Fluorescence techniques membranes

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. 

Table 5.1. Applicability of Fluorescence Techniques to the Study of Membrane Properties...

There have been rather few studies of the location of probes in whole cells. DPH incorporates into most subcellular fractions (see, e.g, Ref. 64), whereas with TMA-DPH, early after introduction only the plasma membranes appear to be labeled/64,65) There is considerable interest in examining the lipid motional properties of living cells by fluorescence techniques. In this type of study the location of the probe has to be carefully checked before conclusions can be drawn. This is carried out by separate measurements of the recovery of probe from intact labeled cells in isolated subcellular fractions and/or by fluorescence microscopy. 

N. L. Thompson, A. G. Palmer, L. L. Wright, and P. E. Scarborough, Fluorescence techniques for supported planar model membranes, Commun. Mol. Cell. Biophys. 5, 109-113 (1988). 

The possibility to carry out conformational studies of peptides at low concentrations and in the presence of complex biological systems represents a major advantage of fluorescence spectroscopy over other techniques. Fluorescence quantum yield or lifetime determinations, anisotropy measurements and singlet-singlet resonance energy transfer experiments can be used to study the interaction of peptides with lipid micelles, membranes, proteins, or receptors. These fluorescence techniques can be used to determine binding parameters and to elucidate conformational aspects of the interaction of the peptide with a particular macro-molecular system. The limited scope of this chapter does not permit a comprehensive review of the numerous studies of this kind that have been carried and only a few general aspects are briefly discussed here. Fluorescence studies of peptide interactions with macromolecular systems published prior to 1984 have been reviewed. 

The use of these natural fluorescence techniques offers not only the possibility of studying the interaction of proteins with membranes, under convective and diffusive conditions, but also they may be easily extended to studies involving proteins and other porous materials such as chromatography media. The areas of application of these techniques will range from polypeptide and protein fractionation to the monitoring of systems where protein-surface interactions are relevant. 

Fluorescence techniques have also been used to determine the localization of molecules in membranes. Using this technique, the localization of the linear dye molecule 3,3 -diethyloxadicarboxyamine iodide (DODCI) in lipid bilayer vesides was determined as a function of lipid chain length and unsaturation. It was found that the fraction of the dye in the interior region of the membrane was decreased as a function of chain length in the order C12 > C14 > C16 > C18. In unsaturated lipids it was Ci4 i > C14 0 > C16 1 > C16 0, which is in agreement with the general observation that the penetration of amphiphilic molecules into the interior of membranes increases with an increase in the fluidity of the membrane structure [59]. 

Equilibrium dialysis is used in a number of examples to analyse the ratio of lipid-bound to free analyte. Kramer et al. (1998) described the use of equilibrium dialysis by separating the liposome suspension and the water phase by a semi-permeable membrane. The analyte is dissolved in the water compartment of the system and diffuses into the liposome compartment. If equilibrium is reached, the remaining concentration of the analyte in the water compartment is determined by means of a quantification method (mainly HPLC or LCMS, fluorescence techniques) and the partition coefficient is calculated. Kramer et al. (1997) used a radio tracer substance as analyte to quantify the compound in both compartments using liquid scintillation counting. 

Using the principle of ion pair formation between ammonium cations and the phosphate anions of lipids, Matile et al.33 prepared 8, an amphiphilic polyamine dendrimer. Rather than acting as a membrane channel, 8 was expected to form reversible membrane defects in the lipid bilayer. The steroid moiety was expected to act as the hydrophobic anchor for bilayer orientation and steric bulk was expected to prevent the polyamine penetrating the bilayer. Proton transport was assessed in unilamellar vesicles using the pH-fluorescence technique in which the external pH was increased to 7.8 relative to the internal pH at 7.4. The results demonstrated that 8 was almost as active as gramicidin, and maximal flux was achieved in ca. 20 s. 

Opiate narcotics are thought to act at specific receptors in the brain since they exhibit stereospecific binding (1) and have been shown by fluorescence techniques to be localized at discrete regions in the central nervous system (2). While much effort has been made to isolate and characterize the opiate receptor C3), relatively little detailed information exists about the nature of the opiate binding site. Some investigators describe the receptor as a membrane bound protein or proteo-lipid (4) while others have used nerve cell components such as cerebroside sulfate or phosphatidyl inositol as models for the opiate receptor (5). 

Gillespie has employed fluorescence techniques to show that smooth muscle membrane can bind norepinephrine, and that this binding can be prevented by prior treatment with the a-adrenergic blocking agent, phenoxybenzamine. 

A potassium-sensitive optode uses a crown ether labeled with an azo dye and immobilized on Amberlite by encapsulating the tip by a porous FIFE membrane. The absorption is modiHed by chelation with potassium ions [74]. All others cation measurements use the fluorescence technique. 

However, because of its far red shifted emission maximum (approximately 1030 nm) until recently it was not possible to measure its fluorescence kinetics on a picosecond time scale. We have investigated the energy transfer kinetics of whole cells, chromatophore membranes, photoreceptor units (quantasomes) and reaction centers in a dark adapted state and in the presence of different redox agents, using single photon timing fluorescence techniques. 

Effects of HO radicals on biological membranes include alterations in membrane proteins, peroxidation of unsaturated lipids accompanied by perturbation of the lipid bilayer polarity. Berroud et al. (1996) have measured radiation-induced membrane modifications using two fluorescent lipophilic membrane probes (TMA-DPH and DPH) by the technique of fluorescence polarization of Chinese hamster ovary K1 and lymphoblastic RPMI 1788 cell lines, ylrradiation from a Co source with dose rates of 0.1 and 1 Gy/min for a final dose of 4 and 8 Gy induced a dose-dependent decrease of... 

Isolated raf hearts subjected to 15 min ischaemia followed by 30 min reperfusion showed concomitant accumulation of free calcium (Indo-1 fluorescence technique) and degradation of membrane phosphoUpids as indicated by an increase of tissue arachidonic acid content (Ivanics et al. 2001). This observation is suggestive for a relationship the Ca -related fluorescence and arachidonic acid accumulation probably due to a calcium-mediated stimulation of phospholipase Aj. 

So far, a large number of low-molar-mass systems have been studied by ultrafast fluorescence techniques in sub-nanosecond time regions [35-39]. Recently, a relatively slow (nanosecond) relaxation process proceeding in mixed low-molar-mass solvents, consisting in redistribution of components of the solvent mixture in the solvate shell of the fluorophore upon the excitation, has also been reported [40-43, 46, 47]. However, an important part of experimental studies is still concerned with relatively slowly relaxing biological systems, such as lipid membranes [48-50], proteins [51, 52], nucleic acids [53], and also colloidal [54] and polymer systems [55-57]. 

Monitoring of Membrane Processes Using Fluorescence Techniques Advances and Limitations... 

This chapter discusses the use of fluorescence techniques for the monitoring of membrane processes, making use of the intrinsic fluorescence properties of the various components involved. This chapter deals with situations where none of these components is labeled with an external reporter, a fluorescence probe, as happens in many other applications described in this book (see Chapters 3,4 and 8). 

This chapter introduces and discusses different fluorescence techniques that do not require the use of external labeling steady-state fluorescence (including 2D fluorescence), fluorescence anisotropy and time-resolved fluorescence and it provides illustrative examples showing how these techniques may be used for the monitoring of membrane processes. 

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