Separation of the components of type A botulinum neurotoxin complex by electrophoresis S.K. Sharma a , M.A. Ramzan b , B.R. Singh a, * a Department of Chemistry and Biochemistry, Center for Marine Science and Technology, University of Massachusetts Dartmouth, 285 Old Westport Road, Dartmouth, MA 02747, USA b IPSEN Ltd, 190, Bath Road, Slough, Berkshire SL1 3XE, UK Received 13 May 2002; accepted 2 October 2002; Abstract Clostridium botulinum neurotoxins (BoNTs) are the most toxic substances known. They exert potent neuroparalysis on vertebrates. C. botulinum produces seven serotypes of neurotoxin (A – G). BoNT/A, found in bacterial cultures of C. botulinum type A, is produced as a complex with a group of neurotoxin associated proteins (NAPs). Botulinum neurotoxin complex is the only known example of a protein complex where a group of proteins (NAPs) protect another protein (BoNT) against the acidity and proteases of the stomach. Here, we used sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS – PAGE) for separation and identification of the constituent proteins of BoNT/A complex. A range of homogenous and gradient SDS – PAGE gels was used to resolve the BoNT/A complex. These gels were run under constant voltage and constant current conditions. The molecular weight and relative amount of each protein band were determined. On a 12.5% homogenous SDS – PAGE under reducing conditions, seven protein bands were identified with average molecular weights of 118, 106, 90, 56, 36, 23 and 17 kDa. The relative amounts of these seven proteins were determined densitometrically as 10, 6, 13, 27, 22, 13 and 8%, respectively. The separation and identification of BoNT/A complex will help in understanding the molecular structure and function of BoNT/A NAPs and their interaction with the toxin, in the toxico-infection process of the botulism diseased state. In particular, the stoichiometry of the individual components is established for a typical preparation of BoNT/A complex. Furthermore, the studies reported here identify the most favorable conditions for the baseline resolution of BoNT/A NAPs proteins for other workers in this field. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Botulinum; Clostridium botulinum; Type A; Electrophoresis; Neurotoxin; Proteins; Reversed-phase 1. Introduction Botulinum neurotoxins (BoNTs) produced in fluid culture or in food are complexes of BoNT with one or more neurotoxin associated proteins (NAPs), resulting in molecular sizes of 12, 16 and 19S (Schantz and Spero, 1967; Sakaguchi et al., 1974; Sugii and Sakaguchi, 1975, 1977). The 16S complex is referred to as ‘large’ or L toxin and 19S is referred as ‘extra large’ or LL toxin. The pure BoNT itself has been referred to as small or 7S toxin (Sakaguchi, 1983). BoNT/A complex can exist in three forms: M, L or LL (Sagane et al., 2002). BoNT/B, /C, /D and /E complexes exist in two forms: L and M (Sakaguchi, 1983; Zhang and Singh, 1995; Li et al., 1997). So far, BoNT/F complex is known to exist only in M form, and BoNT/G complex exists only in L form (Fujita et al., 1995). The M complex consists of the BoNT and the neurotoxin binding protein (NBP) (Singh et al., 1995), which is also commonly referred to as non-toxic non-hemagglutinin (NTNH). The L complex consists of BoNT, NBP, and five other NAPs. Genes of these proteins are topographically located in close proximity to each other, and their expression is regulated in a coordinated manner (East et al., 1994, 1996; Marvaud et al., 1998). It has been clearly demonstrated that the toxic component responsible for the disease botulism is the 7S protein toxin which has the molecular weight of 150 kDa 0041-0101/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. PII: S0041-0101(02)00309-4 Toxicon 41 (2003) 321–331 www.elsevier.com/locate/toxicon * Corresponding author. Tel.: þ1-508-999-8588; fax: þ 1-508- 999-8451. E-mail address: bsingh@umassd.edu (B.R. Singh).