Biocalorimetry

All biological processes depend on specific molecular recognitions. Such interactions can be intermolecular (such as protein binding to DNA, antigen binding to an antibody, or inhibitor binding to an enzyme) or intramolecular (protein folding, for example). The complete characterization of each interaction requires knowledge of structural changes, of the interaction function, and the thermodynamic parameters measured under defined conditions (such as temperature, pH, and the presence of solvents). Thermodynamic parameters are important because every biomolecular interaction involves the formation and breaking of noncovalent bonds, and heat is either absorbed or generated for each interaction.

Biocalorimetry studies molecular interactions and conformational energetics. Calorimetry is the only available method to measure directly the change in heat (enthalpy, AH) for a biological process. Native materials are used in solution, and no chemical modification or immobilization of biomolecules is necessary. If an interaction and/or conformational change is reversible, samples can be recovered. Calorimetry can be used to study turbid solutions and particulate suspensions. For these reasons, calorimetry is considered a universal detector.

Calorimetry of biomolecules is done by two basic methods. Isothermal titration calorimetry (ITC) measures the heat change when a ligand binds to a macromolecule at constant temperature (intermolecular interactions). Differential scanning calorimetry (DSC) measures the heat change in a sample due to structural changes that occur when the temperature is increased (intramolecular interactions). Both methods are used widely in pharmaceutical and biotechnology research, drug discovery, drug screening, product development, formulation development, process monitoring and optimization, and quality control.

Isothermal Titration Calorimetry

In ITC, a syringe containing a ligand solution is titrated into a sample cuvette containing a macromolecular solution. As the two materials interact, heat is either released or absorbed in direct proportion to the amount of binding. As the macromolecules become saturated with added ligand, the heat signal diminishes until only a background heat of dilution is observed. The area under each peak of a resulting thermograph is equal to the total heat released or absorbed for that injection.

Each experiment takes 30-60 minutes, depending on the number of injections and the time interval between injections. By comparing the changes in heat for each injection with the initial concentrations of ligand and macromolecule, a single ITC experiment can measure the stoichiometry of binding (n), and the binding constant of a ligand to a macromolecule (K^sub B), as well as the changes in enthalpy ((Delta)H), free energy ((Delta)G), and entropy ((Delta)S) of the binding event. Enthalpy and entropy can be extremely useful in understanding the mechanisms of molecular interactions.

ITC is useful in the study of a variety of bimolecular interactions: protein-small molecule, protein-protein, proteins-lipid, nucleic acid-protein, enzyme-inhibitors, antibody-antigen, and receptor studies. Applications of this technology include antibody quality control and characterization, rational drug design, drug screening, and enzyme kinetics.

Differential Scanning Calorimetry

For DSC experiments, a solution of biopolymer is added to the sample cuvette. Then buffer is added to a reference cuvette, and the temperature of those cells is increased under control. A thermoelectric device measures the temperature difference between the two cells. When chemical bonds, even noncovalent ones are broken (such as in protein unfolding), heat is absorbed or given off.

Differences in heat energy uptake between the sample and reference cells, required to maintain an equal temperature, correspond to differences in excess heat capacity ((Delta)C^sub p^). That measurement provides information on changes in conformation. The transitional midpoint temperature (T^sub m^ is determined (the transition from native to unfolded protein, for example). The higher the T^sub m^, the more stable the biopolymer. Enthalpy ((Delta)H) for the sample transition is also calculated.

DSC is used to study protein formulation stability (to discover the most stable formulation) by monitoring changes of T^sub m^ when the biopolymer is in different solutions with different additives. Domain structure studies show different T^sub m^'s for different protein structural domains. Ligand binding is studied by evaluating changes in Tm after the event to determine whether a ligand preferentially binds with folded or unfolded protein. The half-lives of low molecular weight compounds can be determined. DSC is also used to study lipid transitions and uncoiling nucleic acids.

New Directions

Over the past 20 years, the sensitivity of calorimetry instruments has dramatically improved - thanks to developments in electronics, materials, and software - to allow precise measurements of heat changes in dilute biomolecule solutions. A recent development is pressure perturbation calorimetry (PPC), which measures volumetric properties of biopolymers. Future developments will increase throughput, allowing more samples to be analyzed in a day.

[Author Affiliation]

Verna Frasca is a technical services scientist at MicroCal, LLC, 22 Industrial Drive East, Northampton MA 01060, 800.633.3155, fax 413.586.0149,vfrasca@microcalorimetry.com, www.microcalorimetry.com.