Biochemical Aspects of a Serine Protease from Caesalpinia echinata Lam. (Brazilwood) Seeds: A Potential Tool to Access the Mobilization of Seed Storage Proteins

Several proteins have been isolated from seeds of leguminous, but this is the first report that a protease was obtained from seeds of Caesalpinia echinata Lam., a tree belonging to the Fabaceae family. This enzyme was purified to homogeneity by hydrophobic interaction and anion exchange chromatographies and gel filtration. This 61-kDa serine protease (CeSP) hydrolyses H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide (K m 55.7 μM) in an optimum pH of 7.1, and this activity is effectively retained until 50°C. CeSP remained stable in the presence of kosmotropic anions (PO4  3−, SO4  2−, and CH3COO−) or chaotropic cations (K+ and Na+). It is strongly inhibited by TLCK, a serine protease inhibitor, but not by E-64, EDTA or pepstatin A. The characteristics of the purified enzyme allowed us to classify it as a serine protease. The role of CeSP in the seeds cannot be assigned yet but is possible to infer that it is involved in the mobilization of seed storage proteins.


Introduction
Peptidases or proteases are essential molecules for the survival of all organisms as they can be involved in many aspects of development, physiology, defense, stress responses, and adaptation to the changes of environment. Also, proteolytic enzymes regulate protein processing and intracellular protein levels and remove either abnormal or damaged proteins from the cell, working as a cellular housekeeper [1].
Serine proteases are the best-characterized group of proteolytic enzymes in mammals and microorganisms. In recent years several of those enzymes have been isolated from various plant species, mainly from their seeds such as trypsin-like peptidases from Glycine max [2] and Canavalia ensiformis [3,4], protease C1 from Glycine max [5], proteinase F from Vigna radiata [6], and subtilisin-like peptidases from Glycine max [7].
Caesalpinia echinata Lam. is a species of the Fabaceae (Leguminosae) family native in Brazilian forests that has been included in the list of species at risk of extinction [8]. Since 2000, our group has studied protein components from C. echinata seeds and we have already purified two serine protease inhibitors from them. C. echinata kallikrein inhibitor (CeKI) is able to inhibit human plasma kallikrein, plasmin, factor XIIa, factor Xa, porcine pancreatic trypsin [9], and human cathepsin G. C. echinata elastase inhibitor (CeEI) inhibits human neutrophil elastase, plasma kallikrein, and cathepsin G, porcine pancreatic elastase and bovine pancreatic chymotrypsin [10]. Inhibitory activity of these 2 The Scientific World Journal molecules upon proteolytic enzymes has been used to study and disclose molecular events involved in a lung edema model, paw edema, and inhibition of cell proliferation in a melanoma model (unpublished data).
It has been recognized that plant protease inhibitors belonging to Kunitz and Bowman-Birk trypsin inhibitor families [11] function as storage proteins in seeds. These proteins are essential for seed germination and normal plant development. The early initiation of storage protein mobilization occurs through the active proteases, such as cysteine, serine, aspartyl, or metallo proteases deposited, during seed maturation, in protein bodies and vacuoles. The start of storage protein mobilization indicates that the mechanisms protecting storage proteins against degradation during middle and late maturation have been overcome. However, only few experiments have been performed with isolated protein bodies to show if the incubation under appropriate conditions leads to the breakdown of internal proteins that could be attributed to the action of these proteases [12,13]. Thus, we were interested to verify the existence of proteases from C. echinata seeds, similar to the ones described, which could be involved in mobilization of seed reserves of this leguminous during early growth. In the present work, we are reporting the purification and characterization of the first serine protease described in C. echinata seeds, named C. echinata serine protease (CeSP).

Enzyme Purification.
A crude enzyme preparation was obtained from C. echinata coatless dry seeds triturated and homogenized in 0.10 M Tris (purchased from Merck KGaA, Darmstadt, Germany) buffer pH 7.5 (1 : 20, w/v). The homogenate was centrifuged for 30 min at 2,300 g and the supernatant was subjected to an (NH 4 ) 2 SO 4 (purchased from Merck, Darmstadt, Germany) fractionation and allowed to stand overnight in cold. The resultant precipitated was recovered by centrifugation at the same conditions described above and dissolved in 50 mM Na 2 HPO 4 /NaH 2 PO 4 (purchased from Merck KGaA, Darmstadt, Germany) buffer, pH 7.0. Proteolytic activity was examined in the hydrolysis of H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroaniline (H-D-Pro-Phe-Arg-pNA) (purchased from Chromogenix, Italy) that was used to monitor the purification procedure. The sample with proteolytic activity was applied to a HiTrap Phenyl column (purchased from GE Healthcare Bio-Sciences AB, Piscataway, NJ, USA) equilibrated in 50 mM phosphate buffer, pH 7.0, 1.0 M (NH 4 ) 2 SO 4 . Proteins were eluted in stepwise procedure with (NH 4 ) 2 SO 4 (0.95, 0.25, and 0.13 M) at a flow rate of 2.0 mL/min. The fractions with enzymatic activity were pooled, dialyzed against 20 mM Tris buffer, pH 7.5, and applied to a Resource Q anion exchange column (purchased from GE Healthcare Bio-Sciences AB, Piscataway, NJ, USA) equilibrated with 50 mM Tris buffer, pH 7.5. Proteins were eluted with a NaCl (purchased from Merck KGaA, Darmstadt, Germany) gradient (0 to 0.50 M) at a flow rate of 2.0 mL/min. The fractions containing enzymatic activity were pooled and submitted to a gel filtration on a Supe-rdex 75 column (purchased from GE Healthcare Bio-Sciences AB, Piscataway, NJ, USA) equilibrated with 50 mM phosphate buffer, pH 7.0, 0.15 M NaCl, using blue dextran 2000 (2,000 kDa), BSA (67.0 kDa), ovalbumin (43.0 kDa), chymotrypsinogen A (25.0 kDa), and ribonuclease A (13.7 kDa) as standards (gel filtration calibration kit purchased from GE Healthcare Bio-Sciences AB, Piscataway, NJ, USA). Proteins were eluted in the same buffer at a flow rate of 0.66 mL/min, and the active fraction was stored at −20 • C for further analysis.

