Interaction of nucleic acids with lipid membranes

The thermodynamics of nucleic acids which were enclosed in reverse-phase evaporation vesicles was studied by thermal denaturation with optical recording. The denaturation curves were recorded with a dual wavelength spectrophotometer. The sum of the hypochromicity of the nucleic acid and of the change in turbidity of the vesicles was measured at 260 nm and was corrected for the change in turbidity at 320 nm. Cloned fragments of double-stranded DNA containing 180 base pairs and poly A:poly U were enclosed in REV with a yield up to every vesicle containing five nucleic acid molecules. Vesicles were prepared from egg-lecithin, and the surface charge of the vesicles was varied by addition of stearic acid, phosphatidyl-glycerol and phosphatidyl-serine. The helix-coil transition of the nucleic acid enclosed in the vesicle could be resolved from that of the free nucleic acid. Due to the enclosure into the egg-lecithin REV the transition is stabilized from 70.5 degrees to 74 degrees C, the transition is broadened from 0.7 degrees C to 2.7 degrees C. Varying the phosphatidyl-serine-lecithin-ratio from 0-100%, an optimum in the yield of enclosure at 20% was obtained, a further broadening of the transition to 5.5 degrees C and a decrease of the stabilization down to a small destabilization at 100% phosphatidyl serine was observed. Qualitatively, similar effects were observed with poly A:poly U. Variation of the ionic strength led to the conclusion that the replacement of the counterions of the phosphate backbone by the surface charge of the membrane, as well as a direct contact between the nucleic acid and the membrane have to be assumed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of lipid membranes on the conformational transitions of nucleic acids

The conformational transitions of nucleic acids which were enclosed in reverse phase evaporation vesicles (REV) were studied by thermal denaturation with optical recording. Cloned fragments of double-stranded DNA containing 179 base pairs and 187 base pairs, respectively, and polyA.polyU were enclosed in REV with a yield up to every vesicle containing 50 nucleic acid molecules. With the 179 base pairs DNA enclosed in the vesicle from egg lecithin two well resolved helix-coil transitions could be measured; one is very similar in the midpoint-temperature Tm and halfwidth delta T1/2 to the transition of the free nucleic acid, and the other transition occurs stabilized at a 3.5 degrees C higher Tm-value and with a broader delta T1/2, 2.7 degrees C instead of 0.6 degree C. Both transitions are from nucleic acids inside the vesicles. Varying the surface charge of the lipid membrane by adding the negatively charged phosphatidylserine or phosphatidylglycerol, an optimum in the yield of enclosure and a maximum in the increase in Tm (4.5 degrees C) and delta T1/2 (5.5 degrees C instead of 1.0 degrees C) was obtained at 20% phosphatidylserine or phosphatidylglycerol. In vesicles from pure negatively charged lipids no second population of nucleic acids was observed. Qualitatively, similar effects were observed with polyA.polyU. Stabilization and broadening of the second transition is higher for nucleic acids inside vesicles from lipids with unsaturated fatty acids, as dioleoyl-phosphatidylcholine, than with saturated fatty acids, dipalmitoyl-phosphatidylcholine. Stabilization and broadening decrease with increasing ionic strength, whereas the relative contributions of both transitions to the total hypochromicity remain unchanged; the second transition coincides with the first at 90 mM Na+. From the experimental results it was concluded that the interaction of nucleic acids and lipid membranes is mainly of electrostatic nature. The nucleic acids exist inside the vesicles in two populations, one behaving like nucleic acid free in solution and one influenced by the contact with the membrane. All results are in accordance with a model in which the interaction between the nucleic acid and the membrane is in competition with the dipole-dipole interaction inside the membrane surface.     

Turbidity changes of lipid vesicles near the phase transition temperature as an indication of fusion

Sonicated liposomes of dipalmitoyl phosphatidylcholine show sharp turbidity changes on heating at two distinct temperatures. A decrease in turbidity at the lower temperature (approx. 37°C) is thought to be associated with the phase transition of small vesicles and a decrease at about 44°C with larger vesicles or multilayer. An increase of turbidity between 38 and 43°C is attributed to the fusion of small vesicles. The turbidity changes were studied under various modes of vesicle preparation to confirm the interpretation of the turbidity data. Alternate interpretations are discussed.     

