Raman band-shape analysis of urea-lysozyme interaction in aqueous solution

Urea is a well-known agent in the denaturation of proteins, although the mechanism involved is far from being understood. The present investigation focuses on the Raman band-shape analysis of the nu(4) mode of urea as a source of information regarding its molecular dynamics and how this is affected by the presence of protein. Lysozyme was chosen since it is a well-characterized protein. From the available data it is possible to observe that in the presence of lysozyme the anisotropic (depolarized) correlation function decays faster than the vibrational correlation function, which is an indication that the former is the most affected, and more specifically that the urea molecules rotate more freely when protein is present. Urea derivatives, namely thiourea and tetramethylurea, have also been studied in order to assess aspects related to hydrogen bonding with water. Guanidinium chloride, a protein denaturant bearing a close chemical structure to urea, has also been assayed. The effect of urea, and of the other above-mentioned compounds, on the Raman water band at about 3300 cm(-1) was investigated. A variation in the band-shape was detected for systems containing urea and guanidinium chloride; this was dependent on the denaturant concentration, reaching a maximum at the highest concentrations investigated (about 8 and 6.7 M for urea and guanidinium chloride, respectively). The occurrence of a high-frequency component in the water stretching band induced by the denaturants suggests the presence of less hydrogen-bonded species in solution. This conclusion was reinforced by the observation of a lower depolarization ratio for the asymmetric species HDO stretching band in concentrated urea solutions. These observations indicate that the water structure is drastically affected under the conditions where the denaturants have their maximum denaturing effect.