Fluorescence Quenching Measurements

In general, reduction potentials of nucleobases have been studied much less than their oxidation potentials, and in particular water-based data are rather lacking [2, 35]. We therefore listed the available polarographic potentials measured in dimethylformamide and data obtained from pulse radiolysis studies or fluorescence quenching measurements. From the data in Table 1, it is evident that the pyrimidine bases are most easily reduced. The reduction potential of the T=T CPD lesion is close to the estimated value of the undamaged thymine base [34, 36]. 

While no spectroscopic evidence of a ground-state complex between anthracene and carbon tetrachloride, naphthalene or 1,2-benzanthracene and carbon tetrabromide has been found, Nemzek and Ware [7] were unable to explain their steady-state fluorescence quenching measurements with the parameters deduced from the determination of the time-dependent rate coefficients unless a ground-state complex was present. This cannot be regarded as a satisfactory and consistent analysis because the time-dependent rate coefficient would be modified by the presence of the initial distribution of quencher and fluorophor in the ground state. 

Cabaniss, S.E. and Shuman, M.S. (1988) Fluorescence quenching measurements of copper-fulvic acid binding. Anal. Chem., 60, 2418-2421. 

In order to extract rate constants for electron transfer from observed fluorescence quenching measurements the following mechanistic scheme is commonly used. In this scheme, presented here in the case of an excited anionic donor, kd and k d are the rate constants for the diffusive formation and dissociation of an encounter complex within which the electron transfer takes place (kel). 

It was of course of interest to review in this context the use of the Rehm-Weller empirical equation, Eq. (6), versus the Marcus model of Eq. (5). The method used to correlate the results for the jl-naphtholate quenchings was thus applied to the important Weller series of fluorescence quenching measurements made in acetonitrile [88] and this shows that 60 values (on a total number of 65) fit quite well with a mean intrinsic activation barrier of 5.5 Kcal. M 1 [96] (see Fig. 3). Moreover, this value is not far from the expected value for the outer contribution (AGJ), as calculated by Eq. (7) and it has often been underlined that the solvent reorganization term is effectively the dominant contribution in... 

The results of fluorescence quenching measurements indicated that a rather large portion of the protein can be penetrated by the physiological ligands of the... 

In Scheme 16, D and A are proton donor molecules and proton acceptor molecules, respectively. The quantity K represents the stability of the complex DH A, formed by hydrogen bonding interactions in the ground state. For the 2-naphthylamine/pyridine system, the K value in hexane (obtained by fluorescence quenching measures) is 0.6 dm3 mol-1. This value is reduced by using benzene as a solvent (K = 0.2 dm3 mol-1) while, in cyclohexane, K = 12. The observed variations confirm the importance of solvents in influencing solute/solute interactions149. 

Experimentally, the most relevant issue for obtaining systematic results is the availability of well-characterized apertures, a point that cannot be overestimated, Obviously, the results depend sensitively on the tip and more precisely on the structure of the aperture. This reveals the importance of the probe in single-molecule near-field fluorescence microscopy in general not only in lifetime measurements. In view of the fluorescence quenching measurements mentioned at the beginning of this... 

Fluorescence Quenching Measurements Addition of an external species, known as a quencher (Q), which is capable of deactivating the excited state through collision can provide information concerning the extent/degree of exposure of a fluorescent species. The process is outlined below... 

Soda Ltd. was used for all measurements but the fluorescence quenching measurements on high salt, 500 mM NaCl, solutions and for the static fluorescence measurements. For die 500 mM NaCl solutions PEO designated Polyox WSR 301 (M =1.5106) was used and for the static fluorescence measurements PEO with M =6 105 from BDH was used. Pyrene (Aldrich) and 2,5-di-tert-butylaniline... 

Quenching rate constants from dynamic fluorescence quenching measurements with -tert-butylaniline as quencher and pyrene as fluorescence probe. 

To apply (B5.3.4) to electron transfer from excited tryptophan residues in proteins, Callis et al. incorporated calculations of H21, AG and A into hybrid classical-quantum mechanical molecular dynamics (MD) simulations [50,68, 88, 98, 99]. (See Box 6.1 for an introduction to MD simulations, and Fig. 6.3 for an illustration of how calculated vibrational motions of a protein can affect the energy gap for electron transfer.) The calculated rate constants for electron transfer to backbone amide groups are in accord with the fluorescence quenching measured in many proteins, and suggest that variations in... 

Once phage or scFv which specifically bind the antigen of interest have been identified it may be necessary to cany out further screening tests to assess which antibodies from the positive population have the highest affinities. There are a variety of ways of achieving this including surface plasmon resonance (BIAcore) screening, fluorescence quench measurement, and competition ELISAs. Descriptions of these protocols are beyond the scope of this chapter. 

---