Site-directed mutagenesis was performed with the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions with the wildtype construct as a template. All constructs were verified by nucleotide sequencing. The hemolytic activity of PLY was determined by lysis of sheep red blood cells (SRBC) (Darji et al., 1995). Purified protein was serially diluted in hemolysis buffer (50 mM sodium phosphate pH 6.6, 150 mM NaCl, 5 mM DTT, 0.1% (v/v) BSA) in final volumes of 50 µl and incubated for 30 min at 37°C with 50 µl of a suspension of SRBC (I08 ml−1). Release of hemoglobin was monitored by recording the absorbance at 405 nm. The amount of toxin necessary to lyse 50% of erythrocytes was determined and expressed as percentage of the value for PLYWT. The absorbance upon incubation with 1% Triton-X100 was used as reference value for 100% lysis of erythrocytes. Three independent measurements were performed for each PLY mutant.