Small Unilamellar Vesicles Of Dipalmitoylphosphatidylcholine Research Articles

Novel type of isoprenoid membrane anchors: an investigation of binding properties with dipalmitoylphosphatidylcholine vesicles

AbstractIn this work, we present a new type of amphiphilic membrane‐anchoring agents that can be easily obtained by the Diels‐Alder reaction between terpene myrcene and N‐substituted maleimides. The interaction between the compounds and small unilamellar dipalmitoylphosphatidylcholine vesicles was investigated using infrared spectroscopy, microgravimetry, and turbidimetry. The ability of the compounds to embed in the phospholipid membrane was shown to be strongly dependent on the charge of their polar group. The insertion of the compounds studied into the lipid bilayer did not lead to disruption of the dipalmitoylphosphatidylcholine vesicles up to the highest tested drug to lipid molar ratio of 0.5 to 0.6. Low lipid solubilization ability of the compounds as well as their rigid nonplanar structure makes them an interesting alternative to the common membrane‐anchoring structural motifs.

1-Naphthol as a Sensitive Fluorescent Molecular Probe for Monitoring the Interaction of Submicellar Concentration of Bile Salt with a Bilayer Membrane of DPPC, a Lung Surfactant

In this study, 1-naphthol has been used as a sensitive ESPT fluorescent molecular probe to investigate the interaction of submicellar concentrations of two physiologically important bile salts, sodium deoxycholate and sodium cholate, with dipalmitoylphosphatidylcholine small unilamellar vesicles in solid gel and liquid crystalline phases. Steady-state and time-resolved fluorescence of the two excited state prototropic forms of 1-naphthol indicate that the incorporation of monomeric bile salt molecules in the lipid bilayer membrane induces appreciable wetting of the bilayer up to the hydrocarbon core region, even at very low (≤1 mM) concentrations of the bile salts.

Differential Effect of Isoflurane on the Anisotropy of Diphenylhexatriene and its Cationic Trimethylamine Analog

The mechanism of action of volatile general anesthetics has not yet been resolved. Recent developments in the understanding of lipid physics, including the discovery of microdomains and computing the lateral pressure profile in simulations, suggest a need to revisit possible indirect effects of lipids on ion channels during anesthesia. We present strong experimental evidence that volatile general anesthetics localize at the headgroup region of a lipid bilayer and therefore increase lateral membrane pressure near the bilayer surface. Theoretically, this increase in pressure in the head group region, in conjunction with a decrease in pressure in the tail group region, may induce conformational changes in ion channels to produce the characteristic effects of volatile general anesthetics. To examine this idea, the anisotropy of fluorophores localized in either the head (trimethylamino-diphenylhexatriene, TMA-DPH) or tail group regions (DPH) of small unilamellar vesicles of dipalmitoylphosphatidylcholine was assessed. These measurements were repeated at multiple temperatures between 20 and 55 °C in the presence or absence of various concentrations of the anesthetic isoflurane. In treated samples, the main phase transition (41.5 °C) was shifted down by 2 to 10 °C depending on the concentration of isoflurane (3.8-13.0 mM). Melting reduced anisotropy by 0.1 (TMA-DPH) or 0.2 (DPH) units. Interestingly, isoflurane caused opposite changes in anisotropy of the two probes in the liquid crystalline phase: DPH anisotropy decreased by ∼0.02 units whereas TMA-DPH anisotropy increased by the same magnitude. This observation suggests that isoflurane partitions into the headgroup region of the bilayer where it increases lateral pressure, while reducing it in the tail region.

