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Purification and characterization of a toxic protein of clostridum botulinum type E, strain Iwanai. Bains, Hardial Singh


Clostridium botulinum type E, strain Iwanai, was grown in a dialysate apparatus, modified from the original apparatus of Vinet and Fredette (1951), using GPB1 medium with 0.5 percent dextrose and 1.0% sodium thioglycollate, for five days at 30° C. The toxin was precipitated from the cell-free toxic filtrate with 0.60 saturated ammonium sulfate at 4° C overnight. The toxic precipitate was obtained by centrifugation at 4,500 R.P.M. at 0° C for 45 minutes and dissolved in 0.01 M sodium acetate buffer at pH 5.5. The preparation was dialysed for 24 hours against the same buffer in versene-treated dialysis sacs and the insoluble material was removed by centrifugation. This preparation was then applied to ion-exchange columns. DEAE (Selectacel) cellulose, suspended and kept in 2 M NaC1 at 4° C, was packed into columns under gravity flow at room temperature, washed with 1N HC1 and equilibrated with 0.01 M sodium acetate buffer at pH 4.5. The toxin, eluted by the same buffer, reprecipitated and rechromatographed, was further analysed in the ultracentrifuge and with electrophoresis. Toxicity of the pure preparation was found to be 7.5 x 10⁶ MLD/mg N, the sedimentation coefficient, S₂₀W = 1.70, and the molecular weight 18,600. Partial amino acid analysis on this preparation was also carried out.

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