Direct Determination of High-Affinity Binding Constants by Continuous Injection Isothermal Titration Calorimetry
- Authors
- Chang, Ji Woong; Mu, Yanyu; Armaou, Antonios; Rioux, Robert M.
- Issue Date
- Dec-2023
- Publisher
- AMER CHEMICAL SOC
- Citation
- JOURNAL OF PHYSICAL CHEMISTRY B, v.127, no.50, pp 10833 - 10842
- Pages
- 10
- Journal Title
- JOURNAL OF PHYSICAL CHEMISTRY B
- Volume
- 127
- Number
- 50
- Start Page
- 10833
- End Page
- 10842
- URI
- https://scholarworks.bwise.kr/kumoh/handle/2020.sw.kumoh/28499
- DOI
- 10.1021/acs.jpcb.3c05650
- ISSN
- 1520-6106
1520-5207
- Abstract
- Isothermal titration calorimetry (ITC) is a method to determine thermodynamic values (Delta G, Delta H, and Delta S) for ligand-receptor binding in biological and abiological systems. It is challenging to directly determine subnanomolar dissociation constants using a standard incremental injection approach ITC (IIA-ITC) measurement. We recently demonstrated a continuous injection approach ITC (CIA-ITC) enables the estimation of thermodynamic parameters in situ. In this work, we demonstrate a label-free and surface modification-free CIA-ITC to determine the complete binding thermodynamics of a ligand with a subnanomolar dissociation constant K D. The K D for desthiobiotin (DTB)-avidin binding was determined to be 6.5 pM with respect to the ligand by CIA-ITC, a quantity unsuccessfully measured with IIA-ITC and surface plasmon resonance spectroscopy (SPR). This value compares well with literature-reported spectroscopic determination of DTB-avidin binding. Criteria with respect to the concentration of the ligand and receptor and flow rate for obtaining true equilibrium dissociation constants without displacement titration are presented.
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