title: A novel form of ficin from Ficus carica latex: Purification and characterization.
abstract: 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% α-helix and 26% β-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 Mg(2+), Ca(2+) and Mn(2+) at a concentration up to 10mM. However, the activity was completely suppressed by Zn(2+) at a concentration of 1mM. 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.
authors.affiliation: Université Libre de Bruxelles, Faculty of Medicine, Protein Chemistry Unit, Campus Erasme (CP 609), 808 route de Lennik, 1070 Brussels, Belgium.
authors.forename: Danielle
authors.initials: D
authors.lastname: Baeyens-Volant
authors.affiliation: University of Liège, Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, Liège, Belgium.
authors.forename: André
authors.initials: A
authors.lastname: Matagne
authors.affiliation: Université Libre de Bruxelles, Faculty of Medicine, Protein Chemistry Unit, Campus Erasme (CP 609), 808 route de Lennik, 1070 Brussels, Belgium.
authors.forename: Rachida
authors.initials: R
authors.lastname: El Mahyaoui
authors.affiliation: University of Mons-Hainaut, Faculty of Sciences, Department of Proteomics and Microbiology, Interdisciplinary Center of Mass Spectrometry (CISMa), Mons, Belgium.
authors.forename: Ruddy
authors.initials: R
authors.lastname: Wattiez
authors.affiliation: Université Libre de Bruxelles, Faculty of Medicine, Protein Chemistry Unit, Campus Erasme (CP 609), 808 route de Lennik, 1070 Brussels, Belgium. Electronic address: mazarkan@ulb.ac.be.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Azarkan
mesh_terms: D002850:Chromatography, Gel; D057056:Cysteine Proteases / Q000187:drug effects / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005361:Ficain / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D030681:Ficus / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007840:Latex / Q000737:chemistry* / Q000302:isolation & purification; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D010618:Phenanthrolines / Q000494:pharmacology; D011092:Polyethylene Glycols; D017433:Protein Structure, Secondary; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D015853:Cysteine Proteinase Inhibitors; D007840:Latex; D010618:Phenanthrolines; D013438:Sulfhydryl Compounds; D011092:Polyethylene Glycols; D057056:Cysteine Proteases; D005361:Ficain; C000606234:ficin E, Ficus; D007930:Leucine; C024974:E 64; C025205:1,10-phenanthroline
keywords: Circular dichroism; Cysteine protease; Ficin; Ficus carica; Latex; Mass spectrometry