Use of intrinsic fluorescence to follow the denaturation of vicilin,a storage protein from Vicia faba

Excitation of native vicilin protein at 274 nm resulted in a single emission peak at 304 nm attributed to tyrosine fluorescence. Changes in the quantum yield of the tyrosine fluorescence with addition of both urea and guanidine hydrochloride (GdnHCl) were only significant at intermediate denaturant concentrations, and not suitable for assessment of conformational change. Emission spectra for the tryptophan residues were obtained by excitation of the vicilin at 295 nm. The wavelength of maximum tryptophan emission shifted from 347 to 353 nm with the addition of denaturants (2.0 M GdnHCl or 3.0 M urea). At higher concentrations of denaturant, the quantum yield values for tryptophan also increased, reflecting a gradual change in the conformation of vicilin. The degree of polarization for both tyrosine and tryptophan fluorescence decreased significantly at denaturant concentrations slightly above those at which the protein showed the first signs of unfolding. This was further indication of a variety of conformational states for vicilin with increasing levels of denaturant.