Diffusion and partitioning of a pesticide, lindane, into phosphatidylcholine bilayers: A new fluorescence quenching method to study chlorinated hydrocarbon-membrane interactions

Chlorinated hydrocarbons, such as the pesticide lindane (γ-hexachlorocyclohexane), quench the fluorescence of carbazole. The observed quenching is a result of the molecular contacts which occur upon diffusional collisions. Because the amount of quenching depends upon the collisional frequency between carbazole and pesticide, this phenomenon provides a measure of both the diffusional rate of lindane and its local concentration. The carbazole fluorophore is localized within phosphatidylcholine bilayers by cosonicating the lipid with a newly synthesized phospholipid, β-(11-(9-carbazole)-undecanoyl)-l-α-phosphatidylcholine. Using this probe in dimyristoyl-l-α-phosphatidylcholine vesicles, and the above mentioned quenching phenomena, we determined the lindane diffusion rate within the bilayer to be 5.7 · 10⁻⁷cm²/s at 37°C. Measurement of the apparent quenching constant at various dimyristoyl phosphatidylcholine concentrations yielded a lipid-water partition coefficient for lindane of 9500, which is in agreement with the value of 8980 obtained by our equilibrium dialysis experiments. Vesicles of dimyristoyl-l-α-phosphatidylcholine become saturated with lindane at a pesticide to lipid molar ratio of approx. 0.28.These results demonstrate the possibility of using the quenching of carbazole fluorescence to investigate the transport and partitioning of pesticides within biological membranes. This ability should prove useful in studies of the interactions of chlorinated hydrocarbons with cell membranes.     

Biosynthetic incorporation of fluorescent carbazolylundecanoic acid into membrane phospholipids of LM cells and determination of quenching constants and partition coefficients of hydrophobic quenchers

A fluorescence quenching method was developed for determining partition coefficients and diffusional rates of small molecules in cell membranes. This method involves quenching the fluorescence of carbazole-labeled membranes by hydrophobic molecules that partition into membranes. Cell membrane phospholipids of mouse LM cells in tissue culture were biosynthetically labeled with the carbazole moiety by supplementing the growth media with 11-(9-carbazolyl)undecanoic acid. Plasma membranes, microsomes, and mitochondria were isolated free of nonmembranous neutral lipids, and the incorporation of the fluorescent probe was characterized. Quenching studies of the carbazole moiety by a series of N-substituted picolinium perchlorate salts showed that the carbazole moiety was located in the hydrophobic interior of the membrane bilayer. The carbazole fluorescence also was quenched by the hydrophobic quenchers lindane, methoxychlor, and 1,1-dichloro-2,2-bis(rho-chlorophenyl)ethylene, indicating that these compounds partitioned into the membrane. Stern-Volmer quenching constants determined by fluorescence lifetime and intensity measurements were identical, as expected for dynamic quenching. The effects of different lipid compositions on quenching constants and partition coefficients were determined by comparing different membrane fractions. These parameters also were measured in membranes from cells in which the phospholipid composition was altered by substituting ethanolamine for choline in the growth medium. Changes in the lipid composition produced changes in the bimolecular quenching constants. For example, bimolecular quenching constants for 1,1-dichloro-2,2-bis(rho-chlorophenyl)ethylene were higher in mitochondrial membranes than in plasma membranes and microsomes. They were also higher in dispersions made from membrane phospholipids as compared with intact membranes or total lipid dispersion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Permeability of lipid bilayers to methylmercuric chloride: Quantification by fluorescence quenching of a carbazole-labeled phospholipid

We investigated the permeabilities of lipid bilayers to the neurotoxin methylmercuric chloride (MMC). This mercurial is an efficient collisional quencher of the fluorescence of N-alkyl carbazole derivatives. Quenching of the fluorescence of β-(3-(9-carbazole)-propionyl-L--phosphatidylcholine (CPA-PC) in vesicles of dimyristoyl phosphatidylcholine and of dioleoyl phosphatidylcholine reveal rapid diffusion of MMC in the alkyl side chain regions of these bilayers. By a combination of (1) the lipid concentration dependence of the apparent quenching constants, (2) the solubility of MMC in concentrated lipid dispersions and (3) the 270 MHz proton magnetic resonance of methylmercury in the presence of lipid bilayers we conclude that the lipid-water partition coefficient of this mercurial is less than or equal to two. Using the fluorescence quenching and the partitioning data we estimate the diffusion coefficient of MMC in these bilayers to range from 0.13 to 0.31 × 10⁻⁵ cm²/sec, or 20–47% of its diffusion coefficient in ethanol. These data indicate that lipid bilayers do not pose a significant permeability barrier to the diffusional transport of MMC.     

