Phospholipid probes

J. R. Lakowicz and D. Hogen, Chlorinated hydrocarbon-cell membrane interactions studies by the fluorescence quenching of carbazole-labeled phospholipids Probe synthesis and characterization ofthe quenching methodology, Chem. Phys. Lipids 26, 1 10 (1980). 

Haest, C.W., Kamp, D., and Deuticke, B., 1997, TransbUayer reorientation of phospholipid probes in the human erythrocyte membrane, Lessions from studies on electroporated and reseated cells, Biochim. Biophys. Acta 1325 17-33. 

Nakatani, Y, Yamamoto, M., Diyizou, Y. et al. (1996). Studies on the topography of biomembranes regioselective photolabelling in vesicles with the tandem use of cholesterol and a photoactivable transmembrane phospholipidic probe. Chemistry - a European Journal, 2, 129-38. 

Grazing incidence excitation of a fluorescent probe in a phospholipid monolayer can also be used to indicate order. The collective tilt of the molecules in a domain inferred from such measurements is indicative of long-range orientational order [222]. 

Although extraction of lipids from membranes can be induced in atomic force apparatus (Leckband et al., 1994) and biomembrane force probe (Evans et al., 1991) experiments, spontaneous dissociation of a lipid from a membrane occurs very rarely because it involves an energy barrier of about 20 kcal/mol (Cevc and Marsh, 1987). However, lipids are known to be extracted from membranes by various enzymes. One such enzyme is phospholipase A2 (PLA2), which complexes with membrane surfaces, destabilizes a phospholipid, extracts it from the membrane, and catalyzes the hydrolysis reaction of the srir2-acyl chain of the lipid, producing lysophospholipids and fatty acids (Slotboom et al., 1982 Dennis, 1983 Jain et al., 1995). SMD simulations were employed to investigate the extraction of a lipid molecule from a DLPE monolayer by human synovial PLA2 (see Eig. 6b), and to compare this process to the extraction of a lipid from a lipid monolayer into the aqueous phase (Stepaniants et al., 1997). 

Sonication of MLV dispersions above the Tc of the lipids results in the formation of SUV (Saunders, et al., 1962). Sonication can be performed with a bath sonicator (Papahadjopoulos and Watkins, 1967) or a probe sonicator (Huang, 1969). During sonication the MLV structure is broken down and small unilamellar vesicles with a high radius of curvature are formed. In case of SUV with diameters of about 20 nm (maximum radius of curvature), the outer monolayer can contain over 50% of the phospholipids and in the case of lipid... 

One major interest in vibrational surface spectroscopy is the ability to directly probe lipid layers. Similarly to the previous case, the structure of the alkyl chains of phospholipids is readily determined from the ratio of the magnitude of the CH2 and CH3 symmetrical stretching modes [136,137]. At the D2O-CCI4 interface, a layer of... 

Four neutral lipid models were explored at pH 7.4 (1) 2% wt/vol DOPC in dode-cane, (2) olive oil, (3) octanol, and (4) dodecane. Table 7.5 lists the effective permeabilities Pe, standard deviations (SDs), and membrane retentions of the 32 probe molecules (Table 7.4). The units of Pe and SD are 10 6 cm/s. Retentions are expressed as mole percentages. Figure 7.22a is a plot of log Pe versus log Kd (octanol-water apparent partition coefficients, pH 7.4) for filters loaded with 2% wt/vol DOPC in dodecane (model 1.0, hlled-circle symbols) and with phospholipid-free dodecane (model 4.0, open-circle symbols). The dashed line in the plot was calculated assuming a UWL permeability (see Section 7.7.6) Pu, 16 x 10-6 cm/s (a typical value in an unstirred 96-well microtiter plate assay), and Pe of 0.8 x 10-6 cm/s... 

The above iso-pH measurements are based on the 2% DOPC/dodecane system (model 1.0 over pH 3-10 range). Another membrane model was also explored by us. Table 7.16 lists iso-pH effective permeability measurements using the soy lecithin (20% wt/vol in dodecane) membrane PAMPA (models 17.1, 24.1, and 25.1) The negative membrane charge, the multicomponent phospholipid mixture, and the acceptor sink condition (Table 7.1) result in different intrinsic permeabilities for the probe molecules. Figure 7.40 shows the relationship between the 2% DOPC and the 20% soy iso-pH PAMPA systems for ketoprofen. Since the intrinsic permeability of ketoprofen in the soy lecithin membrane is about 20 times greater than in DOPC membrane, the flat diffusion-limited transport region of the log Pe... 

Lukac S (1984) Thermally induced variations in polarity and microviscosity of phospholipid and surfactant vesicles monitored with a probe forming an intramolecular charge-transfer complex. J Am Chem Soc 106 4386 -392... 

Klymchenko AS, Duportail G, Mely Y et al (2003) Ultrasensitive two-colour fluorescence probes for dipole potential in phospholipid membranes. PNAS 100 11219-11224... 

Pal, R., Barenholz, Y. and Wagner, R. R. (1988). Pyrene Phospholipid as a biological fluorescent-probe for studying fusion of virus membrane with liposomes. Biochemistry (Mosc). 27, 30-36. 

Sklar, L. A., Hudson, B. S. and Simoni, R. D. (1977). Conjugated polyene fatty-acids as fluorescent-probes - Synthetic phospholipid membrane studies. Biochemistry (Mosc). 16, 819-828. 

