STATUS OF CYS RESIDUES IN THE COVALENT STRUCTURE OF BOTULINUM NEUROTOXIN TYPE-A, TYPE-B, AND TYPE-E

Clostridium botulinum neurotoxin (NT) serotypes A, B, and E have 9, 10 , and 8 Cys residues, respectively, as deduced from nucleotide sequenc es [Whelan et al. (1992), Appl. Environ. Microbiol. 48, 2345-2354]. Ea ch of the 150-kDa NTs has at least one disulfide; but type B, like typ es A and E, may have two disulfides. Using two different chemical reag ents, we studied the status of the Cys residues in these three protein s after (i) the final anion exchange chromatographic step in their pur ification (fresh NT), (ii) 24 hr storage at 8 degrees C, (iii) precipi tation with ammonium sulfate (precipitated NT), and (iv) dissolving th e precipitated NT in 6 M guanidine HCl. In all three NT serotypes the number of Cys residues titrated with 5,5'-dithiobis-2-nitrobenzoic aci d (DTNB) as free -SH groups varied, depending upon the absence or pres ence of EDTA added to the chromatography buffer, storage condition, ag e, and presence of the denaturant. Titration of 9.5-10 and 5.4-6.0 -SH groups in fresh NTs type B and E, respectively, indicated total and p artial absence of disulfide bonds. Fewer titratable -SH groups in the precipitated NT than in the fresh NT suggested formation of disulfide and/or inaccessibility of the -SH groups due to protein's conformation al change(s). When the precipitated NTs were dissolved in 6 M guanidin e . HCl, in the absence of any added reducing agent, all Cys residues of types B and E, and 6.4-8.3 Cys in type A NT were titratable with DT NB. Iodoacetamide modification of precipitated NT types A, B, and E ca rboxymethylated 4, 2, and 2 Cys residues, respectively; these numbers rose to 6, 9.4, and 8 when these proteins were carboxymethylated after dissolving in 6 M guanidine . HCl in the absence of any added reducin g agent. We propose that -S-S- cleavage mediated by the -SH/-S-S- exch ange observed in vitro after unfolding the NTs (also unfolded by 2 M g uanidine . HCl or urea) possibly mimicks a similar exchange process in side the endosomes, where the NTs are thought to undergo conformationa l changes, resulting in the reductive cleavage of the interchain disul fide between the 50-kDa light and 100-kDa heavy chain, which in turn r eleases the light chain and allows its egress out of the endosomes int o the cytosol.