Dioxygenation Product Research Articles

Oxidation of Linoleyl Alcohol by Potato Tuber Lipoxygenase: Possible Mechanism and the Role of Carboxylic Group in Substrate Binding

We have studied the aerobic oxidation of linoleyl alcohol (LAL) by potato tuber lipoxygenase in the presence of 0.02% (w/v) non-ionic detergent Lubrol PX (and its analog C12E10) and 0.1 mM sodium dodecyl sulfate to investigate the role of carboxylic group in substrate binding. While the enzyme displayed a comparable affinity toward LA and LAL, the rate of LAL oxidation was approximately one-fourth of that of linoleic acid. The pH-profile of the reaction suggests that the rate of LAL oxidation is controlled by two ionizable groups with pKavalues of 5.3 and 7.5, with optimal pH being 6.4±0.1. Since LAL is not ionizable at this pH, we conclude that the rate of the reaction is controlled by two ionogenic groups of the enzyme. The primary dioxygenation product(s) of LAL had a maximal absorbance at 233±1 nm. The products have been isolated, catalytically hydrogenated with H2over Pd on carbon, and analyzed by GC-MS. Two major equimolar products were found to be 9- and 13-hydroxystearyl alcohols, indicating that 9- and 13-hydroperoxylinoleyl alcohols are the primary dioxygenation products. Based on these results we propose that the carboxyl group of polyunsaturated fatty acid may not be involved in substrate binding of potato tuber lipoxygenase.

Macrophage-mediated candidacidal activity is augmented by exposure to eosinophil peroxidase: a paradigm for eosinophil-macrophage interaction.

Various disease states are associated with eosinophilia and the release of eosinophil peroxidase (EPO) into the microenvironment. The present study targets the effects of low levels of EPO on macrophage (M phi) phagocytosis and intracellular killing of Candida albicans as well as M phi oxidative activity measured as the luminescence product of luminol dioxygenation. Resident murine peritoneal M phi were exposed to various concentrations of EPO. Chemiluminescence data indicate that nanomolar concentrations of EPO markedly enhanced the dioxygenation activity (respiratory burst) of M phi. In other studies, the exposure of M phi to 0.17 microM EPO for 10 min. enhanced M phi-mediated candidacidal activity 10 fold. The above data indicate that EPO enhances certain M phi functions. Also the results illustrate a previously un-recognized interaction between eosinophils and M phi and implicate yet another possible role for EPO in host defenses against disease.

Oxidation of naphthenoaromatic and methyl-substituted aromatic compounds by naphthalene 1,2-dioxygenase

Oxidation of acenaphthene, acenaphthylene, and fluorene was examined with recombinant strain Pseudomonas aeruginosa PAO1(pRE695) expressing naphthalene dioxygenase genes cloned from plasmid NAH7. Acenaphthene underwent monooxygenation to 1-acenaphthenol with subsequent conversion to 1-acenaphthenone and cis- and trans-acenaphthene-1,2-diols, while acenaphthylene was dioxygenated to give cis-acenaphthene-1,2-diol. Nonspecific dehydrogenase activities present in the host strain led to the conversion of both of the acenaphthene-1,2-diols to 1,2-acenaphthoquinone. The latter was oxidized spontaneously to naphthalene-1,8-dicarboxylic acid. No aromatic ring dioxygenation products were detected from acenaphthene and acenaphthylene. Mixed monooxygenase and dioxygenase actions of naphthalene dioxygenase on fluorene yielded products of benzylic 9-monooxygenation, aromatic ring dioxygenation, or both. The action of naphthalene dioxygenase on a variety of methyl-substituted aromatic compounds, including 1,2,4-trimethylbenzene and isomers of dimethylnaphthalene, resulted in the formation of benzylic alcohols, i.e., methyl group monooxygenation products, which were subsequently converted to the corresponding carboxylic acids by dehydrogenase(s) in the host strain. Benzylic monooxygenation of methyl groups was strongly predominant over aromatic ring dioxygenation and essentially nonspecific with respect to the substitution pattern of the aromatic substrates. In addition to monooxygenating benzylic methyl and methylene groups, naphthalene dioxygenase behaved as a sulfoxygenase, catalyzing monooxygenation of the sulfur heteroatom of 3-methylbenzothiophene.

