0M in 50mM Tris buffer, pH 7.1. The enzymatic assays were performed as described above. The influence of different cations was studied using KCl, NaCl, NH4Cl, MgCl2, and CaCl2 (all purchased from Merck KGaA, Darmstadt, Germany) in the range of 0.10 to 0.70M in the same conditions. 2.7. Inhibition of the http://www.selleckchem.com/products/Roscovitine.html Enzyme ActivityThe effect of inhibitors on the protease amydolytic activity was tested incubating the enzyme (50nM) with irreversible inhibitors as, N��-tosyl-Phe chloromethyl ketone (TPCK��50.0��M [17]), purchased from Fluka BioChemika (Saint Louis, MO, USA), L-trans-epoxysuccinyl-leucylamido-butane (E-64��5.0��M [18]) purchased from Merck KGaA (Darmstadt, Germany), phenylmethylsulphonyl fluoride (PMSF��0.50mM [19]), or N��-tosyl-Lys chloromethyl ketone (TLCK��50.

0��M [17]), both purchased from Sigma-Aldrich Chemical Company Inc. (Saint Louis, MO, USA), in 50mM Tris buffer pH 7.1, for 10min at 37��C. The activity was also tested in the presence of reversible inhibitors as CeKI (21.5 and 215nM [9]) purified by our group, benzamidine (0.50 and 4.0mM [20]) purchased from Sigma-Aldrich Chemical Company Inc., (Saint Louis, MO, USA), pepstatin A (1��M [21]) purchased from USB (Cleveland, OH, USA), aprotinin (92 and 307nM [22]), soybean trypsin inhibitor (SBTI: 49.8 and 498nM [23]), or ethylenediamine-tetraacetic acid (EDTA: 1.0 and 10.0mM [24]), all purchased from Calbiochem (Darmstadt, Germany), in 50mM Tris buffer, pH 7.1, for 10min at 37��C. After the preincubation, 0.40mM H-D-Pro-Phe-Arg-pNA was added, and the substrate hydrolysis was followed for 30min at 37��C at 405nm in the microplate reader.

3. Results and DiscussionIn the recent years, great efforts has been devoted to purification of plant proteases, which appear to be involved in several processes such as germination where specific degradation of cotyledon storage proteins occurs [25]. Also, they are thought to be useful toward understanding of several mechanisms at the subcellular level.This work focuses on the isolation and characterization of a protease of C. echinata seeds��CeSP. The crude protease was obtained by precipitation on 40% (NH4)2SO4. Purification to homogeneity was achieved by hydrophobic interaction and anion exchange chromatographies (Figures 1(a) and 1(b)) and gel filtration (Figure 1(c)). The details of the purification analysis are summarized in Table 1.

The specific activity of the purified enzyme was high (8,424U/mg) as compared to the activity in the crude extract (176U/mg). The total recovery of the activity was 8.2%. Besides, the enzyme was purified 47.8-fold.Figure 1Elution profile of CeSP purification. (a) Hydrophobic interaction chromatography, where a Hitrap GSK-3 Phenyl column was equilibrated with 50mM phosphate buffer, pH 7.0, 1.0M (NH4)2SO4, and proteins were eluted with (NH4)2SO4 (0.95, 0.25, …