Characterization of Immobilized Enzymes by Microcalorimetry

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.