Isolation and identification of two hemolytic forms of streptolysin-O

Streptolysin-O was isolated from culture supernatants of group-A beta-hemolytic streptococci (Richards strain) by ammonium sulfate and polyethylene glycol precipitation, DEAE-ion exchange chromatography, preparative isoelectric focusing, and chromatography on Sephacryl S-300. Two forms of the toxin possessing similar hemolytic capacity were identified. The native toxin was a single polypeptide chain devoid of amino sugars with a sedimentation coefficient of 3.9S and a molecular weight of 69,000, and was isoelectric at pH 6.0 to 6.4. Partial degradation of the native toxin occurred during the isolation procedure, yielding a hemolytically active polypeptide with a molecular weight of 57,000 and a pI of 7.0 to 7.5. Both forms of the toxin generated the typical, heterogeneous, open and closed ring-structured channels in erythrocyte membranes. Structural considerations indicated that between 25 and 100 monomer toxin molecules constituted the individual ultrastructurally recognizable channels. Hemolytic titrations indicated that the presence of 70 to 125 toxin molecules per erythrocyte was required to generate an average of one functional lesion per cell. The data are consistent with the concept that one or very few streptolysin-O channels will cause hemolysis.