An Investigation of the Mechanism of the Luminescent Peroxidation of Luminol by Stopped Flow Techniques

Abstract The luminescent peroxidation of 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) by horseradish peroxidase (HRP) has been investigated by stopped flow and fluorescence polarization techniques. Titration studies show that luminol acts as a 2-electron donor during the reduction of the HRP-H2O2 complexes, while electron paramagnetic resonance measurements indicate the intermediate formation of luminol radicals. Second order rate constants for the reaction between the HRP-H2O2 complex and luminol and between Complex II and luminol were found to be 2.3 x 106 m-1 sec-1 and 7.2 x 104 m-1 sec-1, respectively. An interesting feature of luminol peroxidation is that, in the presence of excess H2O2, light is one of the products. We have made a number of observations relative to the development of an appropriate mechanism for light emission. These are (a) light production occurs in a step subsequent to Complex II formation but prior to the formation of free HRP, (b) fluorescence polarization measurements have shown that Complex II is not a ternary complex and thus does not contain bound luminol, (c) Complex III does not constitute a pathway to luminescence, (d) more than 1 mole of H2O2 per mole of HRP is required for light emission, (e) light production is dependent on the square of the luminol radical concentration, this being confirmed by computer simulation of the luminescence time course, and (f) the effects of both luminol and H2O2 concentration on the initial luminescence rate show normal Michaelis-Menten kinetics when such measurements are done on the stopped flow apparatus. A kinetic scheme has been formulated and a rate equation derived from it that accounts for the data outlined above and which is consistent with current concepts of peroxidase mechanisms.