Air-buffer Interface Research Articles

A thermodynamic study of GPI-anchored and soluble form of alkaline phosphatase films at the air–water interface

Glycosylphosphatidyl inositol (GPI) anchored proteins are localized and clustered on the outer layer of the plasma membranes forming microdomains. Among them, mammalian alkaline phosphatases (AP-GPI) are widely distributed enzymes. They can also exist as soluble proteins without anchor (APs). Using the Langmuir film technique, we study the thermodynamic properties of monolayers for both protein forms at the air–buffer interface. The enzymatic activity is maintained at the interface but the adsorption of the two forms of AP is very different. AP-GPI presents a higher surface activity and a larger molecular area than the soluble form. The molecular area deduced for high surface pressures suggests a different organization of the monolayers for these two forms. APs molecules seem to adsorb as a multilayer at the interface while AP-GPI appear to be orientated with the major axis parallel to the interface. This orientation allows the accessibility of AP-GPI enzymatic sites that are turned in direction of the subphase as in vivo where the active sites must be turned outside of the membrane.

The Surface Activity of Purified Ocular Mucin at the Air-Liquid Interface and Interactions With Meibomian Lipids

Ocular mucins are thought to contribute to the stability of the tear film by reducing surface tension. The purpose of this study was to compare the effect of different mucins and hyaluronic acid (HA) alone and mixed with meibomian lipids on the surface pressure at an air-liquid interface. A Langmuir trough and Wilhelmy balance were used to measure and compare the surface activity of bovine submaxillary gland mucin (BSM), purified BSM, purified bovine ocular mucin and HA, and mixtures of these with meibomian lipids, phosphatidylcholine, and phosphatidylglycerol. Their appearance at the surface of an air-buffer interface was examined using epifluorescence microscopy. Purified ocular mucin had no surface activity even at concentrations that were 100 times more than normally occur in tears. By contrast, commercial BSM caused changes to surface pressure that were concentration dependent. The surface pressure-area profiles showed surface activity with maximum surface pressures of 12.3-22.5 mN/m depending on the concentration. Purified BSM showed no surface activity at low concentrations, whereas higher concentrations reached a maximum surface pressure of 25 mN/m. HA showed no surface activity, at low or high concentrations. Epifluorescence showed that the mucins were located at the air-buffer interface and changed the appearance of lipid films. Purified bovine ocular mucin and HA have no surface activity. However, despite having no surface activity in their own right, ocular mucins are likely to be present at the surface of the tear film, where they cause an increase in surface pressure by causing a compression of the lipids (a reorganization of the lipids) and alter the viscoelastic properties at the surface.

Interaction of Purified Tear Lipocalin with Lipid Membranes

The principal lipid-interacting protein in human tears has been reported to be tear lipocalin (Tlc). Tlc has been suggested to scavenge harmful lipophilic substances from the corneal epithelium and to maintain the integrity of the anterior tear film lipid layer by binding and releasing lipid(s) that are accommodated within the protein. Although lipids can be extracted from Tlc, it is still unclear whether Tlc can actually bind to lipid membranes and accept membrane lipids and whether it possesses lipid transfer activity. The purpose of this study was to explore the interaction of Tlc with neutral, anionic, and cationic lipid membranes and to assess the potential of Tlc to facilitate the transfer of either polar or neutral lipids in a lipid transfer assay. The binding of Tlc to lipid membranes was assessed by a monolayer technique and fluorescence spectroscopy. The polar lipid transfer activity of Tlc was assessed with a radiometric assay based on the transfer of (14)C-phosphatidylcholine (PC) from PC-liposomes to HDL(3). The neutral lipid transfer activity of Tlc was assayed by measuring the transfer of radioactive cholesteryl ester from LDL to HDL(3). Purified Tlc showed significant surface activity as evidenced by an increase in surface pressure at the air-buffer interface. Likewise, it interacted actively with neutral, anionic, and cationic lipid monolayers, as evidenced by an equal increase in surface pressure despite the surface charge. Enhanced quenching of the single tryptophan residue of Tlc by pyrene and I(-) anion suggested that different protein domains are involved in the interaction of Tlc with oppositely charged lipid membranes. Finally, radiometric assays revealed that Tlc does not possess any neutral or polar lipid transfer activity between lipid vesicles or/and lipoproteins. Tlc interacted with lipid membranes composed of neutral, cationic, or anionic membranes, which supports a role for Tlc in the maintenance of the tear film interfaces. Tlc did not show any neutral or polar lipid transfer activity whatsoever. The findings suggest that the notion of the role of Tlc as the major lipid-transferring protein in human tears should be revised.

