Isothermal titration calorimetry
Acronym | ITC |
---|---|
Classification | Thermal analysis |
Manufacturers | TA Instruments, Microcal/Malvern Instruments |
Other techniques | |
Related | Isothermal microcalorimetry Differential scanning calorimetry |
In
The technique was developed by H. D. Johnston in 1968 as a part of his Ph.D. dissertation at Brigham Young University,[5] and was considered niche until introduced commercially by MicroCal Inc. in 1988. Compared to other calorimeters, ITC has an advantage in not requiring any correctors since there was no heat exchange between the system and the environment.
Thermodynamic measurements
ITC is a
(where is the gas constant and is the
For accurate measurements of binding affinity, the curve of the thermogram must be sigmoidal. The profile of the curve is determined by the c-value, which is calculated using the equation:
where is the stoichiometry of the binding, is the
Instrumental measurements
An isothermal titration calorimeter is composed of two identical cells made of a highly efficient thermally conducting and chemically inert material such as
In an
Observations are plotted as the power needed to maintain the reference and the sample cell at an identical temperature against time. As a result, the experimental raw data consists of a series of spikes of heat flow (power), with every spike corresponding to one ligand injection. These heat flow spikes/pulses are integrated with respect to time, giving the total heat exchanged per injection. The pattern of these heat effects as a function of the molar ratio [ligand]/[macromolecule] can then be analyzed to give the thermodynamic parameters of the interaction under study.
To obtain an optimum result, each injection should be given enough time for a reaction equilibrium to reach. Degassing samples is often necessary in order to obtain good measurements as the presence of gas bubbles within the sample cell will lead to abnormal data plots in the recorded results. The entire experiment takes place under computer control.[7]
Direct
Analysis and interpretation
Post-hoc analysis and proton inventory
The collected experimental data reflects not only the binding thermodynamics of the interaction of interest, but any contributing competing equilibria associated to it. A post-hoc analysis can be performed to determine the buffer or solvent-independent enthalpy from the experimental thermodynamics, by simply going through the process of Hess’ law. Below example shows a simple interaction between a metal ion (M) and a ligand (L). B represents the buffer used for this interaction and represents protons.
Therefore,
which can be further processed to calculate the enthalpy of metal-ligand interaction.[10][11] Although this example is between a metal and a ligand, it is applicable to any ITC experiment, regarding binding interactions.
As a part of the analysis, a number of protons are required to calculate the solvent-independent thermodynamics. This can be easily done by plotting a graph such as shown below.
The linear equation of this plot is the rearranged version of the equation above from the post-hoc analysis in a form of y = mx + b:
Equilibrium constant
Equilibrium constant of the reaction is also not independent from the other competing equilibria. Competition would include buffer interactions and other pH-dependent reactions depending on the experimental conditions. The competition from species other than the species of interest is included in the competition factor, Q in the following equation:[11]
where, represents species such a buffer or protons, represents their equilibrium constant, when,
Applications
For the past 30 years, isothermal titration calorimetry has been used in a wide array of fields. In the old days, this technique was used to determine fundamental thermodynamic values for basic small molecular interactions.[12] In recent years, ITC has been used in more industrially applicable areas, such as drug discovery and testing synthetic materials. Although it is still heavily used in fundamental chemistry, the trend has shifted over to the biological side, where label-free and buffer independent values are relatively harder to achieve.[13][14]
Enzyme kinetics
Using the thermodynamic data from ITC, it is possible to deduce
Membrane and self-assembling peptide studies
Drug development
Binding affinity carries a huge importance in medicinal chemistry, as drugs need to bind to the protein effectively within a desired range. However, determining enthalpy changes and optimization of thermodynamic parameters are hugely difficult when designing drugs.[19] ITC troubleshoots this issue easily by deducing the binding affinity, enthalpic/entropic contributions and its binding stoichiometry.
Chiral chemistry
Applying the ideas above, chirality of organometallic compounds can be deduced as well with this technique.[20] Each chiral compound has its own unique properties and binding mechanisms that are comparable to each other, which leads to differences in thermodynamic properties. By binding chiral solutions in a binding site can deduce the type of chirality and depending on the purpose, which chiral compound is more suitable for binding.
Metal binding interactions
Binding metal ions to protein and other components of biological material is one of the most popular uses of ITC, since ovotransferrin to ferric iron binding study published by Lin et al. from MicroCal Inc.
The technique has been well utilized in studying carbon nanotubes to determine thermodynamic binding interactions with biological molecules and graphene composite interactions.[23] Another notable use of ITC with carbon nanotubes is optimization of preparation of carbon nanotubes from graphene composite and polyvinyl alcohol (PVA). PVA assembly process can be measured thermodynamically as mixing of the two ingredients is an exothermic reaction, and its binding trend can be easily observed by ITC.
See also
- Differential scanning calorimetry
- Dual polarisation interferometry
- Sorption calorimetry
- Pressure perturbation calorimetry
- Surface plasmon resonance
References
- ^ ISSN 0003-2700.
- .
- ^ Serdyuk; Zaccai; Zaccai (2017). "Chapter C3 Isothermal Titration Calorimetry". Methods in molecular biophysics: Structure, dynamics, function. Cambridge University Press. pp. 221–233.
- ^ Kuriyan; Conforti; Wemmer (2013). "12.23 Isothermal titration calorimetry allows us to determine the enthalpic and entropic components of the binding free energy.". The molecules of life: Physical and chemical principles. Garland Publishing. pp. 573–577.
- ^ a b Johnston, H. D. (1968) The thermodynamics (log K, ΔH°, ΔS°, ΔCp°) of metal ligand interaction in aqueous solution. Brigham Young University.
- ^ a b Quick Start: Isothermal Titration Calorimetry (ITC) (2016). TA Instrument. New Castle, DE.
- ^ a b c VP-ITC Instruction Manual (2001). Microcal Inc., Northampton, MA.
- ^ a b Stevenson, M. J. (2016) Thermodynamic studies of Cu(I) and other d10 metal ions binding to proteins in the copper homeostasis pathway and the organomercurial detoxification pathway. Dartmouth College.
- S2CID 98752781.
- ^ S2CID 28672066.
- ^ PMID 26794348, retrieved 2023-02-15
- .
- S2CID 206095685.
- S2CID 234472084.
- PMID 33195429.
- PMID 22126339.
- PMID 24119484.
- PMID 18703160.