Cholesterol Monolayers Research Articles

Self-assembled monolayers of cholesterol and cholesteryl esters on graphite.

The molecular arrangements of self-assembled monolayers (SAMs) of cholesterol, cholesteryl laurate, and cholesteryl stearate adsorbed on a graphite surface were studied using scanning tunneling microscopy (STM) at the liquid-solid interface. The STM images of the SAMs showed two-dimensional periodic arrays of bright regions that corresponded to the sterol rings. However, individual sterol rings could not be observed in the bright regions in the STM images of the cholesterol monolayers. Nevertheless, by comparing the STM images and the crystallographic data, it is concluded that the cholesterol molecules are arranged in pairs oriented head-to-head owing to the hydrogen bonds between the hydroxyl groups. These dimers, in turn, are oriented parallel to each other, owing to the interactions between the sterol rings. The STM images of cholesteryl ester monolayers had molecular resolution and showed pairs of cholesteryl ester molecules oriented in an antiparallel manner, with their fatty acid chains located in the central regions. Furthermore, the fatty acid chains of cholesteryl stearate were observed to be oriented in the (1120) zigzag direction of the graphite lattice, whereas those of cholesteryl laurate were oriented in the (1010) armchair direction. These observations reveal that the interactions between the fatty acid chains affect the structure of the SAMs. The molecular arrangements also depend on the lengths of the fatty acid chains of the cholesterol esters and hence on the interactions between the alkyl chains and the graphite surface. The self-assembly at the liquid-solid interface is therefore controlled by the interactions between sterol rings, between alkyl chains, and between alkyl chains and the substrate.

Interaction of Mefloquine Hydrochloride with Cell Membranes Models Studied with Tensiometry and Vibrational Spectroscopy

Mefloquine hydrochloride is a drug used in the treatment of malaria, with a probable effect on cell membrane surfaces. However, the mechanism of action when they interact with lipid surfaces is not sufficiently known so far. For this reason, it is important the understanding at the molecular level of drug-membrane interactions, and using models for biointerfaces membranes is a suitable strategy for this purpose. In this study, we employed Langmuir monolayers of lipids as biointerface models, with the drug incorporated in monolayers of zwitterionic lipids, namely DPPC (dipalmitoyl phosphatidyl choline), negative lipids, namely DPPS (dipalmitoyl phosphatidyl serine), and cholesterol. Combining data on Surface Pressure-Area Isotherms with Polarization Modulation Infrared Reflection-Absorption Spectroscopy (PM-IRRAS), the effect of the mefloquine hydrochloride on lipid monolayers was compared by taking into account the chemical and molecular structure of the lipids and the drug. The adsorption of the drug at the monolayers decreases the order of the lipid film, replaces water molecules from the interface, in mechanisms that involve both polar head groups and alkyl tails from the lipids. A model is then proposed in which mefloquine hydrochloride interacts with lipids at the air-water in such a way that the interactions are maximized owing to geometrical adaptations on behalf of the contact between lipid polar heads and polar drug groups.

Studying Lipid Interactions of Perilipin 3/ Tip 47 using Phospholipid Monolayers

Lipid droplets are found in all eukaryotes and are considered as dynamic cell organelles. In addition to acting as neutral lipid stores, they are also involved in various biological processes such as membrane formation, cell signaling and hormone synthesis. Many aspects of lipid droplet biogenesis and function are not yet fully understood. The basic structure of a lipid droplet consists of a neutral lipid core of triacylglycerols and cholesterol esters surrounded by a monolayer of phospholipids, free cholesterol and proteins.

