Alternatively, luminol dehydrogenation by hypohalite followed by reaction with H2O2 yields dioxygenation consistent with the same reaction order. That reaction is second order for H2O2 is consistent with the non-enzymatic reaction of hypohalite with a second H2O2 to produce singlet molecular oxygen (1O2*) for luminol dioxygenation. Unlike the radical mechanism of common peroxidase, haloperoxidases (XPO) catalyze non-radical oxidation of halide to hypohalite. Haloperoxidase-catalyzed luminol luminescence requires acidity, but HRP action requires alkalinity. For MPO and EPO, reaction is first order for luminol, but for HRP, reaction is greater than first order for luminol. For EPO, reaction is first order for Br−. For MPO, reaction is first order for chloride (Cl−) or bromide (Br−). Horseradish peroxidase (HRP)-catalyzed luminol luminescence is first order for hydrogen peroxide (H2O2), but myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are second order for H2O2. Thus, the reaction order of each participant reactant in luminol luminescence was determined. The velocity of enzyme action is dependent on the concentration of reactants. In addition, it is shown that bicarbonate can participate in secondary oxidation reactions after reacting with ONOO-.Ĭommon peroxidase action and haloperoxidase action are quantifiable as light emission from dioxygenation of luminol (5-amino-2,3-dihydrophthalazine-1,4-dione). These results suggest that ONOO- can be a critical unrecognized mediator of cell-derived luminol chemiluminescence reported in previous studies. NO nor O2.- alone were capable of directly inducing significant luminol chemiluminescence in our assay systems. Luminol radical reacts with O2.- to form the unstable luminol endoperoxide, which follows the light-emitting pathway.
O2.- appears to be formed secondarily to the reaction of a bicarbonate-peroxynitrite complex with luminol, yielding luminol radical and O2.
Chemiluminescence was superoxide dismutase-inhibitable, indicating that O2.- was a key intermediate for chemiexcitation. The quantum chemiluminescence yield of the ONOO- reaction with luminol in bicarbonate was approx.
Peroxynitrite reacted with luminol to yield chemiluminescence which was greatly enhanced by bicarbonate. Vascular endothelial cells, smooth muscle cells, macrophages, neutrophils, Kupffer cells and other diverse cell types generate superoxide (O2.-) and nitric oxide (.NO), which can react to form the potent oxidant peroxynitrite anion (ONOO-).
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