Interaction of phospholipid membranes with poly(ethylene glycol)s

1. The water-soluble polymer, poly(ethylene glycol), causes concentration-dependent increases in the temperature of the gel--liquid crystalline phase transitions of aqueous dispersions of dipalmitoyl phosphatidylcholine and of dipalmitoyl phosphatidylethanolamine. 2. For dipalmitoyl phosphatidylcholine it has been further demonstrated that poly(ethylene glycol) increases the transition enthalpy and entropy while decreasing the cooperativity of the transition. 3. These results are discussed in relation to the possible modes of action of poly(ethylene glycol) in promoting cell fusion.     

Interaction of furosemide with lipid membranes

Summary The interaction of furosemide with different phospholipids was investigated. Its influence on the lipid structure was inferred from its effect on the phase transition properties of lipids and on the conductance of planar bilayer membranes. The thermotropic properties of dipalmitoyl phosphatidylcholine, phosphatidylethanolamine (natural), dipalmitoyl phosphatidylethanolamine, brain sphingomyelin, brain cerebrosides and phosphatidylserine in the presence and absence of furosemide were investigated by differential scanning calorimetry,. The modifying effect of furosemide seems to be strongest on phosphatidylethanolamine (natural) and sphingomyelin bilayers. The propensity of furosemide to decrease the electrical resistance of planar lipid membranes was also studied and it is shown that the drug facilitates the transport of ions. Partition coefficients of furosemide between lipid bilayers and water were measured.Abbreviations DSC differential scanning calorimetry - PLM planar lipid membranes - DPPC dipalmitoyl phosphatidylcholine - DMPC dimyristoyl phosphatidylcholine - PE phosphatidyl ethanol     

Negatively charged phospholipids and their position in the cholesterol affinity sequence

The effects of low concentrations of cholesterol in mixtures of a negatively charged phospholipid (phosphatidylserine or phosphatidylglycerol) and another phospholipid (phosphatidylcholine, sphingomyelin or phosphatidylethanolamine) have been studied by differential scanning calorimetry. Only mixtures which showed a gel phase miscibility gap have been employed. It was demonstrated that in mixtures with phosphatidylethanolamine, cholesterol was preferentially associated with the negatively charged phospholipid, regardless whether this species represented the component with the high or with the low transition temperature in the mixture. In mixtures of a negatively charged phospholipid and phosphatidylcholine, cholesterol associated with the negatively charged phospholipid; when the phosphatidylcholine was the species with the low transition temperature, cholesterol had an affinity for the phosphatidylcholine and for the negatively charged phospholipid as well. Cholesterol, in a mixture of sphingomyelin with a high and phosphatidylserine with a low transition temperature, was preferentially associated with sphingomyelin.From these experiments it is concluded that phospholipids show a decrease in affinity for cholesterol in the following order: sphingomyelin ? phosphatidylserine, phosphatidylglycerol > phosphatidylcholine ? phosphatidylethanolamine.     

A calorimetric study of the thermotropic behaviour of 1,2-dipentadecylmethylidene phospholipids

Differential scanning calorimetry was used to study the thermotropic behaviour of 1,2-dipentadecylmethylidene phospholipids with various head groups. The structural variation in the glycerol backbone region leads to a strong restriction of conformational freedom for the first two methylene segments of the chains, so that dipentadecylmethylidene phospholipids show lower transition temperatures, lower enthalpies and lower cooperativity of the transition from the gel to the liquid crystalline phase. The extreme chemical stability of these lipids in the alkaline pH region enables investigations of phosphatidylethanolamine and phosphatidic acid dispersions at high pH values. Both phospholipids show a decrease in the transition temperature and in the transition enthalpy as they become singly and doubly charged, respectively. A complex behaviour of the transition enthalpy of doubly charged 1,2-dipentadecylmethylidene phosphatidic acid was observed when the NaCl concentration of the dispersion was increased.     

The effect of hashish compounds on phospholipid phase transition

The interaction of hashish compounds, Δ¹-tetrahydrocannabinol and cannabidiol, with dipalmitoyl phosphatidylcholine was investigated using differential scanning calorimetry. Both drugs affect the transition of dipalmitoyl phosphatidylcholine from the gel to liquid crystalline state, decreasing both the melting temperature and the enthalpy of melting. At a drug to dipalmitoyl phosphatidylcholine ratio of approx. 1:5, two peaks appear in the transition profile, suggesting a phase separation in the drug dipalmitoyl phosphatidylcholine mixture.     

