I am not a proper chemist, but I remember that during my college classes my
professor emphasized that ^x is a measured value and not really related to
stoichiometry. If someone has something to add…
Best
Rafael Marques da Silva
Mestre em Física Biomolecular
Universidade de São Paulo
Bacharel em Ciências Biológicas
Universidade Federal de São Carlos
phone: +55 16 99766-0021
"A sorte acompanha uma mente bem treinada"
________________________________________________
De: ABHISHEK SUMAN<mailto:00002fccd9428006-dmarc-requ...@jiscmail.ac.uk>
Enviado:terça-feira, 16 de agosto de 2022 14:41
Para: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Assunto: [ccp4bb] Off-topic question related to ITC binding studies
Hello everyone!
Hope this email finds you well. I have an off-topic question regarding ITC
binding studies, which was asked by a reviewer.
We performed an ITC binding study (using Affinity ITC, TA Instruments) to
evaluate protein-DNA interaction which resulted in a perfect sigmoidal curve.
We used the ‘one set of sites’ binding algorithm (“independent” model) for
curve fitting and to calculate binding and thermodynamic parameters. The study
suggested two copies of the protein binding to a single duplex DNA, i.e., the
stoichiometry of protein:DNA is 2:1 (N=2). The ITC calculated the KD
(equilibrium molar dissociation constant) in μM (micromolar). But the reviewer
is asking to report the KD in (micromolar)^2 instead of micromolar mentioning
that the binding reaction is 2A + B <-> (A2)B and the complex is (A2)B and not
AB. Though we're trying to explain to the reviewer that we couldn't find any
software that can compute the KD in (micromolar)^2 for the stoichiometry of 2
but he is not agreeing to it. We have used the NanoAnalyze software from the TA
instrument. This software does not have a model to measure the KD in
(micromolar)^2.
I would be grateful if you could help me to resolve this problem or at least
let me know what explanation might be appropriate to answer the reviewer’s
concern that it’s a general practice to report the KD in the Molar irrespective
of stoichiometry.
Thanks in advance.
Regards
Abhishek
Dr. Abhishek Suman
Ph.D (Structural Biology)
Indian Institute of Technology Hyderabad
Kandi 502 284 Sangareddy
Telangana INDIA
Contact: +91 91002 74548, +91 80843 11898
Email: abhisheks.i...@gmail.com<mailto:abhisheks.i...@gmail.com>
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