Purification of a plant peroxidase using reversibly soluble ion-exchange polymer

A plant peroxidase localized in the root tissues of Raphanus sativus was purified partially by precipitation using a reversibly soluble/insoluble ion-exchange polymer system of carboxymethyl (CM) cellulose, calcium and polyethylene glycol. The CM-cellulose-Ca2+ -PEG system resulted in a 'negative' purification factor of more than 5 in a single step. The enzyme was further purified and concentrated by thiophilic chromatography to homogeneity. An overall purification factor of 20 was obtained with 66% yield. The final specific enzyme activity was 460 000 U mg(-1) protein. The purified peroxidase showed pH optimum at 5 and temperature optimum at 40 degreesC. The enzyme followed the normal Michelis-Menton kinetics and gave K-m of 4.78 mM with 2,2'-Azinobis [3-ethyl-benzothiazoline-(6)-sulphonic acid] as substrate. (C) 2001 Elsevier Science Ltd. All rights reserved.