2.7. Isothermal Titration Calorimetry Binding Assays

Assays were performed on a ITC200 instrument (MicroCal, Northampton, MA). The experimental titration was carried out in the boric acid buffer system containing 0.3 M NaCl pH 8.3, and the ACE of the 75–125 nm of the buffer was dissolved in the hot pool. The temperature of the sample cell was set at 25∼30°C, the total titration times were 20, and the interval time was 60 s. Catalpol was titrated after the automatic balance of the calorimeter was completed. The thermodynamic parameters of the reaction (ΔG, ΔH, and ΔS) and dissociation constant K_d were calculated using Origin 7.0 with iTC200 MicroCal software.