Purification and characterization of a cationic isoperoxidase from scented-geranium

Abstract

A strongly cationic isoperoxidase named PC3 was purified to homogeneity from scented-geranium ( Pelargonium graveolens) callus by using DEAE-Sephacel chromatography, CM–cellulose chromatography and Sephacryl S-200 gel filtration, respectively. The enzyme was a glycoprotein with M r of ca. 58 kDa estimated by SDS–PAGE and Sephadex G-150 gel filtration. The pI value of the enzyme was 9.1. Kinetic studies revealed that PC3 had a very low K m value for scopoletin of 0.01 mM and could use ascorbate as a substrate. Interestingly, PC3 could not oxidize ferulic acid as a substrate. Chemical modification of the enzyme showed that PC3 was rapidly inactivated by His, Cys, Trp and Lys-specific reagents. The second order rate constants and reaction orders with respect to these inactivations were determined. Notably, ca. 4–7-fold activity boosting of PC3 occurred by adenine and imidazole when anilino substrates, such as o-dianisidine and o-phenylenediamine were oxidized, whereas this activity boosting did not occur when several phenolic substrates were used.