P450 Demethylation Fluorescent Activity Kit

The P450 Demethylase Activity research-use-only kit is a fluorescent activity assay designed for the quantification and detection of P450 demethylating actrivity in liver microsomes or cerosomes such as Cyp P450 3A4, 2B4 and 2D6.

This kit includes black 96-well plate(s), assay buffer, formaldehyde standard, and other components to perform the assay. This kit does not contain P450 systems. Microsome, cerosome, baculosome, or supersome P450 systems, or recombinant P450, need to be supplied by the user. A fluorescence 96-well microplate reader capable of reading fluorescent emission at 510 nm, with excitation at 450 nm, is required for use of this kit.

Performance characteristics
• Assay type: fluorescent activity kit
• Sample types: demethylating P450 systems like liver microsomes or cerosomes such as Cyp P450 3A4, 2B4, and 2D6
• Standard curve range: 1.6–20 pmol/100 μL
• Reactivity: human, rat

Background
The cytochromes P450 (P450s) are a superfamily of heme containing enzymes that display tremendous diversity with regard to substrate specificity and catalytic activity. P450s use a plethora of both exogenous and endogenous compounds as substrates in enzymatic reactions. Usually they form part of multicomponent electron transfer reactions. Catalysis by the eukaryotic P450 enzymes involves a multistep reaction cycle that includes two steps in which electron transfer is accomplished from a redox partner. The diflavin protein, NADPH cytochrome P450 reductase, contains both FAD and FMN and can transfer both electrons needed for the catalytic cycle3. In some P450 reactions, the second electron of the reaction cycle also can be delivered by cytochrome b5.

Lipid plays an important role in the reconstitution of P450-dependent activities after protein purification6. Most in vitro studies for the reconstitution of P450 activities use dilaurylphosphatidylcholine (DLPC) as the lipid component. The reconstitution of enzymatic activity involves a concentrated incubation of P450, its redox partners (NADPH and reductase), and lipid followed by dilution into the final assay components.

Assay principle
The P450 Demethylase Activity kit is designed to quantitatively measure the enzymatic activity of formaldehyde-producing enzymes such as cytochromes P450. The kit is unique in that the fluorescent substrate is not involved in the multicomponent P450 reaction, but measures the product of the demethylation, formaldehyde. No separation or washing is required. The kit has been validated for several P450 systems and should work with any biological system that is producing formaldehyde as a product of demethylation.

The kit provides an optimized buffer for P450, lyophilized vials of the cofactor NADPH for the reaction, a stable formaldehyde standard, the Formaldehyde Detection Reagent (FDR) and two 96-well plates for detecting the generated fluorescent signal. The end user will have to provide the microsomal, baculosome system or the recombinant P450, reductase and cytochrome b5 system and any cofactors necessary for activity, along with any candidate drugs, inhibitors, or activators being tested. The reaction should be carried out in our supplied buffer or a similar PBS based buffer system.

Following the P450 NADPH-induced reaction, the generation of formaldehyde can be stopped by addition of a suitable inhibitor, or the supplied stop solution of acetic acid. The FDR is then added to all the wells. If calibration to formaldehyde is needed (for cross lab comparisons) then a formaldehyde standard curve generated from the supplied standard should be run.

After a short incubation at 37°C for 30 minutes, the fluorescent product is read at 510 nm in a fluorescent plate reader with excitation at 450 nm. The P450 activity is determined based upon formaldehyde production.

Related links
Learn more about ELISA kits
Learn more about other immunoassays