Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen (Germany).
Chemphyschem. 2013 Dec 16;14(18):4094-101. doi: 10.1002/cphc.201300714. Epub 2013 Nov 8.
Electron paramagnetic resonance (EPR) spectroscopy in combination with the rapid freeze-quench (RFQ) technique is a well-established method to trap and characterize intermediates in chemical or enzymatic reactions at the millisecond or even shorter time scales. The method is particularly powerful for mechanistic studies of enzymatic reactions when combined with high-frequency EPR (ν≥90 GHz), which permits the identification of substrate or protein radical intermediates by their electronic g values. In this work, we describe a new custom-designed micro-mix rapid freeze-quench apparatus, for which reagent volumes for biological samples as small as 20 μL are required. The apparatus was implemented with homemade sample collectors appropriate for 9, 34, and 94 GHz EPR capillaries (4, 2, and 0.87 mm outer diameter, respectively) and the performance was evaluated. We demonstrate the application potential of the RFQ apparatus by following the enzymatic reaction of PpoA, a fungal dioxygenase producing hydro(pero)xylated fatty acids. The larger spectral resolution at 94 GHz allows the discernment of structural changes in the EPR spectra, which are not detectable in the same samples at the standard 9 GHz frequency.
电子顺磁共振（EPR）光谱学结合快速冷冻淬火（RFQ）技术是一种成熟的方法，可以在毫秒甚至更短的时间尺度内捕获和表征化学或酶反应中的中间体。当与高频 EPR（ν≥90 GHz）结合使用时，该方法对于酶反应的机制研究特别有效，因为高频 EPR 可以通过其电子 g 值来识别底物或蛋白质自由基中间体。在这项工作中，我们描述了一种新的定制微混合快速冷冻淬火装置，该装置需要的生物样品试剂体积小至 20 μL。该装置配备了适合 9、34 和 94 GHz EPR 毛细管（外径分别为 4、2 和 0.87 毫米）的自制样品收集器，并对其性能进行了评估。我们通过跟踪产生氢（过）氧化脂肪酸的真菌双加氧酶 PpoA 的酶反应来展示 RFQ 装置的应用潜力。在 94 GHz 时更大的光谱分辨率允许辨别 EPR 光谱中的结构变化，而在相同的样品中在标准的 9 GHz 频率下无法检测到这些变化。
