Electron spin resonance study of peroxidase activity and kinetics.
Electron spin resonance study of peroxidase activity and kinetics.
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DOI：
10.1016/0003-9861(92)90242-o
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发表时间：
1992-11
影响因子：
3.9
通讯作者：
K. Moore;M. Moronne;R. Mehlhorn
中科院分区：
文献类型：
--
作者：
K. Moore;M. Moronne;R. Mehlhorn
电子自旋共振（ESR）测定已开发的过氧化物酶活性。该测定测量顺磁性氮氧化物Tempol通过过氧化物酶循环中间体化合物I和II产生的短寿命自由基氧化其羟胺衍生物（TOLH）而形成。以苯酚为过氧化物酶电子给体，ESR法适用于2 μl样品中过氧化物酶活性（~ 0.003U/ml）和H_2O_2微摩尔量的测定。此外，ESR方法可用于连续监测细胞悬液和其他易受光学伪影影响的介质中的活性。TOLH的高膜渗透性也使得有可能估计膜封闭区室中的过氧化物酶活性，条件是TOLH氧化速率可以用外源性过氧化物酶还原剂刺激，例如，苯酚在低电子供体浓度和高浓度H2 O2的条件下TOLH氧化速率的分析也表明底物依赖性抑制和过氧化氢活性增加的明确迹象。计算机模拟结果表明，所得结果与Kohleret等人提出的过氧化物酶反应方案一致。(1988 264，438-449）进行修饰以校正过氧化物酶催化活性的氮氧依赖性刺激。
An electron spin resonance (ESR) assay has been developed for peroxidase activity. The assay measures the formation of the paramagnetic nitroxide Tempol from the oxidation of its hydroxylamine derivative (TOLH) by short-lived radicals produced by peroxidase cycle intermediates, Compounds I and II. Using phenol as a peroxidase electron donor, the ESR approach is suitable for measurements of peroxidase activity (⩾0.003 U/ml) and micromolar quantities of H2O2in sample sizes as small as 2 μl. In addition, the ESR method can be used to continuously monitor activity in cell suspensions and other media that are susceptible to optical artifacts. The high membrane permeability of TOLH also makes it possible to estimate peroxidase activity in membrane-enclosed compartments, provided that TOLH oxidation rates can be stimulated with exogenous peroxidase reductants, e.g., phenol. Analysis of TOLH oxidation rates under conditions of low electron donor concentrations and high concentrations of H2O2also shows clear indications of substrate-dependent inhibition and increased catalatic activity. Computer simulations indicate that the results obtained are consistent with the peroxidase reaction scheme proposed by Kohleret al.(1988,Arch. Biochem. Biophys.264, 438–449) modified to correct for a nitroxide dependent stimulation of peroxidase catalytic activity.