Mechanism of lipoxygenase inactivation by the linoleic acid analogue octadeca-9,12-diynoic acid.

During the irreversible inactivation of soybean Fe(III)-lipoxygenase [Fe(III)-LOX] by octadeca-9,12-diynoic acid (ODYA), significant quantities of 11-oxooctadeca-9,12 diynoic acid (11-oxo-ODYA) are formed [Nieuwenhuizen, W. F., et al. (1995) Biochemistry 34, 10538-10545]. To elucidate the inactivation mechanism, a quantitative study into the relationship between the inactivation and 11-oxo-ODYA formation was carried out. The following observations were made (1) LOX (0.84 microM) was completely inactivated by 10 to 80 microM ODYA. However, at ODYA concentrations greater than 100 microM, LOX was only partially inactivated, and there was no inactivation at all at ODYA concentrations above 750 microM. The average number of turnovers in which 11-oxo-ODYA was formed increased from 1.2 to 12 when the ODYA concentration increased from 1 to 50 microM and then decreased again to 1.2 at 1000 microM ODYA. (2) The enzyme that was not irreversibly inactivated by ODYA was in the Fe(III) form at ODYA concentrations below 10 microM but in the Fe(II) form at ODYA concentrations greater than 100 microM. (3) In the presence of 750 microM ODYA and 25 microM 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid, all of the enzyme was inactivated. On the basis of these results, it is proposed that the dioxygenation product of ODYA is 11-hydroperoxyoctadeca-9,12-diynoic acid (11-HP-ODYA), which can convert Fe(II)-LOX into its Fe(III) form. However 11-HP-ODYA is converted into 11-oxo-ODYA, which cannot perform the oxidation. It is proposed that the inactivating agent is either 11-HP-ODYA or the 11-peroxy-octadeca-9,12-diynoic acid radical (11-peroxy-ODYA radical), formed from the ODYA radical and O2. The oxidation of Fe(II)-LOX into its Fe(III) form as well as the inactivation of Fe(III)-LOX is competitively inhibited by ODYA

Lipoxin A4 induces neutrophil-dependent cytotoxicity for human endothelial cells.

The authors have assessed the capacity of neutrophil granulocytes (PMN) to kill cultured human umbilical-vein endothelial cells (HUVEC) in vitro (as release of 51Cr) in response to the recently described double dioxygenation product of arachidonic acid, lipoxin A4 (LXA4). LXA4 conferred a marked cytotoxicity, whereas formyl-methionyl-leucyl-phenylalanine (fMLP) was less potent. The LXA4 and fMLP effects were dose dependent, with a maximum at 100 nM (which caused 2.7- and 2.3-fold increases of 51Cr release, respectively, relative to buffer-treated controls). The LXA4 and fMLP responses increased with the PMN concentration, depended on the fetal calf serum concentration, incubation temperature and duration and the presence of calcium and magnesium ions.

Isolation and Characterization of (+)-1,1a-Dihydroxy-1-hydrofluoren-9-one Formed by Angular Dioxygenation in the Bacterial Catabolism of Fluorene

Transformation of fluorene by washed cells of fluorene-grown Pseudomonas sp. F274 yielded 1,1a-dihydroxy-1-hydrofluoren-9-one (up to 100 mg/1) as the stable product of angular dioxygenation of 9-fluorenone. Structural identity of the angular keto-diol was established by 13C- and 1H-NMR, gas chromatography- and direct probe-mass spectrometry. Definitive assignment of 1,1a-dioxygenation, but not 4,4a-, was based on the isolation and rigorous identification of 1-hydroxy-fluoren-9-one as the exclusive product of acidic dehydration. Chiral 1H-NMR analysis and optical rotation of isolated 1,1a-dihydroxy-l-hydrofluoren-9-one ([α] D = +132.1°) are indicative of a single enantiomer with an inferred cis-stereochemistiy of the hydroxyl groups. This compound is evidently an intermediate of fluorene catabolism by this strain and not a dead-end product because its formation is transient in washed cell incubations and ultimately it is completely consumed with the formation of acidic metabolites.

