Origin 7 sr4

ITC analysis was used to determine the Ca²⁺ binding properties of EDTA, EGTA and EDDS using Microcal model ITC200 calorimeter (cell volume = 200 μL), with ITC200 software version 1.24.2 (MicroCal Inc., USA). Data acquisition and graphical analysis was achieved using Origin 7 SR4, v7.0552 software (OriginLab Corporation, USA). Water was placed in the reference cell and 10 μCal s⁻¹ selected for reference power. 0.4 mM solutions of EDTA, EGTA and EDDS in 10 mM MES buffer (pH 7.4 ± 0.2) were titrated by CaCl₂ in 10 mM MES buffer (pH 7.4 ± 0.2). Calcium chloride titrant at 6 mM was used for EDTA and EGTA, a higher concentration at 25 mM was used for EDDS in order to reach saturation. A method with an initial injection of 0.4 μL followed by 15 injections of 2 μL with spacing of 150 s; analysis was carried out at 25 °C.

Isothermal titration calorimetry (ITC) experiments were carried out on the MicroCal™ iTC200 instrument (Malvern Panalytical). Experiments were performed in 50 mM Tris-HCl (pH 7.5) and 50 mM NaCl buffer. All titrations were performed at 25°C using 70–120 μM protein. 280 μL of the protein solution in the sample cell were titrated with the dsDNA solution diluted to 35–50 μM depending on the protein concentration (to achieve a 2:1 ratio). The concentrations of proteins and dsDNA were determined by absorbance on a NanoDrop 2000c UV–Vis spectrometer (Thermo Fisher Scientific). Each titration started with a small 0.3 μL injection introduced to compensate the diffusion effects followed by 20×2.0 μL injections with 120 s time spacing between each injection. The steering speed was set to 600 rpm and the reference cell was filled with Milli Q water (Merck). The reference baseline consisting of small peaks of identical size (titration of the corresponding dsDNA into the buffer) was subtracted from the data before fitting to correct for the heat of the dsDNA dilution. ITC data were analyzed using Origin 7 SR4 (OriginLab). The first small-volume data point (used to compensate effects of titrant diffusion across the syringe tip during the equilibration process) was always removed prior to data fitting. “One Set of Sites” fitting model was applied.