Electron Paramagnetic Resonance Tube Research Articles

Simple and effective in situ sample illumination for electron paramagnetic resonance.

Illumination into an electron paramagnetic resonance (EPR) spectrometer is commonly carried out through the optical window, perpendicular to the sample and magnetic field. Here we show that significant improvements can be obtained by using the walls of the EPR tube as a light guide, with the light scattered only around the sample-containing area.

Rapid Freeze-Quench EPR Spectroscopy: Improved Collection of Frozen Particles.

Rapid freeze-quench (RFQ) in combination with electron paramagnetic resonance (EPR) spectroscopy at X-band is a proven technique to trap and characterize paramagnetic intermediates of biochemical reactions. Preparation of suitable samples is still cumbersome, despite many attempts to remedy this problem, and limits the wide applicability of RFQ EPR. We present a method, which improves the collection of freeze-quench particles from isopentane and their packing in an EPR tube. The method is based on sucking the particle suspension into an EPR tube with a filter at the bottom. This procedure results in a significant reduction of the required volume of reactants, which allows the economical use of valuable reactants such as proteins. The approach also enables the successful collection of smaller frozen particles, which are generated at higher flow rates. The method provides for a reproducible, efficient and fast collection of the freeze-quench particles and can be easily adapted to RFQ EPR at higher microwave frequencies than X-band.

Design of a Simple Cryogenic System for Ultraviolet–Visible Absorption Spectroscopy with a Back-Reflectance Fiber Optic Probe

We report a convenient and inexpensive technique for the rapid acquisition of absorption spectra from small samples at cryogenic temperatures using a home built cryostat with novel collection optics. A cylindrical copper block was constructed with a coaxial bore to hold a 4.00 mm diameter electron paramagnetic resonance (EPR) tube and mounted on a copper feed in thermal contact with liquid nitrogen. A 6.35 mm diameter hole was bored into the side of the cylinder so a fiber optic cable bundle could be positioned orthogonally to the EPR tube. The light passing through the sample is reflected off of the opposing surfaces of the EPR tube and surrounding copper, back through the sample. The emergent light is then collected using the fiber optic bundle and analyzed using a dispersive spectrometer. Absorption spectra for KMnO4 were measured between 400 and 700 nm. Absorption intensity at 506, 525, 545, and 567 nm was found to be proportional to concentration, displaying Beer's law-like behavior. The EPR tube had an internal diameter of 3.2 mm; the double pass of the probe beam through the sample affords a central path length of about 6.4 mm. Comparing these measurements with those recorded on a conventional tabletop spectrometer using a cuvette with a 10.00 mm path length, we consistently found a ratio between intensities of 0.58 rather than the anticipated 0.64. These 6% smaller values we attribute to the curvature of the EPR tube and transmission/reflection losses. This system is particularly well-suited to studying the kinetics and dynamics of chemical reactions at cryogenic temperatures. The rapid response (100 ms) and multiplex advantage provided the opportunity of recording simultaneous time courses at several wavelengths following initiation of a chemical reaction with a pulsed laser source.

Formation Mechanism of Cubic Mesoporous Carbon Monolith Synthesized by Evaporation-Induced Self-assembly

The formation mechanism of the cubic mesoporous carbon, FDU-16, synthesized by evaporation-induced self-assembly (EISA) was investigated at the molecular level by electron paramagnetic resonance (EPR) spectroscopic techniques. This material is synthesized using F127 pluronic block copolymer [poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) (PEO106-PPO70-PEO106)] as a structure-directing agent (template) and phenolic resol as a carbon precursor. Using two spin probes derived from pluronics with PEO and PPO chains of different lengths that are designed to sense different regions of the system, we followed the evaporation and thermopolymerization stages of the synthesis in situ. To make such studies possible, we have used a polyurethane foam support, placed in the EPR tube, which allows for the efficient solvent evaporation as required for EISA. We focused on the evolution of the dynamics of the template and its interactions with the resol during the reaction. We observed that during the evaporation stage the resol is distributed throughout the entire PEO blocks, all the way to the PPO–PEO interface, interacting with them via H-bonds, thus hindering the local motion of the PEO chains. At the end of this stage there is no polarity gradient along the PEO blocks, as found for traditional F127 micelles in water or during the synthesis of silica materials, and the mesostructure is not well-defined. A polarity and a resol gradient developed during the thermopolymerization stage where the polymerizing resol is driven out to the outer region of the PEO corona. This produces a corona of resin-pluronic composite and a resol-free PPO core with high mobility of the PEO segments close to the PPO–PEO interface and restricted mobility in the composite corona. During this stage the final structure sets in.

