Raman spectroscopic investigation of the interaction of gramicidin A with dipalmitoyl phosphatidylcholine liposomes

The interaction of gramicidin A with dipalmitoyl phosphatidylcholine liposomes is investigated by Laser-Raman spectroscopy. As revealed by the methylene CH stretching mode the phase transition of the hydrocarbon chains near 40°C is eliminated in the presence of gramicidin A. Liposomes prepared from a mixture of lecithin and cholesterol seem to be unaffected by gramicidin A and show only the normal broadened phase transition.     

Raman spectroscopic investigation of the interaction of gramicidin A with dipalmitoyl phosphatidylcholine liposomes.

The interaction of gramicidin A with dipalmitoyl phosphatidylcholine liposomes is investigated by Laser-Raman spectroscopy. As revealed by the methylene C-H stretching mode the phase transition of the hydrocarbon chains near 40 degree C is eliminated in the presence of gramicidin A. Liposomes prepared from a mixture of lecithin and cholesterol seem to be unaffected by gramicidin A and show only the normal broadened phase transition.     

Interaction kinetics of salmeterol with egg phosphatidylcholine liposomes by surface plasmon resonance

Surface plasmon resonance (SPR) Biacore™ and equilibrium dialysis were applied to investigate the membrane affinities of salmeterol and propranolol and the kinetic interactions of salmeterol with egg phosphatidylcholine liposomes. The two methods revealed similar affinity values; however, they were dependent on the investigated drug concentrations. The kinetic experiments with salmeterol were optimized to obtain pseudo-first-order kinetics that were independent of the drug concentration. The adsorption and desorption phases followed biexponential functions up to pH 8.8 and mono or biexponential functions at higher pH values (i.e., between the two pK_a values). The apparent rate constants of the faster phases of the biexponential functions were beyond the time resolution of the instrument in most measurements. The apparent rate constants of the slower phases ranged from 0.01 to 0.03 s⁻¹ and were pH independent between pH 5.0 and pH 8.0. The rates of the monoexponential kinetics were between 0.08 and 0.12 s⁻¹. We conclude that the biexponential kinetics at physiological pH reflect the partitioning into the outer lipid leaflet and “flip-flop,” respectively, of the cationic species.     

Interaction of phosphatidylcholine liposomes and plasma lipoproteins with sheep erythrocyte membranes: Preferential transfer of phosphatidylcholine containing unsaturated fatty acids

The interaction of sheep erythrocyte membranes with phosphatidylcholine vesicles (liposomes) or human plasma lipoproteins is described. Isolated sheep red cell membranes were incubated with liposomes containing [¹⁴C]phosphatidylcholine or [^3H]phosphatidylcholine in the presence of EDTA. A time-dependent uptake of phosphatidylcholine into the membranes could be observed. The content of this phospholipid was increased from 2 to 5%. The rate of transfer was dependent on temperature, the amount of phosphatidylcholine present in the incubation mixture and on the fatty acid composition of the liposomal phosphatidylcholine. A possible adsorption of lipid vesicles to the membranes could be monitored by adding cholesteryl [¹⁴C]oleate to the liposomal preparation. As cholesterylesters are not transferred between membranes [1], it was possible to differentiate between transfer of phosphatidylcholine molecules from the liposomes into the membranes and adsorption of liposomes to the membranes. The phosphatidylcholine incorporated in the membranes was isolated, and its fatty acids were analysed by gas chromatography. It could be shown that there was a preferential transfer of phosphatidylcholine molecules containing two unsaturated fatty acids.     

Permeability of dimyristoyl phosphatidylcholine/dipalmitoyl phosphatidylcholine bilayer membranes with coexisting gel and liquid-crystalline phases.

The passive permeation of glucose and a small zwitterionic molecule, methyl-phosphoethanolamine, across two-component phospholipid bilayers (dimyristoyl phosphatidylcholine (DMPC)/dipalmitoyl phosphatidylcholine (DPPC) mixtures) exhibit a maximum when gel domains and fluid domains coexist. The permeability data of the two-phase bilayers cannot be fitted to single-rate kinetics, but are consistent with a Gaussian distribution of rate constants. In pure DMPC and DPPC as well as in their mixtures, at the temperature of the maximum excess heat capacity, the logarithm of the average permeability rate constants are linearly correlated with the mole fraction of DPPC in the total system. In addition, in the 50:50 mixture, the excess heat capacity values as well as the apparent fractions of interfacial lipid correlate with the logarithm of the excess permeabilities in the two-phase region. These results suggest that small polar molecules can cross the membrane at the interface between gel and fluid domains at a much faster rate than through the homogeneous phases; the acyl chains located at the domain interface experience lateral density fluctuations that are inversely proportional to their average length, and large enough to allow rapid transmembrane diffusion of the solute molecules. The distribution of the permeability rate constants may reflect temporal and spatial fluctuations of the lipid composition at the phase boundaries.     

