Plasma membrane fluidity gradients of human peripheral blood leukocytes

The shape of the fluidity gradient of the outer hemileaflet of the plasma membrane of normal, living, human white blood cells was determined using a series of n-(9-anthroyloxy) fatty acid probes where n = 2, 3, 6, 7, 9, 11, 12, and 16, to establish a baseline for future studies on the consequences of various pathological states. Fluorescence uptake and steady-state anisotropy values were obtained with a flow cytometer capable of continuous recording over time of vertical and horizontal emission intensities, with the output of these intensities as calculated anisotropy values. The fluorescence uptake of all of the membrane probes was rapid up to about 15 min. The magnitudes of the uptake of fluorescence was, for the n-(9-anthroyloxy) series, in the order 2 greater than 3 greater than 6 greater than 7 greater than 9 greater than 11 = 12 = 16 for neutrophils, lymphocytes, and monocytes. Anisotropy values were constant from 5 to 30 min after addition of the various probes. The orders of the anisotropy magnitudes, indicative of the shapes of the fluidity gradient, were, for neutrophils, 6 greater than 7 greater than 9 greater than 2 = 3 = 11 = 12 greater than 16, for lymphocytes, 7 greater than 6 greater than 9 greater than 11 greater than 2 = 3 greater than 11 = 12 greater than 16, and for monocytes, 9 greater than 7 greater than 6 greater than 11 greater than 2 = 3 greater than 12 greater than 16. The kinetics of anisotropy from 1 to 5 min after addition of the probes differed for each of the three cell types. Probes with an n-value less than or equal to the maxima (n = 6, neutrophils; n = 7, lymphocytes; n = 9, monocytes) rapidly (1.2 min) reached equilibrium, whereas those probes with n-values greater than the maxima took progressively longer times to equilibrate as n increased. This behavior is consistent with the existence of an energy barrier just below the approximate region sensed by the probes, which would correspond to just below 6AS for neutrophils, 7AS for lymphocytes, and 9AS for monocytes.     

Shape of the fluidity gradient in the plasma membrane of living HeLa cells

The shape of the fluidity gradient of the outer hemi-leaflet of the plasma membrane of living HeLa cells was determined using a series of n-(9-anthroyloxy) fatty acid probes where n = 2, 3, 6, 7, 9, 10, 11, 12, and 16. Fluorescence uptake and steady-state anisotropy values were obtained with a flow cytometer capable of continuous recording over time of vertical and horizontal emission intensities, and of the output of these intensities as calculated anisotropy values. The fluorescence uptake of all of the membrane probes was rapid up to about 15 min. The magnitudes of the uptake of fluorescence were, for the n-(9-anthroyloxy) series, in the order 2 greater than 3 greater than 6 greater than 7 greater than 9 greater than 10 greater than 11 = 12 = 16. Anisotropy values were constant from 5 to 30 min after addition of the various probes, and the magnitudes were in the order 7 greater than 6 greater than 9 = 10 greater than 2 = 3 greater than 11 greater than 12 greater than 16, indicative of the shape of the fluidity gradient. No differences were noted between the values obtained with 12-(9-anthroyloxy) stearic acid and 12- (9-anthroyloxy) oleate. The kinetics of anisotropy exhibited by those probes with the anthroyloxy group in positions deeper than 9, where initially higher values declined until equilibrium was reached, were probably indicative of an energy barrier at the approximate depth sensed by 7 AS.     

Membrane structural dynamics of plasma membranes of living human prostatic carcinoma cells differing in metastatic potential

