The Identification and Purification of Multiple Forms of o-Haemolysin (o-Toxin) of Clostridium perfringens Type A

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Abstract
The ø-haemolysin of Clostridium perfringens was purified from culture supernatant fluids of type A strains fractional ammonium sulphate precipitation and isoelectric focusing in narrow pH 5 to 8 gradients. Four components detected on electrofocusing were designated ø1 (pI 6.8 to 6.9), ø2 (pI 6.5 to 6.6), ø3 (pI 6.1 to 6.3) and ø4 (pI 5.7 to 5.9). Specific activities ranged from 0.4 X 106 to 1.2 X 106 haemolytic units/mg protein and 2950 to 3600 LD50/mg protein. Each haemolytic component was activated cysteine hydrochloride, and inactivated cholesterol, addition of sheep erythrocyte ghosts and heating at 60 °C for 10 min; mouse erythrocytes were more resistant than sheep erythrocytes to haemolysis. A reaction of identity was obtained between components in gel diffusion. Sodium dodecyl sulphate polyacrylamide disc gel electrophoresis gave molecular weights in the range 59000 to 62000 for each component. A similar value was obtained for ø1 on density gradient ultracentrifugation. Although the multiple forms were free of 11 factors present in culture supernatants, crossed immunoelectrophoresis and disc gel electrophoresis revealed minor contaminants. These studies reveal that ø-haemolysin has physical properties in common with other oxygen-labile haemolysins.

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