Kinetics of nitroxide spin label removal in biological systems: An in vitro and in vivo ESR study

A systematic study on the disappearance of the electron spin resonance (ESR) signal of nitroxides based on six- or five-membered ring and bearing various charges was carried out in vitro and in vivo. The second-order kinetic rate constants of the reaction of spin probes with ascorbate were measured in vitro at various temperatures in phosphate buffered saline, and the relative activation energies were calculated. Clearance rates of the nitroxide radicals in rat brain homogenates and in blood indicate that the ascorbate contribution to nitroxide removal is about 50–70% in brain and 50–90% in blood. These rates can be easily calculated on the basis of the ascorbate concentration and of the second-order kinetic rate constants measured in phosphate buffered saline. ESR spectra acquired in vivo in rat head and tail, by an L-band resonator, indicated that the nitroxide decay rate is a first-order kinetic process in both domains and that the positively charged nitroxides are not retained in the brain, whereas the anionic and uncharged nitroxides are. Once nitroxides with piperidine ring enter the brain, their decay appears controlled mainly by ascorbate, while the ascorbate has a negligible influence on disappearance in brain of five-membered ring proxyl nitroxides.     

EPR and Quantum Chemical Studies of the pH-sensitive Imidazoline and Imidazolidine Nitroxides with Bulky Substituents

The X- and W-band electron paramagnetic resonance (EPR) spectroscopies were employed to investigate a series of imidazolidine nitroxide radicals with different number of ethyl and methyl substituents at positions 2 and 5 of a heterocycle in liquid and frozen solutions. The influence of the substituents on the line shape and width was studied experimentally and analyzed using quantum chemical calculations. Each pair of the geminal ethyl groups in the positions 2 or 5 of the imidazolidine ring was found to produce an additional hyperfine splitting (hfs) of about 0.2 mT in the EPR spectra of the nitroxides. The effect was attributed to the hfs constant of only one of four methylene hydrogen atoms of two geminal ethyl substituents not fully averaged by ethyl group rotation and ring puckering. In accordance with this assumption, the substitution of hydrogen atoms of CH₂ groups in 2,2,5,5-tetraethyl-substituted imidazolidine nitroxides by deuterium leads to the substantial narrowing of EPR lines which could be useful for many biochemical and biomedical applications, including pH-monitoring. W-band EPR spectra of 2,2,5,5-tetraethyl-substituted imidazolidine nitroxide and its 2,2,5,5-tetraethyl–d₈ deuterium-substituted analog measured at low temperatures demonstrated high sensitivity of their g-factors to pH, which indicates their applicability as spin labels possessing high stability.     

Interactions between Diamagnetic Metal Ions and pH-Sensitive Nitroxides

Specific interactions between pH-sensitive nitroxide radicals and selected diamagnetic metal ions were investigated. To this end, the influence of different metal salts at varying concentrations on the continuous-wave electron paramagnetic resonance spectra of two imidazoline nitroxides was studied. Among the screened metal ions, Zn(II) most significantly affected the spectral profile, analogous to the effect attributed to protonation of the nitroxide imino nitrogen known from pH studies. Simulations showed the acquired spectra to result from the superposition of the signals of the coordinated and the uncoordinated species. The complex formation between Zn(II) and (4-amino-2,5-dihydro-2,2,5,5-tetramethyl-3-imidazoline-1-yloxyl) was modelled by theoretical methods revealing the rather specific selectivity of the nitroxide toward Zn(II). The results suggest imidazoline nitroxides as promising candidates for the development of new specific metal ion probes.     

Observer-selective double electron-electron-spin resonance, a pulse sequence to improve orientation selection

By pulsed double electron-electron resonance (DEER), distances between spin labels in disordered systems up to 8 nm can be measured. In addition, the relative orientation of the interacting radicals can be determined, provided that the bandwidth of the pulses is sufficiently small. On the other hand, the bandwidth has to exceed the dipolar interaction considerably, because otherwise the DEER modulations become distorted and the modulation depth decreases, making distance determination impossible. Therefore, small bandwidths, i.e. long pulses, place a lower limit on the distance that can be determined. Two new pulse sequences, observer-selective DEER (os-DEER) and dead-time free os-DEER, are introduced that make it possible to use long observer pulses with bandwidths that are smaller than the dipolar interaction. The new pulse sequences do not suffer from the distortions caused by the limited bandwidth of the observer pulses, as demonstrated by measurements on a nitroxide biradical. With observer pulses of 140 ns, i.e., significantly longer than the 32 ns used in the conventional DEER sequence, a dipolar interaction of 7.8 MHz has been measured.     

