Dipartimento di Scienze dell'Ambiente e del Territorio, Università degli Studi di Milano-Bicocca, Piazza della Scienza 1, 20126 Milano, Italy.
Bioorg Med Chem. 2011 May 1;19(9):3022-8. doi: 10.1016/j.bmc.2011.02.046. Epub 2011 Mar 2.
In this paper, we use our quantitative (31)P NMR spin trapping methods, already developed for simple oxygen- and carbon-centered radicals, to understand the radical intermediates generated by enzymatic systems and more specifically lipoxygenases. Our methodology rests on the fact that free radicals react with the nitroxide phosphorus compound, 5-diisopropoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide (DIPPMPO), to form stable radical adducts, which are suitably detected and accurately quantified using (31)P NMR in the presence of a phosphorus containing internal standard. This system was thus applied to better understand the mechanism of enzymatic oxidation of linoleic acid by soybean lipoxygenases-1 (LOX). The total amount of radicals trapped by DIPPMPO was detected by (31)P NMR at different experimental conditions. In particular the effect of dioxygen concentration on the amount of radicals being trapped was studied. At low dioxygen concentration, a huge increase of radicals trapped was observed with respect to the amount of radicals being trapped at normal dioxygen concentrations.
在本文中，我们使用已经开发用于简单氧和碳中心自由基的定量（31）P NMR 自旋捕获方法，来了解酶系统产生的自由基中间体，特别是脂氧合酶。我们的方法基于自由基与氮氧化物磷化合物 5-二异丙氧基膦酰基-5-甲基-1-吡咯啉-N-氧化物（DIPPMPO）反应形成稳定的自由基加合物的事实，在含有含磷内标物的情况下，使用（31）P NMR 可以适当地检测和准确地定量这些加合物。该系统因此被应用于更好地理解大豆脂氧合酶-1（LOX）催化亚油酸氧化的机制。在不同的实验条件下，通过（31）P NMR 检测 DIPPMPO 捕获的自由基总量。特别是研究了氧气浓度对自由基捕获量的影响。在低氧浓度下，与在正常氧浓度下捕获的自由基数量相比，观察到捕获的自由基数量有巨大增加。
