Discriminative EPR detection of NO and HNO by encapsulated nitronyl nitroxides

Abstract

Nitric oxide, ^?NO, is one of the most important molecules in the biochemistry of living organisms. By contrast, nitroxyl, NO^?, one-electron reduced analog of ^?NO which exists at physiological conditions in its protonated form, HNO, has been relatively overlooked. Recent data show that HNO might be produced endogenously and display unique biological effects. However, there is a lack of specific and quantitative methods of detection of endogenous HNO production. Here we present a new method for discriminative ^?NO and HNO detection by nitronyl nitroxides (NNs) using electron paramagnetic resonance (EPR). It was found that NNs react with ^?NO and HNO with similar rate constants of about 10⁴ M^{? 1}s^{? 1} but yield different products: imino nitroxides and the hydroxylamine of imino nitroxides, correspondingly. An EPR approach for discriminative ^?NO and HNO detection using liposome-encapsulated NNs was developed. The membrane barrier of liposomes protects NNs against reduction in biological systems while is permeable to both analytes, ^?NO and HNO. The sensitivity of this approach for the detection of the rates of ^?NO/HNO generation is about 1 nM/s. The application of encapsulated NNs for real-time discriminative ^?NO/HNO detection might become a valuable tool in nitric oxide-related studies.     

Unique oxidation of imidazolidine nitroxides by potassium ferricyanide: Strategy for designing paramagnetic probes with enhanced sensitivity to oxidative stress

Abstract

Potassium ferricyanide (PF), routinely employed for the oxidation of sterically-hindered hydroxylamines to nitroxides, is considered to be chemically inert towards the latter. In the present study, we report on an unexpected oxidative fragmentation of the imidazolidine nitroxides containing hydrogen atom in the 4-position of the heterocycle (HIMD) by PF resulting in the loss of the EPR signal. The mechanistic EPR, spectrophotometric, electrochemical and HPLC–MS studies support the assumption that the HIMD fragmentation is facilitated by the proton abstraction from the 4-position of the oxoammonium cation formed as a result of the initial one-electron HIMD oxidation. Increase in steric hindrance around the radical fragment by introducing ethyl substituents decreased the rate of ascorbate-induced HIMD reduction by more than 20 times, but did not affect the rate of ferricyanide-induced HIMD oxidation. This preferential sensitivity of HIMDs to oxidative processes has been used to detect peroxyl radicals in the presence of high concentration of the reducing agent, ascorbate. HIMD-based EPR probes capable to discriminate oxidative and reductive processes might find application in biomedicine and related fields for monitoring the oxidative stress and reactive radical species in biological systems.     

Permeability of liposomes towards amino alcohol and amino acid nitroxides

Electron spin resonance (ESR) allowed the analysis of the encapsulation of amino alcohols and amino acids labeled with nitroxide groups into multilamellar liposomes. The stability and permeability of these phospholipid vesicles were studied in terms of time dependance and structure of these biomolecules.     

Bis(dialkylaminophosphino)ferrocenes: Reactivity and electrochemistry

New compounds containing the 1,1′-bis(dialkylaminophosphino)ferrocene ligands 1,1′-bis(dimethylaminophosphino)ferrocene (dmapf) and 1,1′-bis(diethylaminophosphino)ferrocene (deapf) were prepared and characterized by NMR. The oxidative electrochemistries of these compounds were examined in methylene chloride. The transition metal compounds M_nCl₂(P-P) (M = Pd or Pt, n = 1, P-P = dmapf or deapf; M = Au, n = 2, P-P = dmapf) displayed one-electron, Nernstian waves. The corresponding selenides were prepared and the oxidative electrochemistries, along with those of the previously prepared 1,1′-bis(alkylaminophosphine sulfide)ferrocene, were examined. The sulfides displayed one-electron, Nernstian oxidations. The selenides displayed electrochemically irreversible two-electron waves. The X-ray structures of the sulfides and selenides as well as that of the transition metal compounds [(AuCl)₂(dmapf)] and [PtCl₂(dmapf)] were determined.     

