Immunoblot Analyses of the ElicitedSanguinaria canadensisEnzyme, Dihydrobenzophenanthridine Oxidase: Evidence for Resolution from a Polyphenol Oxidase Isozyme

In our initial purification of dihydrobenzophenanthridine oxidase fromSanguinaria canadensisplant cell cultures, we reported that our most purified preparations contained a major band at 77 kDa and minor lowerMrbands. Here we present evidence on highly purified dihydrobenzophenanthridine oxidase from elicitedS. canadensiscultures to indicate that this enzyme is the 77-kDa protein and that lowerMrbands include an isozyme(s) of the polyphenol oxidase family that copurifies with it. An antibody raised against the 77-kDa protein and an anti-polyphenol oxidase antibody that recognizes a 70-kDa band were used to monitor chromatographic fractions by immunoblot analysis of the oxidases. Oxidase-containing eluates from DEAE–Sephadex, CM, and HiTrap blue were compared to corresponding flow-through fractions. Bands at 77 and 88 kDa were detected with anti-dihydrobenzophenanthridine oxidase antibody in eluates displaying high dihydrobenzophenanthridine oxidase activity. Polyphenol oxidase specific activity and immunoreactivity partitioned both in flow-through and eluate fractions of the CM and HiTrap columns. Estimation of the dihydrobenzophenanthridine oxidase and polyphenol oxidase specific activities for each step showed increasing enrichment of alkaloidal enzyme accompanied by variable dihydrobenzophenanthridine oxidase/polyphenol oxidase activity ratios. Taken together these observations indicate that the dihydrobenzophenanthridine and polyphenol oxidases haveMrvalues of 77 and 70 kDa, respectively, and the two enzymes are different entities.