Freeze quench method

Coupling of rapid chemical quench or freeze-quench methods with ERR, MS, or NMR has been a very useful strategy for detecting reaction intermediates that are paramagnetic such as transition metal complexes in metalloenzymes and radical species " " or to determine the chemical structure of transient reaction intermediates or reaction products." ... 

Write a brief report on a recent research article in which at least one of the following techniques was used to study the kinetics of a biochemical reaction stopped-flow techniques, flash photolysis, chemical quench-flow methods, or freeze-quench methods. Your report should be similar in content and extent to one of the Case studies found throughout this book. 

The ways of getting around this problem involve increasing the lifetime of the radicals by some physical or chemical means. One such approach involves stabilizing the radicals by immobilization, for example, by freeze-quenching a reaction mixture [80]. The disadvantage of this method is that an immobilized radical is generally much harder to characterize and identify than one in fluid solution. Other approaches make use of the chemical reactivity of radicals, for example, their ability to add to the double bonds in nitrones and nitroso compounds. This has led to the development of the spin-trapping procedure [81,82], in which a transient radical is reacted with the... 

As discussed further in the following sections, there are other variations of rapid mixing/quench methods in which the enzymatic reaction is terminated by freezing the reaction mixture with liquid isopropane. The frozen sample is then analyzed hy electron paramagnetic resonance (EPR), solid-state NMR, or other spectroscopic techniques such as resonance Raman spectroscopy that can accommodate a solid sample. Perhaps the major limitation for implementation of this methodology is the sensitivity of the spectroscopic method and the requirement for large amounts of enzyme. ... 

Scheme 10-5 shows the simplest version for a possible mechanism of enzyme activation. However, this mechanism does not reveal much about the control of the S-C bond cleavage within the intermediate sulfuranyl radical. Spontaneous cleavage of this radical within the active site is not unlikely if one takes the great instabihty of such radicals into account. Furthermore it has to be emphasized that no direct evidence for intermediate free radicals, such as the sulfuranyl radical or the 5 -deoxyadenosyl radical, is available yet. Whether these radicals are true intermediates or not need to be demonstrated, by using rapid freeze quench EPR methods for example. 

Kirk and coworkers have studied the reaction of DMSOR with (CH3)2NO. At the B3LYP/6-31G level, they found only a single transition state at 67 kJ mol for this substrate. They also studied a freeze-quench trapped Mo(v) intermediate with spectroscopic and computational methods. 

The rapid-quench method [78] was used in Ref. 83 to analyze the mechanism of a bacterial phenylalanine hydroxylase, a mononuclear nonheme iron protein that uses tetrahydropterin as the source of the two electrons needed to activate O2 for the hydroxylation of phenylalanine to tyrosine. Mossbauer spectra of samples prepared by freeze-quenching the reaction of the complex enzyme— Fe(ll)-phenylalanine-6-methyltetrahydropterin with O2 revealed the accumulation of an intermediate at short reaction times (20-100 ms). The Mossbauer parameters of the intermediate (3 = 0.28mms, A q= l.26mms ) suggested it to be a high-spin Fe(IV) complex similar to those that have previously been detected in the reactions of other mononuclear Fe(ll) hydroxylases. 

The freeze fracture method has been used to study the structure of colloidal particles in water-oil mixtures stabilized by polymer emulsifiers. Microemulsions consisting of water, toluene and graft copolymer composed of a polystyrene backbone and a poly(ethylene oxide) graft were deposited onto a small gold plate, quenched in liquid nitrogen in equilibrium with its own solid phase [436]. Replicas of the fractured surfaces were washed with tetrahydrofuran, which showed the micellar structure of the copolymers. A similar method was used for the preparation of polystyrene polymer latexes for TEM study of the size distribution [437]. In this case, the frozen droplet was microtomed, with a cold knife at -100 to -120°C, etched for up to 90 s and then a platinum-carbon replica was prepared. Etching was found to be unnecessary and a potential cause of error. The remaining latex was dissolved away before examination of the replica. Such replicas can reveal the size distribution and structure of the latex particles. 

Bray who has pioneered the rapid freeze method for studying fast kinetics, has recently used this method in conjunction with XAS The procedure involves quenching reacting solutions by squirting them into a cold imiscible solvent and allows the preparation of a series of samples, each frozen at a particular reaction time (from about 3 ms upwards). Bray and co-workers used this method to Study the structural... 

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