Red-edge excitation spectroscopy

A comparison with the results of model studies indicates that the behavior of these probes bound to proteins differs fundamentally from their behavior in liquid media in which the position of their fluorescence spectra with ordinary excitation is similar to that for the protein complexes. In the latter case, the red-edge effect is always absent. 

The fluorescent probe 2,6-TNS and other similar aminonaphthalene derivatives (1,8-ANS, DNS) were considered to be indicators of the polarity of protein molecules, and they were assumed to be bound only to hydrophobic sites on the protein surface. The detection of considerable spectral shifts with red-edge excitation has shown that the reason for the observed short-wavelength location of the spectra of these probes when complexed to proteins is not the hydrophobicity of their environment (or, at least, not only this) but the absence of dipole-relaxational equilibrium on the nanosecond time scale. Therefore, liquid solvents with different polarities cannot be considered to simulate the environment of fluorescent probes in proteins. 

These data may be explained in terms of the above mechanism of the long-wavelength shift of fluorescence spectra for red-edge excitation. The properties of the environment of the tryptophan residues in the proteins studied are such that during the lifetime of the excited state, structural relaxation of the surrounding dipoles fails to proceed. Studies of the dependence of the 

In using the method of the red-edge shift in UV fluorescence spectroscopy, we should take into account the possibility of emission not only of tryptophan but also of tyrosine residues. In many tryptophan-containing proteins, tyrosine fluorescence is not observed. However, it is considerable in serum albumin, and the decrease in its intensity is responsible for the long-wavelength shift of the spectra recorded at Aex 290 nm. At Aex 292 nm, the tyrosine component should be completely absent. 

Among multitryptophan proteins emitting light around 330 nm, we have observed the largest red-edge effect (estimated from the difference between the maxima of the fluorescence spectra obtained at 290- and 305-nm excitation) for papain in the active and inactive forms (13 and 10 nm, respectively). Large shifts were also observed for rabbit muscle asparagyl- and valyl-RNA synthetases (8 nm). For rabbit aldolase A, the observed shift was 6 nm, for skeletal muscle myosin, 4.5 nm, for chymotrypsin, 2.5 nm, and for carbonic 

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A. P. Demchenko, Red-edge-excitation spectroscopy of single-tryptophan proteins, Eur. Biophys. J. 16, 121-129 (1988). 

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