11-Hydroperoxyeicosatetraenoic acid is the major dioxygenation product of lipoxygenase isolated from hairy root cultures of Solanum tuberosum

The profile of primary dioxygenation products of arachidonic acid catalyzed by lipoxygenase isolated from hairy root cultures of Solanum tuberosum treated with a fungal elicitor was compared to that obtained for the enzyme from potato tubers. 11-Hydroperoxyeicosatetraenoic acid (11-HPETE) was the most abundant dioxygenation product formed followed by 8- and 5-HPETEs in the decreasing order of abundance. In contrast, 5-HPETE is the predominant oxidation product of lipoxygenase from potato tubers. Differences in the defense requirements of storage tuber as compared to roots may be the basis of the differences in regiospecificity demonstrated in this work.

Angioplasty triggers intracoronary leukotrienes and lipoxin A4. Impact of aspirin therapy.

Percutaneous transluminal coronary angioplasty (PTCA) is a widely used and important method of reperfusing coronary arteries. However, it is also associated with serious complications such as acute reocclusion and accelerated restenosis. The factors as well as the mechanisms involved in PTCA-associated complications remain to be fully elucidated. Because peptidoleukotrienes and lipoxins are potent vasoactive compounds, the formation of which is not inhibited by aspirin (ASA) treatment in vitro, it is possible that these eicosanoids are involved in PTCA-associated untoward events. To test this, we determined the intracoronary levels of peptidoleukotrienes and lipoxin A4 (LXA4) as well as thromboxane (TX) and 5S,12S-dihydroxyeicosatetraenoic acid (5S,12S-DiHETE; a product of double dioxygenation) after plaque rupture and evaluated the impact of ASA therapy. PTCA was performed on 12 patients with coronary artery disease, six undergoing ASA therapy and six without ASA therapy, for at least 2 weeks before PTCA. By means of a technique that permitted sampling of intracoronary blood at the plaque site in situ, samples were taken immediately before and 10 seconds after initiation of plaque rupture. Lipoxygenase (LO)-derived products, including LXA4 and 5S,12S-DiHETE, and a marker of cyclooxygenase activity, i.e., TXB2, were quantitated after extraction and chromatography using deuterium-labeled internal standards and electron capture negative ion chemical ionization mass spectrometry. Peptidoleukotrienes (LTC4 and LTD4) were quantitated after reverse-phase high-performance liquid chromatography coupled with radioimmunoassay. Intracoronary blood taken before PTCA showed no detectable levels of these eicosanoids (the minimum limits of detection were within the picomole range). In contrast, each of these LO products was detected after PTCA. Patients undergoing ASA treatment showed elevated levels of each LO product examined compared with those not receiving ASA. Eicosanoid levels were (mean +/- SEM): LTC4, 7.10 +/- 1.22 ng/ml (ASA) versus 0.48 +/- 0.10 ng/ml; LTD4, 4.92 +/- 0.56 ng/ml (ASA) versus 1.17 +/- 0.48 ng/ml; LXA4, 24.98 +/- 4.11 ng/ml (ASA) versus 15.83 +/- 2.43 ng/ml; 5S,12S-DiHETE, 19.47 +/- 3.98 ng/ml (ASA) versus 11.98 +/- 1.83 ng/ml; TXB2, complete blockage (ASA) versus 31.04 +/- 7.38 ng/ml (p less than 0.05 for LTC4 and LTD4). To distinguish between dilatation of whole blood versus dilatation of whole blood and atheroma for contribution of eicosanoids, we also monitored their formation in Gore-tex grafts. Upon balloon inflation, TXB2 was generated, but LO products were not detected. In contrast, injection of platelet- and leukocyte-directed agonists within the graft led to both peptidoleukotriene and lipoxin formation. The results indicate that PTCA triggers the intraluminal release of peptidoleukotrienes and LXA4 and that ASA therapy enhances their appearance in intracoronary blood. In addition, they provide direct evidence for LO products (LTC4, LTD4, and LXA4) in a local milieu in vivo. Moreover, the presence of the double dioxygenation product 5S,12S-DiHETE (a potential marker of 5- and 12-LO interactions) suggests that transcellular metabolic events can contribute to eicosanoid formation in vivo.

