Hello,
I am using NanoITC-Low Volume from TA Instruments to study protein-ligand binding. I have an unnecessarily complex ITC situation due to ligand solubility.
Since the ligand is very hydrophobic, I have set up a protein (in syringe) into ligand (in cell) experiment. Due to the high concentration of the protein in the syringe, the protein solution dimerizes within the syringe.
Once it is injected, the first 8-14 injections (depending on concentration) contain a dimer dissociation event, along with dilution.
The dissociation of the dimer is endothermic, the dilution is exothermic and the binding is exothermic. The peaks don't just add up to become a single peak with incorporated heats but produce chimeric peaks as (I suspect): Heat of dilution (exo) - dimer dissociation (endo) - binding (exo). The binding peak seems to grow within the large dimer dissociation peaks.
My first question is, how do you integrate such peaks with both exo and endo? I have been drawing a straight-line baseline from the start of injection to the end of the last peak (whether exo/endo).
Second question is, how would you model this, because at the first ~10 injections, the protein is mostly monomeric due to the low concentration of the protein, while in the second half, the protein is probably dimeric again.
I think you are trying to kill two birds with one shot. I suggest you to first characterize the energetics of the dissociation equilibrium of the dimer by doing a dilution titration calorimetry experiment (Biophys. J. (2011) 101:1423-1431), which will give you also information about the heat of dilution of the protein.
good luck!
I think that getting reliable net heat for each peak is the easy part of your problem. If you can estimate the baseline before and after, and if you can trust it to continue smoothly through the peaks, then just summing the differences across the peak and multiplying by delta t gives a trustworthy estimate of the net heat and probably cannot be improved upon with methods that attempt to decompose the peaks into components. But your model for the process may not fit into the frameworks for any standard models, meaning you or a colleague might need to write your own code for the analysis. You will probably anyway need to get more information about the separate processes you propose, through other experiments, as already suggested.
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