Titration Microcalorimetry

Application to Measurements of Heat of Ca²⁺-Association with Ca²⁺-Binding Proteins

Abstract

A microcomputer-controlled system of a batch microcalorimeter equipped with a titration apparatus was described. Using this system, enthalpy titration profiles of bovine brain calmodulin by Ca²⁺ were observed both in the absence and in the presence of Mg²⁺, and at 5, 15 and 25°C. Titrations of calmodulin with Ca²⁺ in the absence of Mg²⁺ showed that the Ca²⁺-binding reaction is endothermic and thus is driven solely by the entropy change. Following the method of Sturtevant [Proc. Natl. Acad. Sci. U. S. 74, 2236 (1977)], the magnitudes of the hydrophobic and intramolecular vibrational contributions to the entropy change of calmodulin on Ca²⁺-binding were estimated. Ca²⁺-binding to calmodulin gives rise to an increase in hydrophobic entropy and thus to an “assembling” of nonpolar groups. Ca²⁺-binding also gives rise to an increase in vibrational entropy, indicating a “softening” of the overall structure of calmodulin molecule.