Red-edge excitation spectra method

The red-edge excitation spectra method is largely used as a tool for monitoring motions around a fluorophore. The method is very sensitive to the changes that occur in the microenvironment of a fluorophore (Demchenko, 2002). 

We report here die development of a method di allowed us to obtain crystals of ai-acid glycoprotein comple.x to progestenme. rurlliermore. we investigated the mamics of the microenvironments of the two buried Trp residues in the crystals of the protc ni>T( eslerone complex, by the red-edge excitation spectra method. 

Perrin plot and red-edge excitation spectra experiments performed on Trp residues of sialylated and asialylated ai-acid glycoprotein have shown that in both proteins the intrinsic fluorophore displays local motions and are surrounded by a flexible environment. However, the above two mentioned methods yield information on the mean residual motion and can in no way give an indication on the dynamics of each class of Trp residues. In fact, the exposed tryptophan residue should be expected to rotate much more freely than the hydrophobic residues. In order to study the dynamics behavior of each class of Ti p residues, steady-state measurements of emission anisotropy at different temperatures (-45 to + 30°C) can be carried out. This method (the Weber s method) known also as the Y-plot, allows deriving parameters characteristic of the environment of the rotating unit, such as the thermal coefficient of the frictional resistance to the rotation of the fluorophore. 

It is important to mention that the Weber s method allows the measurement of an important parameter, which is the thermal coefficient of the frictional resistance bu indicating that the immediate environment opposes to the rotation of the fluorophore (Weber et al. 1984). This parameter cannot be measured by other methods such as the classical Perrin plot, the anisotropy decay with time or with the red-edge excitation spectra, bu is determined in the proteins by the interactions of the fluorophore with the amino acids in the immediate vicinity. The other methods do not give an exact idea on the definition of the microenvironment of the fluorophore. Is it one, two, few amino acids around the Trp residue or an important region in contact with the fluorophore ... 

Albani, J, 1992, Motions studies of the human ai-acid glycoprotein (orosomucoid) followed by red-edge excitation spectra and polarization of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and of tryptophan residues. Biophysical Chemistry. 44, 129 - 137. Albani, J, 1993, A study of the interaction between two proteins, one containing a flavin mononucleotide. Journal of Biochemical and Biophysical Methods. 26, 105-112. 

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