CYP Activity Assay - (Jan/30/2011 )

Good day everybody. I am a new graduate student at my university and I'm studying the inhibitory effect of compounds on these enzymes. The problem I am having is that when I do the activity assays for the CYP enzymes, the "jump" between my negative control (no CYP activity) and positive control(100% CYP activity) isn't big enough. I've done this assay several times and can't figure out what is wrong. I know enzymes are delicate little things and need the right conditions to be fully active, mainly temperature and pH. But as far as I know, I maintain these two factors while doing the assay. I also know that repeated freezing and thawing of the enzyme will denature it and it will lose activity little by little, but my "trainer" had aliquoted small volumes into microcentrifuge tubes and I have used several of these (3 or 4 so far) and get the same result no matter if it was the first time using that tube or not. My "trainer" also did the assay herself and gave me some tips and I try to follow everything she did. She got a nice difference between her positive and negative when she did it.

So I'm thinking it is my technique. My trainer said I only need to practice, which I have no problem with but I don't want to keep practicing the wrong thing. Can anyone tell me any bad technique skills would cause enzyme activity to drop?

improper or inconsistent pipeting

mdfenko on Mon Jan 31 20:47:33 2011 said:

Thanks for the suggestion, I'm working on that. I just can't believe pipetting would affect my results in that way.

When you say that the difference between the controls is small, does that mean that the negative is too high (mixing in the wrong order) or that the positive is too low (forgetting one of the cofactors)?

I’m not sure which CYP assay you are running or the type of inhibitors you are trying but I’ve found with the fluorescent kits it’s necessary to run complete no-enzyme controls with every compound. Also you might like to discuss with your supervisor the difference between screening for ‘true’ inhibitors or detecting competing substrates.

Good luck

DRT on Tue Feb 1 23:29:38 2011 said:

Thank you, I will keep that in mind when I use inhibitors. I meant that my positive is too low. When I do the activity assay, I don't add any inhibitors to the wells. I'm just trying to see if I can get the enzyme fully active. I posted this on another site, it is my protocol:

Ok, fist of all I stop the reaction with 20% tris base and 80% acetonitrile. As for what I do in my protocol:

Make up enzyme/substrate mix (E/S mix) with phoshate buffer(KPB), DI water, prewarm tube for at least 10 minutes after which I add my substrate then CYP.

I make up NADPH mix during the prewarm time mentioned above, using NADPH, KPB and DI water. I wrap this tube with foil beforehand because NADPH is light sensitive. After making it up, I pour 100microlitres of NADPH in the first 4 columns (A - D) and three rows (so that I get my results in triplicates) in a 96 well microtitre plate then prewarm.

I then do the following:

I use the three wells in A as my positive control; I add 100microlitres of E/S mix to the well, incubate for 15minutes then add 75 microlitres stop solution (the 20%tris/80%acetonitrile).

Well B is my negative control, where I add the E/S mix after adding stop solution. Hence no conversion should accur.

Well C is to test if my stop solution affects fluorescence. In these wells, I replace stop solution with phosphate buffer. I find that my stop solution does not affect fluorescence as A and C give similar readings. Maybe you could use this solution instead of methanol.

Well D is just NADPH.

I try to be quick and not let the E/S mix cool down before adding to wells A and C. My results are usually that my negative control is in the 100s while my positive is only in the 200s or 300s. I should be getting up to 500s or even 700s.

I hope I described my protocol properly.

I can’t spot anything obvious. We include a dehydrogenase to regenerate NADPH but you are already following a protocol that works in your lab. The only other thing that comes to mind is ensuring that the CYP vial is given a gentle mix after it has thawed.

DRT on Wed Feb 2 21:18:19 2011 said:

Ok thank you

I did as you said DRT and got better results. MAYBE that was the reason why I got better results, maybe it wasn't. All I know is that I will continue mixing my reagents before I add them (NADPH, CYP and substrate). My activity was in the high 400s today. Hoping for even better on Monday. I'm also working on my pipetting even though it is hard to know what I am doing wrong without someone telling me. I don't really get how pipetting can be inconsistent unless the pipette itself is not working properly. Anyway, thank you guys.