A reassessment of synchronous fluorescence in the separation of Trp and Tyr contributions in protein emission and in the determination of conformational changes

Synchronous fluorescence spectra are performed by simultaneously scanning both the excitation and emission wavelengths, and are widely used to analyze complex mixtures of fluorophores, since they yield narrower bands than traditional excitation or emission spectra. Many recent studies claim that synchronous spectra are able to separate tryptophan (Trp) and tyrosine (Tyr) emission in proteins, and use this approach to analyze conformational transitions induced by ligand binding. Here, the reliability of this method is reassessed, studying mixtures of the two intrinsic protein fluorophores in different solvents, as well as a real protein (bovine serum albumin). Unfortunately, synchronous spectra were found to be unreliable in the separation of Trp and Tyr emission components in proteins. A simple alternative approach based on the deconvolution of emission spectra is presented. In addition, an equation predicting the synchronous spectrum of a specific fluorophore from its excitation and emission spectra has been derived. (C) 2014 Elsevier B.V. All rights reserved.