The complex between hydrogenase-maturation proteins HypC and HypD is an intermediate in the supply of cyanide to the active site iron of [NiFe]-hydrogenases

Carbamoylphosphate has been shown to be the educt for the synthesis of the CN ligands of the NiFe metal centre of hydrogenases from Escherichia coli. In the absence of carbamoylphosphate, cells accumulate a complex of two hydrogenase maturation proteins, namely HypC and HypD for the synthesis of hydrogenase 3. A procedure for the purification of wild-type HypD protein or of a biologically active derivative carrying the Strep-tagII((R)) at the N terminus has been developed. HypD is a monomeric protein possessing about 4 mol of iron per mol of protein. Electron paramagnetic resonance (EPR) and Mossbauer spectroscopy demonstrated that the iron is present as a diamagnetic [4Fe-4S](2+) cluster. The complex between HypC and HypD can be cross-linked by a number of thiol and primary amine-specific linkers. When HypD and HypC were overproduced side-by-side with HypE, the HypC-HypD complex contained substoichiometric amounts of HypE whose proportion in the complex could be augmented when HypF was also overproduced. HypE trapped in this complex could be carbamoylated by protein HypF and after dehydration transferred the cyano group to the HypC-HypD part of the complex. Free HypC and HypD were not cyanated by HypE-CN. An active HypC-HypD complex from anaerobic cells was inactivated by incubation with K(3)[Fe(CN)(6)] but not with K(4)[Fe(CN)(6)]. The results suggest the existence of a dynamic complex between the hydrogenase maturation proteins HypD, HypC, HypE and HypF, which is the site of ligand biosynthesis and attachment to the iron atom of the NiFe site in hydrogenase 3.     

Reactions of Nitric Oxide with Nitronyl Nitroxides and Oxygen: Prediction of Nitrite and Nitrate Formation by Kinetic Simulation

Nitric oxide reacts with nitronyl nitroxides (NNO) to form imino nitroxides (INO) and this transformation can be monitored using electron spin resonance spectroscopy. Recently, Akaike et al., reported that NNO such as 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO) and its derivatives (e.g., carboxy-PTIO) react with nitric oxide (·NO) in a 1:1 stoichiometry forming 2-phenyl-4,4,5,5-tetra-methylimidazoline-1-oxyl (PTI) or the respective product (e.g., carboxy-PTI) together with nitrite and nitrate (Akaike et al., Biochemistry 32, 827-832, 1993). In this paper, we reevaluate their results and show that the stoichiometry of the reaction between PTIO and ·NO is 0.63 ± 0.06:1.0. The reason for this discrepancy is due to an erroneous assumption by Akaike et al., that the stoichiometry for the reaction between ·NO and O₂ is 2:1 in aqueous solution. If the data reported by Akaike et al., were recalculated using a 4:1 stoichiometry established for the aqueous oxidation of ·NO, the reaction between ·NO and PTIO would give a stoichiometry of 0.5:1.0 in closer agreement with our data. We propose a mechanism for the reaction between PTIO and ·NO in aqueous solution. This mechanism predicts that the stoichiometry between carboxy-PTIO and ·NO is dependent on the rate of generation of ·NO and is 1:1 only at low rates of ·NO generation (i.e., 10^-13 M/s). However the stoichiometry approaches 0.5:1.0 at higher rates of ·NO production or when it is added as a bolus. The ratio between nitrite and nitrate also varies as a function of the rate of generation of ·NO. The model agrees with previous experimental observations that the aqueous oxidation of ·NO in air saturated solutions will exclusively form nitrite and predicts that ·NO will only generate substantial amounts of nitrate if it is released at a rate less than 10^-17 M/s. This may have important consequences in cellular systems where the concentration of ·NO is typically measured from nitrite production.     

Taniaphos and Walphos ligands: Oxidative electrochemistry and complexation. Synthesis, characterization, oxidative electrochemistry and X-ray structures of [(Taniaphos/Walphos)MCl2] (M = Pd or Pt)

The oxidative electrochemistry of chiral, bidentate ferrocenylphosphines, five Taniaphos and seven Walphos ligands, was studied in methylene chloride. In general, two waves of varying reversibility were observed. Complexes of the general type [(phosphine)MCl₂] (phosphine = Taniaphos or Walphos; M = Pd or Pt) were prepared and characterized by NMR. Upon coordination, the oxidative electrochemistry of the ligands was greatly simplified. The X-ray structures of a Taniaphos platinum complex as well as a palladium and a platinum complex with a Walphos ligand were determined.     

