Tryptophan-containing peptides fluorescence properties

Fig. 10. Highly schematic representation of the orientation of several tryptophan-containing peptides with respect to calmodulin. (A) With tryptophan in position 1, the indole is located on the hydrophilic side of the helix and is exposed to solvent. Peptides with tryptophan on this face of the helix should exhibit emission maxima near that of indole in water ( 350 nm), a small anisotropy, and a high accessibility for acrylamide quenching. (B) In position 2, the tryptophan is partially exposed at the interface between the peptide and calmodulin. Peptides with a tryptophan in this location should have fluorescence properties that are intermediate between example A and C. (C) The tryptophan is on the hydrophobic side of the helix and is almost entirely buried. The emission maximum should be strongly blue-shifted, the anisotropy should be large, and the accessibility to acrylamide quenching low. Taken from O Neil et al. (1987).

Fig. 11. Variation of the fluorescence properties of a set of tryptophan-containing peptides as a function of the position of the tryptophan in their sequence. The parameter/AVe describes the degree of rigidity and hydrophobicity of the tryptophan s environment it is based on emission maximum, anisotropy, and accessibility to acrylamide. When the values for each of these parameters were similar to those expected for indole in water, a value near 0 was assigned to/, whereas values up to 1.0 were assigned as the fluorescence parameters more closely resembled those observed in very rigid and apolar environments such as the interior of a protein or ethylene glycol at -60°C (Lakowicz, 1983). The values of / calculated for each parameter were then averaged to give /AVe- The dotted curve was generated by fitting a sine wave to the data (period = 3.3 residues). Taken from O Neil et al. (1987).

Although the main part of the chapter covers the chemistry of the tryptophan residue, a section dealing with physico-chemical techniques used in assessing structural characteristics of tryptophan-containing peptides and proteins has been included. Tryptophan residues contribute significantly to the optical, stereooptical and fluorescent properties of proteins and their spectral characteristics are considered to be important gauges of protein conformation. 

Peptides are commonly detected by absorbance at 200-220 nm. However, most of the compounds present in wine may interfere in the ultraviolet detection of peptides when low wavelengths are used. Thus, for the analysis of these compounds it is useful to apply sensitive and selective detection methods. To this end, it is possible to form derivates of the peptides that can be detected at higher and more specific wavelengths. Detection by fluorescence can also be used to detect peptides containing fluorescence amino acids (tyrosine and tryptophan). For peptides without this property, the formation of derivates with derivatizing agents have been proved to be very useful (Moreno-Arribas et al. 1998a). 

Peptides may also be detected by means of a fluorescence detector using the natural fluorescence of certain amino acids or by forming fluorescent derivatives. Tyrosine and tryptophan are fluorescent, and this property can be used to detect peptides that contain these amino acids. The excitation wavelength for these amino acids has been established to be between 220 and 280 nm, with the greatest sensitivity at 220 nm. In alkaline solutions the emission band is 365 nm for tryptophan and 310 nm for tyrosine. Peptides containing both these amino acids have been detected at an emission wavelength of 330 nm (94). 

---