Phospholipid vesicles fluorescent probes

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. 

Since iodide is known to act in phospholipid vesicles as a collisional quencher for AF probes [6,9] by a diffusive process, fluorescence quenching was described by the Stem-Volmer relationship [6] ... 

The NMR rotating frame spin-lattice relaxation time (Tjp) method, which has been used successfully in our laboratory in studies of pressure effects on diffusion in highly viscous liquids, was used in this study to measure lateral diffusion of the phospholipid molecules in DPPC and POPC vesicles. An advantage of this method is that the diffusion coefficient is found directly from measured quantities without estimations of molecular parameters or the effects of the addition of spin or fluorescence probes to... 

Unilamellar vesicles are prepared by cosonication of the phospholipids and the fluorescent probes. 

Mix phospholipids with the fluorescent probe at a mole ratio of 50-100 1 (lipidstprobe) in a vessel resistant to chloroform and suitable for somcation To make 1 mL of a vesicle suspension, use 5-15 pmol of lipids. 

While there are many papers in the literature on the solvent relaxation in solutions of proteins, surfactant micelles, and phospholipid vesicles studied by time-resolved emission spectroscopy [25-27], similar studies focused on block copolymer micelles are scarce. In most cases, amphiphilic fluorescent probes localized in the inner part of the shells of amphiphilic block copolymer micelles in aqueous solutions were used for the studies. The studies reveal the heterogeneity of the binding sites of the probe that manifest itself by multiple-exponential fluorescence decays. In the case of block copolymer micelles, interpretation of the relaxation behavior can be complicated by redistribution of the probe molecules in the micelles during its excited-state lifetime of the probe [28]. The redistribution occurs as a result of the increased polarity of the excited probe as compared with its ground electronic state. 

In this chapter, we overviewed several fluorescence techniques suitable for studies of colloidal particles in aqueous solutions and discussed their application in the research of amphiphilic block copolymer micelles. Unlike surfactant micelles or phospholipid vesicles, amphiphilic block copolymer micelles have no sharp interface between the hydrophobic interior of the particles and the bulk solution, which results in greater heterogeneity of localization sites of fluorescent probes in the polymeric micelles and consequently in more difficult interpretation of data in comparison with surfactant micelles and phospholipid vesicles. 

The effect of branched polypeptides on phospholipid membranes was further investigated using lipid bilayers with DPPC/PG (95/5, 80/20 mol/mol). Two fluorescent probes of different character were used to analyse the effect of polymers on the outer surface (negatively charged, sodium anilino naphthalene sulfonate, ANS) and on hydrophobic core (hydrophobic, l,6-diphenyl-l,3,5-hexatriene DPH) of bilayers. For these studies small imilamellar vesicles were used. 

Neyroz et al. [97] have covalently linked 2NpOH to phos-phatidylethanolamine moiety by the Schiff-base formation between the NH2 of the phospholipid and the aldehyde moiety of 2-hydroxy-1-naphthaldehyde, followed by selective reduction of the imine to obtain a stable secondary amine. This fluorescent phospholipid easily incorporates into DML vesicle membrane and exhibits the typical behavior of ESPT probes. The emission spectrum of this probe inserted in the liposome is similar to that in ethanol medium and is affected by acetate used as a proton acceptor. 

Because the dynamics of phospholipid membranes have been well characterized using AF probes [8-13], fluorescence results obtained with hydrated human SC were compared to aqueous suspensions of unilamellar distearoyl-phosphatidylcholine (DSPC) vesicles. DSPC was also used because its phase transition temperature (55°C) is close to that of SC lipids (65 C). The microenvironment inside DSPC and SC membranes was studied by measuring fluorescence lifetimes, and shifts in emission maxima were compared to excitation maxima (Stokes shifts), along with quenching of a series of AF probes by iodide. Stokes shifts (Av) [6] were calculated as ... 

Membrane-bound probes, which are incorporated directly during vesicle preparation, are commonly fluorescently labeled phospholipids. These probes are mainly used to address more complex changes in membrane structure, thereby providing insights into the structure and mechanism of the transporters. Depending on the specific desired application, various fluorescently labeled phospholipids are available. Popular examples include boron dipyrromethane (BODIPY) probes as FRET acceptors, DOXYL probes as quenchers for parallax analysis, 7-nitro-benzofurazan (NBD) probes such as NBD-POPE for flip-flop assay, and so on (Figure 8). 

Rates of dissociation of retinoids from IRBP are measured by momtonng the process of transfer of the ligands from the protein to unilamellar vesicles of phospholipids. The vesicles contain fluorescent lipid probes that serve as readouts for the arrival of retinoids at the bilayers. The data yield information on rates of dissociation of retinoids from both retmoid-binding sites of IRBP. 

The structural and dynamic properties of polymerized surfactant aggregates such as detergent micelles, vesicles and bilayers have been studied extensively (32). From a biological aspect, it is of interest to determine in which way these structures mimic the properties of natural membranes (33). Most luminescent anisotropy studies of lipid rotation have employed the fluorescence characteristics of incorporated probes (34,35), as the time scales of lipid rotation are usually in the ns regime. However, a recent electron spin study using incorporated phospholipid spin-labels (36), indicated that rotation about the long axis of dimyristoyl-phosphatidylcholine (DMPC) lipids below the phase transition occurrs with time constants of about 60-100 is. Such values lie within the time domain of phosphorescence anisotropy measurements. 

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