Fig. 4.

a Effect of various inhibitors on CmSP activity. The enzyme (0.1 mL/5 μg) was pre incubated with 2 mM concentration of phenylmethylsulfonylfluoride (PMSF), 2-iodoacetic acid, mercuric chloride and ethylenediaminetetraacetic acid (EDTA) and kept at 37 °C for 30 min, followed by addition of 1% casein in 50 mM Tris–HCl buffer of pH 8.0. The residual activities were measured. Trypsin represents 100% activity. CmSP without any inhibitor is used as control. b Activity assay in SDS-PAGE to determine the effect of inhibitors on CmSP. Lane 1—CmSP (without inhibitor), Lane 2—Sample + phenylmethylsulfonylfluoride, Lane 3—Sample + 2-iodoacetic acid, Lane 4—Sample + mercuric chloride, Lane 5—Sample + ethylenediaminetetraacetic acid. 50 µg of CmSP with or without inhibitor is loaded in each well on SDS-PAGE (12.5%) under denaturing conditions. Protein bands were visualized by CBB R- 250 staining. c Effect of various salts on enzyme activity. The enzyme (0.1 mL/5 μg) was pre incubated with 5 mM concentration of salts: FeCl₃, KCl, MgCl₂, NaCl, and CaCl₂ and kept at 37 °C for 30 min, followed by addition of 1% casein in 50 mM Tris–HCl buffer of pH 8.0. The residual activities were measured. Trypsin represents 100% of the protease activity and CmSP without salt shows the activity of purified CmSP