Structural characteristics of dipalmitoylphosphatidylcholine and hydrolysates from sunflower protein isolate mixed monolayers spread at the air-water interface

In this work, surface film balance and Brewster angle microscopy techniques have been used to analyze the structural characteristics (structure, topography, reflectivity, thickness, miscibility, and interactions) of hydrolysates from sunflower protein isolate (SPI) and dipalmitoylphosphatidylcholine (DPPC) mixed monolayers spread on the air-water interface. The degree of hydrolysis (DH) of SPI, low (5.62%), medium (23.5%), and high (46.3%), and the protein/DPPC mass fraction were analyzed as variables. The structural characteristics of the mixed monolayers deduced from the surface pressure (pi)-area (A) isotherms depend on the interfacial composition and degree of hydrolysis. At surface pressures lower than the equilibrium surface pressure of SPI hydrolysate (pi(e)(SPI hydrolysate)), both DPPC and protein are present in the mixed monolayer. At higher surface pressures (at pi > pi(e)(SPI hydrolysate)), collapsed protein residues may be displaced from the interface by DPPC molecules. The differences observed between pure SPI hydrolysates and DPPC in reflectivity (I) and monolayer thickness during monolayer compression have been used to analyze the topographical characteristics of SPI hydrolysates and DPPC mixed monolayers at the air-water interface. The topography, reflectivity, and thickness of mixed monolayers confirm at microscopic and nanoscopic levels the structural characteristics deduced from the pi-A isotherms.     

Comparative study of lipolysis by PLA2 of DOPC substrates organized as monolayers, bilayer vesicles and nanocapsules

The water-soluble lipolytic enzymes act at the interface of insoluble lipid substrates, where the catalytical step is coupled with various interfacial phenomena as enzyme penetration, solubilization of reaction products, loss of mechanical stability of organized assemblies of phospholipids molecule, etc. One biologically relevant example is the enzymatic hydrolysis of DOPC by PLA(2), which results in cleavage of phospholipids molecules into water insoluble lipolytic products, namely oleic acid and lysophospholipid. In general, the enzymatic activity depends on the substrate organization and molecular environment of the catalytic reaction. The lipolysis by phospholipase A(2) of dioleoylphosphatidylcholine substrates organized as monolayer, bilayers vesicles and lipid nanocapsules was studied by measuring the decrease of the surface area at constant surface pressure or increase of the surface pressure at constant area at air-water interface. A kinetic model describing the coupling of the catalytic act with corresponding interfacial phenomena was developed. By using the kinetic model the values for the global hydrolytic kinetic constants were obtained. The obtained value for the monolayer is five orders of magnitude higher than this obtained with small unilamellar vesicles and six orders of magnitude higher then those obtained with lipid nanocapsules. The comparison shows that the enzymatic catalytic act occurring in the lipid environment of the monolayer is more efficacious than at the vesicle and nanocapsules interfaces.     

Action of Humicola lanuginosa lipase on long-chain lipid substrates: 2. Hydrolysis of diolein monolayers

The hydrolysis of 1,2-diolein (DO) monomolecular films by Humicola lanuginosa lipase (HLL) was studied by simultaneous measuring the decrease in the film area and the changes in the surface potential in the “zero-order trough” at constant surface pressure and in the presence of β-cyclodextrin (β-CD). The decrease with time in the film area reflects both the reduction in the area per molecule due to the transformation of substrate DO molecules into the products molecules of monoolein (MO) and oleic acid (OA) and the desorption of the soluble inclusion complexes β-CD–MO and β-CD–OA. The surface potential data were interpreted as an accumulation at the interface of negatively charged products of OA and insoluble β-CD–DO complexes. In the proposed kinetic model, the product solubilization rates in the presence of β-CD and the flux supplied progressively by the moving barrier from the reservoir to the reaction compartment in order to keep the constant surface pressure were taken into account. The surface concentrations of MO and OA transiently present at the interface were determined. The values of the global kinetic constant Q_m′ of hydrolysis of DO to MO were obtained. Comparison with the values of the global kinetic constant of hydrolysis of monoglyceride MO to OA shows that the rates of hydrolysis of diglyceride and monoglyceride by HLL are of the same order of magnitude.     

