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

Koshi Saito, Yoshiyuki Takahashi, and Mizu Wada Department of Applied Chemistry, Faculty of Engineering, Tokai University, 1117 Kitakaname, Hiratsuka-shi, Kanagawa-ken 259-12, Japan Z. Naturforsch. 40c, 819-826 (1985); received August 12, 1985 Carthamin Synthesis, Safflower Seedling, Catalytic Property An enzyme associated with carthamin formation in Carthamus tinctorius L. (carthamin-synthesizing 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)2S 0 4 fractionation, Ca(CH3C 0 2)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 mM 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 carth­ amin 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 0 2 slightly en­ hanced the catalytic rate of carthamin formation. The enzyme was sensitive to phosphorus sub­ stances. Among those compounds tested at 1.2 mM level, orthophosphate showed the most strik­ ing inhibitory action on the enzyme. Metal ions affected on the enzyme activity by different extents. Mn2+ stimulated the enzyme reaction, while Cu2̂ 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.