Activity of type A botulinal toxin and hemagglutinin exposed to proteolytic enzyme

The question of the response of the toxins of Clostridium botulinum to proteolytic enzymes has been inspired by the oral toxicity and protein nature of these toxins. Older reports of the in-nocuousness of proteolytic enzymes for crude preparations of type A toxin are in disagreement with recent reports of detoxification of crystalline toxin by proteolytic enzymes (Halliwell, 1954). Exploration of the question preceded knowledge that the botulinal toxins are associated with separable hemagglutinins (Lowenthal and La-manna, 1953). It is our purpose, therefore, to record observations on the biological activity of both the type A toxin and hemagglutinin upon their exposure in vitro to proteolytic enzymes. Exploratory study of the separation of the hemagglutinin from the toxin has also provided an opportunity to examine some preparations with varying relative contents of neurotoxic and hemagglutinating activities. MATERIALS AND METHODS Toxicity determinations. Two-fold dilutions of toxin solution were prepared in sterile 0.2 per cent gelatin-0.68 per cent phosphate buffer, pH 6.3. One-half ml quantities of a given dilution were injected intraperitoneally into 4 albino mice. LDso values were calculated by the method of Reed and Muench (1938). Hemagglutination determinations. Hemagglu-tination was determined by a spectrophotometric method (Lowenthal and Lamanna, 1953). A unit of hemagglutinating activity is the smallest amount of a solution agglutinating 50 per cent o