Favorable and unfavorable lateral interactions of ceramide, neutral glycosphingolipids and gangliosides in mixed monolayers

Interactions among four natural neutral sphingolipids (ceramide, glucosyl-ceramide, lactosyl-ceramide and asialo-GM1) and six gangliosides (GM3, GM2, GM1, GD3, GD1a and GT1b) were studied in binary Langmuir monolayers at the air–buffer interface in terms of their molecular packing, compressibility, dipole potential and mixing behavior. The changes of surface organization can be grouped into three sets: (a) binary films of neutral GSLs, and of the latter with ceramide, exhibit thermodynamically unfavorable mixing with mean molecular area expansions and dipole moment hyperpolarization; (b) mixed monolayers of ceramide, or of GlcCer, and gangliosides occur with thermodynamically favorable interactions leading to mean molecular area condensation and depolarisation; (c) binary mixtures of LacCer or Gg4Cer with gangliosides, and all ganglioside species among them, revealed molecular immiscibility characterized by additive mean molecular area and dipole potential, with composition-independent constant collapse pressure. These results disclose basic tendencies of GSLs to molecularly mix or demix, leading to their surface segregation, which may underlay vectorial separation of their specific biosynthetic pathways.

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.

Preferential orientation of an immunoglobulin in a glycolipid monolayer controlled by the disintegration kinetics of proteo-lipidic vesicles spread at an air–buffer interface

The insertion of immunoglobulin (IgG) in a glycolipid monolayer was achieved by using the ability of new proteo-glycolipid vesicles to disintegrate into a mixed IgG–glycolipid interfacial film after spreading at an air–buffer interface. The interfacial disintegration kinetics was shown to be directly dependent on the initial vesicle surface density and on the buffer ionic strength. The presence of the immunoglobulin in the glycolipid film was displayed by an increase of the lateral compressibility (Cs) during monolayer compression. Cs magnitude modifications, due to the antibody effect on the monolayer packing, decreases as the spread vesicle density increases. At interfacial saturation, the lateral compressibility profile becomes similar to that of a control monolayer without antibody. However, the careful analysis of the mixed monolayer after transfer by Langmuir–Blodgett technique (ATR-FTIR characterisation, enzyme immunoassociation) clearly demonstrated that the antibody was still present in such conditions and was not completely squeezed out from the interface as compressibility changes could have meant. At nonsaturating vesicle surface density, IgG molecules initially lying in the lipid matrix with the Y-shape plane parallel to the interface move to a standing-up position during the compression, leading to lateral compressibility modifications. For a saturating vesicle surface density, the glycolipid molecules force the IgG molecules to directly adopt a more vertical position in the interfacial film and, consequently, no lateral compressibility modification was recorded during the compression.

Structure and denaturation of adsorbed lysozyme at the air–water interface

Adsorption of lysozyme at the surface of a buffer solution at 25 °C, pH 7, and ionic strength 0.1 is studied under different denaturing conditions using on X-ray reflectometry technique. When the lysozyme is fully denatured with urea and dithiothreitol (DTT), its measured adsorption profile is very well explained by the scale law ( z −4/3) profile theoretically predicted for polymer adsorption. When no denaturing agent is present, a monolayer is also produced, but the adsorption profile cannot be explained by a monolayer of nondenatured lysozyme; furthermore, it is close to the one obtained for lysozyme partially denatured with urea. A PMIRRAS study of native lysozyme adsorbed at the air–buffer interface shows that the secondary structure of the protein is modified: most of the α-helices are replaced by β-sheets. In contrast, when the lysozyme is adsorbed below a monolayer of oleic acid at the air–buffer interface, that is, on a hydrophilic interface, the protein forms a monolayer whose thickness, 3.0 nm, is equal to one dimension of crystallized lysozyme. Under such conditions, the adsorbed protein is not denatured. Thus the hydrophobic nature of the air–water interface yields partial denaturation of the protein upon adsorption, but the disulfur bridges and β-sheets prevent total denaturation.

