Isothermal titration calorimetry (ITC) is widely used to determine the thermodynamics of biologicalinteractions in solution from measurements of the heat detected upon titrating a solution of one reactantinto a solution of the other reactant of a binding interaction. The range of interactions studied by ITC isvery general and covers, although not exclusively, drug-protein, metal ion-protein, sugar-protein,inhibitor-enzyme, DNA-protein, receptor-protein, lipid-protein, antigen-antibody, and protein-proteininteractions. The binding affinity, the binding enthalpy, and the stiochiometry of the binding reaction aredetermined from the fit of a binding model to the binding isotherm generated from the ITC data.Recently, research has focused on the development of standard binding reactions to validate theaccuracy of the ITC measurements. In an ABRF study (1) the interaction of the enzyme bovine carbonicanhydrase II with its inhibitor, 4-carboxybenzenesulfonamide, was investigated by ITC, analyticalultracentrifugation, and surface plasmon resonance. The excellent agreement of the thermodynamicbinding parameters determined from the different methods not only validated the accuracy of the ITCmethod but also showed that this system would be an excellent protein-small ligand standard bindingreaction for evaluating the performance of an ITC. Preliminary results on the development of additionalstandard binding reactions will also be presented and discussed. This discussion will include a proteinproteinbinding reaction between the mutants of barnase and their barstar mutant inhibitors and themulti-binding cofactor NADH to lactate dehydrogenase. The discussion will emphasize the requirementsof an ideal standard binding reaction for evaluation of the operating performance of an ITC.
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