A fluorescence study examining how 14-valerate side chain substitution modulates anthracycline binding to small unilamellar phospholipid vesicles

The intrinsic fluorescence properties of the anthracycline antitumor antibiotics were studied in an effort to understand how 14-valerate side chain substitution modulates drug associations with small unilamellar phospholipid vesicles (SUVs) under near physiological conditions. Drug location and dynamics in fluid-phase dimyristoylphosphatidylcholine (DMPC) bilayers were evaluated for several analogs; accessibilities of bound fluorophores to membrane-impermeable iodide were evaluated in quenching experiments, while the diffusive motions of these agents were studied using lifetime-resolved anisotropy plots. The bulky and hydrophobic valerate substituent was found to further hinder the rotations experienced by a bound drug molecule, with apparent limiting anisotropy (a infinity) values showing increases of 13-82% upon valerate group substitution. In addition, the bimolecular quenching rate constants (unit, 10(9) M-1.s-1) for membrane-bound adriamycin (1.4), N-trifluoroacetyladriamycin (0.4), and their corresponding valerate-substituted analogs (kq values of 1.1 and 0.5, respectively) reveal that the side chain is a weak modulator of fluorophore penetration into the bilayer, with stronger modulation being achieved through amino group substitution. Similar results were obtained for drugs bound to negatively-charged dimyristoylphosphatidylglycerol (DMPG) bilayers. Finally, comparison of the equilibrium binding affinities of the various congeners for electroneutral DMPC versus negatively-charged DMPG bilayers demonstrate that positively-charged parent anthracyclines display high levels of selective binding to negatively-charged phospholipids, unlike valerate-substituted analogs which display no such selectivity. The modulation of anthracycline-membrane interactions achieved through valerate substitution offers potential explanations, at least in part, for some of the novel biological properties of valerate-containing anthracyclines.     

Transverse location of anthracyclines in lipid bilayers. Paramagnetic quenching studies

Quenching of anthracycline fluorescence by a series of spin-labeled fatty acids was used to probe the transverse location of the drug in phosphatidylcholine bilayers in the form of small unilamellar vesicles. Stern-Volmer plots of the quenching data indicate that the fluorophore moiety of the anthracycline is intercalated into the hydrocarbon region of the bilayer, with deeper penetration observed in fluid-phase than in solid-phase vesicles. 31P-NMR parameters (T1 and nuclear Overhauser enhancement (NOE] are unaffected by the presence of drug, consistent with a binding site removed from the interfacial region. Comparison of intensity (F0/F) plots with lifetime (tau 0/tau) data shows that the predominant mechanism of anthracycline quenching by membrane-bound nitroxides is static. Since the membrane-bound drug is also accessible to quenching by I-, the binding site in the membrane must create a channel which is accessible to solvent. Two other fluorescent probes, 12-(9-anthroyloxy)stearate (12-AS) and diphenylhexatriene (DPH), were employed to confirm the results obtained with the anthracyclines, giving quenching data representative of their location in the bilayer.     

Two-dimensional diffusion of amphiphiles in phospholipid monolayers at the air-water interface.

Steady-state and time-resolved fluorescence spectroscopy has been used to examine lateral diffusion in dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) and dimyristoyl-L-alpha-phosphatidylcholine (DMPC) monolayers at the air-water interface, by studying the fluorescence quenching of a pyrene-labeled phospholipid (pyrene-DPPE) by two amphiphilic quenchers. Steady-state fluorescence measurements revealed pyrene-DPPE to be homogeneously distributed in the DMPC lipid matrix for all measured surface pressures and only in the liquid-expanded (LE) phase of the DPPC monolayer. Time-resolved fluorescence decays for pyrene-DPPE in DMPC and DPPC (LE phase) in the absence of quencher were best described by a single-exponential function, also suggesting a homogeneous distribution of pyrene-DPPE within the monolayer films. Addition of quencher to the monolayer film produced nonexponential decay behavior, which is adequately described by the continuum theory of diffusion-controlled quenching in a two-dimensional environment. Steady-state fluorescence measurements yielded lateral diffusion coefficients significantly larger than those obtained from time-resolved data. The difference in these values was ascribed to the influence of static quenching in the case of the steady-state measurements. The lateral diffusion coefficients obtained in the DMPC monolayers were found to decrease with increasing surface pressure, reflecting a decrease in monolayer fluidity with compression.     

