Depolarization energy transfer

Various spectroscopic techniques and probes have been used to investigate solubilization of probe molecules, mostly using UV/visible spectroscopy, fluorescence spectroscopy, ESR spectroscopy [64, 74, 217, 287] and NMR-spectro-scopy [367-369]. Fluorescence spectroscopy is particularly versatile [370], as various static and dynamic aspects can be covered by studying excitation and emission spectra, excimer or exciplex formation, quantum yields, quenching, fluorescence life-times, fluorescence depolarization, energy transfer etc. 

Energy transfer such as that observed between two or different chromophores, for examples between two tryptophan residues and / or from tryptophan to heme, is a source of depolarization. Energy transfer in hemoproteins between tryptophans and heme is commonly observed. The high energy transfer efficiency in hemoproteins can be put into evidence by plotting the Perrin plot by varying the polarization or the anisotropy as a function of the temperature. 

Any factor that affects the size or shape of a molecule, the hindered movement of a fluorophore within a molecule, or the energy transfer within the molecule will affect the measured depolarization of its fluorescence emission. Therefore, the conformation of humic fractions in solution can be studied as a function of pH, ionic strength, temperature, and other factors by depolarization measurements. The principle of the method is that excitation of fluorescent samples with polarized light stimulates... 

Steady-State Fluorescence Depolarization Spectroscopy. For steady state depolarization measurements, the sample is excited with linearly polarized lig t of constant intensity. Observed values of P depend on the angle between the absorption and emission dipole moment vectors. In equation 2 (9), Po is the limiting value of polarization for a dilute solution of fluorophores randomly oriented in a rigid medium that permits no rotation and no energy transfer to other fluorophores ... 

The intercept, 1/Po, is called the anisotropy of the molecule and is an indication of the nonrotational depolarization of the molecule. This intrinsic depolarization is due to the segmental motion of the fluorophores within the molecule the depolarization due to energy transfer and the angular difference in transition dipole moments of the absorbing and emitting states. 

Homo-FRET is a useful tool to study the interactions in living cells that can be detected by the decrease in anisotropy [106, 107]. Since commonly the donor and acceptor dipoles are not perfectly aligned in space, the energy transfer results in depolarization of acceptor emission. Imaging in polarized light can be provided both in confocal and time-resolved microscopies. However, a decrease of steady-state anisotropy can be observed not only due to homo-FRET, but also due to rotation of the fluorescence emitter. The only possibility of discriminating them in an unknown system is to use the variation of excitation wavelength and apply the... 

F. Perrin Discussion on Jean Perriris diagram for the explanation of the delayed fluorescence by the intermediate passage through a metastable state First qualitative theory of fluorescence depolarization by resonance energy transfer... 

Otherwise, depolarization would also be a result of energy transfer between probe molecules. Because the transition moments of two interacting probes are unlikely to be parallel, this effect is indeed formally equivalent to a rotation. Moreover, artefacts may arise from scattering light that is not totally rejected in the detection system. 

Figure 2.4. Schematic representation of processes which lead to fluorescence depolarization in proteins rotation of the protein molecule as a whole with correlation time rotation of the fluorophore with correlation time d, and excitation energy transfer, represented by the wavy arrow.

In order to avoid complications caused by excitation energy transfer between tryptophan residues, most investigations have been performed with proteins containing one tryptophan residue per molecule. When studying protein solutions, there are difficulties in separating the effects of rotation of entire protein molecules and of the chromophores themselves relative to their environment in the protein matrix. It is usually assumed that intramolecular motions are more rapid and manifest themselves as short-lived components of anisotropy decay curves or in depolarization at short emission lifetimes. 

Most interesting applications of intramolecular energy transfer between nonconjugated chromophores are found in the conformational studies of biomolecules like nucleic acids and proteins. The experiments on rotational depolarization of emission from intrinsic fluorescent groups on externally attached fluorescent probes, have resulted in a vast store of knowledge which has helped to enrich the subject of photobiology. 

ANS (see Basic Protocol 1) has been the most popular hydrophobic probe for the determination of surface hydrophobicity of proteins. Its dimeric form bis-ANS, which has a greater quantum yield in nonpolar environments by binding more strongly with proteins than the ANS monomer, is occasionally used for the same purpose (Das and Surewicz, 1995). These effects permit the observation of depolarization by energy transfer among the bound fluoropho-res, which can be used to estimate the distribution of the ligands among the protein molecules (Farris et al., 1978). 

However, during the excited-state lifetime, energy transfer to neighboring molecules and/or local and global motions of the fluorophores can be observed. These two phenomena induce reorientation of the emission dipole, thereby depolarizing the system. Therefore, the value of the measured polarization P will be lower than intrinsic polarization value PQ (Weber, 1952). 

Energy transfer is a source of depolarization. For example, a high energy-transfer efficiency in hemoproteins can be evidenced by plotting Perrin plot at different temperatures. 

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