Abstract
High enterotoxin-producing substrains of Clostridium perfringens type A were selected reproducibly as colonies having toxin-antitoxin haloes on agar plates of Duncan-Strong medium containing antitoxin serum. Enterotoxin from these sub strains was subjected to rapid purification by high performance liquid chromatography (HPLC). For this, the toxin was extracted by sonication from sporulating bacteria grown in Duncan-Strong sporulation medium, fractionated by ammonium sulfate (40% saturation) precipitation and differential solubilization and then purified by HPLC: gel permeation chromatography through a G2000SW column and ion-exchange chromatography on a Mono Q column. Purified toxin preparations had a similar specific activity (4.2 x 102 mouse MLD/mg protein) and homogeneity on polyacrylamide gel-electrophoresis to preparations obtained by conventional gel permeation through a Sephadex-G200 column. By further HPLC on a Mono Q column, minor nontoxin proteins were separated from the toxin without loss of the toxicity on a protein basis. The final yield of the purified toxin was about 15° of that in the bacterial extract. The two HPLC procedures each took only one hour.
Similar content being viewed by others
References
BarnhartH.M., BullermanL.B., BallE.M. and WagnerF.W. (1976): Isolation and purification of Clostridium perfringens enterotoxin by affinity chromatography - J. Food Sci., 41: 903–905.
DavisB.J. (1964): Disc electrophoresis IL method and application to human serum proteins. - Ann. N.Y. Acad. Sci., 121: 404–427.
DuncanC.L. (1970): Clostridium perfringens food poisoning. - J. Milk Food. Technol., 33: 35–41.
DuncanC.L. and SomersE.B. (1972): Quantitation of Clostridium perfringens type A enterotoxin by electroimmunodiffusion. - Appl. Microbiol., 24: 801–804.
DuncanC.L. and StrongD.H. (1968): Improved medium for sporulation of Clostridium perfringens. -Appl. Microbiol., 16: 82–89.
DuncanC.L. and StrongD.H. (1971): Clostridium perfringens type A food poisoning. - Infect. Immun., 3: 167–170.
DuncanC.L., StrongD.H. and SebaldM. (1972): Sporulation and enterotoxin production by mutants of Clostridium perfringens. - J. Bact., 110: 378–391.
EndersG.L. and DuncanC.L. (1977): Preparative polyacrylamide gel electrophoresis purification of Clsotridium perfringens enterotoxin. - Infect. Immun., 17: 425–429.
GenigeorgisC. (1975): Public health importance of Clostridium perfringens. - J. Am. Vet. Med. Assoc., 167: 821–827.
GenigeorgisC., SakaguchiG. and RiemannH. (1973): Assay method for Clostridium perfringens type A enterotoxin. - Appl. Microbiol., 26: 111–115.
GranumP.E. and SkjelkvaleR. (1977): Chemical modification and characterization of enterotoxin from Clostridium perfringens type A. - Acta Path. Microbiol. Scand. Sect. B, 85: 89–94.
GranumP.E. and WhitakerJ.R. (1980): Improved method for purification of enterotoxin from Clostridium perfringens type A. - Appl. Environ. Microbiol., 39: 1120–1122.
HauschildA.H.W. and HilsheimerR. (1971): Purification and characteristics of enterotoxin of Clostridium perfringens type A. - Can. J. Microbiol., 17: 1425–1433.
HauschildA.H.W., HilsheimerR. and MartinW.G. (1973): Improved purification and further characterization of Clostridium perfringens type A enterotoxin. -Can. J. Microbiol., 19: 1379–1382.
HauschildA.H.W., WalcroftM.T. and CampbellW. (1971): Emesis and diarrhea induced by enterotoxin of Clostridium perfringens type A in monkeys. -Can. J. Microbiol., 17: 1141–1143.
LabbeR.G. (1980): Relationship between sporulation and enterotoxin production in Clostridium perfringens type A. - Food Technol., 34: 88–90.
LaurellC.B. (1965): Antigen-antibody crossed electrophoresis.- Anal. Biochem., 10: 358–361.
LowryO.H., RosenbroughN.J., FarrA.L. and RandallR.J. (1951): Protein measurement with a Folin phenol reagent. - J. Biol. Chem., 193: 265–275.
McDonelJ.L. (1980): Clostridium perfringens toxins (type A, B, C, D, E). - Pharmac. Ther., 10: 617–655.
PopoffM.R. (1984): Clostridium perfringens type A enterotoxin: a rapid for preparation of a specific antiserum using an enterotoxin purified by the polyacrylamide gel electrophoresis technique. -FEMS Microbiol. Lett., 21: 1–5.
ReynoldsD., TranterH.S. and CapelB.J. (1984): Large-scale production and purification of Clostridium perfringens type A enterotoxin - Biochem. Soc. Trans., 12: 262–263.
SakaguchiG., UemuraT. and RiemannH.P. (1973): Simplified method for purification of Clostridium perfringens type A enterotoxin. - Appl. Microbiol., 26: 762–767.
ScottV.N. and DuncanC.L. (1975): Affinity chromatography purification of Clostridium perfringens enterotoxin. - Infect. Immun., 12: 536–543.
StarkR.L. and DuncanC.L. (1971): Biological characteristics of Clostridium perfringens type A enterotoxin. -Infect. Immun., 4: 89–96.
StarkR.L. and DuncanC.L. (1972): Purification and biochemical properties of Clostridium perfringens type A enterotoxin. - Infect. Immun., 6: 662–673.
ToddE.C.D. (1978): Foodborne disease in six countries —a comparison. - J. Food Protect., 41: 559–565.
YotisW.W. and CatsimpoolasN. (1975): Scanning isoelectric focusing and isotachophoresis of Clostridium perfringens type A enterotoxin - J. Appl. Bact., 39: 147–156.
Author information
Authors and Affiliations
Additional information
Corresponding author.
Rights and permissions
About this article
Cite this article
Sugimoto, N., Ozutsumi, K. & Matsuda, M. Purification by high performance liquid chromatography of Clostridium perfringens type a enterotoxin prepared from high toxin producers selected by a toxin-antitoxin halo. Eur J Epidemiol 1, 131–138 (1985). https://doi.org/10.1007/BF00141806
Issue Date:
DOI: https://doi.org/10.1007/BF00141806