2P210 固液界面に形成したコレステロールとコレステロールエステルからなる自己組織化単分子膜の研究(13A. 生体膜・人工膜:構造・物性,ポスター,第52回日本生物物理学会年会(2014年度))

2P210 固液界面に形成したコレステロールとコレステロールエステルからなる自己組織化単分子膜の研究(13A. 生体膜・人工膜:構造・物性,ポスター,第52回日本生物物理学会年会(2014年度))

Autophagosomes contribute to intracellular lipid distribution in enterocytes

Enterocytes, the intestinal absorptive cells, have to deal with massive alimentary lipids upon food consumption. They orchestrate complex lipid-trafficking events that lead to the secretion of triglyceride-rich lipoproteins and/or the intracellular transient storage of lipids as lipid droplets (LDs). LDs originate from the endoplasmic reticulum (ER) membrane and are mainly composed of a triglyceride (TG) and cholesterol-ester core surrounded by a phospholipid and cholesterol monolayer and specific coat proteins. The pivotal role of LDs in cellular lipid homeostasis is clearly established, but processes regulating LD dynamics in enterocytes are poorly understood. Here we show that delivery of alimentary lipid micelles to polarized human enterocytes induces an immediate autophagic response, accompanied by phosphatidylinositol-3-phosphate appearance at the ER membrane. We observe a specific and rapid capture of newly synthesized LD at the ER membrane by nascent autophagosomal structures. By combining pharmacological and genetic approaches, we demonstrate that autophagy is a key player in TG targeting to lysosomes. Our results highlight the yet-unraveled role of autophagy in the regulation of TG distribution, trafficking, and turnover in human enterocytes.

Effect of cholesterol nanodomains on monolayer morphology and dynamics.

At low mole fractions, cholesterol segregates into 10- to 100-nm-diameter nanodomains dispersed throughout primarily dipalmitoylphosphatidylcholine (DPPC) domains in mixed DPPC:cholesterol monolayers. The nanodomains consist of 6:1 DPPC:cholesterol "complexes" that decorate and lengthen DPPC domain boundaries, consistent with a reduced line tension, λ. The surface viscosity of the monolayer, ηs, decreases exponentially with the area fraction of the nanodomains at fixed surface pressure over the 0.1- to 10-Hz range of frequencies common to respiration. At fixed cholesterol fraction, the surface viscosity increases exponentially with surface pressure in similar ways for all cholesterol fractions. This increase can be explained with a free-area model that relates ηs to the pure DPPC monolayer compressibility and collapse pressure. The elastic modulus, G', initially decreases with cholesterol fraction, consistent with the decrease in λ expected from the line-active nanodomains, in analogy to 3D emulsions. However, increasing cholesterol further causes a sharp increase in G' between 4 and 5 mol% cholesterol owing to an evolution in the domain morphology, so that the monolayer is elastic rather than viscous over 0.1-10 Hz. Understanding the effects of small mole fractions of cholesterol should help resolve the controversial role cholesterol plays in human lung surfactants and may give clues as to how cholesterol influences raft formation in cell membranes.

Sum Frequency Generation Vibrational Spectroscopy of Cholesterol in Hybrid Bilayer Membranes

The assignment of the vibrational spectrum of cholesterol is surprisingly incomplete for such a fundamental molecule. To improve our understanding, a new investigation of the spectra of cholesterol in the C-H stretching region has been undertaken using the surface specific technique of Sum Frequency Generation (SFG) vibrational spectroscopy and the complementary technique of Reflection Absorption Infrared Spectroscopy (RAIRS). They were used to record the spectra of monolayers of cholesterol in hybrid bilayer membranes (HBMs). In addition to cholesterol, spectra were recorded of HBMs comprising monolayers of the partially deuterated cholesterol isotopologues d6-cholesterol and d7-cholesterol, and the cholesterol analogues cholestanol and androstanol to aid assignment of the spectra. Monolayers of each of the five molecules were used to form the distal leaflet of HBMs with the proximal leaflet consisting of a monolayer of deuterated mercaptoundecanoic acid (d-MUA) self-assembled on a gold substrate. Although cholesterol has five methyl groups and eleven methylene groups, by using molecules in which certain groups were either deuterated or entirely absent, it was possible to assign vibrational bands to specific sets of methyl or methylene groups either in the alkyl chain or sterol ring system of the molecule. Analysis of the spectra showed that the alkyl chains of cholesterol are orientated away from the substrate, which is opposite to their orientation in HBMs when the proximal leaflet is a hydrophobic self-assembled monolayer of octadecane thiol (ODT) adsorbed on gold. Additionally it was shown that in the d-MUA HBM, the α-face of the cholesterol ring is inclined toward the layer of air above the film, and the β-face is inclined toward the gold substrate.

