Plieth Christoph
Zentrum für Biochemie und Molekularbiologie (BiMo), Universität Kiel, Am Botanischen Garten 9, 24118 Kiel, Germany.
ACS Omega. 2019 Feb 14;4(2):3268-3279. doi: 10.1021/acsomega.8b03564. eCollection 2019 Feb 28.
The peroxidation of luminol yields bright luminescence when the reaction is catalyzed by heme proteins. However, an excess of peroxide leads to less light and altered luminescence kinetics, an effect commonly referred to as "suicide inactivation". The aim of this study is to present the molecular processes causing this effect. A comprehensive set of data reported here demonstrates that suicide inactivation is due to a peroxide-induced liberation of iron from its coordinating porphyrin. Liberated iron launches catalysis of the reaction at much lower efficiency. The light-yielding efficiencies of different organic and inorganic catalysts are precisely quantified and compared. It is shown that the catalysis by free iron involves superoxide. This is explained by the formation of a ferryl-oxo-iron complex. In this context, a complete reaction mechanism involving a modified Fenton-Haber-Weiss cycle is proposed for the first time. The switch from the highly efficient biogenically catalyzed luminescence to a less efficient inorganically catalyzed reaction is accompanied by a transition from "flash-type" to "glow-type" luminescence kinetics. Ethylenediaminetetraacetic acid-mediated chelation of iron is used to demonstrate this effect and to separate both kinetics. The explanation of kinetic heterodyning is underpinned by mathematical modeling. The results are able to explain the as yet unexplained phenomena discussed in the less recent literature and to settle disputes about them. It is concluded that peroxide concentrations exceeding the level tolerated by the catalyzing heme protein negatively impact performance and precision of luminol-based assays, where the light yield is used as a quantitative measure for analyte concentrations.
当反应由血红素蛋白催化时，鲁米诺的过氧化作用会产生明亮的发光现象。然而，过量的过氧化物会导致发光减少且发光动力学发生改变，这种效应通常被称为“自杀失活”。本研究的目的是揭示导致这种效应的分子过程。此处报告的一系列全面数据表明，自杀失活是由于过氧化物诱导铁从其配位卟啉中释放出来。释放出的铁以低得多的效率引发反应的催化作用。精确量化并比较了不同有机和无机催化剂的发光效率。结果表明，游离铁的催化作用涉及超氧化物。这可以通过形成铁氧-铁络合物来解释。在此背景下，首次提出了一个涉及修正的芬顿-哈伯-维伊斯循环的完整反应机制。从高效的生物催化发光转变为效率较低的无机催化反应，伴随着从“闪光型”到“辉光型”发光动力学的转变。利用乙二胺四乙酸介导的铁螯合作用来证明这种效应并区分这两种动力学。动力学外差现象的解释通过数学建模得到了支持。这些结果能够解释早期文献中讨论的尚未得到解释的现象，并解决关于它们的争议。得出的结论是，过氧化物浓度超过催化血红素蛋白所能耐受的水平，会对基于鲁米诺的检测的性能和精度产生负面影响，在这种检测中，发光量被用作分析物浓度的定量指标。
