Radical Electron Paramagnetic Resonance Signal Research Articles

Computational engineering of previously crystallized pyruvate formate-lyase activating enzyme reveals insights into SAM binding and reductive cleavage

Radical S-adenosyl-l-methionine (SAM) enzymes are ubiquitous in nature and carry out a broad variety of difficult chemical transformations initiated by hydrogen atom abstraction. Although numerous radical SAM (RS) enzymes have been structurally characterized, many prove recalcitrant to crystallization needed for atomic-level structure determination using X-ray crystallography, and even those that have been crystallized for an initial study can be difficult to recrystallize for further structural work. We present here a method for computationally engineering previously observed crystallographic contacts and employ it to obtain more reproducible crystallization of the RS enzyme pyruvate formate-lyase activating enzyme (PFL-AE). We show that the computationally engineered variant binds a typical RS [4Fe-4S]2+/+ cluster that binds SAM, with electron paramagnetic resonance properties indistinguishable from the native PFL-AE. The variant also retains the typical PFL-AE catalytic activity, as evidenced by the characteristic glycyl radical electron paramagnetic resonance signal observed upon incubation of the PFL-AE variant with reducing agent, SAM, and PFL. The PFL-AE variant was also crystallized in the [4Fe-4S]2+ state with SAM bound, providing a new high-resolution structure of the SAM complex in the absence of substrate. Finally, by incubating such a crystal in a solution of sodium dithionite, the reductive cleavage of SAM is triggered, providing us with a structure in which the SAM cleavage products 5'-deoxyadenosine and methionine are bound in the active site. We propose that the methods described herein may be useful in the structural characterization of other difficult-to-resolve proteins.

Identifying the chloroperoxyl radical in acidified sodium chlorite solution.

The present study identified the active radical species in acidic sodium chlorite and investigated the feasibility of quantifying these species with the diethylphenylenediamine (DPD) method. Electron spin resonance (ESR) spectroscopy was used to identify the active species generated in solutions containing sodium chlorite (NaClO2). The ESR signal was directly observed in an acidified sodium chlorite (ASC) aqueous solution at room temperature. This ESR signal was very long-lived, indicating that the radical was thermodynamically stable. The ESR parameters of this signal did not coincide with previously reported values of the chlorine radical (Cl●) or chlorine dioxide radical (O = Cl●-O and O = Cl-O●). We refer to this signal as being from the chloroperoxyl radical (Cl-O-O●). Quantum chemical calculations revealed that the optimal structure of the chloroperoxyl radical is much more thermodynamically stable than that of the chlorine dioxide radical. The UV-visible spectrum of the chloroperoxyl radical showed maximum absorbance at 354 nm. This absorbance had a linear relationship with the chloroperoxyl radical ESR signal intensity. Quantifying the free chlorine concentration by the DPD method also revealed a linear relationship with the maximum absorbance at 354 nm, which in turn showed a linear relationship with the chloroperoxyl radical ESR signal intensity. These linear relationships suggest that the DPD method can quantify chloroperoxyl radicals, which this study considers to be the active species in ASC aqueous solution.

Trapping and Electron Paramagnetic Resonance Characterization of the 5'dAdo• Radical in a Radical S-Adenosyl Methionine Enzyme Reaction with a Non-Native Substrate.

