2.5. ITC determination of equilibrium dissociation constant (KDITC)

Isothermal titration calorimetry (ITC) was used to measure thermodynamic parameters for ScpAS512A-rhC5a binding in solution. Measurements were performed using an iTC200 calorimeter (MicroCal, U.S.A.) equipped with a 200 μL sample cell (GE Healthcare). Purified protein samples were extensively dialysed against 50 mM Hepes-KOH buffer, pH 7.5 containing 150 mM NaCl. The reference power was set to 4.5 μcal/sec and the instrument was equilibrated to 25 °C. ScpAS512A (18 μM) was placed in the sample cell and titrated with 170 μM rhC5a in 2.5 μL injections with 120 s interval between the injections and stirring (1000 rpm). Titration data were collected in triplicate and analysed with the ORIGIN software package (MicroCal, U.S.A.). The peaks areas were integrated and corrected for the heat of rhC5a dilution. Plots of kcal/mol of injectant versus molar ratio were analyzed with the ‘one set of sites’ model to obtain values for the association binding constant (KA), stoichiometry (N), and the enthalpy of binding (ΔHbind), entropy of binding (ΔSbind) were determined. The reported equilibrium dissociation constant (KDITC) was derived from KA.

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