高效液相色谱法测定家蝇细胞色素P450O-脱甲基活性  

High Performance Liquid Chromatographic Assay for p-Nitroanisole O-Demethylation by Cytochrome P450 Enzymes in Musca domestica L.

作　　者：艾国民[1] 王庆敏[1] 邹东云[1] 高希武[1] 李富根[1] 

机构地区：[1]中国农业大学农学与生物技术学院昆虫学系,北京100193

出　　处：《分析化学》2009年第8期1157-1160,共4页Chinese Journal of Analytical Chemistry

基　　金：国家973计划(No.2006CB102003)资助项目

摘　　要：建立了以乙酸乙酯/正己烷为终止剂(含1%H3PO4)和萃取剂,利用高效液相色谱,ZORBAXEclipseXDBC18色谱柱,梯度洗脱,在316nm检测,外标法分析对硝基苯酚的生成量,表征家蝇粗酶液中细胞色素P450催化的对硝基苯甲醚O-脱甲基活性的新方法。本方法的检出限为0.1ng;分析精密度的RSD为1.02%;0.351,1.755和8.755μg对硝基苯酚3个添加水平的回收率为92.34%～87.60%;回收率的RSD为2.64%～5.90%;对硝基苯酚在9.72～486ng范围内,线性关系良好,r=0.9995。应用本方法比较了家蝇吡虫啉抗性品系与敏感品系细胞色素P450的活性差异。结果表明,家蝇抗性品系的P450O-脱甲基活性是敏感品系的3.34倍;细胞色素P450活性的提高是家蝇对吡虫啉产生抗性的一个重要机制。A novel sensitive method was developed for measuring p-nitroanisole o-demethylation catalyzed by cytochrome P450 enzymes in crude homogenates of Housefly, Musca domestica （Diptera： Muscidae） , using HPLC with diode array detector. The method is based on using liquid-liquid extraction and HPLC with reversed-phase Cis column, and gradient UV detection at 316 nm. The detection limit （S/N = 3 ） of p-nitrophenol was 0.1 ng. The relative standard deviation （ RSD ） of peak area response of the HPLC was 1.0%. The average recoveries of 0.351, 1. 755 and 8. 755 μg ofp-nitrophenol added to incubation mixtures ranged from 92.3% to 87.6% and the RSD of recoveries of three added levels were in the range of 2.6% - 5.9%. The relationships between peak areas and concentration were linear in the range of 9.72 -486 ng for p-nitrophenol with a correlation coefficient of r = 0. 9995. This method is sensitive, precise, reproducible, and is also applicable to the assay of p-nitroanisole o-demethylases which have low catalytic activities, present in other insects or organisms. With this newly developed HPLC method, comparison of P450 o-demethylation activity of an imidacloprid resistant strain with a susceptible strain of Musca domestica （ L. ） was conducted to identify the roles of P450 enzymes involved in the resistance to imidacloprid. The results showed that the resistance mechanism of Musca domestica to imidaeloprid was intensely assisted with elevated P450 activities.

关 键 词：高效液相色谱 O-脱甲基 细胞色素P450 家蝇 吡虫啉 

分 类 号：Q966[生物学—昆虫学]