Avoiding Buffer Interference in ITC Experiments: A Case Study from the Analysis of Entropy-Driven Reactions of Glucose-6-Phosphate Dehydrogenase

Methods in Enzymology
M Lucia Bianconi

Abstract

Isothermal titration calorimetry (ITC) is a label-free technique that allows the direct determination of the heat absorbed or released in a reaction. Frequently used to determining binding parameters in biomolecular interactions, it is very useful to address enzyme-catalyzed reactions as both kinetic and thermodynamic parameters can be obtained. Since calorimetry measures the total heat effects of a reaction, it is important to consider the contribution of the heat of protonation/deprotonation that is possibly taking place. Here, we show a case study of the reaction catalyzed by the glucose-6-phosphate dehydrogenase (G6PD) from Leuconostoc mesenteroides. This enzyme is able to use either NAD(+) or NADP(+) as a cofactor. The reactions were done in five buffers of different enthalpy of protonation. Depending on the buffer used, the observed calorimetric enthalpy (ΔH(cal)) of the reaction varied from -22.93 kJ/mol (Tris) to 19.37 kJ/mol (phosphate) for the NADP(+)-linked reaction, and -11.67 kJ/mol (Tris) to 7.32 kcal/mol or 30.63 kJ/mol (phosphate) for the NAD(+) reaction. We will use this system as an example of how to extract proton-independent reaction enthalpies from kinetic data to ensure that the reported accurately represe...Continue Reading

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