Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

122 Lopez and Makhatadze
Fig. 1. Isothermal titration calorimetric study of the interactions of Ca 2+ -binding
protein S100P with the 29 amino acid residue peptide melittin (Guzman-Casado, M.
and Makhatadze, G. I., personal communication). (A) Experimental profile of 29
injections of 10 µL each of melittin (1.9 mM) into the cell containing 22 µM S100P
obtained at 32°C using VP-ITC (upper profile). The heat of dilution of melittin was
measured by injecting 10 µL of melittin (1.9 mM) into the cell containing just the
50 mM Tris-HCl, 2 mM EDTA, pH 7.5 buffer (lower profile). Note that the peaks
corresponding to the melittin injection into the buffer are similar to the peaks observed
at the last several injections of melittin into S100P. (B) The integral heat of interactions
(normalized per mole of melittin) between S100P and melittin obtained from the
experiment shown on (A), i.e., injection of S100P into melittin are shown as solid
triangles (�). In the parallel experiment, mellitin was injected into the solution of
S100P (actual data not shown) and the integral heat effect (normalized per mole of
S100P) is shown as solid squares (�). If the stoichiometry of interactions would have
been 1:1 both experiments would overlap. Otherwise the ratio of the heat effects at the
saturations will give the stochiometry of the interactions which in the shown example
is two molecules of melittin per molecule of S100P, with the equilibrium constant K d ≈
80 µM.
protein concentration in the in the cell, the weaker the signal. This means that
there is an upper limit in the values of the affinity constants that can be measured.
For example, the sensitivity of VP-ITC (Microcal Inc.) is 0.1 µcal and
accurate measurements require 3–5 µcal per injection into 1.3-mL cell. Thus,
binding constants in the order of 10 7 M –1 can be easily obtained for reactions
with ∆H = –10 Kcal/mol (or even higher if ∆H is larger). For the binding reactions
with very high affinity (≥10 9 M –1 ) the equilibrium constant cannot be
measured and only the enthalpy of binding, ∆H, can be estimated (see Fig. 1).