Isolation and characterization of two peroxidases from Cucumis sativus.
Two heme peroxidases of 35.2 and 36.5 kDa have been isolated from cucumber (Cucumis sativus) peelings and characterized through electronic and 1H NMR spectra in the pH range 3.5-10.5. Their spectroscopic and catalytic properties, which are closely similar, are characteristic of highly homologous isoenzymes. Both proteins, as isolated, exist as a mixture of two ferric forms containing a high-spin and a low-spin heme in an approximately 2:1 molar ratio. The latter form likely contains a hydroxide ion axially coordinated to the heme iron and is proposed to be the result of partial irreversible protein inactivation due to the purification procedure. Both proteins in the reduced form are fully high-spin. The high-spin ferric form is sensitive to two acid-base equilibria with apparent pKa values of approximately 5 and 8.5, which have been assigned to the distal histidine and the arginine adjacent to it, respectively. These equilibria also affect the catalytic activity and the interaction with inorganic anions such as azide and fluoride. The reactivity of both proteins is closely similar to that of other plant peroxidases, primarily horseradish peroxidase; however, they also show spectroscopic properties similar to those of cytosolic ascorbate peroxidase. Therefore, overall, these two species show molecular, spectroscopic and catalytic features which are rather peculiar among plant peroxidases.
Battistuzzi
G
G
Department of Chemistry, University of Modena and Reggio Emilia, Italy.
D'Onofrio
M
M
Loschi
L
L
Sola
M
M
D001386:Azides / Q000737:chemistry; D002384:Catalysis; D002845:Chromatography; D018553:Cucumis sativus / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010544:Peroxidases / Q000737:chemistry*; D013053:Spectrophotometry; D013997:Time Factors
D001386:Azides; D006418:Heme; D006861:Hydrogen Peroxide; D007501:Iron; D010544:Peroxidases