Quenching of fluorescence of pyrene-substituted lecithin by tetracyanoquinodimethane in liposomes.

In this work we have applied a kinetic scheme derived from fluorescence kinetics of pyrene-labeled phosphatidylcholine in phosphatidylcholine membrane to explain the fluorescence quenching of 1-palmitoyl-2-(10-[pyrenl-yl]-sn-glycerol-3-phosphatidylchol ine (PPDPC) liposomes by tetracyanoquinodimethane (TCNQ). The scheme was also found to be applicable to neat PPDPC and the effect of the quencher could be attributed to certain steps of the proposed mechanism. The TCNQ molecules influence the fluorescence of pyrene moieties in PPDPC liposome in two ways. Firstly, an interaction between the quencher molecule and the pyrene monomer in the excited state quenches monomer fluorescence and effectively prevents the diffusional formation of the excimer. Secondly, an interaction between the quencher molecule and the excited dimer quenches the excimer fluorescence. The TCNQ molecule does not prevent the formation of the excimer in pyrene moieties aggregated in such a way that they require only a small rotational motion to attain excimer configuration. The diffusional quenching rate constant is calculated to be 1.0 x 10(8) M-1 s-1 for the pyrene monomer quenching and 1.3 x 10(7) M-1 s-1 for the pyrene excimer quenching. The diffusion constant of TCNQ is 1.5 x 10(-7) cm2 s-1 for the interaction radii of 0.8-0.9 nm. The TCNQ molecules are practically totally partitioned in the membrane phase.     

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.     

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.     

Light-induced electron transfer in porphyrin-calixarene conjugates.

The fluorescence from a set of porphyrin-calixarene complexes is quenched upon addition of benzo-1,4-quinone (BQ) in fluid solution. In N,N-dimethylformamide solution, fluorescence quenching involves both static and dynamic interactions but there are no obvious differences between porphyrins with or without the appended calixarene. Under such conditions, the static quenching behaviour is attributed to pi-complexation between the reactants and it is concluded that the calixarene cavity does not bind BQ. An additional static component is apparent in dichloromethane solution. This latter effect involves partial fluorescence quenching, for which the intramolecular rate constant can be obtained by time-resolved fluorescence spectroscopy. The derived rate constants depend on molecular structure in a manner consistent with fluorescence quenching being due to electron transfer. In all cases, however, the dominant quenching step involves diffusional contact between the porphyrin nucleus and a non-bound molecule of BQ.     

Effects of hydrostatic pressure on the location of PRODAN in lipid bilayers: a FT-IR study

The effects of hydrostatic pressure on the location of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN), an environmentally sensitive fluorescent probe, in phosphatidylcholine lipid bilayers have been studied by Fourier-transform infrared spectroscopy (FT-IR) over the pressure range of 0.001-25 kbar. The results derived from the PRODAN C = O stretching band, the correlation field splitting of the methylene scissoring mode, and the methylene symmetric stretching mode as well as the absorption of the naphthalene ring show that in the sample of 4% (w/w) PRODAN in dimyristoyl-L-alpha-phosphatidylcholine (DMPC) at pH 6.8, most of the PRODAN molecules are embedded in the bilayers. In contrast, at pH 3.0, PRODAN was found to reside either on the membrane surface or dispersed in water. Compared to DMPC, egg yolk phosphatidylcholine (egg PC), which contains a substantial amount of unsaturated fatty acyl chains, is more susceptible to PRODAN permeation. The present study shows that the pressure dependence of the location of PRODAN in lipid membranes is different from that of tetracaine, a local anesthetic, in lipid bilayers. The model regarding the PRODAN location in lipid bilayers derived from the present infrared data has been compared with that obtained with previous fluorescence studies.     

