Interactions between trypsin and its peptidic inhibitors studied by isothermal titration calorimetry (ITC)

Abstract

Isothermal titration calorimetry (ITC) technique was used to study the interactions of trypsin with bicyclic sunflower-derived trypsin inhibitor (SFTI-1) as well as with its new monocyclic (with disulphide bridge only) analogues (C 3 H 5 O)-SFTI-1 and (C 8 H 15 O)-SFTI-1. ITC measurements were run in 50 mM buffer solution of HEPES or Tricine of pH 8, containing 20 mM CaCl 2 at 298.15 K. Based on calorimetric data, the equilibrium constants for the inhibitor-enzyme-binding processes, K, the binding stoichiometry, N (inhibitor-to-enzyme molar ratio), as well as thermodynamic parameters (DG, DH, DS) for the reactions were determined. The study revealed that the stoichiometry of the resulting complexes equals 1:1. The negative binding enthalpy (D ITC H) and favourable entropy factor (TD ITC S) suggest an important contribution of hydrogen bonding as well as hydrophobic interactions to the inhibitor-enzyme affinity. Furthermore, the relationship between the modification of the peptide structure, the experimental conditions and the thermodynamic parameters has been discussed.

Origin and Formation Mechanism Investigation of Compound Precipitation from the Traditional Chinese Prescription Huang-Lian-Jie-Du-Tang by Isothermal Titration Calorimetry

“…Isothermal titration calorimetry (ITC) was used in order to explore the mechanism of formation of the CFP [ 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 ]. ITC is a physical technique used to determine the thermodynamic parameters of interactions in solution.…”

“…By analysing the relationship between Δ H and T Δ S , the driving form of precipitation could be identified. A negative enthalpy and positive entropy suggest an advantageous contribution to reactions [ 16 ]. A huge energy change was observed in the reaction process between the decoctions of Scutellaria baicalensis and Coptis chinensis and the changes found were very large, indicating the existence of chemical reactions between the main components of Scutellaria baicalensis and Coptis chinensis .…”

Tissue Kallikrein Inhibitors Based on the Sunflower Trypsin Inhibitor Scaffold – A Potential Therapeutic Intervention for Skin Diseases

“…Due to the potential flaws in IC 50 values, it is a common practice in early stage drug discovery to validate hits by orthogonal assays and determine the true K d of optimized lead molecules using standard techniques such as ITC. Interestingly, a recent report has determined the ITC K d of native SFTI for bovine trypsin to be 7-13nM [40], which underlines the fact that assay values can differ greatly depending on the assay conditions and measurement technique used.…”

“…Under the test conditions (1xPBS pH 8.0 at 25°C), the binding of all the tested SFTI-1 analogues to KLK5 was endothermic. This is an interesting contrast to the binding of native SFTI with bovine trypsin, which was found to be exothermic [40] albeit the two serine proteases share a highly conserved structural scaffold. A small positive enthalpy (ΔH) but large positive entropy (ΔS) change implies their binding was predominantly entropically driven.…”

Buffer contribution to formation enthalpy of copper(II)–bicine complex determined by isothermal titration calorimetry method

“…Moreover, if a process of complex formation is accompanied with proton transfers, the equivalent number of protons is taken up or released by the buffer. It generates an additional heat that is proportional to the enthalpy of buffer ionization [12,13]. In this case, the enthalpy measured during the ITC experiment reflects both the buffer ionization and complex formation [14].…”