Interactions between phospholipids and barbiturates.

The effects of a number of barbiturates on the temperature of the lipid phase transition have been studied using chlorophyll a as a fluorescence probe. The barbiturates cause a reduction in the temperature of the phase transitions of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylethanolamine, the effects being greatest at lower pH values where more of the barbiturate is present in the uncharged form. There was no significant interaction between the barbiturates and dipalmitoyl phosphatidylserine. These and other observations on the actions of local anaesthetics are used to develop a model for local anaesthesia. It is suggested that the sodium channel is surrounded by an annulus of lipid in the gel state, this rigid microenvironment preventing the sodium channel relaxing from its active configuration to an inactive one. Local anaesthetics, which reduce the temperature of lipid phase transitions, trigger a change of the annular lipid from the gel to the liquid-crystalline state, with a consequent relaxation of the sodium channel to an inactive configuration, in which the sodium current is reduced or blocked.     

The effect of dolichol on the structure and phase behaviour of phospholipid model membranes

The effect of dolichol C₉₅ on the structure and thermotropic phase behaviour of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine and stearoyloleoylphosphatidylethanolamine has been examined by synchrotron X-ray diffraction and differential scanning calorimetry. The presence of dolichol C₉₅ had no detectible effects on the temperature of either the gel to ripple or the ripple to liquid-crystal phase transition of dipalmitoylphosphatidylcholine. A proportionate increase of a few degrees in the temperature of the gel to lamellar liquid-crystal phase transition is observed in dispersions of dipalmitoylphosphatidylethanolamine and significantly there is a decrease in the temperature of the lamellar to non-lamellar phase transition of stearoyloleoylphosphatidylethanolamine. There was no significant change in the bilayer repeat spacing of all three mixed dispersions in gel phase in the presence of up to 20 mol% dolichol C₉₅. Electron density calculations showed that there was no change of bilayer thickness of dipalmitoylphosphatidylcholine with incorporation of up to 7.5 mol% dolichol C₉₅. These data suggest that effect of dolichol on the phospholipid model membranes depend on both the head group and the hydrocarbon chains of the phospholipid molecules. The presence of dolichol in phosphatidylcholine bilayers conforms to a model in which the polyisoprene compound is phase separated into a central domain sandwiched between the two monolayers in gel phase. In bilayers of phosphatidylethanolamines dolichol tends to stabilize the bilayers in gel phase at low temperatures and destabilize the bilayers in lamellar disordered structure at high temperatures. Non-lamellar structures coexist with lamellar disordered phase over a wide temperature range suggesting that dolichol is enriched in domains of non-lamellar structure and depleted from lamellar phase. These findings are useful to understand the function of dolichol in cell membranes.     

Effect of melittin on thermotropic lipid state transitions in phosphatidylcholine liposomes.

We have examined the Raman scattering due to CH stretching vibrations, as well as to v(-C=C-) and v(=C-C=) of beta-carotene, of liposomes composed of phosphatidylcholine (egg, dimyristoyl, dipalmitoyl) +/- cholesterol, beta-carotene or melittin in the temperature range of -10 degrees C to 45 degrees C. (2) Plots vs. temperature of the intensities of the 2885 cm-1 and 2930 cm-1 CH stretching bands relative to the intensity of the thermally stable 2850 cm-1 band, i.e. the I2885/I2850 and I2930/I2850 ratios, reveal a sharp discontinuity in cholesterol-free phosphatidylcholine liposomes; this coincides with the gel leads to liquid-crystal transition temperature of the fatty acyl chains. In cholesterol/phosphatudylcholine liposomes the change in I2885/I2850 occurs over a very broad temperature range and I2930/I2850 remains stable. (3) I1527/I1158, i.e. the intensity of v(-C=C-) relative to that of v(=C-C-) in beta-carotene/phosphatidylcholine liposomes, changes discontinuously at the gel leads to liquid-crystal transition temperature. The values above the transition temperature approach those of the carotenoid in organic solution. (4) The transitions reported in I2885/I2850 for phosphatidylcholine/melittin liposomes (25-56; 1, M/M) are shifted to much higher temperatures than observed in phosphatidylcholine liposomes. In the case of dimyristoyl phosphatidylcholine/melittin the changes in I2930/I2850 also occurs at a higher temperature (28 degrees C) than without melittin (21 degrees C), but the temperature shift is less than the +13 degrees C observed for I2885/I2850. It appears that the apolar moiety of melittin organizes phospholipids adjacent to and more remote from the peptide moiety, to form complexes with an elevated lipid transition temperature. The effect of the peptide moiety is greater on the methylene segments (I2885/I2850) than on the methyl termini (I2930/I2850).     

