Modification of acetylcholinesterase with the fluorescent thiol reagent S-mercuric-N-dansylcysteine.
11s acetylcholinesterase (EC 3.1.1.7) from Torpedo californica electroplax, purified by a combination of affinity and gel chromatography was found to react stoichiometrically with S-mercuric-N-dansylcysteine. Approximately four mols of reagent per mol of enzyme were incorporated when the modification was carried out in 1.0 mM Tris-C1, pH 7.5, either in the presence or absence of 0.1 M NaCl. Prior incubation of the enzyme with 1.0 x 10(-4) M Zn2+ allowed the incorporation of about six mols of reagent per mol of enzyme. Binding of the reagent produced shifts in the emission and excitation wavelength maxima which were similar for all reaction conditions; however the enhancement of fluorescence intensity which accompanied binding of reagent was dependent on the ionic composition of the reaction medium. The modified enzyme remained active towards the active site titrant 7-(dimethylcarbamoyloxy)-N-methylquinolinium and retained its sensitivity towards inactivation by Zn2+. The results suggest that acetylcholinesterase as prepared contains several accessible thiol groups, and that the bound reagent may prove to be a useful probe of ligand-induced conformational changes in acetylcholinesterase.
D000818:Animals; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D002800:Cholinesterase Inhibitors; D003545:Cysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004557:Electric Organ / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D014101:Torpedo
D002800:Cholinesterase Inhibitors; D013439:Sulfhydryl Reagents; C028374:S-mercuric-N-dansylcysteine; D003545:Cysteine