Enzyme-bound pentadienyl and peroxyl radicals in purple lipoxygenase

Samples of purple lipoxygenase prepared by addition of either 13-hydroperoxy-9,11-octadecadienoic acid or linoleic acid and oxygen to ferric lipoxygenase contain pentadienyl and/or peroxyl radicals. The radicals are identified by the g values and hyperfine splitting parameters of natural abundance and isotopically enriched samples. The line shapes of their EPR spectra suggest the radicals are conformationally constrained when compared to spectra of the same radicals generated in frozen linoleic acid. Further, the EPR spectra are unusually difficult to saturate. The radicals are stable in buffered aqueous solution at 4 degrees C for several minutes. All of this implies that these species are bound to the enzyme, possibly in proximity to the iron. Only peroxyl radical is seen when the purple enzyme is generated with either hydroperoxide or linoleic acid in O2-saturated solutions. Addition of natural abundance hydroperoxide under 17O-enriched O2 leads to the 17O-enriched peroxyl radical, while the opposite labeling results in the natural abundance peroxyl radical, demonstrating the exchange of oxygen. Both radicals are detected in samples of purple lipoxygenase prepared with either linoleic acid or hydroperoxide under air. Addition of the hydroperoxide in the absence of oxygen favors the pentadienyl radical. We propose that addition of either linoleic acid or hydroperoxide to ferric lipoxygenase leads to multiple mechanistically connected enzyme complexes, including those with (hydro)peroxide, peroxide, peroxyl radical, pentadienyl radical, and linoleic acid bound. This hypothesis is essentially identical with the proposed radical mechanism of oxygenation of polyunsaturated fatty acids by lipoxygenase.     

The specificity of lipoxygenase-catalyzed lipid peroxidation and the effects of radical-scavenging antioxidants

The oxidation of low density lipoprotein (LDL) by lipoxygenase has been implicated in the pathogenesis of atherosclerosis. It has been known that lipoxygenase-mediated lipid peroxidation proceeds in general via regio-, stereo- and enantio-specific mechanisms, but that it is sometimes accompanied by a share of random hydroperoxides as side reaction products. In this study we investigated the oxidation of various substrates (linoleic acid, methyl linoleate, phosphatidylcholine, isolated LDL, and human plasma) by the arachidonate 15-lipoxygenases from rabbit reticulocytes and soybeans aiming at elucidating the effects of substrate, lipoxygenase and reaction milieu on the contribution and mechanism of random oxidation and also the effect of antioxidant. The specific character of the rabbit 15-lipoxygenase reaction was confirmed under all conditions employed here. However, the specificity by soybean lipoxygenase was markedly dependent on the conditions. When phosphatidylcholine liposomes and LDL were oxygenated by soybean lipoxygenase, the product pattern was found to be exclusively regio-, stereo-, and enantio-random. When free linoleic acid was incorporated into PC liposomes and oxidized by soybean lipoxygenase, the free acid was specifically oxygenated, whereas esterified linoleate gave random oxidation products exclusively. Radical-scavenging antioxidants such as alpha-tocopherol, ascorbic acid and 2-carboxy-2,5,7,8-tetramethyl-6-chromanol selectively inhibited the random oxidation but did not influence specific product formation. It is assumed that the random reaction products originate from free radical intermediates, which have escaped the active site of the enzyme and thus may be accessible to radical scavengers. These data indicate that the specificity of lipoxygenase-catalyzed lipid oxidation and the inhibitory effects of antioxidants depend on the physico-chemical state of the substrate and type of lipoxygenase and that they may change completely depending on the conditions.     

Superoxide and peroxyl radical generation from the reduction of polyunsaturated fatty acid hydroperoxides by soybean lipoxygenase.

