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] On Thu, Oct 3, 2019 at 11:51 AM Bernhard Rupp <[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]> *On Behalf Of *Keller, > Jacob > *Sent:* Thursday, October 3, 2019 17:41 > *To:* [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]> *On Behalf Of *Bernhard > Rupp > *Sent:* Thursday, October 3, 2019 11:06 AM > *To:* [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$> > > [email protected] > > +1 925 209 7429 > > +43 676 571 0536 > > ------------------------------------------------------ > > Many plausible ideas vanish > > at the presence of thought > > ------------------------------------------------------ > > > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > <https://urldefense.com/v3/__https:/www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1__;!oCotSwSxbw8!SE9mT6grUy1tHFSKLCraXt4bhlDri03OEMEyqQUCLAVSLsg3vwn0GTQtxbStkIPgsQE$> > > > ------------------------------ > > 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
