人血清对氧磷酶的部分纯化及解毒效应  

Partial purification of paraoxonase from human serum and its detoxication effect

作　　者：宁江海 王玉霞[1] 王青定[1] 池木根[1] 孙曼霁[1] 

机构地区：[1]军事医学科学院毒物药物研究所,北京100850

出　　处：《中国药理学与毒理学杂志》2002年第1期60-65,共6页Chinese Journal of Pharmacology and Toxicology

摘　　要：目的　寻求对氧磷酶 (paraoxonase ,EC 3.1.8.1)作为外源性抗毒剂对抗G类有机磷毒剂的可能性。方法　采用亲和凝胶柱层析进行了人血清对氧磷酶的部分纯化 ,测定了其对于不同底物的米氏常数 (Km)以及钙离子对于酶活性的影响 ,比较了不同种属的对氧磷酶活性 ,采用体外解毒 ,体内染毒的方法检测了对氧磷酶对于不同的底物的作用。结果部分纯化的对氧磷酶对于梭曼和对氧磷均有较好的催化水解作用。以对氧磷为底物时 ,Km 值为 0 .2 2mmol·L- 1,比活性为 375 μmol·min- 1·g- 1蛋白 ;以梭曼为底物时 ,Km 值为 0 .6 0mmol·L- 1,比活性为 4 34μmol·min- 1·g- 1蛋白。不同种属来源的血清对氧磷酶的活性差异较大 ,是造成不同种属动物对有机磷毒剂中毒敏感性差别的原因之一。部分纯化的对氧磷酶在含 1mmol·L- 1CaCl2 的缓冲液中活性良好 ,加入乙二胺四乙酸 (EDTA)后 ,无论以对氧磷或梭曼为底物 ,酶活性均受到明显抑制 ,表明人血清对氧磷酶是钙离子依赖性酶。EDTA的pI50 约为 1.8mmol·L- 1。人血清对氧磷酶能有效地水解对氧磷、梭曼、沙林、塔崩及敌敌畏 ,但对VX及乐果无效 ,表明其对P F、P CN及P O键有选择性 ,对P S键无作用。AIM To explore the possibility of paraoxonase as exogeneous antidote against G type organophosphate nerve agents. METHODS Paraoxonase is partially purified by affinity gel column chromatography. The K m value of paraoxonase of different substrate is determined. The effect of calcium ion on the enzyme activity is measured. Paraoxonase activities from different species are compared. Effect of paraoxonase on different substrates is studied by in vitro hydrolysis and in vivo poisoning. RESULTS The human serum paraoxonase was partially purified using affinity gel column chromatography, it was found that the partially purified paraoxonase was able to well catalyze the hydrolysis of paraoxon and soman. The K m while using paraoxon as the substrate, came to 0.22 mmol·L -1 with a specific activity of 375 μmol·min -1 ·g -1 , whereas using soman as substrate, the K m came to 0.60 mmol·L -1 with a specific activity of 434 μmol·min -1 ·g -1 . The difference of paraxonase activity in various animal species is ascribed to the difference of susceptibility to organophosphate poisoning of various animals. In the presence of 1 mmol·L -1 CaCl 2, the partially purified paraoxonase manifested well the enzyme activity. However, the enzyme activity was profoundly inhibited by the addition of EDTA, when soman or paraoxon was used as substrate. It suggests that human serum paraoxonase be a calcium ion dependent enzyme. The pI 50 of EDTA came to about 1.8 mmol·L -1 . The human serum paraoxonase could efficiently catalyze the hydrolysis of soman, sarin, tabun, paraoxon and dichlorvos, however, it is inefficient to degrade VX and dimethoate. It indicates that human serum paraoxonase selectively hydrolyzes the P F, P CN and P O bonds but not the P S bond. CONCLUSION Paraoxonase is a potential antidote against G type nerve agents.

关 键 词：水解酶类 对氧磷酶 有机磷化合物 沙林 塔崩 维埃克斯 敌敌畏 梭曼 

分 类 号：R969[医药卫生—药理学]