Spin Labels in Biology

At present the use of imidazoline nitroxides as spin labels for molecular biology is in its initial stage, though radicals of this type have a diverse reactivity (see Section 3) that is not associated with the presence of a radical center and a greater stability than radicals of other types within a wide pH range. In this section we give examples of imidazoline nitroxides that are and can be used as spin labels and spin probes. 

Easy transformation of 1,2-hydroxylaminoketones to l-hydroxy-3-imidazolines and then to nitroxides allowed introduction of a nitroxyl group into molecules containing a ketone group, similar to the method suggested by Keana et al (1967). The method of Keana is based on the condensation of a compound containing a ketone group with 2-amino-2-methylpropanol-l in the presence of p-toluenesulfonic acid and subsequent oxidation of the resulting oxazolidine derivative with m-chloroper- 

Of particular interest as spin labels with regard to the carbonyl group are hydrazohes 69a,b (Grigor ev and Volodarsky, 1975 c Schlude, 1975) 

Carboxylic acids 33 and 46 are not only acylating spin labels but were also used as spin probes for studying the process of mitochondrial respiration (Section 5). 

As alkylating agents haloidalkylnitrones 64, 65, and 72a and iodo-enaminoketone 122 can be used. Alkylation with iodoenaminoketone 122 of 2,6-diaminoacridine yielded a convenient fluorescent spin label for nucleic acids 123 (Reznikov and Volodarsky, 1979). 

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Hustedt, E. J., and Beth, A. H. (2000). Structural Information from CW-EPR spectra of dipolar coupled nitroxide spin labels. In Biological Magnetic Resonance Distance Measurements in Biological Systems by EPR, (L. J. Berliner, S. S. Eaton, and G. R. Eaton, eds.), Vol. 19. Kluwer Academic/Plenum Publisher, New York. 

Nitroxide radicals are widely used as spin labels in biology, biochemistry and biophysics to gain information about the structure and the dynamics of biomolecules, membranes, and different nanostructures. Their widespread use is related to an unusual stability, which allows researchers to label specific sites and to detect the most informative EPR parameters (g and hyperfme tensors) that are very sensitive to interactions with the chemical surroundings. Figure 2.1 collects all the radicals used in the following to illustrate the different aspects mentioned in the preceding section. 

Although nitroxide radicals do not seem to exist in naturally biological systems, they are relatively non-toxic, and survive as such for considerable periods after administration. This property makes them important as spin-labels in biological systems, ESR spectroscopy being an ideal technique for studying their behaviour. However, in these studies their radical nature is not significant chemically it is the presence of an unpaired electron, and the remarkable stability of these species that is utilized. However, nitroxides can act as electron donors and acceptors, and we compare them here with ascorbate radicals (Section 1.4). 

Griffith, O. H. Jost, P. C. Lipid spin labels in biological membranes. In Spin Labeling Theory and Application Berliner, L. J., Ed. New Yoik-San Francisco-London, Acad. Press, 1979,489-569. 

Marsh D (1994) Spin labelling in biological systems. Specialist Periodical Reports, Electron Spin Resonance 14 166-202. 

Marsh, D. 1981. Electron spin resonance Spin labels. In Membrane Spectroscopy. Molecular Biology, Biochemistry, and Biophysics, ed. E. Grell, Vol. 31, pp. 51-142. Berlin, Germany Springer-Verlag. 

The above historical outline refers mainly to the EPR of transition ions. Key events in the development of radical bioEPR were the synthesis and binding to biomolecules of stable spin labels in 1965 in Stanford (e.g., Griffith and McConnell 1966) and the discovery of spin traps in the second half of the 1960s by the groups of M. Iwamura and N. Inamoto in Tokyo A. Mackor et al. in Amsterdam and E. G. Janzen and B. J. Blackburn in Athens, Georgia (e.g., Janzen 1971), and their subsequent application in biological systems by J. R. Harbour and J. R. Bolton in London, Ontario (Harbour and Bolton 1975). 

Likhtenstein, G.I. (1974) Method of spin labels in molecular biology. Moscow Nauka. 

In solving problems of enzyme catalysis, molecular biophysics of proteins, biomembranes and molecular biology it is necessary to know the spatial disposition of individual parts. One must also know the depth of immersion of paramagnetic centers in a biological matrix, i.e. the availability of enzyme sites to substrates, distance of electron tunneling between a donor and an acceptor group, position of a spin-label in a membrane and in a protein globule, distribution of the electrostatic field around the PC, etc. 

