Hello!
I want to quantify HMGCS2 enzymatic activity in a simple way and I wondered if someone knows of an in vitro enzymatic activity protocol using spectrophotometry or similar.
Thank you!
Hi Silvia,
It should be fairly simple to detect the synthase activity. The enzyme takes acetyl-CoA and acetoacetyl-CoA and makes HMG-CoA + free CoA. The simplest way is to measure free CoA using a thiol reagent like DTNB or CPM. I can go into this in more detail if you need.
Another fluorogenic reagent that can be used to measure CoA production is called ThioGlo-1. This assay and the chromogenic DTNB (Ellman's reagent) assay are not compatible with thiol-based reducing agents like dithiothreitol and 2-mercaptoethanol.
I was thinking about this. The thiol detection is somewhat non-specific since any thiols in the sample will give a signal. Background should be constant with a rising signal due to CoA but if the background is too high, it can potentially interfere. An alternative method would be to add NADPH and some HMG-CoA reductase and observe the decrease in signal at 340 nm. Again, depending upon the species you are working with and what else is in your samples, you could have interference from NADPH consumption by non-HMGCR sources but they could be easily corrected for by observing signal in the absence of a HMGCS2 substrate.
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