Pigment bleaching, carbonyl production and antioxidant effects during the anaerobic lipoxygenase reaction

Abstract

Bleaching of the plant pigments β-carotene and chlorophyll a occurs during the lipoxygenase-mediated oxidation of unsaturated fatty acids. The type-1 lipoxygenase from soybeans is a poor pigment-bleacher under aerobic (air or oxygen-saturated) conditions. However, under anaerobic (argon) conditions, lipoxygenase-1 is a powerful pigment bleaching agent in the presence of substrate fatty acid and product hydroperoxide. When β-carotene or chlorophyll a was bleached, absorbance at 285 nm increased, indicating the formation of keto- or oxodienes. Simultaneously, the conjugated diene hydroperoxide present in the reaction mixture disappeared. The rate of bleaching of carotene was faster than for chlorophyll. The addition of antioxidants (butylated hydroxyanisole, butylated hydroxytoluene, α-tocopherol, propyl gallate, or tertiary butylated hydroxyquinone) affected the rates of pigment bleaching and carbonyl production. Butylated hydroxyanisole, butylated hydroxytoluene and α-tocopherol were slightly more effective than propyl gallate in inhibiting pigment bleaching, while tertiary butylated hydroxyquinone was completely inhibiting. The production of carbonyl compounds continued essentially unchanged in the presence of butylated hydroxyanisole, butylated hydroxytoluene and α-tocopherol, while tertiary butylated hydroxyquinone and propyl gallate reduced the rate to zero. Based on these results, a scheme for the anaerobic bleaching of plant pigments during the lipoxygenase-catalyzed reaction is proposed. The formation of an enzyme-fatty acid radical complex which reacts with the plant pigment appears to be necessary for bleaching to occur. Certain antioxidants (tertiary butylated hydroxyquinone) preferentially react at this point in the pathway, thus effectively blocking chlorophyll and carotene bleaching.