Investigation of the structure and proteolytic activity of papain in aqueous miscible organic media
The stability of papain was studied in aqueous–organic mixtures by means of residual proteolytic activity along with various spectroscopic analyses (fluorescence and ATR-FTIR combined with isotopic exchange with D2O). The investigated systems contained 1 or 10% (v/v) of an aqueous buffered solution...
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Autores principales: | , , , , |
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Formato: | Articulo |
Lenguaje: | Inglés |
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2012
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Acceso en línea: | http://sedici.unlp.edu.ar/handle/10915/112868 |
Aporte de: |
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I19-R120-10915-112868 |
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institution |
Universidad Nacional de La Plata |
institution_str |
I-19 |
repository_str |
R-120 |
collection |
SEDICI (UNLP) |
language |
Inglés |
topic |
Biología Papain Aqueous–organic media Catalytic stability Structural stability Thermal stability Fluorescence spectroscopy ATR-FTIR spectroscopy |
spellingShingle |
Biología Papain Aqueous–organic media Catalytic stability Structural stability Thermal stability Fluorescence spectroscopy ATR-FTIR spectroscopy Llerena Suster, Carlos Rafael José, Carla Collins, Sebastián E. Briand, Laura Estefanía Morcelle del Valle, Susana Raquel Investigation of the structure and proteolytic activity of papain in aqueous miscible organic media |
topic_facet |
Biología Papain Aqueous–organic media Catalytic stability Structural stability Thermal stability Fluorescence spectroscopy ATR-FTIR spectroscopy |
description |
The stability of papain was studied in aqueous–organic mixtures by means of residual proteolytic activity along with various spectroscopic analyses (fluorescence and ATR-FTIR combined with isotopic exchange with D2O). The investigated systems contained 1 or 10% (v/v) of an aqueous buffered solution (pH 8.0) in acetonitrile (ACN), methanol (MeOH) or dimethyl formamide (DMF). The results evidenced that papain retained almost all its catalytic activity after 24 h of incubation in the presence of ACN, and a more compact conformation of the enzyme was detected. Papain suffered an important loss of enzymatic activity (ca. 80%) after 24 h incubation in MeOH although, no global conformational change and minor secondary structure rearrangements were detected. This observation suggests that somehow the active site region was altered. On the other hand, papain suffered a complete inactivation when exposed to those media containing DMF. Fluorescence analyses revealed that an irreversible conformational change took place after 24 h incubation, and a moderate increase in b-sheet and b-turn structures was the most relevant finding when secondary structure was analyzed. The evidences demonstrated that the organic solvents induce a more rigid and compact structure of papain regardless of the organic:buffer ratio investigated. In turn, these modifications affect the active catalytic site in the particular case of MeOH and DMF. These findings were in agreement with the thermo-stability of the enzyme performed after heating at 353 K in all the studied media, that is the presence of ACN did not substantially affect the secondary structure of papain. Nevertheless, the a-helix domain demonstrated to be less thermally stable than the b-sheet domain, turning into aggregated structures after heating, especially in the presence of MeOH and DMF. |
format |
Articulo Articulo |
author |
Llerena Suster, Carlos Rafael José, Carla Collins, Sebastián E. Briand, Laura Estefanía Morcelle del Valle, Susana Raquel |
author_facet |
Llerena Suster, Carlos Rafael José, Carla Collins, Sebastián E. Briand, Laura Estefanía Morcelle del Valle, Susana Raquel |
author_sort |
Llerena Suster, Carlos Rafael |
title |
Investigation of the structure and proteolytic activity of papain in aqueous miscible organic media |
title_short |
Investigation of the structure and proteolytic activity of papain in aqueous miscible organic media |
title_full |
Investigation of the structure and proteolytic activity of papain in aqueous miscible organic media |
title_fullStr |
Investigation of the structure and proteolytic activity of papain in aqueous miscible organic media |
title_full_unstemmed |
Investigation of the structure and proteolytic activity of papain in aqueous miscible organic media |
title_sort |
investigation of the structure and proteolytic activity of papain in aqueous miscible organic media |
publishDate |
2012 |
url |
http://sedici.unlp.edu.ar/handle/10915/112868 |
work_keys_str_mv |
AT llerenasustercarlosrafael investigationofthestructureandproteolyticactivityofpapaininaqueousmiscibleorganicmedia AT josecarla investigationofthestructureandproteolyticactivityofpapaininaqueousmiscibleorganicmedia AT collinssebastiane investigationofthestructureandproteolyticactivityofpapaininaqueousmiscibleorganicmedia AT briandlauraestefania investigationofthestructureandproteolyticactivityofpapaininaqueousmiscibleorganicmedia AT morcelledelvallesusanaraquel investigationofthestructureandproteolyticactivityofpapaininaqueousmiscibleorganicmedia |
bdutipo_str |
Repositorios |
_version_ |
1764820445968728064 |