Dr.Soroka

Hello all, I was hoping as fellow scientists you could help me with a quandary. I am a molecular biologist/ biochemist reseraching functional foods and their bioactive compounds. Currently, we are investigating constituents of ginger and their bioactivity. I've set up an assay in CYP2A6 Human Liver Supersomes to test an unknown inhibitor's inhibition properties on this enzyme. In my experimental set-up, I've used coumarin as a substrate, hoping the 7-hydroxy coumarin levels after incubation/metabolism would help me gauge if our compound inhibits CYP2A6 or not. I've set up several standard curves with 7-hydroxycoumarin in different organic solvent systems and read its fluorescence on a plate reader at 360em/460ex. The standard curves have been coming out nicely. However, I've performed the experiment with CYP2A6, which should supposedly hydrolyze coumarin to 7-HC in the absense of inhibitor and have gotten no response in my no-inhibitor control. My buffer solution is potassium phosphate and water and I stop the reaction with 1:1 acetonitrile, with 2% acetic acid. Any thoughts on why I couldn't be getting a response? LC/MS also showed no response and HPLC wasn't helpful either. Thank you so much! Dr. Soroka