The influence of succinylation on lysozyme is studied using circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. The spectroscopic data reveal that at room temperature the structures of succinylated lysozyme and native lysozyme are similar. However, the calorimetric results show that the thermal stability of succinylated lysozyme is lower than that of native lysozyme. For succinylated lysozyme, the denaturation temperature (Td) varies in the range of 325-333 K (52-60°C) and the associated denaturation enthalpy (ΔdenH) varies between 225 and 410 kJ/mol. For lysozyme, Td is 342-349 K (69-76°C) and ΔdenH is 440-500 kJ/mol. From these data, the change in the heat capacity (ΔdenCp) upon thermal denaturation is derived. For lysozyme, ΔdenCp is 7.5 kJ/mol/K, and for succinylated lysozyme, it is 16.7 kJ/mol/K. The value of Δ denCp for lysozyme is comparable to previously reported values. The high value of ΔdenCp for succinylated lysozyme is explained in terms of an extended degree of unfolding of the secondary structure and exposure of the apolar parts of the succinyl groups. Furthermore, the Gibbs energy of denaturation, as a function of temperature, derived from the thermodynamic analysis of the calorimetric data, indicates a cold-denaturated state of succinylated lysozyme below 20°C. However, because a denatured state at low temperatures could not be detected by CD or fluorescence measurements, the native state may be considered to be metastable at those conditions. © 2005 American Chemical Society. Chemicals/CAS: lysozyme, 9001-63-2; succinic acid, 110-15-6; Muramidase, EC 3.2.1.17; Succinic Acid, 110-15-6