The Separation of Alpha Toxin (Lecithinase) from Filtrates of Clostridium Welchii

Abstract

Alpha toxin has been separated from filtrates of Cl. welchii type A by fractionation in methanol-water mixtures under controlled pH, ionic strength and temperature. By means of four fractionation steps, the toxin was purified 130 fold on the basis of Lb activity per mg of non-dialysable nitrogen. Lecithinase combining activity, lethal activity, and alpha (hot-cold) hemolysin were found to be proportional in the four fractions, indicating that one substance may be responsible for all these activities. However, a homogeneous protein has not been isolated as yet, and conclusive proof that the three activities are functions of a single agent is still lacking. The major contribution of the present study was the effective chemical separation of alpha from theta toxin. Earlier attempts to separate alpha from theta toxin by ultrafiltration (13) and ammonium sulfate fractionation (6) were unsuccessful. PIV #253 was highly lethal and the ratio of Lb to M.L.D. per mg N remained constant in both the filtrate and in PIV. This is additional evidence that alpha toxin and not theta is the lethal component of Cl. welchii type A infections. It should be mentioned that purification studies were greatly retarded by low potency parent filtrates. It appears that much work remains to be done to produce filtrates rich in alpha toxin on a defined medium, if further progress is to be made in purification of the biologically and immunologically active components in Cl. welchii culture filtrates.