Luminol oxidation by hydrogen peroxide catalyzed by tobacco anionic peroxidase: steady-state luminescent and transient studies

• Published in: Photochemistry and photobiology 1998, Vol.67 (1), p.106-110
• Main Authors: Gazaryan, I.G, Rubtsova, M.Y, Kapeliuch, Y.L, Rodriguez-Lopez, J.N, Lagrimini, L.M, Thorneley, R.N.F
• Format: Article
• Language: English
• Subjects: transgenic plants
• ISSN: 0031-8655; 1751-1097

The properties of a newly isolated anionic tobacco peroxidase from transgenic tobacco plants overexpressing the enzyme have been studied with respect to the chemiluminescent reaction of luminol oxidation. These were compared to the properties of horseradish peroxidase in the cooxidation of luminol and p-iodophenol, the enhanced chemiluminescence reaction. The pH, luminol and hydrogen peroxide concentrations were optimized for maximum sensitivity using the tobacco enzyme. The detection limit for the latter under the optimal conditions (2.5 mM luminol, 2 mM hydrogen peroxide, 100 mM Na-borate buffer, pH 9.3) was about 0.1 pM, which is at least five times lower than that for horseradish peroxidase in enhanced chemiluminescence with p-iodophenol. The rate constants for the elementary steps of the enzyme-catalyzed reaction have been determined: k1 = 4.9 x 10(6) M-1 s-1, k2 = 7.3 x 10(6) M-1 s-1, k3 = 2.1 x 10(6) M-1 s-1 (pH 9.3). The similarity of these rate constants is unusual for plant peroxidases. The high catalytic activity of tobacco peroxidase in the luminescent reaction is explained by the high reactivity of its Compound II toward luminol and the high stability of the holoenzyme with respect to heme dissociation. This seems to be a unique property of this particular enzyme among other plant peroxidases.