Nuclear magnetic resonance and thermal studies on the interaction between salicylic acid and model membranes

DSC and (1H and 31P) NMR measurements are used to investigate the perturbation caused by the keratolytic drug, salicylic acid (SA) on the physicochemical properties of the model membranes. Model membranes (in unilamellar vesicular (ULV) form) in the present studies are prepared with the phospholipids, dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylethanolamine (DPPE), dipalmitoyl phosphatidic acid (DPPA) and mixed lipid DPPC-DPPE (with weight ratio, 2.5:2.2). These lipids have the same acyl (dipalmitoyl) chains but differed in the headgroup. The molar ratio of the drug to lipid (lipid mixture), is in the range 0 to 0.4. The DSC and NMR results suggest that the lipid head groups have a pivotal role in controlling (i) the behavior of the membranes and (ii) their interactions with SA. In the presence of SA, the main phase transition temperature of (a) DPPE membrane decreases, (b) DPPA membrane increases and (c) DPPC and DPPC-DPPE membranes are not significantly changed. The drug increases the transition enthalpy (i.e., acyl chain order) in DPPC, DPPA and DPPC-DPPE membranes. However, the presence of the drug in DPPC membrane formed using water (instead of buffer), shows a decrease in the transition temperature and enthalpy. In all the systems studied, the drug molecules seem to be located in the interfacial region neighboring the glycerol backbone or polar headgroup. However, in DPPC-water system, the drug seems to penetrate the acyl chain region also.     

Physicochemical characterization of 1,2-diphytanoyl-sn-glycero-3-phosphocholine in model membrane systems

We report here on a series of studies aimed at characterization of the structural and dynamical properties of the synthetic lipid diphytanoyl phosphatidylcholine, in multilamellar dispersions and vesicle suspensions.This lipid exhibits no detectable gel to liquid crystalline phase transition over a large temperature range (?120°C to +120°C).Examination of proton nuclear magnetic resonance (NMR) free induction decays obtained from multilayer dispersions of diphytanoyl phosphatidylcholine provided an estimate of the methylene proton order parameter. The estimated magnitude of 0.21 is comparable to those determined for other phospholipids.Sonication of aqueous dispersions of diphytanoyl phosphatidylcholine led to formation of bilayer vesicles as determined by the measurement of the outer/inner choline methyl proton resonances, vesicle sizes in electron micrographs, and comparison of proton NMR linewidths between multilayer and sonicated dispersions. Ultracentrifugation studies of diphytanoyl phosphatidylcholine vesicles in H²O and ²H₂O media yielded a value of 1.013 ± 0.026 ml/g for the partial specific volume of this lipid.We have measured spin lattice relaxation rates for the methyl and methylenemethyne protons of the hydrocarbon chains of diphytanoyl phosphatidylcholine in bilayer vesicles over a range of temperatures and at two NMR frequencies (100 and 220 MHz). The observed relaxation rates for the methylene protons in this system were approximately twice those previously reported for dipalmitoyl phosphatidylcholine at comparable temperatures and resonance frequencies, whereas the relaxation rates measured for the methyl protons were greater than those of the straight chain lipid by an order of magnitude.Measurement of the spin lattice relaxation rates of the hydrocarbon protons of the diphytanoyl phosphatidylcholine in a 10 mol% mixture of the branched-chain lipid in a deuterated host lipid, diperdeuteropalmitoyl phosphatidylcholine, showed a discontinuity in the temperature dependence of the proton NMR longitudinal relaxation rates of the branched-chain lipid in the region of the gel to liquid crystalline phase transition temperature of the deuterated dipalmitoyl phosphatidylcholine host lipid. This result may be taken as evidence of lateral phase separation of a liquid cyrstalline phase enriched in diphytanoyl phosphatidylcholine from a gel phase enriched in diperdeuteropalmitoyl phosphatidylcholine at temperatures below the phase transition temperature of deuterated host lipid. This conclusion is supported by the observation of an abrupt change in the hydrocarbon methylene linewidth (at 100 MHz) of 10 mol% diphytanoyl phosphatidylcholine in diperdeuteropalmitoyl phosphatidylcholine over the temperature range where lateral phase separation is taking place according to differential thermograms.     

