Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Fluorescence Methods for Ca 2+ Exchange 95
3.4. Measurements of Ca 2+ On-Rates
Ca 2+ on-rates can be measured in proteins like F19W by observing the rate of
the Ca 2+ -induced increase in TRP fluorescence as a function of increasing [Ca 2+ ].
1. In order to measure Ca 2+ on rates, the protein should be Ca 2+ free. If it is not, this
is achieved by incubating approx 30 µM of protein with chelex resin (generally
1 mL of resin for each 4 mL of protein) for approx 4 h in a plastic test tube. The
solution should be shaken, not stirred during the incubation. Stir bars can fragment
the resin. After incubation, the resin is allowed to settle to the bottom or is
pelleted by low-speed centrifugation and the supernatant decanted. The chelexed
protein can then be diluted into chelexed buffer and tested for Ca 2+ occupancy.
This is done by diluting 2 µM of the protein into 1 mL of a chelexed buffer
(10 mM MOPS, 90 mM KCl, pH 7.0) and running a fluorescence spectra, as
shown in Fig. 1. After taking the initial spectra, add 100 µM Ca 2+ to the protein
and run the spectra of the protein in the Ca 2+ saturated state. Finally, add 2 mM
EGTA to this 1-mL solution to produce the Ca 2+ free state and run a spectra. By
comparing the fluorescence intensity in the original state, the + Ca 2+ state and the
+EGTA state, the percent saturation of the chelexed protein can be easily determined.
If the protein is found to be Ca 2+ free, then it can be used for determining
Ca 2+ on rates (see Note 10).
2. Fill syringe A with 2 µM protein (F19W) in 10 mM MOPS, 90 mM KCl at pH 7.0.
Fill syringe B with the same chelexed (if required) buffer.
3. After three shots to clear the mixing chamber of wash solution, average 5–7 shots.
Because we are introducing little or no Ca 2+ to protein in these control shots,
there should be little time dependent increase in F19W fluorescence.
4. Keeping syringe A the same, now add increasing amounts of Ca 2+ (4, 6, 8, 10, 15,
and 20 µM) to syringe B and average 5–7 shots at each [Ca 2+ ]. The [Ca 2+ ] points
to be used can be determined from the Ca 2+ titration of the protein (see Fig. 1).
The rate of the increase in F19W TRP fluorescence should increase as a function
of increasing [Ca 2+ ] as shown in Fig. 3 inset. F19W TRP fluorescence increases
at a rate of 495/s for 2 µM Ca 2+ (after 1:1 dilution of 4 µM Ca 2+ ), at 658/s for 4 µM
Ca 2+ and at 1138/s for 10 µM Ca 2+ . Figure 3 shows a plot of the rate (K obs) of the
Ca 2+ -induced increase in F19W fluorescence as a function of increasing [Ca].
This linear plot exhibits a slope which is equal to the Ca 2+ association rate
8 × 10 7 M/s and an intercept on the y-axis (approx 320/s) should equal to the
Ca 2+ off-rate. For further verification of this on rate see Note 11.
4. Notes
4.1. Fluorescence Methods for Determining Ca2+ Affinity
Two major advantages of using fluorescence changes to follow Ca2+ -binding
are: The ease and reproducibility of the Ca2+ titrations; (each titration takes
approx 30 min and error bars of