Solubilisation of dipalmitoylphosphatidylcholine bilayers by sodium taurocholate: A model to study the stability of liposomes in the gastrointestinal tract and their mechanism of interaction with a model bile salt

In order to better understand the mechanism of destabilization of liposomes used as drug carriers for oral administration by bile salts, the insertion and partition of sodium taurocholate (TC) into small unilamellar vesicles (SUV) and multilayers (ML) of dipalmitoylphosphatidylcholine (DPPC) were examined by continuous turbidity analysis and DSC. Optical density was recorded during the progressive solubilisation of DPPC SUV and ML into DPPC/TC mixed micelles by varying the rate of TC addition and the temperature. The results show that the insertion and diffusion of TC in the DPPC membrane is a slow process influenced by the polymorphism of the lipid, independently of its organisation. This dynamic study mimics physiological phenomena of the digestion of liposomes. In the gastrointestinal tract, DPPC SUV would be more resistant to TC than egg phosphatidylcholine (EPC) SUV [K. Andrieux, L. Forte, S. Lesieur, M. Paternostre, M. Ollivon, C. Grabielle-Madelmont, Insertion and partition of sodium taurocholate into egg phosphatidylcholine vesicles, Pharm. Res. 21 (2004) 1505–1516] because of the lower insertion of TC into DPPC bilayer at 37 °C at low TC concentration in the medium (fasted conditions). At high TC concentration (postprandially or after lipid absorption), the use of DPPC to prepare liposomes will delay or reduce the liberation of a drug encapsulated into liposomes in the gastrointestinal tract. As a conclusion, the addition of DPPC appears an attractive strategy to formulate orally administered liposomes.

Fluorescence Light Microscopy of Pulmonary Surfactant at the Air-Water Interface of an Air Bubble of Adjustable Size

The structural dynamics of pulmonary surfactant was studied by epifluorescence light microscopy at the air-water interface of a bubble as a model close to nature for an alveolus. Small unilamellar vesicles of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, a small amount of a fluorescent dipalmitoylphosphatidylcholine-analog, and surfactant-associated protein C were injected into the buffer solution. They aggregated to large clusters in the presence of Ca2+ and adsorbed from these units to the interface. This gave rise to an interfacial film that eventually became fully condensed with dark, polygonal domains in a fluorescent matrix. When now the bubble size was increased or decreased, respectively, the film expanded or contracted. Upon expansion of the bubble, the dark areas became larger to the debit of the bright matrix and reversed upon contraction. We were able to observe single domains during the whole process. The film remained condensed, even when the interface was increased to twice its original size. From comparison with scanning force microscopy directly at the air-water interface, the fluorescent areas proved to be lipid bilayers associated with the (dark) monolayer. In the lung, such multilayer phase acts as a reservoir that guarantees a full molecular coverage of the alveolar interface during the breathing cycle and provides mechanical stability to the film.

Effect of Cardiolipin on Proton Permeability of Phospholipid Liposomes: The Role of Hydration at the Lipid–Water Interface

The effect of cardiolipin on the proton permeability of dipalmitoyl-phosphatidylcholine small unilamellar vesicles was examined by utilizing the pH-dependent fluorescence emission of 5- (and 6-) carboxyfluorescein. It has been found that the proton permeability of the phospholipid bilayer was greatly enhanced in the presence of cardiolipin, an acidic phospholipid mainly found in the inner mitochondrial membranes. In the presence of bovine heart cardiolipin, the bilayer surface hydration, as assessed with the fluorescence lifetime of 1-anilinonaphthalene-8-sulfonic acid, was increased, while hydration in the acyl chain region was not altered. In addition, the bilayer fluidity was also not affected. Taken together, these results suggest that the lipid–water interface is the major energy barrier for proton permeation of the bilayer vesicles, and alteration to properties of this interface by cardiolipin headgroup appears to be responsible for the enhanced proton permeability.

