Demonstration and isolation of phallolysin, a haemolytic toxin from Amanita phalloides

Phallolysin, a high-molecular weight, thermo- and alcohol-labile toxin with haemolytic activity, was demonstrated in Amanita phalloides. Phallolysin was isolated from cold aqueous toadstool extracts by precipitation with 40% ammonium sulphate, ion-exchange chromatography on DEAE cellulose, gel chromatography on Biogel P-30 and electrofocussing in Ampholine. Electrofocussing in a flat pH gradient resulted in the separation of the gelchromatographically homogeneous phallolysin into phallolysin A (I.P. 8.06) and phallolysin B (I.P. 7.49). Phallolysin A exhibited 70% purity with respect to its behaviour on polyacrylamide gel electrophoresis when stained with Coomassie Brilliant Blue. On the average 0.62 mug, at least 0.20 mug of protein was equivalent to 1 haemolytic unit (HU). The i.p. LD50 for mice was found to be 1060 (930--1210) HU/kg.