Rigidity of the outer hemileaflet of the plasma membrane of two prostatic carcinoma cell lines with different metastatic potential, 1-LN and 1-LN-EMS-10, was assessed by steady-state anisotropy, using a battery of fluorescent probes. The "bulk" membrane rigidity sensed by diphenylhexatriene, trimethylammonio-DPH, 1-palmitoyl-2-[DPH-ethylcarbonyl]-phosphatidylcholine, and 10-pyrenedecanoic acid indicated slightly higher rigidity in the membrane of the highly metastatic line (1-LN). This was accompanied by 26% greater mole fraction of cholesterol and 9% lower phospholipid, resulting in 40% greater cholesterol/phospholipid ratio. Phosphatidylethanolamine was increased 12%, but corresponding decreases in phosphatidylserine and phosphatidylinositol resulted in no significant change in molar ratio of choline/noncholine phospholipids. Whereas unsaturation index was slightly higher in 1-LN, fatty acids of 1-LN plasma membranes contained 15% more 18:1, 43% more 20:4, 26% more 22:4, and 38% less 18:2. Anisotropy gradients were determined for the two cell lines using a series of n-(9-anthroyloxy) fatty acid probes with n = 2, 3, 6, 7, 9, 12, and 16. Gradients differed only in position of anisotropy maxima, which occurred with n = 6, in 1-LN, and n = 7, in 1-LN-EMS-10. Possible relationships between observed anisotropy gradients and differences in membrane cholesterol and fatty acid composition are discussed.     

Properties and the locations of a set of fluorescent probes sensitive to the fluidity gradient of the lipid bilayer

The synthesis and properties of a set of four fluorescent probes (n-(9-anthroyloxy) fatty acids, n = 2, 6, 9, 12) sensitive to the fluidity gradient of the lipid bilayer are described. Fluorescent quenching experiments show that the probes locate at a graded series of depths in the bilayer. A fifth probe, methyl-9-anthroate, locates near the bilayer centre. As an example of their application, the probes are used to study the phase transitions of dipalmitoyl phosphatidyl-choline. Changes in the rotational relaxation times of the probes across the transitions are more pronounced at the centre of the bilayer than at the surface.     

Fluorescence resonance energy transfer between lipid probes detects nanoscopic heterogeneity in the plasma membrane of live cells

Fluorescence resonance energy transfer (FRET) between matched carbocyanine lipid analogs in the plasma membrane outer leaflet of RBL mast cells was used to investigate lateral distributions of lipids and to develop a general method for quantitative measurements of lipid heterogeneity in live cell membranes. FRET measured as fluorescence quenching of long-chain donor probes such as DiO-C18 is greater with long-chain, saturated acceptor probes such as DiI-C16 than with unsaturated or shorter-chain acceptors with the same chromophoric headgroup compared at identical concentrations. FRET measurements between these lipid probes in model membranes support the conclusion that differential donor quenching is not caused by nonideal mixing or spectroscopic differences. Sucrose gradient analysis of plasma membrane-labeled, Triton X-100-lysed cells shows that proximity measured by FRET correlates with the extent of lipid probe partitioning into detergent-resistant membranes. FRET between DiO-C16 and DiI-C16 is sensitive to cholesterol depletion and disruption of liquid order (Lo) by short-chain ceramides, and it is enhanced by cross linking of Lo-associated proteins. Consistent results are obtained when homo-FRET is measured by decreased fluorescence anisotropy of DiI-C16. These results support the existence of nanometer-scale Lo/liquid disorder heterogeneity of lipids in the outer leaflet of the plasma membrane in live cells.     

Localization of adriamycin in model and natural membranes. Influence of lipid molecular packing

The interaction of adriamycin with lipids was studied in model (monolayers, small unilamellar vesicles, large multilamellar vesicles) and natural (chinese hamster ovary cell) membranes by measurement of fluorescence energy transfer and fluorescence quenching. 2-APam, 7-ASte, 12-ASte and anthracene-phosphatidylcholine were used as fluorescent probes in which the anthracene group is well located at graded depths in the membrane. Egg-yolk phosphatidylcholine and a 1/1 mixture of it with bovine brain phosphatidylserine were used in model membrane systems. Large fluorescence energy transfer was observed between these molecules as donors and the drug as acceptor. With liposomes, at pH 7.4 and over an adriamycin concentration range of 0-100 microM, the efficiency of energy transfer was 12-ASte greater than 7-ASte greater than 2-APam, with 100% energy transfer for 12-ASte above a drug concentration of 30 microM. At pH 5, where the fatty acids are buried deeper (0.45 nm) in the lipid bilayer due to protonation of the carboxyl group, the order of energy transfer 7-ASTe greater than 12-ASte = 2-APam was observed. Measurements of fluorescence quenching using the non-permeant Cu2+ ion as quencher and spectrophotometric assays indicated that around 40% of the adriamycin molecules were deeply embedded in the lipid bilayer. Adriamycin molecules thus appear to penetrate the lipid bilayer, with the aminoglycosyl group interacting with the lipid phosphate groups and the dihydroanthraquinone residue in contact with the lipid fatty acid chains. In contrast, fluorescence energy transfer and quenching studies on CHO cells showed that adriamycin penetrated the plasma membrane of these cells to a much more limited extent than in the model membrane systems. This can be related to the squeezing out of the drug from a film of phosphatidylcholine which was observed in monolayers by means of surface pressure, potential and fluorescence experiments. These observations indicated that the penetration of adriamycin into lipid bilayers strongly depends on the molecular packing of the lipid.     

