A more realistic approach is to inject a solution with a high
heat of dilution (such as 1% v/v ethanol or methanol) into
water in the cell. This method includes the effects of injection
speed and stirring on the measurement. The final portion of
the heat peak produced as the signal recovers to baseline should
fit an exponential that is the time constant of the ITC. It is
likely that injection and stirring speed will influence the values
obtained, and it has been noted that cell cleanliness seems to
surprisingly have a significant effect [6]
16. On removing the syringe to reload while leaving the cell contents in situ, it is important to recalculate the concentrations of
protein and ligand present. During the initial titration, material
is eliminated from the cell and does not re-equilibrate as discussed in Note 3. However, the removal of the syringe to
reload will remix the contents of cell and loading tube giving
a new set of starting conditions for a subsequent titration.
References
1. Sophianopoulos AJ, Vanholde KE (1964)
Physical studies of muramidase (lysozyme).
II. pH-dependent dimerization. J Biol Chem
239:2516–2524
2. Turnbull WB, Daranas AH (2003) On the
value of c: can low affinity systems be studied
by isothermal titration calorimetry? J Am
Chem Soc 125(48):14859–14866. https://
doi.org/10.1021/ja036166s
3. Kantonen SA, Henriksen NM, Gilson MK
(2018) Accounting for apparent deviations
between calorimetric and van’t Hoff enthalpies. Biochim Biophys Acta, Gen Subj 1862
(3):692–704.
https://doi.org/10.1016/j.
bbagen.2017.11.020
4. Kantonen SA, Henriksen NM, Gilson MK
(2017) Evaluation and minimization of uncertainty in ITC binding measurements: heat
error, concentration error, saturation, and stoichiometry. Biochim Biophys Acta, Gen Subj
1861(2):485–498. https://doi.org/10.1016/
j.bbagen.2016.09.002
5. Brautigam CA (2015) Fitting two- and threesite binding models to isothermal titration
calorimetric data. Methods 76:124–136.
https://doi.org/10.1016/j.ymeth.2014.11.
018
6. Dumas P, Ennifar E, Da Veiga C, Bec G,
Palau W, Di Primo C, Pineiro A, Sabin J,
Munoz E, Rial J (2016) Extending ITC to
kinetics with kinITC. Methods Enzymol
567:157–180. https://doi.org/10.1016/bs.
mie.2015.08.026
7. Le VH, Buscaglia R, Chaires JB, Lewis EA
(2013) Modeling complex equilibria in isothermal titration calorimetry experiments:
thermodynamic parameters estimation for a
three-binding-site model. Anal Biochem 434
(2):233–241. https://doi.org/10.1016/j.ab.
2012.11.030
8. Vega S, Abian O, Velazquez-Campoy A (2015)
A unified framework based on the binding
polynomial for characterizing biological systems by isothermal titration calorimetry. Methods 76:99–115. https://doi.org/10.1016/j.
ymeth.2014.09.010
9. Zhao H, Piszczek G, Schuck P (2015) SEDPHAT--a platform for global ITC analysis and
global multi-method analysis of molecular
interactions. Methods 76:137–148. https://
doi.org/10.1016/j.ymeth.2014.11.012
10. Freiburger L, Auclair K, Mittermaier A (2015)
Global ITC fitting methods in studies of protein allostery. Methods 76:149–161. https://
doi.org/10.1016/j.ymeth.2014.12.018
11. Olsson TS, Ladbury JE, Pitt WR, Williams MA
(2011) Extent of enthalpy-entropy compensation in protein-ligand interactions. Protein Sci
20(9):1607–1618. https://doi.org/10.1002/
pro.692
12. Chodera JD, Mobley DL (2013) Entropyenthalpy compensation: role and ramifications
in biomolecular ligand recognition and design.
