African Journal of Biotechnology Vol. 6 (7), pp. 923-928, 2 April 2007   

ISSN 1684–5315 © 2007 Academic Journals        

Full Length Research Paper

Optimum conditions for cotton nitrate reductase extraction and activity measurement

Justin Yatty KOUADIO¹, Hilaire Tanoh KOUAKOU¹, Mongomaké KONE¹, Michel  ZOUZOU² and Pierre Abo ANNO²

¹Université d’Abobo-Adjamé, UFR des Sciences de la Nature, Laboratoire de Biologie et Amélioration des Productions Végétales. 02 BP 801 Abidjan 02, Côte d’Ivoire.

²Université de Cocody, UFR Biosciences, Laboratoire de Physiologie Végétale, 22 BP 582 Abidjan 22, Côte d’Ivoire.

*Corresponding authors E-mail: yatjust@yahoo.fr.  Tel : (225) 20 30 42 60. Fax: (225) 20 37 81 18.

Accepted 17 June, 2006

Conditions of nitrate reductase extraction and activity measurement should be adapted to plant species, and to the organs of the same plant, because of extreme weaknesses and instabilities of the enzyme. Different extraction and reaction media have been compared in order to define the best conditions for cotton callus nitrate reductase activity measurement. The use of potassium phosphate buffer, at pH 7.5  allowed for a better stabilization of the enzyme during extraction. The addition of 15 mM of glutamine in the extraction buffer stimulates significantly, in vitro, the reduction of nitrate. Enzyme activity is moreover optimal when 1 M of exogenous nitrate, as substrate, is added to the reaction medium. At these optimum conditions of nitrate reductase activity determination,  the substrate was completely reduced after 20 min of enzyme incubation, and the greatest velocity of nitrate transformation into nitrite  (µg of NO₂^-/min/g F.W) is observed when incubation period of enzyme is short (1 to 5 min).

Key words: Extraction, dosage, nitrate reductase activity, callus, cotton.