High-throughput assay for detection of soybean lipoxygenase-1

A high-throughput assay was developed to detect soybean lipoxygenase 1 (LOX-1) using a multilabel plate reader. The assay was also adapted to a single cell fluorometer. Fluorescein is degraded by linoleic hydroperoxide produced from soybean lipoxygenase and linoleic acid. The decrease in fluorescence is measured over time, and the area-under-the-curve (AUC) is used to quantify the LOX-1 content of soybean extract. A dose-dependent response is seen with varied dilutions of pure LOX enzyme or soybean extracts. Percent recovery was between 97% and 108%, and relative standard deviation was 4.3%. Advantages of the assay include the reduced preparation time of samples and reduced use of reagents in the high-throughput assay. Multiple samples can be measured in a single run with a multilabel plate reader.     

Dual positional and stereospecificity of lipoxygenase isoenzymes from germinating barley (green malt): biotransformation of free and esterified linoleic acid

The lipoxygenase isoenzymes LOX1 and LOX2 from green malt were separated by isoelectric focusing, and their catalytic properties regarding complex lipids as substrates were characterized. The regio- and stereoisomers of hydroperoxy octadecadienoates (HPODE) resulting from LOX1 and LOX2 enzymatic transformations of linoleic acid, methyl linoleate, linoleic acid glycerol esters monolinolein, dilinolein, and trilinolein, and 1-palmitoyl-2-linoleoyl-glycero-3-phosphocholine (PamLinGroPCho) were determined. In addition, biotransformations of polar and nonpolar lipids extracted from malt were performed with LOX1 and LOX2. The results show that LOX2 catalyzes the oxidation of esterified fatty acids at a higher rate and is more regioselective than LOX1. The dual position specificity of LOX2 (9-HPODE:13-HPODE) with trilinolein as the substrate (6:94) was higher than the resultant ratio (13:87) when free linoleic acid was transformed. A high (S)-enantiomeric excess of 13-HPODE was analyzed with all esterified substrates confirming the formation of 13-HPODE through the LOX2 enzyme; however, 9-HPODE detected after LOX2 biotransformations showed (R)-enantiomeric excesses. PamLinGroPCho was oxygenated by LOX1 with the highest regio- and stereoselectivities among the applied substrates.     

Electron spin resonance and luminescence spectroscopic observation and kinetic study of chemical and physical singlet oxygen quenching by resveratrol in methanol

Electron spin resonance (ESR) spectroscopy and near-infrared (NIR) fluorescence spectroscopy were performed to observe singlet oxygen quenching by resveratrol. Resveratrol greatly decreased the 2,2,6,6-Tetramethyl-4-piperidone-N-oxyl radical signal as determined by ESR spectroscopy. Resveratrol also efficiently decreased luminescence emission at 1268 nm as studied with a NIR spectrofluorometer, showing positive evidence of singlet oxygen quenching by resveratrol. The total singlet oxygen quenching rate constant (kr+kq) of resveratrol in methanol was determined to be 2.55×10(7) M(-1) s(-1). The singlet oxygen chemical quenching rate constant (kr) of resveratrol was calculated by measuring its reaction rate with singlet oxygen relative to that of α-terpinene in the same solution under light illumination. The kr value of resveratrol was 1.15×10(6) M(-1) s(-1). The percent partition of chemical quenching over total singlet oxygen quenching (kr×100)/(kr+kq) for resveratrol was 5.11%. The results showed that resveratrol quenches singlet oxygen almost exclusively through the mechanism of physical quenching. Resveratrol showed a protective activity similar to that of BHA on the methylene blue sensitized photooxidation of α-terpinene. This unambiguously explains the mechanism of how resveratrol protects tissues and cells in biological systems or important nutrients in food systems against their photosensitized oxidations.     

