Fluorescence techniques complexes

Intercalation of BPDE. Several groups have studied the reversible intercalative binding of BPDE to DNA. The fluorescence quantum yield of BPDE is much lower than that of BP derivatives which do not contain an epoxide group and fluorescence techniques have not been widely used to study BPDE physical binding to DNA (4). Association constants for the DNA intercalation of BPDE have been obtained by measuring red shifts in the UV absorption spectra of BPDE which occur upon the formation of intercalated complexes and from fluorescence studies (8) of the kinetics of DNA catalyzed hydrolysis of BPDE. The hydrolysis reaction is conveniently monitored by following the fluorescence of the hydrolysis product, BPT, which has a quantum yield many times greater than BPDE. 

The broad field of nucleic acid structure and dynamics has undergone remarkable development during the past decade. Especially in regard to dynamics, modem fluorescence methods have yielded some of the most important advances. This chapter concerns primarily the application of time-resolved fluorescence techniques to study the dynamics of nucleic acid/dye complexes, and the inferences regarding rotational mobilities, deformation potentials, and alternate structures of nucleic acids that follow from such experiments. Emphasis is mainly on the use of time-resolved fluorescence polarization anisotropy (FPA), although results obtained using other techniques are also noted. This chapter is devoted mainly to free DNAs and tRNAs, but DNAs in nucleosomes, chromatin, viruses, and sperm are also briefly discussed. 

The reader is referred to other reviews for detailed discussions of the electronic states and luminescence of nucleic acids and their constituents/0 fluorescence correlation spectroscopy/2) spectroscopy of dye/DNA complexes/0 and ethidium fluorescence assays/4,0 A brief review of early work on DNA dynamics as well as a review of tRNA kinetics and dynamics have also appeared. The diverse and voluminous literature on the use of fluorescence techniques to assay the binding of proteins and antitumor drugs to nucleic acids and on the use of fluorescent DNA/dye complexes in cytometry and cytochemistry lies entirely outside the scope of this chapter. 

The possibility to carry out conformational studies of peptides at low concentrations and in the presence of complex biological systems represents a major advantage of fluorescence spectroscopy over other techniques. Fluorescence quantum yield or lifetime determinations, anisotropy measurements and singlet-singlet resonance energy transfer experiments can be used to study the interaction of peptides with lipid micelles, membranes, proteins, or receptors. These fluorescence techniques can be used to determine binding parameters and to elucidate conformational aspects of the interaction of the peptide with a particular macro-molecular system. The limited scope of this chapter does not permit a comprehensive review of the numerous studies of this kind that have been carried and only a few general aspects are briefly discussed here. Fluorescence studies of peptide interactions with macromolecular systems published prior to 1984 have been reviewed. 

Oyama et al. [23] have pointed out that the fluorescence technique is more sensitive to the formation of intermolecular complexes than the conventional... 

However, the fluorescence technique was able to detect complexation between PAA and PEO at a PEO molecular weight as low as 4800. It is worth noting that Chen and Morawetz [13] and Bednar et al. [15], who used dansyl-labeled PAA, observed only a small change in fluorescence intensity at PEO molecular weight of 8000 and no change at 3400. 

Minis Label IT , green emission) was used, which attaches covalently to the DNA in the CL/DNA complex (Figure 10.11C). These preliminary experiments demonstrated that the double-fluorescence technique will allow the imaging of position of CL/DNA complexes inside the cell at different times. 

Similar very good agreement was found for a quantitative mixing analysis when determining the diffusive widths at various locations along the mixing channel and for various flow rates [70], Thus, the use of the confocal fluorescence technique for complex mixing analysis was demonstrated and compared with the potential of... 

In a recent crossed molecular beam experiment. Alagia et til. [36] measured the total DCS and product translational energy distribution for the N( D)+D2 reaction at 165 and 220 meV collision energies. They found an exact forward-backward symmetry which is consistent with an insertion mechcuiism rmd the existence of an intermediate complex. Using a laser-induced fluorescence technique. Umemoto [37] measured nascent rotational distributions and concluded that only the insertion mcchtmism is important in the N( D)- -H2 reaction at low mid medium cnergj-. This result has been recentlj confirmed by the measure of the product vibrational population for NH(f = 0,1.2,3,4) [38]. 

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. 

The use of fluorescence techniques for the characterization of the behavior of chain molecules in solution offers a number of important advantages (a) Since fluorescence can be detected at extreme dilution of the emitting species, labeling polymers with fluorophores need not result in significant modifications of their other properties. (b) Since the lifetime of the excited chromophore, I, Is generally of the order of 10 ns, the kinetics of fast processes whose relaxation times are comparable to t may be studied by fluorescence techniques. nonradiative energy transfer over relatively long distances may be used to characterize the spatial relation of donor and acceptor labeled chain molecules, (d) The medium sensitivity of the emission from the "dansyl" label has been used to study polymer complex formation and the transition of chain molecules from a contracted to an expanded state. 

Miniaturization and integration of sensors into lab on a chip total analysis systems are likely to have very important implications in medical diagnostics and microanalysis of complex mixtures. The breakthroughs here have involved microfluidics and laser-induced fluorescence techniques, as well as ultramicroelectrodes tor ultrasensitive electrochemical analysis. 

When the bulk behavior is checked with fluorescence techniques, much smaller CAC values are observed, an order of magnitude smaller than for a more hydrophilic polyelectrolyte, sodium polyacrylate.4 Bulk association could explain why surface tension changes are only appreciable at high polymer concentration. In this case, the simple adsorption model valid for PAMPS would evidently not work, because it does not account for bulk complexes. 

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