Number: 2001-030-1-100
Title: Recommendations on the measurement and
analysis of results obtained on biological substances with isothermal
titration calorimetry
Task Group
Chairmen: Frederick
P. Schwarz and Hans-Jurgen
Hinz
Completion Date: 2008 - project completed
Objective:
Recommendations for isothermal titration calorimetry measurement procedures
on biological substances, calibration procedures, and procedures for
testing the performance of isothermal titration calorimeters (ITCs)
with a biological test solution are described. Recommendations for
the analysis and reporting of the results will be presented to facilitate
universal comparability of ITC data from different laboratories.
Description:
Isothermal titration calorimeters (ITCs) have been widely used
in the biotechnological and pharmaceutical industry and in academia
to determine the thermodynamics of binding reactions of biological
substances, including DNA-DNA, DNA-RNA, protein-protein, protein-ligand,
and DNA-intercalating drug interactions. ITCs monitor the power exchanged
between a reference vessel and a solution vessel as aliquots of a
titrant solution are injected into the solution vessel, isothermally.
The enthalpy change, binding affinity, and the stoichiometry for the
binding reaction are obtained from an analysis of the power exchanged
per each injection and the total concentrations of the reactants in
the solution vessel. In addition, the temperature dependence of these
quantities can be determined in terms of a heat capacity change for
the binding reaction. Thus, the thermodynamics of the binding reaction
can be completely characterized over a wide temperature range.
Published results sometimes lack a systematic reporting
and analysis of the data and often employ a variety of notation inconsistent
with IUPAC recommendations. An important aspect of this project is
the fabrication of a test solution for determination of ITC binding
data. ITC test solutions will be distributed to members of the working
party to run in their ITCs so that a consensus set of binding data
can be developed on a particular system under a specified set of operating
conditions. This consensus data can then be used to determine if a
particular ITC is being operated properly. This is particularly important
for new and in-experienced researchers employing ITC for the first
time. The results will also be shared with the MIRG committee of the
Association of Biomolecular Research Facilities (ABRF). MIRG is involved
in evaluating how well binding affinities determined by ITC, Biacore
SPR, and Ultra-analytical centrifugation are in agreement.
Progress:
June 2005 - Measurements are complete for a working standard
NAD/NADH binding to a protein, lactate-dehydrogenase for checking
the performance of isothermal titration calorimeters. The first draft
of the report is will be complete by December 2005. The round-robin
ITC results from 12 laboratories on the binding of 4 carboxybenzene
sulfonamide to carbonic anhydrase are complete and they are being
evaluated for inclusion in the IUPAC Recommendations.
Sep 2008 - Project completed - IUPAC Technical Report published in Pure Appl. Chem., 2008, Vol. 80, No. 9, pp. 2025-2040
Last update: 19 September 2008