S-Adenosyl methionine (SAM) is employed as a [4Fe-4S]-bound cofactor in the superfamily of radical SAM (rSAM) enzymes, in which one-electron reduction of the [4Fe-4S]-SAM moiety leads to homolytic cleavage of the S-adenosyl methionine to generate the 5′-deoxyadenosyl radical (5′dAdo•), a potent H-atom abstractor. HydG, a member of this rSAM family, uses the 5′dAdo• radical to lyse its substrate, tyrosine, producing CO and CN that bind to a unique Fe site of a second HydG Fe–S cluster, ultimately producing a mononuclear organometallic Fe-l-cysteine-(CO)2CN complex as an intermediate in the bioassembly of the catalytic H-cluster of [Fe–Fe] hydrogenase. Here we report the use of non-native tyrosine substrate analogues to further probe the initial radical chemistry of HydG. One such non-native substrate is 4-hydroxy phenyl propanoic acid (HPPA) which lacks the amino group of tyrosine, replacing the CαH-NH2 with a CH2 at the C2 position. Electron paramagnetic resonance (EPR) studies show the generation of a strong and relatively stable radical in the HydG reaction with natural abundance and 13C2-HPPA, with appreciable spin density localized at C2. These results led us to try parallel experiments with the more oxidized non-native substrate coumaric acid, which has a C2=C3 alkene substitution relative to HPPA’s single bond. Interestingly, the HydG reaction with the cis-p-coumaric acid isomer led to the trapping of a new radical EPR signal, and EPR studies using cis-p-coumaric acid along with isotopically labeled SAM reveal that we have for the first time trapped and characterized the 5′dAdo• radical in an actual rSAM enzyme reaction, here by using this specific non-native substrate cis-p-coumaric acid. Density functional theory energetics calculations show that the cis-p-coumaric acid has approximately the same C–H bond dissociation free energy as 5′dAdo•, providing a possible explanation for our ability to trap an appreciable fraction of 5′dAdo• in this specific rSAM reaction. The radical’s EPR line shape and its changes with SAM isotopic substitution are nearly identical to those of a 5′dAdo• radical recently generated by cryophotolysis of a prereduced [4Fe-4S]-SAM center in another rSAM enzyme, pyruvate formate-lyase activating enzyme, further supporting our assignment that we have indeed trapped and characterized the 5′dAdo• radical in a radical SAM enzymatic reaction by appropriate tuning of the relative radical free energies via the judicious selection of a non-native substrate.

Radical-lanthanide ferromagnetic interaction in a TbIII bis-phthalocyaninato complex

Recent studies have highlighted the importance of organic ligands in the field of molecular spintronics, via which delocalized electron-spin density can mediate magnetic coupling to otherwise localized 4f moments of lanthanide ions, which show tremendous potential for single-molecule device applications. To this end, high-field/high-frequency electron paramagnetic resonance (EPR) spectroscopy is employed to study a neutral terbium bis-phthalocyaninato metalorganic complex, [TbPc2]0, with the aim of understanding the magnetic interaction between the Ising-like moment of the lanthanide ion and the unpaired spin density on the coordinating organic radical ligand. The measurements were performed on a previously unknown [TbPc2]0 structural phase crystallizing in the Pnma space group. EPR measurements on powder samples of [TbPc2]0 reveal an anisotropic spectrum, which is attributed to the spin-12 radical coupled weakly to the EPR-silent TbIII ion. Extensive double-axis rotation studies on a single crystal reveal two independent spin-12 signals with differently oriented (albeit identical) uniaxial g-tensors, in complete agreement with x-ray structural studies that indicate two molecular orientations within the unit cell. The easy-axis nature of the radical EPR spectra thus reflects the coupling to the Ising-like TbIII moment. This is corroborated by studies of the isostructural [YPc2]0 analog (where Y is nonmagnetic yttrium), which gives a completely isotropic radical EPR signal. The experimental results for the terbium complex are well explained on the basis of an effective model that introduces a weak ferromagnetic Heisenberg coupling between an isotropic spin-12 and an anisotropic spin-orbital moment, J=6, that mimics the known, strong easy-axis TbPc2 crystal-field interaction.

Studies on the antioxidant activity of some chromonylrhodanine derivatives.

Fifteen chromonylrhodamine derivatives (CRs) were synthesized and the antioxidant activity levels were evaluated for the first time. The antioxidant activity potencies of these chromone derivatives were evaluated towards superoxide anion radicals, hydroxyl radicals and 2,2-diphenyl-1-picrylhydrazyl radicals. Also, the total antioxidant capacity of the tested compounds was measured using the ferric-ferrozine assay. The antioxidant activities were investigated using a chemiluminescence (CL) assay, spectrophotometry measurements, direct electron paramagnetic resonance (EPR) and the EPR spin-trapping technique. The 5,5-dimethyl- 1-pyrroline-1-oxide (DMPO) was applied as spin trap. Eleven of the 15 chromone compounds exhibited a decrease in the CL accompanying the superoxide anion radical produced in anhydrous dimethylsulfoxide (DMSO), ranging from 71-94% at concentration of 1 mmol /L; four of these compounds enhanced light emission in the range 231-672%. Similarly, these compounds caused 28-58% inhibition in the intensity of the DMPO-OOH radical EPR signal and the DMPO-OH radical (from 12-48%). Furthermore, three of these compounds showed very good antioxidant response towards the DPPH radical (EC50 : 0.51-0.56 µmol/L) and the high reduction potentials. These findings demonstrate that the chromone compounds tested may be considered as effective free radicals scavengers, a finding that is of great pharmacological importance.

