12.3.3 Applications
There are many and highly varied applications for fluorescence despite the fact that
relatively few compounds exhibit the phenomenon. The effects of pH, solvent com-
position and the polarisation of fluorescence may all contribute to structural elucida-
tion. Measurement of fluorescence lifetimes can be used to assess rotation correlation
coefficients and thus particle sizes. Non-fluorescent compounds are often labelled
with fluorescent probes to enable monitoring of molecular events. This is termed
extrinsic fluorescenceas distinct from intrinsic fluorescence where the native com-
pound exhibits the property. Some fluorescent dyes are sensitive to the presence of
metal ions and can thus be used to track changes of these ions inin vitrosamples, as
well as whole cells.
Since fluorescence spectrometers have two monochromators, one for tuning the
excitation wavelength and one for analysing the emission wavelength of the fluoro-
phore, one can measure two types of spectra: excitation and emission spectra. For
fluorescenceexcitation spectrummeasurement, one sets the emission monochromator
at a fixed wavelength (lem) and scans a range of excitation wavelengths which are then
recorded as ordinate (x-coordinate)oftheexcitationspectrum;thefluorescenceemission
atlemis plotted as abscissa. Measurement ofemission spectrais achieved by setting a
fixed excitation wavelength (lexc) and scanning a wavelength range with the emission
monochromator. To yield a spectrum, the emission wavelengthlemis recorded as
ordinate and the emission intensity atlemis plotted as abscissa.Intrinsic protein fluorescence
Proteins possess three intrinsic fluorophores: tryptophan, tyrosine and phenylalanine,
although the latter has a very low quantum yield and its contribution to protein fluores-
cence emission is thus negligible. Of the remaining two residues, tyrosine has the lower
quantum yield and its fluorescence emission is almost entirely quenched when it becomes
ionised, or is located near an amino or carboxyl group, or a tryptophan residue. Intrinsic
protein fluorescence is thus usually determined by tryptophan fluorescence which can be
selectively excited at 295–305 nm. Excitation at 280 nm excites tyrosine and tryptophan
fluorescence and the resulting spectra might therefore contain contributions from both
types of residues.
The main application for intrinsic protein fluorescence aims at conformational
monitoring. We have already mentioned that the fluorescence properties of a fluoro-
phore depend significantly on environmental factors, including solvent, pH, possible
quenchers, neighbouring groups, etc.
A number of empirical rules can be applied to interpret protein fluorescence spectra:- As a fluorophore moves into an environment with less polarity, its emission
spectrum exhibits a hypsochromic shift (lmaxmoves to shorter wavelengths) and
the intensity atlmaxincreases. - Fluorophores in a polar environment show a decrease in quantum yield with
increasing temperature. In a non-polar environment, there is little change. - Tryptophan fluorescence is quenched by neighbouring protonated acidic groups.
497 12.3 Fluorescence spectroscopy