Determination of partition and lateral diffusion coefficients of ubiquinones by fluorescence quenching of n-(9-anthroyloxy)stearic acids in phospholipid vesicles and mitochondrial membranes

The quenching of fluorescence of n-(9-anthroyloxy)stearic acids and other probes by different ubiquinone homologues and analogues has been exploited to assess the localization and lateral mobility of the quinones in lipid bilayers of model and mitochondrial membranes. The true bimolecular collisional quenching constants in the lipids together with the lipid/water partition coefficients were obtained from Stern-Volmer plots at different membrane concentrations. A monomeric localization of the quinone in the phospholipid bilayer is suggested for the short side-chain ubiquinone homologues and for the longer derivatives when cosonicated with the phospholipids. The diffusion coefficients of the ubiquinones, calculated from the quenching constants either in three dimensions or in two dimensions, are in the range of (1-6) X 10(-6) cm2 s-1, both in phospholipid vesicles and in mitochondrial membranes. A careful analysis of different possible locations of ubiquinones in the phospholipid bilayer, accounting for the calculated diffusion coefficients and the viscosities derived therefrom, strongly suggests that the ubiquinone 10 molecule is located within the lipid bilayer with the quinone ring preferentially adjacent to the polar head groups of the phospholipids and the hydrophobic tail largely accommodated in the bilayer midplane. The steady-state rates of either ubiquinol 1-cytochrome c reductase or NADH:ubiquinone 1 reductase are proportional to the concentration of the quinol or quinone substrate in the membrane. The second-order rate constants appear to be at least 3 orders of magnitude lower than the second-order constants for quenching of the fluorescent probes; this is taken as a clear indication that ubiquinone diffusion is not the rate-determining step in the quinone-enzyme interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of the lateral diffusion coefficients of ubiquinones in lipid vesicles by fluorescence quenching of 12-(9-anthroyl)stearate

The lateral diffusion coefficients of some ubiquinone homologues have been measured in phospholipid vesicles exploiting the fluorescence quenching of the probe 12-(9-anthroyl)stearate by the quinones. Diffusion coefficients higher than 10(-6) cm2 X s-1 have been found at 25 degrees C, compatible with the localization of the ubiquinones in the low-viscosity midplane region of the bilayer.     

Characteristics of self-quenching of the fluorescence of lipid-conjugated rhodamine in membranes

Self- or concentration quenching of octadecylrhodamine B (C18-Rh) fluorescence increases linearly in egg phosphatidylcholine (PC) vesicles but exponentially in vesicles composed of egg PC:cholesterol, 1:1, as the probe concentration is raised to 10 mol%. Cholesterol-dependent enhancement of self-quenching also occurs when N-(lissamine-rhodamine-B-sulfonyl)dioleoylphosphatidylethanolamine is substituted for C18-Rh and resembles that in dipalmitoylphosphatidylcholine vesicles below, as opposed to above, the phase transition. These effects are not due to changes in dimer:monomer absorbance. Stern-Volmer plots indicate a dependence of quenching on nonfluorescent dimers both in the presence and absence of cholesterol. Decreases in fluorescence lifetimes with increasing probe concentration parallel decreases in residual fluorescence of C18-Rh with increasing probe concentration in PC and PC + cholesterol membranes, respectively. Decreases in the steady-state polarization of C18-Rh fluorescence as its concentration is raised to 10 mol% indicate energy transfer with emission between probe molecules in PC and to a lesser extent in PC + cholesterol membranes. The calculated R0 for 50% efficiency of energy transfer from excited state probe to monomer was 55-58 A and to dimer was 27 A. Since lateral diffusion of C18-Rh is probably too slow to permit collisional quenching during the lifetime of the probe, even if C18-Rh were concentrated in a separate phase, C18-Rh self-quenching appears to be due mainly to energy transfer without emission to nonfluorescent dimers.     

