COMPARATIVE GEL FILTRATION OF TOXIN PRECURSOR AND TRYPSIN-ACTIVATED TOXIN OF CLOSTRIDIUM BOTULINUM TYPE E

Abstract

Precursor of type E botulinus toxin was highly purified from bacterial cells by extraction, ammonium sulfate precipitation, ribonuclease digestion, and chromatography on CM-Sephadex. The sample free from ribonucleic acid had a toxicity of 5.1 x 105 LD50 per mg of nitrogen before activation and 8.3 x 107 LD50 per mg of nitrogen after activation. This precursor and its activated product were subjected to gel filtration on a column of Sephadex G-200. No evidence for smaller fractions was obtained. Both precursor and trypsin-activated toxin were eluted in the void volume with 0.05 or 1 [image] acetate buffer (pH 6.0) or with 0.05 or 0.5 [image] phosphate buffer (pH 7.5). Intact trypsin and its degradation products were separated from toxin. The toxins eluted with the acetate buffers had potencies of 1.2 x 108 and 1.3 x 108 LD50 per mg of N, while those eluted with the phosphate buffers showed lower toxicities. Possible mechanisms involved in the activation process are discussed.