Positional specificity of a Lupinus Albus lipoxygenase in relation to enzyme concentration and effect of a double dioxygenation product of arachidonic acid

When arachidonic acid was incubated with Lupinus Albus lipoxygenase, a trienoic fatty acid, 8,15-dihydroperoxy-5,9,11,13-eicosatetraenoic acid was formed in addition to the three HPETEs, 15-HPETE, 8-HPETE and 5-HPETE. The formation of the two major monohydroperoxy acids (15- and 5-HPETE) has been shown to depend on enzyme concentration. At low enzyme concentration (< 1 unit), Lupinus Albus lipoxygenase displays its highest regiospecificity at pH 5.8, resulting in the formation of 5-HPETE as the major product (80%). As the enzyme concentration increased, the proportion of 5-HPETE decreased, while the one of 15-HPETE increased. On the other hand, the preincubation of the enzyme with 8,15-diHPETE led to the gradual inactivation of the lipoxygenase activity. The inhibitory effect of 8,15-diHPETE was abolished by using high enzyme concentration. Based on these observations, it is proposed that the trienoic fatty acid, 8,15-diHPETE stommed from further lipoxygenation of 15-HPETE, blocked the formation of the 15(S) hydroperoxide by Lupinus Albus lipoxygenase but not the formation of 5(S) hydroperoxide.

Conversion of dihomo-γ-linolenic acid to mono- and dihydroxy acids by potato lipoxygenase: Evidence for the formation of 8,9-leukotriene A 3

Evidence for the formation of a positional isomer of leukotriene (LT) C 3 (8,9-LTC 3) from dihomo-γ-linolenic acid has been published (Hammarström, S. J. Biol. Chem. 256, 7712–7714, 1981). This report describes the conversion of dihomo-γ-linolenic acid to a postulated intermediate in former reaction, 8,9-LTA 3, by purified lipoxygenase from potato tubers. 8( S)-Hydroperoxyeicosatrienoic acid (8( S)-HPETrE) was the most abundant dioxygenation product formed followed by 11-, 15-, and 12-HPETrEs (in decreasing order of abundance). In addition, 8( S),15( S)- plus 8( S), 15( R)-dihydroperoxyeicosatetraenoic acid (DiHPETrE) (EZE), and 8( S),15( S)- plus 8( S),15( R)-dihydroxy-eicosatetraenoic acid (DiHETrE) (EEE) were generated. Under anaerobic conditions only the latter two isomers of 8,15-DiHETrE (EEE) were obtained from 8-HPETrE. The results suggest that 8,9-LTA 3 is synthesized by the sequential action of 8- and 11-lipoxygenase activities associated with the potato enzyme.

Formation of 6,13-dihydroxyoctadecatrienoic acid isomers from γ-linolenic acid

Incubation of γ-linolenic acid with soybean lipoxygenase initially at pH 9.3 and sibsequently at pH 7.9 gave rise to the conjugated triene dioxygenation product (λ max = 267 nm, > 50% yield), which was reduced to form 9-cis isomer of 6,13-dihydroxyoctadecatrienoic acid (6,13- diHOT) accompanied by minor isomers. Meanwhile, hemoglobin converted 13-hydroperoxyoctadecatrienoic acid into two major 9-trans isomers of 6,13-diHOT and two 9-cis isomers as minor products. The four isomers of 6,13-diHOT methyl ester were separated from each other on SP-HPLC, and characterized by chromatographic, spectrometric and cis → trans isomerization analyses.

Rearrangement of 5S, 12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-eicosatetraenoic acid during gas chromatography: formation of a cyclohexadiene derivative

The 5S,12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-eicosatetraenoic acid, a product of double dioxygenation of arachidonic acid by lipoxygenases, undergoes severe decomposition during gas chromatography-mass spectrometric (GC-MS) analysis of the trimethylsilyl ether methyl ester derivative. The decomposition product was studied by GC-MS and identified as a cyclohexadiene derivative of the parent compound formed by ring closure at C 6 and C 11. Under identical GC conditions, two stereoisomers, i.e. 5S,12R-dihydroxy-6,8,10,14-(Z,E,E,Z)-eicosatetraenoic acid (leukotriene B 4), and 6- trans -leukotriene B 4 showed excellent chromatographic properties. These data indicated that the 5,12-dihydroxy derivative of arachidonic acid carrying the transcis-trans triene unit selectively undergoes cyclization during GC. These studies also provided an explanation to the controversial GC-MS data reported for this lipoxygenase product.

