Advantages of performing a global fitting of several Isothermal Titration Calorimetry isotherms

We have remarked the relevance of global fitting as an indispensable tool to perform a robust analysis of complex binding interactions. Actually, global fitting is a very advantageous tool for the analysis of simpler binding modes like standard 1:1 interactions and it can be used to identify the source of potential differences observed between experiment repeats.
Here we will see how to use global fitting together with the parameters rA and rM of AFFINImeter to identify the source of discrepancies between repeats of the same experiment, to ultimately end up with reliable results.
The isotherms of figure 1 are replicates of the same ITC experiment where a 1:1 interaction between the titrant (A) and the titrate (M) is monitored, but individual fitting of each isotherm to the simple model M + A ↔ MA (Figure 2) yield different K_A (association constant) and ΔH values. In fact, from a visual inspection of the isotherms one can presume that  the saturation degree reached in replicate 1 is less than in replicate 2 and that the mid titration point is below the expected stoichiometric equivalence point (A_t/M_t = 1), most probably because the titrant (A) and/or titrate (M) concentration was not accurately determined in this experiment.

 

Figure 1. A)  Global fitting 2 where K, ΔH  and rA are global parameters and rM is individual between isotherms. B) Global fitting 1 where KA, ΔH and rM are global parameters and rA is individual between isotherms.

 

A global fitting of the two isotherms was performed in which K_A, ΔH  and rA are shared fitting parameters amongst the isotherms; moreover, rM* is set as individual fitting parameter to check for potential deviations between nominal and true concentration of M in replicate 1; similarly, a global fitting was performed in which K_A, ΔH and rM are shared parameters, and rA* is an individual fitting parameter to check for potential deviations between nominal and true concentration of A (figure 1).

Figure 2. Representation of the experiment using the AFFINImeter AM code.

 

Comparison of the two global fittings clearly shows that a good result is only achieved when deviations in the concentration of A is considered. The value of rA obtained indicates that the active concentration of A is 79% times the nominal concentration (table).

Table: Results obtained from global fitting 2.

 

This protocol using AFFINImeter has already been proven to be useful in drug discovery programs, working with ligand samples of inaccurate concentration.

We would like to thank Dr. Eric Ennifar (University of Strasbourg) for kindly providing the ITC experiments described herein.

 

*rM, rA, are scaling parameters employed to correct for potential differences between the nominal and true concentration of compounds M and A, respectively.

Following the last part of this course, we want to introduce the second fitting approach that can be performed with AFFINImeter:

The Stoichiometric equilibria approach.

This approach uses binding models based on equilibria between stoichiometric species and yields stoichiometric binding constants. In AFFINImeter, traditional and tailored stoichiometric binding models can be easily generated with the “model builder” tool.
Stoichiometric binding models are applicable to a great variety of complex interactions like:

• A ligand binding to a multi-site receptor with known stoichiometry, where the sites can be independent or cooperative.
• Competing ligands binding to the same receptor.
• Oligomerization.

….among others.

The appropriate design and use of binding models in AFFINImeter pass through an understanding of the nomenclature that the software uses to describe the species located in the calorimetric cell and in the syringe.
In the following link you can visualize a quick video about the Nomenclature of the Model model builder:

Nomenclature of Model Builder.

The last video of this course is about how to use the stoichiometric equilibria approach to perform an individual and global analysis.

The course about ITC data analysis comes to an end so if you want to try the versatility of AFFINImeter you can register to get a free trial during 1 month:

 

Get a free trial

Isothermal Titration Calorimetry (ITC) is widely considered the gold standard technique for the direct determination of binding thermodynamics.
Nowadays, getting kinetic information directly from the thermogram is also possible thanks to the method KinITC implemented in AFFINImeter. Besides KinITC, AFFINImeter offers a number of advanced analytical tools to take the most out of ITC data.

Aiming to show researchers how to make use of these tools, we have prepared a course on ITC data analysis, consisting of a series of video tutorials imparted by Eva Muñoz (Senior Scientist at AFFINImeter) and that we will release in three parts.

The following picture shows the scheme of the course:

The first part, we present three video tutorials dedicated to the analysis of ITC data with KinITC and the multi-temperature tools. Here we are attaching the video tutorials.

1. Introduction-KinITC automatic processing
2. KinITC Manual process
3. Multi-temperature analysis

In the following parts of this course, we will be focused on the design of two binding models for ITC curve fitting with AFFINImeter:

• Based on Independent site approach.
• Based on Stoichiometric equilibria approach.