Use of alanine-silicone pellets for electron paramagnetic resonance gamma dosimetry.

Silicone is proposed as an alternative binding substance in the production of D-L alanine pellets used in electron paramagnetic resonance (EPR) dosimetry of gamma rays. The dosimeters are manufactured at room temperature, making the production simple. Examination by EPR silicone-alanine pellets irradiated with 60Co gamma rays in the dose range 10 to 10(6) Gy shows that the proposed silicone binder does not affect typical alanine dose-response curves. Thermal stability of the pellets below 40 degrees C is good, but their pre-dose EPR signal amplitude is slightly higher than for nonirradiated alanine.     

Alanine radicals,part 4: relative amounts of radical species in alanine dosimeters after exposure to 6-19 MeV electrons and 10 kV-15 MV photons

The amino acid l-alpha-alanine can be used for high-precision dosimetry over a wide dose range, using EPR spectroscopy for monitoring radical concentrations. It is important, however, to understand the underlying composition of the observed EPR spectrum. In previous work, it was shown that the EPR signal from irradiated alanine consists of at least three different radical species, with the relative importance of each of these being almost independent of absorbed dose. However, it was not known whether the relative importance of each radical is independent of the radiation quality responsible for the EPR signal. In the present work, the relative contributions of the different radical species to the total EPR signal from alanine dosimeters irradiated with 6-19 MeV electrons and 10 kV-15 MV photons at a dose of 10 Gy were examined. By spectrum reconstruction using benchmark spectra generated from a simulation procedure, the relative amounts of the three different radical species were shown to be virtually independent of these radiation beam qualities.     

EPR studies of 5-bromouracil crystal after irradiation with X rays in the bromine K-edge region

Radicals induced in a single crystal of 5-bromouracil (BrUra) by synchrotron soft X rays in the bromine K-edge region (13.461-13.482 keV) were investigated using the X-band EPR method. The crystal was irradiated at three peak energies of the absorption spectrum at room temperature or at 80 K. A hydrogen abstraction radical derived from N1 of the pyrimidine ring was commonly observed for all of the energies used, though with some variation in quantity. Similar characteristics were also observed in the EPR signal for the off-K-edge low-energy (13.42 keV) and (60)Co gamma rays used for comparison. When irradiated at 80 K, a much larger exposure (roughly 10 times) of soft X rays was needed to obtain the same signal intensity as that observed at room temperature. EPR signals were not detectable with gamma irradiation at liquid nitrogen temperature.     

Alanine radicals,part 3: properties of the components contributing to the EPR spectrum of X-irradiated alanine dosimeters

The amino acid l-alpha-alanine has attracted considerable interest for use in radiation dosimetry and has been formally accepted as a secondary standard for high-dose and transfer dosimetry. Recent results have shown that the alanine EPR spectrum consists of contributions from three different radicals. A set of benchmark spectra describing the essential spectral features of these three radical components was used for reconstructions of the experimental spectra. In the present work, these basis spectra have been used to investigate the differential effects of variations in radiation doses and microwave power, as well as the dependence upon temperature annealing and UV illumination. The results presented here, based solely on relatively low-energy (60-80 keV) X rays, indicate that the three components behave very similarly with respect to radiation dose at room temperature. However, with respect to the thermal annealing/fading behavior and microwave power saturation properties, the three species behave significantly differently. It is concluded that even if it is now realized that three different radicals contribute to the composite EPR alanine spectrum, this has a minor impact on the established protocols for present-day applications (high-dose) of EPR/alanine dosimetry. However, some care should be exercised when e.g. constructing calibration curves, since fading and power saturation behavior may vary over the dose range in question. New results from UV-illumination experiments suggest a possible procedure for experimental spectral separation of the EPR signals due to the three radicals.     

