Hi everyone,

I am attempting a citrate synthase reactivation assay modeled after several previous assays I have found in literature. In short, I am using oxaloacetate and Acetyl-CoA to determine the activity of my commercial citrate synthase. I am using Ellman's reagent (DTNB) which reacts with Coenzyme A, produced by the citrate synthase reaction and monitoring an increase in absorbance at 412nm.

The issue that I am having, is that my Acetyl CoA that we purchased ( >96% purity) appears to be laced with CoA, as my reagent controls always have the highest absorbance, far above, or the same as, reactions containing citrate synthase. I need to be able to observe the activity of citrate synthase to produce CoA, but I can't achieve this when my Acetyl CoA + DTNB is the only combination that is producing the increase in absorbance.

I have tried increasing CS concentrations, decreasing Acetyl CoA concentrations, and the combination of both and I still can't separate out the activity of citrate synthase. Any suggestions are welcome!! Thank you in advance!