Physical Biochemistry Isothermal Titration Calorimetry (ITC)

Structural detail is not sufficient to describe the energetic components that drive molecular
interactions. In order to understand function and to modulate cellular activities it is useful to fully
understand the thermodynamic and kinetic factors involved in these interactions.

Isothermal Titration Calorimetry

We need to measure quantitatively the affinities of interactions

ITC is a true ‘in-solution’ technique – no immobilisation is required

Does not require labelling or chromophores
Directly measures sub-millimolar to nanomolar binding constants (10 2 to 109 M-1)

ITC is the only technique that can directly yield parameters such as…
 Binding constant (Kb)
 Reaction stoichiometry (n) or the number of sites for a ligand in a receptor
 Enthalpy (ΔH) and entropy (ΔS) of binding

Together, these parameters provide a complete thermodynamic profile of a molecular interaction

in a single experiment…
ΔG = – RT ln Kb – TΔS = ΔG – ΔH

In combination with structural data derived from NMR etc. ITC is essential to…
 Identify binding sites / interaction surfaces
 Analyse the effect of mutations / ligand variability
 Interpret the effects of hydration and conformational changes
All of which are important in drug development and design

ITC measures the change in heat associated with a chemical reaction that is triggered by the
mixing of two reagents.
The chemical reaction carried out by each injection of reagent either releases of absorbs a certain
amount of heat (qi), which is proportional to…
 The amount of ligand that binds to the substrate in a particular injection
(v × ΔLi)
 The characteristic enthalpy change (ΔH) for the reaction

Qi = v × ΔH × ΔLi

1 cal = the amount of heat required to raise the temperature of 1g of water by

1C at 1atm

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Physical Biochemistry Isothermal Titration Calorimetry (ITC)

Enthalpy (ΔH):
Net heat associated with making and breaking non-covalent bonds in/between molecules and/or
solvent. Also heat associated with intermolecular associations – conformational changes and
solvent rearrangement.

Despite the large number of bonds contributing to ΔH, key interactions may be identified with high
resolution structural data (e.g. analyse free and bound forms of interacting molecules).
Calorimetry measures enthalpic changes only. Processes that a driven by a purely entropic
component are not recorded (or athermal reactions).

Entropy (ΔS):
A measure of molecular mobility, or the tendency of a system to disorder
Correlates with the burial of hydrophobic surface area
e.g. Release of water in an unfolded  folded transition

Gibbs Free Energy (ΔG):

A measure of the tendency of a chemical change to occur spontaneously

Based on a number of different contributions, such as intermolecular contacts, conformational

changes, hydrophobic transfer, changes in rotational / translational motions.

ΔG = ΔH – TΔS

Allows comparison of affinities at a defined set of conditions

e.g. Ligand binding of WT vs. mutants, measure ΔΔG’s

ITC Preparation:
10μM is the typical sample concentration in a reaction cell (1.4ml)
Ligand (titrant) concentrations should be at least 10× higher that the sample concentration
All interacting components must be degassed and dialysed extensively in the same buffer to avoid
large heats of dilution / mixing which will interfere with the reaction of interest.

Accurate / reproducible ITC data require careful determination of macromolecule and ligand
concentrations, which can be determined using UV/VIS absorbance from known
 values.
 = molar extinction coefficient

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Physical Biochemistry Isothermal Titration Calorimetry (ITC)

Optimisation of the experiment may require several ITC scans, but material is easily recoverable
as the technique is non-destructive
Change in Heat Capacity (ΔCp):
Dependence of ΔH on temperature at constant pressure

Choice of Buffer Solution:

An appropriate buffer system needs to be selected
There will be different effects depending on the ΔH of protonation of the buffer
Degradation of certain compounds in buffers will produce a drift in the raw data baseline
e.g. Oxidation of DTT; use TCEP of -ME instead

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Physical Biochemistry Isothermal Titration Calorimetry (ITC)

Parameters to determine by ITC:

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Physical Biochemistry Isothermal Titration Calorimetry (ITC)

Determination of binding constants:

[P] × Kb = c
c must be lower than 1000 for the reaction to be measured directly by ITC

[P] = Protein concentration

Kb = Binding constant
c = A parameter known as sigmoidicity

In practical terms, this restriction sets an upper limit of ~10 9 M-1 for Kb

Data Analysis:
For a reaction involving a single binding event

K = binding constant
n = # of binding sites
Vo = active cell volume
Mt and [M] = bulk and free concentration of macromolecule in Vo
Xt and [X] = bulk and free concentration of ligand
Θ = fraction of sites occupied by ligand X

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Physical Biochemistry Isothermal Titration Calorimetry (ITC)

By combining these equations…

The heat derived from an injection (Q) is related to the change of enthalpy by the following
equation…

MORE EQUATIONS?

Further modifications are made to the final equation in order to correct for changes in volume after
each injection. The process of fitting experimental data then involves…
1. Initial guesses (which can often be made accurately using Origin software) of n,
Kb and ΔH
2. Calculation of ΔQi (corrected) for each injection and comparison of these values with the
measured heat for the corresponding experimental injection
3. Improvement in the initial values of n, Kb and ΔH by standard Marquardt methods
4. Iteration of the above procedure until no further significant improvement occurs
5. Once Kb is known, work out…

ΔG = – RT ln Kb – TΔS = ΔG – ΔH

Thermodynamic Discrimination:
ITC can help us to discriminate the type of energetic contributions that drive different binding
events

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