Enzymatic Activity Assay of rSeCP

The enzymic activity of rSeCP was also determined by cleavage of the fluorescent substrate Z-carbobenzoxy-L-phenylalanyl-L-arginine-(7-amino-4-methylcoumarin) (Z-Phe-Arg-AMC, Sigma) as previously described [34 ]. Briefly, the substrate was prepared at a concentration of 2 mM dissolved in dimethylsulfoxide (DMSO). The reaction was carried out in a volume of 240 μl of standard assay buffer (100 mM sodium acetate, 5 mM dithiothreitol (DTT); pH 5.5) coupled with an appropriate quantity of enzyme. The rSeCP was pre-incubated in assay buffer at a volume of 160 μl at 37°C for 30 min, while substrate at a final concentration of 3 μM was pre-incubated in assay buffer as well. The reaction was started by mixing the two samples together. The fluorescence intensity was continuously measured with spectrophotofluorometry (Synergy H1, BioTek, USA) using excitation and emission wavelengths of 355 nm and 460 nm, respectively. The pH activity profiles were established using the following buffers: 100 mM sodium acetate buffer (pH 3.0–5.5), 100 mM sodium phosphate (pH 6.0–7.5), and 100 mM Tris–HCl (pH 8.0–8.5) containing 5 mM DTT. The optimal temperature for rSeCP activity was assayed at 10°C, 20°C, 28°C, 37°C, 45°C and 50°C. The residual activity in the samples and the control (without reagents) was also determined. The highest enzyme activity was used as the control (100% of relative activity). The influence of metal ions on rSeCP activity was determined and different concentrations of Cu⁺⁺, Mn⁺⁺ and Zn⁺⁺ metal ions (0.01 mM, 0.1 mM, 1 mM, 10 mM, and 100 mM) were added to the assay in the form of CuCl₂, MnCl₂ and ZnCl₂, respectively. The relative fluorescence unit (RFU) was used to express catalytic activity. rSeCP was also pre-incubated with inhibitor at 37°C for 30 min. Substrate was added, and incubated at 37°C for 30 min. Inhibitors included PMSF, phenylmethylsulfonyl fluoride; AEBSF, 4-(2-Aminoethyl) benzenesulfonyl fluoride; EDTA, ethylenediaminetet-raacetate; E-64, and L-trans-epoxysuccinyl-leucylamide (4-guanidino) butane. Percentages are based on activity in the presence of 10 mM DTT without inhibitors.