An adder–mixer for adding a few microliters of reagent into an electron paramagnetic resonance quartz tube

In electron paramagnetic resonance (EPR) experiments, it is very difficult to add a few microliters of reagent into the solution in an EPR tube and to mix it efficiently. This report explains how we could overcome this problem.

Protection of NADH‐linked Fe‐S clusters in cardiac mitochondria by ranolazine

Mitochondrial complex I (CI), composed of Fe‐S clusters numbered N1‐N5, is susceptible to damage by reactive oxygen species during cardiac ischemia reperfusion (IR). We used electron paramagnetic resonance (EPR) to assess how IR injury affects these Fe‐S clusters and if ranolazine (RAN), an antianginal drug, attenuates these effects. Guinea pig hearts were perfused with Krebs Ringer (KR) buffer and subjected to one of the following protocols: a) KR perfusion for 30 min, b) 30 min ischemia (I), c) 5 μM RAN for 1 min before ischemia (RAN+I), d) 30 min ischemia followed by 10 min reperfusion (IR) and e) 5 μM RAN for 1 min before 30 min ischemia and 10 min reperfusion (RAN+IR). Hearts were immediately ground in liquid N2 and the powder packed into EPR tubes. Changes in signal intensities at g=1.94, 1.86, and 1.89, assigned to N1b, N3, and N4, respectively, were recorded at 10°K (liquid He) at 9.6 GHz, 5 mW. Versus time control (100%), N1b signal decreased to 0% in the I hearts and was restored to 40%, 100%, and 70% in IR, RAN+I and RAN+IR hearts, respectively. N4 signal was 67% for I, 183% for RAN+I, 117% for IR, and 100% in RAN+IR hearts. N3 signal was 50% for both IR and RAN+IR hearts, and unaltered for I and RAN+I hearts (100%). These data suggest that RAN treatment before IR protects electron flux only through some Fe‐S centers. This finding may underlie a possible mechanism of RAN to attenuate electron flux in distinct Fe‐S clusters of CI.This work was supported in part by grants from NIH HL089514 and HL073246; VA 8204‐05P, and AHA 0855940G.

Molecular Details of Bax Activation, Oligomerization, and Membrane Insertion

Bax and Bid are pro-apoptotic members of the Bcl-2 protein family. Upon cleavage by caspase-8, Bid activates Bax. Activated Bax inserts into the mitochondrial outer membrane forming oligomers which lead to membrane poration, release of cytochrome c, and apoptosis. The detailed mechanism of Bax activation and the topology and composition of the oligomers are still under debate. Here molecular details of Bax activation and oligomerization were obtained by application of several biophysical techniques, including atomic force microscopy, cryoelectron microscopy, and particularly electron paramagnetic resonance (EPR) spectroscopy performed on spin-labeled Bax. Incubation with detergents, reconstitution, and Bid-triggered insertion into liposomes were found to be effective in inducing Bax oligomerization. Bid was shown to activate Bax independently of the stoichiometric ratio, suggesting that Bid has a catalytic function and that the interaction with Bax is transient. The formation of a stable dimerization interface involving two Bcl-2 homology 3 (BH3) domains was found to be the nucleation event for Bax homo-oligomerization. Based on intermolecular distance determined by EPR, a model of six adjacent Bax molecules in the oligomer is presented where the hydrophobic hairpins (helices alpha5 and alpha6) are equally spaced in the membrane and the two BH3 domains are in close vicinity in the dimer interface, separated by >5 nm from the next BH3 pairs.