Synthesis, location, and lateral mobility of fluorescently labeled ubiquinone 10 in mitochondrial and artificial membranes

To explore the influence of the long isoprene chain of ubiquinone 10 (UQ) on the mobility of the molecule in a phospholipid bilayer, we have synthesized a fluorescent derivative of the head-group moiety of UQ and measured its lateral diffusion in inner membranes of giant mitochondria and in large unilamellar vesicles. The diffusion coefficients, determined by the technique of fluorescence redistribution after photobleaching, were 3.1 X 10(-9) cm2 s-1 in mitochondria and 1.1 X 10(-8) cm2 s-1 in vesicles. Similar diffusion rates were observed for fluorescently labeled phosphatidylethanolamine (PE) with the same moiety attached to its head group (4-nitro-2,1,3-benzooxadiazole: NBD). Fluorescence emission studies carried out in organic solvents of different dielectric constants, and in vesicles and mitochondrial membranes, indicate that NBDUQ is located in a more hydrophobic environment than NBDPE or the starting material IANBD (4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitro-2,1,3- benzoxadiazole). Fluorescence quenching studies carried out with CuSO4, a water-soluble quenching agent, also indicate that NBDUQ is located deeper in the membrane than NBDPE. These results suggest that ubiquinone and PE are oriented differently in a membrane, even though their diffusion rates are similar. Conclusions regarding whether or not diffusion of UQ is a rate-limiting step in electron transfer must await a more detailed knowledge of the structural organization and properties of the electron transfer components.     

Partitioning of (+-)-5,6-dihydro-6-phenyl-2-n-alkyl-imidazo- [2,1-b]thiazoles into large unilamellar liposomes: a steady-state fluorescence quenching study.

The interaction of the tetramisole derivative (+-)-5,6-dihydro-6-phenyl-imidazo[2,1-b]thiazole and a number of its 2-n-alkyl homologues (-ethyl through -n-pentyl and -n-heptyl) with large unilamellar phosphatidylcholine/phosphatidylethanolamine/dipalmitoylphosphatidic acid (2:1:0.06, w/w) vesicles was studied by means of steady-state fluorescence quenching using 8-(2-anthryl)octanoic acid as membrane probe. Linear Stern-Volmer plots were obtained for each derivative, indicating dynamic quenching. The slopes of the plots decreased with increasing liposomal concentration. For four short-chain homologues (-H, -ethyl, -n-propyl and -n-butyl), the respective membrane partition coefficients Kp and bimolecular quenching rate constants kq were determined from the plots of the reciprocal of the apparent quenching rate constant (kappq)-1 against the lipid volume fraction alpha L of the liposomes. The partition coefficients increased with increasing chain-length of the tetramisoles. A linear relationship was found between the free energy of partitioning and the number of methylene units of the homologues (-delta G degrees per methylene group = 1.6 +/- 0.1 kJ mol-1). For the n-pentyl and n-heptyl derivatives, the fluorescence quenching technique did not allow one to determine their membrane partition coefficients. Analysis of the fluorescence intensity measurements with Scatchard plots gave further evidence for the partitioning nature of the tetramisole derivatives' association with the liposomal membranes.     

A method for the determination of diffusion coefficients for small molecules in aqueous solution

A method is described for determining the diffusion coefficients of small solutes in limited volumes (approximately equal to 4-9 ml) of fluid. Diffusion is measured in a three-chamber diffusion cell across a central unstirred compartment. Compartments are separated by nitrocellulose membranes. The instantaneous concentration gradient and the instantaneous flux of solute into the dilute end compartment are derived from changes in the concentration of solute in the two stirred end compartments through time. The diffusion coefficient is calculated from the slope of the least-squares regression line relating the magnitude of the instantaneous solute flux to that of the instantaneous concentration gradient. The apparatus is calibrated with a solute of known diffusivity (KCl). Diffusion coefficients thus determined in water at 25 degrees C for CaCl2 (7.54 X 10(-6) cm2.s-1), Na2-ATP (7.01 X 10(-6) cm2.s-1), 2-deoxyglucose (5.31 X 10(-6) cm2.s-1), and D-Na-lactate (5.62 X 10(-6) cm2.s-1) differed by an average of 3.7% from literature values. The method described results in accurate estimates of diffusion coefficients by a simple and relatively rapid procedure.     

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.     

