The in vitro cytotoxicity assay of the purified protease of S. typhimurium against HT29 human adenocarcinoma cell line (Merck, Darmstadt, Germany) was performed on a 96-well plate by incubation for 24 h. Per 1 × 105 HT29 cells per milliliter, fifteen micrograms of the enzyme were used. By the end of incubation, the percent of cell death of HT29 was assessed by the standard MTT assay16 (link). For negative blanks, physiological saline was used instead of the active protease preparations, while for blanks, a medium without cells was used. The idea of this assay is that the remaining surviving cells can convert the yellow tetrazolium MTT reagent into the purple formazan complex with a characteristic absorption at A540 nm, therefore indicating HT29 viability. The intensity of the purple color is in direct relation to the number of surviving cells after exposure to the UcB5.
Furthermore, an assay of cell-damaging activity against RBCs (hemolytic activity) due to the purified enzyme was done. This was achieved by vortexing identical volume sizes of 15 µg protease/mL and 4% (v/v) washed human RBCs suspended in 0.1 M borate buffer, pH 7.5. Incubation was done at 37 °C for 90 min, thereafter, the quantity of released hemoglobin was measured colorimetrically. For comparison, a complete hemolysis treatment was done by mixing RBCs suspension with 1% (v/v) triton X-100 solution.
Also, an in vivo screening of cell-damaging activity was done and the LD50 value was calculated. For this, BALB/c mice weighing 22–25 g were acclimatized to the laboratory conditions for one week and retained at relatively fixed nutritional and physical conditions. They were then divided into six groups of six per cage. The first group was represented as the universal blank group. Mice in this group were intraperitoneally inoculated with an equal volume of a heat-denatured enzyme preparation at a concentration of 60 µg/body weight. While the other five groups were intraperitoneally injected with the active protease preparation at various concentrations (60, 30, 15, 8, and 4 µg protease/body weight) in a total volume of 1 ml solution. Animals were then observed at time intervals throughout 48 h for the LD50 calculation according to the method of Karber. Livers of affected and blank mice were removed instantly after death and fixed in 5% (v/v) glutaraldehyde then 1% (w/v) OsO4 solution. Before dissection of a blank mouse, it was anaesthsized by the inhalant gas sevoflurane. Ultrathin sections of 70 nm were sliced by RMC ultramicrotome and loaded on standard-grade TEM support grids made-up of copper for examination under JEOL 1010 TEM.
Furthermore, an assay of cell-damaging activity against RBCs (hemolytic activity) due to the purified enzyme was done. This was achieved by vortexing identical volume sizes of 15 µg protease/mL and 4% (v/v) washed human RBCs suspended in 0.1 M borate buffer, pH 7.5. Incubation was done at 37 °C for 90 min, thereafter, the quantity of released hemoglobin was measured colorimetrically. For comparison, a complete hemolysis treatment was done by mixing RBCs suspension with 1% (v/v) triton X-100 solution.
Also, an in vivo screening of cell-damaging activity was done and the LD50 value was calculated. For this, BALB/c mice weighing 22–25 g were acclimatized to the laboratory conditions for one week and retained at relatively fixed nutritional and physical conditions. They were then divided into six groups of six per cage. The first group was represented as the universal blank group. Mice in this group were intraperitoneally inoculated with an equal volume of a heat-denatured enzyme preparation at a concentration of 60 µg/body weight. While the other five groups were intraperitoneally injected with the active protease preparation at various concentrations (60, 30, 15, 8, and 4 µg protease/body weight) in a total volume of 1 ml solution. Animals were then observed at time intervals throughout 48 h for the LD50 calculation according to the method of Karber. Livers of affected and blank mice were removed instantly after death and fixed in 5% (v/v) glutaraldehyde then 1% (w/v) OsO4 solution. Before dissection of a blank mouse, it was anaesthsized by the inhalant gas sevoflurane. Ultrathin sections of 70 nm were sliced by RMC ultramicrotome and loaded on standard-grade TEM support grids made-up of copper for examination under JEOL 1010 TEM.
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