Resorufin benzyl ether is a fluorogenic enzyme substrate applicable for the detection of CYP3A4 enzyme activity; after modification with boronic acid recognition groups, this compound enables the detection of ONOO⁻ via a self-immolative mechanism. Ex/Em=530-570 nm/590 nm.

CYP3A4:530–570 nm / 590 nm (Ex/Em)

Determination of CYP3A4 Activity [2]
1.Prepare the reaction solution
a. Prepare a 1 mM stock standard solution with Resorufin benzyl ether as the fluorescent substrate: Take 5 mg of Resorufin benzyl ether and dissolve it in a mixed solution of 1 mL of 2% w/v Poloxamer 188 , 500 μL of dimethyl sulfoxide and 3.5 mL of acetonitrile.
b. Freshly prepare the CYP3A4 enzyme solution: Take 5 mL of 1 mM stock enzyme solution and dilute it with 995 μL of buffer to prepare a 5 nM enzyme solution.
2.Perform the CYP3A4 activity assay
a. Conduct the reaction in a 96-well plate: Add 99 μL of buffer mixture and 1 mL of 1 mM Resorufin benzyl ether to each well, and adjust the substrate to a final concentration of 5 mM.
b. Add 100 μL of 5 nM enzyme solution, then incubate at 37℃ for 30 minutes.
c. Set the excitation wavelength (λex=570 nm) and emission wavelength (λem=590 nm), and determine the enzyme activity by fluorometric assay.
d. Factors affecting the determination of CYP3A4 activity include buffer type (phosphate buffer, Tris-HCl buffer), buffer concentration (50-200 mM) and incubation time (0-50 min).
Determination of CYP3A4 Metabolic Activity [3]
1.Add CYP3A4 enzyme to the reaction system and adjust its final concentration to 5 pmol per well.
2.Add 50 pM substrate and 200 mM potassium phosphate buffer to the reaction system of each well.
3.After co-incubating with BzRes for 45 minutes, detect the fluorescence value of metabolites with the excitation wavelength (Ex)=530 nm and emission wavelength (Em)=590 nm.
4.Calibrate the concentration of the full-strength extract as 100% (diluted at a ratio of 1:4 in the final assay system).
5.Perform two serial 1:3 dilutions on the 100% extract, and calculate the mean and standard deviation of the fluorescence values.

DMF: 0.15 mg/mL (0.49 mM), Sonication is recommended.