Quencher iodide

One difficulty frequently encountered in fluorescence is that of fluorescence quenching by many substances. These are substances that, in effect, compete for the electronic excitation energy and decrease the quantum yield (the efficiency of conversion of absorbed radiation to fluorescent radiation— see below). Iodide ion is an extremely effective quencher. Iodide and bromide substituent groups decrease... 

Quenching of fluorescence by known quenchers (iodide, nitrate, cesium ion. acrylamide) also provides relevant information. Fluorescence due to whichever of the fluorescing amino acids is quenched, that amino acid should be on the surface. 

The Dp and Dq are the diffusion coefficients of probe and quencher, respectively, N is the number molecules per millimole, andp is a factor that is related to the probability of each collision causing quenching and to the radius of interaction of probe and quencher. A more detailed treatment of fluorescence quenching including multiexponential intensity decays and static quenching is given elsewhere/635 There are many known collisional quenchers (analytes) which alter the fluorescence intensity and decay time. These include O2/19 2( 29 64 66) halides,(67 69) chlorinated hydrocarbons/705 iodide/715 bromate/725 xenon/735 acrylamide/745 succinimide/755 sulfur dioxide/765 and halothane/775 to name a few. 

In addition to the sensors dealt with in Section 3.3.1.1, which could equally have been included in this Section as they use consumable immobilized reagents and regenerable fluorophores, Frei et al. developed a sensor for HPLC determinations based on the solid-state detection cell depicted in Fig. 3.38.B, where they immobilized 1-bromonaphthalene for measuring phosphorescence quenchers. Experiments demonstrated the sensor s usefulness for determining nitrate with a detection limit of ca. 10" M and an RSD of 4% for an analyte concentration of M. However, the scope of application of this sensor to chromatographically separated anions is rather narrow owing to the low sensitivity of the quenched phosphorescence detection for iodide and other halides [268]. 

Despite the high energy of the excitation, fluorescence emission may be quenched by a range of quenchers, such as iodide (I- and Cu ). The net effect of a quencher is that the observed fluorescence emission reduces as the quencher concentration increases (typically milli-molar concentrations are employed). This is due to an energy transfer between the excited fluorophore and the quencher. The quencher thus provides an alternative relaxation pathway for the excited molecule. Not surprisingly the effect of the... 

The rate of relaxation by nonradiative pathways can be increased by addition of quenchers. Quenching of fluorescence occurs by several mechanisms, many of which involve collision of the excited chro-mophore with the quenching molecule. Some substances such as iodide ion are especially effective quenchers. The fluorescence efficiency of a substance in the absence of a quencher can be expressed (Eq. 23-lb) in terms of the rate constants for fluorescence (fcf), for nonradiative decay (km), and for phosphorescence ( r )=... 

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. 

The most common quenchers are oxygen, acrylamide, iodide, and cesium ions. The kq value increases with probability of collisions between the fluorophore and quencher. Oxygen is a small and uncharged molecule, so it can diffuse easily. Therefore, the bimolecular diffusion constant kq observed for oxygen in solution is the most important between all cited quenchers. 

When a protein possesses two or several Trp residues, when quenchers such as iodide, cesium, or acrylamide are used, and if all Trp residues are not accessible to the quencher, the Stern-Volmer equation yields a downward curvature. In this case, we have selective quenching (Figure 10.5b). From the linear part of the plot, we can calculate the value of the Stern-Volmer constant corresponding to the interaction between the quencher and accessible Trp residues. Upon complete denaturation and loss of the tertiary structure of a protein, all Trp residues will be accessible to the quencher. In this case, the Stern-Volmer plot will show an upward curvature. In summary, inhibition of the protein fluorescence with two or several Trp residues can yield three different representations for the Stern-Volmer equations, depending on the accessibility of the fluorophore to the quencher. 

The fluorescence yield of a molecule is suppressed by perturbations. These perturbations may be within the molecule itself. For example, in the case of fluorescein dyes increasing extent and polarizability of halogen substituents markedly decrease the fluorescence yield. Nitro groups are also very effective quenchers, e.g., eosin B, a nitro substituted eosin is non-fluorescent whereas eosin itself is strongly fluorescent. Quenching of fluorescence can also be achieved by external means. A particularly effective quencher for aqueous systems is iodide ion. Nitrobenzene is often a good fluorescence quencher for non-aqueous systems. 

Apart from iodide ion, radicals are efficient quenchers of excited states of molecules [16] the processes of quenching of excited states of various molecules by radicals were studied earlier in detail [17 - 19]. It was shown that the triplet states of usual cyanine dyes are mainly quenched by the mechanism of acceleration of the intersystem crossing to the ground state (T-So). In this case, the quenching process is described by the following scheme ... 

A comparison of the experimental data on quenching of triplet states of dyes in solutions in the absence and in the presence of DNA permits estimation of the steric complexation effect on the quenching process and conclusions about the structure of the dye-DNA complexes formed. In the case of dye K4, we may conclude that complexation with the biopolymer has relatively weak effect on the kq value. This is probably due to the fact that the quenching process for K4 occurs in the kinetic regime (kc k a, see reaction (2)), and diffusion of the quencher to dye molecules boimd to DNA exerts no substantial effect on kq (another assumed reason for this phenomenon could be partial decomposition of the dye(T)-DNA complex and the presence of free triplet dye molecules in the solution however, the experiments on quenching of the K4 triplet state by iodide ion considered above reject this possibility). 

In summary, the triplet decay kinetics of KI-K4 in the presence of DNA are biexponential the two observed components are attributed to two different complexes between the dye and DNA. Using nitroxyl radical and iodide ion as quenchers, we have shown that cyanine dyes form with DNA two types of complexes formed by binding of the dye in the groove of a DNA molecule and by intercalation of the dye between base pairs. 

Some small molecules or ions, such as oxygen, acrylamide, iodide or thiocyanate ions, are able to convert the energy of the excited state into heat, by a process of collisional quenching, whose efficiency is proportional to the concentration of quencher kQ cQ. The processes which contribute to the loss of energy from the excited state can be incorporated into a kinetic equation for the lifetime of the excited state ... 

Since iodide is known to act in phospholipid vesicles as a collisional quencher for AF probes [6,9] by a diffusive process, fluorescence quenching was described by the Stem-Volmer relationship [6] ... 

Langner and S. W. Huie. Iodide penetration into lipid bilayers as a probe of membrane lipid organization. Chem. and Phys. of Lipids 60 127-132 (1991). Chalpin and A. M. Kleinfeld. Interaction of fluorescence quenchers with the n-(9-anthroyloxy) fatty acid membrane probes. Biochim. et Biophys. Acta 73I 465-A14 (1983). 

The thin layer chromatography (TLC) plates that we buy are slide glasses coated with silica gel. In order to visualize UV-active organic compounds, the silica gel is coated with a layer of fluorescent dye Flur. Therefore, UV-active compounds can be detected under UV light. Occasionally, non-UV-active compounds are also seen. One case is iodides, which are UV-light quenchers, while another case is inorganic salts, often seen on the baseline the salts are visualized under UV simply because they cover the fluorescent dye "Flur."... 

As an alternative approach, r0 (p0) may be derived by extrapolation of r 1 (p 1) to zero Tf while tc is held constant. This can be achieved by the addition of relatively small concentrations of a quencher capable of dynamically deactivating the excited state [110]. / 1 is subsequently plotted against 7/7° or t/t° to allow derivation of rn 1 from the intercept. For example, this form of extrapolation for ACE-labeled PMAA at pH 12.7 using iodide as a quencher results in an r l value of 8.7 [111]. 

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