A photophysical study of the polyene antibiotic filipin: Self-aggregation and filipin–ergosterol interaction

Filipin, a macrolide polyene antibiotic, is known to interact selectively with ergosterol, a constituent of fungi membranes. In this work, the fluorescence resonance energy transfer (FRET) between a fluorescent analog of ergosterol, dehydroergosterol (DHE), and filipin was measured in small unilamellar vesicles of dipalmitoylphosphatidylcholine at 25°C. The time-resolved FRET results were rationalized in the framework of the mean concentration model, and were complemented with steady-state fluorescence intensity, anisotropy and absorption measurements. The results point to the formation of both DHE–filipin aggregates (evidence from static quenching of DHE fluorescence by filipin) and filipin–filipin aggregates (evidence from: (i) the FRET acceptor concentration distributions; (ii) spectral changes of filipin absorption in the vesicles, the excitonic interaction suggesting a stack arrangement; (iii) filipin fluorescence self-quenching), even in presence of DHE and low antibiotic mole fractions (<1 mol%). These results point out that apparently contradictory biochemical models for the action of filipin (some based on the presence of sterols, others not) can be equally valid. Moreover, since results (ii) and (iii) are also observed when a sterol is present, both models of action can actually coexist in membranes with a low sterol content.

Interaction of Ubiquinone-10 with Dipalmitoylphosphatidylcholine and Their Formation of Small Dispersed Particles

Stable aqueous dispersions of ubiquinone-10 (UQ) were obtained by cosonication with dipalmitoylphosphatidylcholine (DPPC) in the UQ mole fraction range 0.1–0.7. To clarify the dispersal mechanism, the dispersed particles were characterized, and the interaction between UQ and DPPC was investigated using several physicochemical techniques. Dynamic light scattering (DLS) measurements showed that the diameter of the dispersed particles was 50–70 nm. A limited amount of UQ was incorporated into DPPC bilayer membranes (approximately 5 mol%). The trapped aqueous volume inside the particles was determined fluorometrically using the aqueous space marker calcein, and the volume in the UQ/DPPC particles decreased remarkably with the addition of UQ into small unilamellar vesicles of DPPC. The decline in the fraction of vesicular particles was also confirmed by fluorescence quenching of N-dansylhexadecylamine in the DPPC membrane by the addition of the quencher CuSO4. These results indicate that the excess UQ separated from the DPPC bilayers is stabilized as emulsion particles by the DPPC surface monolayer.

A Direct Method for the Correction of Pressure-Induced Scrambling of Polarized Fluorescence Intensities

A simple and direct method for the simultaneous correction of steady-state polarized fluorescence intensities, depolarized (or scrambled) by the effects of applied hydrostatic pressure, is described. In the method discussed here, it is not necessary to first determine the scrambling factors from a separate experiment with a dye immobilized in a rigid medium. Rather correction for depolarizing effects of the high-pressure spectroscopy cell windows is achieved by direct recalculation of the measured polarized data obtained for the sample of interest at the time of data collection. This method of correction is tested for common fluorescent dyes 1,6-diphenyl-1,3,5-hexatriene (DPH) and 9,10-diphenylanthracene in glycerol where their rotational behavior is well understood. In addition, the pressure-induced “melt” profile for the more complicated biologically relevant system of DPH imbedded within dipalmitoylphosphatidylcholine small unilamellar vesicles has been reexamined. While the method discussed here is used for the correction of steady-state polarized data, it may be easily adapted for use in time-resolved polarized fluorescence measurements. Advantages and limitations of the new correction method are discussed.

Formation and structure of stably dispersed particles composed of retinal with dipalmitoylphosphatidylcholine: coexistence of emulsion particles with bilayer vesicles