Influence of the 524-VAAEIL-529 sequence of annexins A6 in their interfacial behavior and interaction with lipid monolayers

Annexin A6 (AnxA6), a calcium- and membrane-binding protein, is expressed in mammalian cells in two isoforms: AnxA6-1 and AnxA6-2, the latter lacking the 524-VAAEIL-529 sequence at the start of repeat 7. The different intracellular localization of these two isoforms suggests distinct function in membrane dynamics. The aim of this work was to analyze the behavior of AnxA6 isoforms at the air/water interface alone and in the presence of membrane mimicking lipid monolayers. Using Langmuir technique showed that AnxA6-2 was less adsorbed to the neat air–water interface than AnxA6-1 at acidic pH and minor differences in their PM-IRRAS spectra were observed. Both isoforms exhibited similar behavior towards cholesterol monolayer. However, the interactions of AnxA6-2 with cholesterol ester monolayer were most favorable compared to AnxA6-1. Our experimental data are discussed in relation with the different intracellular localization of the two isoforms and with our constructed model of AnxA6-2 with the known crystal structure of AnxA6-1 showing the persistence of the 516–529 α-helix in AnxA6-2 despite the absence of the 524-VAAEIL-529 sequence.

Chitosan as a subphase disturbant of membrane lipid monolayers. The effect of temperature at varying pH: II. DPPC and cholesterol

In this part of our work aimed at describing temperature-mediated effects of chitosan on membrane lipids in monolayers at different pHs, we studied the monolayers of dipalmitoylphosphatidylcholine (DPPC) and cholesterol. The lipids are typical components of eukaryotic cell membranes. The compression surface pressure–area (π–A) isotherms of the lipid monolayers were recorded in the Langmuir film balance, in the presence of chitosan dissolved in the subphase (0.1–1mg/mL) at different temperature (15–37°C) and pH conditions (3.5 and 6.0). Structural and thermodynamic parameters of the monolayers were estimated and compared to those of the pure lipid films. The overall work was performed with the general aim to examine if antibacterial activity of chitosan consists of the loosening of the bacterial membrane structure. It was ascertained in this study that unlike on DPPG monolayer, chitosan produced a minor disturbing effect on DPPC films. The data obtained suggest that the interactions in the system responsible for this weak effect should be primarily non-electrostatic. Further, the effect was found to be only slightly promoted by increasing temperature and importantly, it appeared to be a little stronger at pH 6.0 than at 3.5. This finding is of consequential significance for the possible application of chitosan as an antibacterial agent, which is because in addition to overall weak disturbance of DPPC monolayer compared to DPPG, the preferred condition for its occurrence is pH 6.0 which is opposite to that of DPPG at pH 3.5. Finally, the expansions of cholesterol monolayers were ascribed to the insertion of chitosan to the monolayer, an effect possible due to hydrophobic interactions and hydrogen bonding. Although not particularly prominent, these expansions are important in that they prove that hydrophobic interactions and hydrogen bonding play an important role in the cell membrane disturbing activity of chitosan.

Mixed DPPC–cholesterol Langmuir monolayers in presence of hydrophilic silica nanoparticles

Langmuir monolayers of Cholesterol (Chol) and a mixture of Chol with 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), at a ratio of 17:83 in weight, spread on pure water and on silica nanoparticle dispersions, have been investigated measuring the compression isotherms as well as the surface pressure response to harmonic area variation of the monolayer. Aim of this study was to evaluate the effects of the interaction of silica nanoparticles with Chol and the conditions for the incorporation in the monolayer. In previous works on different kind of lipid monolayers, it has been shown that hydrophilic silica nanoparticles dispersed in the sub-phase may transfer into the monolayer, driven by the interaction with the lipid molecules that make them partially hydrophobic. The results here obtained indicate that also for Chol and Chol–DPPC mixtures the presence of silica nanoparticles may have important effects on the phase behaviour and structural properties of the monolayer. As confirmed by complementary structural characterisations, BAM, AFM and ellipsometry, the principal effect of the nanoparticle incorporation is the disruption of the monolayer packing, owing to the alteration of the cohesive interactions of lipid components.