Free Radical Generation and Electron Flux in Mitochondrial Fe-S Centers During Cardiac Injury; Changes with Mitochondrial Protective Drug Ranolazine

Some TCA cycle enzymes, like aconitase, are more susceptible to ischemia reperfusion (IR) injury. Ranolazine (RAN) is cardioprotective against IR injury. It is a late Na+ current blocker that may also limit lipid peroxidation and complex I activity. It is unknown if RAN alters the redox state of Fe-S clusters or free radical generation (FRG) to underlie its protection. Here we examined how IR injury affects FRG and Fe-S clusters of aconitase and succinate dehydrogenase, using electron paramagnetic resonance (EPR), and if RAN alters these effects. Guinea pig hearts (n=8) were isolated and perfused with Krebs Ringer buffer and exposed to: a) control, b) 30 min global ischemia, c) 10 μM RAN for 10 min just before ischemia, or d) ischemia and 10 min reperfusion. Hearts were immediately ground in liquid N2 and packed into EPR tubes. We examined changes in signal intensity in liquid He (10°K) of assigned g=2.016 (aconitase 3Fe-4S), g=1.93 (succinate dehydrogenase 2Fe-2S), g=2.006 (free radical), and g=6.0 (Fe group of cytochrome c). Versus time control (100%), the signal for aconitase Fe-S at the end of ischemia was 46%, suggesting oxidative damage; this was partially restored by 10 min reperfusion to 91% and after I+RAN treatment to 55% of control. Signal intensity for succinate dehydrogenase was unaltered by IR or RAN+IR. The presumptive ubisemiquinone radical signal increased 19% after ischemia, suggesting increased FRG, but only by 4% at 10 min reperfusion. I+RAN treatment decreased the signal by 19%. The signal for cytochrome c (g=6.0) increased 730% after IR, but was only 81% after I+RAN. These data suggest that RAN treatment partially restores electron flow through some Fe-S centers and reduces FRG, which may partially underlie its cardioprotective effects.

Demonstration That CobG, the Monooxygenase Associated with the Ring Contraction Process of the Aerobic Cobalamin (Vitamin B12) Biosynthetic Pathway, Contains an Fe-S Center and a Mononuclear Non-heme Iron Center

The ring contraction process that occurs during cobalamin (vitamin B(12)) biosynthesis is mediated via the action of two enzymes, CobG and CobJ. The first of these generates a tertiary alcohol at the C-20 position of precorrin-3A by functioning as a monooxygenase, a reaction that also forms a gamma lactone with the acetic acid side chain on ring A. The product, precorrin-3B, is then acted upon by CobJ, which methylates at the C-17 position and promotes ring contraction of the macrocycle by catalyzing a masked pinacol rearrangement. Here, we report the characterization of CobG enzymes from Pseudomonas denitrificans and Brucella melitensis. We show that both contain a [4Fe-4S] center as well as a mononuclear non-heme iron. Although both enzymes are active in vivo, the P. denitrificans enzyme was found to be inactive in vitro. Further analysis of this enzyme revealed that the mononuclear non-heme iron was not reducible, and it was concluded that it is rapidly inactivated once it is released from the bacterial cell. In contrast, the B. melitensis enzyme was found to be fully active in vitro and the mononuclear non-heme iron was reducible by dithionite. The reduced mononuclear non-heme was able to react with the oxygen analogue NO, but only in the presence of the substrate precorrin-3A. The cysteine residues responsible for binding the Fe-S center were identified by site-directed mutagenesis. A mechanism for CobG is presented.

