Purification and Characterization of an Anionic Isoperoxidase from Scented‐Geranium Callus

Abstract

Secretory anionic isoperoxidase (EC 1.11.1.7), named PA1, was 68‐fold purified from scented‐geranium (Pelargonium graveolense) callus by using ion exchange chromatography and gel filtration. Isoperoxidase PA1 was a glycoprotein with an isoelectric point (pI) of 4.0. The molecular weight of PA1 was approximately 42.5 and 44 kDa, estimated by SDS–PAGE and Sephadex G‐150 gel filtration, respectively. The optimum pH of the enzyme was 5.0 for guaiacol and H2O2, and the K m values for guaiacol and H2O2 were 1.96 and 8.5 mM, respectively. Substrate studies in terms of optimum pHs and K m values with various synthetic and naturally occurring phenolic compounds were performed. In comparison with cationic isoperoxidase, PC3, which has been already characterized, anionic isoperoxidase PA1 had much lower K m values for synthetic phenolic compounds and much higher K m values for naturally occurring phenolic compounds than PC3. Moreover, anionic isoperoxidase PA1 could utilize ferulic acid as a substrate very well, while cationic isoperoxidase PC3 could not utilize ferulic acid as a substrate.