Soybean lipoxygenase is shown to catalyze the breakdown of polyunsaturated fatty acid hydroperoxides to produce superoxide radical anion as detected by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In addition to the DMPO/superoxide radical adduct, the adducts of peroxyl, acyl, carbon-centered, and hydroxyl radicals were identified in incubations containing linoleic acid and lipoxygenase. These DMPO radical adducts were observed just prior to the system becoming anaerobic. Only a carbon-centered radical adduct was observed under anaerobic conditions. The superoxide radical production required the presence of fatty acid substrates, fatty acid hydroperoxides, active lipoxygenase, and molecular oxygen. Superoxide radical production was inhibited when nordihydroguaiaretic acid, butylated hydroxytoluene, or butylated hydroxyanisole was added to the incubation mixtures. We propose that polyunsaturated fatty acid hydroperoxides are reduced to form alkoxyl radicals and that after an intramolecular rearrangement, the resulting hydroxyalkyl radical reacts with oxygen, forming a peroxyl radical which subsequently eliminates superoxide radical anion.     

Isolation and identification of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone radical adducts formed by the decomposition of the hydroperoxides of linoleic acid, linolenic acid, and arachidonic acid by soybean lipoxygenase.

alpha-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) radical adducts, which are formed in the reactions of soybean lipoxygenase with linoleic acid, arachidonic acid, and linolenic acid, were isolated using HPLC-ESR spectroscopy. Both linoleic acid and arachidonic acid gave one radical adduct, whereas in the case of linolenic acid, two radical adducts were isolated. These radical adducts all showed virtually identical uv spectra with lambda max at 292 and 220 nm in hexane. The absence of absorbance with lambda max at 234 nm indicates that a conjugated diene structure is not contained in these radical adducts. The mass spectra of the radical adducts formed from linoleic and arachidonic acids were identical and contained a molecular ion of m/z 264, consistent with the trapping of the pentyl radical by 4-POBN. Indeed, authentic 4-POBN pentyl radical adduct obtained from the reaction between pentylhydrazine and 4-POBN gave the same mass spectrum as the product obtained from the reaction of linoleic acid and arachidonic acid with 4-POBN. The two 4-POBN radical adducts formed in the linolenic acid reaction were shown by mass spectrometry to be isomers of pentenyl radicals. The 4-POBN-pentyl radical adduct was also detected in the reaction mixture of 13-hydroperoxy-linoleic acid, soybean lipoxygenase, and 4-POBN, indicating that the pentyl radical and pentenyl radical are formed by the decomposition of the hydroperoxides.     

A search for oxygen-centered free radicals in the lipoxygenase/linoleic acid system

Studies of the oxygenation of linoleic acid by soybean lipoxygenase utilizing electron spin resonance spectroscopy and oxygen uptake have been undertaken. The spin trap, alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN) was included in the lipoxygenase system to capture short-lived free radicals. Correlation of radical adduct formation rates with oxygen uptake studies indicated that the major portion of radical adduct formation occurred when the system was nearly anaerobic. Incubations containing [17O]oxygen with nuclear spin of 5/2 did not have additional ESR lines as would be expected if an oxygen-centered 4-POBN-lipid peroxyl radical adduct were formed indicating that the trapped radical must be reassigned as a carbon-centered species. To establish the presence of [17O2]oxygen in our incubations, a portion of the gas from the lipoxygenase/linoleate experiments was used to prepare the 4-POBN-superoxide radical adduct utilizing a superoxide producing microsomal/paraquat/NADPH system.     

Spin trapping of polyunsaturated fatty acid-derived peroxyl radicals: reassignment to alkoxyl radical adducts

Polyunsaturated fatty acid (PUFA) peroxyl radicals play a crucial role in lipid oxidation. ESR spectroscopy with the spin-trapping technique is one of the most direct methods for radical detection. There are many reports of the detection of PUFA peroxyl radical adducts; however, it has recently been reported that attempted spin trapping of organic peroxyl radicals at room temperature formed only alkoxyl radical adducts in detectable amounts. Therefore, we have reinvestigated spin trapping of the linoleic, arachidonic, and linolenic acid-derived PUFA peroxyl radicals. The slow-flow technique allowed us to obtain well-resolved ESR spectra of PUFA-derived radical adducts in a mixture of soybean lipoxygenase, PUFA, and the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). However, interpretation of the ESR spectra was complicated by the overlapping of the PUFA-derived alkoxyl radical adduct spectra. In order to understand these spectra, PUFA-derived alkoxyl radical adducts were modeled by various alkoxyl radical adducts. For the first time, we synthesized a wide range of DMPO adducts with primary and secondary alkoxyl radicals. It was found that many ESR spectra previously assigned as DMPO/peroxyl radical adducts based on their close similarity to the ESR spectrum of the DMPO/superoxide radical adduct, in conjunction with their insensitivity to superoxide dismutase, are indeed alkoxyl radical adducts. We have reassigned the PUFA alkylperoxyl radical adducts to their corresponding alkoxyl radical adducts. Using hyperfine coupling constants of model DMPO/alkoxyl radical adducts, the computer simulation of DMPO/PUFA alkoxyl radical adducts was performed. It was found that the trapped, oxygen-centered PUFA-derived radical is a secondary, chiral alkoxyl radical. The presence of a chiral carbon atom leads to the formation of two diastereomers of the DMPO/PUFA alkoxyl radical adduct. Therefore, attempted spin trapping of the PUFA peroxyl radical by DMPO at room temperature leads to the formation of the PUFA alkoxyl radical adduct.     