Comparative kinetics of phospholipase A2 action on liposomes and monolayers of phosphatidylcholine spread at the air-water interface

A detailed kinetic model describing the surface transformation of spread liposomes along with their enzymatic hydrolysis was developed. The model was applied to the hydrolysis of the long-chain phosphatidylcholine generating reaction products which remain at the interface and to medium-chain substrates from which the products desorb rapidly into the bulk phase. The overall kinetic constants of the hydrolysis in liposomal systems were compared with those obtained with monolayers under barostatic conditions. The values of the interfacial Michaelis-Menten constant were estimated.     

Hydrolysis reaction analysis of L-alpha-distearoylphosphatidylcholine monolayer catalyzed by phospholipase A2 with polarization-modulated infrared reflection absorption spectroscopy

The hydrolysis reaction of L-alpha-distearoylphosphatidylcholine (DSPC) monolayers catalyzed by phospholipase A2 (PLA(2)) has been studied using polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) with film balance measurements. The PM-IRRAS analysis provides quantitative information about the reaction efficiency at different surface pressures. It was found that the reaction efficiency of L-DSPC monolayer hydrolysis catalyzed by PLA(2) decreased with increasing surface pressure. At zero pressure (lift-off point), the hydrolysis reaction efficiency has the highest value of 45%. Increasing surface pressure leads to the decrease of the hydrolysis efficiency. Since the surface pressure is above 20 mN/m, the hydrolysis reaction nearly stopped. PM-IRRAS technique provides a powerful means to study the hydrolysis process catalyzed by phospholipase A2 at the air/water interface.     

Induced removal of dipalmitoyl phosphatidylcholine by the exclusion of fibrinogen from compressed monolayers at air/liquid interfaces

The induced removal of dipalmitoyl phosphatidylcholine (DPPC) by the exclusion of fibrinogen from mixed DPPC/fibrinogen monolayers at compressed air/liquid interfaces was analyzed. The surface pressure-area hysteresis curves of the monolayers at interfaces were obtained by a Langmuir trough. The hysteresis curves of equilibrium fibrinogen adsorption layers suggest that fibrinogen desorption during the area compression stage became significant at a higher bulk concentration of 1000 ppm. For mixed monolayers of DPPC with fibrinogen, the fibrinogen molecules were expelled from the interface upon compression due to the presence of insoluble DPPC molecules. The squeeze-out of fibrinogen molecules evidently removed a significant number of DPPC molecules from the interface, with the extent depending on fibrinogen surface concentration. During the subsequent area expansion stage, fibrinogen molecules entered the interface and participated in the rise of surface pressure. The induced loss of free DPPC molecules at the interface by the expelled fibrinogen molecules during the area compression stage was then evaluated from the hysteresis curves.     

Action of Humicola lanuginosa lipase on mixed monomolecular films of tricaprylin and polyethylene glycol stearate

The hydrolysis catalyzed by Humicola lanuginosa lipase (HLL) of pure tricaprylin (TC) or stearate of polyethylene glycol 1500 (PEG-St) as well as their mixtures spread as monomolecular films were studied. The catalytic transformation of the two substrates TC or PEG-St into their respective reaction products was detected by measuring simultaneously the decrease in the film area and the surface potential using the "zero order" trough at constant surface pressure. A kinetic model describing the enzymatic hydrolysis was developed. The surface concentrations of the two substrates and their respective reaction products as well as the values of the global kinetic constants of hydrolysis were determined. The experimentally obtained global kinetic constants of the catalytic action of HLL against TC and PEG-St present in mixed monolayers of TC/PEG-St are approximately the same as in the case of pure monolayers. These obtained results give some indications that the activity of enzyme is not significantly affected by the different molecular environments in the mixed monolayers.     

Kinetics and layer organization during the polymerization of coniferyl alcohol at the air-water interface

The initial stage of the polymerization of coniferyl alcohol catalyzed by a peroxydase was studied at the air-water interface. The properties of the monolayers were investigated at constant area and at constant surface pressure by surface pressure, surface potential and ellipticity measurements. On the basis of the results obtained at constant surface area, it is proposed that the formation of a 2D layer occurs up to the inflection point of the surface pressure-area isotherm, and that for larger surface pressures a 3D structure is formed during the polymerization. If the barostat is set at the inflection point (the surface pressure is constant while the surface area increases), the 2D organization of the monolayer is retained. A kinetic model describing the adsorption of the reaction products in a 2D layer is proposed. The kinetic constants were determined from the surface potential, ellipsometry, surface pressure and surface area data.     

