正交试验法确定测定意大利蜜蜂头部乙酰胆碱酯酶反应的最佳条件

用正交试验法研究了酶浓度、底物浓度、反应体系的pH值、反应温度和反应时间5个因素对测定意大利蜜蜂Apis mellifera ligustica Spinola头部乙酰胆碱酯酶(AChE)活性的影响, 并从试验组合中选出最佳条件。蜜蜂AChE活性的测定采用Gorun(1978)改进的Ellman方法, 以碘化硫代乙酰胆碱(ATCI)为底物, 5, 5' 二硫双硝基苯甲酸(DTNB)为显色剂, 测定反应物在412 nm波长下的光密度值, 用考马斯亮蓝G-250法测定蛋白质含量, 经计算得到蜜蜂头部AChE的比活力。对正交试验结果进行极差分析和方差分析, 结果表明各因素对实验结果影响的大小顺序为：温度＞pH值＞时间＞酶浓度＞底物浓度。并得出测定蜜蜂头部AChE活性的最佳条件是：酶终浓度0.2头/mL、底物终浓度0.8 mmol/L、pH值7.5、温度40℃及反应时间5 min。

The optimization of conditions for assaying activity of acetylcholinesterase from the Italian honeybee (Apis mellifera ligustica Spinola) by orthogonal matrix method

The optimal conditions of assaying the activity of acetylcholinesterase (AChE) from the Italian honeybee (Apis mellifera ligustica Spinola) heads were determined by orthogonal matrix method. The concentrations of acetylcholinesterase and substrate,  pH and temperature of the reaction system,  and reaction time were optimized. L₂₅ (5⁶) orthogonal matrix was adopted,  without considering interaction. The specific activity of AChE was determined by colorimetric method of Ellman modified by Gorun (1978),  using acetylthiocholine iodide (ATCI) as substrate and DTNB (5, 5′-dithio-bis-2-nitrobenzoic acid) as chromogen. The optical density was measured by spectrophotometric detection (at 412 nm) of the increase of yellow color produced from thiocholine when reacted with 5,  5′-dithio-bis-2 nitrobenzoate ions. Protein concentrations in homogenate supernatants were determined by the method of Bradford (1976). The results revealed that temperature of the reaction system was the most important factor for measuring the activity of AChE from honeybee heads, and the measuring was also remarkably affected by the other four factors. The effects on the activity of acetylcholinesterase were in the order of temperature＞pH＞time＞concentration of AChE＞concentration of substrate. The optimal conditions were: 0.2 heads/mL, 0.8 mmol/L substrate, pH 7.5, 40℃, 5 minutes.