Studies on the antioxidant activities of some new chromone compounds

Recent reviews evidence that the naturally occurring compounds containing the chromone skeleton exhibit antiradical activities, providing protection against oxidative stress. The antioxidant activities of 13 new synthesized chromonyl-2,4-thiazolidinediones, chromonyl-2,4-imidazolidinediones and chromonyl-2-thioxoimidzolidine-4-ones were evaluated using in vitro antioxidant assays, including superoxide anion radical (O2(-•)), hydroxyl radical (HO(•)), 2,2-diphenyl-1-picryl-hydrazyl free radical (DPPH(•)) scavenging capacity and total antioxidant capacity ferric ion reducing activity. Superoxide anion radical was produced using potassium superoxide/18-crown-6-ether dissolved in dimethylsulfoxide, and the Fenton-like reaction (Fe(II) + H2O2) was a generator of hydroxyl radicals. Chemiluminescence, spectrophotometry, electron paramagnetic resonance (EPR) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin trap were the measurement techniques. The results showed that the majority of the chromone derivatives tested showed a strong scavenging effect towards free radicals, similar to the chemiluminescence reaction with superoxide anion radical with a high activity, inhibition of the DMPO-OOH radical EPR signal (24-58%), the DMPO-OH radical EPR signal (4-75%) and DPPH radical EPR signal (6-100%) at 1 mmol/L. Several of the examined compounds exhibited the high reduction potentials. The results obtained show that the new synthesized chromone derivatives may directly scavenger reactive oxygen species and thus may play a protective role against oxidative damage.

Regulation of Influenza Virus-Caused Oxidative Stress by Kegan Liyan Oral Prescription, as Monitored by Ascorbyl Radical ESR Signals

To study the oxidative stress level of the influenza virus A FM1 subset-infected mouse in intranasal inhalation as a model, we employ an ascorbyl radical's ESR (electron spin resonance) spectrum as an oxidative stress biomarker. These infected mice were pretreated with Ribavirin, ascorbic acid, superoxide dismutase (SOD) or Kegan Liyan oral prescription (KGLY, proprietary Chinese medicine for influenza and common cold) in the stomach tube for 3 days, and then followed by the virus-infecting for 4 days. On the 4th day, samples were collected. It is recognized the strength of ascorbyl radical's ESR signal (A(-.)) (a(H4 = 0.177) Gauss, g = 2.00517) denotes oxidative stress level in vivo and in vitro. The magnitude of ESR spectrum (28.65 +/- 10.71 AU) in mice infected with influenza virus was significantly higher than those of healthy control mice (19.10 +/- 3.61 AU). Serum A(-.) in mice treated with Ribavirin, ascorbic acid, SOD and KGLY declined to 19.70 +/- 6.05, 18.50 +/- 2.93 and 16.25 +/- 3.59, 18.40 +/- 2.14 AU respectively. It is close to A(-.) signal height in healthy controls via down-regulation of the influenza virus-caused oxidative stress level getting decline in the lung index of pneumonia as compare to those of untreated healthy and the influenza virus infected mice pneumonia. It is well known that SOD can prevent the influenza virus pneumonia enhancing mouse survival rate; Ribavirin can treat viral diseases. Data from this study suggested that KGLY may indirectly relieve influenza virus-infected pneumonia via down- regulation of virus caused oxidative stress coupled with a redox reaction cascade as ribavirin, ascorbic acid and SOD.

