Interaction of rabbit C-reactive protein with phospholipid monolayers at air/water interface

C-reactive protein (CRP) is a major acute phase reactant in most mammalian species. The structure of CRP has been previously established by crystallography, and the significance of its interaction with lipid membranes is accepted in the literature. However, the nature of the interaction between CRP and phospholipids is not yet well understood. In this paper we use monolayer technique to study the characteristics of the interaction of rabbit C-reactive protein (rCRP) with the phospholipid membranes. The results show that rCRP is surface active and can spontaneously insert into the lipid monolayers. The critical pressure for rCRP inserting into the phospholipid monolayers is about 34.5 mN/m, which is not sensitive to the types of the lipid headgroups and the presence of calcium ions in the subphase. The findings of this paper may provide a clue to the further understanding of the mechanism of the interactions between rCRP and the biological membranes.     

Incorporation of blood clotting factor X into phospholipid model membranes: fluorescence microscopy imaging and subphase effects surrounding the lipid phase transition region

Factor X is a blood clotting protein that associates at membrane surfaces to become activated during the coagulation cascade. A molecular level understanding of the protein-membrane phospholipid interactions has not been reached, although it is thought that the protein binds to phospholipids in the presence of calcium through a bridge with the Gla (gamma-carboxyglutamic acid) domain on the protein. In this work, phospholipid Langmuir monolayers have been utilized as model membranes to study factor X association with phospholipid membrane components. Surface pressure measurements indicate that subphase addition of sodium, magnesium, and calcium ions enhances protein penetration of the lipid monolayer, with the largest association found with calcium ions in the subphase. Fluorescence microscopy images collected after protein penetration of lipid monolayers indicate monolayer condensation in the presence of sodium and magnesium ions. Aggregation of lipid domains is induced when calcium is in the subphase, indicating binding-induced flocculation of surface lipid aggregates. Calcium binding to factor X likely causes a conformational change which allows protein-membrane interaction via hydrophobic association with lipid molecules.     

Mixed micelle formation between amino acid-based surfactants and phospholipids

The mixed micelle formation in aqueous solutions between an anionic gemini surfactant derived from the amino acid cystine (C(8)Cys)(2), and the phospholipids 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC, a micelle-forming phospholipid) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, a vesicle-forming phospholipid) has been studied by conductivity and the results compared with the ones obtained for the mixed systems with the single-chain surfactant derived from cysteine, C(8)Cys. Phospholipid-surfactant interactions were found to be synergistic in nature and dependent on the type of phospholipid and on surfactant hydrophobicity. Regular solution theory was used to analyse the gemini surfactant-DHPC binary mixtures and the interaction parameter, β(12), has been evaluated, as well as mixed micelle composition. The results have been interpreted in terms of the interplay between reduction of the electrostatic repulsions among the ionic head groups of the surfactants and steric hindrances arising from incorporation of the zwitterionic phospholipids in the mixed micelles.     

Effect of the molecular structure of more common amphiphilic molecules on their interactions with dioleoyl-phosphatidylcholine monolayers

A large number of surface-active or amphiphilic compounds interact with biological membranes and their various models. The surface-active properties of these compounds have been cited as a fundamental factor which determines the tendency of these molecules to bind to, to cross and to perturb the lipid structure of biomembranes and biomembranes models. As well as surface activity, the extent of interaction of a compound with a membrane has been correlated with its lipophilicity. This study shows that the surface activity and lipophilicity of a compound are not the only factors which determine its interaction with a lipid membrane structure. Experiments have been carried out looking at the effect of compounds of different molecular structure on their type of association with self-assembled phospholipid monolayers on mercury electrodes. The paper concludes by demonstrating the properties of different compounds which facilitate their interaction with the supported lipid monolayer.     

The antimalarial agent halofantrine perturbs phosphatidylcholine and phosphatidylethanolamine bilayers: a differential scanning calorimetric study.

