Purification, Characterization, and Oral Toxicity of Botulinum Type G Progenitor Toxin

  • Masafumi Nukina
  • Yumi Mochida
  • Tsutomu Miyata
  • Sumiko Sakaguchi
  • Genji Sakaguchi

Abstract

Clostridium botulinum type G (or C. argentinense) strain 2470 was grown overnight at 37°C in chopped meat glucose medium and transferred to a medium consisting of proteose peptone 4%, yeast extract 1%, glucose 1%, and L-cysteine 0.2%, pH 7.3, which was incubated for 6 days at 30°C. The 10-L culture with a potential toxicity of 1.3 x 108 mouse i.p. LD50 was brought to pH 4.0 with sulfuric acid and kept overnight at 4°C. The precipitate was packed by centrifugation and extracted twice with 0.2 M phosphate buffer, pH 6.0. The extract was treated with 0.5% trypsin (Difco 1:250) at 37°C for 60 min. The extract recovered 1.3 × 108 LD50. The residual cells were sonicated and treated with trypsin in a similar way, which recovered 1.1 × 108 LD50. The two extracts were combined, the toxicity of which was set as 100%. Ammonium sulfate was added to the combined extracts to a 0.5 saturation. The precipitate was dissolved in 150 ml of 0.2 M phosphate buffer, pH 6.0, which was clarified by centrifugation. This extract was dialyzed against 0.05 M acetate buffer, pH 4.0, which caused precipitation of the toxin. The precipitate was dissolved in 100 ml of 0.5 M NaC1-0.05 M acetate buffer, pH 4.5. It was clarified by centrifugation, concentrated by salting out, followed by dialysis. The extract of the second salting out recovered 191 mg of protein and 3.3 × 108 LD50 (118%). The extract, divided into 20-ml portions, was subjected to gel filtration on Sephadex G-200 (2.5 × 90 cm). The effluent in the void volume contained 96.3 mg protein and 2.8 × 108 LD50 (100%). The fraction was dialyzed against 0.5 M NaC1-0.05 M acetate buffer, pH 4.0, and added were the same volume of 0.5 M NaCl-0.05 M citrate buffer, pH 4.5, and 0.01% protamine. This was clarified by centrifugation and percolated through SP-Sephadex C-50 (2.5 × 30 cm) equilibrated with 0.5 M NaC1-0.05 M acetate buffer, pH 4.5. The percolate was diluted 2.5-fold with 0.05 M acetate buffer, pH 4.0, applied again to SP-Sephadex C-50 (1.6 × 12 cm) equilibrated with 0.2 M NaC1-0.05 M acetate buffer, pH 4.0, and eluted with NaC1 gradient from 0.2 to 0.7 M. The fractions eluted at 0.34 to 0.48 M NaC1 contained 48.0 mg of protein and 1.9 × 108 LD50 (68%). The toxin fraction was concentrated by salting out and subjected to the second gel filtration on Sephadex G-200 (2.5 × 90 cm) with the same buffer. A single protein peak eluted contained 22.9 mg of protein and 1.1 × 108 LD50 (39%). The specific toxicity was 3.0 × 107 LD50/mg N.

Keywords

Acetate Buffer Oral Toxicity Clostridium Botulinum Molecular Dissociation Toxin Molecule 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

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Copyright information

© Springer Science+Business Media New York 1993

Authors and Affiliations

  • Masafumi Nukina
    • 1
  • Yumi Mochida
    • 2
  • Tsutomu Miyata
    • 1
  • Sumiko Sakaguchi
    • 2
  • Genji Sakaguchi
    • 2
  1. 1.Public Health Research Institute of Kobe CityChuo-ku, KobeHyogo 650Japan
  2. 2.College of AgricultureUniversity of Osaka Prefecture Sakai-shiOsaka 591Japan

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