EPR evidence of two structurally different diferric sites in Mycobacterium tuberculosis R2-2 ribonucleotide reductase protein

Mixed-valent species were generated in the diiron site of active (with tyrosyl free radical) and met (without radical) forms of protein R2-2 in a class Ib ribonucleotide reductase from Mycobacterium tuberculosis by low temperature reduction (γ-irradiation) at 77 K. The primary mixed-valent EPR signal is a mixture of two components with axial symmetry and g_av<2.0, observable at temperatures up to 77 K, and assigned to antiferromagnetically coupled high spin ferric/ferrous sites. The two components in the primary EPR signal can be explained by the existence of two structurally distinct μ-oxo-bridged diferric centers, possibly related to structural heterogeneity around the iron site, and/or different properties of the two polypeptide chains in the homodimeric protein after the radical reconstitution reaction. Annealing of the irradiated R2-2 samples to 143 K transforms the primary EPR signal into a rhombic spectrum characterized by g_av<1.8 and observable only below 25 K. This spectrum is assigned to a partially relaxed form with a μ-hydroxo-bridge. Further annealing at 228 K produces a new complex rhombic EPR spectrum composed of at least two components. An identical EPR spectrum was observed and found to be stable upon chemical reduction of Mycobacterium tuberculosis RNR R2-2 at 293 K by dithionite.     

EPR evidence of two structurally different diferric sites in Mycobacterium tuberculosis R2-2 ribonucleotide reductase protein

Mixed-valent species were generated in the diiron site of active (with tyrosyl free radical) and met (without radical) forms of protein R2-2 in a class Ib ribonucleotide reductase from Mycobacterium tuberculosis by low temperature reduction (γ-irradiation) at 77 K. The primary mixed-valent EPR signal is a mixture of two components with axial symmetry and g_av<2.0, observable at temperatures up to 77 K, and assigned to antiferromagnetically coupled high spin ferric/ferrous sites. The two components in the primary EPR signal can be explained by the existence of two structurally distinct μ-oxo-bridged diferric centers, possibly related to structural heterogeneity around the iron site, and/or different properties of the two polypeptide chains in the homodimeric protein after the radical reconstitution reaction. Annealing of the irradiated R2-2 samples to 143 K transforms the primary EPR signal into a rhombic spectrum characterized by g_av<1.8 and observable only below 25 K. This spectrum is assigned to a partially relaxed form with a μ-hydroxo-bridge. Further annealing at 228 K produces a new complex rhombic EPR spectrum composed of at least two components. An identical EPR spectrum was observed and found to be stable upon chemical reduction of Mycobacterium tuberculosis RNR R2-2 at 293 K by dithionite.     

Accumulation of magnetic nanoparticles in plants grown on soils of Apsheron peninsula

The disclosure of magnetic nanoparticles in five plant species growing in Apsheron peninsula have been detected by the EPR method. The EPR spectra of these nanoparticles proved to be similar to those of synthesized magnetic nanoparticles. The result demonstrated that plants are capable of absorbing magnetic nanoparticles from the soil. The accumulation of nanoparticles in plants is confirmed by the presence of a broad EPR signal whose maximum position of the low-field component changes from g = 2.38 and halfwidth of the signal of 32 mT at room temperature to g = 2.71 and 50-55 mT at 80 K. The intensity of the broad EPR signal for plants grown in radioactively contaminated areas (170-220 mkR per h) was substantially lower compared with plants grown on clean soil. The parameters of the broad EPR signal and its dependence on the temperature of recording were identical with those for synthetic magnetic nanoparticles. The photosynthetic activity and changes in the genome of irradiated plants by the analysis of PCR products were studied.     

Photoreduction of copper chromophores in blue oxidases.

