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~
~Justin
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
----
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!
F
---------------------------------------------
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder
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