Purification and characterization of a new cationic peroxidase from fresh flowers of Cynara scolymus L.
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)=42300+/-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 (epsilon=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 x 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.
López-Molina
Dorotea
D
Grupo de Investigación de Enzimología (GENZ), Departamento de Bioquímica y Biología Molecular A, Facultad de Biología, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain.
Heering
Hendrik A
HA
Smulevich
Giulietta
G
Tudela
José
J
Thorneley
Roger N F
RN
García-Cánovas
Francisco
F
Rodríguez-López
José Neptuno
JN
D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000032:analysis; D031943:Cynara scolymus / Q000201:enzymology* / Q000235:genetics; D035264:Flowers / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009569:Nitric Oxide / Q000032:analysis / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013379:Substrate Specificity
D002241:Carbohydrates; D009569:Nitric Oxide; D010544:Peroxidases