Figure 4
Effect of incubation of SOD with proteolytic enzymes.
SOD was incubated in the absence (-) and presence (+) of 1/20 w/w of trypsin, chymotrypsin or papain in 50 mM potassium phosphate buffer, pH 7.8 and 37°C for 1, 2 and 3 h and run on a SDS-PAGE (a). M, molecular weight marker procured from Fermentas, USA; the protein markers were- β-galactosidase (11 6000 Da), bovine serum albumin (66 200 Da), Ovalbumin, 45 000 Da), lactate dehydrogenase (35 000 Da), REase Bsp981 (21 000), β- lactoglobulin (18 400) and lysozyme (14 400 Da)]. Full-length gels are shown in Supplementary Fig. S9a. Activity staining of protease treated SODs is shown in panel “b”. Protease treated SODs were run on native rather than SDS containing poly-acrylamide gel. Therefore, the migration pattern of SODs shows some variation between the panels (a) and (b). SOD activity was not detected for C56A and C145A and hence corresponding SOD activity stained gels are not shown. Full-length gels are shown in Supplementary Fig. S9b. Panel (c) depicts the effect of proteolytic enzymes on SOD activity; values are mean ± SE of three separate replicates. WT, wild type; C56A, cysteine at position 56 substituted with alanine; C95A, cysteine at position 95 substituted with alanine; C145A, cysteine at position 145 substituted with alanine.