Localization of cyanine dye binding to brush-border membranes by quenching of n-(9-anthroyloxy) fatty acid probes

The location and orientation of 3,3'-dipropylthiodicarbocyanine (diS-C3-(5)) binding sites in renal brush-border membrane vesicles was examined from the quenching of n-(9-anthroyloxy) fatty acid (n-AS) fluorescence. Based on previous kinetic studies (Cabrini, G. and Verkman, A.S. (1986) J. Membrane Biol. 90, 163-175) monomeric aqueous diS-C3-(5) binds to brush-border membrane vesicles (BBMV) by an initial 6 ms association to form bound monomer, a 30-40 ms equilibrium between bound monomer (M) and bound dimer (D), and a 1-1.3 s translocation of D from the outer to inner membrane leaflet. Based on Stern-Volmer and lifetime analyses, M and D quench the fluorescence of the n-AS probes by a collisional mechanism. At low [diS-C3-(5)]/[BBMV] (R), where M predominates, the n-AS quenching efficiencies (Q) are similar (n = 2-16); at high R, where D predominates, Q increases with n (16 greater than 12 much much greater than 6 greater than 2), suggesting that M is oriented parallel, and D perpendicular, to the phospholipid chains deep within the membrane. Mixture of diS-C3-(5) with brush-border membrane vesicles containing n-AS in a stopped-flow apparatus gave a biexponential fluorescence decrease (excitation 390 nm, emission above 450 nm) with time constants 30-40 ms and 1-1.5 s; there was no 6 ms quenching process. These findings are incorporated into a model in which diS-C3-(5) adheres loosely to the outer membrane surface in 6 ms, binds parallel to the membrane phospholipid in 30-40 ms, dimerizes and rotates by 90 degrees in much less than 30 ms, and translocates to the opposite half of the bilayer in 1-15 s.     

Comparisons of steady-state anisotropy of the plasma membrane of living cells with different probes

We have used an extended Perrin equation which was in agreement with literature data for steady-state anisotropy (rSS) for a wide variety of artificial and isolated biological membranes labeled with various probes (Van der Meer et al. (1986) Biochim. Biophys. Acta 854, 38-44 to obtain the static component (r infinity) for the intact plasma membranes of living cells. We show that lipid structural order parameters can be obtained for DPH and TMA-DPH in the plasma membranes of intact cells. We have examined the relationship between 'fractional limiting hindered anisotropy', r infinity/r0, which is related to the lipid structural order parameter, of DPH, TMA-DPH, DPHpPC, and a series of depth-dependent probes (n-(9-anthroyloxy) fatty acids, with n = 2-16), using data from 19 cell types. There was a linear relationship between r infinity/r0 values of DPH and TMA-DPH, but the relationship between either of these probes was non-linear with respect to DPHpPC or the series of fatty acid probes. The relationship between r infinity/r0 values of DPHpPC and the series of fatty acid probes was linear, suggesting that they not only undergo similar motions in the membrane, but also experience similar types of restriction to motion, a type which is different from that experienced by DPH and TMA-DPH. We show that for the plasma membranes of living cells, 'second degree' order parameters can be estimated for DPH and TMA-DPH, and propose that the parameter r infinity/r0, or the 'fractional limiting hindered anisotropy', analogous to a 'first degree' order parameter, can be estimated for DPHpPC and the depth-dependent fatty acid probes to evaluate the density of membrane packing.     

The penetration of local anesthetics into the red blood cell membrane as studied by fluorescence quenching.

