Description of a new Gill titration calorimeter for the study of biochemical reactions. I: assembly and basic response of the instrument

Abstract

A detailed account of the assembly of a thermopile heat-conduction isothermal titration calorimeter to be used in the study of biomolecular interactions is given. The instrument is built according to the twin-cell principle with a sensitivity of 5 pJ. Its reaction vessel volume is only 200 {il. Stirring heats of the reaction vessel are below 5 pJ.

Measurement of Biochemical Affinities with a Gill Titration Calorimeter

“…A detailed description of the isothermal reaction calorimeter used and its operation can be found elsewhere (5,6). The calorimeter is of the thermopile heat-conduction type, built according the twin-cell principle, with two identical reference and reaction cells and a sensitivity of about 1 mcal.…”

“…[5] and pH at which a maximum amount of protons, DN, is [6] is related to the known total macromolecule, [M] T,i , absorbed in the reaction, so that Dn can be measured and ligand, [L] T,i , concentrations in the reaction vessel more precisely. In a typical experiment, 1.5 ml of 0.29 by the expression mM ribonuclease A was placed in a thermostatized glass container at 25 { 0.05ЊC and titrated with 25-ml additions from a Hamilton syringe of 3.1 mM 2-CMP.…”

“…[2] by Eq. [6] and [5], respectively, tion solvent and afterward water-saturated nitrogen the experimental heats, q i , are fitted to obtain the unwas bubbled through the solution during 30 min to known binding parameters n, k, and DH 0 . The nonlineliminate carbon dioxide.…”

“…Immediately after the enIn this paper we study the last model of the titration zyme is collected and in order to keep its activity, the calorimeters designed by Gill and coworkers (5,6) and pH is lowered to 3 by adding 1.5 M sodium formate. The evaluate its capacity to determine simultaneously (in active site concentration in b-trypsin was measured a single experiment) the equilibrium constant and the by titration with p-nitrophenyl-p-guanidinobenzoate enthalpy change of a biomolecular reaction.…”

The role of backlash in the “first injection anomaly” in isothermal titration calorimetry

“…The dependence of the heat q on extent of reaction constitutes a titration curve, the analysis of which yields the enthalpy change DH°and the equilibrium constant K for the reaction [1][2][3]. In ITC experiments, it is common practice to initiate each run with a small ''throwaway'' injection of titrant.…”