Fluorescence intensity quenching

The Ksv value shows the importance of fluorophore accessibility to the quencher, while the value of A q gives an idea of the importance of the diffusion of the quencher within the medium. Figure 10.1 shows a Stern-Volmer plot of fluorescence intensity quenching with iodide of flavin free in solution and of flavin bound to flavocytochrome ba- The Ksv values found are 39 and 14.6 M-1 for free and bound flavins, respectively, i.e., values of kq equal to 8.3 x 109 and 3.33 x 109 M-1 s-1, respectively. Accessibility of flavin to KI is more important when it is free in solution, and the presence of protein matrix prevents frequent collisions between iodide and FMN thereby decreasing the fluorophore accessibility to the quencher. Also, as revealed by the Aq values, diffusion of iodide in solution is much more important than in flavocytochrome b2. The protein matrix inhibits iodide diffusion, thereby decreasing the A q value. 

Figure 10.2 shows a Stern-Volmer plot for the fluorescence intensity quenching by oxygen of zinc protoporphyrin IX embedded in the heme pocket of apomyoglobin (Mbdes Fe). The slope of the plot yields Ksv and A q values of 15.96 M-1 s-1 and 7.6 x 109 M-1 s-1,... 

Figure 10.1 Stern-Volmer plots of fluorescence intensity quenching with iodide of FMN free in solution (plot b) and of FMN bound to flavocytochrome b2 (plot a). Reproduced from Albani, J.R., Sillen, A., Engel borghs, Y. and Gervais, M. (1999). Photochemistry and Photobiology, 69, 22-26, with the permission of the American Society for Photobiology.

Figure 10.4 Fluorescence intensity quenching of L-Trp by Kl. Stock solution = 4 M Kl + 10 3 Na2SC>4.

Figure 10.9 Stern-Volmer plots of the fluorescence intensity quenching of by oxygen. The Arg...

Figure 13.6 Fluorescence intensity quenching of fluorescein bound to LVA, as a result of STF-LCA (a) and of LCF-LCA (b) interactions. [LCA] = 0.7 /xM. Source Albani, J. R., Debray, H., Vincent, M. and Gallay, J. (1 997). Journal of Fluorescence, 7, 293-298. Albani, J.R., Debray, H., Vincent, M. and Gallay, J. (1997). Journal of Fluorescence, 7, 293-298. Figure No. 1. With kind permission of Springer Science and Business Media (1, 2, and 3).

Figure 14.2 shows fluorescence intensity quenching of 4,6-diamidino-2-phenylindole (DAPI) complexed to DNA in the presence of two concentrations of Acridine Orange. In fact, one can see that while the fluorescence intensity of DAPI decreases, that of Acridine... 

Figure 15.9 Stern-Volmer plot of fluorescence-intensity quenching of free Trp in solution with TNS.

Figure 2 Fluorescence intensity quenching [(/q//)- 1] of AF probes in SC as a function of iodide (KI) concentration. Data represents the average S.D. and are obtained from three different pieces of skin. Symbols solid triangles (2-AF) open triangles (6-AF) solid circles (9-AF) open circles (12-AF) solid squares (16-AF).

The amount of fluorescence intensity quenching can be quantitatively related to the partial pressure of oxygen in a sample from the simplified Stern-Volmer equation ... 

Titration curves of HS fluorescence quenching versus concentration of added metal quencher have been used to obtain the CC values of HS ligands and the stability constants of HS-metal complexes (Saar and Weber, 1980, 1982 Underdown et al., 1981 Ryan et al., 1983 Weber, 1983 Dobbs et al., 1989 Grimm et al., 1991 Hernandez et al., 2006 Plaza et al., 2005, 2006). Two fluorescence techniques, lanthanide ion probe spectroscopy (LIPS) and fluorescence quenching of HSs by Cu-+, have been used in conjunction with a continuous distribution model to study metal-HS complexation (Susetyo et al., 1991). In the LIPS technique, the HS samples are titrated by Eu-+ ions, and the titration plot of the ratio of the intensities of two emission lines of Eu + is used to estimate the amount of bound and free species of the probe ion. In the other technique, titration curves of fluorescence intensity quenched by Cu versus the logarithm of total added Cu2+ are used. 

