Expression and functional activity of IsSMase. A, Purified recombinant IsSMase was resolved by 10% discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue dye. Lane 1, molecular weight standard (kDa); Lane 2, flow-through; Lane 3, final wash; Lanes 4-8, eluates containing purified IsSMase (indicated by arrow). B, Functional activity of IsSMase was assessed by monitoring the rate of sphingomyelin hydrolysis at 37°C under standard conditions. C and D, effect of metal ions and pH on IsSMase activity. The sphingomyelinase activity of the IsSMase was recorded at multiple time points using excitation in the range of 530-560 nm and emission detection at 590 nm. Data plotted represent means ± SE (n=3). C, Activity of IsSMase at different pH.