Fluorescence quenching fatty acids

Let us recall the micellar aqueous system, as this procedure is actually the basic one. The chemistry is based on fatty acids, that build micelles in higher pH ranges and vesicles at pH c. 8.0-8.5 (Hargreaves and Deamer, 1978a). The interest in fatty acids lies also in the fact that they are considered possible candidates for the first prebiotic membranes, as will be seen later on. The experimental apparatus is particularly simple, also a reminder of a possible prebiotic situation the water-insoluble ethyl caprylate is overlaid on an aqueous alkaline solution, so that at the macroscopic interphase there is an hydrolysis reaction that produces caprylate ions. The reaction is very slow, as shown in Figure 7.15, but eventually the critical micelle concentration (cmc) is reached in solution, and thus the first caprylate micelles are formed. Aqueous micelles can actually be seen as lipophylic spherical surfaces, to which the lipophylic ethyl caprylate (EC) avidly binds. The efficient molecular dispersion of EC on the micellar surface speeds up its hydrolysis, (a kind of physical micellar catalysis) and caprylate ions are rapidly formed. This results in the formation of more micelles. However, more micelles determine more binding of the water-insoluble EC, with the formation of more and more micelles a typical autocatalytic behavior. The increase in micelle population was directly monitored by fluorescence quenching techniques, as already used in the case of the... 

Amplified photochemical quenching of carbazolyl fluorescence was observed in mixed LB films containing pure CUA and long chain fatty acids [12,14], A pure CUA was synthesized from 2-nitrobiphenyl and 11-bromoundecanoic acid methyl ester [12,13], Two monolayers of mixtures of CUA (fc = 0.02 to 0.50) and PA were deposited on five monolayers of cadmium arachidate at 15°C and 20 mN m 1 at pH 6.3. 

Typical hydrolysis rates are pmoles/min. They can be determined from the increase in pyrene fluorescence and a calibration plot obtained with pyrene lipid standards (fatty acids) whose fluorescence is not quenched. Under proper conditions, the measured activities depend linearly on the amount of enzyme used. 

Similarly, l-anilinonaphthalene-8-sulfonic acid (ANS) only emits in a hydrophobic environment, being almost completely quenched in aqueous solution. ANS and some other dyes, including 6-(p-toluidinyl)naphthalene-2-sulfo-nate, pyrene, l,6-diphenyl-l,3,5-hexatriene, fluorescein, and rhodamine derivatives attached to long acyl chains or to fatty acids that localize in the cellular membranes were used as probes for hydrophobic sites in proteins, protein folding, imaging of membranes of the cell, and solvent polarity. Pyrene-labeled fatty acids were used to detect the fusion of two membranes. When present in a membrane at sufficiently high concentrations, pyrene excimers (excited-state dimers) are formed that emit at 470 nm. Upon fusion with other membranes, probe concentration decreases, and excimer fluorescence is replaced by monomer fluorescence at 400 nm. This process can be monitored by ratiometric detection of pyrene labels. 

The fluorescence of each sample was compared with that of the reference sample where the acceptor is absent. This fluorescence is totally quenched in case (1), half quenched in case (2) if the Ci6 fatty acid is used as the spacer-layer, and totally unquenched in case (3) [28]. 

Fig. 17 Quenching of fluorescence of dye II (D) by electron acceptor I (A) in the arrangement of Fig. 15, case (b). Log (/o//) — 1 is plotted against the spacer layer thickness d for fatty acids with 14-22 C atoms.

Atoms or molecules adsorbed on a metal surface do not fluoresce because of highly effective quenching via creation of electron-hole pairs in the substrate. However, their fluorescence can be observed if they are separated from a metal by a dielectric spacer layer. The Langmuir-Blodgett technique allows one to deposit a fatty acid layer of a well-controlled thickness and thus provides an opportunity to study fluorescence of an adsorbate versus its distance from the surface (see Fig. 5.9). 

Faria, J. L. Berberan-Santos, M. Prieto, M. J. E. A comment on the localization of cyanine dye binding to brush-border membranes by the fluorescence quenching of n-(9-anthroyloxy) fatty acid probes. Biochim. Biophys. Acta, Biomembr. 1990, 1026, 133-134. 

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