Protein Quantification.
Protein content was quantified according to the Bradford method [14] and using a standard curve of bovine serum albumin (BSA, purchased from Sigma-Aldrich Chemical Company Inc, Saint Louis, MO, USA).

Molecular Mass Determination.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 12% slab gels according to Laemmli method [15]; the bands of reduced protein (50 μg) were stained with Coomassie brilliant blue, and compared to the molecular mass markers from Kaleidoscope Prestained Standards (purchased from Bio-Rad Laboratories Hercules, CA, USA).

Optimum pH and Thermal Stability Determination.
The optimum pH for the protease activity was determined in the H-D-Pro-Phe-Arg-pNA hydrolysis in a mixture of buffers (50 mM acetate/borate/phosphate-all components purchased from Merck KGaA, Darmstadt, Germany) to cover the pH range of 2.0 to 11.5. The enzyme (50 nM) was kept in each pH for 10 min at 37 • C. After that, the substrate (0.40 mM) was added and the hydrolysis in each pH was followed by the absorbance at 405 nm (ε pNA 8,990 M −1 cm −1 [16]) for 20 min in a Synergy HT microplate reader (purchased from Biotek Instruments, Winooski, VT, USA). For the thermal stability determination, the purified enzyme (50 nM) was first kept in 50 mM Tris buffer pH 7.1 for 30 min at different temperatures (20-60 • C). Afterward, the temperature was kept at 37 • C and H-D-Pro-Phe-Arg-pNA (0.40 mM) was added and the hydrolysis followed for 20 min at this temperature by the absorbance at 405 nm in the microplate reader.

Kinetic Characterization.
Michaelis-Menten constants (K m ) for the protease activity were obtained in the hydrolysis of H-D-Pro-Phe-Arg-pNA, H-D-valyl-L-leucyl-L-lysinep-nitroanilide (H-D-Val-Leu-Lys-pNA), and N-benzoyl-Lisoleucyl-L-glutamyl (γ-OR)-glycyl-L-arginine-p-nitroanilide (Bz-Ile-Glu(γ-OR)-Gly-Arg-pNA) (all purchased from Chromogenix, Italy). The enzyme (50 nM) was kept in 50 mM Tris buffer, pH 7.1, for 2 min at 37 • C. After that, a specific substrate was added in different concentrations (0.020 mM to 1.2 mM) and the hydrolysis of each substrate was followed by the absorbance at 405 nm, for 30 min at 37 • C in the microplate reader. Two different experiments were performed in triplicate. The mean ± SD was obtained by statistical analysis using the commercial program GraphPad Prism version 5 (GraphPad Softaware Inc, San Diego, CA, USA). One-way analysis of variance with Bonferroni posttest was used.

Salt
Influence on Catalytic Activity. The influence of different anions on the enzyme activity was determined using NaCl, NaBr, NaI, Na 2 SO 4 , NaH 2 PO 4 , and NaC 2 H 3 O 2 (all purchased from Merck KGaA, Darmstadt, Germany) in the range of 0.50 to 1.0 M in 50 mM Tris buffer, pH 7.1. The enzymatic assays were performed as described above. The influence of different cations was studied using KCl, NaCl, NH 4 Cl, MgCl 2 , and CaCl 2 (all purchased from Merck KGaA, Darmstadt, Germany) in the range of 0.10 to 0.70 M in the same conditions.

