Fluorescence polarization interactions

Polarization of the Emission. We have sought support for the weakly interacting chain segment model from measurements of room temperature fluorescence polarization (19) on dilute solutions of 1 in 3-methylpentane. An independent preliminary report of similar measurements on a dilute glassy solution at 77K and on a neat polymer has also appeared (21). In the latter case, the analysis is complicated by inter-chain energy transfer. 

The versatility of luminescence goes beyond intensity-, wavelength- and kinetic-based measurements. Fluorescence polarization (or anisotropy) is an additional parameter still largely unexplored for optical sensing yet widely used in Biochemistry to study the interaction of proteins, the microfluidity of cell membranes and in fluorescence immunoassays. Although only a few optosensors based on luminescence polarization measurements can be found in the literature, elegant devices have recently been reported to measure chemical parameters such as pFI or O2 even with the bare eye41. 

Fluorescence polarization is also well suited to equilibrium binding studies when the free and bound species involved in the equilibrium have different rotational rates (Scheme 5.1). Most molecular interactions can be analyzed by this method. It should be emphasized that, in contrast to other methods using tracers, fluorescence polarization provides a direct measurement of the ratio of bound and free tracer without prior physical separation of these species. Moreover, measure-... 

Different techniques have been applied to study the protein-protein, protein-ligand and, in particular, MIP-ligand interactions. They may serve to estimate or determine the binding constant and the number of independent binding sites (N) of a ligand-to-receptor (MIP or antibody) interaction. The range of affinity constants that can be calculated depends on the sensitivity of the assay and, in those cases where the separation of the bound and free species is a step of the assay, perturbation of the equilibria in the separation step will also be important [22]. Direct nonseparation techniques such as spectroscopic techniques (e.g., SPR or fluorescence polarization) can be used as well as indirect separation techniques such as radiolabeling [22]. 

Protein-ligand interactions can not only be secreened or selected in vitro, but also can be directly characterized for particular interaction partners. Nemoto et al. (1999) applied the mRNA-peptide fusion technology to fluorescently label the displayed proteins in order to study protein-protein interactions by fluorescence polarization measurements. 

Circular polarization of luminiscence, stopped-flow fluorescence, fluorescence-monitored chemical relaxation, the evaluation of relative orientation by polarized excitation energy transfer, time-resolved fluorescent polarization ( nanosecond polarization ), and other new techniques have become valuable means for studying protein structures, their interactions and structural changes in relation to various treatments (e.g. denaturation). New fluorescent probes and quenchers have enabled the research field to expand from isolated proteins to more complicated systems such as membranes, muscle and nerve components and other subcellular structures (see also 7.3). 

In addition to biosensor experiments, the interaction YTXs-PDEs was also confirmed by eheek-ing the variations in the fluorescence polarization (FP) of PDEs labeled with eatboxy-fluoreseein. FP units change when the molecule is bound to the toxin. With this technique, a good relationship of toxin concentration to FP units can be observed (Alfonso et al. 2005), whieh again eonfirms PDEs-YTXs binding. 

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