磷酸酶抑制比色法快速测定贝类中腹泻性贝毒素

Rapid Determination of Diarrhetic Shellfish Poisoning in Shellfish by Colorimetric Protein Phosphatase Inhibition Assay

摘要:

基于腹泻性贝毒素（Diarrhetic shellfish poisoning，DSP）的致腹泻性组分大田软海绵酸（okadaic acid，OA）及其衍生物鳍藻毒素（dinophysistoxins，DTXs）能抑制蛋白磷酸酶活性的特点，建立了快速测定贝类中DSP的磷酸酶抑制比色分析方法。采用对硝基苯磷酸二钠（p-nitrophenyl phosphate disodium，p-NPP）为底物，其与蛋白磷酸酶2A（protein phosphatase 2A，PP2A）反应生成的黄色水解产物在碱性条件下于405 nm波长处有强烈的吸收峰，根据吸光度值计算抑制剂的浓度。酶抑制法继承了小鼠生物法能建立剂量-效应关系的优势，直接反映毒素的相对毒性大小，测定的是DSP毒素致腹泻性成分的总量，以OA浓度计。本研究优化了样品前处理方法并考察了基质浓度的影响。方法的筛选检出限为80 μg/kg。采用该方法进行加标回收实验，回收率在90.43%~118.52%范围内，相对标准偏差（RSD）为6.85%~13.93%。该方法操作简便、快捷，回收率高，重现性好，可作为快速筛查工具用于贝毒的日常监控。

Abstract:

Okadaic acid (OA) and its derivatives, the components of diarrhetic shellfish poisoning (DSP), are potent inhibitors of protein phosphatases. Based on the mechanism of action, a protein phosphatase 2A (PP2A) inhibition assay for the rapid determination of DSP toxins in bivalve was developed. In this study, p-nitrophenyl phosphate (p-NPP) was used as the substrate and hydrolyzed by PP2A, then the product was measured at 405 nm. The total DSP content (calculated by OA) in samples could be detected according to the standard dose-effect curve developed with a series of OA standard solutions. The experimental conditions of sample preparation were optimized, and shellfish matrix loading limits for the protein phosphatase inhibition assay were established according to the shellfish species. The detection limit was 80 μg/kg, andthe spiked recoveries for OA in shellfish samples were between 90.43% and 118.52%, with relative standard deviations (RSD) ranged from 6.85% to 13.93%. The colorimetric protein phosphatase inhibition assay was simple, rapid and showed good recovery and reproducibility, demonstrating this proposed method could be used as an efficient analysis tool for rapid screening of DSP in shellfish and suit for daily monitoring to control shellfish toxicity.