In a Isothermal Titration Calorimetry experiment, If the injected volume or any of the concentrations is too small, or if the ratio between both concentration values is not appropriate, then the signal-to-noise ratio will be low and the uncertainty of any result will be high.
Determinant factors of poor quality results in Isothermal Titration Calorimetry Experiments
The control of these factors might be limited by the available amount or the corresponding solutes (they can be expensive or difficult to synthesize/purify). If the total number of titrations is low then the solute in the sample cell will not be saturated and the quality of the results will be poor. Additionally, in reactions for which multiple chemical species can be formed it is always better to simultaneously fit several experimental data series, each focusing the sampling in a different concentration region which is more sensitive to any of the species.
Advantages of prior Isotherm Simulation
The simulator tool provided by AFFINImeter allows you to test the effect of the parameters listed above on your experiment in order to optimize its design, thus saving time and samples.
Isothermal Titration Calorimeters have been designed to determine the thermodynamic parameters corresponding to physicochemical processes that take place in a solution: protein-ligand binding, enzyme assay, drug encapsulation, dissociation/aggregation, micellization, etc. The reliability of the thermodynamic parameters obtained from Isothermal titration calorimetric experiments depends critically on:
(i) the volume injected in the sample cell upon each titration
(ii) the concentration of the solution in the sample cell
(iii) the concentration of the solution in the injection system
(iv) the number of titrations
(v) the number and relative concentration of the different chemical species that can be formed upon mixing of both solutions
(vi) the heat involved in the formation of those chemical species
(vii) the order in which the solutions are considered, i.e., which solution is introduced into the sample cell and which is in the injection system.
ITC displacement titrations offer an attractive alternative to standard assays when working with ultra high- or ultra low- affinity interacting systems. The method requires the fitting of at least two isotherms that share various adjustable parameters. AFFINImeter counts with advanced tools, like the global fitting of multiple dataseries and the analysis of isotherms registered under unusual experimental design, which can facilitate the analysis and expand the range of applications of isothermal titration calorimetry experiments. As an illustration, herein we present a displacement titration assay to determine the thermodynamics of HIV-protease with indinavir, a high affinity binder, and with acetyl-pepstatin, a weaker ligand. Using AFFINImeter a global analysis of four isotherms was performed describing: HIV-protease binding to indinavir (I) or to acetyl-pepstatin (II): HIV-protease binding to indinavir incorporating acetyl-pepstatin in the cell (III) or in the syringe (IV).
Isothermal Titration Calorimetry is one of the most commonly used approaches to obtain affinity and thermodynamic data of molecular interactions and has become a routine method in the pharmaceutical industry.1 Isothermal titration Calorimetry is applicable to numerous interacting systems, as long as a detectable heat change is produced during complexation, covering an important range of binding affinities (106 ≤ KA ≤108 M-1). Nevertheless, standard ITC experiments present some limitations in the case of very low- or very high-affinity interactions (i.e. affinities in the low millimolar or high nanomolar range, respectively). High affinity interactions (KA ≥ 109 M-1) yield square-shaped isotherms whose fitting yield accurate values of the binding enthalpy but only estimates of the association constant. Attempts to recover a sigmoidal shape requires the use of very low concentrations of the interactants that, in most cases, is not feasible in the practice (the minimum concentration that will typically cause a confidently measurable heat change for a 1:1 interaction is about 10 μM). On the opposite, low affinity interactions should be studied at high concentrations and this requirements is often a serious limiting step due to various potential reasons like limited solubility and/or availability of the sample molecule, or the existence of aggregation processes at the required concentration. In both high- and low affinity systems these experimental drawbacks can be circumvented by using the ITC displacement method.2,3 Here, the receptor is titrated with a high affinity ligand, but in the presence of a weaker ligand in the sample cell that competes for the complexation with the receptor (figure 1). With this experimental set up the apparent affinity of the strong ligand is “artificially” lowered, obtaining a sigmoidal isotherm that yields more accurate binding data. When the goal is to obtain the thermodynamic parameters of an ultra highaffinity system, a titration with the weaker binder is performed first to obtain the corresponding affinity constant and enthalpy (KA-weak and H-weak). These values are required for the analysis of the ITC displacement experiment, where a competitive binding model is used to estimate the thermodynamic parameters of the tight binding (KA-tight and H-tight). Analogously, when the goal is to obtain information of an ultra low- affinity system a direct ITC titration of the receptor alone with a ligand of higher affinity is performed. The resulting KA-tight and H-tight are then incorporated in the analysis of the isotherm from the ITC displacement assay.
This case study exemplifies the potential advantages of using AFFINImeter in ITC displacement assays. The software offers unique advanced tools that enhance the robustness of the method and makes it more versatile, facilitating the acquisition of reliable thermodynamic data from ultra-high of ultra-low affinity systems. Thus, it opens a door for new applications of the displacement assay. 1 G. Holdgate, S. Geschwindner, A. Breeze, G. Davies, N. Colclough, D. Temesi, L. Ward, Biophysical Methods in Drug Discovery from Small Molecule to Pharmaceutical. Protein-Ligand Interactions. In Methods in Molecular Biology 2013, 1008, pp 327-355. 2 A. Vellazquez-Campoy and E. Freire, Isothermal titration calorimetry to determine association constants for highaffinity ligands. Nature protocols 2006, 1, pp 186-191. 3 W. B. Turnbull, Divided we fall? Studying low-affinity fragments of ligands by ITC. GE Healthcare Life Sciences protocol.
AFFINImeter attended the Development in Protein Interaction Analysis (DiPIA) Conference in La Jolla, CA this June 2014, where we were part of the leading scientists working in protein interactions.
We are glad to tell you that our Poster of AFFINImeter: a new tool for analysis of Isothermal Titration Calorimetry experiments has been awarded with the 4th prize of the poster competition. Thanks to our representative J. Sabín of doing such a good job presenting AFFINImeter.
If you haven’t registered yet to try the beta version visit our webpage www.affinimeter.com or contact us at email@example.com
This weekend we are joining the leading scientists at the Development in Protein Interaction Analysis (DiPIA) conference. A representative of AFFINImeter will present a new tool to analyze Isothermal Titration Calorimetry Experiments.
New generations of Isothermal Titration Calorimetry (ITC) equipments have significantly improved their performance in terms of higher sensitivity and lower sample size requirements. Thus, physicochemical processes that were undetectable in the past by ITC are now sensed with high precision. In contrast to the progress achieved in instrumentation, ITC data analysis is still limited in many aspects. Researchers often face complicated situations that cannot be represented by standard binding models i.e. the formation of higher order complexes, the presence of multiple candidates to bind the host molecule and/or the simultaneous occurrence of binding and aggregation processes.
AFFINImeter allows to design personalized binding models directly in chemical language, through an original user-friendly interface that allows the design of advanced models.