Soluble lipoxygenase isoforms from tomato fruit

Abstract

Extraction of maximum lipoxygenase activity from tomato fruits was shown to require the presence of 0.1% Triton X-100. The detergent appeared to have a dual role improving recovery and preventing enzyme inactivation, especially during mechanical homogenization. Ultracentrifugation of total lipoxygenase activity revealed that the majority (96%) was of a soluble form, with very little associated with the membrane fraction. By ammonium sulphate fractionation and anion-exchange chromatography, soluble lipoxygenase was purified 46-fold. Separation of lipoxygenase activity by isoelectric focusing and detection by in-gel activity staining resulted in the identification of two predominant isoforms with pI values of 4.8 and 5.0, one of which appears to be ripening-specific. On examination of reaction products by HPLC, both isoforms appeared to exclusively produce 9-hydroperoxides.