Synthesis and structure of the first sulfur-donor adduct of a diruthenium tetracarboxylate

The first sulfur-donor adduct of a diruthenium tetracarboxylate, [Ru₂(μ-O₂CCH₃)₄(THT)₂]PF₆ (1), where THT=tetrahydrothiophene, has been synthesized and characterized by X-ray diffraction, IR and UV-Vis spectroscopy, magnetic susceptibility and electrochemistry. Complex 1 displays a typical RuRu bond length of 2.285(4) Å but a very long RuS axial bond of 2.627(13) Å. The redox potential for the [Ru₂(μ-O₂CCH₃)₄(THT)₂]^+/0 couple is −322 mV (vs. Fc/Fc⁺) which is comparable to other moderately strong Lewis base adducts. Axial ligand binding is driven by σ-donation with little π-backdonation from the metal centers.     

Effects of different detachment procedures on viability,nitroxide reduction kinetics and plasma membrane heterogeneity of V‐79 cells

Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V‐79 cells in the plateau phase of growth (arrested in G₁). We have measured cell viability by a dye‐exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin‐probe MeFASL(10,3) (5‐doxylpalmitoyl‐methylester), which partitions mainly in cell membranes and the hydrophilic spin‐probe TEMPONE (4‐oxo‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin‐probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin‐treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V‐79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged.     

Resonance Raman scattering and optical absorption of adrenodoxin and selena-adrenodoxin

Raman spectra have been recorded for native and selenium substituted adrenodoxin in dilute solution. Adrenodoxin shows three bands at 397, 350 and 297 cm^?1, all polarized, which can be associated with the iron-sulfur core. Selenium substitution leaves the 350 cm^?1 band essentially unshifted, but the other two bands disappear and are replaced by new bands at 355 and 263 cm^?1. The 350 cm^?1 band is assigned to stretching of iron-sulfur (cysteine) bonds, while the 397 and 297 cm^?1 bands are associated with vibrations of the labile sulfur atoms. The iron-selenium charge transfer bands were observed at 438 and 480 nm for the oxidized form and at 580 nm for the reduced form. The reduced selena-adrenodoxin displayed absorption maxima at 4, 450 and 5, 550 cm^?1, which can be assigned to the d-d transitions of high-spin ferrous ion. From this data and the reported g-values of electron paramagnetic resonance signals, the spin-orbit coupling constants were calculated to be 170 and 210 cm^?1 for the respective d-d transitions.     

14N-ENDOR evidence for imidazole coordination in copper proteins

¹⁴N-ENDOR studies of simple nitrogen-coordinated copper(II) complexes in frozen aqueous solutions show that the nitrogen hyperfine constants, A_″ and A_⊥, of imidazole are much more isotropic ($\text{R =}\text{A}{}_{\mathrm{″}}\text{A}{}_{\mathrm{\perp }}\text{= 1.05}$) than those of the other biologically-related ligand nitrogens. From this result, combined with ¹⁴N-ENDOR results of some copper proteins containing imidazoles as ligands, it is concluded that R < 1.10 for nitrogen hyperfine constants can be employed as an empirical criterion for demonstration of the existence of imidazole coordination in copper proteins.     

Synthesis, spectral and electrochemical properties of cyclotriphosphazene appended with six metalloporphyrins

The metal derivatives (Co^II, Ni^II, Cu^II, Zn^II and Sn^IV) of hexaporphyrinato cyclotriphosphazene systems were prepared by treating the hexaporphyrin assembly on cyclotriphosphazene ring with the appropriate metal salt under standard metallation conditions. The complete metallation of all six porphyrin units in hexaporphyrin assembly required 10-12 h reflux as judged by the absorption spectroscopy. The metal derivatives were confirmed by molecular ion peak in mass spectra for all compounds. ³¹P and ¹H NMR spectra for Ni(II), Zn(II) and Sn(IV) derivatives and ESR spectra for Cu(II) derivative also confirmed the complete metallation of all six porphyrin units. The hexametalloporphyrin assemblies are freely soluble in common organic solvents. The spectral, electrochemical and fluorescence studies indicate that the six porphyrin units in assemblies interact very weakly and retain their individual characteristic features in the ground and excited states.     

