title: Isothermal titration calorimetry for drug design: Precision of the enthalpy and binding constant measurements and comparison of the instruments.
abstract: Isothermal titration calorimetry (ITC) is one of the most robust label- and immobilization-free techniques used to measure protein - small molecule interactions in drug design for the simultaneous determination of the binding affinity (ΔG) and the enthalpy (ΔH), both of which are important parameters for structure-thermodynamics correlations. It is important to evaluate the precision of the method and of various ITC instrument models by performing a single well-characterized reaction. The binding between carbonic anhydrase II and acetazolamide was measured by four ITC instruments - PEAQ-ITC, iTC200, VP-ITC, and MCS-ITC and the standard deviation of ΔG and ΔH was determined. Furthermore, the limit of an approach to reduce the protein concentration was studied for a high-affinity reaction (Kd= 0.3 nM), too tight to be measured by direct (non-displacement) ITC. Chemical validation of the enthalpy measurements is discussed.
authors.affiliation: Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio 7, Vilnius, LT, 10257, Lithuania.
authors.forename: Vaida
authors.initials: V
authors.lastname: Linkuvienė
authors.affiliation: Department of Molecular Biophysics, University of Kaiserslautern, Kaiserslautern, Germany; B CUBE-Center for Molecular Bioengineering, Technische Universität Dresden, Dresden, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Krainer
authors.affiliation: Department of Chemical and Materials Engineering, National Central University, Taiwan.
authors.forename: Wen-Yih
authors.initials: WY
authors.lastname: Chen
authors.affiliation: Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio 7, Vilnius, LT, 10257, Lithuania. Electronic address: matulis@ibt.lt.
authors.forename: Daumantas
authors.initials: D
authors.lastname: Matulis
mesh_terms: D002151:Calorimetry / Q000379:methods; D024402:Carbonic Anhydrase II / Q000737:chemistry*; D015195:Drug Design; D006801:Humans; D013816:Thermodynamics
chemical_list: D024402:Carbonic Anhydrase II
keywords: Acetazolamide; Calorimeter; Carbonic anhydrase; Enthalpy; Isothermal titration calorimetry (ITC); Nitpic and Sedphat; Protein – ligand binding