My drug is inhibitor and I am measuring its Ki against trypsin, but my substrate is not soluble in same buffer. If I use DMSO to dissolve that, then do I need to dialyze it with buffer as well? What other things I have to consider before titration?
You are asking two questions: 1) Has anyone measured Ki using ITC?; and 2) What concerns do arise when using DMSO in ITC experiments?
The answer to the first question is YES.
In ITC you can perform equilibrium experiments and kinetic experiments.
In the equilibrium experiments you get the dissociation constant Kd for the enzyme/inhibitor complex, which in general (not always) can be considered equal to the inhibition constant Ki for the inhibitor, by titrating the inhibitor into the enzyme (or viceversa).
In the kinetic experiments you get the kinetic constants (Km and kcat) and the Ki by performing injections of substrate into the enzyme in the presence/absence of inhibitor. Please, check this publication:
http://www.ncbi.nlm.nih.gov/pubmed/11554713
On the other hand, regarding the second question... YES, you must take some precautions when performing ITC experiments in the presence of DMSO. Please, check the following RG post:
https://www.researchgate.net/post/Has_anyone_ever_performed_Isothermal_Titration_Calorimetry_ITC_experiments_with_DMSO
Thanks Adrian.This information is really useful to me. I tried with BAPNA substrate and dissolved it in 5%DMSO. I maintained 5% DMSO in trypsin solution and buffer followed method of single injection of substrate. I performed at 25 C and at 37C , But my result was so strange. I tried to compare with the one paper you have suggested. I saw rapid decrease in instrumental thermal power but the power did not returned to initial baseline. I don't understand that either there was any mismatching in instrumental parameters or substrate or enzyme concentration was not enough. What do you suggest? I performed using Microcal ITC 200.
The power did not returned to the initial baseline value, but, how different was it? How big was the injection effect? Sometimes it is a matter of signal-to-noise ratio... that is, for small heat effects, the noise or the baseline drift may seem too big, while the same noise or baseline drift looks negligible for larger heat affects.
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