The interaction of halofantrine with phosphatidylcholine and phosphatidylethanolamine bilayers has been investigated by differential scanning calorimetry. Halofantrine caused a broadening of the gel to liquid crystalline phase transition endotherm of the phosphatidylcholines. A decrease in the transition temperature Tm and enthalpy (delta H) of transition was also observed. This varied with the chain length of the phospholipid and was more pronounced with short chain members. Halofantrine-induced changes to the thermotropic characteristics of dipalmitoylphosphatidylcholine (DPPC)/cholesterol bilayers suggested that the penetration of halofantrine into the bilayer was diminished in the presence of cholesterol. A more complex calorimetric profile was observed in the interaction of halofantrine with phosphatidylethanolamines and the results suggested that halofantrine did not disrupt the cooperativity of the phosphatidylethanolamine bilayers to the same extent as that observed with the phosphatidylcholines. Halofantrine caused significant perturbation of phospholipids and this property might have an important bearing on its pharmacodynamic effects.     

Transverse location of new fluorene based depth dependent fluorescent probes in membranes--quenching studies with 9,10-dibromostearic acid

Fluorescence quenching technique has been used to determine the transverse location of the fluorescent fluorenyl fatty acids in single bilayer vesicles prepared from phosphatidylcholine. The fluorenyl fatty acids used here are 2-fluorenyl acetic, butyric, hexanoic and octanoic acid. In addition a new type of fluorescent probe, 7-n-butyl-fluorene-2-butyric acid, wherein a hydrophobic tail is attached to 2-fluorenyl-butyric acid has also been used to study its effect on alignment of these probes in the membrane. The association properties of the quencher 9,10-dibromostearic acid have been analysed. It is observed that the quencher association involves partitioning into the vesicles and does not involve any binding to the vesicles. The absolute partition coefficient of the 9,10-dibromostearic acid which partitions between the aqueous and the lipid phases of the phospholipid dispersion has been evaluated. Using this information the corrected Stern-Volmer plots were drawn and the bimolecular quenching constant evaluated.     

Trichosanthin's interfacial interactions with phospholipids: a monolayer study

Lipid monolayer at the air/water interface, as half a membrane, was used here to investigate the interaction between trichosanthin (TCS), a ribosome inactivating protein, and phospholipid membrane. First, the protein adsorption experiments showed that the negatively charged DPPG caused obvious enrichment of TCS beneath the monolayer, indicating electrostatic attraction between TCS and the negatively charged phospholipid. Second, when TCS was incorporated into the phospholipid monolayer, it could not be completely squeezed out until the monolayer collapsed. The results were demonstrated to be irrelative with the phospholipid headgroup, suggesting a strong hydrophobic force between TCS and phospholipid hydrocarbon chain was involved in the interaction. Third, the protein/membrane interaction was further studied with fluorescence microscope. The results showed that TCS could penetrate into both the condensed and the fluid phase of the DPPG monolayer under low pH condition and eventually resulted in a homogeneous phospholipid phase. The breakage of ordered packing of phospholipid by TCS may be responsible for this homogenizing effect.     

Cratylia mollis lectin at the air–aqueous solution interface: adsorption and lectin–lipid interactions

The interfacial behaviour of Cratylia mollis (Cra) at the air/water interface and its penetrant ability into spread phospholipid monolayers (Lipoid E80 and Epicuron 200) has been monitored by surface tension measurements. The first-order rate constants defining adsorption and rearrangement obtained from surface tension kinetics data reveal that Cra is a rather stable protein which exhibits characteristic protein adsorption patterns in which the breaking points separating diffusion–penetration and rearrangement profiles could have been easily distinguished. The penetration of Cra into Lipoid E80 and Epicuron 200 phospholipid monolayers has been inferred in terms of penetration pressure increments (ΔΠ) versus time relationships. The data clearly showed that penetrant ability of the lectin was, to a large extent, dependent on monolayer compressibilities. Thus, for Lipoid E80, which contained a rather high percentage of phosphatidylethanolamine (DPPE) in the mixture with phosphatidylcholine (DPPC), penetration of Cra at the high monolayer compression (20 mN m⁻¹) was lower than that observed for Epicuron 200, which did not contain DPPE. Indeed, in the middle of the Π-A isotherm, DPPE was markedly less compressible than DPPC. However, at the low monolayer surface coverage (3 mN m⁻¹), the rates of Cra penetration into both Lipoid E80 and Epicuron 200, although much higher for the latter at the beginning of adsorption, yielded similar limiting values of ΔΠ. This has been attributed to the occurrence of a hydrophobic interaction between the lectin and hydrophobic phospholipid chains that have the same length for both Lipoid E80 and Epicuron 200.     

