Isolation and characterization of buckwheat ( Fagopyrum esculentum M.) chalcone synthase and its polyclonal antibodies

Abstract

Chalcone synthase was isolated from illuminated buckwheat ( Fagopyrum esculentum M.) hypocotyls and purified to electrophoretic homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using (NH) 4SO 4 fractionation, gel filtration on AcA 44, ion exchange chromatography on DEAE-Bio-Gel, and HPLC on hydroxylapatite. The properties of the enzyme were pH optimum, 8.0; M r ∼83,000 ± 1000; M r subunit, ∼41,500 ± 500; isoelectric point, pH 5.2; K m , 1 × 10 −6 m for malonyl-CoA, and 0.6 × 10 −6 m for p-coumaryl-CoA. Buckwheat chalcone synthase used p-coumaryl-CoA as substrate and also utilized caffeyl-CoA and ferulyl-CoA at 20 and 80%, respectively, of the rate of p-coumaryl-CoA in the chalcone synthase reaction. Antibodies against the buckwheat chalcone synthase were developed in a New Zealand white rabbit and characterized for specificity by enzyme-linked immunosorbent assay, Ouchterlony double immunodiffusion, and Western blotting.