Effective coupling of rapid freeze-quench to high-frequency electron paramagnetic resonance

E Gabriele Panarelli,Edgar J J Groenen,Peter Gast,Harmen Van Der Meer

doi:10.1371/journal.pone.0232555

E Gabriele Panarelli, Edgar J J Groenen + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0232555

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Abstract

We report an easy, efficient and reproducible way to prepare Rapid-Freeze-Quench samples in sub-millimeter capillaries and load these into the probe head of a 275 GHz Electron Paramagnetic Resonance spectrometer. Kinetic data obtained for the binding reaction of azide to myoglobin demonstrate the feasibility of the method for high-frequency EPR. Experiments on the same samples at 9.5 GHz show that only a single series of Rapid-Freeze-Quench samples is required for studies at multiple microwave frequencies.

Highlights

Determination of reaction rates and detection of short-lived intermediates of fast chemical reactions are an important goal in those fields that involve molecular chemistry
A Rapid Freeze-Quench (RFQ) sample contained in a quartz tube is transferred from liquid nitrogen into dry ice for a few minutes to ensure the softening of the content
Fig 1. 3D rendering of the home-built assembly used for cold loading of RFQ sample tubes into the cavity of the 275 GHz spectrometer: (A), the probe head containing the 275 GHz cavity; (B), the assembly that is clamped onto the probe head with the sliding path for the metal block; (C), the metal block, which holds the sample tube, with the three lids to cover the sample tube in order to keep the sample cold

Summary

Introduction

Determination of reaction rates and detection of short-lived intermediates of fast chemical reactions are an important goal in those fields that involve molecular chemistry. The reactant mixture is freezequenched, and the sample is “scraped” off the wheels and collected by tapping with a capillary suitable for 130 GHz EPR Such approach introduces the advantage of not having to handle static frozen particles floating in isopentane, the packing factor the authors report is limited to 0.5. In the aforesaid methods a large amount of sample is required, which is often a disadvantage when working with biological samples An improvement in this respect is introduced by Kaufmann et al [4] and Pievo et al [5], following a development by Cherepanov and de Vries [6] at low microwave frequency. We demonstrate that EPR experiments at multiple microwave frequencies require only one series of RFQ samples

Materials

Sample preparation

Sample packing and loading for 275 GHz EPR

EPR measurements

Results and discussion

Conclusions