A peroxidase isoenzyme secreted by turnip (Brassica napus) hairy-root cultures: inactivation by hydrogen peroxide and application in diagnostic kits.
We have purified various peroxidase isoenzymes from roots and hairy-root cultures of turnip (Brassica napus) which could potentially be used for commercial applications such as an enzyme immunoassays, diagnostic test kits, wastewater treatment and soil remediation. One of them, a basic peroxidase called HR2, was secreted into the medium of turnip hairy-root cultures. HR2 had a pI of 9.6, a molecular mass of 39.3 kDa and showed great thermostability. The inactivation of HR2 by H2O2 in the absence of reductant substrates was studied. Under these conditions H2O2 acted as a suicide substrate. The kinetic constants calculated have been compared with those of a basic isoperoxidase from horseradish (Armoracia sp.) roots (HRP-C), which is commonly used in commercial kits. The results for HR2 indicated that it was more resistant to inactivation because it presented a lower inactivation efficiency and a higher value for the partition ratio (r=1250) than those described for HRP-C. These results make turnip peroxidase HR2 suitable for use in systems in which high H2O2 concentrations are found. Such an application is demonstrated, namely an enzymic diagnostic kit for determination of uric acid in which HR2 was found to be as efficient as the enzyme originally included in standard kits.
Agostini
Elizabeth
E
Departamento de Biología Molecular, Facultad de Ciencias Exactas Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, CP 5800 Río Cuarto, Córdoba, Argentina.
Hernández-Ruiz
Josefa
J
Arnao
Marino B
MB
Milrad
Silvia R
SR
Tigier
Horacio A
HA
Acosta
Manuel
M
D029688:Brassica napus / Q000033:anatomy & histology / Q000201:enzymology*; D046508:Culture Techniques; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004795:Enzyme Stability; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010084:Oxidation-Reduction; D018517:Plant Roots / Q000201:enzymology; D011933:Reagent Kits, Diagnostic; D014527:Uric Acid / Q000032:analysis
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011933:Reagent Kits, Diagnostic; D014527:Uric Acid; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase