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Botulinum neurotoxin B (BoNT/B) serotype specifically cleaves between the amino acids glutamine and phenylalanine (Q and F bond) in position 76–77 of synaptobrevin (VAMP2). We evaluated peptides that contain the QF cleavage site but are not identical in primary structure to the VAMP2 sequence surrounding the QF site for both inhibition of BoNT/B proteolytic activity and as substrates for BoNT/B. A reverse‐phase high‐performance liquid chromatography (RP‐HPLC) method was used to measure digested peptides. A dose as high as 600 μM of substance P, and 11‐amino acid peptide containing the QF bond, was neither a substrate nor inhibitor of BoNT/B in our assay, suggesting that more than the QF bond is required to be recognized by BoNT/B. Buforin I (B‐I, QF site 24–25) is 39 amino acids in length, and sequence comparison of B‐I and VAMP2 indicated a similarity of 18% for conserved amino acids around the QF site. Furthermore, computer‐aided secondary structure computations predict α‐helical structures flanking the QF site for VAMP2 and for the upstream sequence of B‐I. Although predictions for the downstream sequence give nearly equal tendencies for α‐helical and β‐sheet structures, Yi et al. showed that the downstream sequence is likely to be the α‐helix based on their examination of buforin II (B‐II, a 21‐amino acid subset of B‐I (16–36)), which includes the QF site and the downstream sequence of B‐I. Buforin I was found not to be a substrate for BoNT/B; however, B‐I dose dependently and competitively inhibited BoNT/B activity, yielding IC50 = 1 × 10−6 M. In contrast, B‐II was not a substrate for BoNT/B and exhibited only 25% of the B‐I inhibition of BoNT/B. Two additional B‐I deletion peptides were tested for inhibition of BoNT/B proteolysis: peptide 36 (36 mer; containing B‐I amino acids 1–36) and peptide 24 (24 mer; B‐I amino acids 16–39). Peptide 24 had a similar inhibitory effect to B‐II (ca. 25% of B‐I) but peptide 36 was almost 50% as potent as B‐I. These findings suggest that the buforin tertiary structure is important for the inhibitory activity of these peptides for BoNT/B.

Monoclonal antibodies were generated against fetal bovine serum acetylcholinesterase and fetal bovine serum acetylcholinesterase inhibited by diisopropyl fluorophosphate or 7‐(methylethoxyphosphinyloxy)‐1‐methylquinolinium iodide. Six monoclonal antibodies inhibited 70 to >98% of the catalytic activity of fetal bovine serum acetylcholinesterase. Inhibition of serum acetylcholinesterase from several mammalia by four monoclonal antibodies showed broad cross‐reactivity. In all cases, monoclonal antibodies bound to the native form of acetylcholinesterases. None reacted with serum butyrylcholinesterases from various species. Although all monoclonal antibodies inhibited catalytic activity of acetylcholinesterases, the site of interaction with acetylcholinesterase appeared to differ for several antibodies. Two types of acetylcholinesterase:monoclonal antibody complexes were formed: one between tetrameric forms and another between catalytic subunits within the tetramer. Monoclonal antibodies that inhibited acetylcholinesterase activity at >98% also considerably slowed binding of diisopropyl fluorophosphate and other organophosphorus compounds to the acetylcholinesterase:monoclonal antibody complex. Binding of these monoclonal antibodies to acetylcholinesterase influenced function of the enzyme's peripheral anionic site. None of the antibodies bound to the esteratic site of acetylcholinesterase. Monoclonal antibodies caused changes in catalytic activity of acetylcholinesterase by interaction at a site remote from the catalytic site, presumably at the entrance to the active site gorge.