Measurement of Binding Constants and Heats of

Binding using Isothermal Titration Calorimeter

Wei Li
Department of Chemistry
University of Victoria

Winter, 2013
Acknowledgement

Professor Hof
Classmates
Outline

Thermodynamic parameters
Isothermal titration calorimeter (ITC)
ITC development history
Advantages of using ITC
Basic configuration of an ITC
Sample preparation
Raw data and plot of processed data
Systematic errors
References
Thermodynamic Parameters

When characterizing interactions between a biological macromolecule M and a

small ligand X, or between two macromolecules,

M +X = MX
The single-site binding constant K, H,
MX + X = MX2 and the number of sites n are the
. independent variables of thermodynamic
interest.

MXn-1 + X = MXn, G and S of binding are dependent

variables obtained by the calculation.
K = [MXn]/[MXn-1] X]

G = G + RTlnK (Standard conditions: 1 mole of P and 1 mole of L at PH7 and 25 C)

At equilibrium, under standard conditions, G = 0.
G = -RTlnK = H - TS
Thermodynamic Parameters

Entropy n = stoichiometry
Hydrophobic interactions Number of protein binding sites reflects
Water release the purity and the functional integrity of a
Ion release protein preparation if the measure and
fitted stoichiometry can be compared to
Conformational changes the known, previously determined
stoichiometry
Enthalpy
Hydrogen bonding
Protonation events
ITC Development History

1960s built in the second half to study chemical reactions

1970s sensitivity: mJ
metal + ligand complex
1980s sensitivity: J
ligand binding processes and micelle formation

1990s number of published papers increased due to new commercial

calorimeters became available. Evolved from a specialist method to a
widely used technique

2000s widely employed in the design and discovery of new drugs and
for the study of liquid mixtures

2009 374 out of 432 papers on protein interaction with other proteins,
small molecules, metal ions, lipids, nucleic acids, and carbohydrates as
well as on nucleic acid interactions with small molecules.
Advantages of using ITC

No labeling required
In-solution
No molecular weight limitations
Optical clarity unimportant
Minimal assay development
Using heat as signal
In one single experiment, K, H, and Stoichiometry n of
interaction between two or more molecules in solution can be
determined
G and S can be calculated
Basic Configuration of an ITC

VP-ITC micro calorimeter

Reference cell and sample cell in an adiabatic jacket.

Reference cell filled with buffer or water. Sample cell filled with protein.
Both cells are heated in such a way that the temperature is almost
constant, i.e. T < 10 x (E-6) C at all times.
Syringe device holds the ligand. This device titrates the ligand into the
sample cell and also acts as a stirrer.

Isothermal titration calorimetry (ITC) is a biophysical technique that allows a

thermodynamic characterization of an interactive system.
Basic Configuration of an ITC

Reference and sample cells containing

identical buffer are located within an
adiabatic jacket, and the latter contains
macromolecule of interest.
A small, constant power is applied to the
reference cell, which activated the cell
feedback circuit to drive T to 0.
No reaction the feedback power
remains constant at the resting baseline
value.
Exothermal reactions temporarily and
endothermic reactions temporarily
feedback power. The reaction heats are
readily obtained by computer integration
of these deflections from the resting
baseline.
Sample Preparation

General consideration
Experiment performed at reasonable PH
Protein has to be extensively dialysed and the ligand has to be dissolved
in the buffer recovered from the last protein dialysis step

Protein preparation
As pure as possible
Concentration should be correctly estimated.

Ligand preparation
As pure as possible
Accurate concentration

Buffers
Buffers with low ionization enthalpy should be considered.
Raw Data and Plot of Processed Data

Top: Raw ITC data showing an

exothermic binding reaction. Each peak
corresponds to an injection of ligand into
a protein solution in the sample cell; the
area under the peak is proportional to
the amount of heat released in the
binding reaction.
When the protein becomes saturated,
the DP signal diminishes until only the
background heat of dilution is observed.

Bottom: Binding isotherm obtained by

integrating the area of each peak. The
heat released per mole of ligant is
plotted against the molar ratio of the two
reactants.
Systematic Errors

Blank experiment
Heat of dilution of ligand
Heat of dilution of macromolecule
Buffer to buffer experiment
Heat of proton ionization

Degassing
Generation of bubbles during an ITC experiment will generate spurious heat signals.

Measurements of the concentrations

Accuracy of the measurements are imperative for a good ITC experiment.
Sample concentrations also need to be within a proper range to get reliable data.
Small molecules impurities and PH mismatches in the buffer will cause artifacts.
References

Nunez, S.; Venhorst, J.; Kruse, C. G. Targetdrug interactions: First

principles and their application to drug discovery. Drug Discov. Today
2012, 17, 1022.
Wiseman T. et al, Rapid measurement of binding constants and heats of
binding using a new titration calorimeter. Anal Biochem 179: 131-137,
1989.
Mark A. Williams and Tina Daviter (eds), Protein-Ligand Interactions:
methods and applications. Springer Science and Business Media, New
York, 2013.
Joel Tellinghuisen, John D. Chodera, Systematic errors in isothermal
titration calorimetry: concentrations and baselines, Analytical
Biochemistry 414 (2011) 297-299.
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