The interaction of hypochlorite with fatty acid hydroperoxides results in the generation of free radicals

The interaction of hypochlorite with linoleic acid hydroperoxides was studied by the coumarin C-525-enhanced chemiluminescence and ESR spin trapping techniques. Linoleic acid hydroperoxide was obtained in the reaction of lipoxygenase and linoleic acid. Alpha-(4-pyridyl-1-oxyl)-N-tert Butylnitron was used as a spin trap. It was shown that the addition of hypochlorite to the incubation media containing linoleic acid and lipoxygenase resulted in an intensive chemiluminescence flash. The intensity of this flash correlated with the hydroperoxide concentration. The analysis of ESR spectra of spin adducts produced in the reaction of hypochlorite with linoleic acid hydroperoxide showed the presence of O-centered, most likely peroxyl, radical with the splitting constants alphabetaH = 0.260 mT aN = 1.662 mT and C-centered penthyl radical with the splitting constants alphabetaH = 0.260 mT; aN = 1.662 mT. These data suggest that hypochlorite produced by phagocytes in vivo can induce the generation of free O- and C-centered radicals, promoters of free radical processes.     

Formation of acyl radical in lipid peroxidation of linoleic acid by manganese-dependent peroxidase from Ceriporiopsis subvermispora and Bjerkandera adusta.

Lipid peroxidation by managanese peroxidase (MnP) is reported to decompose recalcitrant polycyclic aromatic hydrocabon (PAH) and nonphenolic lignin models. To elucidate the oxidative process, linoleic acid and 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid [13(S)-HPODE] were reacted with MnPs from Ceriporiopsis subvermispora and Bjerkandera adusta and the free radicals produced were analyzed by ESR. When the MnPs were reacted with 13(S)-HPODE in the presence of Mn(II), H2O2 and tert-nitrosobutane (t-NB), the ESR spectrum contained a sharp triplet of acyl radical (aN = 0.81 mT). Formation of acyl radical was also observed in the reactions of Mn(III)-tartrate with 13(S)-HPODE and with linoleic acid, but the latter reaction occurred explosively after an induction period of around 30 min. Reactions of MnP with linoleic acid in the presence of Mn(II), H2O2 and t-NB gave no spin adducts while addition of t-NB after preincubation of linoleic acid with MnP/Mn(II)/H2O2 for 2 h gave spin adducts of carbon-centered (aN = 1.53 mT, aH = 0.21 mT) and acyl (aN = 0.81 mT) radicals. In contrast to linoleic acid, methyl linoleate and oleic acid were not peroxidized by MnP and chelated Mn(III) within a few hours, indicating that structures containing both the 1,4-pentadienyl moiety and a free carboxyl group are necessary for inducing the peroxidation in a short reaction time. These results indicate that MnP-dependent lipid peroxidation is not initiated by direct abstraction of hydrogen from the bis-allylic position during turnover but proceeds by a Mn(III)-dependent hydrogen abstraction from enols and subsequent propagation reactions involving the formation of acyl radical from lipid hydroperoxide. This finding expands the role of chelated Mn(III) from a phenol oxidant to a strong generator of free radicals from lipids and lipid hydroperoxides in lignin biodegradation.     

Electron spin resonance studies on the lipoxygenase reaction by spin trapping and spin labelling methods.

