15M NaCl, using blue dextran 2000 (2,000kDa), BSA (67.0kDa), ovalbumin (43.0kDa), chymotrypsinogen selleck chem Vandetanib A (25.0kDa), and ribonuclease A (13.7kDa) 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 0.66mL/min, and the active fraction was stored at ?20��C for further analysis.2.2. Protein QuantificationProtein 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).2.3.

Molecular Mass DeterminationSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 12% slab gels according to Laemmli method [15]; the bands of reduced protein (50��g) 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).2.4. Optimum pH and Thermal Stability DeterminationThe optimum pH for the protease activity was determined in the H-D-Pro-Phe-Arg-pNA hydrolysis in a mixture of buffers (50mM acetate/borate/phosphate��all components purchased from Merck KGaA, Darmstadt, Germany) to cover the pH range of 2.0 to 11.5. The enzyme (50nM) was kept in each pH for 10min at 37��C. After that, the substrate (0.40mM) was added and the hydrolysis in each pH was followed by the absorbance at 405nm (��pNA 8,990M?1cm?1 [16]) for 20min in a Synergy HT microplate reader (purchased from Biotek Instruments, Winooski, VT, USA).

For the thermal stability determination, the purified enzyme (50nM) was first kept in 50mM Tris buffer pH 7.1 for 30min at different temperatures (20�C60��C). Afterward, the temperature was kept at 37��C and H-D-Pro-Phe-Arg-pNA (0.40mM) was added and the hydrolysis followed for 20min at this temperature by the absorbance at 405nm in the microplate reader.2.5. Kinetic CharacterizationMichaelis-Menten constants (Km) for the protease activity were obtained in the hydrolysis of H-D-Pro-Phe-Arg-pNA, H-D-valyl-L-leucyl-L-lysine-p-nitroanilide (H-D-Val-Leu-Lys-pNA), and N-benzoyl-L-isoleucyl-L-glutamyl (��-OR)-glycyl-L-arginine-p-nitroanilide (Bz-Ile-Glu(��-OR)-Gly-Arg-pNA) (all purchased from Chromogenix, Italy). The enzyme (50nM) was kept in 50mM Tris buffer, pH 7.1, for 2min at 37��C.

After that, a specific substrate was added in different concentrations (0.020mM to 1.2mM) and the hydrolysis of each Cilengitide substrate was followed by the absorbance at 405nm, for 30min 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.2.6.