MELTING-POINTS OF LYSOZYME AND RIBONUCLEASE-A CRYSTALS CORRELATED WITH PROTEIN UNFOLDING - A RAMAN-SPECTROSCOPIC STUDY

The effects of a temperature increase on monoclinic and tetragonal lys ozyme single crystals were investigated by polarizing microscopy, X-ra y diffraction and laser Raman spectroscopy. To prevent dissolution, th e mother liquor was removed, and the crystals were covered by the oil poly-(chlorotrifluoroethylene). Upon heating, their macroscopic shape was stable beyond 453 K but a change (or loss) of birefringence was ob served around 352 and 367 K for the tetragonal and monoclinic crystal forms, respectively, which is associated with tighter packing and high er crystal forces in monoclinic lysozyme. Raman spectral changes in th e amide I and amide III regions indicated denaturation of the protein within the crystalline environment at temperature when birefringence c hanges, and differences in the S-S band suggest that in monoclinic lys ozyme, denaturation is accompanied with disruption of some S-S bonds. Comparison with thermal denaturation and gel formation (beta-aggregati on) of lysozyme in solution indicates that intermolecular interactions are mainly involved in the stabilization of the denatured lysozyme cr ystals. The behavior of ribonuclease A is very different. This protein unfolds and refolds reversibly in solution and its crystals melt at t he unfolding temperature at 333 K, i.e. loss of structure induces brea kdown of crystal lattice and macroscopic shape. Although the crystal l attice of proteins is stabilized by only few intermolecular contacts, its breakdown with increasing temperature is primarily a result of the rmal unfolding of the polypeptide chains.