Preparative studies on the isolation of an enzyme associated with carthamin synthesis in Carthamus tinetorius L.

Abstract

Preliminary operations for the isolation of an enzyme associated with cathamin synthesis (carthamin-synthesizing enzyme) were done with a soluble extract of safflower seedlings. Ethanol and acetone seriously affected the solubility and the activity of the enzyme. Ammonium sulfate fractionation, calcium acetate precipitation and protamine sulfate treatment were all useful techniques for removing inert protein from the crude extract however, the activity of the carthamin-synthesizing enzyme could not be separated from that of native polyphenol oxidase or peroxidase. Partial purification of the carthamin-synthesizing enzyme could be achieved by filtration of a crude extract through Sephadex G-100 gel after treating it with ammonium sulfate, calcium acetate and protamine sulfate. -the purified enzyme reacted possitively with precarthamin at pH 4.8 in 50.0 mM acetate buffer. The synthesized product was identified as carthamin by checking its R_f values on silica gel plates developed with three different developing solvents and UV absorption spectral pattern in methanol. The enzyme sample was unstable on storage and stabilization of the sample was tested by applying many procedures. Carthamin-synthesizing enzyme had low affinity to celite 535. No carthamin was found in an incubation medium containing precarthamin and an authentic sample of polyphenol oxidase or peroxidase. Carthamin could not be converted to any blackish catabolites through polyphenol oxidase- or peroxidase-catalyzed oxidative reaction. Changes in the activities of three different enzymes, carthamin-synthesizing enzyme, polyphenol oxidase and peroxidase, were investigated in the extract from both etiolated and green seedlings of safflower and the data were discussed in relation to the catalytic properties of the enzymes.