Sample solution is provided at 25 µL, 10mM.
|Cell experiment :|
Human normal lymphocytes and MEC-1 leukemia cells
Cells were irradiated with UV radiation (290 – 315 nm). Then add 20 μl of BMPO to 80μ l cell suspension (20 mM), then mixed in an eppendorf tube. After that, transferred the mixture to microhaematocrit capillaries and sealed with Paraffin. Then put it into EPR Pyrex tubes and inserted into the cavity of a Bruker EMX-131 X-band spectrometer.
The final concentration of BMPO is 100 mM. Samples were exposed to UVB radiation (290-315 nm) and to UVA radiation (315-400 nm) for 3 and 10 min at 47.7 and 159 mJ/cm2 and 53.7 mJ/cm2. The spectrometer was operated at ~9.5 GHz, while the spectra were recorded at room temperature.
BMPO combined with EPR spectroscopy provides detection and identification of cellular ROS. Which might contribute to the understanding of some fundamental mechanisms leading to oxidative stress or damage in biological systems.
. Tepe Çam S, et al. Tea extracts protect normal lymphocytes but not leukemia cells from UV radiation-induced ROS production: An EPR spin trap study. Int J Radiat Biol. 2015 Aug;91(8):673-80.
BMPO. is a cyclic nitrone spin trap agent, it is a water-soluble white solid which makes BMPO purification easier than other spin trap agents. Even after prolonged storage at 220°C, there was no artificial signal formation from aqueous solutions containing BMPO. (25–100 mM). BMPO. offers several advantages over the existing spin traps in the detection and characterization of thiyl radicals, hydroxyl radicals, and superoxide anions in biological systems. One of the perceived advantages of BMPO. is that the BMPO./•OOH adduct does not readily decay nonenzymatically to the BMPO./•OH adduct. 
The in vitro experiment took advantage of Rabbit aortic segments, which were treated with ADR and incubation with 0.1 M BMPO., a novel solid superoxide spin trap. Results showed that BMPO.-hydroxyl radical adduct was detected in supernatants upon 10-min incubation of aortic segment with incremental doses of ADR. Time-dependent changes in the ESR spectra of superoxide adducts of BMPO. was generated in a xanthine/xanthine oxidase incubation system. BMPO. superoxide adducts did not decay to the corresponding hydroxyl adducts. Results also indicate that the BMPO. superoxide adduct is persistent. The decay kinetics of BMPO./•OOH also demonstrate this feature. In this system, the ESR spectrum of the BMPO./•OOH adduct could be detected even up to 35 min. Although BMPO./•OOH is intrinsically more stable, it is likely to be enzymatically reduced to BMPO./•OH in biological systems.
. Zhao H, Joseph J, Zhang H, Karoui H, Kalyanaraman B. Synthesis and biochemical applications of a solid cyclic nitrone spin trap: a relatively superior trap for detecting superoxide anions and glutathiyl radicals. Free Radic Biol Med. 2001 Sep 1;31(5):599-606.
. Duquaine D, Hirsch GA, Chakrabarti A, Han Z, Kehrer C, Brook R, Joseph J, Schott A, Kalyanaraman B, Vasquez-Vivar J, Rajagopalan S. Rapid-onset endothelial dysfunction with adriamycin: evidence for a dysfunctional nitric oxide synthase. Vasc Med. 2003 May;8(2):101-7.