Transplantation of the uterus in the sheep: oxidative stress and reperfusion injury after short-time cold storage

To study the effects of cold ischemia and reperfusion after transplantation of the sheep uterus and to compare the preservation solution Perfadex (Vitrolife, Mölndal, Sweden) with Ringer's acetate. Experimental animal study. University hospital. Adult, female sheep. One uterine horn with the common uterine cavity and cervix of sexually mature ewes was auto-transplanted after 1 hour of cold ischemic storage in either Perfadex (n = 5) or Ringer's acetate (n = 5). During 3 hours of reperfusion, uterine venous blood was collected and analyzed for several parameters that were indicative of oxidative stress and reperfusion injury. A biopsy was taken for histological analysis at the end of the experiment. Lipid peroxidation and ascorbyl radicals in uterine venous blood during reperfusion. Light microscopy and quantification of neutrophils in tissue after 3 hours of reperfusion. A decline in pH and a rise in lactate and pCO(2)-pO(2), as well as an elevation of antioxidant capacity, lipid peroxidation, and intensity of ascorbyl radical electron spin resonance signal, was found that was more prominent after storage in Ringer's acetate. The histological analysis revealed mild inflammation in both study groups. Short-time cold ischemic storage of the sheep uterus does not induce any severe reperfusion damage, but the use of the protective buffer Perfadex decreases oxidative stress and inflammation when compared with a more simple solution.

ATP inhibits hydroxyl radical formation and the inflammatory response of stimulated whole blood even under circumstances of severe oxidative stress

We recently reported that Adenosine-5′-triphosphate (ATP) is able to inhibit the inflammatory reaction in stimulated whole blood. Many diseases, in which inflammatory reactions are involved, are associated with oxidative stress. In the present study, we therefore, investigated the effect of ATP on cytokine release in stimulated whole blood under conditions of oxidative stress, as simulated by pre-incubation of blood with hydrogen peroxide (H2O2). In the presence of H2O2, ATP at concentrations of 100 and 300 μM inhibited Tumour Necrosis factor-alpha (TNF-α) release and stimulated IL-10 release in LPS-PHA stimulated whole blood. Moreover, electron spin resonance (ESR) measurements showed that ATP and its breakdown product Adenosine-5′-diphosphate (ADP) attenuated spin trap-hydroxyl radical adduct formation in the Fenton reaction. Our results demonstrate that even in circumstances of severe oxidative stress, ATP has marked anti-inflammatory properties in stimulated whole blood. Moreover, the inhibition of the hydroxyl radical ESR signal indicates a direct attenuation of oxidative stress by ATP.

Scavenging of reactive oxygen species by a novel glucurinated flavonoid antioxidant isolated and purified from spinach

NAO is a natural water soluble antioxidant that was isolated and purified from spinach leaves. Using HPLC, NMR, and CMR spectroscopy, the main components were identified as flavonoids and p-coumaric acid derivatives. The NAO was found to be a very effective antioxidant in several in vivo and in vitro biological systems. In the present study, the antioxidant activity of the novel antioxidant glucurinated flavonoid (GF) isolated and characterized from NAO, is compared to well-known antioxidants. In addition, the direct free radical scavenging properties of the purified component GF were studied using the electron spin resonance (ESR) technique. GF and NAO were found to be superior to EGCG and NAC and to the Vitamin E homologue Trolox in inhibiting reactive oxygen species (ROS) formation in the autooxidation system of linoleic acid and in fibroblasts exposed to metal oxidation. GF and NAO were found to inhibit the ESR signal intensity of DMPO-O 2 radical formation during the riboflavin photodynamic reaction. 10 mM GF caused approximately 90% inhibition in the intensity of the ESR signal, while NAO at a concentration of 60 μg/ml caused an inhibition of about 50%. Using the Fenton reaction, GF and NAO were found to inhibit DMPO-OH radical formation. A concentration of 2 mM GF caused a 70% inhibition in the intensity of the DMPO-OH radical ESR signal, while propyl gallate at the same concentration caused only 50% inhibition. Furthermore, both GF and NAO also inhibited the 1O 2 dependent TEMPO radical generated in the photoradiation TPPS4 system. About 80% inhibition was obtained by 4 mM GF. The results obtained indicate that the natural antioxidants derived from spinach may directly affect the scavenging of ROS and, as a consequence, may be considered as effective sources for combating oxidative damage.

