Principles of Fluorescence Spectroscopy

536 PROTEIN FLUORESCENCE
Figure 16.10. Emission spectra of proteins that lack tryptophan
residues. Neutral pH. Revised from [48–49].
multiple tryptophan residues, and the residues contribute
unequally to the total emission. There have been attempts to
divide proteins into classes based on their emission spectra.
56–59 The basic idea is that the tryptophan emission spectrum
should reflect the average environment of the tryptophan.
For tryptophan in a completely apolar environment a
blue-shifted structured emission characteristic of indole in
cyclohexane can be observed. As the tryptophan residue
becomes hydrogen bonded or exposed to water, the emission
shifts to longer wavelengths (Figure 16.11). In fact,
individual proteins are known that display this wide range
of emission spectra. 60–61 For example, later in this chapter
we will see that azurin displays an emission spectrum characteristic
of a completely shielded tryptophan residue. The
emission from adrenocorticotropin hormone (ACTH) is
characteristic of a fully exposed tryptophan residue.
The emission maximum and quantum yield of tryptophan
can vary greatly between proteins. Denaturation of
proteins results in similar emission spectra and quantum
yields for the unfolded proteins. Hence, the variations in
tryptophan emission are due to the structure of the protein.
We are not yet able to predict the spectral properties of proteins
using the known structures, but some efforts are
underway. 61 One might expect that proteins that display a
blue-shifted emission spectrum will have higher quantum
yields (Q) or lifetimes (τ). Such behavior is expected from
Figure 16.11. Effect of tryptophan environment on the emission spectra. The emission spectra are those of apoazurin Pfl, ribonuclease T 1 , staphylococcal
nuclease, and glucagon, for 1 to 4, respectively. Revised from [59] and [60].