Microperoxidase 8 adsorbed on a roughened silver electrode as a monomeric high-spin penta-coordinated species: characterization by SERR spectroscopy and electrochemistry

Microperoxidase 8 (MP8), a heme octapeptide obtained by hydrolytic digestion of cytochrome c, was adsorbed at the surface of a roughened silver electrode in order to provide a new supported biomimetic system for hemoproteins. A combination of two techniques was used to study its redox and coordination properties: electrochemistry and surface-enhanced resonance Raman (SERR) spectroscopy. This allowed us to show that MP8 could be adsorbed as a monolayer at the surface of the roughened silver electrode, where it could undergo a reversible electron transfer. Under those conditions, a redox potential of –0.4 V vs. SCE (–0.16 V vs. NHE) was measured for MP8, which was almost identical to that reported for N-acetyl-MP8 in aqueous solution. In addition, whereas MP8 appeared to aggregate in solution, and led to a mixture of high-spin penta-coordinated (5cHS) and low-spin hexa-coordinated (6cLS) iron(III) or iron(II) species, it was recovered almost exclusively as a monomeric high-spin penta-coordinated species at the surface of the electrode, both in the reduced and in the oxidized states. This then allowed a free coordination site on the iron, on the distal face of MP8 accessible to ligands. Accordingly, experiments performed in the presence of potassium cyanide demonstrated that MP8 adsorbed on a silver electrode could be ligated by a sixth CN^– ligand. Thus there is the possibility of binding several kinds of ligands such as O₂ or H₂O₂, which will open the way to biocatalysis of oxidation reactions at the surface of an electrode, or ligands such as drugs which will lead to the design of new biosensors for molecules of biological interest.     

The heme environment of mouse neuroglobin: histidine imidazole plane orientations obtained from solution NMR and EPR spectroscopy as compared with X-ray crystallography

The ¹H NMR chemical shifts of the heme methyl groups of the ferriheme complex of metneuroglobin (Du et al. in J. Am. Chem. Soc. 125:8080–8081, 2003) predict orientations of the axial histidine ligands (Shokhirev and Walker in J. Biol. Inorg. Chem. 3:581–594, 1998) that are not consistent with the X-ray data (Vallone et al. in Proteins Struct. Funct. Bioinf. 56:85–94, 2004), and the EPR spectrum (Vinck et al. in J. Am. Chem. Soc. 126:4516–4517, 2004) is only marginally consistent with these data. The reasons for these inconsistencies appear to be rooted in the high degree of aqueous solution exposure of the heme group and the fact that there are no strong hydrogen-bond acceptors for the histidine imidazole N–H protons provided by the protein. Similar inconsistencies may exist for other water-soluble heme proteins, and ¹H NMR spectroscopy provides a simple means to verify whether the solution structure of the heme center is the same as or different from that in the crystalline state.     

Interactions of Chloride and Formate at the Donor and the Acceptor Side of Photosystem II

Chloride is required for the maximum activity of the oxygen evolving complex (OEC) while formate inhibits the function of OEC. On the basis of the measurements of oxygen evolution rates and the S₂ state multiline EPR signal, an interaction between the action of chloride and formate at the donor side of PS II has been suggested. Moreover, the Fe₂+Q–A EPR signals were measured to investigate a common binding site of both these anions at the PS II acceptor side. Other monovalent anions like bromide, nitrate etc. could influence the effects of formate to a small extent at the donor side of PS II, but not significantly at the acceptor side of PS II. The results presented in this paper clearly suggest a competitive binding of formate and chloride at the PS II acceptor side.     

基于电子顺磁共振技术的谷胱甘肽检测方法

目的 谷胱甘肽（GSH）是细胞中巯基（―SH）含量最丰富的非蛋白质化合物,在提供还原当量、直接中和有毒反应物质以及维持细胞氧化还原平衡等方面发挥着至关重要的作用,因此,准确检测组织中的GSH含量具有重要意义。鉴于电子顺磁共振（EPR）技术在GSH检测方面的应用报道相对较少,本文提出了一种基于EPR技术的GSH检测方法。方法 首先,将ABTS（2,2\"-联氮-双-3-乙基苯并噻唑啉-6-磺酸）溶液与K2S2O8溶液混合并在避光条件下反应12~16 h制备ABTS?⁺溶液,借助UV-Vis对ABTS?⁺溶液进行浓度测定。然后,基于ABTS?⁺的EPR信号变化来检测GSH浓度,在此基础上探究最适的反应时间和温度,建立ABTS?⁺的EPR信号强度和GSH 浓度之间的标准方程。最后,采用计算得到的标准曲线对C57BL/6J 小鼠全血的GSH浓度进行定量分析,并与文献结果进行比较,验证方法的准确性。结果 实验结果显示,本方法对GSH的检测范围（50 nmol/L~15 μmol/L）分布超过两个数量级,检测限（LOD）低至0.50 nmol/L,检测到的小鼠全血中GSH含量为（10 660±706）nmol/g（每克血红蛋白所含GSH纳摩尔数）,与文献报道结果（（11 200±237）nmol/g）接近。此外我们还使用类似的方法进一步发展了对GSSG的检测方法。结论 本文提出了一种基于EPR技术,使用ABTS?⁺快速检测GSH的方法。得益于EPR技术的独特优势,相比于传统的比色法,本方法具有更高的检测灵敏度,更宽的线性范围。我们进一步拓展了其在血液样品中的检测应用,可以快速准确地检测全血中GSH的含量,为衡量组织中氧化还原平衡状态提供数据支持,具有重要意义。     

