Reference : Selective and reversible thiol-pegylation, an effective approach for purification and ch...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/2268/97122
Selective and reversible thiol-pegylation, an effective approach for purification and characterization of five fully active ficin (iso)forms from Ficus carica latex.
English
Azarkan, Mohamed [> > > >]
Matagne, André mailto [> > > >]
Wattiez, Ruddy [> > > >]
Bolle, Laetitia [Université de Liège - ULg > Département des sciences de la vie > Enzymologie et repliement des protéines]
Vandenameele, Julie mailto [> > > >]
Baeyens-Volant, Danielle [ > > ]
2011
Phytochemistry
72
14-15
1718-31
Yes (verified by ORBi)
International
0031-9422
1873-3700
United States
[en] ficin ; ficus carica ; cysteine protease ; circular dichroism ; mass spectrometry ; thiol-pegylation
[en] The latex of Ficus carica constitutes an important source of many proteolytic components known under the general term of ficin (EC 3.4.22.3) which belongs to the cysteine proteases of the papain family. So far, no data on the purification and characterization of individual forms of these proteases are available. An effective strategy was used to fractionate and purify to homogeneity five ficin forms, designated A, B, C, D1 and D2 according to their sequence of elution from a cation-exchange chromatographic support. Following rapid fractionation on a SP-Sepharose Fast Flow column, the different ficin forms were chemically modified by a specific and reversible monomethoxypolyethylene glycol (mPEG) reagent. In comparison with their un-derivatized counterparts, the mPEG-protein derivatives behaved differently on the ion-exchanger, allowing us for the first time to obtain five highly purified ficin molecular species titrating 1mol of thiol group per mole of enzyme. The purified ficins were characterized by de novo peptide sequencing and peptide mass fingerprinting analyzes, using mass spectrometry. Circular dichroism measurements indicated that all five ficins were highly structured, both in term of secondary and tertiary structure. Furthermore, analysis of far-UV CD spectra allowed calculation of their secondary structural content. Both these data and the molecular masses determined by MS reinforce the view that the enzymes belong to the family of papain-like proteases. The five ficin forms also displayed different specific amidase activities against small synthetic substrates like dl-BAPNA and Boc-Ala-Ala-Gly-pNA, suggesting some differences in their active site organization. Enzymatic activity of the five ficin forms was completely inhibited by specific cysteine and cysteine/serine proteases inhibitors but was unaffected by specific serine, aspartic and metallo proteases inhibitors.
http://hdl.handle.net/2268/97122
10.1016/j.phytochem.2011.05.009
Copyright (c) 2011 Elsevier Ltd. All rights reserved.

File(s) associated to this reference

Fulltext file(s):

FileCommentaryVersionSizeAccess
Restricted access
Azarkan(2011).pdfPublisher postprint808.78 kBRequest copy

Bookmark and Share SFX Query

All documents in ORBi are protected by a user license.