Separation and Characterization

Clostridium botulinum type C toxin consists of a heavy and a light chain with molecular weights of 98,000 and 53,000, respectively, which are linked by one disulfide bond. The two components were separated from each other by quaternary aminoethyl Sephadex A-50 column chromatography by stepwise elution with NaCl in 21.5 mM borax-45 mM sodium dihydrogen phosphate buffer, pH 8.0, containing 5% 2-mercaptoethanol at 0°C. The purified components had different amino acid compositions and antigenicities, and the toxicity of the toxin was neutralized completely by either antiheavy chain Fab or anti-light chain Fab. The two components could be reconstituted to form an active molecule with recovered toxicity which varied according to the method used. Maximum recovery was obtained in a system in which the intersubunit SS bond was first formed in the presence of high concentration of neutral salts, after which the concentration of salt was gradually decreased. The reconstituted preparation was highly toxic and had the same properties as the parental toxin on chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunodiffusion. By the use of three perturbants, the fractions of exposed tryptophans and tyrosines of the preparation were found to be almost the same as that of the parental toxin.