The local anesthetics procaine and tetracaine were found to quench the fluorescence of the probes N-octadecyl naphthyl-2-amine 6-sulfonic acid and 12-(9-anthroyl)stearic acid in the presence of erythrocyte membranes. This quenching was shown to be due to the aromatic amine of the procaine and tetracaine molecules. Lidocaine, an active anesthetic that does not contain an aromatic amine in the same position as does procaine and tetracaine did not quench either of the fluorophores. The preferential quenching of the fluorescent probes by procaine and tetracaine indicated a greater accessibility of tetracaine than of procaine to the hydrocarbon region of the membrane and a greater accessibility of procaine than of tetracaine at the membrane's surface. The addition of calcium was found to reverse the quenching of 12-(9-anthroyl)stearic acid by tetracaine in the presence of red cell membranes.     

Relative location of anthroyl derivatives of stearic acid in low density lipoproteins

Effective quenching constants (K'sv) of 2-, 7- and 12-(9-anthroyloxy)stearic acid (n-AS) fluorescence in LDL were determined. Spin probes I(m, n) (n = 3, 7, 10, 14) and I- anions were used as quenchers. Quenching of 2-AS and 12-AS fluorescence by I(m, n) was the more effective, the deeper spin probe nitroxyl fragment was located (the greater n was); maximal K'sv value corresponded to I(1,14). By contrast, for 7-AS the quenching by I(12,3) was the most effective. 2-AS and 12-AS spectra maxima and fluorescence polarization were similar. We concluded that the 2-AS chromophore was located deeper in LDL phospholipid monolayer than chromophore of 7-AS (as was the case for 12-AS).     

Effects of ethanol on the Escherichia coli plasma membrane.

The effects of ethanol on the fluidity of Escherichia coli plasma membranes were examined by using a variety of fluorescent probes: 1,6-diphenyl-1,3,5-hexatriene, perylene, and a set of n-(9-anthroyloxy) fatty acids. The anthroyloxy fatty acid probes were used to examine the fluidity gradient across the width of the plasma membrane and artificial membranes prepared from lipid extracts of plasma membranes. Ethanol caused a small decrease in the polarization of probes primarily located near the membrane surface. In comparison, hexanol decreased the polarization of probes located more deeply in the membrane. Temperature had a large effect on probes located at all depths. The effects of ethanol on E. coli membranes from cells grown with or without ethanol were also examined. Plasma membranes isolated from cells grown in the presence of ethanol were more rigid than those from control cells. In contrast to plasma membranes, artificial membranes prepared from lipid extracts of ethanol-grown cells were more fluid than those from control cells. These differences are explained by analyses of membrane composition. Membranes from cells grown in the presence of ethanol are more rigid than those from control cells due to a decrease in the lipid-to-protein ratio. This change more than compensates for the fluidizing effect of ethanol and the ethanol-induced increase in membrane C18:1 fatty acid which occurs during growth. Our results suggest that the regulation of the lipid-to-protein ratio of the plasma membrane may be an important adaptive response of E. coli to growth in the presence of ethanol.     

Fluorescent analogues of N,N'-dicyclohexylcarbodiimide as structural probes of the bovine mitochondrial proton channel

N-Cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]carbodiimide (NCD-4) and N-cyclohexyl-N'-(1-pyrenyl)carbodiimide (NCP) are two novel fluorescent analogues of the mitochondrial inhibitor dicyclohexylcarbodiimide (DCCD). Although nonfluorescent in aqueous media, both compounds form fluorescent conjugates with mitochondrial electron transport particles (ETPH) or purified H+-ATPase (F1-F0) vesicles. DCCD prevents the reaction of ETPH with both NCD-4 and NCP. The fluorescent probes are effective inhibitors of ATPase activity and ATP-driven membrane potential, although their reaction rates are considerably slower than that of DCCD. The fluorescence of NCD-4- or NCP-treated H+-ATPase is quenched by hydrophobic spin-label nitroxide derivatives of stearic acid (chi-NS) in the order 16-NS greater than 12-NS greater than 7-NS approximately equal to 5-NS, whereas membrane-impermeant iodide ions have negligible effect. The quenching behavior of 16-NS (the most effective quencher) suggests that a small fraction of labels remain inaccessible to the quencher. It is concluded that the DCCD-binding sites are oriented toward the membrane lipids and are located in the lipid bilayer ca. 18 A from the membrane surface.     

