title: 1-Anilino-8-naphthalene sulfonate (ANS) is not a desirable probe for determining the molten globule state of chymopapain.
abstract: The molten globule (MG) state of proteins is widely detected through binding with 1-anilino-8-naphthalene sulphonate (ANS), a fluorescent dye. This strategy is based upon the assumption that when in molten globule state, the exposed hydrophobic clusters of protein are readily bound by the nonpolar anilino-naphthalene moiety of ANS molecules which then produce brilliant fluorescence. In this work, we explored the acid-induced unfolding pathway of chymopapain, a cysteine proteases from Carica papaya, by monitoring the conformational changes over a pH range 1.0-7.4 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). The spectroscopic measurements showed that although maximum ANS fluorescence intensity was observed at pH 1.0, however protein exhibited ∼80% loss of secondary structure which does not comply with the characteristics of a typical MG-state. In contrast at pH 1.5, chymopapain retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii and nearly 30-fold increase in ANS fluorescence with respect to the native state, indicating that MG-state exists at pH 1.5 and not at pH 1.0. ITC measurements revealed that ANS molecules bound to chymopapain via hydrophobic interaction were more at pH 1.5 than at pH 1.0. However, a large number of ANS molecules were also involved in electrostatic interaction with protein at pH 1.0 which, together with hydrophobically interacted molecules, may be responsible for maximum ANS fluorescence. We conclude that maximum ANS-fluorescence alone may not be the criteria for determining the MG of chymopapain. Hence a comprehensive structural analysis of the intermediate is essentially required.
authors.affiliation: Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India.
authors.forename: Atiyatul
authors.initials: A
authors.lastname: Qadeer
authors.forename: Gulam
authors.initials: G
authors.lastname: Rabbani
authors.forename: Nida
authors.initials: N
authors.lastname: Zaidi
authors.forename: Ejaz
authors.initials: E
authors.lastname: Ahmad
authors.forename: Javed M
authors.initials: JM
authors.lastname: Khan
authors.forename: Rizwan H
authors.initials: RH
authors.lastname: Khan
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D002151:Calorimetry; D002917:Chymopapain / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008962:Models, Theoretical; D017510:Protein Folding; D017433:Protein Structure, Secondary
chemical_list: D000817:Anilino Naphthalenesulfonates; D002917:Chymopapain