Electron spin label nitroxide free radical

The solution structures, dynamics, and interactions of and between biological macromolecules are topics of widespread interest in biochemistry. The chapter on electron spin labels, by Millhauser et al. illustrates how the EPR spectra of the stable nitroxide free radical can be used to address such problems. The chemistry of the nitroxides and their modes of attachment to the host molecules are discussed first. The details of the EPR spectra and of the spin Hamiltonian are then presented, showing how the intrinsic tensorial nature of the EPR spectrum of the reporter group is affected by motion. Such dynamic information is then extracted from some small peptides. The interaction between pairs of nitroxides is used to extract structural information. Finally, an example of Fourier transform EPR is introduced. ... 

Normally, biological macromolecules which lack unpaired electrons can not be studied by e.s.r. because they do not resonate. These macromolecules can however be spin labelled (spin labelling involves the attachment of a stable and unreactive free radical to the macromolecule) and studied. Thus, lateral diffusion of glycerophosphatides in plasma membranes has been studied by labelling glycerophosphatides with a stable nitroxide free radical. 

Electron spin resonance (ESR), often called electron paramagnetic resonance (EPR), requires the presence of unpaired electrons in the sample [5, 15, 16, 135]. Since most organic molecules do not carry free radieals, the most common method is to simply dissolve stable free radieals as spin probes in the sample. Alternatively, free radical groups can be ehemically bonded to the molecule of interest providing a spin label. Probably the most frequently used spin probes are nitroxide free radicals like 2,2,5,5-tetra-methyl-piperidinoxide (TEMPO). 

Since RNA is diamagnetic, EPR studies of RNA require incorporation of unpaired electrons into the biopolymer. Nitroxides in five- or six-membered rings that are flanked by methyl groups are stable organic free radicals that are commonly used for spin-labeling (Fig. 15.1). For a free... 

Flavodoxins have a natural spin label, but, unlike the nitroxides, the flavin-free radical suffers from electron de-localisation, which interferes with distance calculations. The broadening of protein resonances in the spectra of the semiquinone forms of flavodoxins from Clostridium MP and Peptostreptococcus elsdenii is highly selective [99], and peaks from groups in the vicinity of the radical can be assigned. 

Nitroxides are persistent free radicals [1] which can often be isolated and handled as kinetically stable species. Nitroxides react rapidly with carbon free-radical intermediates [2] with well-characterized rate constants [3], and can thus be used as kinetic and mechanistic probes, as well as to trap carbon radicals in synthetic processes. They are easily oxidized or reduced, and thus have a rich redox chemistry that has been utilized for a variety of oxidations. As nitroxides have an unpaired electron, they are paramagnetic and thus ESR active, making them valuable as spin labels for biomolecules [4] and as spin traps for transient radicals [5]. In addition, nitroxides have been developed as organic ferromagnetic materials [6]. The synthesis of nitroxides has been reviewed in 1994 [7]. This review will focus on the synthetic applications of nitroxides. 

Electron spin resonance (ESR) spectroscopy has been widely used to obtain information about the molecular dynamics of polymers. The method requires the introduction of a stable free-radical reporter group, such as a nitroxide, into the system. Nitroxide spin labels can be covalently attached to the polymer of interest, and can therefore serve as probes of the local backbone dynamics of the polymer, providing information on the local orientation, stracture, dynamics, and enviromnent. " A commonly used nitroxide is shown in Eig. 1. Depending on the ESR frequency, motion on time scales between 10 and 10 ° s may be investigated by this method, making it ideal to study the dynamics of macromolecules and macromolecular structures or assemblies. 

A technique which is similar to radioimmunoassay, the difference being that the antigen is labelled with a free radical such as nitroxide. When the antibody combines with the labelled antigen, the free radical is immobilized and broad spectral peaks are observed on an electron spin resonance spectrometer. When the labelled antigen is displaced by unlabelled antigen from the test sample, a sharp peak is produced. In this way, the amount of antigen in the patient s sample can be measured. 

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