fluctuations (noise) in the feedback circuitry. Time constants of
3–10 s or slower, depending on ITC model, are typically the
maximum that can be obtained for “instant” heat signals (either
electrical signals or dilution experiments; see Note 15). Processes
that are slower than this instrumental response, which might
include conformational rearrangements, polymerization, or aggregation, will lead to broadening of the ITC peak shape. This can
even generate very unusual peak profiles with opposing endothermic and exothermic phases in the same injection once the slower
events become uncoupled from the faster mechanical and dilution
heats that are inherent to every injection. The binding itself can also
involve multiple steps, such as complex formation that then triggers
a conformational change, and these may also have disparate kinetic
time windows and result in unusual peak profiles.
More generally, there is the potential for changes in the rate of
heat release during every ITC titration because each injection
changes the concentrations of reactants, and the equilibrium
between bound and unbound will change following each injection
with kinetics that are determined by contributions from forward
and reverse rate constants (see Eq. 2). As the effective equilibrium
concentration of free protein decreases during sequential addition
of ligand because of binding, then the bimolecular rate component
will also decrease leading to a reduction in the overall rate of
relaxation of the equilibrium. Where this process leads to slowing
beyond the time constant of the ITC there will be a broadening of
the ITC peak shape reflecting this kinetic limitation. This phenomenon leads to the possibility of extracting kinetic on and off rate
constants from ITC data during analysis in addition to the standard
thermodynamic binding parameters [6, 14, 15]. It is certainly not
uncommon to observe this kinetic broadening of ITC peak shapes
as the heat effect of adding ligand decreases when the experiment
approaches saturation, but the ability of this analysis to yield reliable
kinetics will depend on the experimental design, the magnitude of
the individual rate constants, and the configuration of the ITC
instrument.
3.10 Not Just
Protein–Ligand
Interactions
The nature of ITC as a label-free nonoptical method gives it distinct
advantages over many other techniques under challenging conditions (such as high absorbance solutions or crude cell extracts) or
for more complex binding interactions. Competition (displacement) binding experiments can be easily performed to confirm
whether different binding partners share the same site. Where
ligands compete for an identical site, competitive titrations can
increase the upper and lower range of affinities that ITC can measure. An economical way to perform this type of experiment for a
high-affinity interaction (low nM or tighter) is to measure a weaker
binding ligand in an initial titration and leave this end point in the
ITC cell. The syringe is then reloaded with the tighter binding
Isothermal Titration Calorimetry
151
3–10 s or slower, depending on ITC model, are typically the
maximum that can be obtained for “instant” heat signals (either
electrical signals or dilution experiments; see Note 15). Processes
that are slower than this instrumental response, which might
include conformational rearrangements, polymerization, or aggregation, will lead to broadening of the ITC peak shape. This can
even generate very unusual peak profiles with opposing endothermic and exothermic phases in the same injection once the slower
events become uncoupled from the faster mechanical and dilution
heats that are inherent to every injection. The binding itself can also
involve multiple steps, such as complex formation that then triggers
a conformational change, and these may also have disparate kinetic
time windows and result in unusual peak profiles.
More generally, there is the potential for changes in the rate of
heat release during every ITC titration because each injection
changes the concentrations of reactants, and the equilibrium
between bound and unbound will change following each injection
with kinetics that are determined by contributions from forward
and reverse rate constants (see Eq. 2). As the effective equilibrium
concentration of free protein decreases during sequential addition
of ligand because of binding, then the bimolecular rate component
will also decrease leading to a reduction in the overall rate of
relaxation of the equilibrium. Where this process leads to slowing
beyond the time constant of the ITC there will be a broadening of
the ITC peak shape reflecting this kinetic limitation. This phenomenon leads to the possibility of extracting kinetic on and off rate
constants from ITC data during analysis in addition to the standard
thermodynamic binding parameters [6, 14, 15]. It is certainly not
uncommon to observe this kinetic broadening of ITC peak shapes
as the heat effect of adding ligand decreases when the experiment
approaches saturation, but the ability of this analysis to yield reliable
kinetics will depend on the experimental design, the magnitude of
the individual rate constants, and the configuration of the ITC
instrument.
3.10 Not Just
Protein–Ligand
Interactions
The nature of ITC as a label-free nonoptical method gives it distinct
advantages over many other techniques under challenging conditions (such as high absorbance solutions or crude cell extracts) or
for more complex binding interactions. Competition (displacement) binding experiments can be easily performed to confirm
whether different binding partners share the same site. Where
ligands compete for an identical site, competitive titrations can
increase the upper and lower range of affinities that ITC can measure. An economical way to perform this type of experiment for a
high-affinity interaction (low nM or tighter) is to measure a weaker
binding ligand in an initial titration and leave this end point in the
ITC cell. The syringe is then reloaded with the tighter binding
Isothermal Titration Calorimetry
151