Carboxyfluorescein-5,6 self-quenching

Fig. 12. Phospholipase A2 mediated release of self-quenched carboxyfluorescein from unpolymerized and polymerized mixed vesicles of 15 and DPPC (1 1) at 30 °C. (A) Monomeric and polymerized vesicles in the absence of enzyme. (B) Polymerized vesicles after the addition of phospholipase A2 (30 pg/ml). (C) Unpolymerized vesicles after the addition of phospholipase A2 (30 pg/ml).

Conveniently, one monitors the increase of the fluorescence intensity of an encapsulated self-quenched dye, eg 50 mM 6-carboxyfluorescein (6-CF) (160) or calcein (161), as it permeates the vesicles. pHJGlucose is more generally applicable because it does not interact with charged lipid membrane surfaces and can be used at low ionic strength. Alternatively, a dilute solution of 6-CF (10 /am) is encapsulated and Co + (500 /am) is added to the external solution which quenches the fluorescence. Permeation of either Co + ions or the dye across the membranes causes a reduction in fluorescence intensity. 

CF assay A transport assay that reports the release of self-quenched 5(6)-carboxyfluorescein from LUVs as the recovery of fluorescence. 

Pol3nnerization of liposomes affects their membrane stability. In contrast to monomeric liposomes the polymerized membrane systems remain stable for weeks without precipitation. Entrapped substances are released much slower from polymeric vesicles thah from the corresponding monomers. This has been studied in the case of the diene lipid (LI) entrapped 6-carboxyfluorescein (6-CF) in high concentration exhibits self-quenching release into the surrounding aqueous medium results in strong fluorescence due to dilution (24), At room temperature vesicles made from DPPC (dipal-mitoylphosphatidylcholine) are below the phase transition temperature showing 8% release after 40 hours (fig. 10). 

The Carboxyfluorescein concentration of the vesicle suspension Cv and after destroying the vesicles with the surfactant Triton X 100 C/ were calculated from the fluorescence intensity of the diluted aqueous solutions. The turbidity of the vesicle solution declined within seconds after detergent addition (vesicle busting), and we obtained then a clear, aqueous solution. Typical results of these measurements are summarized in Fig. 12. Due to Ihe self-quenching properties the destruction of the vesicles with a high inner Carboxyfluorescein concentration (0.05-0.2 mol/1) led to an increase of the Fluorophore concentration in the outer phase. This occurred if a large amount of emulsion droplets coalesced with the lower water phase, thus releasing their Carboxyfluorescein content. 

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