Hi! I am performing and enzymatic assay, where Acetoacetyl-CoA is the substrate. I have gotten the Acetoacetyl-CoA powder that SIGMA sells (5 mg). I think it would be easier to handle if I just make a solution with all the powder, and store it in aliquotes for later use. However, I am not sure which solvent should I use to dissolve Acetoacetyl CoA. The buffer for my enzymatic assay is a phosphate buffer pH 7. SIGMA only says that this compound is soluble on water, but I have read that the Acetoacetyl-CoA solution should be in a pH between 4 and 6 to be stable for several months. Any suggestions? I need to use a solvent that will not interfere with my enzymatic assay. Thanks! :)
Hi Kelly,, it is advisable that you retain your substrate as powder until you are ready for your assay. Besides finding the right solvent for it, degradation will set in the moment you dissolve it and by the time you are ready to do the assay, the actual content of your acetoacetyl CoA could be much less than you have initially dissolved. However if you insist, an acetate buffer will be suitable to keep your sample near its native pH of 4-5. And you will be able to know exactly the total acetate ion concentration in your buffered solution perhaps your reaction will entail hydrolysis of your substrate. You might want to account for the additional acetate released from your substrate to determine the rate of the hydrolysis.
Best of luck.
Do not include a reducing agent in the buffer you use to dissolve acyl-CoA because it will cause loss of the acyl group. Also, when running your reaction, keep the reducing agent concentration as low as practicable for the same reason.
Hi there,
Acidic buffers like acetate or citrate will do as long as the dilution factor is large enough when preparing the assay to ensure the final pH will actually be 7. It might be these buffers interfere with the assay as they are structurally related to the substrate. You can also consider phosphate buffer at pH5.8 or 6 as we are still in the buffering range of pH (roughly pKa-1)...
Maybe it makes sense to weigh the total amount of AcCoA exactly, dissolve and aliquot the material and the dry it imeediately (e.g. by SpeedVac, vacuum cabinet or lyophilization). Then store desiccated and maybe at -80° until use.
I would simply diluteyour acetoacetyl CoA powder in cold water and freeze your aliquots. In that state they should be stable until you need them. Check the pH to see if the solution is mildly acidic. If not you can adjust with a drop of dilute HCl. This way of handling it means that you don't need to worry about buffer solutions interfering with your assay.
If you don't feel 100% secure about stability over time, you can always check using a quantitative assay with beta-hydroxxybutyrate DH and NAD+.
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