Routine Fluorescence
The three aromatic amino acid residues (tryptophan, tyrosine, and phenylalanine) may contribute to the intrinsic fluorescence of a protein. In practice, however, the fluorescence spectrum is usually dominated by emissions due to the tryptophan residue. The intensity (quantum yield) and wavelength maximum of the tryptophan fluorescence is dependant on the environment surrounding the residue. The fluorescence spectrum shifts to shorter wavelength and the intensity of the fluorescence increases as the polarity of the solvent surrounding the tryptophan residue decreases. Tryptophan residues that are exposed to water, have maximum fluorescence intensity at a wavelength of about 340-350 nm, while totally buried residues fluoresce at about 330 nm.
The steady state fluorescence emission from the tryptophan has been widely used as a probe to monitor, for instance, the folding and unfolding of proteins in response to stimuli such as temperature or varying concentrations of chemical denaturant. It should be noted that the fluorescence spectrum can alter as a result of changing the solvent environment even in the absence of any protein structural changes.
With a custom built rotating stage Avacta Analytical can also provide front face fluorescence measurements
Avacta instrumentation includes the Shimadzu RF 5301-PC spectrofluorimeter.