When interpreting effects observed in fluorescence experiments, one has to consider
carefully all possible molecular events. Forexample, a compound added to a protein
solution can cause quenching of tryptophan fluorescence. This could come about by
binding of the compound at a site close to the tryptophan (i.e. the residue is surface-
exposed to a certain degree), or due to a conformational change induced by the compound.
The comparison of protein fluorescence excitation and emission spectra can yield
insights into the location of fluorophores. The close spatial arrangement of fluoro-
phores within a protein can lead to quenching of fluorescence emission; this might be
seen by the lower intensity of the emission spectrum when compared to the excitation
spectrum (Fig. 12.10).Extrinsic fluorescence
Frequently, molecules of interest for biochemical studies are non-fluorescent. In many
of these cases, an external fluorophore can be introduced into the system by chemical
coupling or non-covalent binding. Some examples of commonly used external fluoro-
phores are shown in Fig. 12.11. Three criteria must be met by fluorophores in this
context. Firstly, it must not affect the mechanistic properties of the system under
investigation. Secondly, its fluorescence emission needs to be sensitive to environ-
mental conditions in order to enable monitoring of the molecular events. And lastly,
the fluorophore must be tightly bound at a unique location.
A common non-conjugating extrinsic chromophore for proteins is 1-anilino-8-
naphthalene sulphonate (ANS) which emits only weak fluorescence in polar environ-
ment, i.e. in aqueous solution. However, in non-polar environment, e.g. when bound to
hydrophobic patches on proteins, its fluorescence emission is significantly increased
and the spectrum shows a hypsochromic shift;lmaxshifts from 475nm to 450nm. ANS200 250 300 350 400 450350
300
250
200
150
100
50
0Fluorescence emission in a.u.(a) (b)
l(nm) l(nm)500 200 250 300 350 400 450250200150100500Fluorescence emission in a.u.
500Fig. 12.10Comparison of fluorescence excitation and emission spectra can yield insights into internal
quenching. Excitation spectra with emission wavelength 340 nm are shown in dark green. Emission spectra with
excitation wavelength 295 nm are shown in light green; emission spectra with excitation wavelength 280 nm
are grey. (a) PDase homologue (Escherichia coli). (b) CPDase (Arabidopsis thaliana); in this protein, the
fluorophores are located in close proximity to each other which leads to the effect of intrinsic quenching,
as obvious from the lower intensity of the emission spectrum as compared to the excitation spectrum.498 Spectroscopic techniques: I Photometric techniques