Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

100 Johnson and Tikunova
cence intensity and bound Ca 2+ . These calibration studies should be done directly
after the Ca 2+ off-rate experiments, using the same instrument conditions, to
reduces changes in intensity or amplitude produced by time dependent changes
in lamp output. By comparing the amplitude of the increase in Quin fluorescence
in the Ca 2+ off-rate experiments to the increase in Quin fluorescence when it is
reacted with a specific [Ca], we calculate that two moles of Ca 2+ were dissociated
per mole of F19W in Fig. 2. Thus, Quin allows a rapid determination of not only
the Ca 2+ off-rate, but also the number of sites from which Ca 2+ dissociated.
9. Quin will report the Ca 2+ dissociation rate from any contaminating chelator that
is with the protein. Thus, it is important to thoroughly dialyze your purified protein
to remove chelator. If the protein is contaminated with EGTA or EDTA you
will see Ca 2+ dissociation at 0.55/s and 0.7/s, respectively, at 10°C, because of
Ca 2+ dissociation from chelators (15).
4.3. Measurement of Ca 2+ On-Rates
10. While chelex can generally be used to remove Ca 2+ from the protein, for highaffinity
Ca 2+ -binding proteins some residual Ca 2+ may remain. In these cases,
small amounts of EGTA can be used to remove the remaining Ca 2+ . It should be
noted that EGTA can affect the measured on rate for proteins that have slower on
rates. This is because EGTA can bind some of the Ca 2+ which is being reacted
with the protein at a rate of 1.3 × 10 6 M/s (see ref. 14) and at higher [EGTA] this
effect is substantial .
11. A quick approximation of the Ca 2+ on-rate can be obtained before shooting all of
the [Ca 2+ ] shown in Fig. 3 by simply mixing a 10- to 20-fold molar excess of Ca 2+
with the protein and determining the rate of increase in protein fluorescence.
When Ca 2+ is in sufficient excess of protein, the reaction approaches pseudofirstorder
and the on-rate can be approximated by the ratio of the observed on rate
(1138/s) to the concentration of Ca 2+ reacted with the protein (10 µM), yielding
an on-rate of 1.1 × 10 8 M/s. Further verification of the Ca 2+ on-rate can be
achieved by confirming that the off-rate (approx 320/s) determined as the intercept
on the y-axis in the linear plot of Fig. 3 inset, is similar to the actual measured
off-rate (310/s in Fig. 2).
Because the K d is the ratio of the off rate to the on rate, the on rate can also be
calculated from K on = K off/K d. For F19W the K off is 310/s and the K d (from Fig. 1) is
4 × 10 –6 M, indicating a K on of 8 × 10 7 M/s, identical to the actual measured Ca 2+
on-rate. Thus, by using Quin and TRP fluorescence we can completely characterize
the Ca 2+ affinity and the Ca 2+ exchange rates of a Ca 2+ -binding protein.
4.4. Other Methods for Determining Ca2+ Binding and Ca2+ Exchange Rates with Proteins
12. Ca2+ -binding to proteins can also be completely analyzed in terms of affinity,
number of sites, and cooperativity of sites by using 45Ca2+ and equilibrium or
flow dialysis (as discussed by M. Yazawa in Chapter 1) and by the use of Ca2+