H2O2-mediated Cross-linking between Lactoperoxidase and Myoglobin: ELUCIDATION OF PROTEIN-PROTEIN RADICAL TRANSFER REACTIONS

The H(2)O(2)-dependent reaction of lactoperoxidase (LPO) with sperm whale myoglobin (SwMb) or horse myoglobin (HoMb) produces LPO-Mb cross-linked species, in addition to LPO and SwMb homodimers. The HoMb products are a LPO(HoMb) dimer and LPO(HoMb)(2) trimer. Dityrosine cross-links are shown by their fluorescence to be present in the oligomeric products. Addition of H(2)O(2) to myoglobin (Mb), followed by catalase to quench excess H(2)O(2) before the addition of LPO, still yields LPO cross-linked products. LPO oligomerization therefore requires radical transfer from Mb to LPO. In contrast to native LPO, recombinant LPO undergoes little self-dimerization in the absence of Mb but occurs normally in its presence. Simultaneous addition of 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) and LPO to activated Mb produces a spin-trapped radical electron paramagnetic resonance signal located primarily on LPO, confirming the radical transfer. Mutation of Tyr-103 or Tyr-151 in SwMb decreased cross-linking with LPO, but mutation of Tyr-146, Trp-7, or Trp-14 did not. However, because DBNBS-trapped LPO radicals were observed with all the mutants, DBNBS traps LPO radicals other than those involved in protein oligomerization. The results clearly establish that radical transfer occurs from Mb to LPO and suggest that intermolecularly transferred radicals may reside on residues other than those that are generated by intramolecular reactions.

Electron paramagnetic resonance spectrometry evidence for bioreduction of tirapazamine to oxidising free radicals under anaerobic conditions

Tirapazamine (SR 4233) is a bioreductive antitumour drug in Phase III clinical trial which is activated in hypoxic tumour regions to generate a cytotoxic species. Electron paramagnetic resonance (EPR) spectrometry was used to investigate directly the formation of free radicals as the result of tirapazamine reduction by NADPH-supplemented liver microsomes. Under anaerobic conditions, the tirapazamine nitroxide free radical EPR signal was not evident over a range of rat or human liver microsomal protein (1–5 mg) concentrations. However, in combination with 1,1′,5,5′-dimethylpyrolline-1-N-oxide (DMPO), a spin trap for short-lived free radicals, tirapazamine resulted in formation of a 1:1:1:1:1:1 spectrum with hyperfine splitting A N = 15.8 G A H = 22.3 G consistent with generation of DMPO-R, a carbon-centered radical adduct. Addition of DMSO increased the signal intensity of the carbon-centred radical by at least twofold. The hyperfine splitting constants associated with DMPO-R could be indicative of a tirapazamine carbon-centred radical per se or, more likely, carbon radicals from endogenous materials (or DMSO) in the biological matrix as a result of oxidative attack by the tirapazamine primary radical. Formation of DMPO-OH, the hydroxyl radical spin adduct, by tirapazamine in the absence of air indicates that liberation of a hydroxyl radical may be a consequence of tirapazamine bioreduction under anaerobic conditions. The reactivity of tirapazamine free radicals with endogenous microsomal substances to generate reactive carbon-centred radicals indicates that tirapazamine may disrupt a wide range of cellular activities.

Electron paramagnetic resonance and electron nuclear double resonance spectroscopic identification and characterization of the tyrosyl radicals in prostaglandin H synthase 1.

The tyrosyl radicals generated in reactions of ethyl hydrogen peroxide with both native and indomethacin-pretreated prostaglandin H synthase 1 (PGHS-1) were examined by low-temperature electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies. In the reaction of peroxide with the native enzyme at 0 degrees C, the tyrosyl radical EPR signal underwent a continuous reduction in line width and lost intensity as the incubation time increased, changing from an initial, 35-G wide doublet to a wide singlet of slightly smaller line width and finally to a 25-G narrow singlet. The 25-G narrow singlet produced by self-inactivation was distinctly broader than the 22-G narrow singlet obtained by indomethacin treatment. Analysis of the narrow singlet EPR spectra of self-inactivated and indomethacin-pretreated enzymes suggests that they reflect conformationally distinct tyrosyl radicals. ENDOR spectroscopy allowed more detailed characterization by providing hyperfine couplings for ring and methylene protons. These results establish that the wide doublet and the 22-G narrow singlet EPR signals arise from tyrosyl radicals with different side-chain conformations. The wide-singlet ENDOR spectrum, however, is best accounted for as a mixture of native wide-doublet and self-inactivated 25-G narrow-singlet species, consistent with an earlier EPR study [DeGray et al. (1992) J. Biol. Chem. 267, 23583-23588]. We conclude that a tyrosyl residue other than the catalytically essential Y385 species is most likely responsible for the indomethacin-inhibited, narrow-singlet spectrum. Thus, this inhibitor may function by redirecting radical formation to a catalytically inactive side chain. Either radical migration or conformational relaxation at Y385 produces the 25-G narrow singlet during self-inactivation. Our ENDOR data also indicate that the catalytically active, wide-doublet species is not hydrogen bonded, which may enhance its reactivity toward the fatty-acid substrate bound nearby.