In order to develop an intravenous formulation of all- trans-retinal (vitamin A aldehyde, VAA) for the treatment of night blindness, VAA and dipalmitoylphosphatidylcholine (DPPC) were sonicated and the dispersions in the VAA mole fraction range of 0.1–0.7 were stable at room temperature for 3 days. In order to clarify the dispersal mechanism, the dispersed particles were characterized and the interaction between VAA and DPPC was investigated using several physicochemical techniques. Dynamic light scattering measurements showed that the diameter of the dispersed particles was 50–70 nm. A limited amount of VAA is incorporated into DPPC bilayer membranes (approximately 5 mole%). The trapped aqueous volume inside the particles was determined fluorometrically using the aqueous space marker calcein and the volume in the VAA/DPPC particles was decreased remarkably with the addition of VAA into small unilamellar vesicles of DPPC. The decline in the fraction of vesicular particles was also confirmed by fluorescence quenching of N-dansylhexadecylamine in the DPPC membrane by the addition of the quencher CuSO 4. These results indicate that the excess VAA separated from the DPPC bilayers is stabilized as emulsion particles by the DPPC surface monolayer. The monolayer–bilayer equilibrium of VAA/DPPC mixtures was estimated by measurement of spreading and collapse pressures. The results showed that the coexistence of emulsion particles (surface monolayer of DPPC+ core of VAA) with vesicular particles (bilayer) was critically important for the formation of the stably dispersed particles of the lipid mixture.

Interaction of retinol with dipalmitoylphosphatidylcholine and their formation of small dispersed particles

Stable aqueous dispersions of all- trans-retinol (vitamin A, VA) were obtained by sonication with dipalmitoylphosphatidylcholine (DPPC) in the VA mole fraction range 0.1–0.7. In order to clarify the dispersal mechanism, the dispersed particles were characterized and the interaction between VA and DPPC was investigated using several physicochemical techniques. Dynamic light scattering measurements showed that the diameter of the dispersed particles was 50–70 nm. A limited amount of VA was incorporated into DPPC bilayer membranes (approximately 5 mol%). The trapped aqueous volume inside the particles was determined fluorometrically using the aqueous space marker calcein and the volume in the VA/DPPC particles was decreased markedly with the addition of VA into small unilamellar vesicles of DPPC. The decline in the fraction of vesicular particles was also confirmed by fluorescence quenching of N-dansylhexadecylamine in the DPPC membrane by the addition of the quencher CuSO 4. These results indicate that the excess VA separated from the DPPC bilayers is stabilized as emulsion particles by the DPPC surface monolayer.

Interaction of Soybean Oil with Phosphatidylcholine and Their Formation of Small Dispersed Particles

Stable aqueous dispersions of soybean oil (SO) were obtained by cosonication with dipalmitoylphosphatidylcholine (DPPC) in the SO mole fraction range 0.1–0.8. To clarify the dispersal mechanism, the dispersed particles were characterized, and the interaction between SO and DPPC was investigated using several physicochemical techniques. Dynamic light scattering (DLS) measurements showed that the diameter of the dispersed particles was 40–60 nm. The trapped aqueous volume inside the particles was determined fluorometrically using the aqueous space marker calcein. The trapped volume in the SO/DPPC particles decreased remarkably with the addition of SO into small unilamellar vesicles of DPPC. The decline in fraction of vesicular particles was also confirmed by fluorescence quenching of N-dansylhexadecylamine in the DPPC membrane by the addition of the quencher CuSO4. These results indicate that the excess SO separated from the DPPC bilayers is stabilized as emulsion particles by the DPPC surface monolayer. Monolayer-bilayer equilibrium of SO/DPPC mixtures was estimated by measurement of spreading and collapse pressures. The results showed that the coexistence of emulsion particles (surface monolayer of DPPC + core of SO) with vesicular particles (bilayer) was critically important for the formation of stably dispersed particles of the lipid mixture.

Interaction of -.ALPHA.Tocopherol Acetate with Phosphatidylcholine and Their Formation of Small Dispersed Particles.

