LUMINOL OXIDATION BY HYDROGEN-PEROXIDE CATALYZED BY TOBACCO ANIONIC PEROXIDASE - STEADY-STATE LUMINESCENT AND TRANSIENT KINETIC-STUDIES

The properties of a newly isolated anionic tobacco peroxidase from tra nsgenic tobacco plants overexpressing the enzyme have been studied wit h respect to the chemiluminescent reaction of luminol oxidation, These were compared to the properties of horseradish peroxidase in the coox idation of luminol and p-iodophenol, the enhanced chemiluminescence re action, The pH, luminol and hydrogen peroxide concentrations were opti mized for maximum sensitivity using the tobacco enzyme, The detection limit for the latter under the optimal conditions (2.5 mM luminol, 2 m M hydrogen peroxide, 100 mM Naborate buffer, pH 9.3) was about 0.1 pM, which is at least five times lower than that for horseradish peroxida se in enhanced chemiluminescence with p-iodophenol, The rate constants for the elementary steps of the enzyme-catalyzed reaction have been d etermined: k(1) = 4.9 x 10(6) M-1 s(-1), k(2) = 7.3 x 10(6) M-1 s(-1), k(3) = 2.1 x 10(6) M-1 s(-1) (pH 9.3), The similarity of these rate c onstants 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 stabil ity of the holoenzyme with respect to heme dissociation, This seems to be a unique property of this particular enzyme among other plant pero xidases.