Mutus B, Duncan D V, Tomlinson G
Biochem Biophys Res Commun. 1983 May 16;112(3):941-7. doi: 10.1016/0006-291x(83)91708-4.
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.
通过亲和色谱和凝胶色谱相结合的方法纯化得到的来自电鳐（Torpedo californica）电板的11s乙酰胆碱酯酶（EC 3.1.1.7），被发现能与S-汞-N-丹磺酰半胱氨酸发生化学计量反应。当在1.0 mM Tris-Cl，pH 7.5中进行修饰时，无论有无0.1 M NaCl，每摩尔酶大约结合四摩尔试剂。酶先与1.0×10⁻⁴ M Zn²⁺预孵育，使得每摩尔酶能结合约六摩尔试剂。试剂的结合导致发射和激发波长最大值发生位移，所有反应条件下的位移相似；然而，伴随试剂结合的荧光强度增强取决于反应介质的离子组成。修饰后的酶对活性位点滴定剂7-（二甲基氨基甲酰氧基）-N-甲基喹啉仍有活性，并保留了对Zn²⁺失活的敏感性。结果表明，所制备的乙酰胆碱酯酶含有几个可及的巯基，并且结合的试剂可能被证明是乙酰胆碱酯酶中配体诱导构象变化的有用探针。