Stable aqueous dispersions of α-tocopherol acetate (α-TA) were obtained by cosonication with dipalmitoylphosphatidylcholine (DPPC) in the α-TA mole fraction range of 0.1-0.8. In order to clarify the dispersal mechanism, the dispersed particles were characterized and the interaction between α-TA and DPPC was investigated using several physicochemical techniques. Dynamic light scattering measurements showed that the diameter of the dispersed particles was 50-75 nm. The trapped aqueous volume inside the particles was determined fluorometrically using the aqueous space marker calcein. The trapped volume in the α-TA/DPPC particles decreased remarkably with the addition of α-TA into small unilamellar vesicles of DPPC. The decline in the fraction of vesicular particles was also confirmed by fluorescence quenching of N-dansylhexadecylamine in the DPPC membrane by the addition of the quencher CuSO4. These results indicate that the excess α-TA separated from the DPPC bilayers is stabilized as emulsion particles by the DPPC surface monolayer. The monolayer-bilayer equilibrium of α-TA/DPPC mixtures was estimated by measurement of spreading and collapse pressures. The results showed that the coexistence of emulsion particles (surface monolayer of DPPC+core of α-TA) with vesicular particles (bilayer) was critically important for the formation of the stably dispersed particles of the lipid mixture.

Simulation of the gel-fluid transition in a membrane composed of lipids with two connected acyl chains: application of a dimer-move step

Phospholipids have been treated as dimers on a hexagonal lattice, and a move has been introduced that allows the dimers to move and change their orientation on the lattice. Simulations have been performed in which phospholipid chains have been treated as being either independent or infinitely coupled thermodynamically with regard to their conformational state. Both types of simulation have reproduced well experimental heat-capacity curves of dipalmitoyl phosphatidylcholine small unilamellar vesicles. Apart from a different gel-fluid interaction parameter and a different number of unlike nearest-neighbor contacts, most of the averages and thermodynamic quantities were essentially the same in the two types of simulation. These results indicate that the transition is not first order and validate those of previous Monte Carlo simulations that have neglected the dimeric nature of phospholipids in the sense that they show that for the thermotropic transition the approximation of phospholipids as monomers is valid.

Stopped-flow fluorometric study of the interaction of melittin with phospholipid bilayers: importance of the physical state of the bilayer and the acyl chain length

Stopped-flow fluorometry has been employed to study the effects of melittin, the major protein component of bee venom, on dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) small unilamellar vesicles (SUVs) on the millisecond time scale, before melittin-induced vesicle fusion takes place. Use is made of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), which is an oriented fluorescent probe that anchors itself to the bilayer-water interface and is aligned parallel to the normal to the bilayer surface; its fluorescence anisotropy reports on the "fluidity" of the bilayer. For DMPC bilayers, melittin is found to decrease their fluidity only at their melting transition temperature. This perturbation appears to be exerted almost instantaneously on the millisecond time scale of the measurements, as deduced from the fact that its rate is comparable to that obtained by following the change in the fluorescence of the single tryptophan residue of melittin upon inserting itself into the bilayer. The perturbation is felt in the bilayer over a distance of at least 50 A, with measurements of transfer of electronic energy indicating that the protein is not sequestered in the neighborhood of TMA-DPH. The length of the acyl chains is found to be an important physical parameter in the melittin-membrane interaction: unlike the case of DMPC SUVs, melittin does not alter the fluidity of DPPC SUVs and has a considerably greater affinity for them. These results are discussed in terms of the concept of elastic distortion of the lipids, which results from a mismatch between the protein and the acyl chains that are attempting to accommodate it. Melittin is also found to cause a small (approximately 10%) enhancement in the total fluorescence intensity of TMA-DPH, which is interpreted as indicating a reduction in the degree of hydration of the bilayer.

Porphyrins as fluorescent probes for monitoring phase transitions of lipid domains in biological membranes. Factors influencing the microenvironment of haematoporphyrin and protoporphyrin in liposomes

