Haas A, Geldmacher-von Mallinckrodt M
Childrens Hospital, University of Düsseldorf, Federal Republic of Germany.
Electrophoresis. 1990 Aug;11(8):621-6. doi: 10.1002/elps.1150110807.
A new approach is described for studying the polymorphism of paraoxon hydrolyzing serum esterases after isoelectric focusing of native sera. Enzyme visualization is performed by a modified sandwich procedure which is faster and also affords higher resolution and considerably improved sensitivity. Up to seven paraoxon splitting isoenzymes can be visualized and clearly distinguished from arylesterases and phosphatases by using 3-naphtyl acetate, paraoxon, 5-bromo-4-chloro-3-indolyl acetate and 5-bromo-4-chloro-3-indolyl phosphate as substrates. The new technique is also able to differentiate between paraoxonase isoenzymes sensitive to EDTA and those which are EDTA-stable. Immunofixation with anti-human serum albumin-antibodies revealed similar isoelectric points for these isoenzymes, although they are not assumed to be identical. The new technique may prove useful in other applications of enzyme visualization where diffusion of enzymes and/or cleavage products is the major problem.
本文描述了一种新方法，用于研究天然血清经等电聚焦后对氧磷水解血清酯酶的多态性。酶可视化通过改良的夹心程序进行，该程序更快，分辨率更高，灵敏度也显著提高。使用乙酸-3-萘酯、对氧磷、5-溴-4-氯-3-吲哚乙酸酯和5-溴-4-氯-3-吲哚磷酸作为底物，可观察到多达七种对氧磷裂解同工酶，并能将它们与芳基酯酶和磷酸酶清晰区分开来。新技术还能够区分对EDTA敏感的对氧磷酶同工酶和对EDTA稳定的同工酶。用抗人血清白蛋白抗体进行免疫固定显示，这些同工酶具有相似的等电点，尽管它们并不被认为是完全相同的。这项新技术可能在酶可视化的其他应用中有用，在这些应用中，酶和/或裂解产物的扩散是主要问题。
