The detection of linoleic acid radicals in the anaerobic reaction of lipoxygenase

Groot, J.J.M.C. de; Garssen, G.J.; Vliegenthart, J.F.G.; Boldingh, J.

doi:10.1016/0005-2760(73)90254-3

Cited by 151 publications

(43 citation statements)

References 7 publications

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“…ESR spectra were recorded by using a Varian Associates model E-9 spectrometer. The spin traps 2-methyl-2-nitroso-1-propanol (MNPOL) and 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) were prepared according to the methods outlined by DeGroot et aL (12) and Bonnett et a! (13).…”

Section: Methodsmentioning

confidence: 99%

Spin trapping of free radicals during hepatic microsomal lipid peroxidation.

Rosen¹,

Rauckman²

1981

Proc. Natl. Acad. Sci. U.S.A.

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We have used spin-trapping techniques to identify radical species formed during the NADPH-stimulated peroxidation of rat hepatic microsomes. Using 5,5-dimethyl-1-pyrroline-1-oxide, we have confirmed the presence of substantial quantities of superoxide but found evidence for the formation of only small quantities of hydroxyl radical. Use of the spin traps Ntert-butyl-a-4-pyridylnitrone-l-oxide and 2-methyl-2-nitroso-lpropanol have allowed us to determine that lipid peroxyl radicals are the predominant "lipid-type" radical found in peroxidizing microsomes under aerobic conditions. These data suggest that microsomal lipid dienyl radicals react with molecular oxygen at near diffusion-controlled rates. can be spin trapped even though other radical species, such as superoxide and lipid hydroperoxyl radicals, have been shown to be generated during lipid peroxidation (1).Here we describe experiments showing that, in the presence ofrat liver microsomes and NADPH, superoxide, hydroxyl, and lipid peroxyl radicals (LOO-) are spin trapped; we could not find any evidence for the existence ofa carbon-centered dienyl lipid radical (L-). General Comments. Hemin, N,amino]ethyl)glycine (DETAPAC), soybean lipoxygenase, and NADPH were purchased from Sigma. Linoleic acid was obtained from Nu Chek Prep (Elysian, MN) and was >99% pure. 2-Methyl-2-nitropropane was purchased from Fluka/ Tridom. Chelex-100 was obtained from Bio-Rad. ESR spectra were recorded by using a Varian Associates model E-9 spectrometer. The spin traps 2-methyl-2-nitroso-1-propanol (MNPOL) and 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) were prepared according to the methods outlined by DeGroot et aL (12) and Bonnett et a! (13). The spin trap N-tert-butyl-a-4-pyridylnitrone-l-oxide (4-POBN) was a gift from William Yamanashi (Department of Ophthalmology, Emory University, Atlanta, GA). Because the rate of spin trapping of some free radicals is slow [e.g., the rate of spin trapping superoxide by DMPO is 10 M-1sec-1 (8)], high concentrations of the probes are essential. For this reason, we chose to use the water-soluble spin traps DMPO, 4-POBN, and MNPOL (Fig. 1). Unless otherwise indicated, all buffers used were passed through a ChelexAbbreviations: EPR, electron paramagnetic resonance; LOO-, lipid peroxyl radical; L, dienyl lipid radical; DMPO, 5,5-dimethyl-1-pyrroline-1-oxide; DMPO-OH, 5-hydroxy-2,2-dimethyl-1-pyrrolidinyloxyl; DMPO-OOH, 5-hydroperoxy-2,2-dimethyl-1-pyrrolidinyloxyl; 4-POBN, N-tert-butyl-a-pyridylnitrone-l-oxide; 4-POBN-OH, tert-butyl(hydroxy)-4-pyridylmethyl nitroxide; 4-POBN-OOH, tertbutyl(hydroperoxy)-4-pyridylmethyl nitroxide; MNPOL, 2-methyl-2-nitroso-l-propanol; MNPOL-H, 2-methylpropyl-1-nitroxide; DETA-PAC, NN-bis(2-[bis(carboxymethyl)amino]ethyl)glycine. MATERIALS AND METHODS PBN 4-POBNThe publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be -hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Section: Methodsmentioning

confidence: 99%

Spin trapping of free radicals during hepatic microsomal lipid peroxidation.

