Linoleic reaction with lipoxygenase

Verhagen, j., G. A. Veldink, M. R. Egmond, J. F. G. Vliegenthart, J. Boldingh, and J. Van Der Star Steady-State Kinetics of Anaerobic Reaction of Soybean Lipoxygenase-I with Linoleic Acid and I3-L-HydroperoxylinoIeic Add. Biochem. Biophys. Acta 529, 369 (1978). 

In our study, the reusability of LOX immobilized in calcium-alginate beads was determined by repeatedly using the same beads for five successive reactions with linoleic acid (LA) (Fig. 2). The LOX activity was measured after each cycle, and the beads were recovered and washed with sodium borate buffer before reuse. To initiate the next cycle of oxidation, LA was added to the incubation mixture containing the recovered beads. The data (Fig. 2) demonstrates that LOX immobilized in beads can be reused at least five times without substantial loss in enzyme activity. In contrast, free lipoxygenase is typically inactivated by hydroperoxide accumulation and the partial anaerobic condition that develops in the reaction mixture (8). 

Lipoxygenase [EC 1.13.11.12] catalyzes the reaction of linoleate with dioxygen to produce (9Z,11 )-(135 )-13-hydroperoxyoctadeca-9,ll-dienoate. This iron-depen-dent enzyme can also oxidize other methylene-interrupted polyunsaturated fatty acids. See also specific enzyme... 

Formation of Secondary Products and Lipohydroperoxide Destruction. As early as 1945 Holman and Burr (132) found that crude soybean lipoxygenase acting on a number of substrates produced carbonyl-containing material in addition to diene. Holman, as noted above (107), used his crystalline enzyme and found that it was difficult to establish a correspondence between O2 consumption and diene conjugation. The diene concentration always tended to be too low. Privett et al. 123) found that the reaction products varied with enzyme concentration and method of addition. Vioque and Holman 133) identified 9-keto-ll,13- and 13-keto-9,ll-octadecadienoate with the usual hydroperoxides in a reaction carried out with linoleic acid and a relatively large amount of crude soybean lipoxygenase at pH 9. 

Spin-trapping techniques have been used to study reactions of lipoxygenase with linoleic acid ( ). It was proposed that the formation of dimeric linoleic acid required the involvement of hydroperoxylinoleic acid ( ). The ESR spectrum from the interactions of the PUFA radical and the spin-trap indicated this involvement occurred Similar spin-trapping techniques could be used to investigate the possibility that DPE s may ultimately induce formation of various fatty acid dimers (e.g., dimers of linolenic acid). Formation of such polymers should dramatically affect membrane function. 

An additional method for the assessment of adequacy of blanching is based on the detection of residual lipoxygenase. The lipoxygenase catalyzes a reaction between linoleic acid and oxygen to form a hydroperoxide. This product reacts with potassium iodide to form iodine, giving a brown coloration which can be measured spectrometrically. 

The biological function of a lipoxygenase is closely connected with the type of the reaction catalysed and with the kind of reaction product formed. From a biological point of view, it appears to be reasonable to classify the reactions of lipoxygenases in two groups (Table 1), i.e. those with free polyenoic fatty acids as substrate and those with more complex substrates in which the polyenoic fatty acid is esterified. As far as the reactions with free polyenoic fatty acids are concerned, owing to their abundance linoleic and a-linolenic acids are the most relevant... 

Lipoxygenases (Lox) are selective towards polyunsaturated fatty acids containing the c/, c/ -l,4-pentadiene moiety to produce either the 9(5)-hydroperoxide, 13(i )-hydroperoxide, or a mixture of both from linoleic and linolenic acids. These enzymes contain an iron atom in their active center. They are activated by hydroperoxides, and the Fe + is oxidized to Fe +, according to a scheme in which the pentadiene radical of linoleic acid becomes bound to the enzyme, reacts with oxygen, and the peroxyl radical formed is reduced by the enzyme to produce a hydroperoxide after reaction with a proton (1). 

Schnurr et al. [22] showed that rabbit 15-LOX oxidized beef heart submitochondrial particles to form phospholipid-bound hydroperoxy- and keto-polyenoic fatty acids and induced the oxidative modification of membrane proteins. It was also found that the total oxygen uptake significantly exceeded the formation of oxygenated polyenoic acids supposedly due to the formation of hydroxyl radicals by the reaction of ubiquinone with lipid 15-LOX-derived hydroperoxides. However, it is impossible to agree with this proposal because it is known for a long time [23] that quinones cannot catalyze the formation of hydroxyl radicals by the Fenton reaction. Oxidation of intracellular unsaturated acids (for example, linoleic and arachidonic acids) by lipoxygenases can be suppressed by fatty acid binding proteins [24]. 

Holmes et al. (1998) performed two enzymatic reactions, the lipase-catalyzed hydrolysis of y>-nitrophenol butyrate and lipoxygenase-catalyzed peroxidation of linoleic acid, in w/c microemulsions stabilized by a fluorinated two-chained sulfosuccinate surfactant (di-HCF4). The activity of both enzymes in the w/c microemulsion environment was found to be essentially equivalent to that in a water/heptane microemulsion stabilized by Aerosol OT, a surfactant with the same headgroup as di-HCF4. The buffer 2-(A-morpholino)ethanesulfonic acid (MES) was used to fix the pH in the range 5-6. 

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