The rate of oxygenation and that of trapping linoleic acid free radicals in the lipoxygenase [EC 1.13.11.12] reaction were measured in the presence of linoleic acid, oxygen, and nitrosobenzene at various concentrations, with a Clark oxygen electrode and ESR spectroscopy. The results were interpreted under the assumption that the free radical of linoleic acid, an intermediate of the lipoxygenase reaction, reacts competitively with oxygen or nitrosobenzene. The oxidation of the iron in the active site of lipoxygenase caused by the spin label reagent, 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinyloxyl, was also observed by ESR- and fluorescence-spectroscopy.     

Lipid peroxyl radical intermediates in the peroxidation of polyunsaturated fatty acids by lipoxygenase. Direct electron spin resonance investigations

Lipid peroxyl radicals resulting from the peroxidation of polyunsaturated fatty acids by soybean lipoxygenase were directly detected by the method of rapid mixing, continuous-flow electron spin resonance spectroscopy. When air-saturated borate buffer (pH 9.0) containing linoleic acid or arachidonate acid was mixed with lipoxygenase, fatty acid-derived peroxyl free radicals were readily detected; these radicals have a characteristic g-value of 2.014. An organic free radical (g = 2.004) was also detected; this may be the carbon-centered fatty acid free radical that is the precursor of the peroxyl free radical. The ESR spectrum of this species was not resolved, so the identification of this free radical was not possible. Fatty acids without at least two double bonds (e.g. stearic acid and oleic acid) did not give the corresponding peroxyl free radicals, suggesting that the formation of bisallylic carbon-centered radicals precedes peroxyl radical formation. The 3.8-G doublet feature of the fatty acid peroxyl spectrum was proven (by selective deuteration) to be a hyperfine coupling due to a gamma-hydrogen that originated as a vinylic hydrogen of arachidonate. Arachidonate peroxyl radical formation was shown to be dependent on the substrate, active lipoxygenase, and molecular oxygen. Antioxidants are known to protect polyunsaturated fatty acids from peroxidation by scavenging peroxyl radicals and thus breaking the free radical chain reaction. Therefore, the peroxyl signal intensity from micellar arachidonate solutions was monitored as a function of the antioxidant concentration. The reaction of the peroxyl free radical with Trolox C was shown to be 10 times slower than that with vitamin E. The vitamin E and Trolox C phenoxyl radicals that resulted from scavenging the peroxyl radical were also detected.     

Isolation and characterization of the initial radical adduct formed from linoleic acid and alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone in the presence of soybean lipoxygenase.

The spin trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) was used to trap the initial radical formed from [U-14C]linoleic acid in the reaction with soybean lipoxygenase. By using low levels of enzyme and relatively short incubation times it was possible to avoid the formation of secondary oxidation products and polymers. The adduct was extracted after methyl esterification, and isolated by a combination of open column chromatography on silicic acid and high pressure liquid chromatography on Spherisorb S5 CN with non-aqueous solvents. The 1:1 POBN-linoleate adduct was characterized by UV, IR and ESR spectra of the appropriate HPLC column fraction, by the ratio of the UV absorption to 14C content, and by mass spectrometry of the reduced (hydroxylamine) form. The results indicated that POBN trapped a linoleic acid carbon-centered radical such that POBN was attached to the fatty acid chain at C-13 or C-9 (two isomers), the linoleate double bonds having become conjugated in the process. The exact locations of the bridges in the two isomers were only tentatively determined. There was no evidence for the presence of oxygen-bridged adducts. The trapped linoleoyl radical adduct provides evidence for the production of a free radical as part of the enzymatic mechanism of soybean lipoxygenase.     

Identification of spin trapped carbon-centered radicals in soybean lipoxygenase-dependent peroxidations of omega-3 polyunsaturated fatty acids by LC/ESR,LC/MS,and tandem MS