Ligand-dependent conformational equilibria of serum albumin revealed by tryptophan fluorescence quenching

Ligand-dependent structural changes in serum albumin are suggested to underlie its role in physiological solute transport and receptor-mediated cellular selection. Evidence of ligand-induced (oleic acid) structural changes in serum albumin are shown in both time-resolved and steady-state fluorescence quenching and anisotropy measurements of tryptophan 214 (Trp214). These studies were augmented with column chromatography separations. It was found that both the steady-state and time-resolved Stern-Volmer collisional quenching studies of Trp214 with acrylamide pointed to the existence of an oleate-dependent structural transformation. The bimolecular quenching rate constant of defatted human serum albumin, 1.96 x 10(9) M-1 s-1, decreased to 0.94 x 10(9) M-1 s-1 after incubation with oleic acid (9:1). Furthermore, Stern-Volmer quenching studies following fractionation of the structural forms by hydrophobic interaction chromatography were in accordance with this interpretation. Time-resolved fluorescence anisotropy measurements of the Trp214 residue yielded information of motion within the protein together with the whole protein molecule. Characteristic changes in these motions were observed after the binding of oleate to albumin. The addition of oleate was accompanied by an increase in the rotational diffusion time of the albumin molecule from approximately 22 to 33.6 ns. Within the body of the protein, however, the rotational diffusion time for Trp214 exhibited a slight decrease from 191 to 182 ps and was accompanied by a decrease in the extent of the angular motion of Trp214, indicating a transition after oleate binding to a more spatially restricted but less viscous environment.     

Permeation of tetracyclines through membranes of liposomes and Escherichia coli.

Uptake of tetracycline (tc), 2-tetracyclinonitrile (CN-tc), and 9-(N, N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline (DMG-DMDOT) by liposomes containing Tet repressor (TetR) and by Escherichia coli cells overexpressing TetR was examined. TetR specifically binds to tetracyclines, enhances their fluorescence and thereby allows selective detection of tetracyclines that have crossed the membranes. Analysis of the diffusion of tc and DMG-DMDOT into liposomes yielded permeation coefficients of (2.4 +/- 0.6) x 10-9 cm.s-1 and (3.3 +/- 0.8) x 10-9 cm.s-1, respectively. Similar coefficients were obtained for uptake of these tetracyclines by E. coli, indicating that diffusion through the cytoplasmic membrane is the rate-limiting step. The permeation coefficients translate into half-equilibration times of approximately 35 +/- 15 min and explain how efflux pumps can mediate resistance against tetracyclines. Furthermore, diffusion of CN-tc into liposomes was at least 400-fold slower than that of tc, indicating that the carboxamide group at position C2 is required for efficient permeation of tc through lipid membranes and thereby explaining the lack of antibiotic activity of CN-tc.     

The susceptibility of membrane vesicles to interaction with ethanol

The susceptibility of membranes to interaction with ethanol is an important consideration in the further understanding of the ethanol-membrane interaction. Interaction of membrane vesicles, including passive diffusion of ethanol across membranes, leakage of internal molecules out of membranes and membrane-membrane interaction, were examined systematically using two populations of fluorescent probe-encapsulated phospholipid bilayer vesicles, each prepared with 1,2-dimyristoyl phosphatidylcholine, cholesterol and a fluorescent probe. Fluorescence quenching experiments with these vesicles were performed in a medium containing a wide range of ethanol concentrations (0.30-3.5 M). In the presence of a lower concentration of ethanol in the external medium, passive diffusion of ethanol across membrane vesicles occurred. This was demonstrated by an interaction of ethanol with the encapsulated fluorescence probe molecules inside the vesicles, resulting in an increase in the fluorescence intensity and a shift of the fluorescence emission spectrum to a shorter wavelength. While, in the presence of a higher concentration of ethanol in the external medium, a strong perturbation of lipid bilayers by ethanol was found, leading to an over expansion of membranes and consequently causing the membrane leakage. As a result of this, the initially encapsulated probe molecules leaked out of the vesicles so as to interact with the other probe molecules in the external medium. Consequently, fluorescence quenching was observed. Moreover, studies of the mixture of two populations of fluorescence probe-encapsulated membrane vesicles revealed that ethanol acted on individual membranes and did not promote membrane-membrane interactions. The implication of the present results to the alcohol-mediated expansion of membranes is discussed.     

Localization and mobility of coenzyme Q in lipid bilayers and membranes.

We have studied the mobility of coenzyme Q (CoQ) in lipid bilayers and mitochondrial membranes in relation to the control of electron transfer activities. A molecular dynamics computer simulation in the vacuum yielded a folded structure for CoQ10, with a length of only 21 A. Using this information we were able to calculate diffusion coefficients in the range of 10(-6) cm2/s in good agreement with those found experimentally by fluorescence quenching of pyrene derivatives. To investigate if CoQ diffusion may represent the rate-limiting step of electron transfer, we reconstituted complexes I and III and assayed the resulting NADH-cytochrome c reductase activity in presence of different CoQ10 levels and at different distances between complexes; the experimental turnovers were higher than the collision frequencies calculated using diffusion coefficients of 10(-9) cm2/s but compatible with values found by us by fluorescence quenching. Since the experimental turnovers are independent of the distance between complexes, we conclude that CoQ diffusion is not rate-limiting for electron transfer.     