The electron paramagnetic resonance characterisation of a copper-containing extracellular peroxidase from Thermomonospora fusca BD25

The actinomycete Thermomonospora fusca BD25 contains a peroxidase with a high activity over a broad range of temperature and pH and a high stability against denaturing agents. Unusually this peroxidase (PO) is a non-haem enzyme. As prepared PO is characterised by two electron paramagnetic resonance (EPR) signals, detected at liquid helium temperature, a free radical signal ( g=2.0045) and a broad signal at g=2.056. The peroxidase activity of the purified enzyme was assayed using H 2O 2 and 2,4-dichlorophenol (DCP). The intensity of the free radical EPR signal correlated with the peroxidase activity in a variety of enzyme preparations. Furthermore, when DCP and H 2O 2 were added to PO a significant increase of both the free radical signal and the broad signal at g=2.056 was observed. We associate the increase of the broad signal with the oxidation of the preparation since a similar increase can be achieved by the addition of ferricyanide. The high intensity of the broad signal in the ferricyanide treated PO allowed us to deconvolute the signal into several components using the difference in their relaxation characteristics: two distinct copper signals were detected, one of which was similar to a type 2 centre. Furthermore a symmetrical singlet was detected at g=2.059, consistent with the presence of an iron complex with a high degree of symmetry and weakly coordinated ligands.

Redox cycles of caffeic acid, alpha-tocopherol, and ascorbate: implications for protection of low-density lipoproteins against oxidation.

This study addresses the dynamic interactions among alpha-tocopherol, caffeic acid, and ascorbate in terms of a sequence of redox cycles aimed at accomplishing optimal synergistic antioxidant protection. Several experimental models were designed to examine these interactions: UV irradiation of alpha-tocopherol-containing sodium dodecyl sulfate micelles, one-electron oxidations catalyzed by the hypervalent state of myoglobin, ferrylmyoglobin, and autoxidation at appropriate pHs. These models were assessed by ultraviolet (UV) and electron paramagnetic resonance (EPR), entailing direct- and continuous-flow experiments, spectroscopy and by separation and identification of products by HPLC. The alpha-tocopheroxyl radical EPR signal generated by UV irradiation of alpha-tocopherol-containing micelles was suppressed by caffeic acid and ascorbate; in the former case, no other EPR signal was observed at pH 7.4, whereas in the latter case, the alpha-tocopheroxyl radical EPR signal was replaced by a doublet EPR spectrum corresponding to the ascorbyl radical (A*-). The potential interactions between caffeic acid and ascorbate were further analyzed by assessing, on the one hand, the ability of ascorbate to reduce the caffeic acid o-semiquinone (generated by oxidation of caffeic acid by ferrylmyoglobin) and, on the other hand, the ability of caffeic acid to reduce ascorbyl radical (generated by autoxidation or oxidation of ascorbate by ferrylmyoglobin). The data presented indicate that the reductive decay of ascorbyl radical (A*-) and caffeic acid o-semiquinone (Caf-O*) can be accomplished by caffeic acid (Caf-OH) and ascorbate (AH-), respectively, thus pointing to the reversibility of the reaction Caf-O* + AH- <--> Caf-OH + A*-. Continuous-flow EPR measurements of mixtures containing ferrylmyoglobin, alpha-tocopherol-containing micelles, caffeic acid, and ascorbate revealed that ascorbate is the ultimate electron donor in the sequence encompassing transfer of the radical character from the micellar phase to the phase. In independent experiments, the effects of caffeic acid and ascorbate on the oxidation of two low-density lipoprotein (LDL) populations, control and alpha-tocopherol-enriched, were studied and results indicated that alpha-tocopherol, caffeic acid, and ascorbate acted synergistically to afford optimal protection of LDL against oxidation. These results are analyzed for each individual antioxidant in terms of three domains: its localization and that of the antioxidant-derived radical, its reduction potential, and the predominant decay pathways for the antioxidant-derived radical, that exert kinetic control on the process.

