Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

126 Lopez and Makhatadze
pendence of Q on [L] t/[P] t (5). This approach is used by the VP-ITC ORIGIN
software and presents certain advantages when complicated binding models need
to be used.
2. Identical and noninteracting binding sites. In this case more than one molecule of
ligand binds to one molecule of protein (n >1). The Eq. 3 now becomes:
Q = i∑Qi = n · ∆H · V · Ka · [L] b
(5)
and can be calculated as described by Eq. 3.
3. Nonidentical and noninteracting binding sites. This is the most general case in
which there are “i” different sets of binding sites and each set has its own number
of binding sites ni, enthalpy ∆Hi, and equilibrium constant Ki a. The heat involved
in the binding process can be written as:
i i Q = ∑Qi = V · ∑ni · ∆Hi · V · Ka · [L]b (6)
i
i
and nonlinear regression allows estimated of the binding parameters to the each
type of the binding site.
4. Notes
1. To avoid long equilibration times, keep the washing buffer and protein solution at
a lower temperature than the temperature at which you want to perform the titration.
2. Keep always the buffer from the last change of the dialysis under the same conditions
as your protein and ligand. Even if the protein and ligand are stable at 4°C,
try to use them as soon as possible to ensure that the properties of the samples
and buffer remain the same. If samples are not used within several days, consider
re-dialysis with one change of buffer.
3. Special care has to be taken with the needle from the stirring syringe. Any small
bending of the needle will disturb the baseline of the instrument and will make
the syringe unusable.
References
1. Makhatadze, G. I. (1998) Heat capacities of amino acids, peptides and proteins.
Biophys. Chem. 71, 133–156.
2. Gill, S. C. and von Hippel, P. H. (1989) Calculation of protein extinction coefficients
from amino acid sequence data. Anal. Biochem. 182, 319–326.
3. Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. (1995) How to measure
and predict the molar absorption coefficient of a protein. Protein Sci. 4, 2411–2423.
4. Winder, A. F. and Gent, W. L. (1971) Correction of light-scattering errors in spectrophotometric
protein determinations. Biopolymers 10, 1243–51.
5. Wiseman, T., Williston, S., Brandts, J. F., and Lin, L. N. (1989) Rapid measurement
of binding constants and heats of binding using a new titration calorimeter. Anal.
Biochem. 179, 131–137.