A new spectrofluorimetric method more sensitive than the Ellman method was developed for determination of both acetylcholinesterase and butyrylcholinesterase activity and for kinetic analysis of these enzymes and their mutants. Two selected mutants of human butyrylcholinesterase (E197Q and E197G) were included in this work. As for the Ellman's method, substrates are thiocholine esters, but the chromogenic reagent, DTNB (dithio-bisnitro benzoic acid) is replaced by a fluorogenic probe, "Calbiochem Probe IV", (3-(7-Hydroxy-2-oxo-2H-chromen-3-ylcarbamoyl)acrylic acid methylester). Compared to the classical Ellman's method, the sensitivity of this new spectrofluorimetric assay is 2 orders of magnitude higher. The method allows measurement of activity in media containing <10-11 M of cholinesterase active sites at low substrate concentrations, either under first order conditions, [S] << Km, or under conditions where kinetics obeys the Michaelis-Menten model, i.e. at [S] < 1 mM for wild-type enzymes. The method adapted to titration plate reader assays is suitable for clinical and toxicological routine analyses, for high throughput screening of novel cholinesterase mutants and screening of inhibitor libraries of pharmacological interest.

中文翻译：

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一种新的灵敏的荧光光谱法，用于测定胆碱酯酶的活性和动力学。

为了测定乙酰胆碱酯酶和丁酰胆碱酯酶的活性，并对这些酶及其突变体进行动力学分析，开发了一种比Ellman方法更灵敏的新的荧光光谱法。人丁酰胆碱酯酶的两个选定突变体（E197Q和E197G）包括在这项工作中。至于Ellman方法，底物是硫代胆碱酯，但是发色试剂DTNB（二硫代-双硝基苯甲酸）被荧光探针“ Calbiochem Probe IV”取代，（3-（7-Hydroxy-2-oxo-2H -铬-3-基氨基甲酰基）丙烯酸甲酯）。与经典的Ellman方法相比，这种新的荧光光谱测定法的灵敏度高出2个数量级。该方法可以测量包含< 在低底物浓度下，在一级条件[S] << Km或动力学符合Michaelis-Menten模型的条件下，即在野生型[S] <1 mM的条件下，10-11 M的胆碱酯酶活性位点酶。适用于滴定板读数器测定的方法适用于临床和毒理学常规分析，适用于高通量筛选新型胆碱酯酶突变体和筛选具有药理作用的抑制剂库。