Quencher molecules

The energy transfer may result in a reactive or nonreac-tive quencher molecule ... 

Proteases are one of the largest families of enzymes and are involved in a multitude of vital processes. Due to their biological relevance and diversity, multiple fluorescent reporters monitoring their activity have been designed and successfully applied in vitro and in vivo [112-114]. Standard small molecule FRET probes for proteases consist of an amino acid sequence flanked by a FRET pair, consisting of two fluorophores or one fluorophore and a quencher molecule. Upon cleavage of the peptide sequence, the emission of the donor fluorophore is dequenched and the intensity increases whereas the emission of the acceptor decreases and vanishes more or less completely in those cases where the acceptor is fluorescent (see Fig. 6.11). 

When the probability of finding a quencher molecule within the encounter distance with a molecule M is less than 1, this situation is relevant to static... 

When M and Q cannot change their positions in space relative to one another during the excited-state lifetime of M (i.e. in viscous media or rigid matrices), Perrin proposed a model in which quenching of a fluorophore is assumed to be complete if a quencher molecule Q is located inside a sphere (called the sphere of effective quenching, active sphere or quenching sphere) of volume Vq surrounding the fluorophore M. If a quencher is outside the active sphere, it has no effect at all on M. Therefore, the fluorescence intensity of the solution is decreased by addition of Q, but the fluorescence decay after pulse excitation is unaffected. 

The above considerations can be generalized to complexes of the type M Q ( > 1). The probability that a molecule M is in contact with n quencher molecules can be approximately expressed by the Poisson distribution (Eq. 4.21). Perrin s equation (4.23) is then found again. 

Let us consider a fluorescent probe and a quencher that are soluble only in the micellar pseudo-phase. If the quenching is static, fluorescence is observed only from micelles devoid of quenchers. Assuming a Poissonian distribution of the quencher molecules, the probability that a micelle contains no quencher is given by Eq. (4.22), so that the relationship between the fluorescence intensity and the mean occupancy number < > is... 

Method II Dynamic quenching by totally micellized immobile quenchers It is assumed that the probability of quenching of a fluorescent probe in a given micelle is proportional to the number of quenchers residing in this micelle. The rate constant for de-excitation of a probe in a micelle containing n quencher molecules is given by... 

Quenching of fluorescence of tryptophan residues, coenzyme fluoro-phores, or extrinsic probes buried in the interior of proteins by colli-sional quencher molecules diffusing through the protein matrix/7,25 27)... 

Table 3.3. Survey of Quencher Molecules of NATA and Parvalbumin Phosphorescence"...

There is a thermally activated structural rearrangement of the protein such that channels appear and the quencher molecules are able to penetrate the protein—the penetration model.(67) This model distinguishes between external diffusion, kd(ext), and diffusion within the protein as follows ... 

The quencher molecules that are near the excited molecule in the moment of excitation have a much higher probability for depleting the excited molecules than the other quencher molecules. For that reason the rate coefficients used to describe the decay of excited molecules often have a so-called transient term, l a nDt) the transient term... 

Reactions (30) and (31) may give the same products. In (31) the polarization energy decreases the energy demand for temporal charge separation and it can be exothermic when B has a considerable electron affinity. For aromatic hydrocarbon quenchers (e.g., anthracene) such mechanism leads to dissipation of the excitation energy on the vibrational levels. When the quencher molecules contain Cl or Br atom in the intermediate step, Cl or Br elimination is expected, e.g., with benzyl chloride additive ... 

In the previous section, the diffusion equation for motion of a large number of particles was developed [see eqn. (211)]. When the solvent is at rest and both hydrodynamic forces and inter-reactants potential energy terms can be ignored, the equation becomes much simpler. This equation provides the basis for the analysis by Wilemski and Fixman. They chose to consider just one excited A molecule in a volume, V, together with m quencher molecules. The co-ordinates of all these molecules are rA and rQj, rQ2... rQ,n at a time f. Initially, thefluorophorand quencher molecules were positioned at rA° and rqj... r m. As a shorthand notation, these co-ordinates are called (r) and r0, respectively. The fluorophor was excited at time f°. 

On the right-hand side, the terms represent the rate of loss A and all m quencher molecule density by diffusion of A and of each of the Q quencher molecules and, finally, by reaction of each quencher with A. The boundary conditions on the density n are different from those of the Smoluchowski model. No loss of any particle can occur on. the outer surface (Vnj , -> 0 etc.), i.e. a closed system such as a glass beaker Where the quencher and fluorophor can interpenetrate each other, there is no net... 

Let /0 be the fluorescence intensity of pyrene in the absence of Q and let Iq be the intensity in the presence of Q (both measured at the same concentration of micelles). The quotient /Q//0 must be e Q, which is the probability that a micelle does not possess a quencher molecule. Substituting Q = [Q]/[M] gives... 

Based on steady-state and time-resolved emission studies, Scaiano and coworkers have concluded that silicalite (a pentasil zeolite) provides at least two types of sites for guest molecules [234-236], The triplet states of several arylalkyl ketones and diaryl ketones (benzophenone, xanthone, and benzil) have been used as probes. Phosphorescence from each molecule included in silicalite was observed. With the help of time-resolved diffuse reflectance spectroscopy, it has been possible to show that these triplet decays follow complex kinetics and extend over long periods of time. Experiments with benzophenone and arylalkyl ketones demonstrate that some sites are more easily accessed by the small quencher molecule oxygen. Also, diffuse reflectance studies in Na + -X showed that diphenylmethyl radicals in various sites decay over time periods differing by seven orders of magnitude (t varies between 20/is and 30 min) [237]. 

If the quencher molecule Q has an excited state Q lower than M the excitation energy can be transferred according to... 

In a rigid system such as a glass or a polymer, the molecules M and Q are distributed at random and do not move, at least within the lifetimes of excited states. The distance distribution follows the Perrin law which is based on a very simple model. Take any excited molecule M, and ask if one quencher molecule Q happens to be within the volume of action defined by the centre-to-centre distance r. Should any molecule Q be found within this action volume, the molecule M is quenched instantaneously, but if there is no quencher Q within this space, then M emits as if no quenchers at all were present. Figure 3.39 gives a picture of the Perrin model. The mathemat-... 

Figure 8.21 Diagram of a multilayer Langmuir-Bloggett film. F, metal or glass support LI, layer of chromophoric molecules M S, spacer layer L2, layer of quencher molecules...

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