ESR studies of ascorbic acid-dependent recycling of the vitamin Ehomologue Trolox by coenzyme Q0 in murine skin homogenates

The recycling of Trolox, a water-soluble vitamin E homologue, by coenzyme Q0 (CoQ0) during Cu2+-initiated oxidation of ascorbate in mouse skin homogenates was investigated using electron spin resonance (ESR) spectroscopy. In a mixture containing CoQ0, Cu2+ and mouse skin homogenates, the ESR signal of CoQ0 semiquinone radical (CoQ0.-) appeared and declined with time; addition of Trolox accelerated the CoQ0.- signal decay. Only after the disappearance of the CoQ0.- signal was the appearance of the Trolox phenoxyl radical signal observed. In addition, the lifetime of the CoQ0.- signal and the length of the lag period during which the Trolox radical ESR signal could not be detected were dependent on the presence of Trolox, CoQ0 or Cu2+. The resultsm suggest that CoQ0.-, formed by the interaction between CoQ0 and endogenous ascorbic acid (AscH-) in skin homogenates, regenerates Trolox from its phenoxyl radical.

The Fate of the Oxidizing Tyrosyl Radical in the Presence of Glutathione and Ascorbate: IMPLICATIONS FOR THE RADICAL SINK HYPOTHESIS

Cellular systems contain as much as millimolar concentrations of both ascorbate and GSH, although the GSH concentration is often 10-fold that of ascorbate. It has been proposed that GSH and superoxide dismutase (SOD) act in a concerted effort to eliminate biologically generated radicals. The tyrosyl radical (Tyr.) generated by horseradish peroxidase in the presence of hydrogen peroxide can react with GSH to form the glutathione thiyl radical (GS.). GS. can react with the glutathione anion (GS-) to form the disulfide radical anion (GSSG-). This highly reactive disulfide radical anion will reduce molecular oxygen, forming superoxide and glutathione disulfide (GSSG). In a concerted effort, SOD will catalyze the dismutation of superoxide, resulting in the elimination of the radical. The physiological relevance of this GSH/SOD concerted effort is questionable. In a tyrosyl radical-generating system containing ascorbate (100 microM) and GSH (8 mM), the ascorbate nearly eliminated oxygen consumption and diminished GS. formation. In the presence of ascorbate, the tyrosyl radical will oxidize ascorbate to form the ascorbate radical. When measuring the ascorbate radical directly using fast-flow electron spin resonance, only minor changes in the ascorbate radical electron spin resonance signal intensity occurred in the presence of GSH. These results indicate that in the presence of physiological concentrations of ascorbate and GSH, GSH is not involved in the detoxification pathway of oxidizing free radicals formed by peroxidases.

Interaction of UV light-induced alpha-tocopherol radicals with lipids detected by an electron spin resonance prooxidation effect.

