Separation of Toxin and Hemagglutinin from Crystalline Toxin of Clostridium botulinum Type A by Anion Exchange Chromatography and Determination of Their Dimensions by Gel Filtration

Crystalline preparations of Clostridium botulinum type A toxin were fractionated on DEAE-cellulose columns with Tris-HCl buffers at pH 8.0. The isolated toxic fraction was free of hemagglutinating activity and contained 5 times the specific activity of the crystalline toxin. The second fraction was a powerful hemagglutinin but was only feebly toxic; it emerged from the column as one or more peaks, under different elution conditions. By rechromatography and immunological tests, the toxicity of the second fraction was shown to be due to contamination with traces of the toxic fraction. By several criteria, the toxic and hemagglutinating components appeared to be at least two different proteins. Most of the information obtainable from the ultracentrifugal analysis of these substances was also obtained by gel filtration on a Sephadex G-200 column and with much smaller protein concentrations. At physiological pH, the toxic fraction had a molecular weight of 150,000 and a Stokes radius of 48 A; these dimensions were in agreement with those established in vivo by other investigators. The hemagglutinin appeared to exist in three forms of aggregation with molecular weights of 290,000, 500,000, and 900,000.