Inactivation and cleavage of radish peroxidase by various reducing agents

The inactivation and cleavage of Korean radish (Raphanus sativus L.) peroxidase (EC. 1.11.1.7) by three reducing agents, dithiothreitol (DTT), ascorbate and 2-mercaptoethanol (2ME) was examined. Upon incubation of the enzyme with DTT, inactivation followed by degradation and cleavage of the anionic isoperoxidase A(1) (M-r: 43 k) was observed, whereas, inactivation and degradation took place almost simultaneously in the presence of ascorbate. Nevertheless, the cleaved products generated from the inactivated enzyme showed similar M-r values of about 30 k, 23 k and 18 k in both cases. When 2ME was used, the formation of larger M-r aggregates of 66 k and 69 k was observed without cleavage of smaller peptides. When the enzyme was inactivated by DTT and ascorbate, dimethyl sulfoxide and ethanol protected the enzyme inactivation almost completely in both cases, but these scavengers did not reverse the inactivation significantly when 2ME was used as a reducing agent. The singlet oxygen scavenger histidine prevented the enzyme from inactivation by ascorbate and 2ME to some degree, but it failed to inhibit enzyme inactivation by DTT. These results suggest that there might be different inactivation and cleavage mechanisms of radish peroxidases, depending upon the reducing agents used. (C) 1998 Published by Elsevier Science Ltd. All rights reserved.