Properties of Clostridium Perfringens (Welch II) Type-A  -Toxin (Phospholipase c) Purified By Electrofocusing

SUMMARY Clostridium perfringens type-A α-toxin (phospholipase C) was purified on a preparative scale by a simple, rapid, two-stage procedure involving precipitation of culture-supernatant fluids with ammonium sulphate at 35 to 50% saturation, followed by isoelectric focusing in a pH 4.6 gradient. Milligram yields of highly purified α-toxin with 10 to 15% recovery of activity were obtained in single fractions. Two forms of α-toxin were identified by electrofocusing. The major peak of activity possessed a pI of 5.49±0.06(α A) and the minor, a pI of 5.25(α B). The former was free from detectable collagenase (pI 4.54), hyaluronidase (pI 4.73), θ-toxin (pI 6.56) and neuraminidase. It gave a single precipitin arc on immunoelectrophoresis, but analysis by SDS polyacrylamide disk-gel electrophoresis at high protein-loading revealed a major protein component of molecular weight 53,800 and two minor protein bands. Atomic emission spectro-scopy did not detect the presence of zinc in such preparations. The latter showed a single line of identity with α A in Ouchterlony gel diffusion tests and contained a protein component with the same molecular weight as that of α A. Fractions α A and α B both possessed hot-cold haemolytic, phospholipase-C, and lethal activities. Both hydrolysed lecithin and sphingomyelin. Electro-focusing of α-toxin in the presence of 6m urea resulted in the detection of only one component, α urea, with a pI identical to α A. It also possessed all three biological activities of α-toxin. Removal of the urea and refocusing was accompanied by the reappearance of α B. The occurrence and formation of α B could not be interpreted in terms of artefactual causes of multiple forms of proteins identified by isoelectric focusing. These studies provided evidence favouring the suggestion that α A and α B could be related as conformers, although aggregation or polymerisation could not be entirely excluded as possible alternative explanations.