8/13/2023 0 Comments Red edge control![]() ![]() The quenching of a fluorophore buried in a macromolecule requires diffusion of the quencher through the closely packed macromolecule. Collisional quenching of fluorescence can reveal the dynamics of biopolymers because contact between the fluorophore and quencher is required for quenching. These are the permeability of the macromolecule to quenchers and the rotational diffusion of proteins or membrane-bound fluorescent probes. Underlying these diverse approaches has been the use of basically two phenomena. A variety of fluorescence methods have been employed, such as steady-state, time-resolved, and phase-shift methods. This comparison indicates rapid spectral relaxation of TNS bound to lipid vesicles and a somewhat slower relaxation around TNS bound to the heme site of apomyoglobin.ĭuring the past 10 years, fluorescence spectroscopic methods have been widely utilized to investigate the dynamic properties of proteins and membranes. By comparison of the data obtained for TNS in solvents and bound to the macromolecules, one may estimate the relaxation rate of the environment. ![]() ![]() The spectral shifts and lifetime data indicate that red-edge excitation results in the selective excitation of “solvent-relaxed” fluorophores. We used this phenomenon to investigate the dynamic properties of reference solvents, model membranes, and the protein apomyoglobin labeled with 6-( p-toluidinyl)-2-naphthalenesulfonic acid (TNS). Consequently, the magnitude of the excitation red shifts depends upon the dynamic properties of the environment surrounding the fluorophore, as well as upon the solvent polarity and the sensitivity of the fluorophore to the polarity of the solvent. The observation of excitation red shifts requires that these enhanced dipole–dipole interactions are maintained in the photoselected population during the lifetime of the excited state. Red shifts occur because long-wavelength excitation results in photoselection of those fluorophores which are interacting most strongly with the polar solvent molecules. In moderately polar and viscous solvents, the emission spectra of fluorophores often shift to longer wavelengths as the excitation wavelength is increased toward the long-wavelength (red) side of the absorption. ![]()
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