Rapid Freeze-Quench EPR Spectroscopy: Improved Collection of Frozen Particles.

Faezeh Nami,Edgar J J Groenen,Peter Gast

doi:10.1007/s00723-016-0783-7

Abstract

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.

Highlights

Detection and characterization of intermediates involved in enzymatic reactions is an important step to understand the mechanism of such reactions
We present a method, which improves the collection of freeze-quench particles from isopentane and their packing in an electron paramagnetic resonance (EPR) tube
The Rapid freeze-quench (RFQ) technique has been mostly combined with electron paramagnetic resonance (EPR) spectroscopy, because enzymatic reactions often involve a radical or a transition metal ion [4,5,6]

Summary

Introduction

Detection and characterization of intermediates involved in enzymatic reactions is an important step to understand the mechanism of such reactions. Elucidating the reaction kinetics and the structure of enzymatic intermediates commonly requires a combination of spectroscopic techniques in view of the complexity of the systems [1]. Except for the optical stopped-flow technique, spectroscopic characterization necessitates to trap intermediates before observation. The RFQ technique has been mostly combined with electron paramagnetic resonance (EPR) spectroscopy, because enzymatic reactions often involve a radical or a transition metal ion [4,5,6]. The mechanism of a large number of enzymatic reactions, in particular copper- and iron-containing enzymes, has been studied by RFQ EPR techniques [7,8,9,10,11,12,13,14]

Methods

Results

Conclusion