Ficin; Ficus carica; Cysteine protease; Latex; Mass spectrometry; Circular dichroism
Abstract :
[en] A novel ficin form, named ficin E, was purified from fig tree latex by a combination of cation-exchange chromatography on SP-Sepharose Fast Flow, Thiopropyl Sepharose 4B and fplc-gel filtration chromatography. The new ficin appeared not to be sensitive to thiol derivatization by a polyethylene glycol derivative, allowing its purification. The protease is homogeneous according to PAGE, SDS–PAGE, mass spectrometry, N-terminal micro-sequencing analyses and E-64 active site titration. N-terminal sequencing of the first ten residues has shown high identity with the other known ficin (iso)forms. The molecular weight was found to be (24,294 ± 10) Da by mass spectrometry, a lower value than the apparent molecular weight observed on SDS–PAGE, around 27 kDa. Far-UV CD data revealed a secondary structure content of 22% a-helix and 26% b-sheet. The protein is not glycosylated as shown by carbohydrate analysis.
pH and temperature measurements indicated maxima activity at pH 6.0 and 50 °C, respectively.
Preliminary pH stability analyses have shown that the protease conserved its compact structure in slightly acidic, neutral and alkaline media but at acidic pH (<3), the formation of some relaxed or molten state was evidenced by 8-anilino-1-naphtalenesulfonic acid binding characteristics. Comparison with the known ficins A, B, C, D1 and D2 (iso)forms revealed that ficin E showed activity profile that looked like ficin A against two chromogenic substrates while it resembled ficins D1 and D2 against three fluorogenic substrates. Enzymatic activity of ficin E was not affected by Mg2+, Ca2+ and Mn2+ at a concentration up to 10 mM. However, the activity was completely suppressed by Zn2+ at a concentration of 1 mM. Inhibitory activity measurements clearly identified the enzyme as a cysteine protease, being unaffected by synthetic (Pefabloc SC, benzamidine) and by natural proteinaceous (aprotinin) serine proteases inhibitors, by aspartic proteases inhibitors (pepstatin A) and by metallo-proteases inhibitors (EDTA, EGTA). Surprisingly, it was well affected by the metallo-protease inhibitor o-phenanthroline. The enzymatic activity was however completely blocked by cysteine proteases inhibitors (E-64, iodoacetamide), by thiol-blocking compounds (HgCl2) and by cysteine/serine proteases inhibitors (TLCK and TPCK). This is a novel ficin form according to peptide mass fingerprint analysis, specific amidase activity, SDS–PAGE and PAGE electrophoretic mobility, N-terminal sequencing and unproneness to thiol pegylation.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Baeyens-Volant, Danielle; Université Libre de Bruxelles - ULB
Matagne, André ; Université de Liège > Département des sciences de la vie > Enzymologie et repliement des protéines
El Mahyaoui, Rachida; Université Libre de Bruxelles - ULB
Wattiez, Ruddy; Université de Mons-Hainaut - UMH
Azarkan, Mohamed; Université Libre de Bruxelles - ULB
Language :
English
Title :
A novel form of ficin from Ficus carica latex: Purification and characterization
Publication date :
2015
Journal title :
Phytochemistry
ISSN :
0031-9422
eISSN :
1873-3700
Publisher :
Pergamon Press, New York, United States - New York
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