The distribution properties of haematoporphyrin (HP) and protoporphyrin (PP) (concentration range 0.5–15 μM) in small unilamellar vesicles (SUVs) of dimiristoylphosphatidylcholine (DMPC), sometimes mixed with cholesterol (Chol) or cardiolipin (Card), were compared with those already reported for HP and PP in SUVs of dipalmitoylphosphatidylcholine (DPPC). In the latter system the study was extended to vesicles modified by addition of high concentrations of Chol (up to 55 mol% of total lipids) or Card (up to 37% w/w). These lipids are determinants for most biological membranes. The studies were performed by following the temperature dependence of the porphyrin fluorescence polarization and the quenching of porphyrin fluorescence by methyl viologen. In all lipid systems HP interacts with very polar, solvent-accessible regions of the inner monolayer. In DMPC liposomes HP is partitioned at the phospholipid headgroup-water interfaces over the whole concentration range. In contrast, discrimination of different areas (lipid-headgroup and headgroup-water interfaces) in the polar regions of the inner monolayer of DPPC liposomes is possible by modulating the dye concentration. HP shows no tendency to diffuse into pure lipid domains: a significant loading of the outer monolayer is observed only after an increase in the fluidity of the system by addition of Card or by increasing the temperature. A limit situation is obtained in DPPC liposomes enriched with high concentrations of Chol: in spite of the very low affinity of HP for Chol-rich domains of lipid membranes, porphyrin molecules do not move to the external monolayer but instead accumulate in mixed DPPC-Chol regions at the water-lipid inner interface. In all lipid systems PP is preferentially distributed in the most buried, hydrophobic regions of the lipid bilayer. A shift of the porphyrin molecules towards more quencher-accessible lipid domains is possible only in mixed DMPC-Chol or DMPC-Card liposomes brought to the fluid state with high temperatures.

Porphyrins as fluorescent probes for monitoring phase transitions of lipid domains in biological membranes. Factors influencing the microenvironment of haematoporphyrin and protoporphyrin in liposomes

The distribution properties of haematoporphyrin (HP) and protoporphyrin (PP) (concentration range 0.5–15 μM) in small unilamellar vesicles (SUVs) of dimiristoylphosphatidylcholine (DMPC), sometimes mixed with cholesterol (Chol) or cardiolipin (Card), were compared with those already reported for HP and PP in SUVs of dipalmitoylphosphatidylcholine (DPPC). In the latter system the study was extended to vesicles modified by addition of high concentrations of Chol (up to 55 mol% of total lipids) or Card (up to 37% w/w). These lipids are determinants for most biological membranes. The studies were performed by following the temperature dependence of the porphyrin fluorescence polarization and the quenching of porphyrin fluorescence by methyl viologen. In all lipid systems HP interacts with very polar, solvent-accessible regions of the inner monolayer. In DMPC liposomes HP is partitioned at the phospholipid headgroup-water interfaces over the whole concentration range. In contrast, discrimination of different areas (lipid-headgroup and headgroup-water interfaces) in the polar regions of the inner monolayer of DPPC liposomes is possible by modulating the dye concentration. HP shows no tendency to diffuse into pure lipid domains: a significant loading of the outer monolayer is observed only after an increase in the fluidity of the system by addition of Card or by increasing the temperature. A limit situation is obtained in DPPC liposomes enriched with high concentrations of Chol: in spite of the very low affinity of HP for Chol-rich domains of lipid membranes, porphyrin molecules do not move to the external monolayer but instead accumulate in mixed DPPC-Chol regions at the water-lipid inner interface. In all lipid systems PP is preferentially distributed in the most buried, hydrophobic regions of the lipid bilayer. A shift of the porphyrin molecules towards more quencher-accessible lipid domains is possible only in mixed DMPC-Chol or DMPC-Card liposomes brought to the fluid state with high temperatures.

Regulation by gangliosides and sulfatides of phospholipase A 2 activity against dipalmitoyl- and dilauroylphosphatidylcholine in small unilamellar bilayer vesicles and mixed monolayers