Electron paramagnetic resonance and Mössbauer spectroscopy of intact mitochondria from respiring Saccharomyces cerevisiae

Mitochondria from respiring cells were isolated under anaerobic conditions. Microscopic images were largely devoid of contaminants, and samples consumed O(2) in an NADH-dependent manner. Protein and metal concentrations of packed mitochondria were determined, as was the percentage of external void volume. Samples were similarly packed into electron paramagnetic resonance tubes, either in the as-isolated state or after exposure to various reagents. Analyses revealed two signals originating from species that could be removed by chelation, including rhombic Fe(3+) (g = 4.3) and aqueous Mn(2+) ions (g = 2.00 with Mn-based hyperfine). Three S = 5/2 signals from Fe(3+) hemes were observed, probably arising from cytochrome c peroxidase and the a(3):Cu(b) site of cytochrome c oxidase. Three Fe/S-based signals were observed, with averaged g values of 1.94, 1.90 and 2.01. These probably arise, respectively, from the [Fe(2)S(2)](+) cluster of succinate dehydrogenase, the [Fe(2)S(2)](+) cluster of the Rieske protein of cytochrome bc (1), and the [Fe(3)S(4)](+) cluster of aconitase, homoaconitase or succinate dehydrogenase. Also observed was a low-intensity isotropic g = 2.00 signal arising from organic-based radicals, and a broad signal with g (ave) = 2.02. Mössbauer spectra of intact mitochondria were dominated by signals from Fe(4)S(4) clusters (60-85% of Fe). The major feature in as-isolated samples, and in samples treated with ethylenebis(oxyethylenenitrilo)tetraacetic acid, dithionite or O(2), was a quadrupole doublet with DeltaE (Q) = 1.15 mm/s and delta = 0.45 mm/s, assigned to [Fe(4)S(4)](2+) clusters. Substantial high-spin non-heme Fe(2+) (up to 20%) and Fe(3+) (up to 15%) species were observed. The distribution of Fe was qualitatively similar to that suggested by the mitochondrial proteome.

Concerted Nitric Oxide Formation and Release from the Simultaneous Reactions of Nitrite with Deoxy- and Oxyhemoglobin

Recent studies reveal a novel role for hemoglobin as an allosterically regulated nitrite reductase that may mediate nitric oxide (NO)-dependent signaling along the physiological oxygen gradient. Nitrite reacts with deoxyhemoglobin in an allosteric reaction that generates NO and oxidizes deoxyhemoglobin to methemoglobin. NO then reacts at a nearly diffusion-limited rate with deoxyhemoglobin to form iron-nitrosyl-hemoglobin, which to date has been considered a highly stable adduct and, thus, not a source of bioavailable NO. However, under physiological conditions of partial oxygen saturation, nitrite will also react with oxyhemoglobin, and although this complex autocatalytic reaction has been studied for a century, the interaction of the oxy- and deoxy-reactions and the effects on NO disposition have never been explored. We have now characterized the kinetics of hemoglobin oxidation and NO generation at a range of oxygen partial pressures and found that the deoxy-reaction runs in parallel with and partially inhibits the oxy-reaction. In fact, intermediates in the oxy-reaction oxidize the heme iron of iron-nitrosyl-hemoglobin, a product of the deoxy-reaction, which releases NO from the iron-nitrosyl. This oxidative denitrosylation is particularly striking during cycles of hemoglobin deoxygenation and oxygenation in the presence of nitrite. These chemistries may contribute to the oxygen-dependent disposition of nitrite in red cells by limiting oxidative inactivation of nitrite by oxyhemoglobin, promoting nitrite reduction to NO by deoxyhemoglobin, and releasing free NO from iron-nitrosyl-hemoglobin.