Structural basis of anthracycline selectivity for unilamellar phosphatidylcholine vesicles: an equilibrium binding study

Fluorescence anisotropy titration was used to determine the equilibrium binding affinities of several anthracycline antitumor antibiotics for sonicated dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) vesicles at 27.5 degrees C. Eight daunomycin analogues, all differing from the parent by one structural change in the aglycon portion of the molecule, as well as four anthracycline congeners modified in the amino sugar were studied. Double-reciprocal plots were used to determine overall binding affinities (K). It was shown that structural changes in both the aglycon and amino sugar portions of the daunomycin molecule strongly modulated K values for DMPC and DPPC bilayers. For modifications in the aglycon portion of an anthracycline, a correlation between drug hydrophobicity and membrane affinity was observed. The number of binding sites per phospholipid molecule (n) and the apparent association constant (Kapp) where K = nKapp, were determined at several temperatures for adriamycin, daunomycin, and carminomycin. The n values were found to be independent of temperature for fluid-phase DMPC or solid-phase DPPC bilayers. The Kapp values (25 degrees C) ranged from (0.82-4.4) X 10(5) M-1 for DMPC vesicles to (4.4-7.3) X 10(5) M-1 for DPPC vesicles. Although the Kapp values for the three drugs were similar for a particular bilayer, major differences were noted in the values of n and, therefore, in the overall vesicle affinities (nKapp). van't Hoff plots showed that anthracycline binding was exothermic; in all cases but one binding was accompanied by a decrease in entropy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Iodide penetration into lipid bilayers as a probe of membrane lipid organization.

The quenching efficiency of iodide as a penetrating fluorescence quencher for a membrane-associated fluorophore was utilized to measure the molecular packing of lipid bilayers. The KI quenching efficiency of tryptophan-fluorescence from melittin incorporated in DMPC bilayer vesicles peaks at the phase transition temperature (24 degrees C) of DMPC, whereas acrylamide quenching efficiency does not depend on temperature. The ability of iodide to penetrate the hydrocarbon region of the bilayer was examined by measuring the fluorescence quenching of the pyrene-phosphatidylcholine incorporated into DMPC vesicles (pyrene was attached to the 10th carbon of the sn-2 chain). The quenching efficiency of pyrene by iodide again shows a maximum at the lipid phase transition. We conclude that iodide penetrates the membrane hydrocarbon region at phase transition through an increased number of bilayer defects. The magnitude of change in quenching efficiency of iodide during lipid phase transition provides a sensitive technique to probe the lipid organization in membranes.     

Anthracycline binding to synthetic and natural membranes. A study using resonance energy transfer

The binding of adriamycin and its two analogues 4'-epidoxorubicin and 4'-deoxydoxorubicin to synthetic and mitochondrial membranes was investigated by using resonance energy transfer between these drugs and two fluorescent probes, diphenylhexatriene (DPH) and tryptophan. The fluorescence of the lipid probe DPH in both types of membranes and tryptophan in mitochondria was quenched by the anthracyclines in a dose-dependent manner. In sonicated, fluid-phase dimyristoyl-L-alpha-phosphatidylcholine (DMPC) vesicles, the half-quenching concentration (K50) of adriamycin was 17 +/- 1 microM, whereas in bilayers containing a 1:1 molar ratio of DMPC to cardiolipin (CL), the value was 8 +/- 1 microM. In liver and heart mitochondria, the K50 values were 8 +/- 2 and 11 +/- 3 microM, respectively. Similar results were obtained for the other two drugs. Replacing a nonionic with an ionic medium or decreasing the pH from pH 7.7 to pH 6.9 increased the K50 value of adriamycin for DPH in DMPC/CL (1:1 molar) liposomes and in mitochondria. Higher concentrations of anthracycline were needed to quench the fluorescence of tryptophan. The results suggest that these drugs interact with both phospholipids and proteins and that the cardiotoxicity of adriamycin is unlikely to be related to the amount of drug bound to heart mitochondria.     

