Measurements of hemolytic activity.

Washed erythrocytes were suspended in HBS to produce an erythrocyte volume fraction of 1.25% in the final test solution. HlyA was added in increasing concentrations for 60 min at 37°C under constant swirling (180 rpm) in 96-well plates. The experiment was terminated by centrifugation of the samples at 2,325 × g for 3 min. The optical density of the supernatant was determined at 540 nm in a plate reader (PowerWave microplate spectrophotometer; Biotek Instruments, Winooski, VT, USA) as a measure of the hemolytic activity. Standard curves with increasing concentrations of HlyA identified the EC₅₀ of the toxin. The EC₅₀ for LtxA was found at a concentration of 0.122 μg ml⁻¹, which is slightly more potent in this purification than what was previously published (13). The EC₅₀ for HlyA varies and has to be determined every experimental week because of the faster degradation of protein activity. The amount of active protein which corresponded to EC₅₀ in our preparation has been determined to be 25 ng ml⁻¹ (4).