Interaction of melittin with dimyristoyl phosphatidylcholine liposomes. Evidence for boundary lipid by raman spectroscopy

The interaction of melittin, a polypeptide consisting of 26 amino acid residues, with dimyristoyl phosphatidylcholine bilayers was investigated by vibrational Raman spectroscopy. Spectral peak height intensity ratios, involving vibrational transitions in both the 3000 cm^?1 acyl chain methylene carbon-hydrogen stretching mode region and the 1100 cm^?1 acyl chain carbon-carbon skeletal stretching mode interval, served as temperature profile indices for monitoring the bilayer order-disorder processes. For a lipid : melittin molar ratio of 14 : 1 two order-disorder transitions were observed. In comparison to a gel to liquid crystalline phase transition of 22.5°C for the pure lipid, the lower transition, exhibiting a 2°C width, is centered at 17°C and is associated with a depression of the main lipid phase transition of dimyristoyl phosphatidylcholine. The second thermal transition, displaying a 7°C interval, occurs at approx. 29°C and is associated with the melting behavior of approximately seven immobilized boundary lipids which surround the inserted hydrophobic segment of the polypeptide. For a lipid : melittin molar ratio of 10 : 1 two thermal transitions are also observed at 11 and 30°C. As before, they represent, respectively, the main gel to liquid crystalline phase transition and the melting behavior of approximately four boundary lipids attached to melittin. From these data alternative schemes are suggested for disposing the immobilized lipids around the hydrophobic portion of the polypeptide within the bilayer.     

A pulsed N.M.R study of D2O bound to 1,2 dipalmitoyl phosphatidylcholine

Spin lattice relaxation times in both the lab and rotating frame, have been measured for deuterons (²H) in a number of unsonicated dispersions of 1,2 dipalmitoyl phosphatidylcholine in D₂O over a range of resonant frequencies from 13 MHz to 1 MHz for temperatures from ?20°C to 65°C.The proton (¹H) spin lattice relaxation time for the lecithin was measured for resonant frequencies of 8.5 MHz, and 40 MHz over a similar range of temperatures.The results agree with broadline measurements by Salsbury et al. [1], and for the liquid crystal phase are consistent with an anisotropic tumbling model of the water molecules bound to the lecithin headgroup. This tumbling occurs with correlation times of ≤10^?10 sec and ≈ 10^?6 sec about axes parallel to and perpendicular to the bisector of the D-O-D angle within a D₂O molecule, hydrogen bonded to the negatively charged phosphate headgroup.     

Physicochemical properties of dipalmitoyl phosphatidylcholine after interaction with an apolipoprotein of pulmonary surfactant

We studied the interaction between an apolipoprotein of pulmonary surfactant and the principal lipid found in this material, dipalmitoyl phosphatidylcholine. The apolipoprotein was extracted from canine surfactant and purified to greater than 90% homogeneity. The apolipoprotein was mixed for 16 h at room temperature with dipalmitoyl phosphatidylcholine dispersed in a buffer containing 0.1 M NaCl and 3mM CaCl2. Unbound lipid, unbound protein, and recombinants of lipid and protein were separated by density gradient centrifugation. 71% of the apolipoprotein was found associated with dipalmitoyl phosphatidylcholine. In comparable experiments using bovine plasma albumin about 13% of the albumin was recovered with the lipid. The physicochemical state of the lipid in the apolipoprotein-lipid complex was modified after binding of the protein. A distinct phase transition at 42 degrees C could no longer be detected, and the rate of adsorption to an air-liquid interface of the apolipoprotein-lipid complex was greater than that of the lipid alone. Surface tension vs. surface area isotherms of the dipalmitoyl phosphatidylcholine-apolipoprotein materials, however, were similar to those exhibited by pure dipalmitoyl phosphatidylcholine. The results suggest a physiological role for this apolipoprotein. It may bind to dipalmitoyl phosphatidylcholine under conditions expected in vivo, and may modify the physical properties of the aggregated dipalmitoyl phosphatidylcholine to form domains of lipid in a liquid-crystalline array. The complex dipalmitoyl phosphatidylcholine and apolipoprotein would have the physical properties necessary for its physiological function, allowing it to absorb to the alveolar interface and reduce its surface tension to less than 10 dynes/cm. Dipalmitoyl phosphatidylcholine, by itself, is in a gel-crystalline array below its phase transition temperature (42 degrees C) and would be incapable of effecting these actions.     