Fluorescence quenching with lindane in small unilamellar l,α-dimyristoylphosphatidylcholine vesicles

The lateral mobility and lipid-water partition of the pesticide lindane was studied by fluorescence quenching of N-isopropylcarbazole (NIPC) and l,-palmitoyl--(N-carbazolyl) undecanoylphosphatidylcholine (PCUPC) in liposomes of dimyristoylphosphatidylcholine at 50°C. In isotropic solvents the quenching reaction was highly inefficient. A scheme for dynamic quenching, in which the monomolecular quenching rate constant is small, was valid. In lipid bilayers the same scheme was applied to describe the quenching results but the rate constant of the backreaction of the excited complex to quencher and excited probe was of comparable magnitude to the monomolecular quenching rate constant. This phenomenon results in biexponential decays of the fluorescent probe in the presence of quencher. All the rate constants of the scheme could be determined. Stern-Volmer plots at different membrane concentrations were obtained from fluorescence intensity and decay time measurements. From these plots the true bimolecular quenching rate constant, k _q , and the rate constant for lateral diffusion, k _d , were determined: . The smaller value of k _q compared to k _d for the quenching reaction of NIPC with lindane indicates that this quenching reaction is not diffusion controlled. The lateral diffusion coefficient D of lindane was found to be 1.7±0.2×10^-6 cm²/s in dimyristoylphosphatidylcholine vesicles at 50°C. The partition coefficient of lindane in these lipid bilayers is very high (>2000).Abbreviations DMPC dimyristoylphosphatidylcholine - lindane 1,2,3,4,5,6-hexachlorocyclohexane (-isomer) - NIPC N-isopropylcarbazole - PCUPC l,-palmitoyl--(N-carbazolyl) undecanoylphosphatidylcholine - SUV small unilamellar vesicles     

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.     

Tryptophan fluorescence study on the interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with model membranes

The interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with different phospholipid vesicles was investigated by fluorescence techniques, using a synthetic mutant signal peptide in which valine at position -8 in the hydrophobic sequence was replaced by tryptophan. First it was established that this mutation in the signal sequence of prePhoE does not affect in vivo and in vitro translocation efficiency and that the biophysical properties of the synthetic mutant signal peptide are similar to those of the wild-type signal peptide. Next, fluorescence experiments were performed which showed an increase in quantum yield and a blue shift of the emission wavelength maximum upon interaction of the signal peptide with lipid vesicles, indicating that the tryptophan moiety enters a more hydrophobic environment. These changes in intrinsic fluorescence were found to be more pronounced in the presence of phosphatidylglycerol (PG) or cardiolipin (CL) than with phosphatidylcholine (PC). In addition, quenching experiments demonstrated a shielding of the tryptophan fluorescence from quenching by the aqueous quenchers iodide and acrylamide upon interaction of the signal peptide with lipid vesicles, a shielding in the case of acrylamide that was more pronounced in the presence of negatively charged lipids. Finally it was found that acyl chain brominated lipids incorporated into phospholipid bilayers were able to quench the tryptophan fluorescence of the signal peptide, with the quenching efficiency in CL vesicles being much higher than in PC vesicles. The results clearly demonstrate that the PhoE signal peptide interacts strongly with different lipid vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monitoring the location profile of fluorophores in phosphatidylcholine bilayers by the use or paramagnetic quenching.

Spin probes differing in the position of their paramagnetic centre are used to quench the fluorescence of pyrene derivatives and chlorophylls incorporated into dimyristoyl phosphatidylcholine membranes. Pyrene butyric acid and pyrene decanoic acid with known orientation relative to the membrane surface are investigated. The quenching efficiency of fatty acid spin probes is dependent on the position of the nitroxide radical group in the fatty acid chain. Using this short range interaction we developed a spectroscopic method to chlorophyll-containing vesicles, we were able to characterize the orientation of the porphyrin ring within the membrane. Moreover, the chlorophyll fluorescence is also quenched by a water-soluble spin label. Therefore the porphyrin ring appears to be orientated in the polar head group region of the lipid layer, but not to be protruding out into the water phase. This conclusion is confirmed by the use of pyrene derivatives. Fluorescence quenching by a water-soluble spin label within the lipid matrix is observed even in the rigid state of the membrane. Fluorescence lifetime measurements suggest the existence of two different quenching mechanisms: (1) a static quenching occurring below the lipid phase transition temperature, and (2) an additional dynamic quenching taking place in the fluid state of the lipid bilayer.     

Comparison of 9-aminoacridine and atebrine induced changes in optical, electrical and mechanical characteristics of lipid bilayers.