Interaction of rifampicin and isoniazid with large unilamellar liposomes: spectroscopic location studies

The location of isoniazid and rifampicin, two tuberculostatics commonly used for the treatment of Mycobacterium tuberculosis and Mycobacterium avium complex infectious diseases, in bilayers of dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and dimyristoyl-L-a-phosphatidylglycerol (DMPG) have been studied by 1H NMR and fluorimetric methods. Steady-state fluorescence intensity and fluorescence energy transfer studies between rifampicin and a set of functionalized probes [n-(9-anthroyloxy)stearic acids, n=2, 12] reveal that, in both systems, isoniazid is located at the membrane surface whereas rifampicin is deeply buried inside the lipid bilayers. Steady-state fluorescence anisotropy studies performed with the probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-diphenylhexa-triene (TMA-DPH), not only corroborate the above results, but also show that no changes in membrane fluidity were detected in either liposome. The 1H NMR results, in DMPC liposomes, confirm the location of rifampicin near the methylene group of the acyl chains of the lipid bilayers.     

Tryptophan fluorescence of mitochondrial complex III reconstituted in phosphatidylcholine bilayers

The tryptophan intrinsic fluorescence of mitochondrial complex III reconstituted in phosphatidylcholine bilayers was examined at different temperatures. Absorption and emission maxima occur at 277 and 332 nm, irrespective of temperature or lipid:protein ratio even if there are indications (from fluorescence quenching) of protein conformational changes as a function of lipid:protein ratio. Low values of Trp fluorescence quantum yield in complex III (0.008-0.010) are probably due to the neighborhood of the heme groups. The temperature-dependent decrease of fluorescence intensity is nonlinear; the corresponding Arrhenius plots show "breaks" or discontinuities that could be interpreted as thermally dependent changes in protein conformation. However, no temperature-dependent changes in fluorescence quenching have been observed that may be related to protein conformational changes. In addition, Arrhenius plots of the fluorescence intensity of simple molecules, such as Trp or 1-anilino-8-naphthalene sulfonate in the presence of aqueous phospholipid dispersions, also show breaks in the same temperature range. Stern-Volmer plots of acrylamide and iodide quenching were also nonlinear, indicating large differences in quenching constants for the various tryptophanyl residues. The quenching results also suggest that, at high lipid:protein ratios, the microviscosity of the protein matrix is higher than that in lipid-poor systems. Comparison of quenching efficiencies of iodide and acrylamide suggest that no significant fraction of the fluorophores occurs in the neighborhood of charged residues.     

Dynamics of bolaamphiphilic fluorescent polyenes in lipid bilayers from polarization emission spectroscopy

The rotational motions of the biamphiphilic polyenes (bolapolyenes) dimethyl all-(E)-octacosa-10,12,14,16,18-pentaenedioate (DE28:5) and dimethyl all-(E)-tetratriaconta-13,15,17,19,21-pentaenedioate (DE34:5), with head-to-head distances of 34 and 42A, respectively, have been examined by fluorescence anisotropy methods. The membrane-spanning bolapolyenes, which contain a central emitting pentaene group tethered to two methoxycarbonyl opposite polar heads by symmetric C(8) (DE28:5) and C(11) (DE34:5) polymethylene chains, were dispersed in lipid bilayers of DPPC or DMPC, and the stationary and picosecond-resolved emission was recorded as a function of temperature. In fluid-phase DMPC bilayers, three relaxation times could be determined, assigned to fast (0.2 and 2ns) single-bond isomerization processes localized on the alkyl chains, and to whole-molecule oscillations ( approximately 11ns), respectively. The anisotropy decay parameters were further analyzed in terms of a diffusive model for wobbling in a Gaussian ordering potential, to assess the anchoring effect of the symmetric polar heads. In this way, the average rotational diffusion constant of the bolapolyenes, D( perpendicular), could be estimated as 0.022-0.026rad(2) ns(-1) (DMPC bilayers, 35 degrees Celsius), a value that is only 1/3 of that corresponding to the related pentaene fatty acid spanning a single membrane monolayer. In contrast, the amplitude of the equilibrium orientational distribution (theta(half-cone) approximately 50 degrees ) is very similar for both the transmembrane and the single-headed polyenes. The reorientational oscillations of the central emitting group in the bolapolyenes necessarily would produce large-amplitude (2-5A) and very fast (ns) translational motions of the polar heads.     

Interactions of angiotensin II non-peptide AT(1) antagonist losartan with phospholipid membranes studied by combined use of differential scanning calorimetry and electron spin resonance spectroscopy.