The modulation by gangliosides GM1 and GD1a, and sulfatide (Sulf) of the activity of porcine pancreatic phospholipase A 2 was studied with small unilamellar vesicles of dipalmitoylphosphatidylcholine ( L-dpPC) and lipid monolayers of dilauroylphosphatidylcholine ( L-dlPC). The presence of Sulf always led to an increase of the maximum rate of the enzymatic reaction, irrespective on whether the vesicles were above, in the range of, or below the bilayer transition temperature. Sulf did not modify the latency period for the reaction that is observed at the bilayer phase transition temperature. Gangliosides inhibited the maximum rate of enzymatic activity against bilayer vesicles in the gel phase but the effect was complex. When the reaction was carried out at a temperature within the range of the bilayer phase transition, the gangliosides inhibited the maximal rate of the reaction in proportion to their content in the bilayer. However, at the same time the latency period observed with vesicles of pure phospholipid at this temperature was shortened in proportion to the mole fraction of gangliosides in the bilayer. At temperatures above the bilayer phase transition, gangliosides stimulated the activity of PLA 2. Preincubation of the enzyme with Sulf or gangliosides did not affect the activity against bilayer vesicles of pure substrate. These glycosphingolipids did not modify the rate or extent of desorption of the enzyme from the interface, nor the pre-catalytic steps for the interfacial activation of PLA 2, or the enzyme affinity for the phospholipid substrate. Also, the activity of the enzyme was not altered irreversibly by glycosphingolipids. Our results indicate that Sulf and gangliosides modulate the catalytic activity of PLA 2 at the interface itself, beyond the initial steps of enzyme adsorption and activation, probably through modifications of the intermolecular organization and surface electrostatics of the phospholipid substrate.

Quenching of perylene fluorescence by Co2+ ions in dipalmitoylphosphatidylcholine (DPPC) vesicles

We report the fluorescence quenching of perylene by CoCl2·6H2O in small unilamellar DPPC vesicles via energy transfer. At the probe-to-lipid ratio of 1∶200 and quencher to lipid ratios of ≥12.5∶1, donor-donor energy transfer between clustered perylene molecules was observed as well as energy transfer from the perylene molecules to cobalt ions bothabove andbelow the main phase transition temperature of the lipid. The fluorescence quenching of perylene by CoCl2·6H2O in the lipid gel state is shown to be well described by Förster long-range energy transfer when both donor-donor and donor-acceptor energy transfer are considered. In the liquid crystalline phase diffusion of the molecules is described as well as energy transfer. The interaction radiusR 0 for energy transfer from perylene to Co(2+) is found to be ∼13.4±1.1 Å in the gel phase at 303 K, in good agreement with the theoretical value forR 0 of 13.9 Å. In the liquid crystalline phase at 323 K the lower value obtained forR 0, ∼11.3±1.6 Å, is attributed to saturation of the Co(2+) ions at the interfacial region of the bilayer. A diffusion coefficient of (1.06±0.15)×10(-6) cm(2) s(-1) is obtained for perylene-cobalt diffusion in the liquid crystalline phase at 323K.

INTERACTION OF THE PEPTIDE HORMONE ADRENOCORTICOTROPIC ACTH(l‐24), WITH A MEMBRANE MODEL SYSTEM: A FLUORESCENCE STUDY*

The peptide hormone adrenocorticotropin and a related peptide were studied in solution and in interaction with a model system of membranes (small unilamellar vesicles of dipalmitoylphosphatidylcholine and 17% dimyristoylphosphatidylglycerol) via fluorescence spectroscopy. In aqueous solution, intramolecular distances between the fluorescent residues R(Tyr2-Trp9) = 9.2 A and R(Trp9-Tyr23) > or = 18 A were obtained, in agreement with molecular models. Interaction of the peptide with the negatively charged membrane is evident from the alteration of the Trp photophysical parameters (quantum yield, fluorescence spectra and anisotropy), with a partition constant between the lipidic and aqueous phase of Kp = 1-2 x 10(3). The existence of two populations of Trp in the membrane, which are distinctly accessed by acrylamide, was concluded from the tryptophan fluorescence quenching study; the two fractions are located near the membrane interface as inferred from its fluorescence quenching by the 5-doxylstearate and 16-doxylstearate lipophilic quenchers. This result is further supported by energy transfer experiments to the 3-(9-anthroyloxyl)stearic acid and 12-(9-anthroyloxyl)stearic acid probes.