The effects of fluorescent probes 9-aminoacridine (9AA) and atebrine (AT) on physical properties of liposomes and planar bilayer lipid membranes (BLM) were studied. The method of fluorescence spectroscopy and the electrostriction method based on measurement of higher current harmonics were used. At low concentrations (10(-5)-5 x 10(-5) mol/l), 9AA increased fluorescence intensity, while in liposomes from soybean phosphatidylcholine fluorescence quenching occurred at higher probe concentration. Fluorescence quenching occurred over the entire concentration range tested (10(-5)-10(-4) mol/l) in liposomes made from a mixture of egg phosphatidylcholine and cardiolipin. In contrast to 9AA, AT, thanks to its hydrophobic chain, penetrates deeper into the hydrophobic membrane moiety; thus, immobilization of the molecule and an increase in fluorescence intensity was always observed. Probes adsorbed to membranes, leaving their electric capacitance effectively unchanged. Adsorption of charged dye particles induced small changes in transmembrane potential. In the presence of 10(-5) mol/l AT, the modulus of elasticity E perpendicular increased somewhat for soft membranes (E perpendicular approximately 2.5 x 10(7) Pa), whereas it decreased for hard membranes (E perpendicular approximately 5 x 10(7) Pa). pH gradient present on the membrane affected the ability of the dyes to incorporate into the membranes. Our results provide evidence against the proposed model of the quenching mechanism introduced by Rottenberg and Lee (1975).     

Use of the parallax-quench method to determine the position of the active-site loop of cholesterol oxidase in lipid bilayers

To elucidate the cholesterol oxidase-membrane bilayer interaction, a cysteine was introduced into the active site lid at position-81 using the Brevibacterium enzyme. To eliminate the possibility of labeling native cysteine, the single cysteine in the wild-type enzyme was mutated to a serine without any change in activity. The loop-cysteine mutant was then labeled with acrylodan, an environment-sensitive fluorescence probe. The fluorescence increased and blue-shifted upon binding to lipid vesicles, consistent with a change into a more hydrophobic, i.e., lipid, environment. This acrylodan-labeled cholesterol oxidase was used to explore the pH, ionic strength, and headgroup dependence of binding. Between pH 6 and 10, there was no significant change in binding affinity. Incorporation of anionic lipids (phosphatidylserine) into the vesicles did not increase the binding affinity nor did altering the ionic strength. These experiments suggested that the interactions are primarily driven by hydrophobic effects not ionic effects. Using vesicles doped with either 5-doxyl phosphatidylcholine, 10-doxyl phosphatidylcholine, or phosphatidyl-tempocholine, quenching of acrylodan fluorescence was observed upon binding. Using the parallax method of London [Chattopadhyay, A., and London, E. (1987) Biochemistry 26, 39-45], the acrylodan ring is calculated to be 8.1 +/- 2.5 A from the center of the lipid bilayer. Modeling the acrylodan-cysteine residue as an extended chain suggests that the backbone of the loop does not penetrate into the lipid bilayer but interacts with the headgroups, i.e., the choline. These results demonstrate that cholesterol oxidase interacts directly with the lipid bilayer and sits on the surface of the membrane.     

Effect of lindane upon the beta-adrenergic stimulation of cyclic AMP accumulation in rat renal cortical tubules caused by alterations in membrane fluidity.

The influence of lindane (gamma-hexachlorocyclohexane) on fluidity and lipid composition in rat renal cortical tubules has been investigated. Lindane increased membrane fluidity as measured by a fluorescence polarization technique using the probe diphenylhexatriene. This effect was dose-dependent and was accompanied by a 70% inhibition of the beta-adrenergic stimulatory activity upon cyclic AMP accumulation after 30 min of preincubation with lindane at 25 degrees C. Experiments with increasing concentrations of isoproterenol indicated that the efficacy, but not the potency, of the beta-adrenergic effect upon cyclic AMP accumulation was affected by lindane. Lindane toxicity could also be associated with variations in the incorporation of acetate into various lipid classes. Lindane increased acetate incorporation into phospholipids and decreased that into cholesterol.     

The 'delta pH'-probe 9-aminoacridine: response time, binding behaviour and dimerization at the membrane

The fluorescence quenching of 9-aminoacridine (9-AA) after imposition of a transmembrane pH gradient (inside acidic) in liposomes has been investigated for a number of different lipid systems. The initial fluorescence decrease after a rapid pH jump, induced in the extravesicular medium by a stopped-flow mixing technique, was ascribed to a response of 9-AA to the imposed pH gradient and not to changes in the vesicular system itself. Time constants for this fluorescence quenching are in the range of several hundred milliseconds at 25 degrees C. Fluorescence recovery which should be correlated to the dissipation of the pH gradient occurs in the 100 s time range and is 10-30-times faster than the delta pH decay monitored with the entrapped hydrophilic pH-indicator dye pyranine. The quenching was severely hindered below the lipid phase transition of dipalmitoylphosphatidylglycerol. No delta pH-induced quenching was obtained in lipid vesicles containing only zwitterionic, net uncharged phosphatidylcholine headgroups. For the occurrence of quenching, the presence of negatively charged headgroups, i.e. phosphatidylglycerol or phosphatidylserine, was necessary. The extent of quenching, at a specific pH difference applied, had a cooperative dependency (Hill coefficient approximately 2) on the number of negative headgroups in the membrane and on the concentration of unquenched (unbound) 9-AA molecules. The concentration of quenched 9-AA molecules was furthermore proportional to the number of dimer-excimer complexes of 9-AA which are formed during the quenching process.     