We used differential scanning calorimetry (DSC) and electron spin resonance (ESR) spectroscopy to investigate the interactions of Losartan, a potent, orally active Angiotensin II AT(1) receptor antagonist with phospholipid membranes. DSC results showed that Losartan sensitively affected the chain-melting behavior of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) bilayer membranes. ESR spectroscopy showed that phosphatidylcholines spin-labeled at the 5-position of the sn-2 acyl chain (n-PCSL with n=5), incorporated either in DMPC or DPPC bilayers containing Losartan, were restricted in motion both in the gel and in the liquid-crystalline membrane phases, indicating a location of the antagonist close to the interfacial region of the phosphatidylcholine bilayer. At high drug concentrations (mole fraction >/= x=0.60), the decrease in chain mobility registered by 5-PCSL in fluid-phase membranes is smaller than that found at lower concentrations, whereas that registered by 14-PCSL is further increased. This indicates a different mode of interaction with Losartan at high concentrations, possibly arising from a location deeper within the bilayer. Additionally, Losartan reduced the spin-spin broadening of 12-PCSL spin labels in the gel-phase of DMPC and DPPC bilayers. As a conclusion, our study has shown that Losartan interacts with phospholipid membranes by affecting both their thermotropic behavior and molecular mobility.     

Pressure effects on the physical properties of lipid bilayers detected by trans-parinaric acid fluorescence decay.

The effects of hydrostatic pressure on the physical properties of large unilamellar vesicles of single lipids dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) and lipid mixtures of DMPC/DPPC have been studied from time-resolved fluorescence of trans-parinaric acid. Additional experiments were carried out using diphenylhexatriene to compare the results extracted from both probes. Fluorescence decays were analyzed by the maximum entropy method. Pressure does not influence the fluorescence lifetime distribution of trans-parinaric acid in isotropic solvents. However, in pressurized lipid bilayers an abrupt change was observed in the lifetime distribution which was associated with the isothermal pressure-induced phase transition. The pressure to temperature equivalence values, dT/dP, determined from the midpoint of the phase transitions, were 24 and 14.5 degrees C kbar-1 for DMPC and POPC, respectively. Relatively moderate pressures of about 500 bar shifted the DMPC/DPPC phase diagram 11.5 degrees C to higher temperatures. The effects of pressure on the structural properties of these lipid vesicles were investigated from the anisotropy decays of both probes. Order parameters for all systems increased with pressure. In the gel phase of POPC the order parameter was smaller than that obtained in the same phase of saturated phospholipids, suggesting that an efficient packing of the POPC hydrocarbon chains is hindered.     

Distribution of tryptophan groups in molecules of troponin T, troponin I-troponin T complex, and alpha-actinin

The method of fluorescence quenching was used to experimentally determine the distribution of tryptophan residues in molecules of troponin T, troponin T-troponin I complexes, and alpha-actinin. Iodide and cesium ions, and acrylamide were used as quenchers. It was shown that cesium ions decrease the fluorescence intensity of troponin T and its complex with troponin I by the mode of dynamic quenching. For alpha-actinin such a dynamic quencher is anionic iodide. By using the modified Stern-Volmer equation, the quenching was found to be about 90% of total fluorescence intensity for troponin T, approximately 70% for the troponin T-troponin I complexes, and 20% for alpha-actinin. The penetration of cesium ions to tryptophan 206 (tryptophan 204) in the troponin T-troponin I complex is hindered, probably due to the participation of this tryptophan in the formation of bonds between troponin subunits.     

Chlorinated hydrocarbon-cell membrane interactions studied by the fluorescence quenching of carbazole-labeled phospholipids: Probe synthesis and characterization of the quenching methodology

