Purification and antigenicity of flavone synthase I from irradiated parsley cells

Flavone synthase I, a soluble 2-oxoglutarate-dependent dioxygenase catalyzi ng the oxidation of flavanones to flavones in several Apiaceae species, was induced in parsley cell cultures by continuous irradiation with ultraviole t/blue light for 20 h. The enzyme was extracted from these cells and purifi ed by a revised purification protocol including the fractionation on hydrox yapatite, Fractogel EMD DEAF, and Mono Q anion exchangers, which resulted i n an apparently homogeneous flavone synthase at approximately 10-fold highe r yield as compared to the previous report. The homogeneous enzyme was empl oyed to raise an antiserum in rabbit for partial immunological characteriza tion. The specificity of the polyclonal antibodies was demonstrated by immu notitration and Western blotting of the crude ammonium sulfate-fractionated enzyme as well as of the enzyme at various stages of the purification. Hig h titer cross-reactivity was observed toward flavone synthase I, showing tw o bands in the crude extract corresponding to molecular weights of 44 and 4 1 kDa, respectively, while only the 41 kDa was detected on further purifica tion. The polyclonal antiserum did not cross-react with recombinantly expre ssed flavanone 3 beta -hydroxylase from Petunia hyhrida or flavonol synthas e from Citrus unshiu, two related 2-oxoglutarate-dependent dioxygenases inv olved in the flavonoid pathway. (C) 2001 Academic Press.