The low temperature (77 K) irradiation of oxidized ceruloplasmin and Rhus vernicifera laccase at the 330 nm absorption which arises from type 3 copper leads to the reduction of type 1 copper as demonstrated by bleaching of the 610 nm chromophore and the decrease of the EPR signal associated with this species. Type 2 copper remains unaffected. Concomitant with the type 1 copper reduction, a new EPR signal which is possibly that of a biradical appears. Upon thawing, type 1 copper is reversibly oxidized and the radical signal disappears. Irradiation of oxidized protein at the absorption band of type 1 copper produces no spectral change. An EPR study at room temperature confirms the wave-length specificity and reversibility of the photoreduction of type 1 copper and radical formation. Radical appearance and disappearance at room temperature are extremely slow (tau1/2 approximately 30 min). Optical studies at room temperature show that upon anaerobic irradiation of laccase in the 330 nm absorption band, both type 3 and type 1 chromophores are slowly reduced. Upon return to the dark and in the presence of O2, both type 3 and type 1 centers are reoxidized. Oxidizing equivalents either from O2 or K3Fe(CN)6 are required for the reoxidation reaction. These studies demonstrate that there is a direct energy transfer between type 3 and type 1 copper sites in blue copper oxidases.     

Comparative study of tyrosine radicals in hemoglobin and myoglobins treated with hydrogen peroxide

The reactions of hydrogen peroxide with human methemoglobin, sperm whale metmyoglobin, and horse heart metmyoglobin were studied by electron paramagnetic resonance (EPR) spectroscopy at 10 K and room temperature. The singlet EPR signal, one of the three signals seen in these systems at 10 K, is characterized by a poorly resolved, but still detectable, hyperfine structure that can be used to assign it to a tyrosyl radical. The singlet is detectable as a quintet at room temperature in methemoglobin with identical spectral features to those of the well characterized tyrosyl radical in photosystem II. Hyperfine splitting constants found for Tyr radicals were used to find the rotation angle of the phenoxyl group. Analysis of these angles in the crystal structures suggests that the radical resides on Tyr151 in sperm whale myoglobin, Tyr133 in soybean leghemoglobin, and either alphaTyr42, betaTyr35, or betaTyr130 in hemoglobin. In the sperm whale metmyoglobin Tyr103Phe mutant, there is no detectable tyrosyl radical present. Yet the rotation angle of Tyr103 (134 degrees) is too large to account for the observed EPR spectrum in the wild type. Tyr103 is the closest to the heme. We suggest that Tyr103 is the initial site of the radical, which then rapidly migrates to Tyr151.     

Electron paramagnetic resonance characterization of tyrosine radical, M+, in site-directed mutants of photosystem II(t).

Photosystem II contains two well-characterized tyrosine radicals, D(.) and Z(.). Z is an electron carrier between the primary chlorophyll donor and the manganese catalytic site and is essential for enzymatic function. On the other hand, D forms a stable radical with no known role in oxygen evolution. D(.) and Z(.) give rise to similar, but not identical, room temperature electron paramagnetic resonance (EPR) signals, which can be distinguished by their decay kinetics. A third room temperature EPR signal has also been observed in site-directed mutants in which a nonredox active amino acid is substituted at the D or Z site. This four-line EPR signal has been shown to have a tyrosine origin by isotopic labeling (Boerner and Barry, 1994, J. Biol. Chem. 269:134-137), but such an EPR signal has never before been observed from a tyrosyl radical. The radical giving rise to this third unique signal has been named M+. Here we provide kinetic evidence that this signal arises from a third redox active tyrosine, distinct from tyrosine D and Z, in the photosystem II reaction center. Isotopic labeling and EPR spectroscopy provide evidence that M is a covalently modified tyrosine.     

EPR spectroscopy of free radical intermediates of antiarrhythmic-antihypoxic drug stobadine, a pyridoindole derivative

Mechanisms of antioxidant action of stobadine, a pyridoindole derivative with cardioprotective and antihypoxic properties, has been probed using EPR spectroscopy. Oxidation of stobadine by PbO2/tBuOOH in benzene results in the formation of nitroxide radical observable directly by EPR spectroscopy at room temperature, indicating conversion of indolic amino group to the corresponding nitroxide.     