Lipoxygenase involvement in ripening strawberry

The enzymatic activity, subcellular localization, and immunolocalization of plant lipoxygenase (LOX) in strawberry fruits (Fragaria x ananassa, Duch) were investigated. Chemical and enzymatic properties of LOX have been characterized, and the LOX capability of oxygenating free and esterified unsaturated fatty acids into C6 volatile aldehydes has been confirmed. Fruits at unripe, turning, and ripe stages were analyzed for LOX activity and protein localization by Western blots, two-dimensional electrophoresis, and immunolocalization analyses. The ability of strawberry tissues to in vivo metabolize linolenic acid or linoleic acid into C6 volatile aldehydes and the LOX products was also analyzed. Analysis of strawberry proteins showed that a number of LOX forms, corresponding to at least two mobility groups of approximately 100 and 98 kDa and pI values ranging between 4.4 and 6.5, were present. Confocal and electron microscopy analyses support the idea that LOX proteins are associated to lipid-protein aggregates. Both exogenously supplied linoleate and linolenate were converted into hexanal and trans-2-hexenal at the three fruit-ripening stages. Our experiments suggest the presence of different LOX isoforms in strawberry fruits and that the lipoxygenase-hydroperoxide lyase pathway plays a role in converting lipids to C6 volatiles during ripening.     

Purification and identification of linoleic acid hydroperoxides generated by soybean seed lipoxygenases 2 and 3

It has been known that lipoxygenase (LOX) isozymes exhibit differences in product formation, but most product information to date is for LOX 1 among soybean (Glycine max) LOX isozymes. In this study, LOXs 2 and 3 were purified and used to generate hydroperoxide (HPOD) products in an in vitro system using linoleic acid as a substrate in the presence of either air or O2. The products were analyzed to determine their stereoisomeric characteristics. The control (no enzyme) showed only low levels of hydroperoxide production and no stereoisomeric specificity. LOX 2 shows high specificity in product formation, producing roughly 4 times more 13-HPOD than 9-HPOD, nearly all of which was 13-S(Z,E)-HPOD. LOX 3 produced more 9-HPOD than 13-HPOD at approximately a 2:1 ratio. No single stereoisomer was predominant among LOX 3 products. These results demonstrate that different isozymes of LOX have characteristic product profiles in in vitro reactions.     

Sclerotiorin, a novel inhibitor of lipoxygenase from Penicillium frequentans

Foods rich with unsaturated fatty acids are prone to enzymatic and nonenzymatic lipid peroxidation; lipoxygenase, a metalloenzyme and a free radical former, oxidizes polyunsaturated fatty acids and is one of the key enzymes in lipid oxidation. Here, we report sclerotiorin, purified from the fermented broth of Penicillium frequentans, as a potent reversible, uncompetitive inhibitor against soybean lipoxygenase-1 (LOX-1) with a half-maximal value (IC50) of 4.2 microM. The inhibitor also showed an antioxidant property by scavenging free radical with an ED50 of 0.12 microM; in addition, nonenzymatic lipid peroxidation was inhibited with a PD50 value of 64 microM and did not show metal chelation. The observations made in this study suggest that sclerotiorin possibly inhibits LOX in two ways: one, by interacting with the enzyme-substrate complex, and two, as an antioxidant by quenching or trapping the free radical intermediates formed in the enzyme reaction. Sclerotiorin compares well with other known natural and synthetic lipoxygenase inhibitors.     

Purification and characterization of soluble Cichorium intybusVar. silvestre lipoxygenase

A water-soluble lipoxygenase enzyme (EC 1.13.11.12; LOX) occurring in the red cultivar produced in the geographical area of Chioggia (Italy) of Cichorium intybus var. silvestre was isolated and characterized. The molecular mass of the enzyme was estimated to be 74,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography. The isoelectric point was pH 6.85. The optimum values of pH, ionic strength, and temperature, shown by isoresponse surface calculated by a randomized multilevel factorial design, were 7.58, 30 mM, and 38.5 degrees C, respectively. The enzyme showed high specificity toward linoleic acid, and the study of the variation of linoleic acid concentration between 30 and 300 microM, in the presence of Tween 20 at a concentration lower than the critical micelle concentration (0.01 v/v), resulted in a typical Michaelis-Mentem curve with KM and Vmax values of 1.49 x 10(-4) M and 2.049 microM min(-1) mg(-1), respectively. The biochemical properties, the kinetic parameters found, and the carotene-bleaching activity shown in aerobic conditions seem to indicate that the isolated enzyme is a lipoxygenase type III according to the indications given for soybean isoenzymes.     