Action of Humicola lanuginosa lipase on long-chain lipid substrates: 1. Hydrolysis of monoolein monolayers

The hydrolysis of 1-monoolein (MO) monomolecular films by Humicola lanuginosa lipase (HLL) was followed by measuring the simultaneous decrease with time in the film area and the surface potential using a ‘zero order trough’ at constant surface pressure (Verger and de Haas, Chem. Phys. Lipids 10 (1973) 127). The decrease with time in the film area reflects both the reduction in the area per molecule as well as the solubilization of the substrate and the product molecules during the transformation of the substrate MO into product of oleic acid (OA). The surface potential changes were interpreted as the results of the accumulation at the interface of negatively charged OA. The surface concentration of OA transiently present at the interface was determined by the surface pressure and the surface potential measurements on the basis of a developed kinetic model. In the proposed model we have taken into account the product and substrate solubilization rates in the presence of β-cyclodextrin (β-CD) as well as the flux supplied progressively by the moving barrier from the reservoir to the reaction compartment in order to keep the constant surface pressure. Values of the global kinetic constant Q_m were obtained. The selective lipolytic product acceptor, β-CD, accelerated considerably the hydrolytic process.     

Roles of gamma-Globulin in the Dynamic Interfacial Behavior of Mixed Dipalmitoyl Phosphatidylcholine/gamma-Globulin Monolayers at Air/Liquid Interfaces

This study investigated the roles of gamma-globulin in the dynamic interfacial behavior of dipalmitoyl phosphatidylcholine (DPPC)/gamma-globulin monolayers at air/liquid interfaces at 25 degrees C. The surface tension behavior demonstrated that gamma-globulin had a large adsorption time scale. Moreover, the surface pressure-area hysteresis behavior of adsorbed gamma-globulin monolayers suggested that no significant desorption occurred during the compression stage, and the respreading of gamma-globulin molecules at the interface during the expansion stage was slow. From the hysteresis behavior of adsorbed gamma-globulin monolayers with spread DPPC molecules, it was found that gamma-globulin molecules were expelled from the interface as DPPC molecules were in a condensed state. The squeeze-out of gamma-globulin molecules seemed to induce the loss of DPPC molecules at the interface with the extent depending on the initial gamma-globulin surface concentration. Furthermore, the expelled gamma-globulin molecules re-entered the monolayer and participated in the surface pressure increase during the following expansion stage. The exclusion of gamma-globulin associated with the removal of DPPC during monolayer compression and the re-entry of gamma-globulin during subsequent monolayer expansion represented a mechanism for DPPC depletion and gamma-globulin enrichment at the interface, which may explain the inhibitory effect of certain proteins on the surface activity of DPPC. Copyright 2000 Academic Press.     

On the interaction of dipalmitoyl phosphatidylcholine with normal long-chain alcohols in a mixed monolayer: a thermodynamic study

This study investigated the mixed monolayer behavior of dipalmitoyl phosphatidylcholine (DPPC) with normal long-chain alcohols at the air/water interface. Surface pressure–area isotherms of mixed DPPC/C₁₈OH and DPPC/C₂₀OH monolayers at 37°C were obtained and compared with previous results for the mixed DPPC/C₁₆OH system. The negative deviations from additivity of the areas and the variation of the collapse pressure with composition imply that DPPC and long-chain alcohols were miscible and formed non-ideal monolayers at the interface. At lower surface pressures, it seems that the attractive intermolecular force was dominant in molecular packing in the mixed monolayers. At higher surface pressures, the data suggest that the molecular packing in mixed DPPC/C₁₆OH monolayers may be favored by the packing efficiency or geometric accommodation. Furthermore, negative values of excess free energy of mixing were obtained and became significant as the hydrocarbon chain length of alcohols increased, which indicates there were attractive interactions between DPPC and long-chain alcohols. In each free energy of mixing–composition curve, there was only one minimum and thus a phase separation did not exist for mixed DPPC/long-chain alcohol monolayers.     