Destabilization of secondary structure in polyadenylic acid by formaldehyde

Results of circular dichroism and ultraviolet absorption measurements of poly A in neutral aqueous formaldehyde solution are presented, which show that the hydroxymethylated residues retain the ability to stack, but the stacking tendency is diminished. The midpoint of the thermal denaturation profile falls from about 38°C to about 18°C on complete hydroxymethylation. Hydroxymethylation, however, appears to have only a small effect on the apparent standard enthalpy and entropy of stacking, both of which become slightly more negative. When present at concentrations greater than about 2 g/100 ml, formaldehyde has a further and somewhat more dramatic denaturing effect on poly A. This ability is probably related to the chemical similarity of aqueous formaldehyde (methylene glycol) and ethylene glycol, which is known to be an effective denaturant for nucleic acids. In the presence of 20 g/100 ml formaldehyde, a very broad thermally induced transition of poly A is observed through the loss of ultraviolet hypochromism. Only the low-temperature part of the transition is observed; it appears to be completed only at temperatures well over 100°C where the hypochromism with respect to adenosine has largely disappeared. The transition cannot be identified with the unstacking reaction usually observed through thermal denaturation experiments in the absence of formaldehyde, although the associated structure may be related in some way. The molecular nature of the structure associated with the transition is not known.     

Biphasic helix–coil transition of the ethidium bromide·poly(dA-dT) and the propidium diiodide·poly(dA-dT) Complexes. Stabilization of base-pair regions centered about the intercalation site

The nonexchangeable base and sugar proton nmr resonances and the 260 and 278-nm uv-absorbance bands of the nucleic acid were utilized to monitor the temperature-dependent duplex-to-strand transition of the alternating purine–pyrimidine deoxyribopolynucleotide poly(dA-dT) in the absence and presence of ethidium bromide (EB) at phosphate/drug = 50, 28, and 15 and propidium diiodide (PI) at P/D = 50, 25, 15, 10, and 5 in 0.1 M salt between 50° and 100°C. The nmr and optical methods monitor a biphasic duplex-to strand transition for the drug–poly(dA-dT) complexes. We have monitored the dissociation of the drug from the complex at the ethidium bromide phenanthridine ring and side-chain proton nmr resonances and the propidium diiodide 494 and 535-nm uv-absorbance bands and demonstrate that dissociation of the drug corresponds to the higher temperature transition in the biphasic nucleic acid melting curves. The lower temperature cooperative transition is assigned to the opening of drug-free AT base-pair regions in the drug–poly(dA-dT) complex and exhibits an increase in transition midpoint and a decrease in cooperativity with increasing drug concentration. The higher temperature cooperative transition is assigned to the opening of AT base-pair regions centered about the bound drug in the complex and exhibits an increase in the transition midpoint on raising the drug concentration. The large upfield shifts of the phenanthridine ring (but not side chain) protons of ethidium bromide on complex formation demonstrate intercalation of the drug between base pairs of the poly(dA-dT) duplex. The nucleic acid base and sugar resonances of poly(dA-dT) in 0.1 M phosphate undergo chemical shift changes between 0° and 50°C indicative of premelting conformational transition(s).     

Hexose and amino acid transport by chicken embryo fibroblasts infected with temperature-sensitive mutant of rous sarcoma virus: Comparison of transport properties of whole cells and membrane vesicles

The effect of transformation on hexose and amino acid transport has been studied using whole cells and membrane vesicles of chicken embryo fibroblasts infected with the temperature-sensitive mutant of the Rous sarcoma virus, TS-68. In whole cells, TS-68-infected chicken embryo fibroblasts cultured at the permissive temperature (37°C) had a 2-fold higher rate of 2-deoxy-d-glucose uptake than the same cells cultured at the non-permissive temperature (41°C). However, both the non-transformed and transformed cells had comparable rates of α-aminoisobutyric acid transport. Membrane vesicles, isolated from TS-68-infected chicken embryo fibroblasts cultured at 41°C or 37°C, displayed carrier-mediated, intravesicular uptake of d-glucose and α-aminoisobutyric acid. Membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 37°C had an approx. 50% greater initial rate of stereospecific hexose uptake than the membrane vesicles from fibroblasts cultured at 41°C. The two types of membrane vesicle had similar uptake rates of α-aminoisobutyric acid. The results of hexose and amino acid uptake by the membrane vesicles correlated well with those observed with the whole cells. K_m values for stereospecific d-glucose uptake by the membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 41 and 37°C were similar, but the V value was greater for the membrane vesicles from TS-68-infected cells cultured at 37°C. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicle. These findings suggest that the membrane vesicles retained many of the features of hexose and amino acid transport observed in whole cells, and that the increased rate of hexose transport seen in the virallytransformed chicken embryo fibroblasts was due to an increase in the number or availability of hexose carriers.     