The interaction of cholesterol with bilayers of phosphatidylethanolamine

The interaction of cholesterol with the glycerol backbone segments of phospholipids was studied in bilayers of phosphatidylethanolamine containing equimolar amounts of cholesterol. Glycerol selectively deuterated at various positions was supplied to the growth medium of Escherichia coli strain 131 GP which is defective in endogeneous glycerol synthesis. The procedure enables the stereospecific labeling of the three glycerol backbone segments of the membrane phospholipids. Phosphatidylethanolamine with wild-type fatty acid composition was purified from E. coli cells and deuterium magnetic resonance spectra were obtained either from dispersions of pure phosphatidylethanolamine or from equimolar mixtures of phosphatidylethanolamine with cholesterol. For comparative purposes 1,2-di[9,10-²H₂]elaidoyl-sn-glycero-3-phosphoethanolamine and [3-α-²H]cholesterol were synthesized in order to monitor the behavior of the fatty acyl chains and of the cholesterol molecule itself. For all deuterated segments the deuterium quadrupole splittings as well as the deuterium spin-lattice (T₁) relaxation times were measured as a function of temperature. The glycerol backbone was found to be a remarkably stable structural element of the phospholipid molecule. The quadrupole splittings of the backbone segments changed only by at most 2 kHz upon incorporation of 50 mol % cholesterol. This was in contrast to the fatty acyl chains where the same amount of cholesterol increased the quadrupole splitting by more than 20 kHz. The glycerol segments exhibited the shortest T₁ relaxation times of all CH₂ segments indicating that the glycerol backbone is the slowest motional moiety of the lipid molecule. Addition of cholesterol has no effect on the backbone motion but the fast reorientation rate of the trans-double bonds in 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine is increased dramatically.     

Influence of basic polypeptides on the phase transition of phospholipid liposomes

Interaction of basic polypeptides (copolymers of lysine with phenylalanine or tyrosine) with phosphatidyserine or dipalmitoyl phosphatidylcholine liposomes was investigated by differential scanning calorimetry. The polypeptides cause a decrease in enthalpy of melting of the phospholipids almost without affecting the midpoint melting temperature.     

Thermotropic and barotropic phase transitions in bilayer membranes of ether-linked phospholipids with varying alkyl chain lengths

The bilayer phase transitions of a series of ether-linked phospholipids, 1,2-dialkylphosphatidylcholines containing linear saturated alkyl chain (C_n = 12, 14, 16 and 18), were observed by differential scanning calorimetry (DSC) under ambient pressure and light-transmittance measurements under high pressure. The thermodynamic quantities of the pre- and main-transitions for the ether-linked PC bilayer membranes were calculated and compared with those of a series of ester-linked PCs, 1,2-diacylphosphatidylcholines. The thermodynamic quantities of the main transition for the ether-linked PC bilayers showed distinct dependence on alkyl-chain length and were slightly different from those of the ester-linked PC bilayers. From the comparison of thermodynamic quantities for the main transition between both PC bilayers, we revealed that the attractive interaction in the gel phase for the ether-linked PC bilayers is weaker than that for the ester-linked PC bilayers. Regarding the pretransition, although changes in enthalpy and entropy for both PC bilayers were comparable to each other, the volume changes of the ether-linked PC bilayers roughly doubled those of the ester-linked PC bilayers. The larger volume change results from the smallest partial molar volume of the ether-linked PC molecule in the interdigitated gel phase. Further, we constructed the temperature-pressure phase diagrams for the ether-linked PC bilayers by using the phase-transition data. The region of the interdigitated gel phase in the phase diagrams was extended by applying pressure and by increasing the alkyl-chain length of the molecule. Comparing the phase diagrams with those for the ester-linked PC bilayers, it was proved that the phase behavior of the ester-linked PC bilayers under high temperature and pressure is almost equivalent to that of the ether-linked PC bilayers in the vicinity of ambient pressure.     

