Formation of carthamin from PC catalyzed by CarS (CtPOD1). (A) The assay mixture for the CarS-catalyzed reaction (assay method II) was analyzed by HPLC (system B). Upper panel, before addition of the purified enzyme; middle panel, 3 min after enzyme addition; lower panel, 10 min after enzyme addition. The inset in each panel shows the appearance of the reaction mixture, showing time-dependent color changes. (B) Peroxidase-catalyzed decolorization of carthamin is circumvented by adsorption of nascent pigment to cellulose. Five nanomoles of PC was incubated with 16 pkat of the purified CarS (CtPOD1) at 30°C for 30 min in 100 μl of 100 mM sodium citrate buffer, pH 5.0, in the absence (1) or presence (2) of a dialysis tubing cellulose membrane (3 mm × 3 mm, six sheets per reaction mixture). Time-dependent color changes of the reaction mixtures were recorded (0, 5 and 30 min). In addition, the reaction mixtures were allowed to stand for 34 d at room temperature.