Preparation and Characterisation of Homogeneous Neurotoxin Type A from Clostridium botulinum

Abstract

1. Large-scale production and purification of complexes between Clostridium botulinum neurotoxin and haemagglutinin have been achieved. 2. Haemagglutinin-free neurotoxic protein of the complexes was purified to high specific neurotoxicity by affinity chromatography, on p-aminophenyl beta-D-thiogalactopyranoside coupled to Sepharose 4B, followed by chromatography on DEAE-Sephacel. 3. The resultant neurotoxin was homogeneous on isoelectric focussing (pI = 6.3) and on dodecylsulphate/polyacrylamide gel electrophoresis under non-reducing conditions when its Mr was 1.4 X 10(5); after reduction two polypeptides (Mr = 9.9 and 5.5 X 10(4) were present. 4. On double-immunodiffusion gels, using antiserum against neurotoxin-haemagglutinin complex, the neurotoxin showed a single, sharp precipitin line that was immunologically distinct from a relatively non-toxic protein (Mr = 1.3 X 10(5), which co-purifies with the neurotoxin but is removed by the ion-exchange chromatography step. 5. Application of the neurotoxin to animals in vitro or in vivo produced near complete and irreversible blockade of neurotransmission. Botulinisation of rat leg muscles reduced spontaneous transmitter release; the amplitude of miniature end-plate potentials was altered from the normal 'bell-shaped" to a skewed distribution. 6. In normal muscle, a large transient increase in frequency of the miniatures was produced by beta-bungarotoxin. In contrast, with botulinised muscle the latter induced a much smaller increase in the absolute frequency; in addition, the mean amplitude was increased somewhat but the distribution remained skewed. The results show botulinisation of muscle modifies the action of beta-bungarotoxin.