Purification and Antigenicity of Flavone Synthase I from Irradiated Parsley Cells

Abstract

Flavone synthase I, a soluble 2-oxoglutarate-dependent dioxygenase catalyzing the oxidation of flavanones to flavones in several Apiaceae species, was induced in parsley cell cultures by continuous irradiation with ultraviolet/blue light for 20 h. The enzyme was extracted from these cells and purified by a revised purification protocol including the fractionation on hydroxyapatite, Fractogel EMD DEAE, and Mono Q anion exchangers, which resulted in an apparently homogeneous flavone synthase at approximately 10-fold higher yield as compared to the previous report. The homogeneous enzyme was employed to raise an antiserum in rabbit for partial immunological characterization. The specificity of the polyclonal antibodies was demonstrated by immunotitration and Western blotting of the crude ammonium sulfate-fractionated enzyme as well as of the enzyme at various stages of the purification. High titer cross-reactivity was observed toward flavone synthase I, showing two bands in the crude extract corresponding to molecular weights of 44 and 41 kDa, respectively, while only the 41 kDa was detected on further purification. The polyclonal antiserum did not cross-react with recombinantly expressed flavanone 3β-hydroxylase from Petunia hybrida or flavonol synthase from Citrus unshiu, two related 2-oxoglutarate-dependent dioxygenases involved in the flavonoid pathway.