Melatonin Binding to Human NQO2 by Isothermal Titration Calorimetry

To ensure the physical interaction between a protein and its ligand, many techniques can be applied. One of them, isothermal titration calorimetry (ITC), measures the heat exchange between a forming molecular complex and its milieu. From this heat exchange, it is possible to acquire the thermodynamic parameters, the binding stoichiometry and the affinity constant (K_a) between the two interacting binding partners, which can then be used to determine the dissociation constant (K_d). We made use of ITC to determine the true K_d of melatonin for its putative receptor MT3, also known as the enzyme quinone reductase 2 (NQO2). In this chapter, we describe the step-by-step procedure for performing this experiment and extend it to 2-iodomelatonin, a melatonin derivative that was used in the initial identification and characterization of MT3. The dissociation constants of melatonin and 2-iodomelatonin toward NQO2 derived from these experiments are in line with data reported previously, albeit using alternative techniques.