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Biotechnology and Applied Biochemistry (2002) 35, (1–7) (Printed in Great Britain)

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A peroxidase isoenzyme secreted by turnip (Brassica napus) hairy-root cultures: inactivation by hydrogen peroxide and application in diagnostic kits

Elizabeth Agostini*, Josefa Hernández-Ruiz†, Marino B. Arnao†, Silvia R. Milrad*, Horacio A. Tigier*¹ and Manuel Acosta†

*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, and †Departamento de Biología (Fisiología Vegetal), Universidad de Murcia, 30100 Murcia, Spain

Key words: diagnostic kits, enzyme inactivation, kinetic studies, suicide substrate, uric acid determination.

Abbreviations used: ABTS, 2,2´-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid); HRP, horseradish peroxidase; HR2, turnip (Brassica napus) cationic peroxidase isoenzyme; HRP-C, horseradish (Armoracia sp.) peroxidase isoenzyme C; SP-, sulphopropyl-.

¹To whom correspondence should be addressed. (e-mail htigier@exa.unrc.edu.ar).

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.

Received 26 June 2001/9 September 2001; accepted 4 October 2001

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