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)     

Translational diffusion of bovine prothrombin fragment 1 weakly bound to supported planar membranes: measurement by total internal reflection with fluorescence pattern photobleaching recovery.

Previous work has shown that bovine prothrombin fragment 1 binds to substrate-supported planar membranes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) in a Ca(2+)-specific manner. The apparent equilibrium dissociation constant is 1-15 microM, and the average membrane residency time is approximately 0.25 s-1. In the present work, fluorescence pattern photobleaching recovery with evanescent interference patterns (TIR-FPPR) has been used to measure the translational diffusion coefficients of the weakly bound fragment 1. The results show that the translational diffusion coefficients on fluid-like PS/PC planar membranes are on the order of 10(-9) cm2/s and are reduced when the fragment 1 surface density is increased. Control measurements were carried out for fragment 1 on solid-like PS/PC planar membranes. The dissociation kinetics were similar to those on fluid-like membranes, but protein translational mobility was not detected. TIR-FPPR was also used to measure the diffusion coefficient of the fluorescent lipid NBD-PC in fluid-like PS/PC planar membranes. In these measurements, the diffusion coefficient was approximately 10(-8) cm2/s, which is consistent with that measured by conventional fluorescence pattern photobleaching recovery. This work represents the first measurement of a translational diffusion coefficient for a protein weakly bound to a membrane surface.     

Acanthamoeba profilin binding to fluorescein-labeled actins.

The binding constants of Acanthamoeba profilin to fluorescein-labeled actin from Acanthamoeba and from rabbit skeletal muscle have been determined by measuring the reduction in the actin tracer diffusion coefficients, determined by fluorescence photobleaching recovery, as a function of added profilin concentration. Data were analyzed using a two-parameter nonlinear regression analysis to determine the profilin-actin dissociation constant Kd and the profilactin diffusion coefficient, DPA. For fluorescein-labeled Acanthamoeba actin, the least-squares estimates for Kd and DPA, along with approximate single standard deviation confidence intervals, are Kd = 48 (36, 63) microM and DPA = 6.72 (6.62, 6.81) X 10(-7) cm2s-1. For fluorescein-labeled skeletal muscle actin, the corresponding values are Kd = 147 (94, 225) microM and DPA = 6.7 (6.3, 7.0) X 10(-7) cm2s-1. These dissociation constants are the first to be determined from direct physical measurement; they are in agreement with values inferred from earlier studies on the effect of profilin on the assembly of actin that had been fluorescently labeled or otherwise modified at Cys 374. These results place important restrictions on the interpretation of experiments in which fluorescently labeled actin is used as a probe of living cytoplasm or cytoplasmic extracts that include profilin.     

Tumor necrosis factor and immune interferon act in concert to slow the lateral diffusion of proteins and lipids in human endothelial cell membranes