Insulin effect on translational diffusion of lipids and proteins in the plasma membrane of isolated rat hepatocytes

The effects of insulin (10(-10)-10(-8) mol/l) on lateral diffusion of three fluorescent lipid probes, 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)aminocaproyl phosphatidylcholine (NBD-PC), 5-(N-hexadecanoyl)aminofluorescein (F-C16), 5-(N-dodecanoyl)aminofluorescein (F-C12), and of fluorescein isothiocyanate-labeled proteins in the plasma membrane of intact rat hepatocytes were studied by the technique of fluorescence recovery after photobleaching. The absolute lateral diffusion coefficients of the lipid analogues NBD-PC, F-C16 and F-C12 at 21 degrees C were 2.5 X 10(-9) cm2/s, 5.4 X 10(-9) cm2/s and 19 X 10(-9) cm2/s, respectively. The diffusion coefficient mean of proteins labeled with fluorescein isothiocyanate was 6.4 X 10(-10) cm2/s. Insulin at 10(-9) and 10(-8) mol/l reduced the lateral diffusion coefficient for F-C12- and F-C16-labeled cells by 20% and for NBD-PC-labeled cells by 30% (P less than 0.025). The insulin effect was specific as tested by cell incubation with proinsulin and desoctapeptide insulin (10(-8) mol/l) and was detectable after 7 min of insulin preincubation. In contrast to lateral diffusion of lipid probes, lateral mobility of unselected membrane proteins was not altered by insulin. The observed modulation of lipid dynamics in the plasma membrane of intact hepatocytes, by which a variety of membrane functions can be influenced, may be an important step in the mechanism of insulin action.     

Perturbation of egg phosphatidylcholine and dipalmitoylphosphatidylcholine multilamellar vesicles by n-alkanols. A fluorescent probe study

The perturbing effects of n-alkanols (pentanol, decanol and tetradecanol) in egg phosphatidylcholine and dipalmitoylphosphatidylcholine multilamellar vesicles were studied with five fluorescent probes, 1-(4'-trimethylaminophenyl)-6-phenylhexa-1,3,5-triene (TMA-DPH), 1,6-diphenyl-1,3,5-hexatriene, and 2-, 7-, and 12-(9-anthroxyloxy)stearic acid (2-, 7-, and 12-AS). These probes localize at various depths in the membrane, enabling study of the membrane-order gradient. Phase-modulation fluorescence spectroscopy was used to measure steady-state anisotropies, excited-state lifetimes and differential polarized lifetimes from which the limiting hindered anisotropies (r infinity) and the logarithm of the rotational rate (log R) were calculated. The probes that localize at about the same depth in the membrane (TMA-DPH and 2-AS, diphenylhexatriene and 12-AS) generally, but not always, showed similar changes in r infinity and log R with added alkanols. However, the absolute values of r infinity and log R were usually different. The inconsistencies are attributed to differences in the probes' sizes, structures, photophysical properties and perturbing abilities. The perturbation of membranes by alkanols is chain-length-dependent. Pentanol disorders the membrane at all depths but is more effective in the membrane center than nearer to the polar headgroups of the phospholipids, tetradecanol can be accommodated into the membrane without effect or with increased order and the effects of decanol are intermediate between pentanol and tetradecanol. Our results with alkanols indicate that: a single perturber can have different effects on membrane order at different depths in the bilayer; the perturbation is observed at and distant from the perturbers' location in the membrane, and the bilayer center is more susceptible to perturbation by alkanols than the region of the bilayer near the phospholipid headgroups.     