Resistance to Nitric Oxide-induced Necrosis in Heme Oxygenase-1 Overexpressing Pulmonary Epithelial Cells Associated with Decreased Lipid Peroxidation

Increased expression of heme oxygenase-1 (HO-1) increases NO resistance in several cell types, although the biochemical mechanism for this protection is unknown. To address this issue, we have measured different molecular markers of nitrosative stress in three stably transfected cell lines derived from the human lung epithelial line A549: two lines that overexpress rat HO-1 (L1 and A4), and a control line with the empty vector (Neo). Compared with the control Neo cells, L1 and A4 cells had, respectively, 5.8- and 3.8-fold greater HO activity accompanied by increased resistance to NO-induced necrosis. Compared with the Neo control, the HO-1-overexpressing cells also showed significantly less lipid peroxide formation and decreased perturbation of transition metal oxidation and coordination states following a cytotoxic NO exposure. These effects were blocked by the HO-1 inhibitors Zn- and Sn-protoporphyrin IX. In contrast, HO-1 overexpression did not significantly affect total reactive oxygen or nitrogen species, the levels of the nucleobase deamination products in DNA (xanthine, inosine, and uracil) following NO exposure, or NO-induced protein nitration. While increased HO-1 activity prevented NO-induced fluctuations in transition metal homeostasis, addition of an iron chelator decreased NO toxicity only slightly. Our results indicate that lipid peroxidation is a significant cause of NO-induced necrosis in human lung epithelial cells, and that the increased NO survival of L1 cells is due at least in part to decreased lipid peroxidation mediated by HO-1-generated biliverdin or bilirubin.

Measuring “free” iron levels in Caenorhabditis elegans using low-temperature Fe(III) electron paramagnetic resonance spectroscopy

Oxidative stress, caused by free radicals within the body, has been associated with the process of aging and many human diseases. Because free radicals, in particular superoxide, are difficult to measure, an alternative indirect method for measuring oxidative stress levels has been used successfully in Escherichia coli and yeast. This method is based on a proposed connection between elevated superoxide levels and release of iron from solvent-exposed [4Fe–4S] enzyme clusters that eventually leads to an increase in hydroxyl radical production. In past studies using bacteria and yeast, a positive correlation was found between superoxide production or oxidative stress due to superoxide within the organism and electron paramagnetic resonance (EPR) detectable “free” iron levels. In the current study, we have developed a reliable and efficient method for measuring “free” iron levels in Caenorhabditis elegans using low-temperature Fe(III) EPR at g = 4.3. This method uses synchronized worm cultures grown on plates that are homogenized and treated with desferrioxamine, an Fe(III) chelator, prior to packing the EPR tube. Homogenization was found not to alter “free” iron levels, whereas desferrioxamine treatment significantly raised these levels, indicating the presence of both Fe(II) and Fe(III) in the “free” iron pool. The correlation between free radical levels and the observed “free” iron levels was examined by using heat stress and paraquat treatment. The intensity of the Fe(III) EPR signal, and thus the concentration of the “free” iron pool, varied with the treatments that altered radical levels without changing the total iron levels. This study provides the groundwork needed to uncover the correlation among oxidative stress, “free” iron levels, and longevity in C. elegans.

Investigation of cu(I)-dependent 2,4,5-Trihydroxyphenylalanine Quinone Biogenesis in Hansenula polymorpha Amine Oxidase

Copper, a mediator of redox chemistries in biology, is often found in enzymes that bind and reduce dioxygen. Among these, the copper amine oxidases catalyze the oxidative deamination of primary amines utilizing a type(II) copper center and 2,4,5-trihydroxyphenylalanine quinone (TPQ), a covalent cofactor derived from the post-translational modification of an active site tyrosine. Previous studies established the dependence of TPQ biogenesis on Cu(II); however, the dependence of cofactor formation on the biologically relevant Cu(I) ion has remained untested. In this study, we demonstrate that the apoform of the Hansenula polymorpha amine oxidase readily binds Cu(I) under anaerobic conditions and produces the quinone cofactor at a rate of 0.28 h(-1) upon subsequent aeration to yield a mature enzyme with kinetic properties identical to the protein product of the Cu(II)-dependent reaction. Because of the change in magnetic properties associated with the oxidation of copper, electron paramagnetic resonance spectroscopy was employed to investigate the nature of the rate-limiting step of Cu(I)-dependent cofactor biogenesis. Upon aeration of the unprocessed enzyme prebound with Cu(I), an axial Cu(II) electron paramagnetic resonance signal was found to appear at a rate equivalent to that for the cofactor. These data provide strong evidence for a rate-limiting release of superoxide from a Cu(II)(O(2)(.)) complex as a prerequisite for the activation of the precursor tyrosine and its transformation for TPQ. As copper is trafficked to intracellular protein targets in the reduced, Cu(I) state, these studies offer possible clues as to the physiological significance of the acquisition of Cu(I) by nascent H. polymorpha amine oxidase.

