Ezio Battistel and Giovanni Rialdi
Direct information on the stability and biological activity of immobilized proteins can be obtained from calorimetry. This technique is flexible enough to give insight on the thermodynamic consequences of the immobilization in most experimental conditions, ranging from multipoint covalent attachment to simple absorption. Different calorimetric techniques can be tailored to study the different aspects of the protein chemistry, depending on the physical environment and the type of confinement. From differential scanning calorimetry experiments the thermodynamic parameters (the middle point temperature, the enthalpy change) of the unfolding transition of either the immobilized and free protein can be derived. Isothermal batch and flow calorimetry can assess the effects of the immobilization, support environment, and the type of entrapment on the active site reflected as differences in the binding capacity of specific ligands. Both techniques are suitable to determine reaction enthalpy changes and equilibrium constants from full protein-ligand titration curves. Selected examples of proteins immobilized by covalent single-point attachment in aqueous solutions and by absorption in organic solvents will be described and discussed in detail.
Key Words: Differential scanning calorimetry; flow and batch titration calorimetry; ligand binding; protein unfolding.
Calorimetry is a useful tool to obtain direct information about the properties of immobilized proteins (1). Unlike other spectroscopic techniques, it has the advantage of being rather insensitive to the presence of the supports that are commonly used to immobilized proteins. Microcalorimetry can precisely address two fundamental questions about immobilized proteins: first, how does the stability of the protein change after immobilization? Second, how much the biological activity is affected? Let us consider these two questions separately.
Concerning the first dilemma, calorimetric results can be related to a true thermodynamic definition of stability, not only for proteins but also for other biological complex systems (i.e., nucleic acids). If stability is defined as an equilibrium between the folded, native (N), and unfolded (U) forms of a protein,
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