Dynamic fluorescence quenching studies on lipid mobilities in phosphatidylcholine-cholesterol membranes

Bimolecular collision rate of 8-anilinonaphthalene-1-sulfonic acid (ANS) and the nitroxide doxyl group attached to various carbons on stearic acid spin labels (n-SASL) in phosphatidylcholine-cholesterol membranes in the fluid phase was studied by observing dynamic quenching of ANS fluorescence by n-SASL's. The excited-state lifetime of ANS and its reduction by the n-SASL doxyl group were directly measured by the time-correlated single photon counting technique to observe only dynamic quenching separately from static quenching and were analyzed by using Stern-Volmer relations. The collision rate of ANS with the n-SASL doxyl group ranges between 1 X 10(7) and 6 X 10(7), and the extent of dynamic quenching by n-SASL is in the order of 5-much much greater than 6- greater than 7- less than 9- less than 10- less than 12- less than 16-SASL (less than 5-SASL) in dimyristoylphosphatidylcholine (DMPC) membranes. Collision rate of 16-SASL is only 10% less than that of 5-SASL. Since the naphthalene ring of ANS is located in the near-surface region of the membrane, these results indicate that the methyl terminal of SASL appears in the near surface area frequently, probably due to extensive gauche-trans isomerism of the methylene chain. The presence of 30 mol% cholesterol decreases the collision rate of ANS with 12- and 16-SASL doxyl groups but not with the 5-SASL doxyl group in DMPC membranes. On the other hand, in egg-yolk phosphatidylcholine membranes, inclusion of 30 mol% cholesterol does not affect the collision of ANS with either 5-SASL or 16-SASL doxyl groups, in agreement with our previous observation that alkyl chain unsaturation moderates cholesterol effects on lipid motion in the membrane (Kusumi et al., Biochim. Biophys. Acta 854, 307-317). It is suggested that dynamic quenching of ANS fluorescence by lipid-type spin labels is a useful new monitor of membrane fluidity that reports on various lipid mobilities in the membrane; a class of motion can be preferentially observed over others by selecting a proper spin label, i.e., rotational diffusion of lipid about its long axis and translational diffusion by using 5-SASL, wobbling motion of the lipid long axis by using 7-SASL or androstane spin label, and gauche-trans isomerism by using 16-SASL.     

Design, synthesis, and fluorescence studies of fluorenyl fatty acids as new depth-dependent fluorescent probes for membranes: getting over the looping-back problem

Fluorescent fatty acids have proved very useful in studying the membrane hydrophobic core. They readily partition into membranes or can be converted to phospholipids, which form integral components of membranes. By attaching the fluorescent chromophore to different positions along the alkyl chain of fatty acids, e.g., an anthroyloxy group attached via an ester linkage to n-hydroxystearic acid, membranes have been probed at different depths. While this is an interesting approach and has been extensively used, relatively little attention has been paid to the molecular design of these probes in order to have minimal membrane perturbation. In the present study we have looked into the general problem of design of such depth-dependent membrane probes. We report here a series of fluorenyl fatty acids with varying fatty acid chain lengths, i.e., (2-fluorenyl)acetic acid, -butyric acid, -hexanoic acid, and -octanoic acid, in order to obtain information at different depths in the membrane hydrophobic core. To see the effect of attachment of a hydrophobic tail on the orientation of such fatty acids in membranes, an n-butyl group was linked to the C-7 position of fluorene in (2-fluorenyl)butyric acid to get 4-(7-n-butylfluoren-2-yl)butyric acid. Further, to assess their ability to act as depth-dependent fluorescent probes, these fatty acids were incorporated in vesicles prepared from egg phosphatidylcholine, and their fluorescence quenching was studied with potassium iodide, Cu(II), 9,10-dibromostearic acid, and 12-bromostearic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

二价金属离子对嵌有线粒体H~+-ATP酶的脂酶体不同层次脂质流动性的影响

本文报道用荧光偏振及顺磁共振两种方法研究Mg~(2+)及其它二价金属离子对嵌有H~+-ATP酶的脂酶体不同层次脂质流动性的影响。 (1)顺磁标记探剂5-、12-、16-氮氧基硬脂酸测定结果表明Mg~(2+)和其它二价金属离子都能降低膜脂双分子层表层的流动性。降低流动性的顺序为Mg~(2+)=Ca~(2+)>Sr~(2+)>Cd~(2+)。较深层脂则无明显变化。 (2)荧光探剂7-、12-(9-蒽酰)硬脂酸及16-(9-蒽酰)棕榈酸的测定结果也表明Mg~(2+)和其它二价金属离子降低了膜脂表层的流动性,尤以Mn~(2+)、Ca~(2+)降低流动性最显著,流动性降低的顺序为;Mn~(2+) Ca>Sr~(2+) Mg~(2+) Cd~(2+)。除Mn~(2+)、Ca~(2+)还能影响膜脂深层的流动性外,其它与对照无明显差异。     

