ligand that displaces the first ligand in a second titration (see Note
16). There are effects on both the observed K d of the second
experiment (bringing it experimentally into a c-value window suitable for measurement) and on the ΔH since the ITC measures the
net heat of both processes, displacement and binding, which frequently have opposing signs. These multiple signatures of displacement binding are useful in applying this methodology to ligand
screening. In this case, titrating an established ligand against a
protein that has been premixed with one or more potential competing compounds gives changes in both ΔH and K d where there is
competition.
ITC can also be used to quantify processes of reversible equilibrium self-association, such as dimerization, using simple buffer
dilution experiments. For systems with a moderate (μM) K d for
forming a dimer (or higher order species), an injection into buffer
and the resulting change in concentration produces some levels of
dissociation. This produces a series of injection peaks (typically
endothermic) that gradually decrease in magnitude since they result
in progressively smaller concentration changes from the stock solution in the syringe as the concentration of protein in the cell
increases. All the protein can be recovered after this titration into
buffer and the data can be fit to give a K d and ΔH for the process.
These two “atypical” applications of ITC rely to some extent
on the technique’s ability to measure heat nonspecifically as the
sum of all events occurring during injection and mixing. This
nonspecificity makes interpretation of ΔH of a single isolated experiment very difficult. But it also enables ITC to be deployed in a wide
range of applications in protein chemistry and in the broader
biological context. Noteworthy among these in the context of
this chapter is ITC’s potential use in studying enzyme kinetics,
where ligand binding is followed by catalytic turnover, as has been
reviewed elsewhere [16, 17].
4 Notes
1. These details refer to the Malvern Panalytical iTC200 instrument. Other models of ITC with different cell geometries,
volumes, and syringe types are available as are instruments
from other manufacturers such as TA Instruments.
2. There can be some ambiguity in the use of the term “ligand”
(meaning “to bind”) across various techniques. Classically, a
receptor ligand would often be much smaller in size than the
protein itself. In ITC, it is common to refer to the component
being titrated from the syringe as the ligand (even if it is a
protein, as in a protein–protein interaction, or physically larger
than the other component). This cell/syringe terminology will
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Christopher M. Johnson
16). There are effects on both the observed K d of the second
experiment (bringing it experimentally into a c-value window suitable for measurement) and on the ΔH since the ITC measures the
net heat of both processes, displacement and binding, which frequently have opposing signs. These multiple signatures of displacement binding are useful in applying this methodology to ligand
screening. In this case, titrating an established ligand against a
protein that has been premixed with one or more potential competing compounds gives changes in both ΔH and K d where there is
competition.
ITC can also be used to quantify processes of reversible equilibrium self-association, such as dimerization, using simple buffer
dilution experiments. For systems with a moderate (μM) K d for
forming a dimer (or higher order species), an injection into buffer
and the resulting change in concentration produces some levels of
dissociation. This produces a series of injection peaks (typically
endothermic) that gradually decrease in magnitude since they result
in progressively smaller concentration changes from the stock solution in the syringe as the concentration of protein in the cell
increases. All the protein can be recovered after this titration into
buffer and the data can be fit to give a K d and ΔH for the process.
These two “atypical” applications of ITC rely to some extent
on the technique’s ability to measure heat nonspecifically as the
sum of all events occurring during injection and mixing. This
nonspecificity makes interpretation of ΔH of a single isolated experiment very difficult. But it also enables ITC to be deployed in a wide
range of applications in protein chemistry and in the broader
biological context. Noteworthy among these in the context of
this chapter is ITC’s potential use in studying enzyme kinetics,
where ligand binding is followed by catalytic turnover, as has been
reviewed elsewhere [16, 17].
4 Notes
1. These details refer to the Malvern Panalytical iTC200 instrument. Other models of ITC with different cell geometries,
volumes, and syringe types are available as are instruments
from other manufacturers such as TA Instruments.
2. There can be some ambiguity in the use of the term “ligand”
(meaning “to bind”) across various techniques. Classically, a
receptor ligand would often be much smaller in size than the
protein itself. In ITC, it is common to refer to the component
being titrated from the syringe as the ligand (even if it is a
protein, as in a protein–protein interaction, or physically larger
than the other component). This cell/syringe terminology will
152
Christopher M. Johnson