No, the law of mass action does not depend on whether A is titrated into B or
vice versa. The heat measured is proportional to the complex formed, relative
to one partner being kept at constant concentration (the referece in the cell).
The law of mass action can be re-written to describe the complex concentration
itself as a function of A, B and Kd. Assuming a one-to-one stoichiometry, the
function contains sums of each variable being substracted by a root function:
AB = 0.5 [ Atotal + Btotal + Kd - sqrt{ (Atotal + Btotal + Kd)^2 -
4*Atotal*Btotal } ]
It is correct that these values should be roughly in the same range. This,
however, is not restricted to ITC solely, but to HSQC or fluorescence-based
titrations too. Its a direct consequence of the law of mass action.
In order to fully saturate either binding partner, say fully saturate A; AB/A
approaching 1, all three factors need to be in the same range. The same would
hold with B in the cell, saturating AB/B. The underlaying mass action is the
same, just the non-ligated reference is exchanged.
For a stochiometry different from 1:1, the mass action does not yield in an
explicit solution like the one written above. To force such a solution, one
concentration is multiplied by a factor, N, assuming N independent,
non-cooperative binding sites for N ligands. Lets assume a 1:2 in your case,
2A+B2 <-> A2B2. Substituting Btotal with N*Btotal would allow to assume a
one-to-one model with N=2. If you decide to exchange the titrant, N now
corrects the other partner, yielding in N=0.5.
Dr. Matthias Barone
AG Kuehne, Rational Drug Design
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin
Germany
Phone: +49 (0)30 94793-284
________________________________
From: CCP4 bulletin board <[email protected]> on behalf of Roger Rowlett
<[email protected]>
Sent: Thursday, October 3, 2019 8:36:27 PM
To: [email protected]
Subject: Re: [ccp4bb] ITC question -dimer vs monomer
Won't this depend on the relative final concentrations of A and B in the two
experiments? If A going into excess B will have different mass action
considerations that B going into excess A. Even if the final concentrations of
A and B are stoichiometric, the initial stages of the titration will have very
different mass action products for A into B vs. B into A. An additional wrinkle
is the concentrations of A and B relative to the dissociation constant Kd. The
titration curve math gets a little more complex when the concentration of the
species is in the same order of magnitude as the Kd. There are quite a few
examples of bollixed binding curves in the literature for tight-binding
equilibria that ignore the relationship between Kd and ligand concentrations.
Cooperativity issues will of course perturb any pure, non-cooperative
statistical analysis based on equilibrium constants.
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
email: [email protected]<mailto:[email protected]>
On Thu, Oct 3, 2019 at 11:51 AM Bernhard Rupp
<[email protected]<mailto:[email protected]>> wrote:
I am not looking for anything yet – I wonder what – if any – the consequences
of doing it one way or the other would be.
I am reasonably certain that any difference affects the analysis.
Thx, BR
From: CCP4 bulletin board <[email protected]<mailto:[email protected]>>
On Behalf Of Keller, Jacob
Sent: Thursday, October 3, 2019 17:41
To: [email protected]<mailto:[email protected]>
Subject: Re: [ccp4bb] ITC question -dimer vs monomer
I don’t understand what you are trying to do—are you trying to show, by the
difference in ITC response, that the predictions you made about the
oligomerization are true?
JPK
+++++++++++++++++++++++++++++++++++++++++++++++++
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+++++++++++++++++++++++++++++++++++++++++++++++++
The content of this email is confidential and intended for the recipient
specified in message only. It is strictly forbidden to share any part of this
message with any third party, without a written consent of the sender. If you
received this message by mistake, please reply to this message and follow with
its deletion, so that we can ensure such a mistake does not occur in the future.
From: CCP4 bulletin board <[email protected]<mailto:[email protected]>>
On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: [email protected]<mailto:[email protected]>
Subject: [ccp4bb] ITC question -dimer vs monomer
Hi Fellows,
please let me ask the respective experts an ITC question: I have 2 proteins,
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer
will form.
Intuitively, it should make a difference whether I titrate the dimer with the
monomer or vice versa.
In the first case, a momomer would initially meet a lot of free dimers, and I
would expect that randomly, a AB2 complex
is more likely to form than a A2B2 (let’s disregard any more complex
colligative/cooperative effects).
If I drip the dimer into the monomer pool, it is quite likely that the B dimer
meets 2 free As, and I get right away a higher population of A2B2s.
Maybe at dilutions of ITC and with sufficient equilibration that is not an
issue at all (again, absent any cooperative effects that might alter the first
Kd vs. the second, despite the sites on the dimer are at least initially
equivalent).
Can someone guide me towards literature about this or perhaps share some
first-hand experience?
Many thanks, BR
------------------------------------------------------
Bernhard Rupp
http://www.hofkristallamt.org/<https://urldefense.com/v3/__http:/www.hofkristallamt.org/__;!oCotSwSxbw8!SE9mT6grUy1tHFSKLCraXt4bhlDri03OEMEyqQUCLAVSLsg3vwn0GTQtxbStgtNvxBs$>
[email protected]<mailto:[email protected]>
+1 925 209 7429
+43 676 571 0536
------------------------------------------------------
Many plausible ideas vanish
at the presence of thought
------------------------------------------------------
________________________________
To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1<https://urldefense.com/v3/__https:/www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1__;!oCotSwSxbw8!SE9mT6grUy1tHFSKLCraXt4bhlDri03OEMEyqQUCLAVSLsg3vwn0GTQtxbStkIPgsQE$>
________________________________
To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
________________________________
To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
________________________________
To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
########################################################################
To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
