Nitroxide probes

Qin, P. Z., et ah (2007). Measuring nanometer distances in nucleic acids using a sequence-independent nitroxide probe. Nat. Protoc. 2, 2354-2365. 

Cai, Q., Kusnetzow, A. K., Hubbell, W. L., Haworth, I. S., Gacho, G., Van Eps, N., Hideg, K., Chambers, E. J., and Qin, P. Z. (2006). Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe. Nucleic Acid Res. 34, 4722-4730. 

The dynamics of the fluorescence relaxation shift of the dual fluorescence-nitroxide probe... 

Figure 1.4. Time resolved fluorescent spectra of a dual dansyl-nitroxide probe 1 incorporated in bovine serum albumin (Likhtenshtein et al., 2000a). Reproduced with permission.

A problem of the experimental measurement of local polarity in the vicinity of donor and acceptor centers incorporated into a protein (bovine serum albumin, BSA) was solved with the use of the dual fluorescence-nitroxide probe (Bystryak et al., 1986 Rubtsova et al., 1993 Fogel et al., 1994 Likhtenshtein, 1993, 1996 Likhtenshtein et al., 2001). In such a hybrid molecule, the photoactive chromophore fragment in the excited singlet state can... 

The proposed method is based upon the quantitative measurement of the contribution of differently charged nitroxide probes to the spin-lattice relaxation rate (1/T i) of protons in a particular molecule, followed by the calculation of local electrostatic potential using the classical Debye equation (Likhtenshtein et al., 1999 Glaser et al., 2000). In parallel, the theoretical calculation of potential distribution with the use of the MacSpartan Plus 1.0 program has been performed. 

Apparent local electrostatic potential U(Ro) can be determined from the experimental dependence of the proton spin-lattice relaxation rate on the concentration of the nitroxide probes [R ]... 

Lozinsky, E., Martin, V.V., Berezina, T.A., Shames, A., Weis, A.L., and Likhtenshtein, G. I. (1999) Dual fluorophore-nitroxide probes for analysis of vitamin C in biological liquids, J. Biochem. Biophys. Meth. 38,29-42. 

Figure 13. Absorption spectra simulations for a nitroxide probe with axially symmetric g tensor and hyperfine tensor. Z> — - Z) , r —f g - P Hq/D is the Bohr magne-...

We start by considering the works of Freed and co-workers [10,59], ESR relaxation studies of small deuterated nitroxide probes have been performed in their laboratory, showing the sensitivity of this spectroscopic... 

From N. K. Adam s observation that many bipolar amphiphatic molecules form gaseous expanded films, it is evident that these nitroxide probe films, with the possible exception of 5-NS and 5-NS(Me), behave as typical bipolar amphiphatic films. The shift to lower areas/molecule for 5-NS and 5-NS (Me) shows that, as the oxazolidine ring moves closer to the primary polar group, the bipolar nature of the molecule diminishes. Thus the carboxyl group and oxazolidine ring for these compounds effectively constitute one polar group. 

The molecular motion of redox couples within polymer-coated electrodes has recently been investigated by making use of both nitroxide spin probes and various cationic spin probes [94-97]. Spin probes, such as the nitroxide probe TEMPO (see Sect. 2.1.2) and its derivatives, have well-defined electrochemistry and their ESR spectra in viscous media exhibit effects due to incomplete rotational averaging of the g and hyperfine coupling constant tensors. Analysis of the spectra [98] allows deductions to be made concerning the molecular rotation. Such analysis has been performed for spin probes incorporated into various polymer films. 

Figure 14.12. ESR spectra for nitroxide probes in different environments.

Popova AM, Kalai T, Hideg K, Qin PZ (2009) Site-specific DNA structural and dynamic features revealed by nucleotide-independent nitroxide probes. Biochemistry-US 48 (36) 8540-8550. doi 10.1021/bi900860w... 

Dynamic ESR spectroscopy, employing free radical spin irobes, has been widely used to study the molecular pro )erties of LCs [8, 69, 70], In particular, nitroxide probes (see Fig. 5) have proven to be ideally suited for this pur x>se [71-74]. One important reason is their exceptional thermal stability. Moreover, the large anisotropy of the nitrogen hyperfine (HF) interaction [75] makes them sensitive probes for the molecular environment. With some computational effort quantitative details about order and motion can be extracted from the ESR experiments. 

Jamil, T., and Jamieson, A. M., Studies of ESR nitroxide probe mobihty in polymer blends, J. Polym. Sci. Polym. Phys., 27, 2553-2560 (1989). 

A synthesized dual stilbene-nitroxide probe was covalently immobilized onto the surface of a quartz plate as an eventual fiber-optic sensor (Figure 1.25) [150]. The immobilization procedure included a cyanogen bromide surface activation followed by smoothing with a protein tether. The rate of fluorescence change was monitored in aqueous glycerol solution of different viscosities and contents of ascorbic acid. 

In such hybrid compounds, the nitroxide moiety quenches the fluorescence of the fluorophore (stilbene moiety). The reduction of nitroxide segment by an antioxidant (ascorbic acid) caused a rise in fluorescence of the fluorophore. The rate constant of the stilbene fragment photoisomerization in such systems depended on the viscosity of the medium. The synthesized dual stilbene-nitroxide probe was covalently immobilized onto the surface of a quartz plate as an eventual sensor. The immobilization procedure included a cyanogen bromide surface activation followed by smoothing with a protein tether. The rate of fluorescence change was monitored in aqueous glycerol solution of different viscosities and content of ascorbic acid. The dependence of kapp on the reciprocal absolute viscosity 1 /T] of the bulk mixture glycerin-water and the dependence of the initial intensity of fluorescence (/o) on solution viscosity were also studied. 

The above considerations suffice to clearly show that a theoretical study aimed to give some insights on the use of nitroxides as spin probes should allow a clear-cut distinction between intrinsic and environmental effects on the EPR parameters of these compounds. Only a reliable assessment of the intra-molecular factors affecting the values of the hfs of nitroxides [12] (nature of the NO substituents, NO conformation...) makes indeed possible to analyze all the environmental effects which govern the magnetic properties of the nitroxide probe polarity and hydrogen bonding power of the solvent, pH, presence of other free radicals, and so on. 

The same cholestane nitroxide probe has been recently utilized by Mangels et al to study alignment of mixed l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and l,2-dihexanoyl-5n-glycero-3-phosphocholine (DHPC) bicelles at 3.4 T magnetic field of W-band EPR." These studies demonstrated that the order parameter for cholestane in DMPC/DHPC bicelles containing 10% cholesterol is S = 0.64 + 0.04 which is very similar to that determined for mechanically aligned bilayers. 

---