A bifunctional luminogenic substrate for two luminescent enzymes: Firefly luciferase and horseradish peroxidase

Horseradish peroxidase (HRP) catalyzes the oxidative chemiluminescent react ion of luminol, and firefly luciferase catalyzes the oxidation of firefly D -luciferin. Here we report a novel substrate, 5-(5'-azoluciferinyl)2,3-dihy dro-1,4-phthalazinedione (ALPDO), that can trigger the activity of HRP and firefly luciferase in solution because it contains both luminol and lucifer in functionalities. It is synthesized by diazotization of luminol and its s ubsequent azo coupling with firefly luciferin. NMR spectral data show that the C5' of benzothiazole in luciferin connects the diazophthalahydrazide. T he electronic absorption and fluorescence properties of ALPDO are different from those of its precursor molecules. The chemiluminescence emission spec tra of the conjugate substrate display biphotonic emission characteristic o f azophthalatedianion and oxyluciferin. It has an optimum pH of 8.0 for max imum activity with respect to HRP as well as luciferase. At pH 8.0 the bifu nctional substrate has 12 times the activity of luminol but has 7 times les s activity than the firefly luciferin-luciferase system. The specific enhan cement of light emission from the cyclic hydrazide part of ALPDO helped in the sensitive assay of HRP down to 2.0 x 10(-13) M and of ATP to 1.0 x 10(- 14) mel. Addition of enhancers such as firefly luciferin and p-iodophenol ( PIP) to the HRP-ALPDO-H2O2 system enhanced the light output. (C) 1999 Acade mic Press.