Interactions of chlorinated hydrocarbon insecticides with membranes

Chlorinated hydrocarbon insecticides quench the fluorescence of N-alkyl derivatives of carbazole. We used phospholipids with covalently attached carbazole as probes for the interactions of chlorinated hydrocarbon insecticides with lipid bilayers, the object being to understand better the toxicities of chlorinated hydrocarbons. Fluorescence quenching measurements revealed the lipid-water partition coefficients of the chlorinated hydrocarbons, their diffusion coefficients in the membranes, and the binding capacities of the membranes for the chlorinated hydrocarbons. Active insecticides were compared with inactive analogues to test whether activities correlated with chlorinated hydrocarbon-membrane interactions. Thus DDT and methoxychlor were compared with inactive DDE, and insecticidal γ-lindane was compared with three less active stereoisomers. The partition coefficients, diffusion coefficients and membrane saturation capacities did not correlate with insecticidal potency. The partition coefficients of these chlorinated hydrocarbons were larger in bilayers containing unsaturated fatty acyl chains as compared to bilayers containing saturated fatty acyl chains. Interestingly, neural membranes are known to contain a large percentage of unsaturated lipids. Our results indicate that the activities of chlorinated hydrocarbons are not a result of specific interactions of these compounds with the lipids of membranes. However, the neurotoxicity of chlorinated hydrocarbons may be amplified by selective partitioning in the unsaturated neural membranes.     

Effects of in vivo and in vitro lindane treatment on rat ventral prostate lipids

The rat ventral prostate accumulated lindane (gamma-hexachlorocyclohexane) (0.59 +/- 0.07 ppm) when this liposoluble toxicant was injected subcutaneously at a concentration of 1 mg of 100 g body weight for 12 days. Total lipids and phospholipids (especially phosphatidylcholine) amounts were augmented in treated rats. Lindane had no significant influence upon cholesterol mass content in the ventral prostate. Using [1-14C]acetate as radioactive precursor, it was possible to conclude that the mass lipid variations caused by lindane treatment were due, at least in part, to a modification of the endogenous biosynthesis of these lipids. No changes were found in the acetate oxidation to CO2 when control rats and lindane-treated rats were compared.     

Fluorescence quenching in model membranes: phospholipid acyl chain distributions around small fluorophores

Fluorescence quenching in lipid bilayers is treated by a new approach based on calculation of the probability distribution of quenching and nonquenching acyl chains around a fluorophore. The effect of acyl lattice site dependence (i.e., correlations of phospholipid sister chain occupancy of neighbor sites) was modeled by use of Monte Carlo simulations of acyl chain occupancy. This explicit accounting of site occupancy correlation was found to fit observed quenching behavior better than did a model wherein phospholipid quenchers are considered to be independent. A key aspect of this approach is to evaluate the rate for quenching in a bilayer composed of pure quenching lipid. In order to evaluate this quenching rate, and also to provide a strong test of the calculated probability distributions, we synthesized lipids with both acyl chains labeled with a quenching moiety (Br or nitroxide), as well as the more usual single-chain quenchers. The fluorescence of tryptophan octyl ester (TOE), and of the 1,6-diphenyl-1,3,5-hexatriene (DPH) derivatives trimethylammonium-DPH (TMA-DPH) and 1-lauroyl-2-(DPH-propionyl)phosphatidylcholine (DPH-PC), was examined. We obtained consistent results with all the fluorophores and quenchers indicating that up to 18 neighboring acyl sites can contribute to quenching, corresponding to two shells of acyl sites on a hexagonal lattice. Calculated discrete distributions of fluorescence intensities were converted into fluorescence lifetimes and compared with Gaussian and Lorentzian continuous lifetime distributions. The fluorescence quenching theory presented here may be used to explain quantitatively the heterogeneity of fluorophore environments in multicomponent membranes.