Purification of flavanone 3 beta-hydroxylase from Petunia hybrida: antibody preparation and characterization of a chemogenetically defined mutant.

Flavanone 3 beta-hydroxylase from Petunia hybrida has been purified to apparent homogeneity utilizing an improved purification protocol including chromatography of the partially purified enzyme on hydroxyapatite, chromatofocusing, and hydrophobic interaction chromatography on phenyl-Superose. The specificity of mouse and rabbit polyclonal antisera directed to flavanone 3 beta-hydroxylase was demonstrated by Western blotting and immunotitration with crude extracts from wild-type flowers of P. hybrida and with the purified enzyme. Cross-reactivity was observed with flavanone 3 beta-hydroxylase from extracts of illuminated parsley cells. A Petunia mutant with white flowers, previously shown to lack 3 beta-hydroxylase activity and to accumulate flavanone glycosides, showed complete absence of the enzyme protein.