Hi Francis,

I might save you some time by telling you up front you should just go back and purify your compound to remove the impurity, you dont even need to read the rest of this, just go.

Along the lines of what Savvas was saying, with any equilibrium binding assay between two direct competitors ("Y" is the impurity and "Z" is your analyte), if you are working at concentrations above the KD then the resultant complexes (XY and XZ) will partition according to their relative association strengths (dG) and concentrations. So, if Y and Z have equivalent dG values, then the concentration of XY ([XY]) and [XZ] will be a function of [Y] and [Z], if [Y]=[Z] in this circumstance then [XY]=[XZ].

If dGy >> dGz or [Y] >> [Z], then you are in the clear. This is why going back to purify Z from Y is a good idea.

Now,the great thing about ITC is of course that you can get dG, dH and -TdS in one experiment, but this is also going to bite you in the butt here since you will simultaneously be determining dG, dH and -TdS for both Y and Z, which leaves you will more unknowns that you have data to solve for unless you independently know [X], [Y], [Z] and dG, dH and -TdS for XY or XZ.

In fact, the circumstance where you know [X], [Y], [Z] and dG, dH and -TdS for XY or XZ is what Savvas is describing with "displacement" assays, and unless I am misunderstanding your situation it sounds like you dont know these parameters. For that reason I would not qualify this as a displacement assay, but instead just as a poorly controlled experiment . Now, you might be able to do the experiment with pure Y binding to X to determine dG, dH and TdS, then perform the proposed experiment with impure Y and Z as a "displacement" binding, but this is going to still be a headache because your uncertainty will be greater, you will not have as accurate a measure of [Y] and [Z] as when they are pure, and since your your direct signal (dH) is going to be from the formation of both XY and XZ (dHtotal = dHy + dHz) S/N will be equal to or less than the experiment with pure Y or pure Z (my nanny used to say 'dont do good experiments with bad reagents, youll just waste time', she was very wise).

Hope that helps, cheers~


Quoting Savvas Savvides <savvas.savvi...@ugent.be>:

Hi Francis
I guess it depends on how much residual high-affinity binder you have in the mixture and what the difference in affinity is between Y and deriv-Y. Another issue is of course whether Y and derY compete for the same binding site and have the same stoichiometry. A well designed displacement ITC experiment and comparisons thereof with ITC data for your high-affinity binder should lead to some good answers. Knowing the ratio of Y vs deriv-Y in your starting compound solution will be an advantage.

A very useful reference in thinking about and carrying out displacement ITC in our group has been the one by Velazquez-Campoy and Freire. This article was specifically written to address the application of displacement titrations in ITC. We have applied this approach to address several types of questions concerning interactions in the uM-pM range.

Velazquez-Campoy A, Freire E.
Isothermal titration calorimetry to determine association constants for high-affinity ligands
Nat Protoc. 2006;1(1):186-91.

Best regards

Savvas Savvides
Unit for Structural Biology @ L-ProBE
Ghent University
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
Ph. +32  (0)472 928 519 http://www.LProBE.ugent.be/xray.html

On 24 Aug 2010, at 17:11, Francis E Reyes wrote:

Hi All

I'm curious the effect of small impurities in commercially synthesized compounds on ITC and its analysis. Say if compound Y is the high affinity binder, but you make a derivative that differs from a single functional group from Y (you used Y to make this new compound) and you never are able to completely get rid of Y. How does this affect the analysis of determining the derivative's affinity by ITC?

References or personal experience is appreciated!


Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder

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