Thermodynamic insights into the binding of ANS with the salt induced molten globule states of cytochrome c

Sharma, Ruchika; Kishore, Nand

doi:10.1016/j.jct.2008.09.011

Cited by 18 publications

(9 citation statements)

References 33 publications

(49 reference statements)

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“…Therefore, we performed ITC experiments in order to explore the dominating interactive forces between ANS molecules and chymopapain under several conditions. The ANS binding parameters obtained from ITC results in this study are comparable to those reported for ANS binding to salt induced MG-state of cytochrome c by Kishore et al [48]. At pH 7.4, we did not observe noticeable binding of ANS molecules with protein which was also in agreement with extrinsic spectroscopic results.…”

Section: Discussionsupporting

confidence: 92%

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

et al. 2012

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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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Section: Discussionsupporting

confidence: 92%

“…It is reported earlier that ANS exhibit great possibility of interacting with protein molecules not only through hydrophobic but also via electrostatic interactions [48], [49]. Therefore, we performed ITC experiments in order to explore the dominating interactive forces between ANS molecules and chymopapain under several conditions.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2012

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“…Thus, in addition to the fluorescence studies, ITC experiments have been used to evaluate the binding of ANS to proteins (Matulis and Lovrien, 1998;Celej et al, 2005;Banerjee and Kishore, 2006;Singh and Kishore, 2006;Kinsley et al, 2008). In these studies, the binding isotherms obtained at acidic pH values show a complex behavior and their analysis is not always possible (Sharma and Kishore, 2009). The origin of this complex behavior would be the dimerization of the ANS at acidic pH values.…”

Section: Introductionmentioning

confidence: 99%

A thermodynamic characterization of the interaction of 8‐anilino‐1‐naphthalenesulfonic acid with native globular proteins: the effect of the ligand dimerization in the analysis of the binding isotherms

Andújar‐Sánchez

Jara‐Pérez

Cobos

et al. 2011

J of Molecular Recognition

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8-Anilino-1-naphthalenesulfonic acid (ANS) is a popular fluorescence probe, broadly used for the analysis of proteins, but the nature of its interaction with proteins and the high increase in the fluorescence intensity that takes place upon such process are still unclear. In the last few years, isothermal titration calorimetry has been used to characterize the nature of the interaction of this dye with proteins. The analysis of the binding isotherms of these studies has not considered the dimerization equilibrium of ANS, which is pH dependent, and it can result in serious errors in the data analysis. In the present work we have developed a suitable data analysis by which this process is taken into account. To study the binding of the dye to proteins at different pH values, we have used the Abl-SH3 domain. Our results suggest that at pH 3 and 5, where the dimerization of the ANS is important, electrostatic interactions are significant for the binding of ANS to the Abl-SH3 domain. However, at pH 7, ANS behaves mostly as monomer and the interaction with the protein is mainly hydrophobic. The pH dependent behavior of the ANS binding to proteins can be explained in terms of ionization states of both, the protein and the ANS.

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“…Our previous studies on the interaction of ANS with the MG states of proteins with different structure and function induced by a variety of co-solutes/co-solvents [3,11,12,[45][46][47] by using ITC have suggested that the binding occurs according to two sets of sites available on the protein. These binding sites can be clusters of hydrophobic residues and availability of charged amino acid residues in the partially folded state of the protein.…”

Section: 4mentioning

confidence: 99%

Thermodynamic analysis of partially folded states of myoglobin in presence of 2,2,2-trifluoroethanol

Talele

Kishore

2015

The Journal of Chemical Thermodynamics

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Thermodynamic insights into the binding of ANS with the salt induced molten globule states of cytochrome c

Cited by 18 publications

References 33 publications

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

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

A thermodynamic characterization of the interaction of 8‐anilino‐1‐naphthalenesulfonic acid with native globular proteins: the effect of the ligand dimerization in the analysis of the binding isotherms

Thermodynamic analysis of partially folded states of myoglobin in presence of 2,2,2-trifluoroethanol

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