Wichmann, O., Gelb, M. H. and Schultz, C. (2007). Probing Phospholipase A(2) with Fluorescent Phospholipid Substrates. Chembiochem 8, 1555-1569. 

Bedzyk and co-workers used the XSW technique to probe the ion distribution in the electrolyte above a charged cross-linked phospholipid membrane adsorbed onto a silicon-tungsten layered synthetic microstructure (LSM) as shown in Figure 2.80(a). The grazing-angle incidence experimental set-up... 

Liposphere formulations are prepared by solvent or melt processes. In the melt method, the active agent is dissolved or dispersed in the melted solid carrier (i.e., tristearin or polycaprolactone) and a hot buffer solution is added at once, along with the phospholipid powder. The hot mixture is homogenized for about 2 to 5 min, using a homogenizer or ultrasound probe, after which a uniform emulsion is obtained. The milky formulation is then rapidly cooled down to about 20°C by immersing the formulation flask in a dry ice-acetone bath, while homogenization is continued to yield a uniform dispersion of lipospheres. 

The studies on phospholipid bilayers with defined amounts of charged component are helpful to explain the partition characteristics in biological membranes. Liposome water partition data of propranolol in lipids from kidney epithelial cells (a common model system in pharmaceutical sciences for the uptake into the gastrointestinal tract) have been successfully described with partition models developed for pure bilayers or defined mixtures [159]. Since lipophilic cations and anions can be used as probes for the membrane potential, their interaction with microbial and mitochondrial membranes has been studied... 

Fig. 1.4. Examples of surfactants, phospholipids and polymers with covalently bound probes. 1 2-(9-anthroyloxy)stearic acid. 2 6-(9-anthroyloxy)stearic acid.

Lipid-protein interactions are of major importance in the structural and dynamic properties of biological membranes. Fluorescent probes can provide much information on these interactions. For example, van Paridon et al.a) used a synthetic derivative of phosphatidylinositol (PI) with a ris-parinaric acid (see formula in Figure 8.4) covalently linked on the sn-2 position for probing phospholipid vesicles and biological membranes. The emission anisotropy decays of this 2-parinaroyl-phosphatidylinositol (PPI) probe incorporated into vesicles consisting of phosphatidylcholine (PC) (with a fraction of 5 mol % of PI) and into acetylcholine receptor rich membranes from Torpedo marmorata are shown in Figure B8.3.1. 

Georgescauld D., Desmasez J. P., Lapouyade R., Babeau A., Richard H. and Winnik M. (1980) Intramolecular Excimer Fluorescence A New Probe of Phase Transitions in Synthetic Phospholipid Membranes, Photochem. Photobiol. 31, 539-545. 

RET can be used to investigate the lateral organization of phospholipids (range of 100 A) in gel and fluid phases. Indeed, information can be obtained on the probe heterogeneity distribution the donors sense various concentrations of acceptor according to their localization. A continuous probability function of having donors with a mean local concentration CA of acceptors in their surroundings should thus be introduced in Eq. (9.36) written in two dimensions ... 

Applications to fluorescent or fluorescently labeled proteins and nucleic acids, and to fluorescent lipid probes in phospholipid bilayers, have been reported. In the latter case, the diffusion coefficients measured above the chain melting temperature were found to be 10 7 cm2 s 1, which is in agreement with values obtained by other techniques. 

A. P. Demchenko and N. V. Shcherbatska, Nanosecond dynamics of the charged fluorescent probes at the polar interface of the membrane phospholipid bilayer, Biophys. Chem. 22, 131-143 (1985). 

The properties of membranes commonly studied by fluorescence techniques include motional, structural, and organizational aspects. Motional aspects include the rate of motion of fatty acyl chains, the head-group region of the phospholipids, and other lipid components and membrane proteins. The structural aspects of membranes would cover the orientational aspects of the lipid components. Organizational aspects include the distribution of lipids both laterally, in the plane of the membrane (e.g., phase separations), and across the membrane bilayer (phospholipid asymmetry) and distances from the surface or depth in the bilayer. Finally, there are properties of membranes pertaining to the surface such as the surface charge and dielectric properties. Fluorescence techniques have been widely used in the studies of membranes mainly since the time scale of the fluorescence lifetime coincides with the time scale of interest for lipid motion and since there are a wide number of fluorescence probes available which can be used to yield very specific information on membrane properties. 

Another factor affecting the lifetime of a membrane fluorophore probe is its proximity to the surface. The lifetimes of the DPH, DPH-phosphatidyl-choline (DPH-PC), and trimethylammonium-DPH (TMA-DPH) probes decrease in the order DPH > DPH-PC > TMA-DPH, as the probe locates nearer to the surface of the lipid bilayer.(7) The same is found for the anthroyl-stearate probes.(8) More recently, it has been shown that with TMA-DPH, the lifetime appears to be fairly sensitive to the differences in lipid bilayer packing induced by differing degrees of unsaturation in the phospholipid fatty acyl chains.(9) This aspect of the use of TMA-DPH and possibly other probes remains to be further exploited. 

The dansyl (dimethylaminonaphthalenesultonyl) group has been attached to various lipids, most notably at the phospholipid head group, where the probe is sensitive to solvent effects (see below). 

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