount of polymers. We calculate the surface pressure of the polymer Langmuir monolayer, implicitly assuming that the monolayer is insoluble, i.e., that the polymer desorption time is much larger than any other characteristic time, in particular, the equilibrium relaxation time of the layer. This work was motivated by experiments done on poly(ethylene oxide)(PEO) and telechelic PEO adsorbed at the air−water interface that present an adsorbed layer structure at certain densities. Our theoretical results explain quite well the shape of the surface pressure isotherms and the influence of the chain length and of the hydrophobic ends.

Interpretation of Mechanochemical Properties of Lipid Bilayer Vesicles from the Equation of State or Pressure−Area Measurement of the Monolayer at the Air−Water or Oil−Water Interface

There are quite a few types of equation of state and abundant π−a curves of various lipid monolayers at the air−water or the oil−water interface in the literature. However, it has been a problem to interpret mechanochemical properties of bilayer vesicles from the π−a information of the monolayer. In fact, even the bilayer surface pressure has not yet been well characterized although the monolayer surface pressure has already been traditionally defined as the lowering in the surface tension from the clean interfacial tension due to the presence of the monolayer. The monolayer−bilayer correspondence problem, therefore, could not be well defined and completely solved despite its importance in practice to apply the monolayer π−a data to elucidate bilayer vesicle properties. In the present analysis, we thus first define the bilayer leaflet pressure as the intrinsic pressure of the lipid layer−water substrate system. This intrinsic surface pressure should be the same function of the lipid density and the temperature for both the monolayer at an interface and a leaflet of bilayer vesicles. Therefore, the difference between a leaflet of a bilayer and the monolayer at an interface is merely that the latter, but not the former, exhibits a microscopic interfacial tension between the air or the oil and the lipid layer. We show that the value of this intrinsic pressure agrees with that of the traditional monolayer pressure if the macroscopic and microscopic hydrophobic effect assumes the same magnitude. We conclude that the equilibrium pressure between the monolayer and bilayer vesicles is equal in magnitude to the microscopic interfacial tension between the water and the monolayer, which, in first approximation, is equal to the macroscopic oil−water interfacial tension, i.e., ca. 49 mN/m. This conclusion agrees with that briefly derived by pioneers (Gruen and Wolfe, 1982; Nagle, 1976, 1986; Jähnig, 1984). We further develop from mechanics and thermodynamics of membranes a procedure to obtain either analytically from a theoretical or empirical equation of state, or graphically from the π−a curve of the monolayer at an interface, mechanochemical properties of this monolayer and bilayer vesicles. The method is exemplified for the monolayer and bilayer vesicles of dilauroyl phosphatidylethanolamine (DLPE).

Fructans interact strongly with model membranes

Bacterial fructans with a high degree of polymerisation cause a very large increase in surface pressure of lipid monolayers at the air–water interface with a broad range of lipids, including phosphatidylethanolamine and several types of phosphatidylcholines. The surface active effect of fructans contrasts strongly with the maximal effects observed for trehalose, sucrose and glucose under comparable conditions (20 and 0.6 mN/m for fructans and the other sugars, respectively). The results demonstrate a profound and specific membrane interaction of the fructans which is probably very different from the effect of the smaller carbohydrates. The fructan concentrations used in this study are within the physiological range observed in fructan-accumulating plants. The suggested water-stress protective effect of fructans may be induced by membrane–fructan interaction which prevent lipid condensation and phase transitions to take place.

Surface Behavior of Spread Sodium Eicosanyl Sulfate Monolayers.: 1. π/AIsotherms Determined on a Langmuir Film Balance and on Drop Surfaces and Brewster Angle Measurements

Spread sodium eicosanyl sulfate (SES) monolayers were characterized by π/Aisotherms and Brewster angle microscopy (BAM). During compression three different states can be distinguished by BAM: a dark expanded phase, a condensed phase, and bright dots. The bright dots are interpreted as traces of eicosanol. π/Aisotherms were determined with a conventional Langmuir film balance and with the pendant drop technique. Depending on the pH SES desorbs from the monolayer. At a constant surface pressure the desorption was characterized by changes in the monolayer area. At pH 3 a reduction of 20% of the monolayer area was observed on a Langmuir film balance after 45 min. In contrast the surface pressure of monolayers spread on surfaces of pendant drops reduces by only about 2 mN/m, even at high surface pressures. This is caused by the small volume of bulk phase which is faster in equilibrium with the monolayer. For the determination of the isotherms on pendant drops, two different procedures were used: stepwise fast compression of the drop surface followed by 5 min of relaxation of each monolayer, and slow compression of the monolayer by evaporation of the drop. The π/Aisotherms at pH 3 determined with the Langmuir film balance and stepwise compression of the drop surface do not show transition points. In contrast however those obtained from slow drop evaporation shows transition points and allows the realization of higher surface pressures. A steep increase in the surface pressure before the monolayer collapse suggests the formation of a solid monolayer phase. At pH 5.5 fast desorption of SES does not allow a monolayer collapse. Slow compression of the monolayer by drop evaporation seems to be an suitable method to determine equilibrium π/Aisotherms.

