Although developed for a binding site at the oligomerization domain, the high affinity of PB-3 with wildtype PLY is possibly supported by a covalent reversible interaction of Cys428 with the olefinic double bond of PB-3. The binding affinity of wildtype PLY was specific for PB-3 compared to other Michael acceptors with similar structures or electrophilic reactivity. Moreover, PB-3 was specific for PLY as it did not react with free L-cysteine and with activated nucleophilic cysteine in the active sites of a tested cysteine protease and protein tyrosine phosphatase, respectively. In that respect PB-3 fulfills essential reactivity and selectivity criteria of clinically admitted covalent drugs. As, however, the Cys-containing undecapeptide (UDP) as well as the oligomerization domain are common features of homologous cholesterol-dependent cytolysins (CDC) and are seemingly involved in the initial step of lipid-membrane insertion and pore-formation, Hemolysis assay. Hemolytic activity of toxins was described in multiple studies. Defibrinated sheep blood was purchased from Thermo Scientific and aliquoted for later use.