Oxygenation of arachidonoyl lysophospholipids by lipoxygenases from soybean, porcine leukocyte, or rabbit reticulocyte

Oxygenation of arachidonoyl lysophosphatidylcholine (lysoPC) or arachidonoyl lysophosphatidic acid (lysoPA) by lipoxygenase (LOX) was examined. The oxidized products were identified by HPLC/UV spectrophotometry/mass spectrometry analyses. Straight-phase and chiral-phase HPLC analyses indicated that soybean LOX-1 and rabbit reticulocyte LOX oxygenated arachidonoyl lysophospholipids mainly at C-15 with the S form as major enantiomer, whereas porcine leukocyte LOX oxygenated at C-12 with the S form. Next, the sequential exposure of arachidonoyl-lysoPC to soybean LOX-1 and porcine leukocyte LOX afforded two major isomers of dihydroxy derivatives with conjugated triene structure, suggesting that 15(S)-hydroperoxyeicosatetraenoyl derivatives were converted to 8,15(S)-dihydroxyeicosatetraenoyl derivatives. Separately, arachidonoyl-lysoPA, but not arachidonoyl-lysoPC, was found to be susceptible to double oxygenation by soybean LOX-1 to generate a dihydroperoxyeicosatetraenoyl derivative. Overall, arachidonoyl lysophospholipids were more efficient than arachidonic acid as LOX substrate. Moreover, the catalytic efficiency of arachidonoyl-lysoPC as substrate of three lipoxygenases was much greater than that of arachidonoyl-lysoPA or arachidonic acid. Taken together, it is proposed that arachidonoyl-lysoPC or arachidonoyl-lysoPA is efficiently oxygenated by plant or animal lipoxygenases, C12- or C15-specific, to generate oxidized products with conjugated diene or triene structure.     

Formation of volatile compounds in model experiments with crude leek (Allium ampeloprasum Var. Lancelot) enzyme extract and linoleic acid or linolenic acid

Three continuous assays are described for lipoxygenase (LOX), hydroperoxide lyase (HPL) and alcohol dehydrogenase (ADH) in leek tissue. The catalytic activity of LOX showed significant difference (significance level 5%) between linolenic acid (9.43 x 10(-)(4) katals per kg protein) and linoleic acid (2.53 x 10(-)(4) katals per kg protein), and the pH-optimum of LOX was 4.5-5.5 against linoleic acid. The catalytic activity of HPL was statistically the same for 9-(S)-hydroperoxy-(10E,12Z)-octadecadienoic acid (1.01 x 10(-)(2) katals per kg protein) and 13-(S)-hydroperoxy-(9Z,11E)-octadecadienoic acid (7.69 x 10(-)(3) katals per kg protein). ADH showed a catalytic activity of 5.01 x 10(-)(4) katals/kg of protein toward hexanal. Model experiments with crude enzyme extract from leek mixed with linoleic acid or linolenic acid demonstrated differences in the amount of produced aroma compounds. Linoleic acid resulted in significantly most hexanal, heptanal, (E)-2-heptenal, (E)-2-octenal, (E,E)-2,4-decadienal, pentanol, and hexanol, whereas linolenic acid resulted in significantly most (E)-2-pentenal, (E)-2-hexenal, (E,Z)-2,4-heptadienal, (E,E)-2,4-heptadienal, and butanol. Leek LOX produced only the 13-hydroperoxide of linoleic acid and linolenic acid.     

Lipoxygenase from banana leaf: purification and characterization of an enzyme that catalyzes linoleic acid oxygenation at the 9-position

