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.
Highlights
The process of protein folding, despite being one of the most intensely investigated areas, remains obscure in terms of its detailed molecular mechanism
The available literature reveals that molten globule state is mostly pinpointed from its maximum anilino-8naphthalene sulfonate (ANS) binding ability under the conditions studied since the other typical features viz pronounced secondary structure and disrupted tertiary contacts often seem to merge with intermediates lying in the vicinity of MG-state [12,15,16,17,18,19,20]
No changes in the ellipticity values were observed in the pH range 3.0– 7.4 though some structure induction was observed at pH 3.0 where MRE222 nm was slightly greater than the native state [Table 1]
Summary
The process of protein folding, despite being one of the most intensely investigated areas, remains obscure in terms of its detailed molecular mechanism. The available literature reveals that molten globule state is mostly pinpointed from its maximum ANS binding ability under the conditions studied since the other typical features viz pronounced secondary structure and disrupted tertiary contacts often seem to merge with intermediates lying in the vicinity of MG-state [12,15,16,17,18,19,20] Such utilization of ANS which is based upon the principle that ANS is practically non fluorescent in water but produces brilliant fluorescence upon binding to hydrophobic sites of protein [21] generally ignores the contribution of sulfonate group that was earlier considered as a mere solublizing agent for otherwise almost water-insoluble anilinonaphthalene moiety. All these observations compel one to ponder whether maximum ANS-fluorescence always indicates the formation of MG-state?
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