Accumulation of ordered ceramide-cholesterol domains in farber disease fibroblasts.

Farber disease is an inherited metabolic disorder caused by mutations in the acid ceramidase gene, which leads to ceramide accumulation in lysosomes. Farber disease patients display a wide variety of symptoms with most patients eventually displaying signs of nervous system dysfunction. We now present a novel tool that could potentially be used to distinguish between the milder and more severe forms of the disease, namely, an antibody that recognizes a mixed monolayer or bilayer of cholesterol:C16-ceramide, but does not recognize either ceramide or cholesterol by themselves. This antibody has previously been used to detect cholesterol:C16-ceramide domains in a variety of cultured cells. We demonstrate that levels of cholesterol:C16-ceramide domains are significantly elevated in fibroblasts from types 4 and 7 Farber disease patients, and that levels of the domains can be modulated by either reducing ceramide or cholesterol levels. Moreover, these domains are located in membranes of the endomembrane system, and also in two unexpected locations, namely, the mitochondria and the plasma membrane. This study suggests that the ceramide that accumulates in severe forms of Farber disease cells is sequestered to distinct membrane subdomains, which may explain some of the cellular pathology observed in this devastating lysosomal storage disease.

Unified treatment and measurement of the spectral resolution and temporal effects in frequency-resolved sum-frequency generation vibrational spectroscopy (SFG-VS)

The lack of understanding of the temporal effects and the restricted ability to control experimental conditions in order to obtain intrinsic spectral lineshapes in surface sum-frequency generation vibrational spectroscopy (SFG-VS) have limited its applications in surface and interfacial studies. The emergence of high-resolution broadband sum-frequency generation vibrational spectroscopy (HR-BB-SFG-VS) with sub-wavenumber resolution [Velarde et al., J. Chem. Phys., 2011, 135, 241102] offers new opportunities for obtaining and understanding the spectral lineshapes and temporal effects in SFG-VS. Particularly, the high accuracy of the HR-BB-SFG-VS experimental lineshape provides detailed information on the complex coherent vibrational dynamics through direct spectral measurements. Here we present a unified formalism for the theoretical and experimental routes for obtaining an accurate lineshape of the SFG response. Then, we present a detailed analysis of a cholesterol monolayer at the air/water interface with higher and lower resolution SFG spectra along with their temporal response. With higher spectral resolution and accurate vibrational spectral lineshapes, it is shown that the parameters of the experimental SFG spectra can be used both to understand and to quantitatively reproduce the temporal effects in lower resolution SFG measurements. This perspective provides not only a unified picture but also a novel experimental approach to measuring and understanding the frequency-domain and time-domain SFG response of a complex molecular interface.

Biochemical Identification of a Linear Cholesterol-Binding Domain within Alzheimer’s β Amyloid Peptide

Alzheimer's β-amyloid (Aβ) peptides can self-organize into amyloid pores that may induce acute neurotoxic effects in brain cells. Membrane cholesterol, which regulates Aβ production and oligomerization, plays a key role in this process. Although several data suggested that cholesterol could bind to Aβ peptides, the molecular mechanisms underlying cholesterol/Aβ interactions are mostly unknown. On the basis of docking studies, we identified the linear fragment 22-35 of Aβ as a potential cholesterol-binding domain. This domain consists of an atypical concatenation of polar/apolar amino acid residues that was not previously found in cholesterol-binding motifs. Using the Langmuir film balance technique, we showed that synthetic peptides Aβ17-40 and Aβ22-35, but not Aβ1-16, could efficiently penetrate into cholesterol monolayers. The interaction between Aβ22-35 and cholesterol was fully saturable and lipid-specific. Single-point mutations of Val-24 and Lys-28 in Aβ22-35 prevented cholesterol binding, whereas mutations at residues 29, 33, and 34 had little to no effect. These data were consistent with the in silico identification of Val-24 and Lys-28 as critical residues for cholesterol binding. We conclude that the linear fragment 22-35 of Aβ is a functional cholesterol-binding domain that could promote the insertion of β-amyloid peptides or amyloid pore formation in cholesterol-rich membrane domains.

