Natural polypeptide-based hydrogels are potentially good candidates for tissue engineering and drug delivery as they meet a majority of the design criteria for biomaterials. An easy way to form biocompatible hydrogels is to exploit the self-assembly of macromolecules, a certain percentage of denatured proteins is required to promote this process . During denaturation process there is a change of the complex structure of proteins, which is the result of interplay among different types of interactions that are strongly affected by the solvent; the balance between intramolecular and protein-solvent attractions determines the equilibrium between folded and unfolded state of the macromolecule .In general, protein systems rearrange to minimize the interaction between hydrophobic residues and the polar solvent . Depending on solvating conditions, the stabilization driving force can favor the folding of the protein system toward its native structure or can lead to aggregation of protein molecules . Such an aggregation process is characterized by different steps in which the protein undergoes conformational rearrangements and intermolecular association to form stable structures of increasing complexity. The b-sheet motif, precursors of amyloid fibrils, is exploited to design responsive peptidic materials, some of which form amyloid-like fibrils while others self-assemble and form hydrogel networks; changing the aggregation condition is possible to modulate hydrogels proprieties . It has been found that for acid solutions of lysozyme through thermal treatments it is possible to produce transparent thermoreverisible gels, suitable for spectroscopic investigations. The uniqueness of this protein is that lysozyme based hydrogels are cytocompatible to living fibroblast cells, suggesting that globular protein-based hydrogels may be useful as scaffolds for tissue engineering. In this work, we have investigated the unfolding, aggregation and gelation processes of highly concentrated solution of lysozyme in denaturing conditions at different temperatures. The selected concentrated conditions have the double benefit of favoring the gelation process and mimicking the situation of crowding of the cytoplasm in living cells. Infrared (IR) and UV Resonant Raman (UVRR) spectroscopy have been used to probe structural properties of both protein and aqueous environment, in concentrated samples during the phase transformations. This study has allowed to develop a methodology for the preparation and characterization of transparent protein hydrogels with different physical characteristics.
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