The affinity of cholesterol for phosphatidylcholine and sphingomyelin.

Erythrocyte ghosts were incubated with sonicated vesicles and the uptake of cholesterol by vesicles allowed to proceed to equilibrium. The experiments were carried out for a series of phospholipids at different temperatures. The equilibrium partition of cholesterol between ghosts and single shelled vesicles provided a measure of the relative affinities of cholesterol for the different phospholipids studied. It was found that the affinity of cholesterol for dipalmitoyl phosphatidylcholine was the same as that for N-palmitoyl sphingomyelin both at temperatures above and below the gel to liquid crystalline transition temperature of these phospholipids.     

The affinity of cholesterol for phosphatidylcholine and sphingomyelin

Erythrocyte ghosts were incubated with sonicated vesicles and the uptake of cholesterol by vesicles allowed to proceed to equilibrium. The experiments were carried out for a series of phospholipids at different temperatures. The equilibrium partition of cholesterol between ghosts and single shelled vesicles provided a measure of the relative affinities of cholesterol for the different phospholipids studied. It was found that the affinity of cholesterol for dipalmitoyl phosphatidylcholine was the same as that for $\text{N-}\text{palmitoyl}$ sphingomyelin both at temperatures above and below the gel to liquid crystalline transition temperature of these phospholipids.     

Phospholipid synthesis in rat liver endoplasmic reticulum after the administration of phenobarbitone and 20-methylcholanthrene

1. Phenobarbitone injection did not affect the concentration of phospholipids in the liver endoplasmic reticulum, but it increased the rate of incorporation of [(32)P]orthophosphate into the phospholipids. 20-Methylcholanthrene caused a transient increase in total phospholipid but a decrease in the turnover rate of the phospholipids. 2. Incorporation of [(32)P]orthophosphate into phosphatidylcholine, compared with that into phosphatidylethanolamine, was increased by phenobarbitone injection but decreased by 20-methylcholanthrene injection. 3. The activity of S-adenosylmethionine-phosphatidylethanolamine methyltransferase increased 12h after phenobarbitone injection, when incorporation of [(32)P]orthophosphate into phosphatidylcholine was a maximum, but at other times, and after 20-methylcholanthrene injection, the activity of the enzyme did not correlate with the rate of phosphatidylcholine synthesis. 4. [(14)C]Glycerol was incorporated more rapidly into phosphatidylcholine than into phosphatidylethanolamine, whereas [(32)P]orthophosphate and [(14)C]ethanolamine were incorporated more rapidly into phosphatidylethanolamine than into phosphatidylcholine. 5. Incorporation of [(32)P]orthophosphate into phosphatidylethanolamine of liver slices incubated in vitro was much more rapid than into phosphatidylcholine, and incorporation into phosphatidylcholine was markedly stimulated by addition of methionine to the medium. Changes in the incorporation of [(32)P]orthophosphate into phospholipids observed in vivo after injection of phenobarbitone or methylcholanthrene could not be reproduced in slices incubated in vitro. 6. It is concluded that phenobarbitone injection causes an increased rate of turnover of total phospholipids in the endoplasmic reticulum and an increased conversion of phosphatidylethanolamine into phosphatidylcholine, whereas 20-methylcholanthrene injection depresses both the turnover rate of total phospholipids and the formation of phosphatidylcholine.     

Phase stability of phosphatidylcholines in dimethylsulfoxide solutions.

The temperature dependence of the phase stability of dispersions of dimyristoyl, dipalmitoyl, and distearoyl derivatives of phosphatidylcholines in excess aqueous dimethylsulfoxide has been examined by differential scanning calorimetry and synchrotron x-ray diffraction methods. There was a close correlation between the enthalpic transitions and the structural changes associated with the pre- and main transitions of the phospholipids in the range of concentrations up to mole fractions of dimethylsulfoxide in water of 0.1333. The temperature of the pre- and main transitions of the three phospholipids were found to increase linearly with increasing mole fraction of dimethylsulfoxide. The difference in phase stability between the lamellar gel and ripple phases induced by increasing dimethylsulfoxide concentration resulted in disappearance of the ripple phase and direct transition between lamellar gel and lamellar liquid-crystal phases. The effect of changing the properties of the solvent by the addition of dimethylsulfoxide on the dimensions of dipalmitoylphosphatidylcholine and solvent layers of the bilayer repeat structure has been determined from electron density distribution calculations. The lamellar repeat spacing recorded at 25 degrees C decreased from 6.36 nm in aqueous dispersion to 6.04 nm in a dispersion containing a mole fraction of 0.1105 dimethylsulfoxide. The results indicate that dipole interactions between solvent and phospholipid and dielectric properties of the solvent are important factors in the determination of the structure of saturated phosphatidylcholines.     