Wetting and electrokinetic properties of cholesterol—Revisited

The important role of cholesterol in human body is well known. Therefore its properties have been investigated in many aspects. However, still some of its surface physicochemical properties are not well understood. Among them are wetting and electrokinetic properties. Therefore the aim of this paper was to study these properties and to compare them with the published literature data. For this purpose the advancing and receding contact angles of water, formamide and diiodomethane on the surface of cholesterol pellets and on its layers deposited on glass from its solution in chloroform were measured and then the surface free energy γS of the layers was calculated from van Oss’ (LWAB) and Chibowski's (CAH) models. Also the images of the surfaces from SEM, confocal and optical microscopes, and profilometer were obtained. It was found that the surface of cholesterol pellets is more hydrophobic than this of the deposited layers, even if up to 200 statistical monolayers have been deposited on glass surface. From the images it was seen that the deposited layers were not uniform and at a larger coverage the needle-like cholesterol crystals were formed on the surface. From the zeta potential of cholesterol as a function of pH in 10−3M NaCl was found that cholesterol shows the isoelectric point at pH 2.65, which is within the range of the literature data 1.8–3. The hydroxyl ions OH–are the potential determining and probably adsorb on OH groups of cholesterol molecules. In the pH range 3–6 the literature and our results of the negative zeta potential of cholesterol are convergent, except for those measured on a cholesterol suspension precipitated in water from its solution in 1-propanol. The zeta potential of silica particles covered with statistical monolayer of cholesterol lie between those of pure silica and cholesterol suspensions. The calculated electrokinetic charge at the cholesterol surface is very low, e.g. at pH 11, where ζ=−50mV, it amounts one excess negative charge (OH−ion) on the surface of 73 cholesterol molecules (σd=0.422μC/cm2). The results obtained in this paper partially confirm those obtained previously and bring new insight into the structures of cholesterol layers deposited on glass.

A Combined Vibrational Sum Frequency Generation Spectroscopy and Atomic Force Microscopy Study of Sphingomyelin–Cholesterol Monolayers

A combination of vibrational sum frequency generation spectroscopy and atomic force microscopy is used to study the changes in morphology and conformational order in monolayers prepared from three natural sphingomyelin (SM) mixtures as a function of surface pressure and cholesterol concentration. The most homogeneous SM gave monolayers with well-ordered acyl chains and few gauche defects with relatively small effects of either increasing surface pressure or cholesterol addition. Heterogeneous SM mixtures with a mixture of acyl chain lengths or with significant fractions of unsaturated acyl chains had much larger contributions from gauche defects at low surface pressure and gave increasingly well-ordered monolayers as the surface pressure increased. They also showed substantial increases in lipid chain order after cholesterol addition. Overall, these results are consistent with the strong hydrogen bonding capacity of SM leading to well-ordered monolayers over a range of surface pressures. The changes in acyl chain order for natural SMs as a function of cholesterol are relevant to formation of sphingolipid-cholesterol enriched domains in cell membranes.