Thermodynamic characteristics and Langmuir-Blodgett deposition behavior of mixed DPPA/DPPC monolayers at air/liquid interfaces

The role of dipalmitoylphosphatic acid (DPPA) as a transfer promoter to enhance the Langmuir-Blodgett (LB) deposition of a dipalmitoylphosphatidylcholine (DPPC) monolayer at air/liquid interfaces was investigated, and the effects of Ca2+ ions in the subphase were discussed. The miscibility of the two components at air/liquid interfaces was evaluated by surface pressure-area per molecule isotherms, thermodynamic analysis, and by the direct observation of Brewster angle microscopy (BAM). Multilayer LB deposition behavior of the mixed DPPA/DPPC monolayers was then studied by transferring the monolayers onto hydrophilic glass plates at a surface pressure of 30 mN/m. The results showed that the two components, DPPA and DPPC, were miscible in a monolayer on both subphases of pure water and 0.2 mM CaCl2 solution. However, an exception occurs between X(DPPA)=0.2 and 0.5 at air/CaCl2-solution interface, where a partially miscible monolayer with phase separation may occur. Negative deviations in the excess area analysis were found for the mixed monolayer system, indicating the existence of attractive interactions between DPPA and DPPC molecules in the monolayers. The monolayers were stable at the surface pressure of 30 mN/m for the following LB deposition as evaluated from the area relaxation behavior. It was found that the presence of Ca2+ ions had a stabilization effect for DPPA-rich monolayers, probably due to the association of negatively charged DPPA molecules with Ca2+ ions. Moreover, the Ca2+ ions may enhance the adhesion of DPPA polar groups to a glass surface and the interactions between DPPA polar groups in the multilayer LB film structure. As a result, Y-type multilayer LB films containing DPPC could be fabricated from the mixed DPPA/DPPC monolayers with the presence of Ca2+ ions.     

Interfacial behavior of lipid nanocapsules spread on model membrane monolayers

The lipid nanocapsules (LNCs) spread at the air–water interface (A/W) undergo destabilization and disaggregation leading to formation of a triglyceride (TG) surface film. The kinetics of reorganization and formation of TG surface film were followed by measuring either the change of surface pressure at constant area or the surface area at constant surface pressure. From the obtained experimental data were determined the effectiveness of TG spreading and the rate of LNC disaggregation at A/W interface covered with preformed model membrane monolayers of DPPC, Curosurf®, and mucus. Partial LNC stabilization due to their interaction with the model membrane monolayers was observed and characterized by atomic force microscopy (AFM). The obtained results demonstrated that the LNCs spread on mucus surface layer, which models the epithelial surface were more stable than if they were spread either on DPPC or Curosurf® surface layers, which emulate the alveolar surface.     

Reorganization of lipid nanocapsules at air-water interface 3. Action of hydrolytic enzymes HLL and pancreatic PLA2

The action of the hydrolytic enzymes humicola lanuginosa lipase (HLL) and pancreatic phospholipase A2 (PLA2) on monolayers formed from lipid nanocapsules (LNC) and model monolayers containing their components, Labrafac, Solutol and Lipoid, is studied by simultaneous measuring the changes in the film area and the surface potential in the "zero order" trough at constant surface pressure (pi). The kinetic models describing the hydrolysis by HLL of the Labrafac, Solutol and their mixtures have been proposed. By using the developed theoretical approach together with the experimental results the surface concentrations of the substrates, hydrolysis products and values of the global kinetic constants were obtained. The comparison between the global kinetic constants in the case of HLL hydrolysis of pure Labrafac, Solutol monolayers and those of the model mixed Labrafac/Solutol monolayers, shows that the rates of hydrolysis are of the same order of magnitude, i.e. an additively of the HLL enzyme action is observed. The composition of the mixed Labrafac/Solutol monolayer, formed after the interfacial LNC destabilization, was estimated.     

髓鞘碱性蛋白对细胞膜脂质分子DPPC、DPPE单层膜影响机理研究

本文通过Langmuir单层膜的表面压力-平均分子面积(π-A)曲线的测定与分析,分别对髓鞘碱性蛋白(MBP)与细胞膜中不同头部基团脂质分子二棕榈酰基磷脂胆碱(DPPC)和二棕榈酰基磷脂酰乙醇胺(DPPE)在空气/液体界面上的相互作用过程进行了系统研究.实验结果表明:(1)当界面上脂质含量一定时,亚相中随着MBP浓度的增大,DPPC、DPPE单层膜的等温线向平均分子面积较大的方向移动;(2)在单层膜表面压力为10 mN/m时,一个MBP分子分别结合140±3个DPPC分子和100±3个DPPE分子,随着表面压力增大,当MBP分子分别与两种磷脂分子相互作用时,MBP插入到磷脂单层界面的个数逐渐减少;(3)随着蛋白质浓度的增加,脂分子形成的单层膜变得较为疏松,且MBP分子易于插入到分子头部较小的DPPE单层膜中;(4)蛋白质的存在使DPPC单层膜的表面压力逐渐减小,且蛋白质浓度越大表面压力降低越多,DPPC被MBP带入到亚相中越多;(5)对于DPPE单层膜,蛋白质通过与DPPE相互作用插入到界面膜中,引起表面压力增大,且蛋白质浓度越高,压力变化量越大.     

