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Protein chemical degradations occur naturally into living cells as soon as proteins have been synthesized. Among these modifications, deamidation of asparagine or glutamine residues has been extensively studied, whereas the intermediate state, a succinimide derivative, was poorly investigated because of the difficulty of isolating those transient species. We used an indirect method, a limited thermal treatment in the dry state at acidic pH, to produce stable cyclic imide residues in hen lysozyme molecules, enabling us to examine the structural and functional properties of so modified proteins. Five cyclic imide rings have been located at sites directly accessible to solvent and did not lead to any changes in secondary or tertiary structures. However, they altered the catalytic properties of lysozyme and significantly decreased the intrinsic stability of the molecules. Moreover, dimerization occurred during the treatment, and this phenomenon was proportional to the extent of chemical degradation. We propose that succinimide formation could be responsible for covalent bond formation under specific physicochemical conditions that could be found in vivo.

Polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS), ellipsometry, and shear elastic constant measurements were used to study the adsorption and the behavior of ovalbumin and S-ovalbumin at the air-water interface at different values of the subphase pH. Native and S-ovalbumin exhibited similar behaviors, with a maximum plateau value of the shear elastic constant near the isoelectric pH of the protein. However, higher surface concentration values were reached with S-ovalbumin in low net charge conditions, which suggest adsorption of aggregates or multilayer adsorption. For both proteins, the statistical analysis of PM-IRRAS spectra demonstrated that the aging of the interfacial film and the increase of the shear elastic constant were correlated with a significant increase in the relative contribution of intermolecular β-sheets in the amide I band with time. This increase was significantly faster at low pH values. At the same pH value and age of the interface, the relative contribution of intermolecular β-sheets was significantly higher for S-ovalbumin.