The Equilibria of Sphingolipid-Cholesterol and Sphingolipid–Sphingolipid in Monolayers at the Air–Water Interface

Monolayers of sphingomyelin (SM), ceramide (Cer) and cholesterol (Ch) and binary mixtures SM–Ch, SM–Cer and Cer–Ch were investigated at the air–water interface. SM, Cer and Ch were used in the experiment. The surface tension values of pure and mixed monolayers were used to calculate π-A isotherms. Surface tension measurements were carried out at 22 °C using a Teflon trough and a Nima 9000 tensiometer. Interactions between sphingolipid and Ch as well as sphingolipid and another sphingolipid result in significant deviations from the additivity rule. An equilibrium theory to describe the behavior of monolayer components at the air–water interface was developed in order to obtain the stability constants and Gibbs free energy values of SM–Ch, SM–Cer and Cer–Ch complexes. We considered the equilibrium between the individual components and the complex and established that sphingolipid and Ch as well as sphingolipid and another sphingolipid formed highly stable 1:1 complexes.

Abstract 46: Cholesteryl Ester Transfer Protein Interactions with Lipoproteins: Insights into Mechanisms by Electron Microscopy and Molecular Dynamics Simulation

Cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids, including cholesteryl esters (CEs) and triglycerides (TGs), between HDL, LDL and VLDL. Lipoprotein particles contain a neutral lipid core composed of CE and TG surrounded by a surface monolayer of phospholipids (PL), free cholesterol (FC), and apolipoproteins, most notably, apo B-100 in LDL and VLDL and apo A-I in HDL. An elevated level of LDL-cholesterol (LDL-C) and/or a low level of HDL-cholesterol (HDL-C) in human plasma are major risk factors for cardiovascular disease (CVD). Since increased CETP can reduce HDL-C concentration and CETP deficiency is associated with elevated HDL-C levels, CETP inhibitors, including torcetrapib, anacetrapib and dalcetrapib have been investigated in clinical trials for treating CVD. Despite the intense clinical interest in CETP inhibition, little is known concerning the molecular mechanisms of CETP-mediated lipid transfer among lipoproteins, or even how CETP interacts with lipoproteins. CETP is a hydrophobic glycoprotein of 476 amino acids (∼53 kDa, before posttranslational modification). Its crystal structure reveals a banana-shaped molecule with N- and C-terminal β-barrel domains, a central β-sheet, and a ∼60 Å-long hydrophobic central cavity. Three CETP neutral lipid transfer hypotheses were proposed more than two decades ago: 1) a shuttle mechanism that involves CETP collecting CEs from one lipoprotein and delivering them through the aqueous phase to another lipoprotein; 2) a tunnel mechanism in which CETP bridges two lipoproteins forming a ternary complex, with lipids flowing from the donor to acceptor lipoprotein through the CETP molecule; and 3) a modified tunnel mechanism implicating a CETP dimer. One difficulty in investigating CETP mechanisms using structural methods is that interaction with CETP can alter the size, shape, and composition of lipoproteins, especially HDL. We validated an optimized negative-staining electron microscopy (NS-EM) protocol in which flash-fixation of lipoprotein particles preserves a near native-state conformation for direct visualization of individual molecular or macromolecular particles. We applied this protocol to study the mechanisms by which CETP interacts with spherical HDL, LDL and VLDL. Three-dimensional (3D) reconstructions of CETP, free and HDL-bound, were obtained by single-particle techniques. In addition, we used inhibitory CETP antibodies to identify the regions of CETP that interact with HDL and LDL. Finally molecular dynamics (MD) simulation was used to assess the molecular mobility of CETP and predict the likely conformational changes that are associated with lipid transfer. We discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into HDL and its C-terminal domain interacting with LDL or VLDL. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.