Fluorescence intensity quenching with iodide can be used to find out whether ethidium bromide added to DNA is intercalated into the double helical DNA or present at the surface of the DNA. In fact, since DNA is negatively charged, addition of iodide ion to a DNA-EB complex will not decrease the fluorescence intensity of the ethidium bromide if the fluorophore is intercalated into the DNA. However, if the fluorophore is bound to the DNA surface, its fluorescence will be quenched by iodide ion. 

Figure 4.9. Stern - Volmer plot for the fluorescence intensity quenching of Trp residues of Vida fava agglutinin, lex = 295 nm and lem = 310 (a), 330 (b) and 350 nm (c).

Thermal Fluorescence intensity quenching of TNS bound tightly to LCA shows two slopes equal to -2.5% per °C and -1.6% per °C. The breaking temperature is equal to 20°C. One can notice that there is no big difference between the two slopes (Fig. 

Figure 4.43. Thermal fluorescence intensity quenching of free FITC (a), of FITC bound to LCA (b), in presence of LCA-LTF complex (c) and in presence of LCA-STF complex (d). Source Albani, J. R. 1998. Biochim. Biophys. Acta. 1425, 405-410.

Fluorescence intensity quenching with iodide (Fig. 4.42) indicates that diffusion of K1 is inhibited by the amino acids of LCA. Binding of LTF to the LCA-FITC complex does not affect the diffusion (kq) and the dynamic constants (Ksv). Thus, the dynamics of the amino acids of LCA do not change in presence of LTF. 

In the fluorescence intensity quenching (thermal and with iodide), it is the fluorescein environment consisting of amino acids (thermal quenching) and of amino acids and solvent dipoles that is relaxing around the excited fluorescein. In the fluorescence anisotropy experiments, on the other hand, the displacement of the emission dipole moment of the fluorescein is monitored. In the first approach, it is the environment that is either fluid or rigid. In the second approach, the restricted reorientational motion of the fluorophore is followed. 

Fluorescence intensity quenching of tyrosine residues by iodide is analyzed with the Stem-Volmer formula... 

When a protein contains two classes of intrinsic fluorophore, one at the surface of the protein and the second embedded in the protein matrix, fluorescence intensity quenching with cesium or iodide allows obtaining the spectra of these two classes. A selective quenching implies that addition of quencher induces a decrease in the fluorescence observables (intensity, anisotropy and lifetime) of the accessible class. At high quencher concentration the remaining observables measured will reflect essentially those of the embedded fluorophore residues. In this case, one can determine the fraction of fluorescence intensity that is accessible (fa). Knowing fa along the emission spectrum will allow us to draw the spectrum of each class of fluorophore (Lehrer, 1971). 

The positions of the two maxima (347 and 326 nm) foimd in our analysis are identical to those (350 and 324 nm) we have already obtained by performing fluorescence intensity quenching with cesium (Fig. 8.26). However, the Ln-normal analysis performed on ai-acid glycoprotein in presence of high calcofluor concentrations is much more accurate than the simple analysis with the sum of different spectra or even... 

Figure 8.53. Fluorescence emission spectra of crystals of ap acid glycoprotein - progesterone complex (a) and of hydrophobic Trp residues in solution in presence of progesterone (b). A ex = 295 nm. Spectrum b was obtained from fluorescence intensity quenching by cesium of a solution of a]- acid glycoprotein - progesterone complex, after extrapolating to [Cs ] = go. Source Albani, J. R. 1998, Journal of Fluorescence, 8, 213-224. Authorization of reprint accorded by Kluwer Academic Publishers.

Figure 9.18. Stem-Vobner plots of the fluorescence intensity quenching of by ojQ gen. Tlic kto values were obtained from the initial slopes of each ploL Source Aliani. J. and Alpert, B. I9S7. Eir. J. Biochem. I t 175-178. Aulltorizaiion of reprini accorded by Blackwell Publishing.

Figure 9.19.Analysis of the fluorescence intensity quenching by oxygen of myoglobin porphyrin with Eq. 4.29. Experiments were performed at 15 (a) and 35°C (b). Only dynamic quenching constants were obtained, equal to 13.2 and 12.3 M at 35 et 15°C, respectively.

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