Vascular endothelial surface-related activities may depend on the lateral mobility of specific cell surface macromolecules. Previous studies have shown that cytokines induce changes in the morphology and surface antigen composition of vascular endothelial cells in vitro and at sites of immune and inflammatory reactions in vivo. The effects of cytokines on membrane dynamic properties have not been examined. In the present study, we have used fluorescence photobleaching recovery (FPR) to quantify the effects of the cytokines tumor necrosis factor (TNF) and immune interferon (IFN-gamma) on the lateral mobilities of class I major histocompatibility complex protein, of an abundant 96,000 Mr mesenchymal cell surface glycoprotein (gp96), and of a phospholipid probe in cultured human endothelial cell (HEC) membranes. Class I protein and gp96 were directly labeled with fluorescein-conjugated monoclonal antibodies; plasma membrane lipid mobility was examined with the phospholipid analogue fluorescein phosphatidylethanolamine (Fl-PE). In untreated, confluent HEC monolayers, diffusion coefficients were 30 x 10(-10) cm2 s-1 for class I protein, 14 x 10(-10) cm2 s-1 for gp96, and 80 x 10(-10) cm2 s-1 for Fl-PE. Fractional mobilities were greater than 80% for each probe. Cultures treated at visual confluence for 3-4 d with either 100 U/ml TNF or 200 U/ml IFN-gamma did not exhibit significant changes in protein or lipid mobilities despite significant changes in cell morphology and membrane antigen composition. In HEC cultures treated concomitantly with TNF and IFN-gamma, however, diffusion coefficients decreased by 71-79% for class I protein, 29-55% for gp96, and 23-38% for Fl-PE. Fractional mobilities were unchanged. By immunoperoxidase transmission electron microscopy, plasma membranes of untreated and cytokine-treated HEC were flat and stained uniformly for class I antigen. "Line" FPR measurements on doubly treated HEC demonstrated isotropic diffusion of class I protein, gp96, and Fl-PE. Finally, although TNF and IFN-gamma retarded the growth of HEC cultures and disrupted the organization of cell monolayers, the slow diffusion rates of gp96 and Fl-PE in confluent doubly treated monolayers were not reproduced in sparse or subconfluent untreated monolayers. We conclude that the slowing of protein and lipid diffusion induced by the combination of TNF and IFN-gamma is not due to plasma membrane corrugations, to anisotropic diffusion barriers, or to decreased numbers of cell-cell contacts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lateral mobility of a lipid analog in the membrane of irreversible sickle erythrocytes

The major feature of sickle cell anemia is the tendency of erythrocytes to sickle when exposed to decreased oxygen tension and to unsickle when reoxygenated. Irreversible sickle cells (ISCs) are sickle erythrocytes which retain bipolar elongated shapes despite reoxygenation. ISCs are believed to owe their biophysical abnormalities to acquired membrane alterations which decrease membrane deformability. While increased membrane surface viscosity has been measured in ISCs, the lateral dynamics of membrane lipids in these cells have not heretofore been examined. We have measured the lateral diffusion of the lipid analog 3,3'-dioctadecylindocyanine iodide (DiI) in the plasma membrane of intact normal erythrocytes, reversible sickle cells (RSCs), and irreversible sickle cells by fluorescence photobleaching recovery (FPR). The diffusion coefficients +/- standard errors of the mean of DiI in intact normal red blood cells (RBCs), RSCs, and ISCs at 37 degrees C are (8.06 +/- 0.29) X 10(-9) cm2 X s-1, (7.74 +/- 0.22) X 10(-9) cm2 X s-1, and (7.29 +/- 0.24) X 10(-9) cm2 X s-1, respectively. A similar decrease in the diffusion coefficient of DiI in the plasma membranes of the three cell types was observed at 4, 10, 17, 23, and 30 degrees C. ANOVA analysis of the changes in DiI diffusion showed significant differences between the RBC and ISC membranes at all temperatures examined. The characteristic breaks in Arrhenius plots of the diffusion coefficients for the RBCs, RSCs, and ISCs occurred at 20, 19, and 18.6 degrees C, respectively. Photobleaching recovery data were used to estimate (Boullier, J.A., Melnykovich, G. and Barisas, B.G. (1982) Biochim. Biophys. Acta 692, 278-286) the microviscosities of the plasma membranes of the three cell types at 25 degrees C. We find significant differences between our microviscosity values and those obtained in previous fluorescence depolarization studies. However, both methods indicate qualitatively similar differences in membrane microviscosity among the various cell types.     

A milling crowd model for local and long-range obstructed lateral diffusion. Mobility of excimeric probes in the membrane of intact erythrocytes.

A new model for lateral diffusion, the milling crowd model (MC), is proposed and is used to derive the dependence of the monomeric and excimeric fluorescence yields of excimeric membrane probes on their concentration. According to the MC model, probes migrate by performing spatial exchanges with a randomly chosen nearest neighbor (lipid or probe). Only nearest neighbor probes, one of which is in the excited state, may form an excimer. The exchange frequency, and hence the local lateral diffusion coefficient, may then be determined from experiment with the aid of computer simulation of the excimer formation kinetics. The same model is also used to study the long-range lateral diffusion coefficient of probes in the presence of obstacles (e.g., membrane proteins). The dependence of the monomeric and excimeric fluorescence yields of 1-pyrene-dodecanoic acid probes on their concentration in the membranes of intact erythrocytes was measured and compared with the prediction of the MC model. The analysis yields an excimer formation rate for nearest neighbor molecules of approximately 1 X 10(7) s-1 and an exchange frequency of approximately greater than 2 X 10(7) s-1, corresponding to a local diffusion coefficient of greater than 3 X 10(-8) cm2 s-1. This value is several times larger than the long-range diffusion coefficient for a similar system measured in fluorescence photobleaching recovery experiments. The difference is explained by the fact that long-range diffusion is obstructed by dispersed membrane proteins and is therefore greatly reduced when compared to free diffusion. The dependence of the diffusion coefficient on the fractional area covered by obstacles and on their size is derived from MC simulations and is compared to those of other theories lateral diffusibility.     