Surface Active Behavior of Hopanoid Lipids: Bacteriohopanetetrol and Phenylacetate Monoester Bacteriohopanetetrol

The hopanoid lipids, bacteriohopanetetrol (tetrol) and phenyl acetate monoester bacteriohopanetetrol (pa-tetrol), were extracted from vesicles of Frankia (actinomycetes) and purified using solvent extraction and high performance liquid chromatography. Surface pressure versus specific area equilibrium isotherms of monolayers of tetrol, pa-tetrol, and mixtures of the two hopanoids were obtained. The collapse pressure for tetrol is about 36 mN m-1, while for pa-tetrol it is about 18 mN m-1. Pa-tetrol has a much more expanded isotherm than tetrol due to the presence of the phenyl acetate group at the interface. Mixtures of tetrol and pa-tetrol are miscible and show an area condensation effect that is most pronounced around 30−70 wt % pa-tetrol as determined from the isotherm data. Langmuir−Blodgett films of tetrol and pa-tetrol were analyzed by Fourier transform infrared spectroscopy. Transmission and grazing-angle absorption−reflection spectra show that tetrol is oriented nearly perpendicular to the interface.

Conformation and lipid binding properties of four peptides derived from the membrane-binding domain of CTP:phosphocholine cytidylyltransferase.

We are probing the mechanism of the lipid selective membrane interactions of CTP:phosphocholine cytidylyltransferase (CT). We have proposed that the membrane binding domain of CT (domain M) consists of a continuous amphipathic alpha-helix between residues approximately 240-295 [Dunne, S. J., et al. (1996) Biochemistry 35, 11975-11984]. This study examined the secondary structure and membrane binding properties of synthetic peptides derived from domain M: a 62mer peptide encompassing the entire domain (Pep62), a 33mer corresponding to the N-terminal portion (PepNH1), and two 33mers corresponding to the three C-terminal 11mer repeats, one with the wild-type sequence (Pep33Ser), and one with the three serines in the nonpolar face substituted with alanine (Pep33Ala). Peptide secondary structure was analyzed by circular dichroism, and lipid interactions were analyzed by a direct vesicle binding assay, by effects of lipid vesicles on peptide tryptophan fluorescence, and by monolayer surface pressure changes. All peptides bound to vesicles as alpha-helices with selectivity for anionic lipids. Binding involved intercalation of the peptide tryptophan into the hydrophobic membrane core. PepNH1, the peptide with the highest positive charge density, showed strong selectivity for anionic lipids. PepNH1 and Pep33Ser did not bind to PC vesicles; however, the more hydrophobic peptides, Pep33Ala and Pep62, did bind to PC vesicles, with apparent partition coefficients for PC that were only approximately 1 order of magnitude lower than those for PC/PG (1/1). Our results suggest that the polar serines interrupting the nonpolar face of the amphipathic helix serve to lower the lipid affinity and thereby enhance selectivity for anionic lipids. Although diacylglycerol is an activator of the enzyme, none of the peptides responded differentially to PC/diacylglycerol vesicles versus pure PC vesicles, suggesting that domain M alone is not sufficient for the enzyme's response to diacylglycerol. Increases in surface pressure at an air-water interface indicated that the domain M peptides had strong surface-seeking tendencies. This supports a binding orientation for domain M parallel to the membrane surface. Binding of CT peptides to spread lipid monolayers caused surface pressure reductions, suggesting condensation of lipids in the formation of lipid-peptide complexes. At low monolayer surface pressures, Pep62 interacted equally with anionic and zwitterionic phospholipids. This suggests that one determinant of the selectivity for anionic lipids is the lipid packing density (area per molecule).

A control system for Langmuir-Blodgett film deposition set-up based on microstepping

A control system for Langmuir-Blodgett (LB) film set-up has been developed based on the principle of microstepping of the stepper motors to drive the barriers and the substrate with precision and smooth motion. The stepper motors and the Wilhelmy plate used to measure the monolayer surface pressure are interfaced to a microcomputer for automatic operation. This home-made LB film set-up has been successfully used to fabricate LB films of cadmium arachidate of high crystalline quality.

Capillary Wave Studies of Multiblock Polypeptide Copolymers at the Air/Water Interface

To observe the changes in the mechanical properties of copolymer interfacial films when controlled changes are imposed on molecular architecture, we have studied capillary waves in spread monolayers of triblock and pentablock copolymers of γ-benzyl-l-glutamate and d,l-glutamic acid having similar molecular weights and hence different block sizes. Using the mechanically generated capillary wave method and the capillary wave dispersion relation, we have extracted the values of viscoelastic parameters including surface pressure, transverse viscosity, longitudinal elasticity, and longitudinal viscosity of copolymer monolayers at the air−water interface, as a function of the interfacial number density of molecules of the triblocks and the pentablocks. The surface pressure, π, shows a clear saturation as a function of the number density for both copolymers. The saturation surface pressure of the pentablocks is not dramatically larger than that of the triblocks, in contradiction to expectations from a steric blocking argument. The longitudinal elasticity increases at a larger rate as the function of the interfacial number density for the pentablocks as compared to the triblocks. We observe negligibly small values of the transverse and the longitudinal viscosity at all number densities of the two copolymers.