Continuous Injection Isothermal Titration Calorimetry for In Situ Evaluation of Thermodynamic Binding Properties of Ligand-Receptor Binding Models.

We utilize a continuous injection approach (CIA) rather than the traditional incremental injection approach (IIA) to deliver ligand (or receptor) to the calorimeter cell to evaluate thermodynamic binding parameters for three common ligand-receptor binding models-single independent, competitive, and two independent binding sites-using isothermal titration calorimetry (ITC). A general mathematical expression for the binding isotherm for any binding stoichiometry under continuous delivery of ligand (or receptor) resulting in an analytical solution for the thermodynamic binding parameters is presented. The advantages of CIA include reduction in experimental time, estimation of thermodynamic binding parameter values, and automation of the experiment since thermodynamic parameters are estimated in situ. We demonstrate the inherent advantages of CIA over IIA for the three binding models. For the single independent site model, we utilized the binding of Ba2+ ions to ethylenediaminetetraacetic acid (EDTA), while competitive binding was captured by titration of Ca2+ ions into a buffered solution of Ba2+ and EDTA. We experimentally simulated a two independent binding site system by injecting Ca2+ into a solution of EDTA and 1,3-diaminopropane-N,N,N',N'-tetraacetic acid (DPTA). The results demonstrate estimation of thermodynamic parameters with greater confidence and simultaneous reduction in the experimental time of 83% and titrating reagent of 50%, as compared to IIA.