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Metastability of Papain and the Molecular Mechanism for its Sequential Acid-Denaturation

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Abstract

Acid unfolding of non-inhibited papain at pH 2 was studied by means of spectroscopic and electrophoresis techniques as well as activity assays. We found a molten globule like species (A state) similar to that previously reported for bromelain and S-carboxy-methyl-papain. We demonstrated that this A state is not thermodynamically stable but a metastable conformer which decays into an unfolded conformation in a few hours. The mechanism of acid unfolding to the A state proved to be completely irreversible, with a biphasic time evolution of spectroscopic signals characteristic of the existence of a kinetic intermediate. This latter species showed properties in-between native and A state such as secondary structure, exposition of hydrophobic area and tryptophan environment, but a native like hydrodynamic radius. Native papain seems to unfold at acid pH through at least two kinetic barriers, being its proregion mandatory to conduct and stabilize its active structure. Computer simulations of acid unfolding, followed by ANS docking, identified three regions of cavity formation induced by acid media which might be used as regions to be fortified by protein engineering in the quest for extreme-resistant proteases or as hot-spots for protease inactivation.

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Abbreviations

CXP:

S-carboxy-methyl papain

SDS:

Sodium dodecyl sulfate

ANS:

8-Anilino-1-naphthalensulfonic acid

DTNB:

5,5′-dithiobis-(2-nitrobenzoic acid)

CD:

Circular dichroism

Fi:

Intrinsic fluorescence

Fans:

ANS fluorescence

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Acknowledgments

We gratefully acknowledge the significant contribution of Dr. Andrés Hernández-Arana to this work and financial support from CONACYT (México) with grants 105532, ECOS-M05S01 and the fellowship 123806 to R.E.F.-Q.

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Authors and Affiliations

  1. Universidad Autónoma de San Luis Potosí, Coordinación Académica Región Altiplano, COARA, Carretera a Cedral Km 5+600, Ejido San José de las Trojes, 78700, Matehuala, San Luis Potosí, Mexico

    Rosa Eréndira Fosado-Quiroz

  2. Departamento de Ciencias Naturales, Universidad Autónoma Metropolitana, Unidad Cuajimalpa, Pedro Antonio de los Santos 84, Col. San Miguel Chapultepec, 11850, Mexico, D.F., Mexico

    Arturo Rojo-Domínguez

  3. Departamento de Química, Universidad Autónoma Metropolitana, Unidad Iztapalapa, Apartado Postal 55-534, 09340, Mexico, D.F., Mexico

    Rosa Eréndira Fosado-Quiroz & Arturo Rojo-Domínguez

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  1. Rosa Eréndira Fosado-Quiroz
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  2. Arturo Rojo-Domínguez
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Correspondence to Arturo Rojo-Domínguez.

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Fosado-Quiroz, R.E., Rojo-Domínguez, A. Metastability of Papain and the Molecular Mechanism for its Sequential Acid-Denaturation. Protein J 30, 184–193 (2011). https://doi.org/10.1007/s10930-011-9319-z

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