The evaluation of new and isotopically labeled isoindoline nitroxides and an azaphenalene nitroxide for EPR oximetry

Isoindoline nitroxides are potentially useful probes for viable biological systems, exhibiting low cytotoxicity, moderate rates of biological reduction and favorable Electron Paramagnetic Resonance (EPR) characteristics. We have evaluated the anionic (5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl; CTMIO), cationic (5-(N,N,N-trimethylammonio)-1,1,3,3-tetramethylisoindolin-2-yloxyl iodide, QATMIO) and neutral (1,1,3,3-tetramethylisoindolin-2-yloxyl; TMIO) nitroxides and their isotopically labeled analogs ((2)H(12)- and/or (2)H(12)-(15)N-labeled) as potential EPR oximetry probes. An active ester analogue of CTMIO, designed to localize intracellularly, and the azaphenalene nitroxide 1,1,3,3-tetramethyl-2,3-dihydro-2-azaphenalen-2-yloxyl (TMAO) were also studied. While the EPR spectra of the unlabeled nitroxides exhibit high sensitivity to O(2) concentration, deuteration resulted in a loss of superhyperfine features and a subsequent reduction in O(2) sensitivity. Labeling the nitroxides with (15)N increased the signal intensity and this may be useful in decreasing the detection limits for in vivo measurements. The active ester nitroxide showed approximately 6% intracellular localization and low cytotoxicity. The EPR spectra of TMAO nitroxide indicated an increased rigidity in the nitroxide ring, due to dibenzo-annulation.     

Nucleotide Spin Labeling for ESR Spectroscopy of ATP-Binding Proteins

Site-directed spin labeling of proteins by chemical modification of engineered cysteine residues with the molecule MTSSL (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl methanethiosulfonate) has been an invaluable tool for conducting double electron electron resonance (DEER) spectroscopy experiments. However, this method is generally limited to recombinant proteins with a limited number of reactive Cys residues that when modified will not impair protein function. Here, we present a method that allows for spin labeling of protein-nucleotide-binding sites by adenosine diphosphate (ADP) modified with a nitroxide moiety on the β-phosphate (ADP-β-S-SL). The synthesis of this ADP analog is straightforward and isolation of pure product is readily achieved on a standard reverse-phase high-performance liquid chromatography (HPLC) system. Furthermore, analyses of isolated ADP-β-S-SL by LC–mass spectrometry confirm that the molecule is very stable under ambient conditions. The crystal structure of ADP-β-S-SL bound to the ATP pocket of the histidine kinase CheA reveals specific targeting of the probe, whose nitroxide moiety is mobile on the protein surface. Continuous wave and pulsed-ESR measurements demonstrate the capability of ADP-β-S-SL to report on active site environment and provide reliable DEER distance constraints.     

Temperature Dependence of Hyperfine Interaction for 15N Nitroxide in a Glassy Matrix at 10–210 K

Principal ¹⁵N hyperfine interaction (hfi) values in ¹⁵N-substituted nitroxide spin probe 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl dissolved in nematic liquid crystal 4-pentyl-4′-cyanobiphenyl (5CB) were measured in a wide temperature range of 10–210 K, for 5CB frozen to a glassy state. X-band continuous-wave electron paramagnetic resonance (CW EPR) and pulse X- and Q-band ¹⁵N electron-nuclear double resonance (ENDOR) techniques were employed. To avoid microwave saturation at low temperatures in CW EPR studies, a holmium complex Ho(Dbm)₃Bpy (where Dbm is dibenzoylmethane and Bpy is 2,2′-bipyridine) was added. X- and Q-band ¹⁵N-ENDOR data have shown that the nitroxide hfi tensor is axially symmetric. The combination of data from all techniques allowed us to obtain the temperature dependence of isotropic and anisotropic parts of the nitroxide hfi tensor. Above ~100 K, a linear dependence of the anisotropic hfi value was observed, whereas below 30 K it was found to be nearly temperature independent. Such a behavior can be interpreted using the model of restricted orientational motions (librations) of a spin probe in a glassy matrix, with quantum effects occurring at low temperature (“freezing” of the librations). The energy quantum for the libration motion estimated from the temperature dependence of hfi of the spin probe is 84 cm^?1. Low-frequency Raman spectra of 5CB were also obtained, which provided the mean vibrational frequency of 76 cm^?1 for glassy 5CB.     

