Refolding of urea denatured ovalbumin with three phase partitioning generates many conformational variants

Three phase partitioning is a process in which mixing t-butanol with ammonium sulphate with a protein solution leads to the formation of three phases. Generally, the interfacial protein precipitate (formed between upper t-butanol rich and lower aqueous phase) can be easily dissolved back in aqueous buffers. In case of ovalbumin, this led to a precipitate which was insoluble in aqueous buffers. This precipitate when solubilized with 8 M urea and subjected to three phase partitioning under various conditions led to many refolded soluble conformational variants of ovalbumin. One of these showed trypsin inhibitory activity, had marginally higher β-sheet content and had higher surface hydrophobicity (both with respect to native ovalbumin). Scanning electron microscopy and Atomic force microscopy of this preparation showed a thread like structure characteristic of amyloid fibrils. The behaviour of ovalbumin during three phase partitioning makes it a valuable system for gaining further understanding of protein aggregation.