Fluorescence depth quenching

The binding of synthetic ion channels and pores to lipid bilayer membranes often causes a change in intra- or intermolecular self-organization that is visible in sufficiently sensitive methods such as fluorescence (e.g. tryptophan emission) [14] or circular dichroism spectroscopy and can be used to determine the partition coefficient. Convenient methods of detection under relevant conditions are fluorescence resonance energy transfer (FRET) or fluorescence depth quenching (FDQ) [3, 4, 6]. Many fluorescent probes for the labeling of both synthetic ion channels/... 

The method of choice to determine, under meaningful conditions, the location of synthetic ion channels and pores in bilayer membranes is fluorescence depth quenching (FDQ) [4, 11]. In this well developed but costly analysis, the position of a quencher in the lipid bilayer is varied systematically. Analysis of the dependence of the efficiency to quench a fluorescent synthetic ion channel or pore on the position of the quencher reveals transmembrane, central or interfacial location (Fig. 11.13b-d). 

NBD probes are often used to assay flip-flop. Flip-flop refers to the reversible transversal diffusion of lipids from one leaflet to the other leaflet of a lipid bilayer membrane. In intact membranes, this transversal diffusion is very slow (fi/2 on the order of hours to days). However, it can be accelerated by biological or synthetic flippases, which are a special class of membrane transporters related to ion carriers. Alternatively, micellar pores are synthetic ion channels and pores with flippase activity and can thus be identified with flip-flop assay (Figure 2 interfacial location of the transporter, as second distinctive characteristic of micellar pores, can be identified by fluorescence depth quenching experiments with DOXYL probes). 

In systems where only dynamic quenching occurs, then steady-state fluorescence intensities can be measured instead of lifetimes/101 103-,07) In experiments where comparisons are being made (i.e., for a comparison of different experimental conditions or types of membrane), it is important that the lifetime of the fluorophore (r0) is not affected by the experimental conditions. Fluorescence intensities can be obtained much more rapidly and without specialized instrumentation. Blatt and Sawyer(101) have employed a relationship essentially the same as Eq. (5.20) in this way. They have pointed out that since the quenching mechanism is collisional, the partition coefficient that is derived is a partition coefficient of the quencher into the immediate environment of the fluorophore and is therefore a local Kp. It is therefore possible to investigate the partition coefficient gradient across the lipid bilayer by using a series of probes, such as the anthroylstearates,(108) located at different depths. In their method, Eq. (5.20) has the form... 

A. Chattopadhyay and E. London, Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipids, Biochemistry 26, 39-45 (1987). 

E. Blatt and W. H. Sawyer, Depth-dependent fluorescent quenching in micelles and membranes, Biochim. Biophys. Acta 822, 43-62 (1985). 

The solution was contained in a cylindrical Pyrex or silica optical cell, 2.5 cm. in diameter and 2 cm. optical depth, fitted with a side arm of diameter about 1 cm. for connection to the vacuum line (see Fig. 10). Both phosphorescence and delayed fluorescence in fluid solution at room temperature are quenched by exceedingly low concentrations of molecular oxygen, and efficient deaeration is of the utmost importance. The following procedure was found to be satisfactory. The cell was first cooled in an acetone/C02 bath and pumped out. It was then isolated from the pump, refluxed to remove dissolved gas, cooled again in the... 

In X-ray fluorescence, self-absorption due to optical quenching of the sample is always observed. For solid samples, radiation can be observed only at a depth of a few micrometres. The intensity P of radiation, after travelling a distance dx in a material which a lineic absorption coefficient p (cm ), will decrease by dP for a penetration angle of 90°. The integrated form of the expression dP = —pP dx is comparable to that described in colorimetry ... 

Strashnikova, N.V., Medvedeva, N., and Likhtenshtein, G.I. (2001) Depth of immersio of fluorescence chromophore in biomembranes studied by quenching with nitrioxideradicals, /. Biochem. Biophys. Meth. 48,43-60. 

The depth of the binding site of small cationic Hgands is at least 11 A as predicted and as demonstrated by fluorescence quenching experiments [12]. 

Lipid peroxidation caused a decrease in phospholipid molecule mobility both in the region of polar heads and in the region of acyl chains till the depth of at least 1.7 nm from water-lipid interface (Panasenko et al. 1991). Under relative high levels of oxidation (>6 pmol malondialdehyde/g LDL phospholipid) the polarity of lipid phase increased. The decrease in efficiency of tryptophan fluorescence quenching by nitroxide fragments incorporated into hydrophobic regions at the depth of 2 nm from water-lipid interface indicated that lipid-protein interaction was disturbed as a result of oxidation of LDL lipids. 

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