Nami Faezeh, Gast Peter, Groenen Edgar J J
Huygens-Kamerlingh Onnes Laboratory, Department of Physics, Leiden University, P.O. Box 9504, 2300 RA Leiden, The Netherlands.
Appl Magn Reson. 2016;47:643-653. doi: 10.1007/s00723-016-0783-7. Epub 2016 Apr 30.
Rapid freeze-quench (RFQ) in combination with electron paramagnetic resonance (EPR) spectroscopy at X-band is a proven technique to trap and characterize paramagnetic intermediates of biochemical reactions. Preparation of suitable samples is still cumbersome, despite many attempts to remedy this problem, and limits the wide applicability of RFQ EPR. We present a method, which improves the collection of freeze-quench particles from isopentane and their packing in an EPR tube. The method is based on sucking the particle suspension into an EPR tube with a filter at the bottom. This procedure results in a significant reduction of the required volume of reactants, which allows the economical use of valuable reactants such as proteins. The approach also enables the successful collection of smaller frozen particles, which are generated at higher flow rates. The method provides for a reproducible, efficient and fast collection of the freeze-quench particles and can be easily adapted to RFQ EPR at higher microwave frequencies than X-band.
快速冷冻淬灭（RFQ）与X波段电子顺磁共振（EPR）光谱相结合是一种成熟的技术，用于捕获和表征生化反应的顺磁中间体。尽管人们多次尝试解决这个问题，但制备合适的样品仍然很麻烦，这限制了RFQ EPR的广泛应用。我们提出了一种方法，该方法改进了从异戊烷中收集冷冻淬灭颗粒并将其填充到EPR管中的过程。该方法基于将颗粒悬浮液吸入底部带有过滤器的EPR管中。此过程可显著减少所需反应物的体积，从而能够经济地使用诸如蛋白质等有价值的反应物。该方法还能够成功收集在更高流速下产生的较小冷冻颗粒。该方法可实现对冷冻淬灭颗粒的可重复、高效且快速的收集，并且可以轻松地应用于高于X波段的更高微波频率的RFQ EPR。