The objective of the present study was to purify and characterize the lipoxygenase (LOX) from banana leaf (Giant Cavendishii, AAA), an unutilized bioresource. LOX was extracted, isolated, and purified 327-fold using 25-50% saturation of ammonium sulfate fractionation, hydroxyapatite column separation, and gel filtration on Superdex 200. The molecular mass of the purified LOX was 85 kDa, K(m) was 0.15 mM, and V(max) was 2.4 microM/min.mg using linoleic acid as substrate. Triton X-100 was required in the extraction medium; otherwise, no LOX activity was detected. LOX activity increased with the concentration of Triton X-100 with an optimum at 0.1%. The optimal pH of the purified LOX from banana leaf was 6.2, and optimal temperature was 40 degrees C. The LOX showed the highest reactivity toward 18:2 followed by 18:3 and 20:4. A very low reaction rate was observed toward 20:5 and 22:6. On the basis of retention time in normal phase HPLC, the products of 18:2 or 18:3 catalyzed by purified LOX were hydroperoxyoctadecadienoic acid or hydroperoxyoctadecatrienoic acid. It seems that 9-LOX is the predominant enzyme in banana leaf. Banada leaf dried at 110 degrees C for 2 h developed algal aroma. Banana leaf extract stored at 10 degrees C for 12 h formed an oolong tea-like flavor. Banana leaf extract reacted with 18:2 or soybean oil pretreated with bacterial lipase produced green and melon-like aroma, whereas the same reaction with 18:3 produced a sweet, fruity, cucumber-like flavor note.     

A Practical Spectrophotometric Approach for the Determination of Lipoxygenase Activity of Durum Wheat

In this study, a practical spectrophotometric approach was used to determine the hydroperoxidation activity of durum wheat lipoxygenase (LOX). As stated in the related literature, the buffered linoleic acid solution used as the reaction medium is not optically clear enough at neutral and lower pH values due to its limited solubility. In our study, the optical clarity was obtained by the formation of sodium-salt of unreacted linoleic acid just before absorbance measurement. The durum wheat LOX was characterized in terms of pH and temperature optima as well as kinetic parameters. The maximum linoleic acid hydroperoxidation activities were determined at pH 5.0 and 6.5 and at 40°C This result can be considered as evidence for the presence of at least two LOX isoforms with respective optima at pH 5.0 and 6.5 in the crude durum wheat extract. The Michaelis constant (K_m) and maximum hydroperoxidation) activity rate (V_max) of durum wheat LOX for linoleic acid were estimated) to be 0.131 ± 0.019 mM and 42.37 ± 3.32 units/mg of protein/min, respectively. The method seems to be useful for the determination of LOX activity in durum wheat and its milling fractions.     

Free radical scavenging properties of conjugated linoleic acids.

Conjugated linoleic acids (CLA) were investigated for free radical scavenging properties against the stable 2,2-diphenyl-1-picryhydrazyl radical (DPPH.) by electron spin resonance (ESR) spectrometry and spectrophotometric methods. ESR results demonstrated that CLA directly reacted and quenched free DPPH radicals in benzene, while spectrophotometric analysis showed the radical scavenging capacity of CLA in ethanol. Dose and time effects of CLA-DPPH. reactions were observed in both tests. The ED(50) of CLA was 18 mg/mL under experimental conditions. CLA are much weaker radical scavengers as compared to vitamin E, vitamin C, and BHT. Kinetics of CLA-DPPH. reactions was different to that of linoleic acid (LA)-DPPH. reactions. CLA reacted and quenched DPPH radicals at all tested levels without a lag phase, while LA had a lag phase and showed no radical quenching activity at levels of 5-80 mg/mL in 30 min. These data indicated that CLA can provide immediate protection against free radicals, but LA cannot.     

Co-oxidation of beta-carotene catalyzed by soybean and recombinant pea lipoxygenases

A number of products including apocarotenal, epoxycarotenal, apocarotenone, and epoxycarotenone generated by lipoxygenase (LOX) catalyzed co-oxidation of beta-carotene have been tentatively identified through the use of GC/MS and HPLC combined with photodiode array detection. Because of the large number of high molecular weight products detected and their probable chemical structures, a co-oxidation mechanism is proposed that involves random attack along the alkene chain of the carotenoid by a LOX-generated linoleoylperoxyl radical. It is suggested that a direct release from the enzyme of the radical, which initiates the co-oxidation of beta-carotene, is greater for pea LOX-3 than for pea LOX-2 or soybean LOX-1. It is proposed that further products may be formed by free radical propagated reactions and that the formation of 1,10- and 1,14-dicarbonyl compounds may arise by secondary oxidation of the primary products.     