The co-ordination of small molecules by manganese(II) phosphine complexes. Part 11. The co-ordination of dioxygen by manganese(II) complexes containing long-chain phosphine ligands

Manganese(II) complexes of long-chain phosphines, [MnX2(phosphine)][X = Cl, Br, or I; phosphine = P(C12H25)3, P(C14H29)3, P(C16H33)3, PPh(C12H25)2, PPh(C14H29)2, or PPh(C16H33)2], have been prepared. They form highly coloured 1 : 1 adducts with dioxygen, [MnX2(phosphine)(O2)], in both toluene and tetrahydrofuran (thf) solution. Infrared and e.s.r. spectra strongly suggest that these [MnX2(phosphine)] complexes are pseudotetrahedral dimers in the solid state and in toluene, contrasting vividly with the previously reported structure of [Mnl2(PPhMe2)]. Some molecular weight data indicate the product of dioxygenation in toluene to be the monomeric [MnX2(phosphine)(O2)] species. Dioxygen-binding curves for toluene and thf solutions have been obtained and equilibrium constants, KO1, for the dioxygenation reaction have been deduced. The general observation that KO2 increases and P50, the partial pressure when 50% of sites are deoxygenated, decreases for changes in halide ligand from Cl to Br to I in both solvents mirrors earlier observations with complexes of short-chain phosphines. The affinity of the complexes for O2 in thf is in the order phenyldialkylphosphines < trialkylphosphines. Hill coefficients, n, are approximately 1 for thf solutions of the chloride and bromide complexes, but for the iodides in thf and all of the complexes in toluene n lies in the range 1.10–1.64 indicating co-operativity for O2 binding, which may be related to the dimeric structure proposed for the complexes in toluene.

Product yield in oxygenation of linoleate by soybean lipoxygenase: The value of the molar extinction coefficient in the spectrophotometric assay

Two iodimetric methods and a gravimetric method were used to determine the spectrophotometric molar absorptivity of the purified product of lipoxygenase-catalyzed dioxygenation of linoleate (13- l s -hydroperoxy- cis,trans-9,11-octadecadienoate). Earlier determinations had led to the use of values varying from 24,000 to 28,000 m −1 cm −1 for ϵ at 235nm. In the current work, the two iodimetric values (spectrophotometric and titrimetric) average 22,500, while gravimetric analysis of scrupulously purified material gives 22,900. Final 235-nm absorbancies for lipoxygenase runs over a wide range of linoleic acid concentrations up to 200 μ m give a constant final percentage completion. If one assumes a 100% reaction, ϵ is 23,600. Each method has less than 1.5% standard error; the average of the three independent methods is 23,000 ± 580 (2.5%), all being lower than the previous values. In the enzyme-catalyzed reaction of linoleate at <200 μ m substrate, only 235-nm-absorbing material is formed. Above 200 μ m linoleate, yields at 235 nm decrease and yields of materials absorbing at 280 nm increase (the latter is known to arise from lipoxygenase-catalyzed reaction of linoleyl hydroperoxide). Below 200 μ m substrate, linoleate purified by HPLC produces only one HPLC-observable product, 13-linoleyl hydroperoxide. At higher substrate concentrations other HPLC peaks arise, again with higher wave-length absorptions. Spectrophotometric data using the ϵ determined here agree with those from the O 2 electrode. It is concluded that at S < 200 μ m, saturating air, and sufficient enzyme, soybean lipoxygenase-1 produces a sole product and the reaction proceeds to completion.

