Biology Reference
In-Depth Information
18.1.3
Binding models
Generally, the list ofmodels includes one-set-of-sites, two-sets-of-sites, and sequential-
binding-sitesmodels. Tochoose the appropriatebindingmodel, reasonable assumptions
should be made, based on preliminary knowledge about the investigated system and/or
the shape of the binding isotherm. The first model, one-set-of-sites, is the most simple
one and can be used for the systems where the ligand has one or more equal independent
(noninteracting) sites on the target molecule. The binding isotherm for such a
system represents a monotonic curve which has no or one inflection point
(
Fig. 18.2
).When concentrationof investigatedmolecule [
M
] in the calorimetric cell
is close to optimal value (10/
NK
a
<
100/
NK
a
), the binding isotherm has a sig-
moid shapewith an inflection point close to themolar ratio corresponding to the stoi-
chiometry of the interaction (
N
)(
Ladbury & Doyle, 2004
). This value should be
[
M
]
<
FIGURE 18.2
Direct and reverse titration by ITC. Raw ITC curves (top panels) and binding isotherms with
fitting curves (bottom panels) of stathmin titration by tubulin (left panels) and tubulin titration
by stathmin (right panels). Both thermograms are monophasic curves with an inflection
point at 2 for direct titration and 0.5 for reverse titration. Thermodynamic parameters of both
titrations are in good agreement. Fitting binding isotherms with model of one-set-of-sites
yielded the same constants for both titrations and the enthalpy of binding around 14 kcal/
(mole of tubulin) for direct binding and 27 kcal/(mole of stathmin) for reverse titration. The last
value corresponds to overall enthalpy of formation of the T2S complex (
DH
T2S
).
