This would be difficult at best. In isothermal titration calorimetry you measure the heat of reaction (Delta H) for binding of a ligand to a protein. If a second ligand also binds, you'd have to distinguish between the heat produced by both processes.

If Delta H for both processes is very different (say, an order of magnitude) then possibly this could be deconvoluted, but not if they are similar.

Note also that ITC requires fairly large amounts of protein per experiment, simply because of the large molecular mass. In your case BSA is fairly cheap, but in general that may not be affordable.

Competitive ligands can be studied by ITC, as well as other binding techniques. In addition, ITC provides two parameters: association (or dissociation) constant and binding enthalpy.

If you have two ligands, A and B, binding to the same protein, several situations may arise: independent ligands, allosteric ligands (heterotropy), competetive ligands.

If both ligands are independent, the binding of A does not alter the binding parameters for B. Then, the association constant and the binding enthalpy of A does not depend on the concentration of B.

If both ligands behave allosterically, the binding of A depends on the presence of B. The association constant will increase (higher affinity) in the presence of B if there is positive cooperativity, and the association constant will decrease (lower affinity) in the presence of B if there is negative cooperativity.

If both ligands behave competitively, this is the extreme case of maximal negative cooperativity.

In particular:

KAapp=KA(1+kKB[B])/(1+KB[B])

where KAapp is the apparent association constant for A in the presence of B at concentration [B], KA is the intrinsic association constant for A, KB is the intrinsic concentration constant for B, and k is the cooperativity constant.

Therefore:

k=1, if A and B are independent ligands.

k>1, if A and B bind with positive cooperativity.

k

Thank you @ Adrian for your cooperation.

in the competitive exp. the cell contain both the protein and ligand B then what should write in cell conc in exp set up?

In a standard ITC experiment (moderate-to-high affinity, sufficient binding enthalpy) you usually employ:

Titrant: 200 micromolar

Titrand: 20 micromolar

In case of low affinity, low binding enthalpy, you should increase the concentrations: increase the titrant solution concentratioin to reach higher saturation levels (if low affinity), or increase both titrant and titrand concentrations (if low enthalpy).

In case of more than one binding site, you should increase accordingly the titrant concentration.

In a ternary experiment (ligand A injected into mix of protein and ligand B), you should plan the same experimental set-up as in the binary experiment (ligand A into protein), but loading a mix of protein and ligand B in the cell. Concentration of B should be sufficient for achieveing a considerable saturation level in the protein and for exerting a significant change in the apparent binding parameters for ligand A compared to its intrinsic binding parameters; that is, the term 1+KB[B] must be considerable larger than 1 (or KB[B]>>1).

Regarding data analysis, in the ternary experiment you do not care about the ligand B: it is there, altering the apparent binding parameters for A, but you analyze the experiment considering just ligand A in syringe and protein in cell (that is, a "quasi-binary" equilibrium).

Of course, there is a way for analyzing the ternary experiment employing an exact model, as in:

Article A unified framework based on the binding polynomial for char...

Article Studying the Allosteric Energy Cycle by Isothermal Titration...

Hi @ Adrian

how to fit site marker exp data ? I want to compare association constant of binary and ternary system in site marker exp. should i fit each exp ( means both binary and ternary ) individually by one site binding model ? or by competitive binding model provided by origin ?

Yes, you perform the two binary experiments and the ternary experiment. Then, you have two options:

1) Analyze all experiments using the single binding site model and compare the apparent binding affinity in the ternary experiment with that in the appropriate binary experiment, as I explained before.

2) Analyze the binary experiments using the single binding site model and the ternary experiment using the cooperativity model (that is, the exact ternary equilibrium). In Origin you will find the competitive binding model, which is not the general cooperativity binding model, but that model restricted to competitive binding (maximal negative cooperativity).

ok thank you @Adrian for your fruitful answer

Hellow @Adrian

This is not related to site marker .

i want to perform enzyme activity in presence of substrate and Inhibitor. I got clear concept about the procedure from ITC manual . but what about the conc issue of all three(enzyme,substrate and inhibitor)? i did this enzyme activity study by time dependent uv measurement. i want to compare b/w this two tech. so if you have any ppr related this kindly share in RG.

Thanks in adv.

Check this:

https://www.ncbi.nlm.nih.gov/pubmed/11554713

And there are more other references in the literature regarding enzyme activity by ITC.

thank you @ Adrian . yes I got many ppr regarding enzyme. but few of them mention concentration issue.

Hi Samima,

Regarding the model(s) used for fitting the competitive experiments, our software AFFINImeter provides the competitive binding model you need and the posibility to perform global fitting of the ITC data of the competitive exp + the ITC data of the individual binding. With these tools you could obtain the thermodynamic profile (K and DH) of each binding event. Here´s some brief info of the competitive binding models and analysis from our blog: http://blog.affinimeter.com/competitive-binding-assay/

Ok. Thank u @ Eva. i will try.