With the combined techniques of on-line liquid chromatography/electron spin resonance (LC/ESR) and on-line liquid chromatography/mass spectrometry (LC/MS), we have previously characterized all classes of lipid-derived carbon-centered radicals (*Ld) formed from omega-6 polyunsaturated fatty acids (PUFAs: linoleic acid and arachidonic acid). In the present study, the carbon-centered radicals formed from two omega-3 PUFAs (linolenic acid and docosahexaenoic acid) resulting from their reactions with soybean lipoxygenase in the presence of alpha-[4-pyridyl 1-oxide]-N-tert-butylnitrone (POBN) were investigated using the combination of LC/ESR and LC/MS techniques. A total of 16 POBN trapped carbon-centered radicals formed from the peroxidation of linolenic acid and 11 formed from the peroxidation of docosahexaenoic acid were detected by LC/ESR, identified by LC/MS, and structurally confirmed by tandem mass analysis (MS/MS). The on-line ESR chromatograms and MS chromatograms obtained from two omega-3 PUFAs closely resembled each other not only because the four major beta-scission products, including an ethyl radical and three isomeric pentenyl radicals, were formed from each PUFA, but also because isomeric POBN adducts of lipid dihydroxyallylic radicals from both PUFAs had almost identical chromatographic retention times.     

Lipid radicals: properties and detection by spin trapping

Unsaturated lipids are rapidly oxidized to toxic products such as lipid hydroperoxides, especially when transition metals such as iron or copper are present. In a Fenton-type reaction Fe2+ converts lipid hydroperoxides to the very short-lived lipid alkoxyl radicals. The reaction was started upon the addition of Fe2+ to an aqueous linoleic acid hydroperoxide (LOOH) emulsion and the spin trap in the absence of oxygen. Even when high concentrations of spin traps were added to the incubation mixture, only secondary radical adducts were detected, probably due to the rapid re-arrangement of the primary alkoxyl radicals. With the commercially available nitroso spin trap MNP we observed a slightly immobilized ESR spectrum with only one hydrogen splitting, indicating the trapping of a methinyl fragment of a lipid radical. With DMPO or 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) adducts were detected with carbon-centered lipid radical, with acyl radical, and with the hydroxyl radical. We also synthesized lipophilic derivatives of the spin trap DEPMPO in order to detect lipid radical species generated in the lipid phase. With all spin traps studied a lipid-derived carbon-centered radical was obtained in the anaerobic incubation system Fe2+/LOOH indicating the trapping of a lipid radical, possibly generated as a secondary reaction product of the primary lipid alkoxyl radical formed. Under aerobic conditions an SOD-insensitive oxygen-centered radical adduct was formed with DEPMPO and its lipophilic derivatives. The observed ESR parameters were similar to those of alkoxyl radical adducts, which were independently synthesized in model experiments using Fe3+-catalyzed nucleophilic addition of methanol or t-butanol to the respective spin trap.     

Characterization of the initial carbon-centered pentadienyl radical and subsequent radicals in lipid peroxidation: identification via on-line high performance liquid chromatography/electron spin resonance and mass spectrometry

The previously reported combination of an on-line high-performance liquid chromatography (LC)/electron spin resonance (ESR) system with mass spectrometric analysis (MS) created a unique technique to identify a variety of lipid-derived radicals ((.)L(d)) formed from in vitro lipid peroxidation (Iwahashi et al. [20]). To improve the sensitivity, resolution, and reliability of this method for in vitro and in vivo studies, we have investigated the effects of mobile phase pH, modifiers, and columns on the chromatographic separation of linoleic acid-derived radical adducts. Using tetrahydrofuran (THF) and 0.1% glacial acetic acid (HOAc) in an H(2)O/acetonitrile (ACN) mobile phase greatly increased the resolution and retention reproducibility of lipid radical adducts in LC/ESR. In addition, these modifications allowed the elimination of an ESR tuning problem and the synchronization of UV and ESR detection of radical adducts in on-line LC/ESR, neither of which had been possible previously. Analyte purity was therefore increased, thus increasing the reliability of radical detection via on-line LC/ESR as well as radical identification via MS analysis. For the first time, POBN adducts of linoleic carbon-centered pentadienyl radicals (L(.)) were detected and identified. The optimization of chromatography in the LC/ESR and MS combination provided a reliable and sensitive way for the detection and identification of expected radical adducts in vitro and in vivo.     