Real-time monitoring of drug-induced changes in the stomach acidity of living rats using improved pH-sensitive nitroxides and low-field EPR techniques

New improved pH-sensitive nitroxides were applied for in vivo studies. An increased stability of the probes towards reduction was achieved by the introduction of the bulky ethyl groups in the vicinity of the paramagnetic NO fragment. In addition, the range of pH sensitivity of the approach was extended by the synthesis of probes with two ionizable groups, and, therefore, with two pKa values. Stability towards reduction and spectral characteristics of the three new probes were determined in vitro using 290 MHz radiofrequency (RF)- and X-band electron paramagnetic resonance (EPR), longitudinally detected EPR (LODEPR), and field-cycled dynamic nuclear polarization (FC-DNP) techniques. The newly synthesized probe, 4-[bis(2-hydroxyethyl)amino]-2-pyridine-4-yl-2,5,5-triethyl-2,5-dihydro-1H-imidazol-oxyl, was found to be the most appropriate for the application in the stomach due to both higher stability and convenient pH sensitivity range from pH 1.8 to 6. LODEPR, FC-DNP and proton-electron double resonance imaging (PEDRI) techniques were used to detect the nitroxide localization and acidity in the rat stomach. Improved probe characteristics allowed us to follow in vivo the drug-induced perturbation in the stomach acidity and its normalization afterwards during 1 h or longer period of time. The results show the applicability of the techniques for monitoring drug pharmacology and disease in the living animals.     

Orientation Selective 2D-SIFTER Experiments at X-Band Frequencies

Frequency-correlated 2D SIFTER with broadband pulses at X-band frequencies can be used to determine the inter-spin distance and relative orientation of nitroxide moieties in macromolecules when the flexibility of the spin-labels is restricted. At X-band frequencies the EPR spectrum of nitroxides is governed by the strongly anisotropic nitrogen hyperfine coupling. For rigid spin-labels, where the orientation of the inter-connecting vector R correlates to the relative orientations of the nitroxide labels, the dipolar oscillation frequency varies over the EPR spectral line shape. Broadband shaped pulses allow excitation of the complete nitroxide EPR spectra. In this case, Fourier transform of the echo signal gives both fast and direct access to the orientation dependent dipole coupling. This allows determination of not only the inter-spin distance R, but also their mutual orientation. Here, we show the application of the frequency-correlated 2D SIFTER experiment with broadband pulses to a bis-nitroxide model compound and to a double stranded DNA sample. In both molecules, there is restricted internal mobility of the two spin-labels. The experimental results are compared to orientation selective pulsed electron double resonance (PELDOR) experiments and simulations based on a simple geometrical model or MD simulations describing the conformational flexibility of the molecules. Fourier transformation of the SIFTER echo signal yields orientation selective dipolar time traces over the complete EPR-spectral range. This leads to an improved frequency resolution and either to a reduced experimental measurement time or a larger span of frequency offsets measured compared to orientation selective PELDOR experiments. The experimental potential and limitations of the 2D SIFTER method for samples containing rigid spin-labels will be discussed.     

Interaction of imidazoline- and imidazolidine-based nitroxides with chloroplasts

In this work, we investigated the interaction of a number of novel pH-sensitive spin probes (imidazoline- and imidazolidine-based nitroxides) with isolated chloroplasts. The light-induced changes in the electron paramagnetic resonance signals of these radicals are determined by several factors: ΔpH-driven accumulation of nitroxides inside the thylakoids, consumption (or evolution) of oxygen by chloroplasts, and irreversible loss of spin probe paramagnetism. A comparative study of these radicals characterized by different pK_a allowed us to conclude that these spin probes can be used as pH indicators for monitoring the intrathylakoid pH- and light-induced changes in oxygen concentration in chloroplast suspension.     

Dead-time free measurement of dipole-dipole interactions between electron spins. 2000

A four-pulse version of the pulse double electron–electron resonance (DEER) experiment is presented, which is designed for the determination of interradical distances on a nanoscopic lengthscale. With the new pulse sequence electron–electron couplings can be studied without dead-time artifacts, so that even broad distributions of electron–electron distances can be characterized. A version of the experiment that uses a pulse train in the detection period exhibits improved signal-to-noise ratio. Tests on two nitroxide biradicals with known length indicate that the accessible range of distances extends from about 1.5 to 8 nm. The four-pulse DEER spectra of an ionic spin probe in an ionomer exhibit features due to probe molecules situated both on the same and on different ion clusters. The former feature provides information on the cluster size and is inaccessible with previous methods.     

Nitroxide radicals

The proton hyperfine constants have been measured, using N.M.R., for a series of sterically hindered alkyl aryl nitroxides. Computer reconstructions of the electron resonance spectra show that the coupling constants obtained from the pure radical are very similar to those obtained from the electron resonance spectra of dilute solutions. Comparison of methyl and proton splittings in identical positions suggests that the radicals are σ in character although the molecular-orbital calculations are unable to distinguish between σ or π radicals.     

