Investigation of the structure and proteolytic activity of papain in aqueous miscible organic media |
| |
Authors: | Carlos R Llerena-SusterCarla José Sebastián E CollinsLaura E Briand Susana R Morcelle |
| |
Institution: | a Laboratorio de Investigación de Proteínas Vegetales (LIPROVE), Depto. de Cs. Biológicas, Fac. Cs. Exactas, Universidad Nacional de La Plata, Calle 47 y 115, La Plata, Argentina b Centro de Investigación y Desarrollo en Ciencias Aplicadas-Dr. Jorge J. Ronco. Universidad Nacional de La Plata, CONICET, CCT La Plata, Calle 47 No 257, B1900AJK La Plata, Buenos Aires, Argentina c Instituto de Desarrollo Tecnológico para la Industria Química INTEC, Universidad Nacional del Litoral, CONICET, Güemes 3450, 3000 Santa Fe, Santa Fe, Argentina |
| |
Abstract: | 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 β-sheet and β-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 α-helix domain demonstrated to be less thermally stable than the β-sheet domain, turning into aggregated structures after heating, especially in the presence of MeOH and DMF. |
| |
Keywords: | Papain Aqueous-organic media Catalytic stability Structural stability Thermal stability Fluorescence spectroscopy ATR-FTIR spectroscopy |
本文献已被 ScienceDirect 等数据库收录! |
|