Topography of dipalmitoyl-phosphatidyl-choline monolayers penetrated by β-casein

In this work we have analyzed the topography by atomic force microscopy (AFM) of dipalmitoyl-phosphatidyl-choline (DPPC) monolayers previously spread at the air–water interface and penetrated by β-casein. AFM images of β-casein–DPPC monolayers were taken from Langmuir–Blodgett films deposited onto hydrophilic mica substrates at different initial surface pressures (π_i) and after the compression of the mixed films. The monolayer topography depends on the initial structure of the phospholipid:liquid expanded (LE) at 3 mN/m, coexistence between LE and liquid condensed (LC) structures at 7 mN/m, at the end of the LE–LC transition at 10 mN/m, and with a LC structure at 15 mN/m. The area occupied by DPPC domains in the mixed film increases with the π_i value, especially for DPPC with a LC structure at 15 mN/m. At this surface pressure the thickness of the film is at a maximum. After the film compression at 25 mN/m, which is above the equilibrium spreading pressure of β-casein (), this protein is displaced from the interface by DPPC and the topography of the mixed monolayer depends on the initial structure of the DPPC monolayer. A notable feature of the topography of these mixed monolayers is the presence of multilayers of β-casein and DPPC of high thickness (50–70 nm) at the lower π_i values. Although the film is dominated by DPPC at the highest surface pressures (at 25 mN/m), β-casein is not displaced totally from the interface and coexists as β-casein collapsed domains within the network of the DPPC structure.     

Interfacial behavior of N-nitrosodiethylamine/bovine serum albumin complexes at the air-water and the chloroform-water interfaces by axisymmetric drop tensiometry

Interfacial properties of N-nitrosodiethylamine/bovine serum albumin (NDA/BSA) complexes were investigated at the air-water interface. The interfacial behavior at the chloroform-water interface of the interaction product of phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), dissolved in the chloroform phase, and NDA/BSA complex, in the aqueous phase, were also analyzed by using a drop tensiometer. The secondary structure changes of BSA with different NDA concentrations were monitored by circular dichroism spectroscopy at different pH and the NDA/BSA interaction was probed by fluorescence spectroscopy. Different NDA/BSA mixtures were prepared from 0, 7.5 x 10(-5), 2.2 x 10(-4), 3.7 x 10(-4), 5 x 10(-4), 1.6 x 10(-3), and 3.1 x 10(-3) M NDA solutions in order to afford 0, 300/1, 900/1, 1 500/1, 2 000/1, 6 000/1, and 12 500/1 NDA/BSA molar ratios, respectively, in the aqueous solutions. Increments of BSA alpha-helix contents were obtained up to the 2 000/1 NDA/BSA molar ratio, but at ratios beyond this value, the alpha-helix content practically disappeared. These BSA structure changes produced an increment of the surface pressure at the air-water interface, as the alpha-helix content increased with the concentration of NDA. On the contrary, when alpha-helix content decreased, the surface pressure also appeared lower than the one obtained with pure BSA solutions. The interaction of DPPC with NDA/BSA molecules at the chloroform-water interface produced also a small, but measurable, pressure increment with the addition of NDA molecules. Dynamic light scattering measurements of the molecular sizes of NDA/BSA complex at pH 4.6, 7.1, and 8.4 indicated that the size of extended BSA molecules at pH 4.6 increased in a greater proportion with the increment in NDA concentration than at the other studied pH values. Diffusion coefficients calculated from dynamic surface tension values, using a short-term solution of the general adsorption model of Ward and Tordai, also showed differences with pH and the NDA concentration. Both, the storage and loss dilatational elastic modulus were obtained at the air-water and at the chloroform-water interfaces. The interaction of NDA/BSA with DPPC at the chloroform-water produced a less rigid monolayer than the one obtained with pure DPPC (1 x 10(-5) M), indicating a significant penetration of NDA/BSA molecules at the interface. At short times and pH 4.6, the values of the storage elastic modulus were larger and more sensible to the NDA addition than the ones at pH 7.1 and 8.4, probably due to a gel-like network formation at the air-water interface.     