Simultaneous acquisition of pulse EPR orientation selective spectra

High resolution pulse EPR methods are usually applied to resolve weak magnetic electron-nuclear or electron-electron interactions that are otherwise unresolved in the EPR spectrum. Complete information regarding different magnetic interactions, namely, principal components and orientation of principal axis system with respect to the molecular frame, can be derived from orientation selective pulsed EPR measurements that are performed at different magnetic field positions within the inhomogeneously broadened EPR spectrum. These experiments are usually carried out consecutively, namely a particular field position is chosen, data are accumulated until the signal to noise ratio is satisfactory, and then the next field position is chosen and data are accumulated. Here we present a new approach for data acquisition of pulsed EPR experiments referred to as parallel acquisition. It is applicable when the spectral width is much broader than the excitation bandwidth of the applied pulse sequence and it is particularly useful for orientation selective pulse EPR experiments. In this approach several pulse EPR measurements are performed within the waiting (repetition) time between consecutive pulse sequences during which spin lattice relaxation takes place. This is achieved by rapidly changing the main magnetic field, B(0), to different values within the EPR spectrum, performing the same experiment on the otherwise idle spins. This scheme represents an efficient utilization of the spectrometer and provides the same spectral information in a shorter time. This approach is demonstrated on W-band orientation selective electron-nuclear double resonance (ENDOR), electron spin echo envelope modulation (ESEEM), electron-electron double resonance (ELDOR)--detected NMR and double electron-electron resonance (DEER) measurements on frozen solutions of nitroxides. We show that a factors of 3-6 reduction in total acquisition time can be obtained, depending on the experiment applied.     

An EPR study of lipid vesicles as paramagnetic agent vectors

Paramagnetic nitroxides have been proposed as probes in electron paramagnetic resonance (EPR) imaging and in clinical diagnosis. However, nitroxides are rapidly reducedin vivo to hydroxylamines, diamagnetic EPR-inactive species. Reduction occurs in blood via soluble agents such as ascorbic acid, as well as in the cells via enzymatic and non-enzymatic endocellular systems. To prevent the reduction, a water soluble nitroxide, i.e., potassium peroxylamine disulfonate, is entrapped in reverse phase evaporation vesicles. The loaded liposomes have a high entrapment capacity, and vesicles with the encapsulated agent are stable for days, even at room temperature. The vesiclesin vitro can almost completely prevent the reduction of the entrapped nitroxide by ascorbic acid. In blood of a rat, enriched with a homogenate of rat liver proteins, the vesicles are able to greatly prolong the life time of the nitroxide. In particular, the encapsulated nitroxide has a half-life of more than one hour, compared to two minutes for free nitroxide under the same conditions. Due to these protective effects, the lipid vesicles might be useful as a delivery system for paramagnetic agents.     

Determination of the 14N quadrupole coupling constant of nitroxide spin probes by W-band ELDOR-detected NMR

Nitroxide spin probe electron paramagnetic resonance (EPR) has proven to be a very successful method to probe local polarity and solvent hydrogen bonding properties at the molecular level. The g(xx) and the (14)N hyperfine A(zz) principal values are the EPR parameters of the nitroxide spin probe that are sensitive to these properties and are therefore monitored experimentally. Recently, the (14)N quadrupole interaction of nitroxides has been shown to be also highly sensitive to polarity and H-bonding (A. Savitsky et al., J. Phys. Chem. B 112 (2008) 9079). High-field electron spin echo envelope modulation (ESEEM) was used successfully to determine the P(xx) and P(yy) principal components of the (14)N quadrupole tensor. The P(zz) value was calculated from the traceless character of the quadrupole tensor. We introduce here high-field (W-band, 95 GHz, 3.5 T) electron-electron double resonance (ELDOR)-detected NMR as a method to obtain the (14)N P(zz) value directly, together with A(zz). This is complemented by W-band hyperfine sublevel correlation (HYSCORE) measurements carried out along the g(xx) direction to determine the principal P(xx) and P(yy) components. Through measurements of TEMPOL dissolved in solvents of different polarities, we show that A(zz) increases, while |P(zz)| decreases with polarity, as predicted by Savitsky et al.     

