Affinity nano-ITC: affinity nano-isothermal titration calorimetry

Affinity ITC measures the heat released or absorbed by the stepwise additions of a molecule solution (titrant) to a solution containing a specific (bio)macromolecule (Fig. 1A). The heat exchanged is registered as a peak in the enthalpogram, and the area of the peak normalized for the injected quantity (ΔH, kJ/mol) generates a point of the binding isotherm (Fig. 1B). A single measurement allows for the determination of the reaction stoichiometry and the thermodynamic parameters (affinity constant Ka, ΔH, ΔS) characterizing the binding affinity. Titrations at different temperatures or different buffers allow to obtain the change in heat capacity associated to the ligand binding (ΔCp) or the number of exchange protons, respectively.

Regarding the titration cell (Fig. 1C), the combination of shape (cylindrical), volume (1 mL) and material (Au) has many advantages, including avoiding death volume and trapped bubbles commonly present in competitive design (i.e. coin cell), titrant efficiently mixing with generation of sharper peaks and very stable baseline, high thermal conductivity and inert chemical property. Moreover, thanks to the stirrer (FlexSpin) shape and injection cannula position, stirring speed to mix titrant and solution is 10X lower than competitive design, preserving the integrity of delicate macromolecular structures. The injection system (Fig. 1D) provides several advantages including high accuracy of measurement, minimization of the inject diffusion, decrease in the equilibration time by 40%, semi-automated procedures.

Theory and Applications – Instrumentation Basics
Marco Coletti (Senior Manager · EMEA Scientific Operations · TA Instruments)