Characterisation of molten globule-like state of sheep serum albumin at physiological pH

Sheep serum albumin (SSA) is a 583 amino acid residues long multidomain monomeric protein which is rich in cysteine and low in tryptophan content. The serum albumins (from human, bovine and sheep) play a vital role among all proteins investigated until now, as they are the most copious circulatory proteins. We have purified SSA from sheep kidneys by a simple and efficient two-step purification procedure. Further, we have studied urea-induced denaturation of SSA by monitoring changes in the difference absorption coefficient at 287 nm (δε₂₈₇), intrinsic fluorescence emission intensity at 347 nm (F₃₄₇) and mean residue ellipticity at 222 nm ([θ]₂₂₂) at pH 7.4 and 25 °C. The coincidence of denaturation curves of these optical properties suggests that urea-induced denaturation is a bi-phasic process (native (N) state ↔ intermediate (X) state ↔ denatured (D) state) with a stable intermediate populated around 4.2-4.7 M urea. The intermediate (X) state was further characterized by the far-UV and near-UV CD, dynamic light scattering (DLS) and fluorescence using 1-anilinonaphthalene-8-sulfonic acid (ANS) binding method. All denaturation curves were analyzed for Gibbs free energy changes associated with the equilibria, N state ↔ X state and X state ↔ D state in the absence of urea.