Hey guys I couldn't find much literature on this. I'm working with an enzyme that may be covalently attached to coenzyme A and I'm looking to determine if A: the cofactor is present and B: if so in what percentage of the enzyme purified contains this cofactor.
Thanks for your help guys
Harry
If the CoA is covalently attached to the protein, it should be readily apparent by protein mass spectrometry. This should also be able to tell you what fraction of the enzyme has CoA attached if you see peaks with the mass of the apo-enzyme and the acyl-enzyme.
Coenzyme A might sometime formed unusual covalent complex between cysteine residue of the protein and the coenzyme this can be detected by mass-spectrometeric and reduction studies good luck
Thanks guys! Unfortunately we'll be waiting roughly a month for mass spec. I was wondering is there a way to denature the protein, separate COA from the protein, use dialysis to separate the protein from the cofactor and then quantify the COA present.
You can try to treat your enzyme with urea in the presence of strong reducing agent such as DTT then precipitate the denatured protein by ammonium sulfate assay the supernatant for Coenzyme A using BioVision CoenzymeA colorimetric Kits . This might work while you waiting for mass spect. Good luck again
If the CoA is covalently attached to the enzyme, the CoA will not separate from the dentaured protein. If the bond is labile in the presence of thiol reducing agent or acid, then it might work if you add such a reagent (for example, acetyl CoA will deacetylate in the presence of 10 mM DTT).
You might be able to measure the CoA associated with the protein by its 260 nm absorbance (extinction coefficent about 15,000 M-1cm-1). This is tricky because the protein also absorbs. However, if you know the sequence of the protein, you can calculate the absorbance of the urea-denatured protein with an on-line calculator, then see if there is a higher-than-expected absorbance at 260 nm.
Another approach would be to digest the protein to completion with trypsin and do a peptide map on HPLC, with 260 nm/280 nm absorbance ratio >1 as the indication for when the CoA-modified peptide elutes. Authentic CoA could be used as a standard for quantitation.
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