星豹蛛头胸部乙酰胆碱酯酶最佳反应体系及其药剂敏感度

Establishment of optimization of assaying activity of acetylcholinesterase from cephalothorax of Pardosa astrigera and their sensitivity to pesticides

利用正交试验法研究5个因素对测定星豹蛛(Pardosa astrigera)头胸部乙酰胆碱酯酶(AChE)活性的影响,以建立酶活测定的最佳反应体系。结果表明,各因素对AChE的比活力影响由大到小的顺序为反应体系的pH值、反应时间、酶质量浓度、反应温度和底物浓度。测定AChE活性的最佳反应条件是:酶质量浓度为12 g·L^(-1),底物浓度为0.6 mmol·L^(-1),反应体系pH值为7.0,反应温度为30℃,反应时间为5 min。此外,4种杀虫剂对星豹蛛头胸部AChE活性的影响试验结果表明,灭多威、辛硫磷、高效氯氰菊酯、毒死蜱对AChE的抑制中浓度(IC_(50))分别为7.76×10^(-5)mol·L^(-1)、1.76×10^(-4)mol·L^(-1)、4.12×10^(-4)mol·L^(-1)和4.94×10^(-4)mol·L,并且上述4种杀虫剂对AChE的抑制具有明显的剂量关系。

The present paper is aimed to introduce our study results on setting-up the optimum testing activity response system of acetyl- cholinesterase to the cephalothorax of pardosa astrigera and its sensitivity to the applied pesticides. For our research purpose, we have adopted the orthogonal experiment by choosing five factors for our experiments so as to test the activity of acetylcholinesterase （ACHE） from the cephalothorax of Pardosa astrigena. In proceeding with our experiments, we have determined the concentrations of acetyl- cholinesterase and substrate, pH and temperature of the reaction system and optimized our reaction time. And then we have determined the specific activity of AChE by using Ellman＇ s colorlmetric method modified by Gorun（1978）. As a result, the effects on the activity of aeetylcholinesterase can be shown in the order of the concentration of pH 〉 time 〉 AChE 〉 temperature 〉 concentration of substrate. The optimum conditions prove to be： 12 g· L^-1 enzyme, 0.6 mmol·L^- 1 substrate, pH = 7.0, 30 ℃, 5 minutes. Next, we have determined the inhibition of the four insecticides to the AChE from the cephalothorax of Pardosa astrigena. Furthermore, we have also found that the IC50 values of Methomyl, Phoxim, Beta cypermethrin, Chlorpyrifos （which inhibit the cephalothorax of Pardosa astrigena ） have beenfound to be 7.76×10^-5 mol·L^-1, 1.76×10^-4 mol·L^-1, 4.12 ×10^-4 mol· L^-1 and 4.94 × 10^-4 mol·L^-1, respectively. The tendency of the four insecticides in inhibiting the cephalothorax AChE activities has shown a positive relation dependent on the concentrations of the insecticides. The above results suggest that the cephalothorax AChE of Pardosa astrigena is expected to be Used as a biochemical marker to monitor the insecticides in the environment.