title: Synthesis and characterization of a heterobifunctional mercurial cross-linking agent: incorporation into cobratoxin and interaction with the nicotinic acetylcholine receptor.
abstract: The heterobifunctional organomercurial reagents 3-(acetoxymercurio)- and 3-(chloromercurio)-5-nitrosalicylaldehyde were prepared, characterized in model studies, and used to probe the interaction between cobratoxin, purified from the venom of the Thailand cobra (Naja naja siamensis), and the affinity-purified nicotinic acetylcholine receptor (AcChR) from Torpedo california electroplax. These reagents may also be useful in introducing chemically well-defined heavy metal atoms into proteins containing no reactive thiols. Model reagent adducts were prepared in situ by reductive amination with N-butylamine and N alpha-acetyllysine-N-methylamide. The nitrophenolic pKaS of the amine adducts were similar to those of the aldehyde reagents through reduced by 1.3-1.5 units when compared with the hydroxylmethyl reduction product. Reaction of either mercuriosalicylaldehyde with cobratoxin led to a single major modification product incorporating 1 mol of the reagent into cobratoxin at Lys 23. The Lys 23 modified toxin had a reduced binding affinity for the AcChR over that of the native toxin (Kd 2.75 nM cf. 0.3 nM). Reduction of the purified AcChR with 1 mM dithiothreitol (DTT) followed by removal of excess thiol led to cross-linking reactions with the Lys 23 modified cobratoxin to both the alpha and beta subunits of the AcChR complex. Reaction of DTT-treated AcChR with N-ethylmaleimide (NEM) blocked cross-linking, while treatment of the initially cross-linked toxin-AcChR complex with mercaptoethanol leads to reversal of cross-linking. These observations strongly support cross-linking mediated by the formation of a mercury-sulfur bond and further lend support the identity of the respective interacting sites in AcChR and toxin.
authors.affiliation: Department of Medicinal and Biological Chemistry, College of Pharmacy, University of Toledo, Ohio 43606.
authors.forename: E R
authors.initials: ER
authors.lastname: Wohlfeil
authors.forename: R A
authors.initials: RA
authors.lastname: Hudson
mesh_terms: D000109:Acetylcholine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001345:Autoradiography; D002851:Chromatography, High Pressure Liquid; D003039:Cobra Neurotoxin Proteins / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000138:chemical synthesis* / Q000378:metabolism; D004229:Dithiothreitol / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009941:Organomercury Compounds / Q000138:chemical synthesis* / Q000378:metabolism; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011978:Receptors, Nicotinic / Q000378:metabolism*; D013057:Spectrum Analysis; D014101:Torpedo; D014357:Trypsin
chemical_list: D000596:Amino Acids; D003039:Cobra Neurotoxin Proteins; D003432:Cross-Linking Reagents; D009941:Organomercury Compounds; D011978:Receptors, Nicotinic; D014357:Trypsin; D000109:Acetylcholine; D005033:Ethylmaleimide; D004229:Dithiothreitol