title: Use of ESR and HPLC to follow the anaerobic reaction catalysed by lipoxygenases.
abstract: The measurement of the 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) consumption by using ESR allows to follow the anaerobic reaction between linoleic acid (LH) and its 13-hydroperoxide (LOOH) catalysed by lipoxygenase. During this reaction, two types of radicals are initially obtained, alkyl (L) and alkoxyl (LO) radicals which formed two types of adducts (LT and OLT) with TEMPOL as characterised by HPLC. The stoichiometry of the adduct formation is two mole of TEMPOL consumed for one mole of LH and one mole of LOOH. Using ESR, the kinetic parameters and the mechanism of the anaerobic reaction have been determined at pH 6.5 for three different lipoxygenases, soybean, horse bean and wheat and compared to the values obtained at pH 9 for soybean lipoxygenase. Wheat lipoxygenase is very weakly active compared to the other enzymes. An uncompetitive inhibition of the anaerobic reaction catalysed by soybean and horse bean lipoxygenases was observed with 2,6-di-tert-butyl-4-methylphenol (BHT).
authors.affiliation: AgroParisTech, Ingénierie Procédés Aliments, UMR1145, Massy, France; INRA, Ingénierie Procédés Aliments, UMR1145, Massy, France; CNAM Ingénierie Procédés Aliments, UMR1145, Massy, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Brandicourt
authors.affiliation: AgroParisTech, Ingénierie Procédés Aliments, UMR1145, Massy, France; INRA, Ingénierie Procédés Aliments, UMR1145, Massy, France; CNAM Ingénierie Procédés Aliments, UMR1145, Massy, France.
authors.forename: Jacques
authors.initials: J
authors.lastname: Nicolas
authors.affiliation: AgroParisTech, Ingénierie Procédés Aliments, UMR1145, Massy, France; INRA, Ingénierie Procédés Aliments, UMR1145, Massy, France; CNAM Ingénierie Procédés Aliments, UMR1145, Massy, France.
authors.forename: Aline
authors.initials: A
authors.lastname: Boussard
authors.affiliation: AgroParisTech, Ingénierie Procédés Aliments, UMR1145, Massy, France; INRA, Ingénierie Procédés Aliments, UMR1145, Massy, France; CNAM Ingénierie Procédés Aliments, UMR1145, Massy, France. Electronic address: anne-marie.riquet@agroparistech.fr.
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Riquet
mesh_terms: D000693:Anaerobiosis; D055162:Biocatalysis; D002084:Butylated Hydroxytoluene; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D019787:Linoleic Acid / Q000737:chemistry; D058945:Lipoxygenases / Q000378:metabolism*; D010636:Phenols / Q000737:chemistry; D013025:Glycine max / Q000201:enzymology
chemical_list: D010636:Phenols; D002084:Butylated Hydroxytoluene; D019787:Linoleic Acid; D006861:Hydrogen Peroxide; D058945:Lipoxygenases
keywords: Anaerobic reaction; ESR; HPLC–DAD–MS; Linoleic acid; Lipoxygenases; Spin scavenging