The effect of gramicidin A on the temperature dependence of water permeation through liposomal membranes prepared from phosphatidylcholines with different chains lengths.

The permeation of water through liposomal membranes composed of various saturated phosphatidylcholine plus gramicidin A was studied as a function of temperature. 1. The presence of gramicidin in the liposomal bilayers caused an increase in water permeability. Below the phase transition temperature this effect could be measured quite clearly in all the systems we tested, but the extent of the increase was largely dependent on the length of the hydrocarbon chains. 2. Increasing amounts of gramicidin caused a gradual disappearance of the abrupt change in the rate of water permeation near the gel-liquid crystalline phase transition temperature of dipalmitoyl phosphatidylcholine liposomes. Differential scanning calorimetry analysis of the system containing these relatively small amounts of gramicidin still showed a clear transition from the liquid crystalline to the gel state with only a slight reduction in the enthalpy change. 3. In liposomes composed of dimyristoyl, dipalmitoyl and saturated egg phosphatidylcholine there was a concomitant decrease in the activation energy of water permeation in the presence of gramicidin below and above the phase transition temperature. The activation energy for water permeation through longer chained distearoyl phosphatidylcholine liposomal bilayers was the same with or without gramicidin in the bilayer. 4. It is concluded that the ability of gramicidin to form conducting channels in a gel state bilayer depends on the thickness of the paraffin core.     

Phospholipid phase transitions in homogeneous nanometer scale bilayer discs

Nanoscale protein supported phospholipid bilayer discs, or Nanodiscs, were produced for the purpose of studying the phase transition behavior of the incorporated lipids. Nanodiscs and vesicles were prepared with two phospholipids, dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine, and the phase transition of each was analyzed using laurdan fluorescence and differential scanning calorimetry. Laurdan is a fluorescent probe sensitive to the increase of hydration in the lipid bilayer that accompanies the gel to liquid crystalline phase transition. The emission intensity profile can be used to derive the generalized polarization, a measure of the relative amount of each phase present. Differential scanning calorimetry was used to further quantitate the phase transition of the phospholipids. Both methods revealed broader transitions for the lipids in Nanodiscs compared to those in vesicles. Also, the transition midpoint was shifted 3-4 degrees C higher for both lipids when incorporated into Nanodiscs. These findings are explained by a loss of cooperativity in the lipids of Nanodiscs which is attributable to the small size of the Nanodiscs as well as the interaction of boundary lipids with the protein encircling the discs. The broad transition of the Nanodisc lipid bilayer better mimics the phase behavior of cellular membranes than vesicles, making Nanodiscs a 'native-like' lipid environment in which to study membrane associated proteins.     

Melatonin strongly interacts with zwitterionic model membranes—evidence from Fourier transform infrared spectroscopy and differential scanning calorimetry

Interactions of melatonin with zwitterionic dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes (MLVs) were investigated as a function of temperature and melatonin concentration (1-30 mol%) by using two noninvasive techniques, namely Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). The investigation of the C-H, CO, and PO₂⁻ antisymmetric double stretching modes in FTIR spectra and DSC studies reveal that melatonin changes the physical properties of the DPPC bilayers by decreasing the main phase transition temperature, abolishing the pretransition, ordering the system in the gel phase, and increasing the dynamics of the system both in the gel and liquid crystalline phases. It also causes significant decrease in the wavenumber for the CO stretching and PO₂⁻ antisymmetric double bond stretching bands, which indicates strong hydrogen bonding The results imply that melatonin locates in the interfacial region of the membrane. Furthermore, in the DSC curve, more than one signal is observed at high melatonin concentrations (24 and 30 mol%), which indicates melatonin-induced phase separation in DPPC membranes.