Composition and Molecular Size of Clostridium botulinum Type A Toxin–Hemagglutinin Complex

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Pickett and Perrow have expressed several concerns regarding our recent publication on the stoichiometry of the Clostridium botulinum type A-Hall (Allergan) neurotoxin complex [1, 2]. These concerns demonstrate a misunderstanding regarding the purpose of our publication, which was focused on determining the subunit composition and homogeneity of this complex. Although the results in our publication were obtained with the C. botulinum type A neurotoxin complex used to manufacture BOTOX , it was not our goal to compare various type A neurotoxin complex products currently in clinical use. Based on current literature, knowledge regarding the number of each subunit present in the intact C. botulinum type A neurotoxin complex is extremely limited, even though the complex was purified to homogeneity more than 50 years ago [3, 4]. To date multiple reports exist in the literature suggesting that the 900 kDa complex is a dimer of a 500 kDa complex although there is limited evidence to support this [5, 6]. We established a quantitative procedure to determine the relative subunit composition and to confirm the molecular mass of our neurotoxin complex. As discussed in detail in our publication, we ensured that the established procedure was appropriate for this purpose by verifying both the recovery of the complex subunits as well as the overall consistency of the method. In addition, we clearly described our assumptions regarding the interpretation of the electropherograms, including our approach for handling the co-elution of the HA48 and the light chain subunits. We are, therefore, confident that our capillary electrophoresis method provides a valid procedure for quantifying the subunit stoichiometry of the neurotoxin complex. In contrast, sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS–PAGE) combined with mass spectrometry, as suggested by Pickett and Perrow, would not be suitable for this purpose since such an approach would only provide the molecular mass and the identity of proteins, and provide little if any information regarding the subunit stoichiometry. Analytical methods that evaluate the neurotoxin complex under denaturing conditions, including the capillary electrophoresis method described by us, cannot be used to establish whether the subunits form a single protein complex. We therefore, confirmed the integrity of the neurotoxin complex by size-exclusion HPLC in conjunction with multi-angle laser light-scattering detection. The molecular mass obtained for the neurotoxin complex shows that the different subunits identified in the electropherograms are part of a single complex and the results support the proposed subunit composition. The close agreement between the neurotoxin complex molecular mass values obtained by us and the complex mass values reported in earlier publications, while perhaps ‘‘unremarkable’’, confirms that the Allergan type A neurotoxin complex used to manufacture BOTOX consists of a homogeneous *900 kDa species containing one 150 kDa neurotoxin molecule per complex.