Natural killer cell-mediated lysis involves an hydroxyl radical-dependent step

The role of oxygen radicals in lysis of K562 target cells by human natural killer (NK) cells was determined by addition of scavengers of these free radicals. Lysis was greatly reduced under hypoxic conditions. Superoxide dismutase and cytochrome c, scavengers of superoxide anions, and catalase and scavengers of hypochlorite had no effect on lysis. Of 15 hydroxyl radical scavengers tested, 13 inhibited lysis. These were not toxic, because cell morphology and spontaneous chromium release were not affected and preculture with scavengers was not inhibitory. These scavengers differed widely in structure, but degree of inhibition of lysis correlated with their rate constants (k) for reaction with hydroxyl radical (k vs log inhibitor concentration required to decrease lysis by 50%: r = -0.9202, p less than 0.001), showing that inhibition was due to inactivation of the hydroxyl radical. Target cell binding was not reduced at concentrations that inhibited lysis. Inhibitors of the lipoxygenase pathway also decreased lysis, suggesting this pathway to be the source of hydroxyl radicals. In view of the reported requirements for hydroxyl radical-mediated lipid peroxidation for optimal secretory activity in a number of cell types, it appears that the generation of hydroxyl radicals by NK cells is required for delivery of cytotoxic factors.     

Quantification of lipid alkyl radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition

Lipid alkyl radicals generated from polyunsaturated fatty acids via chemical or enzymatic H-abstraction have been a pathologically important target to quantify. In the present study, we established a novel method for the quantification of lipid alkyl radicals via nitroxyl radical spin-trapping. These labile lipid alkyl radicals were converted into nitroxyl radical-lipid alkyl radical adducts using 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmdeltaP) (a partition coefficient between octanol and water is approximately 3) as a spin-trapping agent. The resulting CmdeltaP-lipid alkyl radical adducts were determined by HPLC with postcolumn online thermal decomposition, in which the adducts were degraded into nitroxyl radicals by heating at 100 degrees C for 2 min. The resulting nitroxyl radicals were selectively and sensitively detected by electrochemical detection. With the present method, we, for the first time, determined the lipid alkyl radicals generated from linoleic acid, linolenic acid, and arachidonic acid via soybean lipoxygenase-1 or the radical initiator 2,2'-azobis(2,4-dimethyl-valeronitrile).     

Comparison of the ability of ferric complexes to catalyze microsomal chemiluminescence, lipid peroxidation, and hydroxyl radical generation

The interaction of microsomes with iron and NADPH to generate active oxygen radicals was determined by assaying for low level chemiluminescence. The ability of several ferric complexes to catalyze light emission was compared to their effect on microsomal lipid peroxidation or hydroxyl radical generation. In the absence of added iron, microsomal light emission was very low; chemiluminescence could be enhanced by several cycles of freeze-thawing of the microsomes. The addition of ferric ammonium sulfate, ferric-citrate, or ferric-ADP produced an increase in chemiluminescence, whereas ferric-EDTA or -diethylenetriaminepentaacetic acid (detapac) were inhibitory. The same response to these ferric complexes was found when assaying for malondialdehyde as an index of microsomal lipid peroxidation. In contrast, hydroxyl radical generation, assessed as oxidation of chemical scavengers, was significantly enhanced in the presence of ferric-EDTA and -detapac and only weakly elevated by the other ferric complexes. Ferric-desferrioxamine was essentially inert in catalyzing any of these reactions. Chemiluminescence and lipid peroxidation were not affected by superoxide dismutase, catalase, or competitive hydroxyl radical scavengers whereas hydroxyl radical production was decreased by the latter two but not by superoxide dismutase. Chemiluminescence was decreased by the antioxidants propylgallate or glutathione and by inhibiting NADPH-cytochrome P-450 reductase with copper, but was not inhibited by metyrapone or carbon monoxide. The similar pattern exhibited by ferric complexes on microsomal light emission and lipid peroxidation, and the same response of both processes to radical scavenging agents, suggests a close association between chemiluminescence and lipid peroxidation, whereas both processes can be readily dissociated from free hydroxyl radical generation by microsomes.     