The reaction rate constants of the interaction between light-induced alpha-tocopherol radicals with unsaturated lipids in a heterogeneous system compared to a homogeneous system are of the same order of magnitude. The decay rates of compartmentalized alpha-tocopherol radicals were significantly reduced by using negatively charged sodium dodecyl sulfate (SDS) micelles. A partially resolved electron spin resonance (ESR) hyperfine structure was observed under the conditions of both high lipid concentrations in comparison to the alpha-tocopherol concentration and of a regular distribution of alpha-tocopherol molecules inside the heterogeneous lipid structures. Alpha-tocopherol radicals have a considerable prooxidation potential at higher concentrations. Ascorbic acid dissolved in the aqueous medium provokes very fast alpha-tocopherol radical recycling through the boundary layer between the aqueous medium and micelles. By contrast, very slow reactions such as those of alpha-tocopherol radicals with glutathione through this boundary layer are measurable. Despite using the heterogeneous SDS micellar system, the decay kinetics of the alpha-tocopherol radical ESR signal is simply compounded. In addition to the known stabilization effect of cholesterol in membrane systems, cholesterol itself acts as a target molecule attacked by free radicals, e.g. alpha-tocopherol radicals. Using stratum corneum extracts that contain unsaturated lipids and cholesterol the alpha-tocopherol radical can prooxidatively react with these compounds. Using focused UV light generates a high radical yield in a relatively short time compared to the lifetime of the alpha-tocopherol radicals. The decay processes after radical induction can be characterized as consecutive reactions. The compartmentalization of radicals induced in SDS micelles and the close proximity of target molecules are essential if very slow one-electron reductions are to be measured.

Enhancement of daunomycin toxicity by the differentiation inducer hexamethylene bisacetamide in erythroleukemia cells

Cytotoxic effects of daunomycin were investigated upon differentiation of Friend erythroleukemia cells induced with hexamethylene bisacetamide, a process during which a 20-fold increase in the hemoglobin content occurred. Daunomycin proved to be more toxic to differentiated Friend cells than to their undifferentiated counterparts. No changes in the daunomycin uptake rates of the two cell types were detectable. Externally added catalase and desferrioxamine mesylate protected against the additional cytotoxicity of daunomycin in differentiated cells, pointing to hydrogen peroxide and iron ions as mediators of the toxic effect. Daunomycin-dependent, cyanide-insensitive oxygen consumption of control and induced cells did not differ significantly, and the rate of formation of the daunomycin semiquinone radical electron paramagnetic resonance signal was similar in both cell types, indicating that the difference in toxicity was not due to increased drug activation by plasma membrane enzymes. Differentiated cells had a lowered catalase content; the cellular iron content was shown to increase by 2.8-fold upon cell differentiation, with hemoglobin-bound iron being about 50% of the total. Altogether, the results suggest increased intracellular hydrogen peroxide generation mediated by hemoglobin, combined with a decrease in catalase activity and an increase in accessible iron, as responsible for the higher sensitivity to daunomycin shown by differentiated Friend cells. This represents the first experimental system where the increase in anthracycline cytotoxicity upon cell differentiation can be attributed to redox activation and the formation of reactive oxygen species.

The effect of the anthrapyrazole antitumour agent CI941 on rat liver microsome and cytochrome P-450 reductase mediated free radical processes: Inhibition of doxorubicin activation in vitro

The anthrapyrazole CI941 is one of a new series of DNA complexing drugs which displays high level broad spectrum antitumour activity in mice. In view of the proposed role of drug free radical formation, Superoxide generation and lipid peroxidation in anthracycline and anthraquinone induced toxicities, the redox biochemistry of CI941 has been investigated. Studies have been performed in vitro using rat liver microsomes and purified cytochrome P-450 reductase. In addition, the ability of CI941 to undergo chemical reduction has been examined. Pulse radiolysis of CI941 demonstrated that the drug can undergo chemical reduction with a one electron reduction potential of E 1 7 = −538 ± 10 mV . However, electron spin resonance (ESR) spectroscopy studies using either NADPH fortified microsomes or cytochrome P-450 reductase, failed to detect a drug free radical signal. Unlike doxorubicin, CI941 (150 μM) inhibited basal rate microsomal NADPH consumption by 45%. Furthermore, CI941 (50–200 μM) antagonised doxorubicin stimulated (1.8-fold) NADPH oxidation by over 50%. CI941 also antagonised the formation of a doxorubicin free radical ESR signal in a concentration dependent manner. CI941 induced minimal Superoxide generation in the presence of either microsomes or cytochrome P-450 reductase and inhibited doxorubicin induced (50 μM) Superoxide formation by up to 80% (50–200 μM CI941). Importantly, CI941 inhibits both basal rate and doxorubicin (100 μM) stimulated lipid peroxidation (52% inhibition at 5μM CI941). These data suggest that CI941 is unlikely to induce free radical mediated tissue damage in vivo. On the contrary, CI941 may have a protective role if used in combination with doxorubicin.