Mutagen–nucleic acid complexes at the polynucleotide duplex level in solution: Intercalation of proflavine into poly(dA-dT) and the melting transition of the complex

The nmr chemical shifts and line widths of the nucleic acid base and sugar proton resonances and the proflavine ring protons can be monitored through the melting transition of the proflavine + poly(dA-dT) complex, phosphate/dye (P/D) ratio = 24 and 8 in 1M salt solution. The nucleic acid and mutagen protons in the complex are in fast exchange between duplex and strand states with the midpoint of the melting transition monitored at the nucleic acid resonances increasing from 72.6°C for poly(dA-dT) to 78.1°C for the P/D = 24 complex and 83.4°C for the P/D = 8 complex in 1M salt solution. The melting transition monitored by the proflavine resonances were 80.0°C for the P/D = 24 complex and 84.3°C for the P/D = 8 complex in 1M salt solution. Since the nucleic acid is in excess at high P/D ratios, the nucleic acid transitions are an average for the opening of mutagen-free and mutagen-bound base-pair regions, while the proflavine transitions monitor the melting of mutagen-bound base-pair regions. The observed 0.75 to 0.95 ppm unfield shift at all four proflavine protons on formation of the complex with poly(dA-dT) provides direct evidence for intercalation of the mutagen between base pairs of the nucleic acid duplex. We have deduced the approximate overlap geometry between the proflavine ring and nearest-neighbor base pairs at the intercalation site from a comparison between experimental proflavine complexation shifts and those calculated for various stacking orientations. The experimental chemical shift of the poly(dA-dT) adenine H-2 resonance in the duplex state in the absence and presence of proflavine suggests that intercalation occurs preferentially at dT-dA sites. The selective chemical shift changes at the sugar H-2′,2″ and H-3′ resonances of the poly(dA-dT) duplex on complex formation demonstrates changes in the sugar pucker and/or torsion angles of the sugar phosphate backbone at the intercalation site.     

Comparison of the enthalpy state of vesicles of different size by their interaction with α-lactalbumin

The characteristics of small unilamellar, large unilamellar and large multilamellar vesicles of dimyristoylphosphatidylcholine and their interaction with α-lactalbumin are compared at pH 4. (1) By differential scanning calorimetry and from steady-state fluorescence anisotropy data of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene it is shown that the transition characteristics of the phospholipids in the large unilamellar vesicles resemble more those of the multilamellar vesicles than of the small unilamellar vesicles. (2) The size and composition of the lipid-protein complex formed with α-lactalbumin around the transition temperature of the lipid are independent of the vesicle type used. Fluorescence anisotropy data indicate that in this complex the motions of the lipid molecules are strongly restricted in the presence of α-lactalbumin. (3) The previous data and a comparison of the enthalpy changes, $\text{ΔH}$, of the interaction of the three vesicle types with α-lactalbumin allow us to derive that the enthalpy state of the small unilamellar vesicles just below 24°C is about 24 kJ/mol lipid higher than the enthalpy state of both large vesicle types at the same temperature. The abrupt transition from endothermic to exothermic $\text{ΔH}$ values around 24°C for large vesicles approximates the transition enthalpy of the pure phospholipid     

The influence of Ca2+ on the turbidity of DPPC-DMPA vesicles within the temperature range of the phase transition

The influence of the addition of Ca2+ on the phase behaviour of vesicles, composed of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidic acid (DMPA) in a ratio of 4 to 1, has been investigated by means of turbidity measurements. As expected one single phase transition for the mixed phospholipids was observed in the absence of Ca2+. Passing through the temperature range of this transition after the addition of Ca2+, conditions appeared to favor fusion of the vesicles. A possible reason for this is that during the transition Ca2+ may permeate through the vesicle membranes and gain access to the inside DMPA binding sites. Therefore it is not unambiguously possible to determine phase transition temperatures from the turbidity changes that occur under these conditions. However, when within the temperature range of the phase transition of the mixed phospholipids the influence of Ca2+ addition to the vesicles was recorded isothermally, at each temperature separately, the final plot of turbidity versus temperature turned out to be far less confused by fusion events and adopted the form of two separate phase transitions. The temperatures at which these two transitions occur closely resemble the phase transition temperatures that may be observed in the absence of Ca2+ for DMPA and DPPC alone, 39 degrees C and 43 degrees C respectively. The results of this study suggest that when Ca2+ has only access to the outside of the vesicle membranes it may segregate the neutral and the acidic phospholipids into separate domains, both domains adopting their proper phase condition at the actual temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of fluorescence energy transfer to distinguish between poly(ethylene glycol)-induced aggregation and fusion of phospholipid vesicles