In recognition of the need to understand better the interactions of the chlorinated hydrocarbon insecticides with cell membranes we investigated the use of fluorescence quenching of membrane-bound fluorophores by these chlorinated hydrocarbons. An extensive survey of potential fluorophores identified the N-alkyl derivatives of carbazole as being especially suitable fluorophores. The fluorescence emission of these derivatives is quenched by a wide variety of commonly-used chlorinated hydrocarbons. This quenching is collisional and does not result in significant photodecomposition.Four structurally distinct carbazole-labeled phospholipids were synthesized, and their structures were confirmed by 270 MHz proton NMR and by chromatographic and chemical means. The carbazole moiety of each labeled phospholipid should be localized at a different depth in lipid bilayer. However, water soluble quenchers indicate that the fluorophores are inaccessible to the aqueous phase, irrespective of their point of attachment to the phospholipids.When incorporated into lipid bilayers, the fluorescence lifetime of these carbazole-labeled phospholipids reveals the collisional frequency between the fluorophore and the chlorinated hydrocarbon. As a result quenching of membrane-bound fluorophores may be used to measure: (1) the diffusional rate of the chlorinated hydrocarbon in the bilayer; (2) the lipid-water partition coefficient; (3) the maximum binding capacity of the membrane for the chlorinated hydrocarbon. Examples of all these measurements are given, and the fluorometric results are confirmed by direct chemical analysis.     

Does diabetes mellitus affect diphenylhexatriene penetration into erythrocyte membrane ghosts?

Diphenylhexatriene transverse distribution has been studied in normal and diabetic erythrocyte membrane ghosts using fluorescence polarization and fluorescence quenching methods. Acrylamide quenched the fluorescence of diphenylhexatriene according to a dynamic mechanism in agreement with Stern-Volmer equation. Nonlinear least-squares analysis based on quenching results has shown greater accessibility of fluorophore to quencher molecules in diabetic ghosts (37.2 +/- 3.2% in normal vs. 67.5 +/- 6.4% in diabetic membranes). Steady-state fluorescence anisotropy measurements evidenced the lowered membrane lipid fluidity in diabetics (anisotropy values: 0.166 +/- 0.011 in normal subjects vs. 0.193 +/- 0.018 in diabetics). A model mechanism is proposed which attributes the lowered capacity of lipid bilayer in diabetes to the increased ordering and more compact structure of membrane phospholipids. The implications of the results for the resolving of steady-state anisotropy data are discussed.     

Analysis of the effect of a peptide sequence of the E2 protein (HGV/GBV-C) on the physicochemical properties of zwitterionic and negatively charged bilayers.

The membrane-interacting properties of a potential epitope of GB virus C/hepatitis G virus located at the region (99-118) of the E2 structural protein were investigated using several fluorescence techniques. SUV of DMPC:DPPC (1:1) or DMPG:DPPC (1:1) zwitterionic and anionic mixtures, respectively, were used as model membranes. FRET with NBD-PE as energy donor and Rho-PE as energy acceptor-labelled SUV indicated that the peptide was able to fuse both zwitterionic and anionic SUVs, the latter requiring lower peptide concentrations. However, the peptide increased the steady-state anisotropy of DPH embedded in the hydrophobic centre of the membrane with zwitterionic headgroups and to a lesser extent in anionic bilayers, suggesting that charge-charge interactions are not required for membrane interactions and also confirming the FRET results. No changes in anisotropy were observed with the probe TMA-DPH located at the surface of the bilayer. Finally, analysis of the intrinsic emission fluorescence of the tryptophan residue, upon incubation with SUV, showed a blue shift in the presence of anionic bilayers, both below and above the main transition temperature (T(m)) (gel to liquid-crystalline state) and, to a lesser extent, with the zwitterionic model membrane.     

Time-resolved fluorescence studies of dityrosine in the outer layer of intact yeast ascospores.

The (time-resolved) fluorescence properties of dityrosine in the outermost layer of the spore wall of Saccharomyces cerevisiae were investigated. Steady-state spectra revealed an emission maximum at 404 nm and a corresponding excitation maximum at 326 nm. The relative fluorescence quantum yield decreased with increasing proton concentration. The fluorescence decay of yeast spores was found to be nonexponential and differed pronouncedly from that of unbound dityrosine in water. Analysis of the spore decay recorded at lambda ex = 323 nm and lambda em = 404 nm by an exponential series (ESM) algorithm revealed a bimodal lifetime distribution with maxima centered at tau 1C = 0.5 ns and tau 2C = 2.6 ns. The relative amplitudes of the two distributions are shown to depend on the emission wavelength, indicating contributions from spectrally different dityrosine chromophores. On quenching the spore fluorescence with acrylamide, a downward curvature of the Stern-Volmer plot was obtained. A multitude of chromophores more or less shielded from solvent in the spore wall is proposed to account for the nonlinear quenching of the total spore fluorescence. Analysis of the fluorescence anisotropy decay revealed two rotational correlation times (phi 1 = 0.9 ns and phi 2 = 30.6 ns) or a bimodal distribution of rotational correlation times (centers at 0.7 ns and 40 ns) when the data were analyzed by the maximum entropy method (MEM). We present a model that accounts for the differences between unbound (aqueous) and bound (incorporated in the spore wall) dityrosine fluorescence. The main feature of the photophysical model for yeast spores is the presence of at least two species of dityrosine chromophores differing in their chemical environments. A hypothetical photobiological role of these fluorophores in the spore wall is discussed: the protection of the spore genome from mutagenic UV light.     

