Purification and Kinetic Properties of Guaiacol Peroxidase in Turnip (Brassica napus var. okapi) Root During Different Growth Stages

In this study, we present changes of peroxidase (E. C. 1.11.1. 7) activities in the root of Brassica napus var. Okapi during rosette stage until ripening of the fruits. Peroxidase activity was determined seasonally over an eight month period. Results showed that total peroxidase activity in the root was highest in mid June and the lowest activities were observed from time of winter dormancy until the beginning of April at which time morphogenic competence of tissues started to increase. Pattern of isoperoxidase bands on poly acrylamide gels (PAGE) showed that there was six inducible isoforms, named as TPA1, TPB1, TPB2, TPC1, TPC2 and TPC3. The isoform TPC3 was purified and partially characterized. Purification of peroxidase from turnip root was achieved by two ammonium sulphate precipitation steps followed by DEAE-sephadex chromatography. The effects of pH and temperature on enzyme activity were determined with guaiacol as electron donor. Highest activity was obtained at pH 6.0-6.5 and at a temperature of 50 °C. The enzyme was active at pH values below 7.0 even after 24 h and remained active after heat treatment at 70 °C for 30 min. It was inhibited by sodium cyanide rather than sodium azide. Loss of TPC3 peroxidase activity in the extracts of non-senescent root tissues suggests that it might play a role in the senescing process