Fluorescence quenching glycerol

Fig. 5.15. Fluorescence quantum yields of bridged and unbridged triphenylmethane dyes in glycerol as a function of temperature/viscosity.211 Not only cationic dyes show the strong fluorescence quenching for lower viscosities, but it is also present for neutral species as shown by the fuchsone DMAF.

Figure 13 Comparison of Pol /3 catalyzed single-nucleotide incorporation at 10% and 35% glycerol, pH 8.3. (a) 2-AP fluorescence stopped-flow assays show that both phases of the fluorescence change are slowed down at increased glycerol concentration, (b) In contrast, rapid chemical quench assays demonstrate that the rate of nucleotide incorporation remains unaffected by the altered glycerol concentration. Adapted with permission from M. Bakhtina M. P. Roettger S. Kumar ...

In such hybrid compounds, the nitroxide moiety quenches the fluorescence of the fluorophore (stilbene moiety). The reduction of nitroxide segment by an antioxidant (ascorbic acid) caused a rise in fluorescence of the fluorophore. The rate constant of the stilbene fragment photoisomerization in such systems depended on the viscosity of the medium. The synthesized dual stilbene-nitroxide probe was covalently immobilized onto the surface of a quartz plate as an eventual sensor. The immobilization procedure included a cyanogen bromide surface activation followed by smoothing with a protein tether. The rate of fluorescence change was monitored in aqueous glycerol solution of different viscosities and content of ascorbic acid. The dependence of kapp on the reciprocal absolute viscosity 1 /T] of the bulk mixture glycerin-water and the dependence of the initial intensity of fluorescence (/o) on solution viscosity were also studied. 

Add 25 xL of the assay mixture that contains 50 mM sodium acetate (pH 5.0), 0.01% Tween 20, 12.5% glycerol, 1 mg/mL BSA, and 12 xM plasmapsin II substrate DABCYL-y-aminobutyric acid-Glu-Arg-Met-Phe-Leu-Ser-Phe-Pro-EDANS into each weii of the 96-well microtiter piate containing dried compounds or empty controi weiis. Sonicate the plates to solubilize the compounds. Initiate the enzymatic reaction with the addition of 25 xL of 8 nM plasmapsin II in an aqueous buffer that contains 50 mM sodium acetate (pH 5.0), 0.01% Tween 20, 1 mg/mL BSA, and 12.5% glycerol. Incubate the assay mixture at 25°C for 10 min and then quench the reaction by the addition of 25 xL of 1 M Tris-HCl (pH 8.5 and containing 50% DMSO). Record the EDANS fluorescence using a Tecan SLT FiuoStar fluorescence plate reader equipped with a 350 nm excitation filter and a 510 nm emission filter. 

---