Figure 2. Serum inhibition of PLY requires cholesterol with a free 3β-hydroxyl.

PLY requires the free 3β-hydroxyl of cholesterol to bind therefore treatment with CHOD will abolish PLY-cholesterol binding. Samples containing PBS, PBS +10% mouse serum or PBS +50% human serum were incubated with and without cholesterol oxidase (CHOD) for 30 minutes at room temperature to oxidize the 3β-hydroxyl. PLY (38 nmol/150 µl) was then serially diluted using untreated or CHOD-treated mouse (A) or human sera (B) and incubated 20′ at 37°C before RBC hemolysis was determined. The EC₅₀ values (toxin concentration required to lyse 50% of RBCs) and the standard deviations (SD) are shown for each sample. The fold difference in the EC₅₀ for CHOD treated and untreated samples versus PLY in the absence of serum (PBS control) is shown in parenthesis. Assays were done in duplicate; the data represent three independent experiments. The EC₅₀ calculation included the starting serum dilutions (mouse serum was initially diluted to a greater extent, as the mouse serum was found to be significantly more inhibitory than human serum).