Inhibition of fish gill lipoxygenase and blood thinning effects of green tea extract

The objective of the present study was to determine whether green tea extracts are inhibitory to lipid oxidations catalyzed by lipoxygenase (LOX) and hemoglobin (Hb) using fish as an animal model. Green tea was extracted with water. LOX was extracted from the gills of grey mullet and tilapia, respectively. The LOX activity was determined using chemiluminescence and high-pressure liquid chromatography. The green tea extract showed inhibitory effects on both LOX-catalyzed and Hb-catalyzed oxidation of arachidonic acid and linoleic acid. Blood thinning effects were observed ex vivo by mixing the green tea extract with fish red blood cells and showed that the flow behavior of fish blood becomes closer to the Newtonian type with a thinner consistency. Similar effects were found on tilapia and grey mullet.     

Regulation of lipoxygenase activity by polyunsaturated lysophosphatidylcholines or their oxygenation derivatives

Lysophosphatidylcholines (lysoPCs) have been known to play a role as lipid mediators in various cellular responses. In this study, we examined whether lysoPC containing linoleoyl, arachidonoyl, or docosahexaenoyl groups or their peroxy derivatives affect lipoxygenase (LOX)-catalyzed oxygenation of native substrates. First, arachidonoyl lysoPC and docosahexaenoyl lysoPC were found to inhibit potato 5-LOX-catalyzed oxygenation of linoleic acid (LA) in a noncompetitive type with Ki values of 0.38 and 1.90 microM, respectively. Likewise, arachidonoyl lysoPC and docosahexaenoyl lysoPC also inhibited 5-LOX activity from rat basophilic leukemia cells-2H3 (RBL-2H3) with IC50 values (50% inhibitory concentration) of 18.5 +/- 3.06 and 30.6 +/- 1.06 microM, respectively. Additionally, arachidonoyl lysoPC and docosahexaenoyl lysoPC also inhibited 15-LOX activity from RBL-2H3 with IC50 values of 16.6 +/- 1.3 and 24.1 +/- 2.4 microM, respectively. In a separate experiment, where lysoPC peroxides were tested for the effect on soybean LOX-1, 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoyl lysoPC and 17(S)-hydroperoxy-4,7,10,13,15,19-docosahexaenoyl lysoPC potently inhibited soybean LOX-1 activity with Ki values of 6.8 and of 1.54 microM, respectively. In contrast, 13(S)-hydroperoxy-9,11-octadecadienoyl lysoPC was observed to stimulate soybean LOX-1-catalyzed oxygenation of LA markedly with AC50 value (50% activatory concentration) of 1.5 microM. Taken together, it is proposed that lysoPCs containing polyunsaturated acyl groups or their peroxy derivatives may participate in the regulation of LOX activity in biological systems.     

Electron spin resonance studies of free radicals in gamma-irradiated soybean paste.

Free radicals in gamma-irradiated soybean paste were investigated by electron spin resonance (ESR) spectroscopy to determine the effect of temperature (77-296 K) and moisture content (1-54%) of samples irradiated at high dose (1-40 kGy). The samples were kept in liquid nitrogen (77 K) during irradiation and subsequent ESR measurements. The spectra shown at 77 K consisted of the hydrogen atom lines at low and high field and complicated symmetric spectrum. By increasing the microwave power, the line shape of ESR spectra altered, which indicated the detection of different paramagnetic centers at different microwave powers. In saturation curves, it was possible to select four types of spectra components which were different in their relaxation times. By the different irradiation doses, the change in free radical concentration showed a curvilinearly increasing relationship with irradiation dose in wet samples, whereas a proportional relationship was observed with dried samples. This might indicate that the indirect process of free radical formation was involved with the existence of free water radicals in the wet samples.     

