BIOPHYSICAL CHARACTERIZATION OF THE STABILITY OF THE 150-KILODALTON BOTULINUM TOXIN, THE NONTOXIC COMPONENT, AND THE 900-KILODALTON BOTULINUM TOXIN COMPLEX SPECIES

Botulinum neurotoxin serotype A is initially released from the bacteri um Clostridium botulinum as a stable 900-kDa complex. The serotype A 9 00-kDa complex is one of the forms of the toxin being used as a therap eutic agent for the treatment of various neuromuscular disorders. Prev ious experiments have demonstrated that the 900-kDa complex form of th e toxin protects the toxin from the harsh conditions of the gastrointe stinal tract. To provide molecular level details of the stability and equilibrium of the 900-kDa complex, the nontoxic component, and the to xic (botulinum neurotoxin) component, the three species have been inve stigated,vith a series of biophysical techniques at the molecular leve l (dynamic light scattering, proteolysis, circular dichroism, pH incub ations, and agglutination assays). These experiments were conducted un der harsh conditions which mimic those found along the gastrointestina l tract. Separately, exposure to denaturing and proteolytic conditions degrades both the botulinum neurotoxin and the nontoxic component. In the 900-kDa complex, the botulinum neurotoxin is protected during exp osure to the gastrointestinal environment and the nontoxic component i s slightly modified. Surprisingly, the toxin protects the ability of t he nontoxic component to agglutinate erythrocytes. Contrary to previou s reports, the purified 900-kDa complex did not have agglutination abi lity until after exposure to the proteolytic conditions. These experim ents provide new evidence and detail for the theory that the nontoxic component and the toxic component protect one another during exposure to harsh conditions, and a molecular model is presented for the passag e of the toxin through the gastrointestinal tract.