Technology

What is ITC?

Isothermal Titration Calorimetry (ITC) is the gold standard for measuring biomolecular interactions.  ITC simultaneously determines all binding parameters (n, K, ∆H and ΔS) in a single experiment – information that cannot be obtained from any other method.

When substances bind, heat is either generated or absorbed.  ITC is a thermodynamic technique that directly measures the heat released or absorbed during a biomolecular binding event.  Measurement of this heat allows accurate determination of binding constants (KB), reaction stoichiometry (n), enthalpy (∆H) and entropy (ΔS), thereby providing a complete thermodynamic profile of the molecular interaction in a single experiment.  Because ITC goes beyond binding affinities and can elucidate the mechanism of the molecular interaction, it has become the method of choice for characterizing biomolecular interactions.

Applications include:

  • Characterization of molecular interactions of small molecules, proteins, antibodies, nucleic acids, lipids and other biomolecules.
  • Lead optimization.
  • Enzyme kinetics.
  • Assessment of the effect of molecular structure changes on binding mechanisms.
  • Assessment of biological activity.

Interactions between any two molecules can be studied with ITC, including:

  • Protein-small molecule
  • Protein-protein
  • Target-drug
  • Enzyme-inhibitor
  • Antibody-antigen
  • Protein-DNA
  • Protein-lipid
  • Small molecule-small molecule

Why ITC?

  • Beyond binding affinities:  True affinity data via heat measurement offers a unique insight into the biology and recognition processes, unobtainable with more limited binding assays and techniques such as surface plasmon resonance (SPR).
  • Directly measure sub-millimolar to nanomolar binding constants (102 to 109 M-1).  Measure nanomolar to picomolar binding constants (109 to 1012 M-1) using the competitive binding technique.
  • Application versatility:  Investigate any biomolecular interaction.
  • True in-solution technique:  No labeling or immobilization required.  No molecular weight limitations or buffer restrictions.  Easily handles colored or turbid solutions and particulate suspensions.
  • Easy to use: Unattended operation after sample loading.  All functions are operated through software to minimize operator involvement and facilitate fast and accurate analyses without the need for expertise in thermodynamics.

Isothermal Titration Calorimetry (ITC) is a powerful analytical tool which measures the binding affinity and thermodynamics between any two biomolecules. ITC is considered the “gold standard” assay for binding.  In a single experiment, ITC can determine:

  • Binding affinity -  Kd in range of millimolar to nanomolar
  • Number of binding sites
  • Can detect multiple and different binding sites
  • Enthalpy (ΔH) and entropy (ΔS) of binding

Thermodynamic data, specifically enthalpy (ΔH) and entropy (ΔS), reveal the forces that drive complex formation and mechanism of action. Thermodynamics provide information on conformational changes, hydrogen bonding, hydrophobic interactions, and charge-charge interactions.  This information is used to describe the function and mechanism at a molecular level. 

The ITC systems from MicroCal are designed and optimized for the biophysical characterization of biomolecules in solution and are widely used at major pharmaceutical, biotech, academic and government institutions worldwide.  There are thousands of literature citations which use ITC to characterize binding interactions.  

ITC Binding Experiment 

In a typical ITC experiment, a solution of a one biomolecule (“ligand” such as.a drug, protein, DNA molecule, etc) is titrated into a solution of its binding partner. The heat released upon their interaction (ΔH) is monitored over time (Figure 1, left panel). Each peak represents a heat change associated with the injection of a small volume of sample into the ITC reaction cell. As successive amounts of the ligand are titrated into the ITC cell, the quantity of heat absorbed or released is in direct proportion to the amount of binding. As the system reaches saturation, the heat signal diminishes until only heats of dilution are observed. A binding curve is then obtained from a plot of the heats from each injection against the ratio of ligand and binding partner in the cell (Figure 1, right panel). The binding curve is analyzed with the appropriate binding model to determine KB, n and ΔH. Note that KB = 1/Kd.

Figure 1: Typical ITC data. Top: Raw ITC data. Bottom: Binding isotherm.
Figure 1: Typical ITC data. Top: Raw ITC data. Bottom: Binding isotherm.

View animation of a typical ITC experiment

Reference Lists

ITC – Reviews Reference List

ITC – Instrumentation and Data Analysis Reference List

How does ITC work?

MicroCal’s ultrasensitive ITC systems use a cell feedback network (CFB) to differentially measure and compensate for heat produced or absorbed between the sample and reference cell. Twin coin-shaped cells are mounted in a cylindrical adiabatic environment, and connect to the outside through narrow access tubes (Figure 1). A thermoelectric device measures the temperature difference between the two cells and a second device measures the temperature difference between the cells and the jacket. As chemical reactions occur in the sample cell, heat is generated or absorbed. The temperature difference between the sample and reference cells (ΔT1) is kept at a constant value (i.e. baseline) by the addition or removal of heat to the sample cell, as appropriate, using the CFB system. The integral of the power required to maintain ΔT1 = constant over time is a measure of total heat resulting from the process being studied. Figure 2 is a schematic drawing of the ITC cells and syringe.

Figure 2: Diagram of ITC cells and syringe. The syringe rotates in place during the ITC experiment. The end of the syringe has been adapted to provide continuous mixing in the ITC cell. The plunger is computer-controlled and injects precise volumes of ligand.

In an ITC experiment, a syringe containing a “ligand” solution is titrated into a cell containing a solution of the “macromolecule” at constant temperature. When the ligand is injected into the cell, the two materials interact, and heat is released or absorbed in direct proportion to the amount of binding. As the macromolecule in the cell becomes saturated with ligand, the heat signal diminishes until only the background heat of dilution is observed (Figure 3).

Figure 3: Representative ITC data. Eighteen injections of ligand solution are added to protein solution in the ITC cell. The area underneath each injection peak (top panel) is equal to the total heat released for that injection. When this integrated heat is plotted against the molar ratio of ligand added to macromolecule in the cell, a complete binding isotherm for the interaction is obtained (bottom panel). The one site model was used to fit the data. The solid red line is the calculated curve using the best-fit parameters. The best values for stoichiometry, binding constant, and enthalpy are shown in the box.

A major advantage of the MicroCal ITC instruments is the availability of three user-selectable modes of operation: high gain, low gain, and passive (US Patent Number 5,967,659). The high gain mode is suggested for most ITC experiments, allowing the fastest re-equilibration between injections, thereby providing the shortest experimental times. The passive mode has the lowest noise, and is useful when examining very small signal changes in systems having slow transients.

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