MOL.865
Biophysikalische Methoden
ITC Tutorial
Isothermal titration calorimetry (ITC) is an accurate method to evaluate heat changes evolving
from
interaction
processes.
In
this
lab
tutorial
the
binding
properties
of
B. subtilis Pdx1 and Pdx2 H170N (glutaminase deficient mutant) are studied in the presence
of 1 mM L-glutamine. A binding isotherm is generated by performing two runs (measurement
and reference run) and data processing is done applying Origin 7.0 software for ITC data
analysis (MicroCal, U.S.A). A VP-ITC MicroCalorimeter (MicroCal, U.S.A) is used to
perform the experiments.
Materials:

200 µM Pdx2 H170N solution

20 µM Pdx1 solution

1 mM L-glutamine

20 mM Tris-HCl, 10 mM NaCl, pH 7.5
Procedure:
Pdx1 and L-glutamine are provided in the cell and Pdx2 H170N is used as titrant and filled
into the 250 µL syringe. In a typical experiment 550 µL of the Pdx2 solution are prepared for
loading the syringe and 2 mL of 20 µM Pdx1 and 1 mM L-glutamine are applied for filling
the cell. In case of the reference measurement 200 µM Pdx2 is titrated into buffer and 1 mM
L-glutamine. All solutions are prepared in the respective tubes and are degassed shortly
before each run. All measurements are performed at 25 °C.
Experimental Setup:
Experimental Parameters
Total Injections
Cell Temperature
Reference Power
Initial Delay
Syringe Concentration
Cell Concentration
Stirring Speed
Feedback Mode / Gain
ITC Equilibration Options
20
25 °C
12 µCal sec-1
60 sec
0.20 mM
0.02 mM
270 rpm
High
Fast Equilibration
Auto
Injection Parameters
1x
Volume
Duration
Spacing
Filter
2 µL
4 sec
250 sec
2 sec
19 x
Volume
Duration
Spacing
Filter
15 µL
29.9 sec
250 sec
2 sec
MOL.865
Biophysikalische Methoden
Results:
Raw data from ITC experiments are processed with Origin 7.0 software for ITC data analysis
(MicroCal, U.S.A). Baseline adjustment is done manually for each measurement. The binding
isotherm is then observed by integrating the obtained peaks and by plotting the resulting heats
of each injection against the ratio of titrant to the Pdx1 protein concentration. Various
thermodynamic parameters (N, Ka, ΔH, ΔS) are determined by applying non linear least
squares fitting. The heats of dilution are considered by subtracting the integrated values for
each injection of the reference measurement from the heats calculated by integration for the
respective experiment.
Kd, which has a direct correlation to the Gibbs free energy ΔG, is calculated from the obtained
Ka value according to the following equation:
Kd 
1
Ka
G   RT ln K a  RT ln K d
Tasks:
1. A measurement to investigate the binding properties of Pdx1 and Pdx2 in presence of
L-glutamine is performed at 25 °C. (see Procedure for a detailed description)
2. A reference measurement has to be performed at 25 °C.
3. Raw Data are analyzed with Origin 7.0 software for ITC data analysis
4. Baseline correction is done manually for both measurements.
5. A binding isotherm has to be generated, where the heats of dilution are considered.
( Reference subtraction)
6. The thermodynamic parameters N, Kd, ΔH, -TΔS and ΔG are determined.