Preparation of an Enzyme Associated with Carthamin Formation in Carthamus tinctorius L

Abstract

Abstract An enzyme associated with carthamin formation in Carthamus tinctorius L. (carthamin-synthe-sizing enzyme) was isolated from the soluble protein extract of the hypocotyl tips of the etiolated seedlings and purified up to 157-fold by the procedures applying (NH4)2SO4 fractionation, Ca(CH3CO2)2 precipitation, protamine sulfate treatment, Celite adsorption chromatography, and Sephadex G-100 gel filtration. Results from atomic absorption spectral analysis of the enzyme protein showed to contain K as a major component, and Ca and Mg as minor ones. Fe, Cu, and Mn could not be detected in the preparation. At pH 4.8 in 50.0 mᴍ acetate buffer, the partially purified enzyme reacted positively with a flame-coloured precarthamin to produce a reddish product in open cuvettes with incubation medium. The reaction product was identified as carthamin by examining its colour, chromatographic mobilities in different developing solvents and spectroscopic properties inclusive shifts, often by comparing with those of an authentic specimen. Anaerobic incubation reduced the enzyme activity, while exogenously applied O2 slightly enhanced the catalytic rate of carthamin formation. The enzyme was sensitive to phosphorus sub­ stances. Among those compounds tested at 1.2 mᴍ level, orthophosphate showed the most striking inhibitory action on the enzyme. Metal ions affected on the enzyme activity by different extents. Mn2+ stimulated the enzyme reaction, while Cu2+ and Mo6+ exhibited reverse effects. Fe2+, Fe3+, Zn2+, Mg2+, and Co2+ were also unfavourable to the enzyme catalyzed carthamin formation. The preparation of the carthamin-synthesizing enzyme showed no activity of polyphenol oxidase or peroxidase under the conditions specifically designed for detecting both enzyme activities.