title: Characterization of a partially folded intermediate of stem bromelain at low pH.
abstract: Equilibrium studies on the acid included denaturation of stem bromelain (EC 3.4.22.32) were performed by CD spectroscopy, fluorescence emission spectroscopy and binding of the hydrophobic dye, 1-anilino 8-naphthalene sulfonic acid (ANS). At pH 2.0, stem bromelain lacks a well defined tertiary structure as seen by fluorescence and near-UV CD spectra. Far-UV CD spectra show retention of some native like secondary structure at pH 2.0. The mean residue ellipticities at 208 nm plotted against pH showed a transition around pH 4.5 with loss of secondary structure leading to the formation of an acid-unfolded state. With further decrease in pH, this unfolded state regains most of its secondary structure. At pH 2.0, stem bromelain exists as a partially folded intermediate containing about 42.2% of the native state secondary structure Enhanced binding of ANS was observed in this state compared to the native folded state at neutral pH or completely unfolded state in the presence of 6 m GdnHCl indicating the exposure of hydrophobic regions on the protein molecule. Acrylamide quenching of the intrinsic tryptophan residues in the protein molecule showed that at pH 2.0 the protein is in an unfolded conformation with more tryptophan residues exposed to the solvent as compared to the native conformation at neutral pH. Interestingly, stem bromelain at pH 0.8 exhibits some characteristics of a molten globule, such as an enhanced ability to bind the fluorescent probe as well as considerable retention of secondary structure. All the above data taken together suggest the existence of a partially folded intermediate state under low pH conditions.
authors.affiliation: Interdisciplinary Biotechnology Unit, Aligarh Muslim University, India.
authors.forename: Soghra Khatun
authors.initials: SK
authors.lastname: Haq
authors.forename: Sheeba
authors.initials: S
authors.lastname: Rasheedi
authors.forename: Rizwan Hasan
authors.initials: RH
authors.lastname: Khan
mesh_terms: D001963:Bromelains / Q000737:chemistry*; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D017510:Protein Folding
chemical_list: D001963:Bromelains; C091760:stem bromelain