Hustedt, EJ. and Beth, A.H. (Structural information from CW-Esr spectra of dipolar coupled nitroxide spin labels, in Berliner, L, Eaton, S., and Eaton, G. (eds.),, Magnetic Resonance in Biology, V. 18, Kluwer Academic Publishers. Dordrecht, pp. 155-184... 

Much of these data on model systems is complementary to information already obtained by other techniques but, as mentioned in other parts of this review, the greatest potential of ESR is in the ability to study complex intact biological systems, and this is true again of the use of spin labels in... 

The method of biosynthetic incorporation of spin label, rather than mechanical addition to isolated material, is a convenient way of ensuring that the results obtained are biologically meaningful and has also been used with such systems as the mould Neurospora crassa [158], Mycoplasma laidlawii [159], human leucocytes, and mouse L cells [160]. The spectra from these two mammalian cells showed distinct similarities for a variety of spin labels, but different spectra were obtained when the labels were incorporated in human erythrocytes. Fractionation of the cell components showed the stearic acid (C, n = 3) spin label in all the major fractions, but by far the largest concentration was in the nuclear membrane. The ESR spectrum underwent a time and temperature dependent decay and spin labels on the surface membrane were reactivated with K3Fe(CN)6. 

Introductory spin-label material appears in Refs. 5 and 13. The two books edited by Berliner [22] contain definitive treatments of selected topics in the field of spinlabeling. Relevant chapters occasionally appear in the Plenum Press series on Biological Magnetic Resonance, also edited by Berliner (see, for example. Ref. 23). The article by Butterfield on Spin-labeling in Disease in Volume 4 of this series will be of interest to many persons. Likhtenshtein [24] provides an overview of many spin-label applications. Several general spin-label chapters that are intended for a biochemical... 

Likhtenshtein, G.I. (1976) Spin Labeling in Molecular Biology, Wiley, New York. 

G. I. Likhtenstein, Method of Spin Labels in Molecular Biology, Nauka, Moscow, 1974. 

Since the first reported study on nitroxide spin labelling in 1965, the technique has found widespread application to biological and chemical problems. The introduction of a small free radical, the spin label, onto the molecule of interest gives environmental information at that point. It has the ability to measure very rapid molecular motion and is usually free from interference, thus making it a very powerful technique that gives useful details about dynamic processes at the molecular level. 

Thus, the interaction of a-haloalkylnitrones 64a,b with amines and hydrazine leads to products that contain no iV-oxide oxygen atom. This allows aldehyde and ketone derivatives of 3-imidazoline nitroxide to be synthesized the latter are of interest as spin labels for biological systems (Schlude, 1973). The interaction of the same compounds (64a,b) with nitrogenous nucleophiles, which contain a more electronegative nitrogen atom than that in amines or hydrazine, as well as the interaction with C-, S-, and O-nucleophiles, proceeds with the preservation of the nitrone group and leads to nucleophilic substitution products. Dehydrobromination of 4-bromoalkyl derivatives leads to reactive a,j8-unsaturated nitrones... 

Spin labeled 5 -deoxyadenosylcobinamide has been used as a cofactor for ethanolamine-ammonia-lyase and the ESR spectrum followed during catalysis (123). This spin labeled coenzyme is biologically active in this enzyme. Enzyme kinetics showed this derivative to have the same Vmax as the cofactor 5 -deoxyadenosylcobinamide, but it has a higher Km value of 5.1 X 10-6 M compared to 4.6 X 10-6 for 5 -deoxyadenosylcobinamide (123). When the spin labeled coenzyme was incubated with apoenzyme to give the enzyme-coenzyme complex, the nitroxide ESR spectrum resembled that of free spin label but the lines are broadened significantly. 

Hyde, J. S. andW. K. Subczynski. 1989. Spin-label oximetry. In Biological Magnetic Resonance. SpinLabeling Theory and Applications. eds. L. J. Berliner and J. Reuben, Vol. 8, pp. 399 125. New York Plenum. 

If the g- and hyperfine anisotropies are known from analysis of a solid-state spectrum, the line-width parameters (1, and yt can be used to compute the rotational correlation time, tr, a useful measure of freedom of motion. Line widths in ESR spectra of nitroxide spin labels, for example, have been used to probe the motional freedom of biological macromolecules.14 Since tr is related to the molecular hydrodynamic volume, Ft, and the solution viscosity, r, by a relationship introduced by Debye 15... 

Applications of spin labels to problems in structural biology have continued to grow over the four decades since McConnell s original proposal. We mention here only two examples, which provided early support for the method. 

Hubbell, W.L., Gross, A., Langen, R., and Lietzow, M.A. 1998. Recent advances in site-directed spin labeling of proteins. Current Opinion in Structural Biology 8 649-656. 

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