the enthalpy change associated with either a binding interaction or a heat-induced conformational transition
for nucleic acids and proteins. These are isothermal titration calorimetry(ITC) and differential scanning
calorimetry(DSC).28–33Many ultra-high sensitivity microcalorimeters can now be obtained.
11.4.4.1 Isothermal Titration Calorimetry. Isothermal titration calorimetry is a powerful and flex-
ible technique that is used to determine directly and independently the change in enthalpy (H) for almost
any bimolecular binding interaction at a fixed and constant temperature. Depending on the magnitude
of the binding constant and solubility of reactants, ITC can also be used to obtain binding isotherms, from
which the equilibrium-binding constant (KbKa) and binding stoichiometry (n) can be determined. This
can therefore yield an almost complete thermodynamic profile for a binding interaction in a single
experiment.
The calorimeter assembly consists typically of two identical cells (one reference and one sample) con-
structed of a metal alloy that has appropriate thermal properties, which is held within an adiabatic jacket.
Detection of heat effects within the cells is achieved by placement of semiconductor thermopiles in
between the cells and the jacket. Following injection of a ligand into the sample cell, the amount of thermal
power that must be applied to actively balance the heat of reaction is measured. Thus the calorimeter moni-
tors the heat effects in the sample cell and then applies appropriate power to maintain a zero temperature
difference between sample and reference cells. The amount of heat applied to the cells is monitored con-
tinuously (cell feedbackor CFB), and the amount of power applied is adjusted so as to drive the measured
thermal power amplitude towards a stable baseline value. This is achieved by interfacing a computer to the
system that continually monitors the output of a nanovoltmeter connected to record the output of the thermo-
piles. The experimentally measured quantity is the applied thermal power as a function of time required to
return CFB to the stable baseline value subsequent to an injection of ligand (or system component), and is
directly proportional to the reaction enthalpy. Modern ultrasensitive ITC instruments, such as the
MicroCal VP-ITC, have baseline stability in the range5ncal s^1 and heat signals in the nanocalorie range
can be detected.
In example data from an ITC experiment (Figure 11.14), Panel A shows primary data, where CFB is
measured in cal sec^1 as a function of time. At equilibrium, CFB is held at a predefined value. Each peak
represents the injection of a defined volume of ligand into a continually stirred sample cell. Addition of
ligand leads to a binding interaction and concomitant heat effects; in this case an exothermic heat is observed.
Each peak is integrated, with respect to time, to yield the binding isotherm in Panel B. After correction of
background heats, due to dilution effects, and correction to a per mole basis the enthalpy is obtained
directly and Kobsand the stoichiometry (n) are obtained by fitting to an appropriate binding model, such
that n is the midpoint of the titration curve and Kobscomes from the lineshape of the binding curve.
11.4.4.2 Differential Scanning Calorimetry. Differential scanning calorimetry involves the con-
tinuous measurement of the apparent specific heat of a system as a function of temperature. Hence, DSC
can be used to examine physicochemical processes initiated by increases or decreases in temperature, for
example phase transitions or conformational changes.
In general terms, a DSC instrument contains two cells, which are suspended in an adiabatic shield and
connected viaa multifunctional thermopile. In a typical experiment, the reference cell is filled with buffer
and the sample cell is filled with buffer containing macromolecule (cell volume 0.5–1.5mL). The temperature
is increased to the 0.1–110°C range by use of electrical heaters that are in good thermal contact with the
cells. During a heat-induced endothermic transition, the temperature of the sample cell falls behind that of
the reference cell, since some of the energy required is used to induce the transition rather than to heat the
solution. This lag is detected by the thermopile, so that extra electrical power is supplied to the sample cell
to maintain it at a temperature identical to that of the reference cell. This additional energy is proportional
to the energy associated with the thermally induced transition. Knowledge of the solute concentration
enables a transformation of the observed electrical energy versustemperature profile into a curve that cor-
responds to an excess heat capacity versustemperature plot.
444 Chapter 11