Calorimetry: Getting the Winning Combination

Ultrasensitive calorimetric instruments for measuring very small heats of reaction at constant temperature (isothermal titration calorimetry [ITC]) and small heat effects induced by temperature (differential scanning calorimetery [DSC]) are becoming increasingly important in biotechnological studies. Ultrasensitive calorimetry has gained much interest among life scientists because it provides information on the energetics of biopolymer structures, and on the interactions of biopolymers with other macromolecules and with small molecules. Since these recognition and binding reactions are central to all biological processes, this information is indispensable for furthering developments in biology, medicine, and the pharmaceutical sciences.

Today’s ultrasensitive calorimeters provide a unique approach to analyzing molecular structures such as proteins, nucleic acids, and their complexes. Our increased understanding of biomolecular structures and interactions provide new possibilities for finding and developing novel biologically active products.

The calorimetric analyses of biomolecules have garnered much attention due to the fact that structural information alone is insufficient for understanding their function. This requires knowledge of the energetics that determine the structures of biomolecules and their complexes. The only way to obtain this information is through direct calorimetric measurements of the thermal properties of the individual molecular objects (proteins, nucleic acids, etc.), i.e., their heat capacities and the heats of their interactions.

Understanding and accurately quantifying such interactions requires characterizing structural and stability changes in biological macromolecules, their mutants, and synthetic analogs. These changes are induced by various external conditions, or by association with their binding partners and ligands. While these processes can be studied by various physical methods, ultrasensitive calorimetry alone provides direct information on the energies involved.

The heat effects arising from macromolecular binding processes are extremely small. Moreover, proteins and nucleic acids are generally available in very limited amounts. In order to measure these heat effects, two types of ultrasensitive microcalorimeters have been developed. Differential scanning calorimeters measure the heat effects induced by temperature variations, and isothermal titration calorimeters measure the heats of interactions at constant temperature. These two types of calorimetric instruments are complementary; only in combination can information on the energetic bases of the studied molecular objects and their interactions be obtained.

Researchers understand that these two tools are complementary: a full thermodynamic description of the interactions between macromolecules (which is the basis of all biological functions) can only be obtained when ITC and DSC are used together to study biomolecular systems. Using both techniques to study a biomolecular system in dilute aqueous solution is important because macromolecular interactions depend on the exact structure and stability of the molecules, which in turn depends on the temperature. Therefore, although ITC provides detailed information about the strength with which two molecules bind, DSC studies are required to understand the thermal properties of the interacting molecules. Attaining this level of understanding will particularly impact the pharmaceutical industry, opening new avenues in the search for active molecular agents with high binding affinity and specificity for their target macromolecule.

Ultrasensitive calorimeters dramatically enhance reliability, baseline reproducibility, and data consistency, resulting in more precise measurements. In many instances, these instruments allow scientists to obtain precise measurements in a single experiment, making it unnecessary to take a multitude of measurements, thereby saving time and money.

The Nano DSC III (Calorimetry Sciences Corp. [CSC], Lindon, UT) enables scientists to maintain very good baselines. They are able to determine the heat capacity of the macromolecule of interest in dilute solution over a broad temperature range, the changes that occur in the protein upon binding a specific ligand, and detect which protein domains are involved in binding a given ligand. Because these measurements are obtained over a range of temperatures, they can be used to make temperature corrections to heat of binding data obtained using the calorimeter. This correction allows correlations between the energetics of binding and the structure of the complex, which is extremely important in analyzing the action of various ligands and in searching for biologically active agents.

While ITC is particularly suitable for following the energetics of association reactions between biomolecules, the combination of ITC and DSC provides a more comprehensive description of the thermodynamics of the system. Both techniques allow researchers to use very small samples. When a system is studied by ITC, the data clearly tell what is happening, and by knowing the fundamentals of this, the user can extrapolate to various conditions. Without the correct DSC data, researchers cannot make the required corrections to the ITC data to extrapolate and work with them.

Essentially, ITC is a thermodynamic technique for monitoring any chemical reaction initiated by the addition of a binding component. It has become the method of choice for characterizing biomolecular interactions. When substances bind, heat is either generated or absorbed. Measuring this heat permits the accurate determination of binding constants, reaction stoichiometry, enthalpy, and entropy, thereby providing a complete thermodynamic profile of the molecular interaction in a single experiment.

DSC is used to study thermally induced conformational changes in biopolymers and lipid membranes, and ligand binding to biopolymers and biomembranes. Changes in heat capacity, enthalpy, and entropy that accompany changes in conformation or binding are commonly determined. Free energies, compressibilities, and thermal expansion coefficients can also be determined by combining DSC with pressure-volume changes.

The limitations of using ITC alone is that researchers are making measurements at multiple temperatures and then trying to get heat capacity changes as a function of temperature from the ITC data. However, they are not taking into account the change in the native protein and its heat capacity change as a function of temperature. Measuring those thermodynamic parameters accurately requires highly sensitive DSC with ultrastable baselines.

Figure 1 - Nano ITC III ultrasensitive calorimeter.

Figure 2 - CSC Nano DSC III and Nano ITC III ultrasensitive calorimeters.

The CSC Nano DSC III and Nano ITC III are power compensation calorimeters with cells that are fixed-in-place (see Figures 1 and 2). These high-precision calorimeters are well suited for biopolymer studies. Because many biopolymers are expensive or difficult to obtain, and may behave differently depending on their concentration in solution, there is a need for scanning and titration instruments that can make reliable measurements with very little sample, often just a few micrograms of biopolymer.

Conclusion

Ultrasensitive ITC and DSC are complementary techniques that enhance our understanding of structural and stability changes as biological molecules interact. Together they form a winning combination that offers a unique approach to analyzing molecular structures such as proteins, nucleic acids, and their complexes.

Mr. Sullivan is a writer based in Hermosa Beach, CA, U.S.A.; tel.: 310-379-0573; e-mail: [email protected].