Study On Extraction, Purification And Sensitivity To Organophosphorus Pesticide Of Crucian AChE

With the banning of organochlorine pesticides,organophosphorus pesticides were used extensively due to their relatively low residue and easy hydrolyzation in the environment.Organophosphorus pesticides residued in foodstuff especially in vegetables is an escalating problem that has aroused public concer over potential healty hazards. Conventional methods for detecting residual pesticides included chromatography,mass spectrometry,spectroscopy and so on,and gas chromatography is national standard method,but it is time consuming,expensive,and not suiTab,for field inspection.So it is imperative to develop a simple and rapid detection method for pesticide residue.Enzyme inhibition method is a rapid detection method.The study of ecotoxicology has displayed that the toxicity of organophosphorus pesticide is commonly attributed to their inhibition effects on acetylcholinesterase（ACHE） in animals,and the degree of inhibition is dependent on the concentration of organophosphorus pesticide.So,the measurement of inhibition of AChE activity provides the analytical basis for enzyme inhibition method. In current research,an enzyme inhibition method of pesticide residues has been developed.The main contents and results of this thesis are as follows:1.The crucian AChE extracting and activity detection conditions are optimized.The best extracting conditions are:pH is 7.5,the quantity volume ratio is 1:4,the extracting solution is phosphate buffer.For brain ACHE,the optimum incubated time was 35min, incubated temperature was 32℃,pH was 7.6,for live ACHE,the optimum incubated time was 28min,incubated temperature was 30℃,pH was 7.6,for muscle ACHE,the optimum incubated time was 31min,incubated temperature was 31℃,pH was 7.6.2.The Km for brain,liver and muscle AChE are respectively 0.548mmol/L, 0.406mmol/L,1.75mmol/L.The results indicate cmcian AChE has good appetency to substrate.3.The IC₅₀ and Ki of dichlorvos to brain,liver and muscle AChE are respectively 1.70μg/mL,3.55μg/mL,4.52μg/mL and 7.40×10³mol/L·min,7.21×10³mol/L·min, 5.94×10³mol/L·min;The IC₅₀ and Ki of phoxim to brain,liver and muscle AChE are respectively 2.45μg/mL,4.05μg/mL,9.46μg/mL and 10.54×10³mol/L·min, 10.11×10³mol/L·min,8.32×10³mol/L·min;The IC₅₀ and Ki of chlorpyrifos to brain, liver and muscle AChE are respectively 10.89μg/mL,14.33μg/mL,17.00μg/mL and 7.81×10³mol/L·min,6.85×10³mol/L·min,4.99×10³mol/L·min.For dichlorvos,phoxim and chlorpyrifos,the order of sensitivity for different AChE are brain AChE＞muscle AChE＞liver ACHE.4.The brain AChE was purified to binary aqueous phase extraction,ion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Sephadex G-200,the activity of brain AChE is raised from 32.14μmol/min·g to 8786.35μmol/min·g,the enzyme is purified 273.4 folds and the enzyme activity recovery ratio is 10.6%.5.Enzyme inhibition method is used to detect organophosphorus pesticides or（and） carbamate pesticide residue in cabbage,green pepper,tomato,vigna and water celery, the results indicate that all the AChE activity are less than 35%,and organophosphorus pesticides or（and） carbamate pesticide residue is substandard in all the vegetable samples.