Purification and some properties of polyphenol oxidase from root-forming carrot callus tissues

1. Polyphenol oxidase (o-diphenol : O2 oxidoreductase; E.C. 1.10.3.1) was isolated from the other phenolases which were present in root-forming carrot callus, and its properties were examined. 2. The enzyme was purified about 45-fold over crude extracts (precipitates between 40–70% saturation widi ammonium sulfate) by a combination of Bio-gel filtration, protein-bag filtration, and carboxymethyl cellulose chromatography. The purified oxidase was homogeneous according to polyacrylamide gel electrophoresis and Sephadex gel filtration. It was confirmed by CM-cellulose chromatography that the enzyme was absent in callus tissues without accompanying redifferentiation. 3. The molecular weight of this oxidase was estimated to be 110,000- 120,000 from molecular weight-mobility profiles on polyacrylamide gels containing sodium dodecyl sulfate and molecular size-elution volume correlations on Sephadex G-150 columns. 4. The enzyme oxidized o-diphenols but showed no detectable activity against monophenols. Pyrocatechol, dopamine, caffeic acid, and chlorogenic acid were effectual substrates of the enzyme with Km values ranging from 10−3 M to 10−5M. The enzyme effectively catalyzed the oxidation of o-diphenols over the range of pH 6.0 to 7.0 and was readily inactivated by heating. The enzyme activity was slightly influenced by increasing ionic strength. The initial rate of the enzymic reaction was enhanced by addition of Cu2+, Co2+ and Mn2+ ions, and was reduced in the presence of DTT, PCMPS, glycylglycine, and DIECA.