titration. This can be seen on close inspection of control heats
as a small linear change in the peak integral with injection
number. In this case these values should be fitted to a linear
function which is used to correct the experimental data prior to
fitting.
13. It is highly informative to examine the signal from an ITC
instrument injecting water from the syringe into water in the
cell as this will give users an idea of the optimal signal-to-noise
that can be expected when there is no binding or dilution heats.
Changes in this signal may be diagnostic of mechanical problems (bent syringe needle) or difficulty with total fill operation
that may arise when the cell requires cleaning. In addition, the
amplitude of the water–water injections will depend on the
volume of the injection, the speed with which the injection is
made and the temperature of measurement in the ITC. This
reflects the fact that the syringe itself is located outside of the
calorimeter cell at room temperature and that the final temperature equilibration between the syringe component and the
solution in the cell takes place as the solution passes down the
needle into the core of the instrument and the measurement
cell. The larger the volume injected, the faster this occurs and
the further from room temperature is the temperature of measurement then the larger will be the background heat from this
process. Having a reference data set of such effects will allow
the selection of optimal ITC parameters for real binding
experiments.
14. Calorimetry provides a unique and direct method for determining the net proton flux associated with an interaction if
measurements are made in buffers of differing ionization
enthalpy but under identical conditions of pH and ionic
strength and temperature. In such cases, the observed ITC
enthalpy is simply plotted against the buffer ionization
enthalpy to yield a slope equal to the net number of protons
exchanging during the interaction with the intercept equal to
the enthalpy of the interaction in the absence of coupled buffer
effects.
15. Determining the time constant (instrument response function)
of an ITC instrument is relatively easy as the sample cell has
electrical calibration heaters installed to facilitate calibration of
the differential power signal. These heaters produce a defined
offset in differential power and this heat is input much faster
than the time constant of the instrument. Therefore, the
change in signal to the new differential power level will occur
as an exponential function that can be fit to yield the time
constant of the ITC.
Isothermal Titration Calorimetry
157
as a small linear change in the peak integral with injection
number. In this case these values should be fitted to a linear
function which is used to correct the experimental data prior to
fitting.
13. It is highly informative to examine the signal from an ITC
instrument injecting water from the syringe into water in the
cell as this will give users an idea of the optimal signal-to-noise
that can be expected when there is no binding or dilution heats.
Changes in this signal may be diagnostic of mechanical problems (bent syringe needle) or difficulty with total fill operation
that may arise when the cell requires cleaning. In addition, the
amplitude of the water–water injections will depend on the
volume of the injection, the speed with which the injection is
made and the temperature of measurement in the ITC. This
reflects the fact that the syringe itself is located outside of the
calorimeter cell at room temperature and that the final temperature equilibration between the syringe component and the
solution in the cell takes place as the solution passes down the
needle into the core of the instrument and the measurement
cell. The larger the volume injected, the faster this occurs and
the further from room temperature is the temperature of measurement then the larger will be the background heat from this
process. Having a reference data set of such effects will allow
the selection of optimal ITC parameters for real binding
experiments.
14. Calorimetry provides a unique and direct method for determining the net proton flux associated with an interaction if
measurements are made in buffers of differing ionization
enthalpy but under identical conditions of pH and ionic
strength and temperature. In such cases, the observed ITC
enthalpy is simply plotted against the buffer ionization
enthalpy to yield a slope equal to the net number of protons
exchanging during the interaction with the intercept equal to
the enthalpy of the interaction in the absence of coupled buffer
effects.
15. Determining the time constant (instrument response function)
of an ITC instrument is relatively easy as the sample cell has
electrical calibration heaters installed to facilitate calibration of
the differential power signal. These heaters produce a defined
offset in differential power and this heat is input much faster
than the time constant of the instrument. Therefore, the
change in signal to the new differential power level will occur
as an exponential function that can be fit to yield the time
constant of the ITC.
Isothermal Titration Calorimetry
157