Interfacial organizations of gel phospholipid and cholesterol in bovine lung surfactant films

Pulmonary surfactants stabilize the lung by way of reducing surface tension at the air-lung interface of the alveolus. 31P NMR, thin-layer chromatography, and electrospray ionization mass spectroscopy of bovine lipid extract surfactant (BLES) confirmed dipalmitoylphosphatidylcholine (DPPC) to be the major phospholipid species, with significant amounts of palmitoyl-oleoylphosphatidylcholine, palmitoyl-myristoylphosphatidylcholine, and palmitoyl-oleoylphosphatidylglycerol. BLES and DPPC spread at the air-water interface were studied through surface pressure area, fluorescence, and Brewster angle microscopy measurements. Langmuir-Blodgett films of monomolecular films, deposited on mica, were characterized by atomic force microscopy. BLES films displayed shape, size, and vertical height profiles distinct from those of DPPC alone. Calcium ions in the subphase altered BLES film domain structure. The addition of cholesterol (4 mol %) resulted in the destabilization of compressed BLES films at higher surface pressures (>40 mN m-1) and the formation of multilayered structures, apparently consisting of stacked monolayers. The studies suggested potential roles for individual surfactant lipid components in supramolecular arrangements, which could be the contributing factors in pulmonary surfactant to attain low surface tension at the air-water interface.     

A monolayer study on interactions of docetaxel with model lipid membranes

Docetaxel (DCT) is an antineoplastic drug for the treatment of a wide spectrum of cancers. DCT surface properties as well as miscibility studies with l-alpha-dipalmitoyl phosphatidylcholine (DPPC), which constitutes the main component of biological membranes, are comprehensively described in this contribution. Penetration studies have revealed that when DCT is injected under DPPC monolayers compressed to different surface pressures, it penetrates into the lipid monolayer promoting an increase in the surface pressure. DCT is a surface active molecule able to decrease the surface tension of water and to form insoluble films when spread on aqueous subphases. The maximum surface pressure reached after compression of a DCT Langmuir film was 13 mN/m. Miscibility of DPPC and DCT in Langmuir films has been studied by means of thermodynamic properties as well as by Brewster angle microscopy (BAM) analysis of the mixed films at the air-water interface, concluding that DPPC and DCT are miscible and they form non-ideally mixed monolayers at the air-water interface. Helmholtz energies of mixing revealed that no phase separation occurs. In addition, Helmholtz energies of mixing become more negative with decreasing areas per molecule, which suggests that the stability of the mixed monolayers increases as the monolayers become more condensed. Compressibility values together with BAM images indicate that DCT has a fluidizing effect on DPPC monolayers.     

Infrared spectroscopy analysis of mixed DPPC/fibrinogen layer behavior at the air/liquid interface under a continuous compression-expansion condition

The mixed layer behavior of dipalmitoyl phosphatidylcholine (DPPC) with fibrinogen at continuously compressed-expanded air/liquid interfaces was analyzed in situ by infrared reflection-absorption spectroscopy (IRRAS). The reflectance-absorbance (RA) intensities and/or wavenumbers of nu(a)-CH2 and amide I bands for a mixed DPPC/fibrinogen layer at the interface were obtained directly by an infrared spectrometer with a monolayer/grazing angle accessory and a removable Langmuir trough. The nu(a)-CH2 RA intensity-area hysteresis curves of a DPPC monolayer indicate a significant loss of free DPPC molecules at the interface during the first compression stage, which is also supported by the corresponding nu(a)-CH2 wavenumber-area hysteresis curves. For a mixed DPPC/fibrinogen layer at the interface, the amide I RA intensity-area hysteresis curves suggest that the fibrinogen molecules were expelled from the interface upon compression, apparently because of the presence of insoluble DPPC molecules. The squeeze-out of fibrinogen evidently removed a pronounced amount of DPPC from the interface, as judged from the corresponding nu(a)-CH2 intensity and wavenumber data. Moreover, significant adsorption of fibrinogen was found during the subsequent interface expansion stage. With the in situ IRRAS analysis of the mixed layer behavior at the interface, the induced loss of DPPC by fibrinogen expulsion from the compressed interface and the dominant adsorption of fibrinogen to the expanded interface were clearly demonstrated.