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 (gamma-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, beta-(11-(9-carbazole)-undecanoyl)-L-alpha-phosphatidylcholine. Using this probe in dimyristoyl-L-alpha-phosphatidylcholine vesicles, and the above mentioned quenching phenomena, we determined the lindane diffusion rate within the bilayer to be 5.7.10-7 cm2/s at 37 degrees 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-alpha-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.     

Lateral diffusion in model membranes is independent of the size of the hydrophobic region of molecules.

We have systematically investigated the probe size and shape dependence of lateral diffusion in model dimyristoyl phosphatidylcholine membranes. Linear hydrophobic polymers, which differ in length by an order of magnitude, were used to explore the effect on the lateral diffusion coefficient of hydrodynamic restrictions in the bilayer interior. The polymers employed are isoprenoid alcohols--citronellol, solanesol, and dolichol. Tracer lateral diffusion coefficients were measured by fluorescence photobleaching recovery. Despite the large difference in lengths, the nitrobenzoxadiazole labelled alcohols all diffuse at the rate of lipid self-diffusion (5.0 x 10(-12) m2 s-1, 29 degrees C) in the liquid crystal phase. Companion measurements in isotropic polymer solution, in gel phase lipid membranes and with nonpolar fluorescent polyaromatic hydrocarbons, show a marked dependence of the lateral diffusion coefficient on the probe molecule size. Our results in the liquid crystal phase are in accord with free area theory which asserts that lateral diffusion in the membrane is restricted by the surface-free area. Probe molecules which are significantly longer than the host phospholipid, seven times longer in the case of dolichol, are still restricted in their lateral motion by the surface properties of the bilayer in the liquid crystal phase. Fluorescence quenching experiments indicate that the nitrobenzoxadiazole label does not reside at the aqueous interface, although it must reside in close proximity according to the diffusion measurements.     

Interaction of anthracyclines with nucleotides and related compounds studied by spectroscopy

Interaction of the antitumour anthracyclines with mononucleotides and related compounds can be assessed through the perturbation of the spectral properties of the drugs. Purine-derived compounds induce spectral changes more efficiently than pyrimidine derivatives. No marked differences are observed when mono-, di- or triphosphate derivatives, deoxy forms, nucleosides or free nitrogen bases are used for the experiments. Visible absorbance data indicate the existence of a drug/purine nucleotide complex in solution. Assuming a simple equilibrium, this complex would be of low affinity (Keq 100 M-1). Circular dichroism spectra of daunomycin in the presence of ATP suggest that the resulting daunomycin/ATP complexes are not comparable to those formed by intercalation of the anthracycline into DNA. 31P-NMR of ATP in the presence of daunomycin does not support the notion that anthracycline/nucleotide complex formation involves interaction through the phosphate group(s) of the nucleotide. Analysis of the quenching of the drug's intrinsic fluorescence in the presence of nucleotides indicates a predominantly collisional, dynamic quenching mechanism. Values in the 2-6 mM and 85-100 mM range, respectively, are estimated for the reciprocal of the Stern-Volmer quenching constant for a variety of purine and pyrimidine derivatives. This indicates that purine derivatives are highly efficient quenchers of the fluorescence of anthracyclines, while pyrimidine derivatives are not. The fluorescence lifetime of daunomycin in the absence of quencher and the Stern-Volmer quenching constants obtained for different nucleotides are used to calculate the apparent bimolecular rate constants for collisions between fluorophore and quencher to occur. Values of (2-3) X 10(11) and 1 X 10(10) M-1 X s-1 are obtained, respectively, for purine and pyrimidine derivatives. This suggests a combination of static and dynamic quenching processes for purine compounds, which is consistent with the drug/purine nucleotide complex formation detected by visible absorbance. Because of the high intracellular concentration of certain nucleotides, particularly ATP, the above processes are predicted to be highly significant 'in vivo'.