Two fluorescence energy transfer assays for phospholipid vesicle-vesicle fusion have been developed, one of which is also sensitive to vesicle aggregation. Using a combination of these assays it was possible to distinguish between vesicle aggregation and fusion as induced by poly(ethylene glycol) PEG 8000. The chromophores used were 1-(4′-carboxyethyl)-6-diphenyl-trans-1,3,5-hexatriene as fluorescent ‘donor’ and 1-(4′-carboxyethyl)-6-(4″-nitro)diphenyl-trans-1,3,5-hexatriene as ‘acceptor’. These acids were appropriately esterified giving fluorescent phospholipid and triacylglycerol analogues. At 20°C poly(ethylene glycol) 8000 (PEG 8000) caused aggregation of l-α-dipalmitoylphosphatidylcholine (DPPC) vesicles without extensive fusion up to a concentration of about 35% (w/w). Fusion occurred above this poly(ethylene glycol) concentration. The triacylglycerol probes showed different behaviour from the phospholipids: while not exchangeable through solution in the absence of fusogen, they appeared to redistribute between bilayers under aggregating conditions. DPPC vesicles aggregated with < 35% poly(ethylene glycol) could not be disaggregated by dilution, as monitored by the phospholipid probes. However, DPPC vesicles containing approx. 5% phosphatidylserine which had been aggregated by poly(ethylene glycol) could be disaggregated by either dilution or sonication. Phospholipid vesicles aggregated by low concentrations of poly(ethylene glycol) appear to fuse to multilamellar structures on heating above the lipid phase transition temperature.     

Interaction of nucleic acids. VI. Interaction of purine with nucleic acids

The interaction of purine with DNA, tRNA, poly A, poly C, and poly A. poly U complex was investigated. In the presence of purine, the nucleic acids in coil form (such as denatured DNA, poly A and poly C in neutral solutions, or tRNA) have lower optical rotations. In addition, hydrodynamic studies indicate that in purine solutions the denatured DNA has a higher viscosity and a decreased sedimentation coefficient. These findings indicate that through interaction with purine, the bases along the poly-nucleotide chain are unstacked and are separated farther from each other, resulting in increased assymmetry (and possibly volume) of the whole polymer. Thus, the de-naturation effect of purine reported previously can be explained by this preferential interaction of purine with the bases of nucleic acids in coil form through a hydrophobic-costacking mechanism. Results from studies on optical rotation and helix-coil transition show that the interaction of purine is greater with poly A than with poly C. The influence of temperature, Mg⁺⁺ concentration, ionic strength, and purine concentration on the effect of purine on nucleic acid conformation has also been investigated. In all these situations the unraveling of nucleic acid conformation occurs at much lower temperatures (20–40°C lower) in the presence of purine (0.2–0.6M).     

Transfer of the polyene antibiotic amphotericin B between single-walled vesicles of dipalmitoylphosphatidylcholine and egg-yolk phosphatidylcholine

Amphotericin B transfer between single-walled vesicles of dipalmitoylphosphatidylcholine (DPPC) and of egg phosphatidylcholine, both containing 10 mol% cholesterol, has been studied concurrently by circular dichroism spectroscopy and permeability measurements. At 22°C amphotericin B is rapidly transferred from DPPC to DPPC vesicles as well as from egg phosphatidylcholine to egg phosphatidylcholine vesicles. On the other hand, although amphotericin B is rapidly transferred from egg phosphatidylcholine to DPPC vesicles, it is not transferred from DPPC to egg phosphatidylcholine vesicles. At 48°C, above the transition temperature of DPPC, transfer occurs rapidly both ways. These results are interpreted in terms of difference of association constant of amphotericin B with vesicle membranes in the gel and liquid-crystalline state.     

Phospholipid exchange between bilayer membrane vesicles.

