Mannens G, Slegers G, Claeys A
Laboratory of Analytical Chemistry, Faculty of Pharmaceutical Sciences, State University of Ghent, Belgium.
Biochim Biophys Acta. 1990 Jan 19;1037(1):1-6. doi: 10.1016/0167-4838(90)90094-v.
A specific assay to measure the activity of the enzyme acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from pigeon liver is described. The assay is based on the HPLC analysis of N-acetylserotonin formed by the enzymatic reaction. A reversed-phase column (Spherisorb 5-microns ODS 2; 150 x 3.2 mm) eluted with 0.1 M sodium acetate (pH 4.75)/methanol (75:25) permits baseline separation of serotonin and N-acetylserotonin within 5.3 min. Several variables on the enzyme reaction were studied to obtain maximum activity. The enzyme is most active in glycine buffer at pH 9.5. The apparent Km value for serotonin (at 0.6 mM CoASAc) is 0.246 mM and 9.9 microM for CoASAc (at 1.5 mM serotonin). To avoid acetyl-CoA or N-acetylserotonin consumption in side-reactions, the enzyme was purified. A two-step purification process (ammonium sulfate fractionation and affinity chromatography on immobilised amethopterin) yielded 60-70% of the initial enzyme activity with a purification factor of 455-560.
本文描述了一种用于测定鸽肝中乙酰辅酶A：芳胺N-乙酰基转移酶（EC 2.3.1.5）活性的特异性检测方法。该检测方法基于对酶促反应生成的N-乙酰血清素进行高效液相色谱分析。使用0.1 M醋酸钠（pH 4.75）/甲醇（75:25）洗脱的反相柱（Spherisorb 5微米ODS 2；150 x 3.2 mm）可在5.3分钟内实现血清素和N-乙酰血清素的基线分离。研究了酶促反应中的几个变量以获得最大活性。该酶在pH 9.5的甘氨酸缓冲液中活性最高。血清素（在0.6 mM CoASAc时）的表观Km值为0.246 mM，CoASAc（在1.5 mM血清素时）的表观Km值为9.9 microM。为避免副反应中乙酰辅酶A或N-乙酰血清素的消耗，对该酶进行了纯化。两步纯化过程（硫酸铵分级分离和固定化氨甲蝶呤亲和色谱）得到了初始酶活性的60 - 70%，纯化因子为455 - 560。
