Title
Characterisation of Molten Globule-like State of Sheep Serum Albumin at Physiological pH
Author
Mohammad Aasif Dar, Wahiduzzaman, Md. Anzarul Haque, Asimul Islam, Md. Imtaiyaz Hassan, Faizan Ahmad
Year
2016
Journal
International Journal of Biological Macromolecules
Abstract
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.
Instrument
J-1500
Keywords
Circular dichroism, Secondary structure, Protein folding, Protein denaturation, Biochemistry