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.