Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Fluorescence Methods for Ca 2+ Exchange 91
Fig. 1. The Ca 2+ -induced increase in F19W tryptophan fluorescence. Ca 2+ titrations
were conducted as described in the methods section. 100% fluorescence corresponds
to a threefold fluorescence increase. Each data point represents the average of three
titrations ±S.E. The inset shows fluorescence emission spectra of F19W before (–Ca
trace) and after (+Ca trace) the addition of Ca 2+ (pCa 4.0) and the spectra of the buffer
without protein (buf trace).
with a Hill coefficient of 2.0. The inset to Fig. 1 shows the fluorescence emission
spectra of F19W in the absence of Ca 2+ (–Ca trace) and in the presence of
pCa 4.0 (+Ca trace).
To conduct these titrations:
1. Be sure the buffer (200 mM MOPS, 90 mM KCl, 2 mM EGTA at pH. 7.0) and
Ca 2+ stocks have equilibrated to the desired temperature. Add 1 mL of the buffer
to a clean cuvet and then add 1 µM of the purified Ca 2+ -binding protein. Place
parafilm over the top of the cuvet and mix by inverting three times, being certain
that no volume is lost during mixing. Visually inspect the contents of the cuvet
and be certain that it has not been contaminated with lint and shows no visible
turbidity.
2. Place the cuvet in the fluorimeter and run an emission spectra by setting the excitation
to the desired wavelength and scanning the emission wavelength from the
excitation wavelength over the wavelength of fluorescence emission. For
example, for tryptophan-containing proteins, you can excite at 285 nm and
because TRP emissions are generally maximal at 320–360 nm, it is necessary to
scan the emission wavelength from 285 to 460 nm, as shown in Fig. 1.
3. Fix the emission wavelength to the wavelength where maximum emission was
obtained on the emission spectra (335 nm in Fig. 1) and record the intensity as a