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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。结论 对氧磷酶可作为G类毒剂的抗毒剂
OBJECTIVE: To investigate the possibility of paraoxonase (EC 3.1.8.1) as an exogenous antidote against G class of organophosphate poisons. Methods Partial purification of human serum paraoxonase was carried out by affinity gel column chromatography. The effect of Km and Ca2 + on the enzymatic activity of different substrates was tested. The effects of different species Paraoxonase activity, in vitro detoxification, in vivo exposure to the method of detecting paraoxonase for different substrates. Results Partially purified paraoxonase had good catalytic hydrolysis for soman and paraoxon. When using paraoxon as substrate, the Km value was 0.22 mmol·L-1, and the specific activity was 375 μmol · min-1 · g-1 protein. When soman was used as substrate, the Km value was 0.6 0mmol·L-1, the specific activity of 4 34μmol · min-1 · g-1 protein. The serum paraoxonase activity varies greatly from one genus to another, which is one of the reasons for the different sensitivities of different animal species to organophosphate poisoning. Partially purified paraoxonase activity in a buffer containing 1mmol·L-1CaCl2 good activity, adding ethylenediaminetetraacetic acid (EDTA), both with paraoxon or soman substrate, enzyme activity were significantly inhibited , Indicating that human serum paraoxonase is a calcium-dependent enzyme. The pI50 of EDTA is about 1.8 mmol·L-1. Human serum paraoxonase is able to hydrolyze paraoxon, soman, sarin, tabun and dichlorvos effectively, but is ineffective against VX and dimethoate, indicating its selectivity for PF, P CN and PO, and for PS No effect. Conclusion Paraoxonase can be used as an antidote to class G agents