Accumulation of magnetic nanoparticles in plants grown on soils of Apsheron peninsula

Magnetic nanoparticles in five plant species growing on the Apsheron peninsula have been detected by the EPR method. The EPR spectra of these nanoparticles proved to be similar to those of synthesized magnetic nanoparticles. The result demonstrated that plants are capable of absorbing magnetic nanoparticles from the soil. The accumulation of nanoparticles in plants is confirmed by the presence of a broad EPR signal whose maximum position of the low-field component changes from g = 2.38 and half-width of the signal of 32 mT at room temperature to g = 2.71 and 50–55 mT at 80 K. The intensity of the broad EPR signal for plants grown in radioactively contaminated areas (170‐220 μR/h) was substantially lower compared with plants grown on clean soil. The parameters of the broad EPR signal and its dependence on the temperature of recording were identical with those for synthetic magnetic nanoparticles. The photosynthetic activity and changes in the genome of irradiated plants by the analysis of PCR products were studied.     

Electron paramagnetic resonance in irradiated fingernails: variability of dose dependence and possibilities of initial dose assessment

The results of electron paramagnetic resonance (EPR) measurements in irradiated fingernails are presented. In total, 83 samples of different fingernails were studied. Five different groups of samples were selected based on the collection time of fingernail samples, their level of mechanical stress, and the number and size of clippings: (1) recently (<24 h) cut, irradiated and measured with EPR without any treatment of samples, and with rigorous control of size and number of clippings (stressed–fresh, controlled); (2) recently (<24 h) cut, irradiated and measured with EPR after application of a special treatment (10 min of water soaking, 5 min of drying time) to reduce the mechanical stress caused by cutting the samples, and with rigorous control of size and number of clippings (unstressed–fresh, controlled); (3) previously (>24 h) cut, stored at room temperature, additionally cut into small pieces immediately prior to study, irradiated and measured with EPR without any treatment of samples, and with rigorous control of size and number of clippings (stressed–old, controlled); (4) previously (>24 h) cut, stored at room temperature, additionally cut into small pieces immediately prior to the study, irradiated and measured with EPR after application of a special treatment to reduce mechanical stress caused by cut, and with rigorous control of size and number of clippings (unstressed–old, controlled); and (5) recently (<24 h) cut, irradiated and measured with EPR after application of a special treatment to reduce the mechanical stress caused by cut, and without rigorous control of size and number of clippings (unstressed–fresh, uncontrolled). Except for the fifth selected group, variability of the dose dependence inside all groups was found to be not statistically significant, although the variability among the different groups was significant. Comparison of the mean dose dependences obtained for each group allowed selection of key factors responsible for radiation sensitivity (dose response per unit of mass and dose) and the shape of dose dependence in fingernails. The major factor responsible for radiation sensitivity of fingernails was identified as their water content, which can affect radiation sensitivity up to 35%. The major factor responsible for the shape of the radiation sensitivity was identified as the mechanical stress. At a significant level of mechanical stress, the shape of the dose dependence is linear in the studied dose range (<20 Gy), and in lesser-stressed samples it is of an exponential growth including saturation, which depends on the degree of mechanical stress. In view of the findings, recommendations are discussed and presented for the appropriate protocol for EPR dose measurements in fingernails.     

Electron transfer in cyanobacterial photosystem I: I. Physiological and spectroscopic characterization of site-directed mutants in a putative electron transfer pathway from A0 through A1 to FX

