Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

82 Weljie and Vogel
4. A series of emission intensities is obtained at various quenching agent concentrations
by incrementally adding the same amount of quenching agent (e.g., 50 µL
in the above example to a final quenching concentration of 2.5 M).
5. The observed intensities must be corrected for dilution effects by multiplying the
observed intensity at each point by the dilution factor (V/Vo where V is the volume
at a given point, and Vo is the initial volume without any quenching agent
present). For example, if 50 µL quenching agent is added to a 3.0-mL sample, the
resultant fluorescence intensity must be multiplied by (3.05/3.0).
6. The Stern-Volmer quenching constants can be derived from a plot that follows
the following equation:
Fo/F = 1 + KSV[Q] where Fo is the fluorescence intensity without any quenching agent present, and
the values of F are the fluorescence intensities at given concentrations of the
quenching agent Q. The slope of the plot of Fo/F vs [Q] will be the Stern-Volmer
quenching constant, KSV (1).
7. Ensure that changes in fluorescence intensity upon dilution are not a result of
concentration changes by repeating Subheading 3.4., steps 4 and 5 with water
alone as a control.
4. Notes
4.1. Emission Spectra and Quenching of Tryptophan Fluorescence
1. The optimal excitation wavelength is dependent on the combination of
fluorophores present in the sample of interest. Tryptophan is generally excited at
295 nm for proteins in order to minimize tyrosine fluorescence, which is
maximally excited at 278 nm. This wavelength provides sufficient excitation to
observe significant signal at low micromolar protein concentrations on an inexpensive
spectrofluorimeter. Lower protein concentrations (up to nanomolar) are
feasible as the detection systems become more complex (and expensive). An
appropriate excitation wavelength can be determined by running a series of excitation
spectra on isolated fluorophores. These profiles will provide an indication
as to which wavelengths provide mutual excitation, and more importantly, where
individual fluorophores can be selectively excited. Often, this wavelength will
not be where a fluorophore exhibits it’s peak extinction coefficient, hence, there
may be a trade-off between selectivity and sensitivity.
2. The choice of quenching agent is dependent on the system under study and tolerable
dilution effects. We have used KI, CsCl, and acrylamide successfully for
quenching of calmodulin-target peptide complexes with equivalent results with
100 mM salt in the buffer. In each of these cases, the quenching agent has a
different charge (I – , Cs + , acrylamide is polar, but neutral), and different quenching
efficiencies. Acrylamide is a very efficient quenching agent, hence smaller
dilution effects will be observed if this is a concern, however caution must be
used as it is a neurotoxin. Also, corrections must be made for the absorption of