Dibenzylfluorescein is a fluorogenic probe that acts as a substrate for specific cytochrome P450 (CYP) isoforms, including CYP3A4, CYP2C8, CYP2C9, CYP2C19, and aromatase (CYP19). [1] [2] [3] Dibenzylfluorescein is dealkylated by these CYP isoforms to produce fluorescein benzyl ether, which is further hydrolyzed to fluorescein by the addition of base (typically 2 M NaOH). [3] Dibenzylfluorescein is typically used near its apparent Km value of 0.87-1.9 &#181M. [1] [2] [3] The fluorescence of fluorescein is evaluated using excitation/emission wavelengths of 485/538 nm. Dibenzylfluorescein is used to detect changes in CYP catalytic activity caused by drugs or disease.[1] [4]

Reference:
[1]. Stresser, D.M., Blanchard, A.P., Turner, S.D., et al. Substrate-dependent modulation of CYP3A4 catalytic activity: Analysis of 27 test compounds with four fluorometric substrates. Drug Metabolism and Disposition 28(12), 1440-1448 (2000).
[2]. Donato, M.T., Jiménez, N., Castell, J.V., et al. Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes. Drug Metab. Dispos. 32(7), 699-706 (2004).
[3]. Salminen, K.A., Leppanen, J., Venalainen, J.I., et al. Simple, direct, and informative method for the assessment of CYP2C19 enzyme inactivation kinetics. Drug Metabolism and Disposition 39(3), 412-418 (2011).
[4]. Moutinho, D., Marohnic, C.C., Panda, S.P., et al. Altered human CYP3A4 activity caused by Antley-Bixler syndrome-related variants of NADPH-cytochrome P450 oxidoreductase measured in a robust in vitro system. Drug Metabolism and Disposition 40(4), 754-760 (2012).