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1-Anilino-8-Naphthalene Sulfonate (ANS) Is Not a Desirable Probe for Determining the Molten Globule State of Chymopapain

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 a...

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Autores principales: Qadeer, Atiyatul, Rabbani, Gulam, Zaidi, Nida, Ahmad, Ejaz, Khan, Javed M., Khan, Rizwan H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3510187/
https://www.ncbi.nlm.nih.gov/pubmed/23209794
http://dx.doi.org/10.1371/journal.pone.0050633
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author Qadeer, Atiyatul
Rabbani, Gulam
Zaidi, Nida
Ahmad, Ejaz
Khan, Javed M.
Khan, Rizwan H.
author_facet Qadeer, Atiyatul
Rabbani, Gulam
Zaidi, Nida
Ahmad, Ejaz
Khan, Javed M.
Khan, Rizwan H.
author_sort Qadeer, Atiyatul
collection PubMed
description 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.
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spelling pubmed-35101872012-12-03 1-Anilino-8-Naphthalene Sulfonate (ANS) Is Not a Desirable Probe for Determining the Molten Globule State of Chymopapain Qadeer, Atiyatul Rabbani, Gulam Zaidi, Nida Ahmad, Ejaz Khan, Javed M. Khan, Rizwan H. PLoS One Research Article 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. Public Library of Science 2012-11-29 /pmc/articles/PMC3510187/ /pubmed/23209794 http://dx.doi.org/10.1371/journal.pone.0050633 Text en © 2012 Qadeer et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Qadeer, Atiyatul
Rabbani, Gulam
Zaidi, Nida
Ahmad, Ejaz
Khan, Javed M.
Khan, Rizwan H.
1-Anilino-8-Naphthalene Sulfonate (ANS) Is Not a Desirable Probe for Determining the Molten Globule State of Chymopapain
title 1-Anilino-8-Naphthalene Sulfonate (ANS) Is Not a Desirable Probe for Determining the Molten Globule State of Chymopapain
title_full 1-Anilino-8-Naphthalene Sulfonate (ANS) Is Not a Desirable Probe for Determining the Molten Globule State of Chymopapain
title_fullStr 1-Anilino-8-Naphthalene Sulfonate (ANS) Is Not a Desirable Probe for Determining the Molten Globule State of Chymopapain
title_full_unstemmed 1-Anilino-8-Naphthalene Sulfonate (ANS) Is Not a Desirable Probe for Determining the Molten Globule State of Chymopapain
title_short 1-Anilino-8-Naphthalene Sulfonate (ANS) Is Not a Desirable Probe for Determining the Molten Globule State of Chymopapain
title_sort 1-anilino-8-naphthalene sulfonate (ans) is not a desirable probe for determining the molten globule state of chymopapain
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3510187/
https://www.ncbi.nlm.nih.gov/pubmed/23209794
http://dx.doi.org/10.1371/journal.pone.0050633
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