Assessment of the role of tryptophan residues in phospholipase A2 by fluorescence quenching

Tryptophan (Trp) fluorescence of two phospholipases A2 (PLA2) from Naja naja atra and Naja nigricollis snake venoms was quenched by acrylamide and iodide. Trp residues in N. naja atra PLA2 were equally accessible to acrylamide and iodide. Iodide quenching studies indicate that there are two classes of Trp fluorophores in N. nigricollis CMS-9. The accessible class consists of Trp-18 and Trp-19. Removal of the N-terminal octapeptide caused a perturbation of the micro-environment of the Trp residues in the PLA2 enzymes. The presence of a substrate lowers the susceptibility of the Trp residues to iodide quenching in N. naja atra PLA2, suggesting that all three Trp residues are at the substrate binding site, but in N. nigricollis CMS-9 Trp-18 and Trp-19 are related to substrate binding.     

Effects of lindane on membrane fluidity: intramolecular excimerization of a pyrene derivative and polarization of diphenylhexatriene.

Fluorescence polarization studies of 1,6-diphenyl-1,3,5-hexatriene (DPH) have been compared with the excimer/monomer fluorescence intensity ratio (I'/I) of 1,3-di(2-pyrenyl)propane, (2Py(3)2Py). This ratio permits evaluation of changes in fluidity of the outer regions of the bilayer, where 2Py(3)2Py preferentially distributes. On the other hand, fluorescence polarization of DPH reports the structural order of the bilayer core. In the fluid phase of DMPC bilayers, for lindane concentrations higher than 25 microM, the excimer/monomer fluorescence intensity ratio (I'/I) decreases, thus reflecting an order increase of the probe environment. However, in the same conditions, the fluorescence polarization of DPH is almost insensitive to any perturbation. Identical results have been obtained in other pure lipid bilayers, namely DPPC and DSPC. However, both probes detect disordering effects of lindane in the gel phase of these lipids. The pyrene probe, unlike DPH, is very sensitive to the pretransitions of DPPC and DSPC, removed in the presence of lindane. Both probes fail to detect any apparent effect of lindane in DMPC bilayers enriched with high cholesterol content (greater than 30 mol%). However, in DMPC bilayers with low cholesterol content (less than 30 mol%), for temperatures below the phase transition of DMPC, both probes detect fluidizing effects induced by lindane. Nevertheless, above the phase transition of DMPC, 2Py(3)2Py detects ordering effects of lindane, whereas DPH detects hardly any effect. These results in DMPC bilayers with low cholesterol content are qualitatively similar to those described for DMPC without cholesterol.     

Effect of free fatty acids on the permeability of 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer at the main phase transition

We measured the influence of saturated and unsaturated free fatty acids on the permeability and partition of ions into 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers. The bilayer permeability was measured using the depletion of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1, 2-dihexadecanoyl-sn-glycero-3-phosphatidylethanolamine (N-NBD-PE) fluorescence as a result of its reduction by dithionite. We observed a distinct increase of dithionite permeability at the main gel-fluid phase transition of DMPC. When vesicles were formed from a mixture of DMPC and oleic acid, the membrane permeability at the phase transition was reduced drastically. Stearic acid and methyl ester of oleic acid have little effect. Similar results in the quenching of pyrene-PC in DMPC vesicles by iodide were obtained. Again, the increase of iodide partition into the lipid phase at the main phase transition of DMPC was abolished by the addition of unsaturated free fatty acids. Free fatty acids, in concentrations up to 5 mol%, do not abolish DMPC phase transition when measured by differential scanning calorimetry. It seems that unsaturated, but not saturated, free fatty acids reduce the lipid bilayer permeability to dithionite and iodide ions at the main phase transition of DMPC, without altering the thermodynamic properties of the bilayer.