Rosen¹,

Rauckman²

1981

Proc. Natl. Acad. Sci. U.S.A.

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“…Electron paramagnetic resonance (EPR) studies by De Groot et al [4] showed that this anaerobic reacAbbreviations. R-13-00H, 13-~,-hydroperoxy-linoleic acid;…”

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confidence: 99%

The Steady-State Kinetics of the Oxygenation of Linoleic Acid Catalysed by Soybean Lipoxygenase

1976

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The steady-state kinetics of the oxygenation of linoleic acid catalysed by soybean lipoxygenase-I were studied. The results showed that lipoxygenase-1 is strongly inhibited by its substrate, linoleic acid. In the presence of the product of the reaction, 13-~,-hydroperoxy-linoleic acid, the substrate inhibition only affects the apparent affinity for O2 and is of a hyperbolic type.A kinetic scheme of the oxygenation reaction is presented, which postulates two substrate-binding sites on the enzyme, one for linoleic acid and one for O,, and a regulatory binding site, which can either bind the product or the fatty acid substrate.Since previous studies indicated that the product of the reaction influences the oxidation state of the iron present in protein, the steady-state kinetics of the native enzyme and of the enzyme pre-incubated with the product were compared.Pre-incubation of the enzyme with the product did not lead to altered steady-state kinetics of the reaction compared to those of the native enzyme.Lipoxygenase type I from soybeans, a mononuclear non-heme-iron(Fe")-containing dioxygenase, catalyzes the conversion of linoleic acid and several other poly-unsaturated fatty acids with a 1,4-cis,cis-pentadiene structure [l] into (n-6)h-hydroperoxy fatty acids. Smith and Lands [2] studied the steady-state kinetics of the formation of the hydroperoxides from several fatty acid substrates in air-saturated solutions.They demonstrated that the enzyme is activated by the product hydroperoxide. Since the reaction is inhibited by trapping of hydroperoxide formed, they proposed, that activation by the product is compulsory.Garssen et al. [3] demonstrated that under anaerobic conditions both the fatty acid substrate (linoleic acid) and the (n-6)~,-hydroperoxide are converted by the enzyme into secondary products, among which are fatty acid dimers, oxodienoic acids and n-pentane. Electron paramagnetic resonance (EPR) studies by De Groot et al. [4] showed that this anaerobic reacAbbreviations. R-13-00H, 13-~,-hydroperoxy-linoleic acid;Enzyme. Lipoxygenase or linoleate: oxygen oxidoreductase (EC EPR, electron paramagnetic resonance.1.13.1 1.12).tion is a radical process, involving linoleic acid freeradicals created by the enzyme. The anaerobic reaction only takes place in the presence of the (n-6)~,-hydroperoxide. Recently it was found that 13-~,-hydroperoxy-linoleic acid (R-13-00H) affects the fluorescence [5], absorbance [6] and EPR spectra [7] of lipoxygenase-1. These studies revealed that an equimolar amount of R-I 3-00H converts the native enzyme from the ferrous state (EPR silent; no absorption maxima in the visible region) into the ferric state (EPR signals aroundg = 6; absorption maximum at about 330nm). Furthermore a molar excess of R-13-00H over enzyme gives rise to the appearance of a second absorption band (at 580 nm) and a strong EPR signal at g=4.3, whereas the signals around g = 6 are reduced in intensity [8]. When linoleic acid is added to the enzyme in the ferric state under anaerobic conditions, the enzyme ...

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“…From the ESR spectra of spin adducts, production of two alkyl radicals (-CH-, signal II and 111) [16][17][18] and one acyl radical (-C=O, signal 1)16) was expected during the reaction. Hyperfine coupling con- signal IV with aN of 16.8G and a H of 11.4G.…”

Section: Resultsmentioning

confidence: 99%