Effects of thermal annealing on the radiation produced electron paramagnetic resonance spectra of bovine and equine tooth enamel: Fossil and modern

The concentration of stable radiation-induced paramagnetic states in fossil teeth can be used as a measure of sample age. Temperature excursions >100 °C, however, can cause the paramagnetic state clock to differ from the actual postmortem time. We have heated irradiated enamel from both fossilized bovid and modern equine (MEQ) teeth for 30 min in 50 °C increments from 100 to 300 °C, measuring the electron paramagnetic resonance (EPR) spectrum after each anneal, to investigate such effects. Samples were irradiated again after the last anneal, with doses of 300–1200 Gy from Co60 photons, and measured. Two unirradiated MEQ samples were also annealed for 30 min at 300 °C, one in an evacuated EPR tube and the other in a tube open to the atmosphere, and subsequently irradiated. The data showed that hyperfine components attributed to the alanine radical were not detected in the irradiated MEQ sample until after the anneals. The spectrum of the MEQ sample heated in air and then irradiated was similar to that of the heat treated fossil sample. We conclude that the hyperfine components are due to sample heating to temperatures/times >100 °C/30 min and that similarities between fossil and MEQ spectra after the 300 °C/30 min MEQ anneal are also due to sample heating. We conclude that the presence of the hyperfine components in spectra of fossil tooth enamel indicate that such thermal events occurred either at the time of death, or during the postmortem history.

Filling factor of a paramagnetic sample in a rectangular cavity: theory and application

A computational method is presented for calculating the filling factor of an electron paramagnetic resonance (EPR) tube in a rectangular TE 102 cavity. The algorithm employs the conventional finite element method. In addition to the filling factor, the algorithm allows to calculate the quality factor and the reflection coefficient of the loaded cavity. This method allows calculating very accurately the EPR signal intensities from which the spin concentration of paramagnetic samples can be determined. A comparison between the predicted EPR signal intensities to several experimental results was found to be satisfactory. The method also allows optimizing the EPR tube dimensions and its glass quality to improve measurement sensitivity.

Quantitative assessment of free-radical generation during ischemia and reperfusion in the isolated rabbit heart

Several investigators have indirectly implicated oxygen free radicals in reperfusion injury following ischemia in the isolated rabbit heart. A quantitative assessment of free-radical production during ischemia and reperfusion was made using electron paramagnetic resonance (EPR) spectroscopy. Serial frozen (77 degrees K) tissue biopsies of the left ventricular wall in isolated rabbit hearts were performed during perfusion, ischemia, and reperfusion. These were pulverized into a fine powder that was filled into EPR tubes. EPR spectra of the tissue were recorded at 77 degrees K. Three predominant signals were seen on EPR analysis: signal A, g = 2.028; signal B, g = 2.005, and signal C, g = 1.940. All three signals were present during the perfusion period and decreased in size during the period of ischemia. After reperfusion, all three signals doubled in size. This study lends direct support to the theory that free radicals are generated during myocardial reperfusion and may result in reperfusion injury.

