Evidence that subunits of type A botulinum toxin need not be linked by disulfide.

Type A neurotoxin of Clostridium botulinum strain 62A was purified by a modification of the procedure of TSE et al. (1982). Electrophoresis in sodium dodecyl sulfate - polyacrylamide gels (SDS - PAGE) indicated the mol. wt of the intact dichain molecule is 140,000 and that of its L subunit is 52,000, both expected from published values. However the mol. wt of 83,000 for the H subunit was lower than the mol. wt of 97,000 in the literature. The purified toxin separated in SDS-PAGE into H and L subunits when pretreated with 2-mercaptoethanol but it unexpectedly behaved similarly without the pretreatment. Specific toxicity (approximately 3 x 10(8) mouse LD50/mg protein) was not affected by the spontaneous molecular change that made dissociation into subunits possible. The subunits of dichain botulinum toxins are believed to be covalently joined by intersubunit disulfide(s) since they have been demonstrated only when samples are treated with 2-mercaptoethanol or dithiothreitol. Since it is not always needed, the pretreatment is apparently not reducing a disulfide that connects the subunits. The strong chelating activity also possessed by the pretreating agents suggest that the subunits may be joined by a metallic divalent cation.