The Photosystem I (PS I) reaction center contains two branches of nearly symmetric cofactors bound to the PsaA and PsaB heterodimer. From the x-ray crystal structure it is known that Trp697PsaA and Trp677PsaB are pi-stacked with the head group of the phylloquinones and are H-bonded to Ser692PsaA and Ser672PsaB, whereas Arg694PsaA and Arg674PsaB are involved in a H-bonded network of side groups that connects the binding environments of the phylloquinones and FX. The mutants W697FPsaA, W677FPsaB, S692CPsaA, S672CPsaB, R694APsaA, and R674APsaB were constructed and characterized. All mutants grew photoautotrophically, yet all showed diminished growth rates compared with the wild-type, especially at higher light intensities. EPR and electron nuclear double resonance (ENDOR) studies at both room temperature and in frozen solution showed that the PsaB mutants were virtually identical to the wild-type, whereas significant effects were observed in the PsaA mutants. Spin polarized transient EPR spectra of the P700+A1- radical pair show that none of the mutations causes a significant change in the orientation of the measured phylloquinone. Pulsed ENDOR spectra reveal that the W697FPsaA mutation leads to about a 5% increase in the hyperfine coupling of the methyl group on the phylloquinone ring, whereas the S692CPsaA mutation causes a similar decrease in this coupling. The changes in the methyl hyperfine coupling are also reflected in the transient EPR spectra of P700+A1- and the CW EPR spectra of photoaccumulated A1-. We conclude that: (i) the transient EPR spectra at room temperature are predominantly from radical pairs in the PsaA branch of cofactors; (ii) at low temperature the electron cycle involving P700 and A1 similarly occurs along the PsaA branch of cofactors; and (iii) mutation of amino acids in close contact with the PsaA side quinone leads to changes in the spin density distribution of the reduced quinone observed by EPR.     

X- and Q-band electron paramagnetic resonance of CO2- in hydroxyapatite single crystals

Both X- and Q-band electron paramagnetic resonance (EPR) research has been conducted using slightly carbonated hydroxyapatite (HAp) single crystals after exposure to ionizing radiation. Below a temperature of 90 K, O(-) and CO(2-) radicals were detected, whereas at room temperature only CO(2-) spectra could be observed. The O(-) ion has previously been investigated in high-purity HAp single crystals, whereas EPR spectra of CO(2-) in HAp single crystals have not been reported. Both paramagnetic defects exhibit EPR angular variations in planes containing the c axis of the crystal from which spin Hamiltonian parameters were derived. Arguments are given for the presence of two CO(2-) defects in the irradiated HAp single crystals.     

E.s.r. of free radicals in irradiated uracil-beta-D-arabinofuranoside.

Electron-spin-resonance measurements have been made on single crystals of uracil-beta-D-arabinofuranoside, which were irradiated by 4-0 MeV electrons at 77 K. At low temperatures, two radicals have been identified, one attributed to a hydrogen abstraction from 05' in the sugar moiety and the other to a radical anion located on the pyrimidine ring. The former is very unstable and seems to act as a precursor to other unidentified radical species stable at 77K. At room temperature, the main resonance is due to hydrogen addition to C5 and is probably produced by protonation of the anion. This same radical is also produced by X-irradiation at room temperature.     

Potentiometric studies on yeast complex III

Potentiometric measurements have been performed on Complex III from bakers' yeast. The midpoint potentials for the b and c cytochromes were measured using room-temperature MCD and liquid-helium temperature EPR. A value of 270 mV was obtained for cytochrome c1, regardless of temperature, while the midpoint potentials found for the two species of cytochrome b varied with temperatures, viz., 62 and -20 mV at room temperature (MCD) compared to 116 and -4 mV at about 10 K (EPR). The midpoint potential of the iron-sulfur center obtained by low-temperature EPR was 286 mV. An abrupt conformational change occurred immediately after this center was fully reduced resulting in a change in EPR line shape. The potentials of the two half-reactions of ubiquinone were measured by following the semiquinone radical signal at 110 K and 23 degrees C. Potentials of 176 and 51 mV were found at low temperature, while values of 200 and 110 mV were observed at room temperature. The midpoint potential of cytochrome c1 was found to be pH independent. The potentials of cytochrome b were also independent of pH when titrations were performed in deoxycholate buffers, while a variation of -30 mV per pH unit was observed for both cytochrome c species in taurocholate buffers. These two detergents also produced different MCD contributions of the two b cytochromes. A decrease in Em of greater than 300 mV was found in potentiometric measurements of cytochrome c1 at high ratios of dye to Complex III. Antimycin does not affect the redox potentials of cytochrome c1 but appears to induce a transition of the low-potential b heme to a high-potential species. This transition is mediated by ubiquinone.     

