I have a protein that decarboxylates malonyl-CoA to form acetyl-CoA as part of it's catalytic activity. I want to directly measure the steady state kinetics for this decarboxylation step in the reaction. I know I can do it using radiometric assays but I am looking to develop a method for a new UPLC instrument that has a diode array, mass spec and evaporative light scattering detectors. These CoA's are hydrophilic thus extracting with organic solvent and concentrating is problematic.
Since CoA has an adenine ring, it can be detected at by its absorbance at 259 nm. It should be possible using reverse phase to separate malonyl CoA from acetyl CoA. The reactions can be quenched with formic or acetic acid.
You can measure the decarboxylation of malonyl-CoA by using one of three methods:
1. Lo MC, Wang M, Kim KW, Busby J, Yamane H, Zondlo J, Yuan C, Young SW, Xiao SH. A highly sensitive high-throughput luminescence assay for malonyl-CoA decarboxylase. Anal Biochem. 2008 May 1;376(1):122-30. doi: 10.1016/j.ab.2008.01.033. Epub 2008 Feb 2.
2. Kerner J, Hoppel CL. Radiochemical malonyl-CoA decarboxylase assay: activity and subcellular distribution in heart and skeletal muscle. Anal Biochem. 2002 Jul 15;306(2):283-9.
3. malonyl-CoA decarboxylase assay kits.
My company (Agilent) has developed a high-throughput MS based assay for the reverse reaction (acetyl-coenzyme A carbaoxylase). This could be used to measure decarboxylation as well.
Do you have experience with spectrophotometric enzyme assays? If acetyl-CoA is formed you might use citrate synthase as a coupling enzyme.
Your decarboxylase forms acetyl-CoA which is utilized by citrate synthase for citrate formation under release of free CoA. The release of CoA can be followed by the reaction with DTNB and an increase in absorption at 412 nm.
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