Pyolysin Hemolysis Activity and Binding

The plo plasmid (pGS59) was a gift from Dr H Jost (University of Arizona), and pyolysin protein was generated as described previously [14 (link), 62 (link)]. The activity of pyolysin was 628,338 HU/mg protein, as determined by hemolysis assay using horse red blood cells (Oxoid, Hampshire, UK), as described previously [14 (link), 63 (link)]. Endotoxin contamination was 1.5 EU/mg protein, as determined by a limulus amebocyte lysate assay (LAL endotoxin quantitation kit; Thermo Fisher Scientific, Hertfordshire, UK). Streptolysin O was purchased from Sigma, stored as 1 mg/ml solution, and activated with 10 mM dithiothreitol according to the manufacturer’s instructions (Sigma). To examine the potential for pyolysin binding to glutamine, 100 HU/ml pyolysin was incubated for 1 h in PBS with vehicle, 2 mM glutamine, or 1 mM cholesterol as a positive control, and a hemolysis assay was conducted.