Effect of soybean lipoxygenase on volatile generation and inhibition of Aspergillus flavus mycelial growth

Volatiles generated from lipoxygenase (LOX) normal and LOX deficient soybean (Glycine max) varieties with and without added lipase inhibited Aspergillus flavus mycelial growth and aflatoxin production. Soybean volatiles were analyzed using a solid phase microextraction (SPME) method combined with gas chromatography-mass spectrometry (GC-MS). Twenty-one compounds, including 11 aldehydes, three alcohols, four ketones, one furan, one alkane, and one alkene were detected in the LOX normal soybean line. However, only nine volatile compounds were observed in the LOX deficient soybean variety. The antifungal aldehydes hexanal and (E)-2-hexenal were observed in both LOX normal and LOX deficient lines and were detected at significantly higher amounts in soybean homogenate with added lipase. These aldehydes may be formed through alternate pathways, other than the LOX pathway, and may account for the inhibition of A. flavus growth observed. Other volatiles detected, particularly the ketones and alcohols, may contribute to the antifungal activity observed in both LOX normal and LOX deficient soybean lines. These results suggest that other factors, other than LOX activity, may better explain why soybeans are generally not as severely affected by A. flavus and aflatoxin contamination as other oilseed crops.     

Purification and partial characterization of lipoxygenase from desert truffle (Terfezia claveryi Chatin) ascocarps

A lipoxygenase from Terfezia claveryi Chatin ascocarp, a mycorrhizal hypogeous fungus, is described for the first time. The higher proportion of PUFA in T. claveryi ascocarps makes lipid rancidity the main factor limiting its storage life. Thus, the studies on LOX from T. claveryi are important because this enzyme, among other roles, may be involved in an alteration of lipids leading to consumer rejection. The enzyme has been purified to apparent homogeneity by phase partitioning in the presence of Triton X-114, followed by two steps of cation-exchange chromatography. The purified T. claveryi LOX preparation consisted of a single major band with an apparent molecular mass of 66 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymic activity exhibited a strong specificity toward linoleic and linolenic acids as substrates, while only 32% activity was observed using gamma-linolenic acid. The pH optimum of this enzyme was pH 7.0. When the enzyme reacted with linoleic acid, it produced a single peak, which comigrated with standard 13-hydroperoxy-octadecadienoic acid; 13-hydroperoxy-octadecatrienoic acid was produced during the reaction with linolenic acid.     

Inhibition of linoleic acid oxidation by interaction with a protein-rich oat fraction

A protein-rich fraction from oat was found to protect linoleic acid against oxidation in an aqueous suspension containing soybean lipoxygenase-1 and micellar linoleic acid. In this system the oat fraction reduced the initial oxidation rate of linoleic acid by 50% when the oat fraction/linoleic acid ratio was 5:1 (w/w). The oat fraction did not act on the lipoxygenase enzyme but reduced the concentration of linoleic acid that serves as a substrate for lipoxygenase-1. To achieve the reduction in the oxidation rate a contact between linoleic acid and the oat fraction was required. The efficiency of the protection was dependent on the duration of this contact: the maximum protection was reached after a 5-min incubation period. However, total cessation of oxidation was not reached with any concentration of the oat fraction, indicating that the oxidizible and non-oxidizible forms of linoleic acid are in equilibrium. Because lipoxygenase-1 prefers the monomeric form of the substrate, the present findings agree with the hypothesis that the oat fraction reduces the concentration of monomolecular form of substrate. In most food systems monomolecular free linoleic acid is liberated slowly and at relatively low concentrations, therefore, even a small amount of the oat fraction would guard the system from oxidative deterioration.     

Improved amperometric method for the rapid and quantitative measurement of lipoxygenase activity in vegetable tissue crude homogenates

An improved amperometric method for rapid (2 min) quantitative determination of lipoxygenase (LOX) activity in vegetable tissue crude homogenates is presented. Measured LOX activity was linear (R(2) > 0.99) throughout the entire activity range for green bean and for corn below 70% activity. The resolution was 0.4% or 1.11 micromol L(-1) s(-1) of oxygen. The limit of detection was 3.43 micromol L(-1) s(-1) of oxygen. The amperometric method was improved by encapsulating linoleic acid (LA) in beta-cyclodextrin (CD) resulting in a stable substrate-buffer solution at a pH below 8.0. Ethanol and Tween 20 were not effective in solubilizing high LA concentrations required by the assay. A prototype benchtop instrument with the potential for use in an industrial environment is also presented.