Alkyl free radicals from the beta-scission of fatty acid alkoxyl radicals as detected by spin trapping in a lipoxygenase system

2-Methyl-2-nitrosopropane (tNB)-radical adducts from incubation mixtures of fatty acids and soybean lipoxygenase in borate buffer (pH 9.0) were measured by electron paramagnetic resonance (EPR). In addition to the previously reported six-line signal of secondary carbon-centered radicals (RCHR'), a weak signal submerged in the baseline was detected after the peroxidation phase was finished. We propose that this radical is a decomposition product formed via beta-scission of fatty acid alkoxyl radicals. EPR spectra of tNB-radical adducts formed in mixtures of either linoleic acid, arachidonic acid, or 15-hydroperoxyeicosatetraenoic acid with lipoxygenase exhibited hyperfine structure characteristic of tNB/.CH2CH2-R with hyperfine coupling constants: aN = 17.1 G; aH beta = 11.2 G (2H); and aH gamma = 0.6 G (2H). In the case of linolenic acid, this radical tNB/.CH=CH-R' with hyperfine coupling constants: aN = 17.1 G; aH beta = 10.9 G (2H); aH gamma = 1.1 G; and aH delta = 0.5 G. In accord with the decomposition scheme of hydroperoxides derived from unsaturated fatty acids, the radical adducts tNB/.CH2CH2-R and tNB/.CH2-CH=CH-R' were assigned as the pentyl and 2-pentenyl radicals, respectively.     

Identification of all classes of spin-trapped carbon-centered radicals in soybean lipoxygenase-dependent lipid peroxidations of omega-6 polyunsaturated fatty acids via LC/ESR,LC/MS,and tandem MS

Using the combined techniques of on-line high performance liquid chromatography/electron spin resonance (LC/ESR) and mass spectrometry (MS), we previously identified spin-trapped adducts of all expected carbon-centered lipid-derived radicals ((*)L(d)) formed in linoleic acid peroxidation. In the present study, spin trapped lipid-derived carbon-centered radicals formed from the reactions of two omega-6 polyunsaturated fatty acids (PUFAs: linoleic and arachidonic acids) with soybean lipoxygenase in the presence of alpha-[4-pyridyl 1-oxide]-N-tert-butyl nitrone (POBN) were identified using a combination of LC/ESR and LC/MS. All expected lipid-derived carbon-centered radicals in lipoxygenase-dependent peroxidations of linoleic acid and arachidonic acid were detected and identified by the combination of LC/ESR and LC/MS with confirmation by tandem mass spectrometry (MS/MS). The five classes of (*)L(d) formed from both omega-6 PUFAs including lipid alkyl radicals (L(*)), epoxyallyic radicals (OL(*)), dihydroxyallyic radicals ((*)L(OH)(2)), and a variety of R(*) and (*)RCOOH from beta-scission of lipid alkoxyl radicals, gave distinct retention times: POBN/(*)L(OH)(2) approximately 4-6 min, POBN/R(*) and POBN/(*)RCOOH approximately 8-22 min, POBN/L(*) and PBON/OL(*) approximately 25-36 min. The major beta-scission products in peroxidations of omega-6 PUFAs were the pentyl radicals. The ratio of beta-scission products, however, varied significantly depending on pH, [PUFA], as well as [O(2)].     

Dual role of oxygen during lipoxygenase reactions

Studying the oxygenation kinetics of (19R/S,5Z,8Z,11Z,14Z)-19-hydroxyeicosa-5,8,11,14-tetraenoic acid (19-OH-AA) by rabbit 15-lipoxygenase-1 we observed a pronounced oxygen dependence of the reaction rate, which was not apparent with arachidonic acid as substrate. Moreover, we found that peroxide-dependent activation of the lipoxygenase depended strongly on the oxygen concentration. These data can be described with a kinetic model that extends previous schemes of the lipoxygenase reaction in three essential aspects: (a) the product of 19-OH-AA oxygenation is a less effective lipoxygenase activator than (13S,9Z,11E)-13-hydroperoxyoctadeca-9,11-dienoic acid; (b) molecular dioxygen serves not only as a lipoxygenase substrate, but also impacts peroxide-dependent enzyme activation; (c) there is a leakage of radical intermediates from the catalytic cycle, which leads to the formation of inactive ferrous lipoxygenase. This enzyme inactivation can be reversed by another round of peroxide-dependent activation. Taken together our data indicate that both peroxide activation and the oxygen affinity of lipoxygenases depend strongly on the chemistry of the lipid substrate. These findings are of biological relevance as variations of the reaction conditions may turn the lipoxygenase reaction into an efficient source of free radicals.