Hello,
I am doing a titration of ligand into protein and am getting good heat releases compared to my ligand into buffer.
I am getting more heat release in later injections. Could cooperativity explain this because I have used varying concentrations of protein and ligand and get the same curve.
Thank you.
Although the ligand into buffer experiment would not be in agreement with my explanation, the simplest explanation is that, for some reason, the background injection heat is quite large, as it can be observed in the last injections. At the beginning of the titration, the interaction heat is of opposite sign and compensates partially the background injection heat.
Then, according to the binding isotherm, the interaction is endothermic and you must introduce the background injection heat in the fitting routine. Remember that one of the most important sources of uncertainty/error in ITC is the incorrect accounting of background injection heat.
Of course, that is the simplest explanation, which may not necessarily be true, but incorporating cooperativity implies a further complexity level that should be further justified.
Hi Adrian Velazquez-Campoy , thanks for your answer, I agree with you that cooperativity might be a stretch. Based on your suggestions, I have a theory that maybe buffer into protein could be whats producing the large heat change because I haven't ran that control yet. I had been told usually ligand into buffer was sufficient for baseline subtraction. If that were the case and the interaction was endothermic I think that could account for the background heat discrepancy "masking" the endothermic curve. Would that be a plausible explanation?
Maybe. You just have to do the experiment (buffer into protein). However, that would be the first time I see the buffer-into-protein gives larger heats than ligand-into-buffer.
Dear Nathan Soper
What if your protein has two binding sites (non-identical)? If so, you do not see the full saturation yet. That would explain the result. Then, you should continue the titration to the higher ratio values.
Regards
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