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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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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Basic Protocol 1: Isothermal Titration Calorimetry

  • 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

  • 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

  • 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

  • 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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  •   FigureFigure 17.8.1 (A) Basic schematic illustration of the ITC instrument, showing the two cells (sample and reference) surrounded by the thermostated jacket, the injection syringe that also works as stirring device, and the computer‐controlled thermostatic and feedback systems (using Peltier and resistor devices as sensor and actuator subsystems). (B) Example of a typical ITC experiment. The top panel shows the sequence of peaks, each one corresponding to each injection of the solution in the syringe. The monitored signal is the additional thermal power needed to be supplied or removed at anytime to keep a constant temperature in the sample cell and as close as possible to the reference cell temperature. This example corresponds to an endothermic binding. The bottom panel shows the integrated heat plot. The areas under each peak, calculated and normalized per mole of ligand injected in each injection, are plotted against the molar ratio (quotient of the total concentrations of ligand and macromolecule in the sample cell). From this plot, and applying the appropriate model, the thermodynamic parameters of the binding can be obtained: binding affinity, binding enthalpy, and stoichiometry.
  •   FigureFigure 17.8.2 Illustration of the effect of the association constant value on the shape of a titration curve. The plots represent three titrations simulated using the same parameters (concentrations of reactants and binding enthalpy), but different binding affinities. Low (A), moderate (B), and high affinity (C) binding processes are shown. To obtain accurate estimates of the binding affinity an intermediate case is desirable (B, 1 < c = Ka × [M]T < 1000). When the parameter c is large enough, a good estimate of the enthalpy can be determined from the y‐axis intercept of the curve (if the heat effect associated with dilution is used as a reference). Such an intercept value is given by ( c/ c+1) × Δ Ha (Indyk and Fisher, ).
  •   FigureFigure 17.8.3 Titration of RNase A with 2′CMP. The experiment was performed in 15 mM potassium acetate, pH 5.5, at 25°C. The concentration of reactants are 76 µM RNase A and 1.13 mM 2′CMP. The solid line corresponds to the theoretical curve with n = 1.02, Ka = 2.9 × 106 M−1 and Δ H = −19.3 kcal/mol.
  •   FigureFigure 17.8.4 Titration of STI with PPT. The experiment was performed in 25 mM potassium acetate, pH 4.5/10 mM calcium chloride, at 25°C. The concentrations of reactants are 21 µM STI (in cell) and 312 µM PPT (in syringe). The inhibitor was placed in the calorimetric cell due to its low solubility. The solid line corresponds to theoretical curve with n = 1.26, Ka = 1.5 × 106 M−1 and Δ Ha = 8.4 kcal/mol.
  •   FigureFigure 17.8.5 Set of calorimetric titrations corresponding to the implementation of the displacement protocol for the estimation of very high affinity. The concentrations of reactants are: (A) HIV‐1 protease 11 µM in cell and nelfinavir 130 µM in syringe; (B) HIV‐1 protease 19 µM in cell and acetyl‐pepstatin 300 µM in syringe; and (C) HIV‐1 protease 10 µM and acetyl‐pepstatin 200 µM in cell, and nelfinavir 130 µM in syringe. The thermodynamic parameters obtained from these experiments are: nelfinavir binding to protease: Δ Ha = 3.1 kcal/mol, n = 1.02, and Ka cannot be reliably obtained; acetyl‐pepstatin binding to protease: Ka = 2.3 × 106 M−1, Δ Ha = 8.0 kcal/mol, n = 0.98; nelfinavir binding to protease pre‐bound to acetyl‐pepstatin Ka = 2.2 × 109 M−1, Δ Ha = 3.3 kcal/mol, n = 0.99.
  •   FigureFigure 17.8.6 Set of calorimetric titrations corresponding to the implementation of the displacement protocol for the estimation of very low affinity. The concentrations of reactants are: (A) RNase A 76 µM in cell and 2′CMP 1.13 mM in syringe; (B) RNase A 76 µM in cell and 5′CMP 1.07 µM in syringe; and (C) RNase A 76 µM and 5′CMP 600 µM in cell, and 2′CMP 1.13 mM in syringe. The first titration corresponds to the one shown in Figure . In the second titration neither the binding affinity nor the binding enthalpy can be estimated. In the third titration, the thermodynamic parameters for the binding of 5′CMP obtained applying the exact analysis are: Ka = 4250 M−1, Δ Ha = −16.3 kcal/mol, n = 0.99. If using the approximation method, the values obtained for the binding of 5′CMP are: Ka = 4300 M−1, Δ Ha = −15.7 kcal/mol, n = 0.98, which are in agreement with the previous ones.
  •   FigureFigure 17.8.7 Temperature dependence of the binding enthalpy for amprenavir and TMC‐126 binding to HIV‐1 protease (Ohtaka et al., ). The experiments were done in 10 mM sodium acetate, pH 5.0/2%DMSO. The slope of the plot is equal to the heat capacity change upon binding and it is related to the burial of accessible surface areas from both the protein and the ligand. The values of the binding heat capacity for amprenavir and TMC‐126 are −440 cal/K·mol and −350 cal/K·mol, respectively.
  •   FigureFigure 17.8.8 Dependence of the binding enthalpy for amprenavir and TMC‐126 binding to HIV‐1 protease on the ionization enthalpy of the buffer used in the experiment (Ohtaka et al., ). The experiments were done at pH 5.0 in 2% DMSO and 10 mM buffer concentration with buffers of different ionization enthalpy (acetate 0.12 kcal/mol, MES 3.72 kcal/mol, ACES 7.51 kcal/mol). The number of protons exchanged (slope) for amprenavir and TMC‐126 is 0.02 and 0.39, respectively. The buffer‐independent binding enthalpy (intercept with y‐axis) for amprenavir and TMC‐126 is −6.9 kcal/mol and −12.0 kcal/mol, respectively. Unlike in the case of amprenavir, where there is no net proton exchange between the ML complex and bulk solution upon binding, for TMC‐126 there is a significant proton transfer.


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