Results and Discussion
In 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% (NH 4 ) 2 SO 4 . 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 (8,424 U/mg) as compared to the activity in the crude extract (176 U/mg).
The total recovery of the activity was 8.2%. Besides, the enzyme was purified 47.8-fold.
About optimum pH and thermal stability, CeSP behaves as other described seed proteases. It exhibits a neutral optimum pH of 7.1 (Figure 3(a)) similar to a protease from soybean seeds, which works at pH 8.0 cleaving between two Arg residues, in the hydrolysis of β-conglycinin, a storage protein [31]. CeSP effectively retains its activity toward H-D-Pro-Phe-Arg-pNA hydrolysis at a temperature range of 20 to 50 • C (Figure 3(b)), and this profile is similar to serine proteases from Cucumis melo [32], Trichosants kirilowi [33], Hordeum vulgare [34], and another leguminous seeds [3,4].
The effect of ions on protein stability may be caused by chemical interactions between proteins and ions to form complexes. The ion specificity was mostly attributed to the ion ability to modify the water structure, thus influences the protein hydration environment. It has been realized that an optimal stabilization of enzymes could be achieved through the use of salts with kosmotropic anions and chaotropic cations [35]. Then, the effect of Hofmeister series on enzyme stability was determined in the enzyme activity in presence of the different salts. The enzyme remained stable in the presence of NaH 2 PO 4 , Na 2 SO 4 , NaC 2 H 3 O 2 , and NaCl, losing its activity in the presence of NaBr and NaI (Figure 4(a)). With the exception of chloride, the pattern of ionic interaction followed the Hofmeister series of salt hydration-PO 4 [35]. The cations influence in the enzyme activity was also studied. CeSP remained stable in the presence of KCl, NaCl, and MgCl 2 , losing its activity in the presence of NH 4 Cl and CaCl 2 (Figure 4(b)). With the exception of ammonium ion, the pattern of ionic interaction followed the Hofmeister series of salt hydration-NH 4 + > K + > Na + > Mg +2 > Ca +2 [35].
In addition to the presented parameters, inhibition studies can provide a first insight into the nature of the enzyme, its cofactor requirement, and the nature of active site. As shown in Table 3 H-D-Pro-Phe-Arg-pNA 55.7 ± 6.7 0 .0053 ± 0.0002 95.2 ± 9.5 Bz-Ile-Glu(γ-OR)-Gly-Arg-pNA 297 ± 45 0.0091 ± 0.0006 30.6 ± 3.1 H-D-Val-Leu-Lys-pNA 2, 09 ± 189 0.031 ± 0.002 14.8 ± 1.0 Hydrolysis of synthetic peptides (0.020 mM to 1.2 mM) by 50 nM CeSP in 50 mM Tris buffer pH 7.1, for 30 min at 37 • C, was followed at 405 nm.  was observed with TLCK, an irreversible inhibitor of serine proteases. It is possible that alkylation of His residue in CeSP by TLCK leads to chemical modification resulting in the inactivation of the specific active site, which is responsible for the cleavage at positively charged residues at P 1 positions of substrates, as reported by Imhoff and Keil-Dlouha [36]. CeSP was also inhibited by benzamidine (about 50%), an inhibitor of serine proteases, showing the CeSP affinity for Arg residues. So, these results may suggest that this protease is a serine protease.
As well as other serine proteases purified from leguminous seeds, C. lineata [4], C. ensiformis [3], and G. max [2], CeSP was not efficiently inhibited by protein inhibitors of serine proteases (aprotinin, SBTI, and CeKI), nor by PMSF (Table 3). Although these macromolecular inhibitors can affect activity of several serine proteases in animal physiological processes, they not necessarily inhibit correlated plant proteases. This result corroborates the fact that the proteins identified as protease inhibitors have others functions in seeds. Regarding to PMSF inability to block CeSP activity, it has been accepted that although serine proteases are stru- cturally conserved enzymes, their catalytic triad may vary slightly and such a variation might account for the observed lack of inhibition of some serine proteases by PMSF.
To corroborate the hypothesis that CeSP is a serine protease, typical inhibitors of aspartyl, metallo, and cysteine protease were tested upon its activity. As shown in Table 3, these inhibitors had no effect on the enzyme activity, indicating that this enzyme did not belong to these classes of proteases.

6
The Scientific World Journal The physiological role of the CeSP was not elucidated yet and additional experiments need to be performed to address this question. However, several reports have documented the involvement of different proteases in the mobilization and cleavage of seed storage proteins upon seed imbibition and germination, which is an essential step in the establishment of the seedling [37]. The protein cleavage makes possible subsequent extensive breakdown catalyzed by other proteases such as endo-and exopeptidases. The proteolytic enzymes that degrade the storage proteins during mobilization identified so far are mostly cysteine proteases [11], but also include serine [6], aspartic, and metallo proteases [38]. However, mobilization of storage protein involves several members of a protease family or members of different protease families, the protease responsible for the initial degradation appears to be a serine protease [5]. This evidence indicates CeSP as a potential enzyme involved in the initial steps of mobilization of storage protein, which would contribute to understanding how plants switch from synthesis and accumulation of storage proteins during development to hydrolysis of those proteins upon germination. Besides addressing elementary questions in plant protein metabolism, studies of the mobilization of seed storage proteins by proteases open the perspective to understand, for example, how these enzymes could be applied toward reduction of food allergens, many of which are storage proteins.

Conclusion
This is the first report of the purification and characterization of a protease from Caesalpinia echinata Lam. seeds. The molecular mass and enzymatic characteristics were allowed us to classify it as a serine protease. The role of CeSP in the seeds cannot be assigned yet but is possible to infer that it is involved in the mobilization of seed storage protein.