Oxygen evolution from hydrogen peroxide in photosystem II: flash-induced catalytic activity of water-oxidizing photosystem II membranes

Water-oxidizing photosystem (PS) II membranes treated with hydrogen peroxide in the dark showed backward transition of their S/sub 0/ state to the S/sub -1/ state, as deduced from the pattern of flash-dioxygen yields. In the presence of hydrogen peroxide, they evolved oxygen after the first flash, as reported for thylakoids. The authors used H/sub 2/ /sup 18/O/sub 2/ and a mass spectrometer that allowed monitoring of the oxygen isotopes in the medium to determine the flash yields. PS II membranes evolved /sup 18/O/sub 2/ and /sup 16/O/sub 2/ in H/sub 2/ /sup 18/O/sub 2/ below 1 mM, but little /sup 16/O/sub 2/ was evolved above 1 mM. No /sup 16/O/sup 18/O was evolved under any conditions. The oxygen yields from hydrogen peroxide at higher concentrations were more than 20-fold that for the PS II reaction center, and the decay of oxygen evolution after the flash was much slower than that from water. By shortening the dark interval between flashes, the evolution of /sup 18/O/sub 2/ was decreased and that of /sup 16/O/sub 2/ increased. Little /sup 18/O/sub 2/ was evolved under continuous light. The flash-induced evolution of dioxygen from hydrogen peroxide in PS II membranes is inferred from the assumptionmore » that the S/sub 0//S/sub 2/-state cycle has catalatic activity with a slow rate and that this activity is lost by the reduction of S/sub 0/ to the S/sub -1/ state, the spontaneous decay of S/sub 2/ to the S/sub 1/ state, and the advancement of S/sub 2/ to the S/sub 3/ state by light. Tris(hydroxymethyl)aminomethane (Tris) treatment of PS II membranes decreased the yield of dioxygen and extinguished the kinetic features that characterize the catalatic production of dioxygen from hydrogen peroxide.« less

Analysis of the stereochemistry of lipoxygenase-derived hydroxypolyenoic fatty acids by means of chiral phase high-pressure liquid chromatography

A chiral phase HPLC method was developed for the simultaneous determination of the positional and optical isomers of the lipoxygenase-derived hydroxypolyenoic fatty acids. With a Bakerbond chiral phase HPLC column (dinitrobenzoyl phenylglycine as chiral phase) the positional and optical isomers of the reduced dioxygenation products (by triphenylphosphine or borohydride) of linoleic acid and arachidonic acid were separated after methylation of the carboxylic groups. No cumbersome chemical derivatization such as conversion to a diastereomer was necessary. As compared with the methods used up till now chiral phase HPLC proved to be simpler and more sensitive. About 10 pmol of hydroxy fatty acids suffice for an analysis. The chiral phase HPLC can be used for the preparative separation of the optical antipodes of the lipoxygenase products. An optical purity of more than 90% can be reached in one preparative run. The method was applied to the determination of the stereochemistry of the dioxygenation products of polyenoic fatty acids formed by the lipoxygenases from soybeans, reticulocytes, pea seeds (isoenzyme I and II), tomato fruits, by the quasilipoxygenase activity of hemoglobin, and by the methylene blue-mediated photooxidation of arachidonic acid.

Leukotriene formation by bovine polymorphonuclear leukocytes

The leukotriene production by bovine polymorphonuclear leukocytes isolated from peripheral blood has been studied. Cells were incubated in the presence of arachidonic acid, glutathione, calcium ionophore A23187 and Ca 2+. The leukotrienes then formed are leukotriene C 4, leukotriene B 4, two all- trans isomers of leukotriene B 4 and the double dioxygenation product 12- epi-6- trans-8- cis-leukotriene B 4. Leukotriene C 4 is formed in such a large quantity by the bovine polymorphonuclear leukocyte that it might constitute an excellent and inexpensive source for the biosynthetic preparation of this spasmogenic leukotriene.

Effects of leukotrienes and f-Met-Leu-Phe on oxidative metabolism of neutrophils and eosinophils.