|Chemical Name||3,4-dihydro-2-methyl-1,1-dimethylethyl ester-2H-pyrrole-2-carboxylic acid-1-oxide|
|Solubility||33mg/mL in ethanol, 25mg/mL in DMSO or in DMF, 10mg/mL in water||Storage||Store at -20°C|
|General tips||For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months.|
|Shipping Condition||Evaluation sample solution : ship with blue ice
All other available size: ship with RT , or blue ice upon request
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BMPO-OOH Spin-Adduct as a Model for Study of Decomposition of Organic Hydroperoxides and the Effects of Sulfide/Selenite Derivatives. An EPR Spin-Trapping Approach
Antioxidants (Basel)2020 Sep 26;9(10):918.PMID: 32993108DOI: 10.3390/antiox9100918
Lipid hydroperoxides play an important role in various pathophysiological processes. Therefore, a simple model for organic hydroperoxides could be helpful to monitor the biologic effects of endogenous and exogenous compounds. The electron paramagnetic resonance (EPR) spin-trapping technique is a useful method to study superoxide (O2•-) and hydroxyl radicals. The aim of our work was to use EPR with the spin trap 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO), which, by trapping O2•- produces relatively stable •BMPO-OOH spin-adduct, a valuable model for organic hydroperoxides. We used this experimental setup to investigate the effects of selected sulfur/selenium compounds on •BMPO-OOH and to evaluate the antioxidant potential of these compounds. Second, using the simulation of time-dependent individual BMPO adducts in the experimental EPR spectra, the ratio of •BMPO-OH/•BMPO-OOH-which is proportional to the transformation/decomposition of •BMPO-OOH-was evaluated. The order of potency of the studied compounds to alter •BMPO-OOH concentration estimated from the time-dependent •BMPO-OH/•BMPO-OOH ratio was as follows: Na2S4 > Na2S4/SeO32- > H2S/SeO32- > Na2S2 ~Na2S2/SeO32- ~H2S > SeO32- ~SeO42- ~control. In conclusion, the presented approach of the EPR measurement of the time-dependent ratio of •BMPO-OH/•BMPO-OOH could be useful to study the impact of compounds to influence the transformation of •BMPO-OOH.
EPR Study of KO 2 as a Source of Superoxide and • BMPO-OH/OOH Radical That Cleaves Plasmid DNA and Detects Radical Interaction with H 2 S and Se-Derivatives
Antioxidants (Basel)2021 Aug 13;10(8):1286.PMID: 34439533DOI: 10.3390/antiox10081286
Superoxide radical anion (O2•-) and its derivatives regulate numerous physiological and pathological processes, which are extensively studied. The aim of our work was to utilize KO2 as a source of O2•- and the electron paramagnetic resonance (EPR) spin trapping 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) technique for the preparation of •BMPO-OOH and/or •BMPO-OH radicals in water solution without DMSO. The method distinguishes the interactions of various compounds with •BMPO-OOH and/or •BMPO-OH radicals over time. Here, we show that the addition of a buffered BMPO-HCl mixture to powdered KO2 formed relatively stable •BMPO-OOH and •BMPO-OH radicals and H2O2, where the •BMPO-OOH/OH ratio depended on the pH. At a final pH of ~6.5-8.0, the concentration of •BMPO-OOH radicals was ≥20 times higher than that of •BMPO-OH, whereas at pH 9.0-10.0, the •BMPO-OH radicals prevailed. The •BMPO-OOH/OH radicals effectively cleaved the plasmid DNA. H2S decreased the concentration of •BMPO-OOH/OH radicals, whereas the selenium derivatives 1-methyl-4-(3-(phenylselanyl) propyl) piperazine and 1-methyl-4-(4-(phenylselanyl) butyl) piperazine increased the proportion of •BMPO-OH over the •BMPO-OOH radicals. In conclusion, the presented approach of using KO2 as a source of O2•-/H2O2 and EPR spin trap BMPO for the preparation of •BMPO-OOH/OH radicals in a physiological solution could be useful to study the biological effects of radicals and their interactions with compounds.
