Quenching resolved emission anisotropy

M. Eftink, Quenching-resolved emission anisotropy studies with single and multitryptophan-containing proteins, Biophys. J. 43, 323-334 (1983). 

The mean fluorescence lifetime is used to calculate the rotational correlation time from the Perrin plot (quenching resolved emission anisotropy experiment). 

When a protein contains fluorophore residues located at the surface and in the core as it is the case for a j-acid glycoprotein, the results obtained from the classical Perrin plot contain contributions from all residues. In order to obtain information on the motion of each class of fluorophore residues, one may follow anisotropy and intensity variations as a function of quencher concentration (Quenching Resolved Emission Anisotropy) or / and anisotropy and lifetime variations with temperature (-50 to +35°C) (Weber method). 

Table 8.7. Comparison of the anisotropies of the two classes of Trp residues of ai-acid glycoprotein and Lens culinaris agglutinin. Measurements were performed at 20°C with the Quenching Resolved Emission Anisotropy method.

Although we haye shown that the three Trp-residues contribute to the global fluorescence of a i-acid glycoprotein (paragraph 5 c of this chapter) quenching resolved emission anisotropy and the Weber method allowed giving a description on the mean local dynamic of the Trp-residues. The dynamic of the surface Trp residue is well separated from the two other Trp residues. 

Quenching resolved emission anisotropy experiments could be performed at emission wavelengths in the blue (< 330 nm) and red (> 330 nm) portions of the spectrum to yield a more consistent data surface. However, this could be possible if at each edge of the fluorescence spectrum the emission occurs mainly from the buried or the surface Trp residues. Unfortunately, this is not the case since for example at 315 nm, the fractional contribution to the total fluorescence of the surface Trp residue is 42%. 

Albani, J. R, 1999, New insights in the conformation of a i-acid glycoprotein (orosomucoid). Quenching resolved emission anisotropy studies. Spectrochimica Acta, Part A. 55,2353-2360. 

In order to put into evidence the Trp residues thet participate in the emission of the protein, quenching resolved emission with cesium were first performed. When a protein contains two classes of intrin fluoropliore. one at the surface of the protein and the second embedded in the protein matrix, fluorescence intensify quenching with cesium allows obtaining the spectra of these two classes. A selective quenching implies that addition of quencher induces a decrease in the fluorescence observables (intensity, anisotropy and lifetime) of the accessible class. At high quencher concentration the remaining observables measured will reflect essentially those of the embedded fluorophore residues. 

There should exist a correlation between the two time-resolved functions the decay of the fluorescence intensity and the decay of the emission anisotropy. If the fluorophore undergoes intramolecular rotation with some potential energy and the quenching of its emission has an angular dependence, then the intensity decay function is predicted to be strongly dependent on the rotational diffusion coefficient of the fluorophore.(112) It is expected to be single-exponential only in the case when the internal rotation is fast as compared with an averaged decay rate. As the internal rotation becomes slower, the intensity decay function should exhibit nonexponential behavior. 

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