Pei L, McGuinn W D, Omburo G, Zitzer A H, Petrikovics I, Way J L
Department of Medical Pharmacology and Toxicology, Texas A&M University, College of Medicine, College Station 77843-1114.
Biotechnol Appl Biochem. 1994 Aug;20(1):35-41.
A method has been developed to continuously measure paraoxonase activity spectrophotometrically in carrier red blood cells (RBCs) containing paraoxonase. This enzyme has a broad substrate specificity that includes parathion, paraoxon, soman, sarin, di-isopropyl fluorophosphate and many other organophosphorus compounds. Paraoxon is hydrolysed by paraoxonase to the less toxic 4-nitrophenol and diethyl phosphate. Determination of enzymic activity was based on the liberation of 4-nitrophenol in the presence of mouse RBCs. Paraoxonase was encapsulated within murine RBCs by hypotonic dialysis with subsequent resealing and annealing. The enzyme within resealed RBCs actively hydrolyses paraoxon in biological fluids to its less toxic metabolites. Paraoxonase incorporated within RBCs, like other enzymes, was found to be quite stable once encapsulated into RBCs and this formed the basis for this spectrophotometric method. Increasing absorbance at 400 nm indicated paraoxon hydrolysis and was the basis employed to determine enzymic activity. The Km of the enzyme within erythrocytes was 0.04 mM. This method offers a convenient, rapid and continuous way to monitor paraoxonase activity inside the carrier cell.
已开发出一种方法，可通过分光光度法连续测定含对氧磷酶的载体红细胞（RBC）中的对氧磷酶活性。该酶具有广泛的底物特异性，包括对硫磷、对氧磷、梭曼、沙林、氟磷酸二异丙酯和许多其他有机磷化合物。对氧磷酶将对氧磷水解为毒性较小的4-硝基苯酚和磷酸二乙酯。酶活性的测定基于在小鼠红细胞存在下4-硝基苯酚的释放。通过低渗透析将对氧磷酶包裹在小鼠红细胞内，随后进行重新密封和退火处理。重新密封的红细胞内的酶可在生物流体中将对氧磷积极水解为毒性较小的代谢产物。与其他酶一样，包裹在红细胞内的对氧磷酶一旦被包裹进红细胞就相当稳定，这构成了这种分光光度法的基础。在400nm处吸光度的增加表明对氧磷发生了水解，这是用于测定酶活性的依据。红细胞内该酶的米氏常数为0.04mM。该方法提供了一种方便、快速且连续的方式来监测载体细胞内的对氧磷酶活性。