Penetration of a GPI-anchored protein into phospholipid monolayers spread at the air/water interface

Mammalian alkaline phosphatases (AP) belong to glycosylphosphatidyl inositol (GPI) anchored proteins family, which are localised and clustered on the outer layer of the plasma membranes forming microdomains. Using Langmuir film and polarisation modulation infrared reflection absorption spectroscopy (PMIRRAS) techniques, the penetration process of the protein into a phospholipid monolayer have been studied at the air–buffer interface. The penetration of AP-GPI in distearoylphosphatidylcholine monolayers (DSPC) induces a more important surface pressure increase than in dioleoylphosphatidylcholine (DOPC) monolayer. However, the exclusion surface pressure rather similar for both lipids, 20.5 and 22 mN m −1 for, respectively, DSPC and DOPC, indicates that the AP-GPI cannot, in similar conditions, insert by itself into bilayer membranes of either biological or mimetic origin. PMIRRAS suggests that the pure acyl chains perdeuterated DSPC (d 70-DSPC) interact with Mg 2+ present into the buffer. AP-GPI inserts progressively into the d 70-DSPC monolayer changing the environment of phospholipid molecules. Amide I band exhibits α helix and β-sheets components with a predominance of the α helix. The shapes, intensities and positions of the amide I and II bands suggest for the α helix an orientation perpendicular to the interface after a period of molecular reorganisation.

Predicting Blood-Brain Barrier Permeability of Drugs: Evaluation of Different In Vitro Assays

The ability of a drug to penetrate the blood-brain barrier (BBB) is essential for its use in the pharmaceutical treatment of CNS disorders. Five different in vitro methods to predict BBB permeability of drugs were compared and evaluated in the present study. All assays were performed with a consistent set of seven compounds and in the same physiological buffer to provide a basis for direct comparison of the results. Octanol-buffer and liposome-buffer partition coefficients were most conveniently obtained but failed to predict BBB permeability for certain drugs. The incorporation of drugs into lipid monolayers at the air-buffer interface was found to be a poor predictor of BBB permeability and was furthermore not considered suitable for screening due to the demanding experimental requirements. Permeability studies using Caco-2 cell monolayers provided a good correlation to an in vitro model of the BBB, which was based on primary cultured porcine brain capillary endothelial cells (PBCEC). However, differences in drug permeability between the intestine and brain derived cells were detected, limiting the advantages of the easy handling of the Caco-2 cell line compared to the more time-consuming primary culture of the BCEC.

The amphiphilic character of glycogenin.

This study describes for the first time the amphiphilicity of the protein moiety of proteoglycogen. Glycogenin but not proteoglycogen associates to phospholipid vesicles and forms by itself stable Gibbs and Langmuir monolayers at the air-buffer interface. The adsorption free energy (-6.7 kcal/mol) and the glycogenin collapse pressure (47 mN/m) are indicative of its high surface activity which can thermodynamically drive and retain the protein at the membrane interface to a maximum equilibrium adsorption surface pressure of 21 mN/m. The marked surface activity of glycogenin is further enhanced by its thermodynamically favorable penetration into zwitterionic and anionic phospholipids with a high cut-off surface pressure point above 30 mN/m. The strong association to phospholipid vesicles and the marked surface activity of glycogenin correspond to a high amphiphilic character which supports its spontaneous association to membrane interfaces, in which the de novo biosynthesis of glycogen was proposed to initiate.

Studies of a weak polyampholyte at the air–buffer interface: The effect of varying pH and ionic strength

We have carried out experiments to probe the static and dynamic interfacial properties of β-casein monolayers spread at the air–buffer interface, and analyzed these results in the context of models of weak polyampholytes. Measurements have been made systematically over a wide range of ionic strength and pH. In the semidilute regime of surface concentration a scaling exponent, which can be linked to the degree of chain swelling, is found. This shows that at pH close to the isoelectric point, the protein is compact. At pH away from the isoelectric pH the protein is extended. The transition between compact and extended states is continuous. As a function of increasing ionic strength, we observe swelling of the protein at the isoelectric pH but contraction of the protein at pH values away from it. These behaviors are typical of a those predicted theoretically for a weak polyampholyte. Dilational moduli measurements, made as a function of surface concentration exhibit maxima that are linked to the collapse of hydrophilic regions of the protein into the subphase. Based on this data we present a configuration map of the protein configuration in the monolayer. These findings are supported by strain (surface pressure) relaxation measurements and surface quasielastic light scattering measurements which suggest the existence of loops and tails in the subphase at higher surface concentrations.

C-Fos Is Surface Active and Interacts Differentially with Phospholipid Monolayers

The transcription factor c-Fos forms stable Gibbs and Langmuir monolayers at the air-buffer interface. Its marked surface activity is enhanced by penetration into phospholipid films above the protein's own maximum adsorption surface pressure to a lipid-free interface. The protein-phospholipid stabilizing interactions at the interface depend on the lipid polar head group and the increases of lateral surface pressure generated are comparable to those of membrane-active proteins. The surface activity of c-Fos is strong enough to thermodynamically drive and retain c-Fos at the membrane interface where it may exert direct or indirect effects.

