Effects of various treatments and pH on hemolytic activity

The purified hemolysin was heated to temperatures between 40 °C and 80 °C for 10 min, cooled on ice, and immediately assayed for hemolytic activity. After incubating the purified hemolysin with 100 μg/mL trypsin (4790 USP units/mg) at 37 °C for 1 h, 300 μg/mL trypsin inhibitor from soybean (3000–6000 USP units/mg) was added, and the hemolytic activity was assayed. The concentrations of EDTA, MgCl2, CaCl2, cholesterol, and sulfur-containing compounds such as l-cysteine, DTT, and 2-mercaptoethanol were determined, and their effect on hemolytic activity was evaluated after 10-min incubation with purified hemolysin at room temperature. The following final concentrations of each reagent were tested for their ability to stimulate the hemolytic activity of the purified hemolysin: 0.1 mM EDTA, 5 mM MgCl2 and CaCl2, 20–50 mM sulfhydryl compounds, and 10 μg/mL cholesterol. The assay was performed as described previously. The purified, untreated hemolysin was used as the control, with an assumed hemolytic activity of 100%. To determine the optimum pH for the induction of hemolytic activity, the activity of purified hemolysin was measured at different pH values ranging from 5.0 to 7.5.

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