Radiation-induced hydroxyl addition to purine molecules: EPR and ENDOR study of hypoxanthine hydrochloride monohydrate single crystals

Three radical species were detected in an EPR/ENDOR study of X-irradiated hypoxanthine.HCl.H2O single crystals at room temperature: RI was identified as the product of net H addition to C8, RII was identified as the product of net H addition to C2, and RIII was identified as the product of OH addition to C8. The observed set of radicals was the same for room-temperature irradiation as for irradiation at 10 K followed by warming the crystals to room temperature; however, the C2 H-addition and C8 OH-addition radicals were not detectable after storage of the crystals for about 2 months at room temperature. Use of selectively deuterated crystals permitted unique assignment of the observed hyperfine couplings, and results of density functional theory calculations on each of the radical structures were consistent with the experimental results. Comparison of these experimental results with others from previous crystal-based systems and model system computations provides insight into the mechanisms by which the biologically important purine C8 hydroxyl addition products are formed. The evidence from solid systems supports the mechanism of net water addition to one-electron oxidized purine bases and demonstrates the importance of a facial approach between the reactants.     

Modifications in heme iron of free and vesicle bound cytochrome c by tert-butyl hydroperoxide: a magnetic circular dichroism and electron paramagnetic resonance investigation

To characterize changes to the heme and the influence of membrane lipids in the reaction of cytochrome c with peroxides, we studied the reaction of cytochrome c with tert-butyl hydroperoxide (tert-BuOOH) by magnetic circular dichroism (MCD) and direct electron paramagnetic resonance (EPR) in the presence and absence of different liposomes. Direct low-temperature (11 degrees K) EPR analysis of the cytochrome c heme iron on exposure to tert-BuOOH shows a gradual (180 s) conversion of the low-spin form to a high-spin Fe(III) species of rhombic symmetry (g = 4.3), with disappearance of a prior peroxyl radical signal (g(o) = 2.014). The conversion to high spin precedes Soret band bleaching, observable by UV/Vis spectroscopy and by magnetic circular dichroism (MCD) at room temperature, that indicates loss of iron coordination by the porphyrin ring. The presence of cardiolipin-containing liposomes delayed formation of the peroxyl radical and conversion to high-spin iron, while dicetylphosphate (DCP) liposomes accelerated these changes. Correspondingly, bleaching of cytochrome c by tert-BuOOH at room temperature was accelerated by several negatively charged liposome preparations, and inhibited by mitochondrial-mimetic phosphatidylcholinephosphatidylethanolaminecardiolipin (PCPECL) liposomes. Concomitant with bleaching, spin-trapping measurements with 5,5-dimethyl-1-pyroline-N-oxide showed that while the relative production of peroxyl, alkoxyl, and alkyl radicals was unaffected by DCP liposomes, PCPECL liposomes decreased the spin-trapped alkoxyl radical signal by 50%. The EPR results show that the primary initial change on exposure of cytochrome c to tert-BuOOH is a change to a high-spin Fe(III) species, and together with MCD measurements show that unsaturated cardiolipin-containing lipid membranes influence the interaction of tert-BuOOH with cytochrome c heme iron, to alter radical production and decrease damage to the cytochrome.     

Attempts at correlation of the radiolytic species of irradiated solid-state captopril studied by multi-frequency EPR and HPLC

The purpose of this study was to provide insight into the processes that occur after the irradiation of solid-state drugs. Electron paramagnetic resonance (EPR) experiments were performed at two different frequencies, X-band (about 9.5 GHz) and Q-band (about 34 GHz), to identify the radicals present in irradiated captopril. The results confirmed that an irradiated drug can trap several main radicals. Moreover, the radical composition varied as a function of the treatment. In addition, non-volatile final products were studied by liquid chromatography coupled to UV and to mass spectrometry (LC-MS). The variation of the radical composition did not influence the profile of the final products; this appears to indicate that, in the case of captopril, the trapped radicals observed by EPR are not the main precursors of the final products. Finally, high-performance liquid chromatography data appear to indicate that radiosterilization of captopril is feasible.