Surface properties of lysozyme aqueous solutions in the presence of reducing agents: the effect of disulfide bonds reshuffling.

The influence of dithiothreitol (DTT) and β-mercaptoethanol (β-MEt) or their mixtures with a chaotropic denaturant, namely guanidine hydrochloride (GuHCl) or urea, on the surface properties of lysozyme aqueous solutions was studied by the methods of dilatational surface rheology and ellipsometry. Adding 0.32 mM DTT to lysozyme solutions led to a considerable increase of the dynamic surface elasticity and a decrease of the dynamic surface tension as compared with the results for native protein solutions. The observed effect was even more pronounced after a preliminary heating of the solutions. The rearrangement of disulfide bonds under the influence of a reducing agent and the subsequent cross-linking of lysozyme molecules resulted in the formation of a dense layer of adsorbed protein aggregates stabilized by intermolecular disulfide bridges at the liquid/gas interface. In the case of lysozyme solutions containing β-MEt, a significantly weaker effect ruled out the dense film formation, yet also assumed some limited perturbations of the protein structure. The influence of reducing agents on the surface properties of lysozyme solutions differed from that of chaotropic denaturants and surfactants. At the same time, the simultaneous addition of both a reducing agent and a chaotropic denaturant led to a decrease of steady-state values of the dynamic surface elasticity due to the slow loosening of the cross-linked layer of lysozyme aggregates. Furthermore, unlike the protein solutions with urea, the molten globule state was not observed for solutions with both urea and a reducing agent, and the surface layer structure in the latter case was presumably similar to that in the layer in solutions containing GuHCl where unfolded protein molecules formed loops and tails in the surface layer. The ellipsometric results corroborated these conclusions and revealed a decrease in the ellipsometric angle Δ in the case of both lysozyme/DTT/GuHCl and lysozyme/DTT/urea solutions.