Applications of pulsed EPR spectroscopy to the study of mitochondrial iron-sulphur clusters and Escherichia coli fumarate reductase

Pulsed methods of electron paramagnetic resonance (EPR) spectroscopy open up new possibilities for studying the structure and properties of paramagnetic centres in biological systems 11 I . The electron spin-echo envelope modulation (ESEEM) technique offers improved resolution of hyperfine interactions between an EPR-detectable species and nuclei of ligand atoms. I t has been shown to be particularly sensitive to weak interactions with quadrupolar nuclei, and is therefore suited for the detection of nuclei, such as 14N, in the vicinity of a paramagnetic centre 121. The spin-echo method allows the accurate determination of time-domain properties, such as the hptn-lattice (T,) and the spin-spin (Tz) relaxation times. Spinspin interactions between the various mitochondrial centres may be observed, and the distances between the centres can then, in principle, be determined. These pulsed methods have so far mainly been applied to the study of transition metal complexes and purified metalloproteins, such as ferredoxins. Our experiments were undertaken to assess the feasibility of pulsed EPR measurements on the more complex proteins of unpurified electron-transferring membranes. The spectrometer used was a Bruker ESP380, fitted with a liquid helium flow cryostat (Oxford Instruments, CS935). Preliminary experiments were done on the, previously studied [3], soluble, 2-subunit form of fumarate reductase (FRD) from Escherichia coli , a membrane protein analogous to the mitochondrial succinate dehydrogenase (SDH). In mitochondria the major EPR-detectable species at low temperatures are the iron-sulphur clusters 141 o f Complex I (Centres Nl-N4), Complex I1 (Centres S 1 S 3 ) and Complex 111 (the Rieske iron-sulphur protein). Submitochondrial particles (SMP) were prepared by sonication of bovine heart mitochondria. The high protein concentration required for pulsed measurements was achieved by centrifuging the SMP suspension inside quartz EPR tubes. Two samples were prepared, and NADH was then added to one tube. Continuous-wave (c.w) EPR spectra were recorded at ISK, with a Bruker ESP300 spectrometer, and verified that each tube contained the expected spectral species; that is, the sample as prepared (oxidized) gave a signal at g=2.01, typical of SDH (a subunit of Complex 11) centre S3, and the NADHreduced sample gave rise to various signals typical of centres N 1 , N2, N3 and N4 of Complex 1. Note that centres S1 and S2 of SDH are probably superimposed on the g=1.94 signal from centre N 1. ESEEM data were collected at 4K (by the Bruker Pulsespel

Spin Trapping of Ibuprofen Radicals: Evidence That Ibuprofen is a Hydroxyl Radical Scavenger

The purpose of this study was to use electron paramagnetic resonance (EPR) spectroscopy to determine if ibuprofen, [2-(4-isobutylphenyl) propanoic acid], a potent nonsterodial anti-inflammatory agent, could modify hydroxyl radicals generation in vitro. Ibuprofen (IBU; 0.1-50 mM) in water or water alone was added to EPR tubes containing ferrous sulfate (0.5-2.0 mM), and either 5,5-dimethyl-1-pyrroline-N-oxide (DMPO; 40 mM) or alpha-phenyl N-tert-butyl nitrone (PBN; 48 mM). Hydrogen peroxide (1 mM) was added to inititate the Fenton reaction, and the systems were then analyzed by EPR spectroscopy to determine the type and relative quantity of free radical(s) produced. IBU caused a dose-dependent decrease of signal intensity of the hydroxyl radical adduct of DMPO (DMPO-OH) which is an indication that IBU either scavenges the hydroxyl radical and/or chelates iron. In addition, other radicals (presumably IBU radicals) produced in these systems were trapped by both DMPO (aN = 16.1 G, aH beta = 24.0 G) and PBN (aN = 15.7 G, aH beta = 4.4 G and aN = 17.0 G, aH beta = 2.1 G). The signal height of these IBU radicals increased in systems containing ferrous sulfate (1 mM), hydrogen peroxide (1 mM), PBN (48 mM), and increasing IBU concentrations. Therefore, we conclude that IBU scavenges the hydroxyl radical. If IBU chelated iron, then less hyroxyl radicals would be generated, less IBU radical formed and the signal height of IBU radicals trapped by PBN would have decreased.(ABSTRACT TRUNCATED AT 250 WORDS)