The role of surface free energy on the formation of hybrid bilayer membranes

The interaction of small phospholipid vesicles with well-characterized surfaces has been studied to assess the effect of the surface free energy of the underlying monolayer on the formation of phospholipid/alkanethiol hybrid bilayer membranes (HBMs). The surface free energy was changed in a systematic manner using single-component alkanethiol monolayers and monolayers of binary mixtures of thiols. The binary surfaces were prepared on gold by self-assembly from binary solutions of the thiols HS-(CH(2))(n)()-X (n = 11, X = CH(3) or OH) in THF. Surface plasmon resonance (SPR), electrical capacitance, and atomic force microscopy (AFM) measurements were used to characterize the interaction of palmitoyl,oleoyl-phosphatidylcholine (POPC) vesicles with the surfaces. For all surfaces examined, it appears that the polar part of surface energy influences the nature of the POPC assembly that associates with the surface. Comparison of optical, capacitance, and AFM data suggests that vesicles can remain intact or partially intact even at surfaces with a contact angle with water of close to 100 degrees. In addition, comparison of the alkanethiols of different chain lengths and the fluorinated compound HS-(CH(2))(2)-(CF(2))(8)-CF(3) that characterize with a low value of the polar part of the surface energy suggests that the quality of the underlying monolayer in terms of number of defects has a significant influence on the packing density of the resulting HBM layer.     

Salt effects on the interaction of an amphiphilic model molecule with immobilized phosphatidylcholine monolayers

The retention of hydrocortisone (used as an amphiphilic model solute) on an immobilized artificial membrane (IAM) column was investigated in relation to the mobile phase concentration of three sodium salts (representing different rankings in the Hofmeister series, i.e. perchlorate, chloride and sulfate) in order to provide insight into the nature of the solute interactions with phospholipid monolayers. The influence of the salt series on solute retention was found to follow the Hofmeister series, emphasizing the role of hydrophobic effect in the solute retention mechanism on phospholipid monolayers. Retention models based on the extended Wyman relations (preferential interaction theory) were developed to analyze more quantitatively the salt effects on the hydrocortisone retention factor. This analysis as well as additional thermodynamic study suggested that the hydrocortisone binding to IAM involved both an insertion into the hydrophobic inside governed by hydrophobic effects and contacts with the interfacial region implying interactions such as van der Waals interactions/hydrogen bonds between the solute hydroxyl groups and the polar headgroups of phospholipidmonolayers.     

Membrane Lipid-Polypeptide Molecular Associations in Non Aqueous Solvent. Effect of Phosphatidylcholine Concentration and Temperature and their Influence on the Gramicidin a Dimer-Monomer Conformational Equilibrium

Abstract

The use of an Ultrastyragel 500 Å column for the study of interactions between phosphatidylcholine and gramicidin A in tetra-hydrofuran is described. Analysis of vacant peak has allowed to establish the influence that eluent lipid composition, concentration of injected gramicidin and temperature have on the interaction. At 20[ddot] C, for the assayed phospholipid concentration range (0.04 to 0.14 %, w/v), the lipid/polypeptide molar binding ratio, BR, varies from 1.3 to 6.9. An increase in temperature from 20 to 40[ddot] C causes a decrease in BR of about 20 %. On the other hand, the interaction releases some of the water bound to the lipid polar head, suggesting that the binding involves at least this phospholipid moiety. It has been also found that the lipid alters the gramicidin dimer-monomer conformational equilibrium resulting in a higher extent of monomeriz-ation, more pronounced as temperature increases. Additional spectro-fluorometric measurements are consistent with the experimental evidence deduced from HPLC analysis.     