We assessed the effects of several leukotrienes and of f-Met-Leu-Phe on oxygen consumption in neutrophils and on the initial burst of chemiluminescence (CL) in both neutrophils and eosinophils. It was found that f-Met-Leu-Phe initiated 2.6 times higher oxygen consumption in neutrophils than did leukotriene B4 (LTB4). f-Met-Leu-Phe also stimulated five to 10 times more CL from both types of granulocytes than LTB4, which was at least five times more potent than its omega-hydroxylated metabolite, 20-OH-LTB4, whereas the corresponding 20-COOH derivative was effective only in eosinophils. The double dioxygenation product 5(S), 12(S)- DHETE caused no CL. Neutrophils from patients with chronic granulomatous disease did not respond with CL to any of the agents. The peak of CL occurred 50 to 60 sec after the addition of fMLP, whereas the LTB4-associated peak occurred after 5 to 6 sec and then rapidly subsided. The treatment of cells with sodium azide to inhibit the myeloperoxidase system did not change the kinetics or the rapid decline of the LTB4-induced CL. The CL response to LTB4 could be inhibited to 85% by 0.5 microgram/ml of superoxide dismutase, to 72% by 200 mg/ml of catalase, and to 50% by 80 microM of mannitol. The corresponding figures for f-Met-Leu-Phe-induced CL were 80, 58, and 16%, suggesting that, although a substantial part of the CL appears to be due to superoxide ion production, other oxygen radicals are involved in luminol-enhanced CL production. Thus, in contrast to some previous reports that leukotrienes do not stimulate an oxidative metabolic response in granulocytes despite their potent activity as chemotactic factors, our studies show that leukotrienes are definite inducers of granulocyte oxidative metabolism.

A novel dioxygenation product of arachidonic acid possesses potent chemotactic activity for human polymorphonuclear leukocytes.

We have found that a novel dioxygenation product of arachidonic acid, 8(S),15(S)-dihydroxy-5,11-cis-9,13-trans-eicosatetraenoic acid (8,15-diHETE), possesses chemotactic activity for human polymorphonuclear leukocytes comparable to that of leukotriene B4. Authentic 8,15-diHETE, identified by gas chromatography-mass spectrometry, was prepared by treating arachidonic acid with soybean lipoxygenase and was purified by reverse-phase high performance liquid chromatography. Using a "leading front" assay, 8,15-diHETE exhibited significant chemotactic activity at a concentration of 5.0 ng/ml. Maximum chemotactic activity was observed at a concentration of 30 ng/ml. The 8,15-diHETE generated by mixed human leukocytes after stimulation with arachidonic acid and the calcium ionophore, A23187, exhibited quantitatively similar chemotactic activity. Two synthetic all-trans conjugated isomers of 8,15-diHETE, however, were not chemotactic at concentrations up to 500 ng/ml. In contrast to its potent chemotactic activity, 8,15-diHETE (at concentrations up to 10 micrograms/ml) was relatively inactive with respect to its ability to provoke either degranulation or generation of superoxide anion radicals by cytochalasin B-treated leukocytes. Both leukotriene B4 and 8,15-diHETE may be important mediators of inflammation.

EPR Spectroscopy of soybean lipoxygenase-1: Description and quantification of the high-spin Fe(III) signals

The interaction of soybean lipoxygenase-1 with 13- L S - hydroperoxy-9-cis,11-trans- octadecadienoic acid, the product of the enzymic dioxygenation of linoleic acid, results in the formation of either a yellow or a purple coloured enzyme form depending on the amount of product used. The composition of the high-spin Fe(III) signals in the EPR spectra of both enzyme forms has been studied and the amount of EPR-visible iron determined by integration and simulation. Sets of g values of the species building up the high-spin Fe(III) signal around g 6 are derived from both third-order perturbation calculation and exact numerical diagonalization of the spin Hamiltonian describing the system. The results of these calculations are generally applicable to systems having S = 5 2 . The iron in the native, colourless enzyme is almost EPR-nondetectable. The yellow form of the enzyme shows a complex EPR signal around g 6 which consists of contributions of at least three species with different ligand symmetry. The signal corresponds to approx. 75% of the total iron content. The g 6 signal of the purple Fe(III) enzyme corresponds roughly to the same amount of iron but the ratio between the different species is not the same as in the yellow enzyme. This enzyme form also shows an additional g 4.3 signal with a large amplitude but a relatively low integrated intensity (approx. 10% of the total iron content). The results are consistent with the suggested mechanism of the catalytic function of iron in lipoxygenase which was based on qualitative EPR results (De Groot, J.J.M.C., Veldink, G.A., Vliegenthart, J.F.G., Boldingh, J., Wever, R. and van Gelder, B.F. (1975) Biochim. Biophys. Acta 377, 71–79).