A discussion on the affecting factors of the fitting procedures' reliability in isothermal titration calorimetry analysis.

The standard isothermal titration calorimetry (ITC) curve, characterized as a typical sigmoid is strictly confined by the so-called c value, which is a ratio of titrand concentration to KD. The proper c value with a range from 5 to 500 is commonly recommended as a standard protocol in routine detection process for acquiring the reliable fitting results in 1:1 binding mode. However, if the c value is less than "1" due to the weak binding or low concentration of analyte, fitting precision gets unstable and susceptible to the data noise. Herein, we first got a deep discussion into the reliability of the fitting procedure for 1:1 binding mode by data simulation, then quantized the effect of several affecting factors on the precision of parameters estimation through mathematical analysis. Finally, we proposed the value of 2~4 times KD for final ligand concentration is optimal for the ITC titration in low c system (c < 1). All the theoretical derivations were further verified by a practical experiment of Magnesium-EDTA binding test.