Annu Rev Biophys 42:121–142. https://doi.
org/10.1146/annurev-biophys-083012130318
158
Christopher M. Johnson
heat of dilution (such as 1% v/v ethanol or methanol) into
water in the cell. This method includes the effects of injection
speed and stirring on the measurement. The final portion of
the heat peak produced as the signal recovers to baseline should
fit an exponential that is the time constant of the ITC. It is
likely that injection and stirring speed will influence the values
obtained, and it has been noted that cell cleanliness seems to
surprisingly have a significant effect [6]
16. On removing the syringe to reload while leaving the cell contents in situ, it is important to recalculate the concentrations of
protein and ligand present. During the initial titration, material
is eliminated from the cell and does not re-equilibrate as discussed in Note 3. However, the removal of the syringe to
reload will remix the contents of cell and loading tube giving
a new set of starting conditions for a subsequent titration.
References
1. Sophianopoulos AJ, Vanholde KE (1964)
Physical studies of muramidase (lysozyme).
II. pH-dependent dimerization. J Biol Chem
239:2516–2524
2. Turnbull WB, Daranas AH (2003) On the
value of c: can low affinity systems be studied
by isothermal titration calorimetry? J Am
Chem Soc 125(48):14859–14866. https://
doi.org/10.1021/ja036166s
3. Kantonen SA, Henriksen NM, Gilson MK
(2018) Accounting for apparent deviations
between calorimetric and van’t Hoff enthalpies. Biochim Biophys Acta, Gen Subj 1862
(3):692–704.
https://doi.org/10.1016/j.
bbagen.2017.11.020
4. Kantonen SA, Henriksen NM, Gilson MK
(2017) Evaluation and minimization of uncertainty in ITC binding measurements: heat
error, concentration error, saturation, and stoichiometry. Biochim Biophys Acta, Gen Subj
1861(2):485–498. https://doi.org/10.1016/
j.bbagen.2016.09.002
5. Brautigam CA (2015) Fitting two- and threesite binding models to isothermal titration
calorimetric data. Methods 76:124–136.
https://doi.org/10.1016/j.ymeth.2014.11.
018
6. Dumas P, Ennifar E, Da Veiga C, Bec G,
Palau W, Di Primo C, Pineiro A, Sabin J,
Munoz E, Rial J (2016) Extending ITC to
kinetics with kinITC. Methods Enzymol
567:157–180. https://doi.org/10.1016/bs.
mie.2015.08.026
7. Le VH, Buscaglia R, Chaires JB, Lewis EA
(2013) Modeling complex equilibria in isothermal titration calorimetry experiments:
thermodynamic parameters estimation for a
three-binding-site model. Anal Biochem 434
(2):233–241. https://doi.org/10.1016/j.ab.
2012.11.030
8. Vega S, Abian O, Velazquez-Campoy A (2015)
A unified framework based on the binding
polynomial for characterizing biological systems by isothermal titration calorimetry. Methods 76:99–115. https://doi.org/10.1016/j.
ymeth.2014.09.010
9. Zhao H, Piszczek G, Schuck P (2015) SEDPHAT--a platform for global ITC analysis and
global multi-method analysis of molecular
interactions. Methods 76:137–148. https://
doi.org/10.1016/j.ymeth.2014.11.012
10. Freiburger L, Auclair K, Mittermaier A (2015)
Global ITC fitting methods in studies of protein allostery. Methods 76:149–161. https://
doi.org/10.1016/j.ymeth.2014.12.018
11. Olsson TS, Ladbury JE, Pitt WR, Williams MA
(2011) Extent of enthalpy-entropy compensation in protein-ligand interactions. Protein Sci
20(9):1607–1618. https://doi.org/10.1002/
pro.692
12. Chodera JD, Mobley DL (2013) Entropyenthalpy compensation: role and ramifications
in biomolecular ligand recognition and design.
Annu Rev Biophys 42:121–142. https://doi.
org/10.1146/annurev-biophys-083012130318
158
Christopher M. Johnson