Adsorption of bovine prothrombin to spread phospholipid monolayers

The interaction of bovine prothrombin with phospholipids was measured, using as the lipid source monolayers spread at the air-buffer interface. Fluorescence spectroscopy was implemented to determine the equilibrium concentration of free prothrombin in the aqueous subphase of the protein-monolayer suspensions, in a continuous assay system. The increase in surface pressure (pi) from the protein-monolayer adsorption was also measured and, with values of the adsorbed protein concentration (c[s]), was used to calculate dc(s)/d(pi). At a particular phosphatidylserine (PS) content of liquid-expanded (LE) phosphatidylcholine (PC)/PS monolayers, dc(s)/d(pi) was independent of the initial surface pressure (pi[i]), when this latter value exceeded 30 mN/m. However, dc(s)/d(pi) varied significantly with the relative PS content of the monolayer. Values of the equilibrium dissociation constants calculated from the concentration dependence of delta(pi) indicated that the affinity of prothrombin for LE monolayers was higher at higher PS contents and lower packing densities. The affinity of prothrombin for liquid-condensed (LC) PC/PS monolayers was found to be much weaker relative to LE monolayers of similar phospholipid composition. This approach, employing spread monolayers to study prothrombin-phospholipid binding, coupled with a simple and accurate method to determine the free protein concentration in protein-monolayer suspensions, offers significant advantages for the investigation of protein-membrane interaction. The equilibrium characteristics that describe the interaction of prothrombin with the different phospholipid monolayers under various conditions also provide support for previous results which indicated that hydrophobic interactions are involved in the adsorption of vitamin K-dependent coagulation and anticoagulation proteins to model membrane systems.

Kinetics of lysozyme adsorption at the air-buffer interface

The adsorption of lysozyme at the air-buffer interface has been monitored previously by a radiotracer method. A major experimental feature of thesestudies is that the amount of protein adsorbed is a significant fraction of the protein in the bulk at the beginning of the adsorption process. In the present study, several kinetic models of adsorption are derived which take into account the bulk protein concentration and a possible attraction of the protein in solution by the protein molecules forming the interfacial adsorption layer. Moreover, the data are analyzed assuming either an exponential regime (the rate of adsorption is controlled by the distance from equilibrium) or a diffusive regime (the rate of adsorption is controlled by the diffusion of protein molecules in the solution). The full model (depletion of protein from the bulk and attraction by the previously adsorbed molecules) leads to an expression for the rate of adsorption of the third degree. Only the third degree gives a good fit to the experimental data where two kinetic phases are obvious. However, the accuracy of the data does not allowus to determine whether the adsorption regimes are diffusive or exponential. It may be concluded that the adsorption of lysozyme at the air-buffer interface is very probably a ‘cooperative’ process.

Properties of human plasma lipid transfer protein in aqueous solution and at interfaces.

Human plasma lipid transfer protein (cholesteryl ester transfer protein) has been characterized for its solution and surface properties. The protein is monomeric in aqueous solution up to 0.62 g/L (11.7 microM) as demonstrated by sedimentation equilibrium. It binds to the surface of a lipid microemulsion having an average diameter of 26 nm made from triolein and egg yolk phosphatidylcholine, with an estimated dissociation constant 1.2 x 10(-8) M, and the maximum saturation binding level is 8 protein molecules per particle regardless of the presence of apolipoprotein A-I. Circular dichroism measurement indicated that the protein in solution is predominantly in the beta-sheet/beta-turn conformation with some alpha-helix, and this profile does not undergo drastic change by its binding to the lipid surface. The analysis of the behavior of the protein in its monomolecular layer at the air-buffer interface indicated that it is also monomeric at the interface. LTP molecules occupied the same area per amino acid as other apolipoproteins in the monolayer but had a higher collapse pressure of its monolayer (18 dyn/cm), and the protein stayed at the interface even after the overcompressing monolayer far beyond the collapsing pressure up to 40 dyn/cm.

