Isothermal titration calorimetry at very low c

In the study of 1:1 binding, M + X right arrow over left arrow MX, isothermal titration calorimetry (ITC) can be used successfully at values of c=K[M](0) well below the value 1.0 that is often considered its lower limit. However, analysis of low-c ITC data may require freezing the stoichiometry parameter n, and that is thought to be prohibitive for biological systems, where n can be poorly known. Here it is noted that the least-squares estimates of the binding constant K are virtually independent of errors in n at low c, permitting reliable determination of K and, from its temperature dependence, DeltaH degrees and n, down to c=10(-4) or lower, ligand solubility permitting.