Modulation of liposomal membrane fluidity by flavonoids and isoflavonoids

The polyphenolic structures of flavonoids and isoflavonoids confer them with the ability to scavenge free radicals and to chelate transition metals, a basis for their potent antioxidant abilities. Another possible contributory mechanism toward their antioxidant activities is their ability to stabilize membranes by decreasing membrane fluidity. In this study, the effects of representative flavonoids, isoflavonoids, and their metabolites on membrane fluidity and their preferential localization in the membrane were investigated using large unilamellar vesicles (LUVs) as the membrane models. These results were compared with those of cholesterol and alpha-tocopherol. Changes in fluorescence anisotropy values for a series of n-(9-anthroyloxy) fatty acid probes (n = 6, 12, 16) upon addition of the test compounds were used to monitor alterations in membrane fluidity at graded depths in lipid bilayer. The results of the study suggest that the flavonoids and isoflavonoids, similar to cholesterol and alpha-tocopherol, partition into the hydrophobic core of the membrane and cause a dramatic decrease in lipid fluidity in this region of the membrane. Localization of flavonoids and isoflavonoids into the membrane interiors and their resulting restrictions on fluidity of membrane components could sterically hinder diffusion of free radicals and thereby decrease the kinetics of free radical reactions.     

Effect of fluorophore linkage position of n-(9-anthroyloxy) fatty acids on probe distribution between coexisting gel and fluid phospholipid phases

The quenching of probe fluorescence by spin-labeled phospholipid has been used to determine the distribution of a series of n-(9-anthroyloxy) fatty acids between coexisting gel and fluid liquid-crystal phases in multilamellar phospholipid vesicles. The phase distribution ratio in every case is found to favor the fluid lipid phase, but is much greater between fluid and Ca2+-induced gel than between fluid and thermal gel. For a given gel type, n-(9-anthroyloxy)stearic acids with n = 3, 6, 9 or 12 as well as 11-(9-anthroyloxy)undecanoic acid all exhibit similar behavior, favoring the fluid phase by about a factor of 4 over thermally-induced lipid gel phase and by 18 over Ca2+-induced gel phase. 16-(9-Anthroyloxy)palmitic acid, with the bulky probe at the terminus of the 16-carbon chain, favors the fluid phase less strongly, by a factor of 1.5 or 11 over thermally-induced or Ca2+-induced gel phase, respectively, indicating better packing of this probe in phospholipid gel phases.     

Mechanism and kinetics of merocyanine 540 binding to phospholipid membranes

The physicochemical mechanism for merocyanine 540 (M540) binding to unilamellar phosphatidylcholine (PC) vesicles was examined by steady-state and dynamic fluorescence and fluorescence stopped-flow methods. At 530-nm excitation, aqueous M540 has an emission peak at 565 nm, which red shifts to 580 nm with formation of membrane-bound monomers (M); bound dimers (D) are nonfluorescent. Equilibrium fluorescence titrations show that 50% of total M540 partitions into the membrane to form D at [M540]/[PC] (Rm/p)_approximately 0.6. M and D concentrations are equal at Rm/p approximately 0.05. For Rm/p less than 0.1, M540 has a single fluorescence lifetime (tau), which decreases with Rm/p [tau-1 (ns-1) = 0.48 + 3.3Rm/p], indicating a rapid collisional rate between M to form D. Dynamic depolarization studies show that hindered rotation of M (r infinity = 0.13 at Rm/p = 0.006) becomes more rapid (rotational rate 0.2-1.9 ns-1) with increasing Rm/p (0.006-0.075). The efficiencies of energy transfer between n-(9-anthroyloxy) fatty acid probes (n = 2, 6, 9, 12, 16) and bound M540 suggest that M is oriented parallel to the phospholipids near the membrane surface; studies of efficiencies of n-AF quenching by D are consistent with an orientation of D perpendicular to the phospholipids. In stopped-flow fluorescence measurements in which M540 is mixed with PC vesicles, there is a rapid (1 ms) followed by a slower (10-50 ms) concentration-dependent fluorescence increase.(ABSTRACT TRUNCATED AT 250 WORDS)