Halothane induced disorder of red cell membranes of subjects susceptible to malignant hyperthermia

Membrane fluidity of red blood cells drawn from malignant hyperthermic pigs and humans was studied using spin-probes and electron paramagnetic resonance technique. The order parameter and rotational correlation time were determined with 12-doxylstearate and 16-doxylstearate, respectively. It was found that halothane decreased both parameters, but that the decrease of these parameters in subjects susceptible to malignant hyperthermia was much greater than that in normal subjects. The differences were most pronounced at 3 mM halothane. A possibility of using blood for a non-invasive screening for malignant hyperthermia is discussed.     

Studies on Leaf Surface Lipid of Tobacco I. Changes in Leaf Surface Lipid and Duvatrienediol during Growth,Senescence and Curing of Tobacco Leaves

Duvatrienediol is a diterpene specifically occurred in tobacco plants and thought to be a precursor of tobacco aroma. Green tobacco leaves contained 0.2~1% of duvatrienediol per dry weight and it was corresponded to 30~60% of leaf surface lipid. Leaves on upper stalk position contained more of leaf surface lipid and duvatrienediol. In leaves on each stalk position, leaf surface lipid and duvatrienediol contents increased with leaf growth and decreased by over-maturation. Production of leaf surface lipid and duvatrienediol was affected by soil conditions or applied amount of nitrogen fertilizer. Both leaf surface lipid and duvatrienediol were decreased during curing of tobacco leaves, but the change in the latter was more drastic. Comparing to leaf surface lipid, changes in cytoplasmic lipid were less during growth and senescence of tobacco leaves.     

Cesium ions influence cultured cell behavior by modifying specific subcellular components: The role of membranes and of the cytoskeleton

The exposure of the epidermoid cell line A431 to different concentrations of CsCl was assessed using different methodological approaches. Two different effects were detected depending upon the concentration of the agent: at low concentrations, cell modification was represented mainly by a very pronounced cell flattening and an alteration of the cell-to-cell contacts, interpreted as an increase in cell adhesion. At higher concentrations, a clear pathogenic effect was observed that allowed the formulation of the hypothesis that specific mechanisms of toxicity at the subcellular level involving mitochondrial and cytoskeletal function can exist. In addition, membrane order parameters, as detected by electron paramagnetic resonance (EPR) spectroscopy, displayed a dose-dependent increase in membrane rigidity. Results reported here seem to suggest that cesium ions can enter the cell, modify plasma membrane integrity and alter some specific cytoplasmic components, e.g. the cytoskeleton. Considering that environmental contamination by cesium as a result of radioactive fallout is of major importance and that few data are available thus far on this matter, this study provides evidence for the possible mechanisms of action of the non-radioactive form of this ion in cells.Abbreviations     

Solubilisation of ferricytochrome c in methanol using a crown ether: absorption, circular dichroism and EPR spectral properties

Ferricytochrome c is normally insoluble in methanol, but its solution is facilitated by complexation with 18-crown-6. Absorption, circular dichroism and EPR spectroscopy indicate that the solubilised protein in MeOH exists in at least three conformational states, all different from the native state in neutral aqueous solution. In two states the haem iron (III) is low spin and in one state it is high spin, but it seems likely that all three forms are globular. The proportion in the high spin form increases at increasing crown ether concentration and on ageing the protein solution. The protein appears to return to its native conformation when it is restored to an aqueous environment.