3-Cyano-7-ethoxycoumarin is cytochrome P450 (CYP) isoforms CYP1A1 and CYP1A2 .

Instructions
1. Solution preparation:
3-Cyano-7-ethoxycoumarin is usually provided in powder form. It can be dissolved in an appropriate solvent (such as DMSO) to prepare a 1-10 mM stock solution.
2. Experimental procedures:
1) Preparation of enzyme reaction system: Add 3-Cyano-7-ethoxycoumarin to the reaction system containing cytochrome P-450 enzyme. The common working concentration is 10-100 µM, but the concentration can be optimized according to the experimental requirements.
2) Appropriate cofactors (such as NADPH) are usually required in the reaction system because cytochrome P-450 requires NADPH as a reducing agent.
3. Enzyme reaction: The reaction system is reacted at an appropriate temperature (usually 37°C) for a period of time, usually 30-60 minutes, and the specific time can be adjusted according to the experimental design.
4. Product detection: After the reaction is completed, the blue fluorescence of the product is detected by a fluorescence spectrophotometer or fluorescence microscope. The excitation wavelength is 410 nm and the emission wavelength is 485 nm, which produces blue fluorescence.
5. Data Analysis:
Based on the intensity of the fluorescence signal, the activity of the P-450 enzyme can be analyzed. Higher fluorescence intensity is usually associated with higher enzyme activity.