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Standard molecular binding isothermal titration calorimetric (ITC) experiments are designed to get thermodynamic information: changes in Gibbs energy, enthalpy, and entropy associated to the studied process. Traditionally, the kinetic information contained in the ITC raw signal has been ignored. For a usual one-step process, this corresponds to the rate constants for the association and the dissociation of the complex (k and k). The availability of highly sensitive ITC instruments with low response time, together with the development of theoretical methods and of public software for the proper analysis of the signal, cancels any reason for not retrieving this kinetic information. Here we describe how to further exploit ITC experiments of simple one-step interactions by using the software AFFINImeter.The method is exemplified using a standard reference system for thermodynamic and kinetic molecular binding analysis: the interaction of carbonic anhydrase (CA) with its inhibitor 4-carboxybenzenesulfonamide (4-CBS) at several temperatures. It is to be emphasized that old experiments initially designed and executed just for thermodynamic analysis can be readily recycled by using AFFINImeter to retrieve the previously ignored kinetic information.
This article was published in the following journal.
Name: Methods in molecular biology (Clifton, N.J.)
Isothermal titration calorimetry (ITC) has become the preferred experimental technique for characterizing intermolecular interactions between biological molecules. Among the several advantages, the us...
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Substances used for the detection, identification, analysis, etc. of chemical, biological, or pathologic processes or conditions. Indicators are substances that change in physical appearance, e.g., color, at or approaching the endpoint of a chemical titration, e.g., on the passage between acidity and alkalinity. Reagents are substances used for the detection or determination of another substance by chemical or microscopical means, especially analysis. Types of reagents are precipitants, solvents, oxidizers, reducers, fluxes, and colorimetric reagents. (From Grant & Hackh's Chemical Dictionary, 5th ed, p301, p499)
Differential thermal analysis in which the sample compartment of the apparatus is a differential calorimeter, allowing an exact measure of the heat of transition independent of the specific heat, thermal conductivity, and other variables of the sample.
The thermodynamic interaction between a substance and WATER.
A method of chemical analysis based on the detection of characteristic radionuclides following a nuclear bombardment. It is also known as radioactivity analysis. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)
Calculation of the energy expenditure in the form of heat production of the whole body or individual organs based on respiratory gas exchange.