Distance determination in human ubiquitin by pulsed double electron-electron resonance and double quantum coherence ESR methods

Recently, distance measurements by pulsed ESR (electron spin resonance) have been obtained using pulsed DEER (double electron-electron resonance) and DQC (double quantum coherence) in SDSL (site directed spin labeling) proteins. These methods can observe long range dipole interactions (15-80A). We applied these methods to human ubiquitin proteins. The distance between the 20th and the 35th cysteine was estimated in doubly spin labeled human ubiquitin. Pulsed DEER requires two microwave sources. However, a phase cycle is not usually required in this method. On the other hand, DQC-ESR at X-band ( approximately 9GHz) can acquire a large echo signal by using pulses of short duration and high power, but this method has an ESEEM (electron spin echo envelope modulation) problem. We used a commercial pulsed ESR spectrometer and compared these two methods.     

CW EPR, echo-detected EPR, and field-step ELDOR study of molecular motions of nitroxides ino-terphenyl glass: Dynamical transition, dynamical heterogeneity and β-relaxation

Continuous-wave electron paramagnetic resonance (CW EPR), echo-detected (ED) EPR, and field-step electron-electron double resonance (FS ELDOR) were simultaneously applied to study molecular motions of nitroxide spin probes of two different types in glassyo-terphenyl. A strong linear temperature dependence of the overall splitting of the CW EPR lineshape was found for nitroxide Tempone and only a weak one for a phenyl-ring-containing imidasoline nitroxide. The linear temperature dependence of the splitting is explained within the model of harmonic librations. The assessed libration frequency for Tempone is of the order of 3·10¹² rad/s. The observed remarkable difference between the two nitroxides is explained by the different strength of interactions between guest and host molecules and by dynamical heterogeneity of the glass. The nonlinear temperature dependence above 250 K is attributed to the onset of anharmonic motion that is postulated in a number of neutron scattering and Mössbauer spectroscopy studies for molecular glasses and proteins (the so-called dynamical transition). Above 245 K also ED EPR spectra change drastically, which may be explained on the same ground. Magnetization transfer was observed in FS ELDOR for nitroxide Tempone, with a time constant around 10^?5 s. It was found to be almost temperature-independent between 160 K and 265 K and was attributed to the Johari-Goldstein β-relaxation process. For the phenyl-ring-containing imidasoline nitroxide this transfer was not observed, which may be explained again by the dynamical heterogeneity of the glass and by small effectivity of the β-relaxation process in this case.     

Probing How Counterion Structure and Dynamics Determine Polyelectrolyte Solutions Using EPR Spectroscopy

In this review article, we describe how methods of electron paramagnetic resonance (EPR) spectroscopy were used to investigate polyion–counterion interactions in polyelectrolyte solutions. This subject is usually treated experimentally by light, X-ray, or neutron scattering techniques. It is shown that a large arsenal of EPR spectroscopic methods–from various sophisticated methods of line shape analysis of continuous-wave EPR, via electron spin echo envelope modulation, nanoscale distance measurements through double electron–electron resonance to high-field pulse EPR–can be used to characterize the intrinsically complicated structures formed in polyelectrolyte solutions. We show that even polymer physical models such as scaling relations can be tested in this way. The distinguishing feature with respect to the numerous scattering studies in this area is that EPR techniques are local methods, and by employing spin-carrying (i.e., EPR-active) probe ions, it is possible to examine polyelectrolytes from the counterions’ point of view.     

Submicrosecond field-jump device for pulsed high-field ELDOR

A field-jump device for fast stepping the electron paramagnetic resonance magnetic field around 3.4 T during pulsed electron-electron double resonance experiments at W-band (95 GHz) is described. Field jumps up to ±160 G and submicrosecond times for the full field-jump cycle allow precession frequency transfer experiments to be made for the determination of the nanometer distance and the orientation of nitroxide spin-label pairs in disordered samples.     

Evaluation of spin labels for in-cell EPR by analysis of nitroxide reduction in cell extract of Xenopus laevis oocytes

Spin-label electron paramagnetic resonance (SL-EPR) spectroscopy has become a powerful and useful tool for studying structure and dynamics of biomacromolecules. However, utilizing these methods at physiological temperatures for in-cell studies is hampered by reduction of the nitroxide spin labels and thus short half-lives in the cellular environment. Consequently, reduction kinetics of two structurally different nitroxides was investigated in cell extracts of Xenopus laevis oocytes using rapid-scan cw-experiments at X-band. The five member heterocyclic ring nitroxide PCA (3-carboxy-2,2,5,5-tetramethylpyrrolidinyl-1-oxy) under investigation features much higher stability against intracellular reduction than the six member ring analog TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxilic acid) and is therefore a suitable spin label type for in-cell EPR. The kinetic data can be described according to the Michaelis–Menten model and thus suggest an enzymatic or enzyme-mediated reduction process.