Purification and characterization of a new cationic peroxidase from fresh flowers of Cynara scolymus L.

Abstract

A basic heme peroxidase isoenzyme (AKPC) has been purified to homogeneity from artichoke flowers ( Cynara scolymus L.). The enzyme was shown to be a monomeric glycoprotein, M r=42 300±1000, (mean±S.D.) with an isoelectric point >9. The native enzyme exhibits a typical peroxidase ultraviolet–visible spectrum with a Soret peak at 404 nm ( ϵ=137 000±3000 M −1 cm −1) and a Reinheitzahl (Rz) value ( A 404nm/ A 280nm) of 3.8±0.2. The ultraviolet–visible absorption spectra of compounds I, II and III were typical of class III plant peroxidases but unlike horseradish peroxidase isoenzyme C, compound I was unstable. Resonance Raman and UV–Vis spectra of the ferric form show that between pH 5.0 and 7.0 the protein is mainly 6 coordinate high spin with a water molecule as the sixth ligand. The substrate-specificity of AKPC is characteristic of class III (guaiacol-type) peroxidases with chlorogenic and caffeic acids, that are abundant in artichoke flowers, as particularly good substrates at pH 4.5. Ferric AKPC reacts with hydrogen peroxide to yield compound I with a second-order rate constant ( k +1) of 7.4×10 5 M −1 s −1 which is significantly slower than that reported for most other class III peroxidases. The reaction of ferric and ferrous AKPC with nitric oxide showed a potential use of this enzyme for quantitative spectrophotometric determination of NO and as a component of novel NO sensitive electrodes.