The turbidity of lipid vesicles, freshly prepared by sonicating purified dimyristoyllecithin (DML) in dilute KCl solutions, was measured as a function of time at various temperatures. A sharp maximum in the rate of increase of turbidity is found just above the crystal:liquid-crystal phase transition temperature (Tm). The initial rate of turbidity increase is first order with respect to DML concentration. Electron and light microscopy reveal large vesicles which are not present before incubation or after incubation at temperatures far from the Tm. When temperature, rather than time, is the independent variable, a sharp drop in turbidity is seen at the Tm. The magnitude of this drop and the temperature at which it occurs were used to measure the rate of lipid transfer between vesicles composed of different lipids. A mixture of DML vesicles and dipalmitoyllecithin (DPL) vesicles exhibits sharp drops in turbidity at 24 and 41 degrees, the corresponding Tm's. With time, the magnitude of the transition at 24 degrees decreases while that which was originally at 41 degrees moves to lower temperatures and increases in magnitude. At equilibrium there is a single transition at 32.5 degrees characteristic of vesicles composed of equimolar DPL and DML. The rate at which equilibrium is approached increases at around 24 degrees and again around 41 degrees. These observations indicate that vesicles are in equilibrium with monomolecular lipid, the concentration of the latter being higher the shorter the lipid acyl group or the smaller the vesicle. DML molecules are therefore lost from small vesicles to large vesicles (DML system) or lost from DML vesicles to DML-DPL vesicles (mixed system). When DML vesicles containing a few percent brain gangliosides were studied, different behavior was observed; the initial rate of increase of turbidity becomes second order in lipid concentration, and the rate constant increases with increasing concentrations of KCl. The kinetic order, coupled with the fact that electrolyte reduces intervesicle electrostatic repulsion, argues that in this situation the mechanism of vesicle growth requires vesicle collision.     

Effect of PEGylated lipid and Lecinol S-10 on physico-chemical properties and encapsulation efficiency of palmitoleate–palmitoleic acid vesicles

A vesicle is a microscopic particle composed of a lipid bilayer membrane that separates the inner aqueous compartment from the outer aqueous environment. Palmitoleate–palmitoleic acid vesicles were prepared and their physico-chemical properties were investigated. Moreover, mixed vesicles composed of palmitoleic acid and PEGylated lipid and/or a mixture of phospholipids were also prepared. The stabilizing effects of these double-chain lipids on the formation of palmitoleate–palmitoleic acid vesicles were studied. Stability of the vesicle suspension was examined using particle size and zeta potential at 30?°C. The magnitude of the zeta potential was relatively lower in the vesicle suspension with the presence of phospholipid. Although some of the mixed vesicles that were formed were not very stable, they displayed potential for encapsulating the active ingredient calcein and the encapsulation efficiencies of calcein were encouraging. The palmitoleate–palmitoleic acid–DPPE-PEG2000 vesicle showed the most promising stability and encapsulation efficiency.     

Author index

Using dynamic light scattering we have been able to determine precisely the hydrodynamic radius of l-α-dimyristoylphosphatidylcholine (DMPC) vesicles as a function of temperature. We have detected a sharp, thermally reversible change in the vesicle radius at a phase transition temperature 24°C, corresponding to an approximate 11% increase in surface are. In the range 10–20°C, the change in radius is less than 1%.     

Preparation of unilamellar lipid vesicles at 37°C by vaporization methods

We have developed a method utilizing low boiling solvents to prepare large, unilamellar vesicles at physiologic temperatures. Solutions of ethyl methyl ether or dichlorofluoromethane (Freon-21) at 4°C containing solubilized lipids were injected into a column of a aqueous buffer at 37°C. Vesicles prepared in this manner have been examined by freeze-fracture, negative stain electron microscopy, and fluorescence microscopy. The principal advantages of this technique are: (1) heat labile substances may be more readily entrapped in the internal vesicle volume without thermal denaturation, and (2) the range of lipids which are soluble in dichlorofluoromethane is greater than that of many other solvents, e.g. diethyl ether.     

