Fluorescence spectroscopy limiting aspects

The possibility to carry out conformational studies of peptides at low concentrations and in the presence of complex biological systems represents a major advantage of fluorescence spectroscopy over other techniques. Fluorescence quantum yield or lifetime determinations, anisotropy measurements and singlet-singlet resonance energy transfer experiments can be used to study the interaction of peptides with lipid micelles, membranes, proteins, or receptors. These fluorescence techniques can be used to determine binding parameters and to elucidate conformational aspects of the interaction of the peptide with a particular macro-molecular system. The limited scope of this chapter does not permit a comprehensive review of the numerous studies of this kind that have been carried and only a few general aspects are briefly discussed here. Fluorescence studies of peptide interactions with macromolecular systems published prior to 1984 have been reviewed. 

A limiting aspect of fluorescence spectroscopy is that quantitative results obtained by different researchers usmg different procedures are generally not comparable (i.e., complexing capacities of HS appear to be dependent on the method of measurement). Also, the source of HS and the procedure used for its isolation, in addition to many experimental factors, including concentration of HS, ionic strength of solution, pH, temperature, and the method of data manipulation for the computation of stability constants, can influence the results (Saar and Weber, 1982). 

In Neunhoeffer s contribution to CHEC-I a large body of detailed data was compiled, for x-ray diffraction, H and C NMR, ESR, UV/VIS, fluorescence, phosphorescence, and photoelectron spectroscopy <84CHEC-i(3)531>. It is not possible within the limited space of this contribution to go back to the roots. There are many individual reports of spectral data on a variety of tetrazine derivatives, but it would be inappropriate to tabulate them here. The principal aspects of different types of spectroscopy have been well described by Neunhoeffer in . A few new results are presented in this chapter. 

In Table 3 the orientation information which can be obtained from these various structural techniques is summarised. This table also shows the part of the molecular structure which is being characterised, and some of the theoretical and experimental limitations of each method. A further technique, that of polarised fluorescence has been added. This technique is exactly analogous in its orientation aspects to Raman spectroscopy. The distinction between the two techniques lies in the fact that in the Raman effect, the lifetime of the process is of the order of the vibrational period ( 10 s) whereas fluorescence occurs after much longer occupancy of the transition state ( 10 s). 

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