Quencher acrylamide

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. 

Other corrections, besides those for static interactions, are important for certain quenchers. For example, acrylamide quenching is often used to help determine the relative solvent accessibility of aromatic residue side chains. In addition to a correction for static quenching,(60,66) acrylamide quenching data for tyrosine residues require both primary and secondary inner filter corrections since acrylamide absorbs both 280- and 305-nm light.(67)... 

Small molecules that act as collisional quenchers may penetrate into the internal structure of proteins, diffuse, and cause quenching upon collision with the aromatic groups. Lakowicz and Weber(53) have shown that the interaction of oxygen molecules with buried tryptophan residues in proteins leads to quenching with unexpectedly high rate constants—from 2 x 109 to 7 x 109 M l s 1. Acrylamide is also capable of quenching the fluorescence of buried tryptophan residues, as was shown for aldolase and ribonuclease 7V(54) A more hydrophobic quencher, trichloroethanol, is a considerably more efficient quencher of internal chromophore groups in proteins.(55)... 

The question of what mechanisms is involved in the case of other quenchers is still unclear. For the quenching of aldolase and ribonuclease Ti by acrylamide, the activation energy is rather high, 40-45 kJ/mol,(54) but the value in the case of cod parvalbumin(60) is lower (27 kJ/mol), being similar to that for oxygen quenching. According to Bushueva et al., the efficiency of... 

The elucidation of the intramolecular dynamics of tryptophan residues became possible due to anisotropy studies with nanosecond time resolution. Two approaches have been taken direct observation of the anisotropy kinetics on the nanosecond time scale using time-resolved(28) or frequency-domain fluorometry, and studies of steady-state anisotropy for xFvarying within wide ranges (lifetime-resolved anisotropy). The latter approach involves the application of collisional quenchers, oxygen(29,71) or acrylamide.(30) The shortening of xF by the quencher decreases the mean time available for rotations of aromatic groups prior to emission. 

The constant K is known as the Stem-Volmer quenching constant /cQ is the rate constant for the quenching reaction, and t0 the lifetime in the absence of quencher. Fluorescence quenching of tryptophan in proteins by acrylamide or 02 has been used to determine whether tryptophan side chains are accessible to solvent or are "buried" in the protein.141 142 The long-lived phosphorescence of tryptophan can be studied in a similar... 

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. 

Acrylamide is an uncharged polar molecule, so it can diffuse within a protein and quenches fluorescence emission of Trp residues. The quencher should be able to collide with tryptophan whether it is on the surface or in the interior of a protein. Nevertheless, Trp residues, mainly those buried within the core of the protein, are not all reached by acrylamide. For a fully exposed tryptophan residue or for Tryptophan free in solution, the highest value of kq found with acrylamide is 6.4 x 109 M-1 s-1. 

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. 

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 ... 

Generation and trapping of radical cations of a,co-diphenylpolyenes within the channels of pentasil zeolites provides an environment which allows these transient species to be spectroscopically characterized . Similarly, complexation of xanthone in cyclodextrin has made it possible for the triplet state of this molecule to be fully characterized . Association and dissociation processes are related to the dipoles developed in the complex and in solution. A unimodal Lorentzian lifetime distribution for 2-anilinonaphthalene-6-sulphonate B-cyclodextran inclusion complexes have been recovered by multifrequency phase-modulation fluorometry in the presence of the quenchers Cu, acrylamide, and I . Both the fluorescence and phosphorescence spectra of benzo[f]quinoline adsorbed on p-cyclodextrin/NaCl have been determined as a function of temperature . 

There can be more specific quenching effects also. Some molecules or groups, such as oxygen (O2), iodide (I ), acrylamide (propenamide) and succinimide (pyrrolidine-2,5-dione), will accelerate non-radiative decay if they come in contact with the excited fluorophore. Such quenching may be dynamic, involving transient collision between the molecules, or static if the quencher molecule (Q) forms a longer-lived complex with the fluorescent group. 

One common approach to fluorescence sensing is to rely on fluorophores which are collisionally quenched by the analyte. There are many known collisional quenchers (andytes) which alter the fluorescence intensity and decay time. These include O2 (27-31), chloride (32-33), chlorinated hydrocarbons (34), iodide (35), bromate (36), xenon (37), acrylamide (38), succinimide (39), sulfur dioxide (40), and halothane (41), to name a few. The quenching process obeys the Stem-Volmer equation ... 

Collisional quenching of fluorescence requires contact between the fluorophore and the quencher. For quenching to occur the quencher must diffuse to and collide with the fluorophore in the excited state. If this occurs the fluorophore returns to the ground state without emission of a photon (Fig. 4). Many small molecules act as collisional quenchers of fluorescence [6,12]. These include iodide, acrylamide, halogenated hydrocarbons and occasionally amines and metal ions. The excited state lifetimes provide ample opportunity for quenching. For instance, acrylamide is known to be an efficient quencher of tryptophan fluorescence [12,13]. Suppose its... 

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