6. Wash the cell-loading syringe, the calorimeter cell, and the
injector syringe extensively with standard assay buffer.
7. Empty the cell and the injector syringe completely by repeated
aspiration to leave them as dry as practicable.
8. Fill the calorimeter cell with the HEWL solution, then the
injector syringe with the NAG3 solution, according to the
procedures recommended by the manufacturer (see Note 31).
9. Insert the injector into the calorimeter cell.
10. Set up the instrumental parameters (see Note 32), enter the
correct concentrations for both species, and start the experiment, making sure to check that the differential power baseline
settles close to the expected value, indicating that the cell is
correctly filled (see Note 33).
3.5.2 ITC Data Analysis
and Typical Results
The aim is to analyze the ITC titration to obtain fitted values for the
dissociation constant K d and the enthalpy of association ΔH.
1. A differential power baseline should be fitted to the raw differential power vs. time data. The curve should be integrated, and
the heats should be converted into molar heats by dividing by
the amount of NAG3 in each injection (see Note 34).
2. The resulting binding isotherm should be fitted by leastsquares nonlinear regression to a 1:1 binding model (see Note
35).
3. Fit the K a (transform the fitted value to K d according to
K a ¼ 1/K d ), the molar enthalpy of association, and either a
stoichiometric ratio (“n value”), an incompetent fraction of
NAG3, or a concentration correction for NAG3 (depending
on the software used for the analysis).
4. The heat of dilution should be accounted for either by measuring it in an independent experiment or estimating the value
from the heats in the final injections, then subtracting it from
the molar heats of injection, or by allowing the asymptotic
value of the molar heat at an infinite concentration of titrant
to be determined in the fit.
Two example datasets are shown in Fig. 6; the data were
acquired using (a) a large cell-volume calorimeter (Malvern Panalytical VP-ITC) and (b) a small cell-volume (Malvern Panalytical
iTC200) calorimeter. The data were integrated and baseline subtracted using the software NITPIC [21, 22] and then individually
fitted to 1:1 binding models using the software Sedphat [23].
The data in Fig. 6a gave a best-fit value for K d of 6.6 μM with a
95% confidence interval of 6.4–6.8 μM and a best-fit value for ΔH
of À13.5 kcal/mol with a 95% confidence interval of À13.6 to
À13.4 kcal/mol. A concentration correction factor of 0.956 for
NAG3 and the heat of dilution were both fitted to the data. The
data in (b) gave a best-fit value for K d of 7.3 μM with a 95%
Interactions by Multiple Methods
65
injector syringe extensively with standard assay buffer.
7. Empty the cell and the injector syringe completely by repeated
aspiration to leave them as dry as practicable.
8. Fill the calorimeter cell with the HEWL solution, then the
injector syringe with the NAG3 solution, according to the
procedures recommended by the manufacturer (see Note 31).
9. Insert the injector into the calorimeter cell.
10. Set up the instrumental parameters (see Note 32), enter the
correct concentrations for both species, and start the experiment, making sure to check that the differential power baseline
settles close to the expected value, indicating that the cell is
correctly filled (see Note 33).
3.5.2 ITC Data Analysis
and Typical Results
The aim is to analyze the ITC titration to obtain fitted values for the
dissociation constant K d and the enthalpy of association ΔH.
1. A differential power baseline should be fitted to the raw differential power vs. time data. The curve should be integrated, and
the heats should be converted into molar heats by dividing by
the amount of NAG3 in each injection (see Note 34).
2. The resulting binding isotherm should be fitted by leastsquares nonlinear regression to a 1:1 binding model (see Note
35).
3. Fit the K a (transform the fitted value to K d according to
K a ¼ 1/K d ), the molar enthalpy of association, and either a
stoichiometric ratio (“n value”), an incompetent fraction of
NAG3, or a concentration correction for NAG3 (depending
on the software used for the analysis).
4. The heat of dilution should be accounted for either by measuring it in an independent experiment or estimating the value
from the heats in the final injections, then subtracting it from
the molar heats of injection, or by allowing the asymptotic
value of the molar heat at an infinite concentration of titrant
to be determined in the fit.
Two example datasets are shown in Fig. 6; the data were
acquired using (a) a large cell-volume calorimeter (Malvern Panalytical VP-ITC) and (b) a small cell-volume (Malvern Panalytical
iTC200) calorimeter. The data were integrated and baseline subtracted using the software NITPIC [21, 22] and then individually
fitted to 1:1 binding models using the software Sedphat [23].
The data in Fig. 6a gave a best-fit value for K d of 6.6 μM with a
95% confidence interval of 6.4–6.8 μM and a best-fit value for ΔH
of À13.5 kcal/mol with a 95% confidence interval of À13.6 to
À13.4 kcal/mol. A concentration correction factor of 0.956 for
NAG3 and the heat of dilution were both fitted to the data. The
data in (b) gave a best-fit value for K d of 7.3 μM with a 95%
Interactions by Multiple Methods
65