Effect of N-alkyl-N,N,N-trimethylamonium bromides on the auto-peroxidation of egg yolk phosphatidylcholine in liposomes.

N-alkyl-N,N,N-trimethylamonium bromides (cnTMA, n = number of carbons in alkyl) stimulate and inhibit the autoperoxidation of egg yolk phosphatidylcholine (EYPC) in liposomes at n less than 12 and n greater than 12, respectively, with maximum stimulation for n = 8. CnTMA intercalate between EYPC molecules (decreasing the yield of ROO. + RH----ROOH+R. reaction, where RH is an unsaturated EYPC acyl chain, R. - EYPC acyl radical, and ROO. - peroxy radical of the EYPC acyl chain) and disorder the hydrophobic region of the bilayer (increasing the oxygen solubility there and thus yield of R. + O2----ROO. reaction). The final level of oxidation is affected by a summation of the EYPC lateral separation and disordering effects.     

The interaction of Bacillus protoplasts with sonicated phosphatidylcholine liposomes

When protoplasts from Bacillus subtilis are incubated with sonicated liposomes made from egg-yolk phosphatidylcholine, this phospholipid is incorporated into the protoplast membranes. Biochemical, fluorescence and ultrastructural data suggest that incorporation occurs through membrane fusion.     

Monodomain samples of dipalmitoyl phosphatidylcholine with varying concentrations of water and other ingredients.

Methods are presented for the preparation of large monodomain phospholipid bilayer arrays containing variable amounts of water approaching the two-phase limit. The optical birefringence of these lamellar phases of dipalmitoyl phosphatidylcholine (DDPC) is measured over a range of temperature and water content, and phase transitions are observed. The techniques employed for pure DPPC and water are extended in order to produce macroscopically aligned samples containing varying concentrations of cholesterol, inorganic salts, antibiotics, and chlorophyll a. Polarization studies of the 670-nm band of chlorophyll a indicate macroscopic orientational order in the chromophore under the same conditions.     

Molecular structure and interaction of dipalmitoyl phosphatidylcholine in multilayers. Comparative study with phosphatidylethanolamine

As a model of phospholipid bilayers in solid an oriented multilayer film (built-up film) of L-α-dipalmitoyl phosphatidylcholine (DPPC) was prepared from the monolayer by the dipping method. Structural analysis has been carried out by measuring infrared dichroism of the built-up film. The results were compared with those of the built-up film of L-α-dipalmitoyl phosphatidylethanolamine (DPPE). The tilting of the hydrocarbon chains is larger for DPPC than for DPPE. The orientation of the bisector of the two non-esterified PO bonds is closer to the film plane for DPPC than for DPPE. The strong hydrogen bonding interaction between the polar head groups was shown for DPPE, but not for DPPC. These features resemble the structural differences between dilauroyl phosphatidylethanolamine (DLPE) and dimyristoryl phosphatidylcholine (DMPC) in crystals. The hydrogen bonding interaction of DPPE found in solid remains even in the presence of water, namely, in the gel state. More closed packing of the hydrocarbon chains of solid DPPE than DPPC in solid was concluded on the basis of infrared and Raman spectra.     

Analysis of dipalmitoyl phosphatidylcholine in amniotic fluid by high-performance liquid chromatography

A novel method for the determination of dipalmitoyl phosphatidylcholine (DPPC) in amniotic fluid by high-performance liquid chromatography (HPLC) is described. Aliquots of 50 μl of amniotic fluid were hydrolyzed with phospholipase C from Bacillus cereus and the resulting dipalmitoylglycerol analyzed by HPLC. Run-to-run and day-to-day precision for DPPC analysis were 4.2 and 6.1%, respectively, and analysis time was 10 min. Recoveries for DPPC ranged between 92 and 98%. In summarizing, this method provides a high precision and fast turnaround time means for the analysis of DPPC in amniotic fluid.     

Pulse NMR study of phase transitions in dipalmitoyl phosphatidylcholine multilayer systems

Pretransition and main transition of aqueous dipalmitoyl phosphatidylcholine (DPPC) dispersions were investigated by pulse NMR. The second moment M2 inter of the proton absorption line shows significant changes at 42 degrees C and about 35 degree C. Over the whole investigated temperature range between 25 and 50 degree C a superposition of at least two distinct second moments assigned to different molecular regions was observed.     

Interaction of ferricytochrome c with liposomes

The binding of ferricytochrome c to liposomes consisting of phosphatidylcholine mixtures with cardiolipin (3:1) or phosphatidylserine (3:1) has been investigated. Experimental data have been analyzed in terms of two-dimensional models of large ligand adsorption. The equilibrium parameters of ferricytochrome c interaction with a phospholipid bilayer are determined.     

