Reorientation of Dipoles in the Fluorophore Environment

The dipole-dipole interactions of the fluorophore in the electronic excited state with the surrounding groups of atoms in the protein molecule or with solvent molecules give rise to considerable shifts of the fluorescence spectra during the relaxation process. These spectral shifts may be observed directly by time-resolved spectroscopic methods. They may be also studied by steady-state spectroscopic methods, but in this case additional data must be obtained by varying factors that affect the ratio between tf and xp. 

The observed spectral shift depends both on the properties of the fluorophore itself (the vectorial difference between the dipole moments in the ground and the excited state, ne — pg) and also on properties of the environment interacting with it. The establishment of dielectric equilibrium with the environment occurs due to the following effects  

Electronic polarization of the environment. This effect is related to the square of the refractive index, n1 2 (dielectric constant at the frequency of light). Here the spectral shift occurs instantly (10 15 s), and its evolution with time is not observed by the kinetic spectroscopic methods. The protein molecule is a medium with a relatively high electronic polarization (n= 1.5 -s-1.6). 

Another aspect of dielectric relaxation in proteins should be considered. If in model solutions of aromatic molecules, dielectric relaxation occurs 

Formation of specific complexes in the excited states ( exciplexes )f 35 52 85) Exciplexes are complexes not present in the ground state that form due to the extensive redistribution of electron density that occurs upon excitation. Among exciplexes, there may be some whose formation does not require substantial nuclear rearrangements and thus occurs rather rapidly even at 77 K. The formation of exciplexes is accompanied by a spectral shift to longer wavelengths. It is postulated that the fluorescence from tryptophan in proteins in a variety of cases is fluorescence from tryptophan exciplexes)35 85) In studies of the effects of environmental dynamics on the spectra, the exciplexes may be considered as individual fluorophores. 

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