Influence of sphingosine on the thermal phase behaviour of neutral and acidic phospholipid liposomes

The physical state of lipids is known to have pronounced effects on membrane functions. We studied the influence of sphingosine, a modulator of diverse cellular processes on the thermal phase behaviour and molecular packing of neutral and acidic phospholipids. Differential scanning calorimetry of multilamellar liposomes as well as the monolayer technique were employed. Inclusion of sphingosine in diacylphosphatidylcholine liposomes increased their pretransition temperature T p until at about 10 mol% sphingosine this transition was abolished. For these liposomes a gradual increase in both the temperature T m and enthalpy ΔH m of the main transition caused by sphingosine was observed. In contrast to diacylphosphatidylcholines, the T p for dihexadecylphosphatidylcholine was lowered by sphingosine, demonstrating that the latter destabilizes the interdigitated gel phase. Inclusion of sphingosine in dimyristoylphosphatidic acid and dipalmitoylphosphatidylserine liposomes first elevated the T m without significant changes in ΔH m, while at sphingosine contents >;50 mol% the appearance of complex melting profiles was evident. The transition temperature for the egg yolk phosphatidic acid was shifted from below 0 to 29°C when mixed with sphingosine in a molar ratio of 1:1. Sphingosine also condensed the eggPA monolayers residing on an air-buffer interface. Accordingly, besides introducing a positive surface charge allowing the binding or activation of some proteins, sphingosine could influence membrane-mediated cellular processes by altering the organization and state of membrane lipids.

Interfacial Properties of Sodium Caseinate/Monoglyceride Mixture; Combined Effects of pH and Surface Area Ratios

Interfacial properties of sodium caseinate (NaCas) in mixture with glycerol monostearate (GMS) was investigated at the air-buffer interface. A Wilhelmy type compression system was used to record the pressure-area (π-A) isotherms of mixed films at four different GMS: NaCas surface ratios (0.2:0.8; 0.4:0.6; 0.6:0.4; 0.8:0.2) and three pHs (5.9; 6.8; 7.7). Results showed no pH effect over the range tested. A condensation of the mixed films was detected in the low pressure region of the isotherms (0-20mN.m−1). Maximum condensation was encountered at an initial surface ratio of 0.6:0.4. In the high pressure region, portion of NaCas remained at the interface and increased the collapse pressure of the film. Results are discussed in relation with the partial miscibility of components at the interface. The importance of these findings with respect to emulsion stability is also emphasized.

Enzymatic synthesis/hydrolysis of cholesteryl oleate in surface films. Inhibition by lecithin and its reversal by bile salts.

The synthesis/hydrolysis of cholesteryl oleate as catalyzed by porcine pancreatic cholesterol esterase has been studied in lipid films at the air-buffer interface. With only reactants and products initially present at the interface, equilibrium is rapidly attained at subphase enzyme concentrations of 4 x 10(-8) M or less. The equilibrium constant for the reaction, 1.4 x 10(-8) mol/cm2, is independent of pH, initial composition, and surface pressure. Lecithin, if present in molar excess relative to the sum of free and esterified cholesterol, is inhibitory. Inhibition is associated with division of the substrate into reactive and unreactive pools which are not exchangeable. Bile salts and other surfactants reverse the inhibition at concentrations one-tenth their critical micelle concentrations. Presumably this occurs through formation of a surfactant surface excess at the lipid-water interface which disrupts the unreactive lecithin-substrate complex. The adsorption of cholesterol esterase to oleic acid monolayers is first order with respect to enzyme and is saturable. At saturation, the enzyme forms a close packed monolayer at the lipid-water interface with a molecular area of 4510 A2. Adsorption of cholesterol esterase to lecithin monolayers is less than one-tenth that to oleic acid monolayers and is proportional to subphase enzyme concentration. With either lipid monolayer, enzyme denaturation at the interface was negligible. In the presence of substrate, differences in enzyme absorption can only partially account for the observed inhibition of catalysis by lecithin, indicating that the reactivity or availability of substrate to the adsorbed enzyme is also affected.

Lipid structure and the behavior of cholesteryl esters in monolayer and bulk phases.

The behavior of cholesteryl esters at the air-buffer interface was studied as a function of molecular area and the presence of noncholesterol-containing lipids (colipids). The data obtained indicate that cholesteryl esters with other than long, saturated acyl groups can be present in surface phases up to packing densities approximately those in natural membranes. Their apparent molecular areas in such phases, which are largely determined by colipid structure, suggest their orientation with the ester function toward the interface. The extent of miscibility in the surface phase is also a strong function of colipid structure. Reversibility of the monolayer to bulk phase transition is determined exclusively by the acyl structure of the cholesteryl ester. Of the esters examined, only those with cis unsaturation collapsed reversibly. Our data predict that cholesteryl esters should be present in small, but finite amounts on the surface of arterial lipid deposits and that a prerequisite for the removal of such deposits is that the bulk lipid phase be in a liquid or liquid crystalline state.