Interaction of merocyanine 540 with charged membranes.

In this work, phospholipid liposomes were used to investigate the influence of lipid negative charge on the interaction of merocyanine 540 (MC540) with model membranes. Liposomes were prepared from a mixture of neutral dimyristoyl lecithin (DMPC) and negatively charged dimyristoyl phosphatidic acid (DMPA). A strong dependence between the presence of charges on the membrane and dye association was found. The affinity of the dye to liposomes was decreased with an increasing content of DMPA in liposomes. Changes in absorption spectra of MC540 suggest that the decrease in affinity of MC540 to charged membranes is accompanied by a hypsochromic solvatochromic shift and changes in monomer/dimer equilibrium of MC540 incorporated in the membrane.     

DIHEMATOPORPHYRIN ETHER-PHOSPHOLIPID INTERACTIONS. THE ROLES OF SURFACE CHARGE and LATERAL PHASE SEPARATIONS

Abstract Fluorescence spectroscopy was utilized to investigate the equilibrium interaction of dihem-atoporphyrin ether(s) (DHE) with binary and ternary phospholipid mixtures of defined composition in order to define the roles of net negative surface charge and lateral phase separations in DHE-membrane partitioning. Binary phospholipid mixtures employed were composed of dimyristoyl-phosphatidylcholine (DMPC) mixed with increasing weight percentages of dimyristoylphosphatidylgly-cerol (DMPG) providing controlled variation of net membrane surface charge. Two types of ternary phospholipid mixtures were utilized. Ternary acid mixtures contained various percentages of palmitoyl-lysophosphatidyl choline (LPC) + palmitic acid (PA) dispersed in DMPC. Ternary alcohol mixtures contained various percentages of LPC + hexadecanol (OL) dispersed in DMPC. The ternary phospholipid mixtures are known to be phase separated. At total DHE concentrations of 0.33 μA/ and using 100% DMPC, the DHE partition coefficient (P) is 250 000. This partition coefficient is to some extent dependent on the DHE concentration. The observed partition coefficients show little dependence on surface charge in DMPC-DMPG mixtures. However, P decreases markedly with increasing phase separation in the ternary lipid mixtures. The fluorescence of membrane-bound DHE is dependent on the composition of the ternary mixtures in a manner suggesting micropartitioning of DHE into the phospholipid bulk phase as well as into the disordered regions between laterally phase separated phospholipid domains.     

Cyclic voltammetric studies of the antipsychotic chlorpromazine using an alkylthiol/phospholipid-modified gold electrode

Self-assembled monolayers of alkanethiols on gold have been reported to be highly stable for voltammetry experiments in aqueous electrolyte. In this work a gold electrode has been modified by first depositing one layer of an alkylthiol (S-C₁₈) and then coating by phospholipid multilayers. Voltammetric oxidation of the antipsychotic chlorpromazine at this two-step modified electrode was followed by means of cyclic voltammetry measurements. The results give important information concerning the behaviour of the pharmacological agent at the lipid-water interface. Measurements made using the pre-concentration method allow good sensitivity improvement after 5 min accumulation time. The ability of chlorpromazine to penetrate inside the phospholipid multilayers has also been investigated under different conditions such as the nature of the phospholipid and the pH of the medium. The accumulation process seems to be closely related to the charge carried by the phospholipid and by the molecule, while the incorporation process seems to be independent of the charge carried by the phospholipid and dependent of the degree of fluidity of their hydrocarbon chains. We found through this work that the acid-base equilibrium of chlorpromazine together with its amphiphilic properties (as compared with the results of similar studies on phenothiazine) could be responsible for governing the principal aspects of the drug's behaviour toward biological membranes. Received: 6 January 1997 / Accepted: 27 February 1997     

Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin

Daptomycin is a cyclic anionic lipopeptide antibiotic recently approved for the treatment of complicated skin infections (Cubicin). Its function is dependent on calcium (as Ca2+). Circular dichroism spectroscopy indicated that daptomycin experienced two structural transitions: a transition upon interaction of daptomycin with Ca2+, and a further transition upon interaction with Ca2+ and the bacterial acidic phospholipid, phosphatidyl glycerol. The Ca2+-dependent insertion of daptomycin into model membranes promoted mild and more pronounced perturbations as assessed by the increase of lipid flip-flop and membrane leakage, respectively. The NMR structure of daptomycin indicated that Ca2+ induced a conformational change in daptomycin that increased its amphipathicity. These results are consistent with the hypothesis that the association of Ca2+ with daptomycin permits it to interact with bacterial membranes with effects that are similar to those of the cationic antimicrobial peptides.     

Adsorption-penetration studies of glucose oxidase into phospholipid monolayers at the 1,2-dichloroethane/water interface.

The interaction between glucose oxidase (GOx) and phospholipid monolayers is studied at the 1,2-dichloroethane/water interface by electrochemical impedance spectroscopy. Electrochemical experiments show that the presence of GOx induces changes in the capacitance curves at both negative and positive potentials, which are successfully explained by a theoretical model based on the solution of the Poisson-Boltzmann equation. These changes are ascribed to a reduced partition coefficient of GOx and an increase of the permittivity of the lipid hydrocarbon domain. Our results show that the presence of lipid molecules enhances the adsorption of GOx molecules at the liquid/liquid interface. At low lipid concentrations, the adsorption of GOx is probably the first step preceding its penetration into the lipid monolayer. The experimental results indicate that GOx penetrates better and forms more stable monolayers for lipids with longer hydrophobic tails. At high GOx concentrations, the formation of multilayers is observed. The phenomenon described here is strongly dependent on 1) the GOx and lipid concentrations, 2) the nature of the lipid, and 3) the potential drop across the interface.     

Interaction of the phospholipid head group with representative quartz and aluminosilicate structures: an ab initio study

Silica dust particles in the form of quartz (but not kaolin) have been hypothesized to promote pulmonary diseases such as silicosis. The hypothesis is that quartz and kaolin have a comparable membranolytic potential on a specific surface area basis, and they have a comparable cytotoxic potential for lavaged pulmonary macrophages. Suppression of the cytotoxic activity occurs when these dust particles are treated with dipalmitoylphosphatidylcholine (DPPC), a common phospholipid component of the lung pulmonary surfactant. However, the enzyme phospholipase A2 is known to digest the phospholipid component more readily in the presence of quartz than kaolin. Since surface silanol (Si-OH) and aluminol (Al-OH) groups may interact differently with the phospholipid, an understanding of the selective removal of phospholipid by PLA2 may explain in vivo differences in cytotoxicity between quartz and kaolin. To develop some insight into this phenomenon, the interaction between a phospholipid and silica particles was examined by performing ab initio DFT calculations on clusters constructed with small (one or two silica tetrahedral units) representative parts of the silicate surface and the phospholipid head group. The clusters consisted of a phospholipid head group or functional groups from the head group complexed with Si(OSiH 3) 3OH, Al(OSiH 3) 3OH (-) or Al(OSiH 3) 3OH 2. Fully optimized geometries of the complexes were used to determine binding energies, -OH vibrational frequency shifts, and NMR chemical shieldings. Results indicate that interaction of the protonated aluminol group (Al-OH 2 (+)) with the phosphate portion of the head group is strongest, while interaction of the -OH 2 (+) group with the trimethyl-choline moiety of the head group is weakest. The presence of the choline moiety increased the magnitude of the -OH vibrational frequency shifts, and the shifts were significantly larger in complexes with protonated aluminol groups relative to silanol complexes. Analysis of ChelpG atomic charges shows that a net transfer of charge occurs from the silica unit to the head group within the complexes.     

