A SIMPLE METHOD FOR PURIFICATION OF TYPE E BOTULINAL TOXIN FROM THE PRECURSOR EXTRACT OF THE BACTERIAL CELLS

Abstract

The toxin of Clostridium botulinum, which gives rise to food poisoning when ingested by man and animals, has been described as a true exotoxin. The toxin of Cl. botulinum type A was obtained in crystalline form (Abrams et al., 1946; Lamanna et al., 1946) and those of other types have been purified (Lamanna and Glassman, 1947; Duff et al., 1957; Cardella et al., 1958; Cardella et al., 1960; Sterne and Wentzel, 1950; Gordon et al., 1957) from cell free culture filtrate or whole culture. These reports also indicated that the toxins of Cl. botulinum types A, B, C, D, and E are simple protein. Accordingly the procedures employed in purifying the toxins were those that had been used in the isolation of proteins.
The recent knowledge on the toxin production by Cl. botulinum tells that the toxin is formed through activation of the atoxic precursor, which is synthesized in bacterial cells (Sakaguchi and Tohyama, 1955 a, b ; Boroff et al., 1952; Bonventre and Kempe, 1960) . Activation is accomplished by certain bacterial enzymes (Sakaguchi and Tohyama, 1955 a, b ; Dolman, 1957; Bonventre and Kempe, 1960) or trypsin (Duff et al., 1956) .
It has also been found that type E toxin has neutral or weakly basic property, whereas the precursor strongly acidic property, which was ascribed to the ribonucleic acid in the precursor molecules (Sakaguchi and Sakaguchi, 1959) .
The authors attempted to purify type E botulinal toxin according to the principle derived from the foregoing knowledge. In the attempt, young bacterial cells were used as the starting material instead of cell free filtrate or whole culture. The extract of the bacterial cells contained a large amount of ribonucleic acid and the precursor. The trypsinization of the extract released the toxin free from ribonucleic acid, which was precipitated with ammonium sulfate. It was found that the toxin purified from the bacterial cells by the method reported here has a potency higher than those described by other workers (cordon et al., 1957; Gerwing et al., 1961; Fiock et al., 1961) .