Purification of progenitor toxin of Clostridium botulinum type B strain Okra was undertaken by sequential steps of acid precipitation, extraction, ammonium sulfate precipitation, ribonuclease digestion, acid precipitation, protamine treatment, sulphopropyl-Sephadex chromatography, and Sephadex G-200 gel filtration. Two different molecular-sized toxins, named large (L) and medium (M) toxins, were obtained. L toxin was centrifugally homogeneous but electrophoretically heterogeneous. It contained 2.5 x 108 to 3.0 x 108 mean lethal doses per mg of nitrogen, and its sedimentation constant was 16S. M toxin was centrif'ugally and electrophoretically homogeneous. It contained 5.5 x 108 to 6.0 x 108 mean lethal doses per mg of nitrogen, and its sedimentation constant was 12S. The presence of both L and M toxins in spent culture was demonstrated. It seems justified, therefore, to call both progenitor toxins. Both consisted of toxic and nontoxic components. The toxic components of L and M toxins appeared to be identical with each other. The nontoxic component of L toxin was 12S and
The pathogenesis of type E botulism is discussed as an aspect of the physicochemical and biological properties of 12S toxins (prototoxin and trypsin-activated 12S toxin) and the Ea and Ef3 components of each 12S toxin. A molecular weight of 350,000 was determined for each 12S toxin and 150,000 for Eca and E3. Owing to the structure comprising the subunits Ect and E3, 12S toxins are much more stable than Ect at low pH values and high temperatures. Such was also the case with type A 19S toxin and its a component. The Ea component alone accounts for the total toxicity of type E toxin. The toxic substance detected in the blood of the animals administered 12S toxins orally or parenterally was identified as Ea from the molecular size and the chromatographic pattern. Prototoxin escaping from detoxification in the stomach owing to the subunit structure may undergo dissociation in the intestine to release the ECa component. After absorption, the activated Ea appeared in the circulating blood without any further signs of dissociation or enzymatic digestion.
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