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Biophys Chem. 2004 May 1;109(2):229-49.

Isothermal acid-titration calorimetry for evaluating the pH dependence of protein stability.

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  • 1Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka 940-2188, Japan.


A new method, which can be called as isothermal acid-titration calorimetry (IATC), was proposed for evaluating the enthalpy of protein molecules as a function of pH using isothermal titration calorimetry (ITC). This measurement was used to analyze the acid-denaturation of bovine ribonuclease A. The enthalpy change by acid-denaturation of this protein was estimated as 310 kJ/mol at pH 2.8 and 40 degrees C. This value agreed well with the enthalpy change obtained by differential scanning calorimetry. The midpoint pH and proton binding-number difference observed by IATC agreed well with those of the acid transition of the three-dimensional structure monitored by circular dichroism spectrometry. The van't Hoff enthalpy of the transition was derived from the temperature dependence of the midpoint pH and the proton binding-number difference. It agreed well with the calorimetric enthalpy change directly observed by IATC, strongly indicating that there was no stable intermediate state during the acid transition of this protein.

Copyright 2003 Elsevier B.V.

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