Role of conformational flexibility in the emulsifying properties of bovine serum albumin.

Although it is well recognized that the conformation state of a protein affects its surface properties, the importance of conformation flexibility to its functionality is still not well understood. This study systemically investigated the influence of protein concentration (c) and disulfide bond (S-S) cleavage with a reducing agent, β-mercaptoethanol (2-ME), on the conformation and emulsifying properties of an ideal globular protein, bovine serum albumin (BSA), with the aim to unravel the role of conformational flexibility in the functionality. The conformations were evaluated using size exclusion chromatography, dynamic light scattering (DLS), extrinsic fluorescence, and derivative UV spectroscopy. The emulsifying properties, including emulsifying ability, extent of droplet flocculation at a specific period of storage, and stability against flocculation and/or coalescence as well as creaming, were characterized using droplet size and creaming index analyses. The results indicated that the tertiary conformation of native BSA was closely dependent on its c (in the range of 0.05-1.0%), and increasing c resulted in a more compacted and rigid conformation. The c dependence largely determined the susceptibility of S-S bridges to reduction and even refolding of reduced BSA molecules. Interestingly, there was approximately a critical c (e.g., 0.25-0.5%) below which the S-S cleavage resulted in a gradual structural unfolding of the molecules and above which the situation was the reverse. On the other hand, the alteration with protein and 2-ME concentrations led to a variety of changes in emulsion size (d4,3; in water or 1% SDS) at 4 and 24 h and creaming index (up to 2 weeks). In general, at a low c value (e.g., 0.25%) increasing the S-S cleavage progressively improved the emulsifying ability and emulsion stability (especially against coalescence and creaming), whereas at c = 0.5 or 0.75%, the S-S cleavage, on the contrary, impaired the emulsifying properties, especially emulsion stability against flocculation and/or coalescence. These results suggest that the conformational flexibility of the protein (in solution) plays a vital role in different aspects of its emulsifying properties, for example, ease of structural unfolding at the interface, lateral interactions between adsorbed proteins, and formation of a viscoelastic interfacial layer (or multilayers). This knowledge could provide an in-depth understanding of the relationships between tertiary conformational flexibility and emulsifying properties of globular proteins.