**1. Introduction**

Spin labeling, as a part of electron paramagnetic resonance (EPR), became one of the most sensitive and adequate methods for investigation the structure, properties of different biological systems, their dynamics, and mechanisms of various processes after opening a new class of chemical reactions of stable nitroxide radicals, in which the unpaired electron remained untouched and retained its paramagnetic properties (Neiman et al., 1962, Rozantsev, 1964, 1970, Rozantzev & Neiman, 1964). Just at once, spin labels, attached to biological (proteins, oligopeptides, polysaccharides, nucleic acids) or synthetic macromolecules, and probes, incorporated into biological or artificial membranes, polymers, solid materials and solutions, have been applied for investigation structural and functional properties of such complex and supra-molecular systems. Harden M. McConnell, O. Hayes Griffith, Gertz I. Likhtenstein, Anatoly L. Buchachenko, Lawrence J. Berliner, Alexander Kalmanson, Geoffrey R. Luckhurst, Jack H. Freed, Andrey N. Kuznetsov, and some others, were first pioneers in this area. Much detailed, the historical aspects of spin label technique are described in chapter written by L. J. Berliner. Great advances have been achieved in practical applications of numerous amount of mono- bi- and poly-radicals synthesized in groups headed by Eduard Rozantsev, John Keana, Andre Rassat, Kalman Hideg, George Sosnovsky, Leonid Volodarsky, Igor Grigor'ev, and their pupils.

First books concerning new method, which are actual up to now, were published approximately in ten years later. Among them, the most cited, are written or edited by Buchachenko & Wasserman, 1973, Likhtenshtein, 1974, Berliner, 1976 & 1979, and Kuznetsov, 1976. Among recent publications, I would like to press attention on several once performing further development and success of this method: Likhtenshtein, 2008, Moebius & Savitsky, 2009, Brustolon & Giamello, 2009, Hemminga & Berliner, 2007, Bender & Berliner, 2006, Schlick, 2006, Webb, 2006. Indeed, high-field (high frequencies) EPR spectroscopy, pulse technique, various double- and multy- resonance methods, timeresolved EPR, etc., enlarged the area of magnetic resonance applications pretty much.

© 2012 Kokorin, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Kokorin, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

One of the most important possibilities giving by EPR methods is connected with distance measurements in chemical, biological and nanostructured systems and materials (Parmon et al., 1980, Berliner et al., 2001, Eaton & Eaton, 2004, Steinhoff, 2004, Webb, 2006, Tsvetkov et al., 2008, Moebius & Savitsky 2009), which allows determine distances, two- and threedimensional distribution of paramagnetic centers, and their mutional orientation in the case of not too long distances.

Stable nitroxide spin probes and labels contain paramagnetic >NO group with the unpaired electron, surrounding usually with four methyl groups in the appropriate piperidine (R6, R6"), pirrolidine (R5) or imidazoline (R5N, R5NO) rings, which have different "tails" with functional residues in fourth or third positions of the ring, by which probes can be attached to macromolecules, surfaces, etc., becoming spin labels. These radicals are shown in Fig. 1.

**Figure 1.** Structures of paramagnetic fragments of nitroxide radicals.

Among spectroscopic methods for determination distances between spin probes, which are discussed in the next section, the simplest one, was developed forty years ago based on empirical parameter d1/d (Fig. 2) characterizing the shape of the X-band EPR spectrum of the nitroxide radical solution frozen at 77 K (Kokorin et al., 1972). d1/d is measured with high precision as the ratio of the summed amplitudes of two lateral (low and high field) lines, recorded as the first derivative of the absorption EPR spectrum, to the amplitude of the central component (Fig. 2). It was shown that d1/d is straightly connected with the efficiency of dipole-dipole coupling between radical paramagnetic >N–O groups, the type of spatial distribution of radicals, as well as with polarity of the surrounding media and temperature of the sample. A methodic procedure of measuring distances is described in detail in Section 3, allows characterize quantitatively the spatial organization of nitroxide biradicals, proteins, nucleic acids, frozen two-component solutions, synthetic polymers, and nanostructured materials. Unfortunately, the most part of the d1/d features and the majority of the resulta obtained have been published in original only in Russian, though translated into English, scientific journals, and are not known well to practical scientists and students.

**Figure 2.** The EPR spectrum of TEMPOL radical at 77 K and *c* = 0.001 M

2

0

2

114 Nitroxides – Theory, Experiment and Applications

of not too long distances.

shown in Fig. 1.

One of the most important possibilities giving by EPR methods is connected with distance measurements in chemical, biological and nanostructured systems and materials (Parmon et al., 1980, Berliner et al., 2001, Eaton & Eaton, 2004, Steinhoff, 2004, Webb, 2006, Tsvetkov et al., 2008, Moebius & Savitsky 2009), which allows determine distances, two- and threedimensional distribution of paramagnetic centers, and their mutional orientation in the case

Stable nitroxide spin probes and labels contain paramagnetic >NO group with the unpaired electron, surrounding usually with four methyl groups in the appropriate piperidine (R6, R6"), pirrolidine (R5) or imidazoline (R5N, R5NO) rings, which have different "tails" with functional residues in fourth or third positions of the ring, by which probes can be attached to macromolecules, surfaces, etc., becoming spin labels. These radicals are

> **N O**


**M e M e**

**M e M e**

**N+**

**N**

**O**

**N**

**O**

**M e M e**

**O**

**R5 NO** **M e M e**

**R5**

**R6**

**Me Me**

Among spectroscopic methods for determination distances between spin probes, which are discussed in the next section, the simplest one, was developed forty years ago based on empirical parameter d1/d (Fig. 2) characterizing the shape of the X-band EPR spectrum of the nitroxide radical solution frozen at 77 K (Kokorin et al., 1972). d1/d is measured with high precision as the ratio of the summed amplitudes of two lateral (low and high field) lines, recorded as the first derivative of the absorption EPR spectrum, to the amplitude of the central component (Fig. 2). It was shown that d1/d is straightly connected with the efficiency of dipole-dipole coupling between radical paramagnetic >N–O groups, the type of spatial distribution of radicals, as well as with polarity of the surrounding media and temperature of the sample. A methodic procedure of measuring distances is described in detail in Section 3, allows characterize quantitatively the spatial organization of nitroxide biradicals, proteins, nucleic acids, frozen two-component solutions, synthetic polymers, and nanostructured materials. Unfortunately, the most part of the d1/d features and the majority of the resulta obtained have been published in original only in Russian, though translated into English, scientific journals, and are not known well to practical scientists and students.

**R5 N**

**M e M e**

**Figure 1.** Structures of paramagnetic fragments of nitroxide radicals.

**M e M e**

**M e M e**

**N**

**N**

**O**

**R6**

**Me Me**

**N O** **H**

**M e M e**

Therefore, the main goal of this chapter is describing the present state of this method and the analysis of regularities and peculiarities of its application to various objects and systems under EPR investigation including molecules, macromolecules and supramolecular systems. Theoretical aspects, experimental details, obtained results and conclusions will be reported in the following sections.