Interaction between the conjugated polyelectrolyte poly{1,4-phenylene[9,9-bis(4-phenoxybutylsulfonate)]fluorene-2,7-diyl} copolymer and the lecithin mimic 1-O-(l-Arginyl)-2,3-O-dilauroyl-sn-glycerol in aqueous solution

In this paper, the interaction between the water-soluble conjugated polyelectrolyte poly{1,4-phenylene[9,9-bis(4-phenoxybutylsulfonate)]fluorene-2,7-diyl} copolymer and the amino acid glyceride conjugate 1-O-(L-arginyl)-2,3-O-dilauroyl-sn-glycerol dichlorohydrate (a mimic for the phospholipid lecithin) has been studied in aqueous solution by electronic spectroscopy (absorption and fluorescence) and small-angle neutron scattering (SANS). A significant increase in the polymer fluorescence and blue shift in its emission are observed on association with the surfactant. This is suggested to be due to breakup of polymer aggregates. In addition, the spectroscopic and photophysical data suggest this is followed by the vesicle to ribbon transition characteristic of this surfactant, leading to incorporation of single chains of the polymer within mixed polymer-surfactant aggregates. Support for this comes from preliminary SANS measurements, from which evidence for polymer dissolution and formation of two-dimensional structures has been obtained.     

Effect of gramicidin on phospholipid-modified monolayers and on ion transfer at a liquid-liquid interface.

The interaction of hybrid lipid/gramicidin A (gA) monolayers with dextran sulfate (DS) and the effect of this interaction on ion transfer at a liquid-liquid interface is reported. The interfacial and physicochemical properties are studied with Langmuir-Blodgett (LB) and electrochemical techniques. The results obtained from compression isotherms demonstrate that the interactions between the different species in the hybrid monolayer vary according to the chemical nature of the lipid (hydrocarbon region and charge of the head group). Interfacial capacitance measured with AC voltammetry indicates that the DS chains form a rather flat and compact layer when adsorbed to either zwitterionic or negatively charged phospholipid monolayers, and that calcium, even at low concentrations, interacts with the monolayers. These results are successfully described by a model based on the solution of the Poisson-Boltzmann equation in the interfacial region. Ion transfer and interactions with the lipid/gA/DS-modified monolayers were also studied with electrochemical techniques. Admittance data show that although the studied ions are not using gA channels for the transfer through the lipid membranes, the incorporation of gA in the lipid domain and the adsorption of DS at the interface have a significant effect on ion transfer across the monolayers. This effect can be explained as a consequence of the modified surface charge and of the compactness of the lipid domain due to its interaction with gA and to calcium and DS adsorption at the interface. The ion-transfer rate, therefore, depends on the composition of the monolayer and the chemical nature of the ion.     

Surface excitations involving headgroup conformations and molecular protrusions. a monte carlo study of surface forces.

The force between two parallel zwitterionic surfaces has been calculated using Monte Carlo computer simulations. The zwitterions are modelled as two oppositely charged hard spheres joined by a string of length L with parameters chosen to mimic a phospholipid system. All centers interact by a homogeneous Coulomb interaction and by a hard sphere exclusion. The anchoring of the negative centers to the surfaces has been treated within two different models. They were either anchored by a parabolic potential or by a protrusion potential, i.e., a potential proportional to the distance between the center and the surface. The latter model gives a more realistic picture of the interaction between amphiphilic surfaces in aqueous solution but here only repulsive forces could be calculated. The first model also allowed the calculation of attractive forces.For distances D between the surfaces, as defined by the location of the negative centers, that are larger than 2L there is an attractive force of the classical van der Waals type. When, on the other hand, D < 2L a strong repulsive force appears, which in the limit D ⪡ 2L is analogous to a double layer force.Recently it was suggested (Israelachvili and Wennerström, Langmuir, 6 (1990) 873 ) that the repulsive so called hydration force observed for biological lipid systems has its origin in confinements of surface excitations induced by a second surface. Here we demonstrate how this mechanism works in a particular microscopic model of the surface and we show that it gives an important contribution to the total force. Although still simplistic several qualitative features of the force in the phospholipid systems are reproduced in the calculations. For example, a reduction of the size of the charged centers leads to a decrease in the repulsive force. This mimics the observed difference between phosphatidyl choline and phosphatidyl ethanolamine.