Is there anyone here who is familiar with calculation of enzyme activity using a radio-enzymatic assay? I am working on enzymes inhibitory activity and I am planning to use radioisotopes. I am getting count per five minutes data in the end after counting the product form by a liquid scintillation counter. Any biochem expert here. need help
Well, one thing I DO remember from my PhD, was the complicated activity calculations (http://darwin.bth.rwth-aachen.de/opus3/volltexte/2006/1629/pdf/Nowicki_Marcin.pdf p.53-56). You need to (1) factor for specific radioactivity in your chemical; (2) recalculate it into the assay / concentration; (3) calculate the product / assay ratio; (4) factor again the ug of protein, time of reaction, and the counts you got.
Also, I was able, at the end of the day, come up with a formula *FOR A GIVEN BATCH OF SUBSTRATE* (specific radioactivity), by keeping substrate conc. and reaction time constant. It came down to sth. on the lines of counts * constant / ug of protein.
When you buy a radioactive compound usally is very hot and at a very low concentration (something micromolar). Usually you need millimolar concentration of substrate, so you prepare the substrate solution at the concentration you need then you add some of the radioactive compound (you may calcyulate the amount utilizing the specific radioactivity indicated by the producer). Then you put a know amount of nanomoles of you mixture in the counter and you will obtain the CPM (count per minutes) , if you divide by the number of nanomoles you put in the vial you will obtain specific radioactivity in terms of CPM/ nmol. Now, depending on your protocol you should separate the radioactive product from the radioactive substrates and you need to have the reaction mixture at 0 time and after 5 minutes. The counter will tell you the radioactivity, at the beginning and at the end of the reaction, that was incorporated in our product, if you divide the CPM obtained from the counter by the specific activity calculated as described above, you will obtain the nanomol of your product generated in 5 minutes divide by 5 and multiply by the dilution operated to perform the assay you will obtain the nanomoles formed per minute which are the milliunits per ml in your enzyme preparation.. If you know the amount of protein in the mixture you can also obtain the specific activity. I hope that this will be of some help to you
See, in my case, I had to synthesize the substrate, so ALL my substrate was hot (labeled). In this case, I was able to drop the 0time point, as ALL my labeled product (separated by TLC, then scraped and counted) could only have arisen from the hot substrate.
Other than that, Maria told you everything perfectly correct. Let us know, how this worked for you! :)
Thanks to Maria Grazia Tozzi and Marcin Nowicki for their valuable advise. It will help me to solve my problem.
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