Impact of SOD-Mimetic Manganoporphyrins on Spin Trapping of Superoxide and Related Artifacts
Appl Magn Reson2011 Feb;40(1):125-134.PMID: 23853422DOI: 10.1007/s00723-010-0188-y
The superoxide dismutase (SOD)-mimetic effectiveness of [meso-tetrakis (R)porphyrinato]manganese with R = 1,3-di-N-ethylimidazolium-2-yl (Mn-TDEIP), 1,3-di-N-methylimidazolium-2-yl (Mn-TDMIP), 1,3-di-N-propylimidazolium-2-yl (Mn-TDPIP), N-ethyl-2-pyridyl (Mn-T2EPyP), 4-sulphonatophenyl (Mn-TSP), 1-methyl-4-pyridyl (Mn-T4PyP), 4-carboxyphenyl (Mn-TBAP), and β-octabromomeso-tetrakis(4-carboxyphenyl porphyrinato)manganese (MnBr8TBAP) was compared with Cu, Zn SOD. Superoxide generated by reaction of xanthine oxidase with hypoxanthine was trapped with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), forming BMPO-OOH, which was monitored by electron paramagnetic resonance. Manganoporphyrins with redox potentials ranging from -0.190 to 0.346 V relative to the standard hydrogen electrode were selected for this study. With 0.1 µM manganoporphyrins and 20 mM BMPO, the effectiveness of the manganoporphyrins in inhibiting formation of BMPO-OOH increases in the order Mn-TSP < Mn-TBAP < MnBr8TBAP < Mn-T4PyP < Mn-T2EPyP < Mn-TDEIP ~ Mn-TDMIP ~ Mn-TDPIP ~ Cu, Zn SOD. However, at higher concentrations of manganoporphyrin and BMPO, a BMPO-OH signal was observed. The formation of BMPO-OH was not inhibited by catalase or dimethylsulfoxide, which demonstrated that it was not produced from hydroxyl radical. The artifactual formation of BMPO-OH is attributed to oxidation of the water adduct of BMPO by the manganoporphyrins or decomposition of BMPO-OOH. Although spin trapping is an effective method for evaluating SOD-mimetic efficacy, caution must be exercised to ensure that artifact signals are not interpreted improperly.
Use of rapid-scan EPR to improve detection sensitivity for spin-trapped radicals
Biophys J2013 Jul 16;105(2):338-42.PMID: 23870255DOI: 10.1016/j.bpj.2013.06.005
The short lifetime of superoxide and the low rates of formation expected in vivo make detection by standard continuous wave (CW) electron paramagnetic resonance (EPR) challenging. The new rapid-scan EPR method offers improved sensitivity for these types of samples. In rapid-scan EPR, the magnetic field is scanned through resonance in a time that is short relative to electron spin relaxation times, and data are processed to obtain the absorption spectrum. To validate the application of rapid-scan EPR to spin trapping, superoxide was generated by the reaction of xanthine oxidase and hypoxanthine with rates of 0.1-6.0 μM/min and trapped with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO). Spin trapping with BMPO to form the BMPO-OOH adduct converts the very short-lived superoxide radical into a more stable spin adduct. There is good agreement between the hyperfine splitting parameters obtained for BMPO-OOH by CW and rapid-scan EPR. For the same signal acquisition time, the signal/noise ratio is >40 times higher for rapid-scan than for CW EPR. Rapid-scan EPR can detect superoxide produced by Enterococcus faecalis at rates that are too low for detection by CW EPR.
Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence
Proc Natl Acad Sci U S A2005 Apr 19;102(16):5727-32.PMID: 15824309DOI: 10.1073/pnas.0501719102
Here we report the structural characterization of the product formed from the reaction between hydroethidine (HE) and superoxide (O(2)(.-)). By using mass spectral and NMR techniques, the chemical structure of this product was determined as 2-hydroxyethidium (2-OH-E(+)). By using an authentic standard, we developed an HPLC approach to detect and quantitate the reaction product of HE and O(2)(.-) formed in bovine aortic endothelial cells after treatment with menadione or antimycin A to induce intracellular reactive oxygen species. Concomitantly, we used a spin trap, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), to detect and identify the structure of reactive oxygen species formed. BMPO trapped the O(2)(.-) that formed extracellularly and was detected as the BMPO-OH adduct during use of the EPR technique. BMPO, being cell-permeable, inhibited the intracellular formation of 2-OH-E(+). However, the intracellular BMPO spin adduct was not detected. The definitive characterization of the reaction product of O(2)(.-) with HE described here forms the basis of an unambiguous assay for intracellular detection and quantitation of O(2)(.-). Analysis of the fluorescence characteristics of ethidium (E(+)) and 2-OH-E(+) strongly suggests that the currently available fluorescence methodology is not suitable for quantitating intracellular O(2)(.-). We conclude that the HPLC/fluorescence assay using HE as a probe is more suitable [corrected] for detecting intracellular O(2)(.-).
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