Pressure-induced Shape Change of Phospholipid Vesicles: Implication of Compression and Phase Transition

A microscopic study has allowed the analysis of modifications of various shapes acquired by phospholipid vesicles during a hydrostatic pressure treatment of up to 300 MPa. Giant vesicles of dimyristoylphosphatidylcholine / phosphatidylserine (DMPC/PS) prepared at 40°C mainly presented a shape change resembling budding during pressure release. This comportment was reinforced by the incorporation of 1,2-dioleyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or by higher temperature (60°C) processing. The thermotropic main phase transition (Lα to Pβ′) of the different vesicles prepared was determined under pressure through a spectrofluorimetric study of 6-dodecanoyl-2-dimethylamino-naphtalene (Laurdan) incorporated into the vesicles’ bilayer. This analysis was performed by microfluorescence observation of single vesicles. The phase transition was found to begin at about 80 MPa and 120 MPa for DMPC/PS vesicles at, respectively, 40°C and 60°C. At 60°C the liquid-to-gel transition phase was not complete within 250 MPa. Addition of DMPE at 40°C does not significantly shift the onset boundary of the phase transition but extends the transition region. At 40°C, the gel phase was obtained at, respectively, 110 MPa and 160 MPa for DMPC/PS and DMPC/PS/DOPE vesicles. In comparing volume data obtained from image analysis and Laurdan signal, we assume the shape change is a consequence of the difference between lateral compressibility of the membrane and bulk water. The phase transition contributes to the membrane compression but seems not necessary to induce shape change of vesicles. The high compressibility of the Lα phase at 60°C allows induction on DMPC/PS vesicles of a morphological transition without phase change.     

Raman spectroscopic measurements in self‐pressurized aqueous solutions above 100°C: The melting of poly(G) and poly(G) · poly(C)

We have studied by Raman spectroscopy the thermal behavior of associated polyguanylic acid [poly(G)] and polyguanylic–polycytidylic acid [poly(G) · poly(C)] in self‐pressurized aqueous solutions contained in sealed capillary tubes. The associated polynucleotides were found to be very resistant to heat, but evidence of thermal degradation was observed after melting of the helical structures. The cooperative melting transition of the four‐stranded complex of poly(G) was located at 141°C in 0.5M KCl, 135°C in 0.5M NaCl, 129°C in 0.5M LiCl, 123°C in 0.1M tetramethylammonium perchlorate, and 105°C in 0.1M tetraethylammonium bromide solutions. The transition was observed at 130°C in poly(G) · poly(C) (in 0.5M NaCl). The results in this case show that a four‐stranded poly(G) complex is formed following the melting of the double helix. © 1999 John Wiley & Sons, Inc. Biopoly 49: 21–28, 1999     

Effect of the phase transition on the transbilayer movement of dimyristoyl phosphatidylcholine in unilamellar vesicles

Dimyristoyl phosphatidylcholine rapidly exchanges between vesicles at 37°C without vesicle fusion.The rate of the transbilayer movement of dimyristoyl phosphatidylcholine in sonicated vesicles has been measured employing ¹³C NMR using N-¹³CH_3? labeled lipids which are introduced into the outer monolayer of non-labeled vesicles by a phosphatidylcholine exchange protein. The rate of transbilayer movement of dimyristoyl phosphatidylcholine shows a distinct maximum (halftime 4 h) in the temperature range at which the hydrocarbon phase transition occurs.The activation energy of the flip-flop rate above the phase transition is 23.7 ± 2.0 kcal/mol.     

Phase transitions of phospholipid single-wall vesicles and multilayers. Measurement by vibrational raman spectroscopic frequency differences

Raman spectroscopic frequency differences between selected carbon-carbon stretching modes of lipid hydrocarbon chains were determined as a function of temperature for use in monitoring lipid phase transition behavior and acyl chain disorder in both multilamellar and single-wall vesicles. Transition temperatues detected by this procedure for pure dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine multilayers were observed at $\text{39±1 °}\text{C}$ and $\text{23±1 °}\text{C}$, respectively. Although the phase transition for unilamellar vesicles of dipalmitoyl phosphatidylcholine occurred at nearly the same temperature as the multilayers, the crystal-liquid crystalline transition for the single-shell vesicles appeared to span a slightly broader temperature range, a characteristic consistent with irregularities in the packing arrangement of the hydrocarbon chains. Within the precision of the Raman spectroscopic method, however, the temperature behavior of both the multilamellar and the unilamellar dimyristoyl phosphatidylcholine assemblies appeared nearly identical. The temperature profile for the Raman frequency differences of an excess water sonicate of 25 mol percent cholesterol in dipalmitoyl phosphatidylcholine served as an example of the effect upon lipid phase transition characteristics of a bilayer component intercalated between the acyl chains. For this particular cholesterol-lipid system the phase transition was broadened over a 30 °C temperature range, in contrast to the narrow 5?4 °C range observed for pure multilayer and single-shell vesicle particles.