Interaction of isopropylthioxanthone with phospholipid liposomes

Isopropylthioxanthone (ITX) is a highly lipophilic molecule which can be released in foods and beverages from the packages, where it is present as photoinitiator of inks in printing processes. Recently it was found in babies milk, and its toxicity cannot be excluded. The structure of the molecule suggests a possible strong interaction with the lipid moiety of biological membranes, and this is the first study of its effects on phospholipid organization, using differential scanning calorimetry (DSC) and spin labelling techniques. The data obtained with multilamellar liposomes of saturated phospholipids of different length, with and without cholesterol, point out that the molecule changes the lipid structure; in particular, in the gel state, behaving like a disordering agent it increases the mobility of the bilayer, while, in the fluid state, tends to rigidify the membrane, in a cholesterol like way. This behavior supports the hypothesis that ITX experiences a relocation process when the lipid matrix passes from the gel to the fluid state.     

Interaction of alpha-chymotrypsin with dimyristoyl phosphatidylcholine vesicles

The amidolytic activity of chymotrypsin for Suc-Ala2-Pro-Phe-MCA was somewhat enhanced by dimyristoyl PC at low ionic strength, but not at high ionic strength. The activity was strongly inhibited by pure egg yolk PA. The inhibition by 200 ng PA was neutralized by addition of 1 microgram dimyristoyl PC or pure egg yolk PC, which formed vesicles with the PA. The Km and kcat (s-1) values of chymotrypsin for hydrolysis of Suc-Ala2-Pro-Phe-MCA changed from 15 microM to 42 microM, 0.1 mM and 0.5 mM, and from 1.5 to 2.7, 3.7, and 1.0 in the presence of 1 microgram dimyristoyl PC, 0.5 micrograms pure egg yolk PE and 0.2 microgram egg yolk PA, respectively. Gel-filtration chromatography showed that dimyristoyl PC formed a complex with chymotrypsin, but did not interact with the substrate, indicating that the basic globular protein, chymotrypsin, interacted with net-neutral PL.     

Fatty-acid chain tilt angles and directions in dipalmitoyl phosphatidylcholine bilayers.

X-ray diffraction has been applied to determine the various tilt angles and directions (if any) which can be assumed by oriented gel phase multilayers of dipalmitoyl phosphatidylcholine (DPPC) as a function of hydration. We report for the first time that oriented DPPC multilayers with a repeat spacing (d-spacing) of 55.2A at 25 degrees C and 0% relative humidity (RH) have hydrocarbon chains tilted at an angle theta of 21.5 degrees with respect to the bilayer normal. In addition, the chains are tilted along one of the bisectors (omega = 0 degrees) of the hexagonal lattice (8 wide-angle maxima, 2 unique), a phase not previously reported in DPPC studies. At 100% RH, the chain tilt angle and d-spacing increased to approximately 29.0 degrees and 58.9A, respectively. Since at 100% RH only 4 wide-angle maxima are observed, we analyze the data on the assumption that the hydrocarbon chains may rotate independently of the hexagonal lattice (omega = 0-30 degrees), at a fixed chain tilt angle theta (Stamatoff, J.B., et al. 1979. Biophys. J. 25:253-262). The largest observed angle phi made by the wide-angle maxima with the equator is 29.5 degrees corresponding to a theta of approximately 32.6 degrees (omega avg. = 24 degrees) and the sample having a d-spacing of 64.0 A (excess water condition). Finally, theta remains relatively constant (approximately 21.5 degrees) up to a RH of approximately 45% and a d-spacing of 57.8A, after which, with increases in RH, theta increases to a maximum of 32.6 degrees.     

Synthesis and interfacial behavior of sulfur-containing analogs of lung surfactant dipalmitoyl phosphatidylcholine

Synthesis methods and initial surface property characterizations are reported for two sulfur-containing phosphonolipids related structurally to dipalmitoyl phosphatidylcholine (DPPC), the major lung surfactant glycerophospholipid. Sulfur linkages in these compounds affect molecular interactions relative to ester linkages, and are structurally resistant to cleavage by phospholipases. The SO₂-linked analog synthesized here had increased adsorption and improved film respreading compared to DPPC, while reaching very low surface tensions (1 mN/m) in cycled interfacial films on both the Wilhelmy balance and the pulsating bubble surfactometer. This compound appears to have potential utility as a component in future phospholipase-resistant synthetic exogenous surfactants for treating clinical forms of inflammatory lung injury.