Isothermal Titration Calorimetry

Adrián Velázquez‐Campoy1, Hiroyasu Ohtaka1, Azin Nezami1, Salman Muzammil1, Ernesto Freire1

1 Johns Hopkins University, Baltimore, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.8
DOI:  10.1002/0471143030.cb1708s23
Online Posting Date:  September, 2004
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Abstract

In the last two decades, isothermal titration calorimetry (ITC) has become the preferred technique to determine the binding energetics of biological processes, including protein‐ligand binding, protein‐protein binding, DNA‐protein binding, protein‐carbohydrate binding, protein‐lipid binding, and antigen‐antibody binding. In this unit several protocols are presented, ranging from the basic ones that are aimed at characterizing binding of moderate affinity to advanced protocols that are aimed at determining very high or very low affinity binding processes. Also, alternate protocols for special cases (homodimeric proteins and unstable proteins) and additional information accessible by ITC (heat capacity and protonation/deprotonation processes coupled to binding) are presented.

Keywords: isothermal titration calorimetry; thermodynamics; binding; macromolecule‐macromolecule interactions

     
 
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Table of Contents

  • Principles of the Technique
  • Instrumentation
  • Basic Protocol 1: Isothermal Titration Calorimetry
  • Support Protocol 1: Data Analysis for ITC Experiments
  • Basic Protocol 2: Macromolecule/Ligand Interaction with Moderate Affinity
  • Basic Protocol 3: Macromolecule/Macromolecule Interaction with Moderate Affinity
  • Alternate Protocol 1: Macromolecule/Ligand Binding with High Affinity
  • Alternate Protocol 2: Macromolecule/Ligand Binding with Low Affinity
  • Alternate Protocol 3: Binding of Homodimeric Proteins
  • Alternate Protocol 4: Binding of Unstable Proteins
  • Alternate Protocol 5: Measuring the Heat Capacity Change Associated with Binding
  • Alternate Protocol 6: Measuring Protonation/Deprotonation Processes Coupled to Binding
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isothermal Titration Calorimetry

  Materials
  • Reactant solutions: macromolecule and ligand
  • Methanol
  • VP‐ITC calorimeter (e.g., Microcal LLC or equivalent)
  • Vacuum pump
  • 2.5‐ml long‐needle syringe (e.g., Hamilton)
  • 12 × 75–mm and 6 × 50–mm glass tubes

Support Protocol 1: Data Analysis for ITC Experiments

  Materials
  • RNase A, lyophilized powder (Sigma)
  • 15 mM potassium acetate buffer, pH 5.5
  • 2′CMP, lyophilized powder (Sigma)
  • 12 × 75–mm and 6 × 50–mm glass tubes
  • 6‐kDa MWCO dialysis membrane
  • 0.22‐µm filter

Basic Protocol 2: Macromolecule/Ligand Interaction with Moderate Affinity

  Materials
  • Porcine pancreatic trypsin (PPT), lyophilized powder (Sigma)
  • 25 mM potassium acetate/10 mM calcium chloride, pH 4.5
  • Soybean trypsin inhibitor (STI), lyophilized powder (Sigma)
  • 10‐kDa MWCO dialysis tubing
  • 0.22‐µm filter

Basic Protocol 3: Macromolecule/Macromolecule Interaction with Moderate Affinity

  Materials
  • Acetyl pepstatin (Bachem)
  • 9 mM NaOH
  • Nelfinavir (Viracept; or any other clinical or experimental inhibitor)
  • 100% DMSO
  • HIV‐1 protease (Todd et al., and Velazquez‐Campoy et al., )
  • 10 mM sodium acetate, pH 5.0/2% (v/v) DMSO
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Figures

Videos

Literature Cited

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