Dibenzylfluorescein

Dibenzylfluorescein (DBF) is a fluorogenic probe (Fluoresecent dye) that acts as a substrate for specific cytochrome P450 (CYP) isoforms, including CYP3A4, CYP2C8, CYP2C9, CYP2C19, and aromatase (CYP19). Dibenzylfluorescein is typically used near its K_m value of 0.87-1.9 µM (Ex=485nm，Em=535nm). Dibenzylfluorescein is used to detect changes in CYP catalytic activity caused by drugs or disease^[1][2][3][4]. In Vitro:The protocol of P450-catalyzed metabolism of Dibenzylfluorescein and effect of base^[3]:
Reaction Process: Dibenzylfluorescein is dealkylated by P450 to form a fluorescein benzyl ester, which is further hydrolyzed to fluorescein by NaOH (if present). Addition of 2 M NaOH causes also decomposition of Dibenzylfluorescein to fluorescein benzyl ether.
1. Incubation mixtures for CYP2C19 enzyme-catalyzed samples each 150 μL contains 0.1 M Tris-HCl buffer (pH 7.4), 10 μM Dibenzylfluorescein, 15 pmol of CYP2C19 enzyme, and 50 μL of NADPH-regenerating system.NADPH-regenerating system contains 1.13 mM NADP, 12.5 mM isocitric acid, 56.33 mM KCl, 187.5 mM Tris-HCl, pH 7.4, 12.5 mM MgCl2, 0.0125 mM MnCl2, and 0.075 U/ml isocitrate dehydrogenase.
2. The samples were incubated for 30-60 min at 37°C. The reactions were terminated by rapid cooling to 4°C and after centrifugation, the supernatants were analyzed by LC-MS.
3. Pure Dibenzylfluorescein, fluorescein benzyl ester, fluorescein benzyl ether, and fluorescein (all 10 μM) were used as standards and were analyzed in the absence and presence of 2 M NaOH.