Second Harmonic Generation to Monitor the Interactions of the Antimicrobial Mycosubtilin with Membrane-Mimicking Interfacial Monolayers

Mycosubtilin is a strong antimicrobial agent belonging to the iturinic lipopeptide family which contains a single tyrosine residue. Its cell target has been shown to be the cytoplasmic membrane. This tyrosine residue has been previously shown to be essential for the biological activity of mycosubtilin. Since we have previously demonstrated that tyrosine, an aromatic amino acid, can be used as an endogenous probe for the frequency doubling process, the presence of a tyrosine residue in mycosubtilin allowed us to investigate the interactions of mycosubtilin with biomimetic lipid monolayers at the air–water interface by second harmonic generation (SHG). Mycosubtilin was added underneath dipalmitoylphosphatidylcholine or cholesterol monolayers at the air–water interface and significant increases in the surface pressure were observed in both cases. This observation demonstrates that mycosubtilin interacts with these biomimetic membranes. A light polarization resolved analysis of the SHG signals recovered for these two systems was then performed and confirmed that those interactions between the tyrosine residue in mycosubtilin and the membranes could be monitored by SHG. Furthermore, the differences exhibited by the nonlinear optical measurements for different membranes showed that these interactions depend on the nature of the biomimetic membrane present at the air–water interface.

The Equilibria of Lipid–K+ Ions in Monolayer at the Air/Water Interface

The effect of K+ ion interaction with monolayers of phosphatidylcholine (lecithin, PC) or cholesterol (Ch) was investigated at the air/water interface. We present surface tension measurements of lipid monolayers obtained using a Langmuir method as a function of K+ ion concentration. Measurements were carried out at 22°C using a Teflon trough and a Nima 9000 tensiometer. Interactions between lecithin and K+ ions or Ch and K+ ions result in significant deviations from the additivity rule. An equilibrium theory to describe the behavior of monolayer components at the air/water interface was developed in order to obtain the stability constants and area occupied by one molecule of lipid–K+ ion complex (LK+). The stability constants for lecithin–K+ ion (PCK+) complex, K_{{{text{PCK}}^{ + } }} = { 3}. 2 6times 10^{ 2} {text{dm}}^{ 3} ,{text{mol}}^{ - 1} , and for cholesterol–K+ ion (ChK+) complex, K_{{{text{ChK}}^{ + } }} = { 1}.00 times 10^{ 3} {text{dm}}^{ 3} ,{text{mol}}^{ - 1} , were calculated by inserting the experimental data. The value of area occupied by one PCK+ complex is 60 Å2 molecule−1, while the area occupied by one ChK+ complex is 40.9 Å2 molecule−1. The complex formation energy (Gibbs free energy) values for the PCK+ and ChK+ complexes are −14.18 ± 0.71 and −16.92 ± 0.85 kJ mol−1, respectively.

Probing the Modes of Antibacterial Activity of Chitosan. Effects of pH and Molecular Weight on Chitosan Interactions with Membrane Lipids in Langmuir Films

Chitosan, a cationic biopolymer derived from chitin, has been described as having antibacterial activity. The modes of this activity, however, have not been established. One mode proposed is that chitosan perturbs bacterial cell membranes. To validate this proposal, in this study we investigated chitosan interactions with lipids in Langmuir monolayers as model membranes. The interactions were assessed by monitoring differences in the shape of the compression isotherms measured in the absence and presence of chitosan in the subphase (acetate buffer). To appraise the contribution of electrostatic interactions versus hydrogen bonding and hydrophobic interactions, three membrane lipids differing in charge were studied-anionic dipalmitoylphosphatidylglycerol (DPPG), zwitterionic dipalmitoylphosphatidylcholine (DPPC), and neutral cholesterol-and the pH of the subphase was varied between 3.5 and 6.0. In addition, the impact of the molecular weight of chitosan on the interactions was assessed at pH 3.5. It was found that while chitosan had a negligible effect on DPPC monolayers over the pH range studied, it distinctly affected DPPG and cholesterol monolayers. The effect on DPPG was found to decrease with increasing pH, that at pH 3.5 being ascribed to the charge-mediating action of chitosan on the local ionic environment and that at higher pHs to the intercalation of chitosan to the monolayers. Practically independent of pH, the effect of chitosan on cholesterol was accounted for by the formation of cholesterol-chitosan hydrogen bonds. Chitosan of lower molecular weight facilitated the interactions with all the three lipids studied. The results obtained may be helpful in identifying the mode of antibacterial activity of chitosan versus other modes that involve the disturbance of cell life cycles.