Freeze‐Quench Kinetics

Abstract

AbstractRapid‐mixing, rapid‐freezing techniques are employed for the study of enzyme or chemical catalytic mechanisms. The techniques aim to provide electronic and molecular structures of transient intermediates formed during the reaction by means of spectroscopic analyses of the rapidly frozen samples. Briefly, the mixing‐sampling procedure consists of the rapid mixing of two reactants, for example, enzyme and substrate, followed by rapid freezing of the mixture in a liquid cryo‐medium or on a cold plate to quench the reaction progress. The resulting frozen powder is subsequently sampled and made available for further low‐temperature spectroscopic analyses such as UV‐Vis spectroscopy, multifrequency Electron Paramagnetic Resonance (EPR) spectroscopy, Electron Nuclear Double Resonance (ENDOR) spectroscopy, Electron Spin Echo Envelope Modulation (ESEEM), Magnetic Circular Dichroïsm (MCD) spectroscopy, Mössbauer spectroscopy, resonance Raman spectroscopy, or X‐ray Absorption Spectroscopy. By preparing a series of samples freeze‐quenched after various times, a full kinetic profile of the catalytic cycle of an enzyme or any other chemical reaction is obtained. The structures of intermediates are assigned in conjunction with (a selection of) the spectroscopic techniques listed above.This article details various aspects of continuous‐flow and stopped‐flow rapid‐mixing techniques, considering mixer design, freeze‐quenching methodology, and sampling. The scope and limitations to (bio)chemical research of the well‐established rapid freeze‐quench (RFQ) and the recently developed microsecond freeze‐hyperquenching (MHQ) technologies are specifically highlighted.