Ammonium sulfate precipitation - loss of activity

[Gearhead26]

Hey guys/gals,

This is my first time going to an internet forum on account of an issue in the lab.

I'm no biochemist, but have decided (for some masochistic reason) that the best way to identify a particular enzyme whose activity can be measured but for which no gene has been identified, is to try to purify this enzyme "from scratch". We have an FPLC, and I've used ion-exchange and gel filtration to purify proteins, however in those cases I always knew the characteristics of those proteins (pI, cofactors, etc) and they were overexpressed.

In this case, I have a sensitive assay for the activity of this "new" enzyme, however I obviously know nothing about its characteristics.

In order to remove bulk impurities, I have tried ammonium sulfate precipitation. The enzyme in question salts out in the 45-55% fraction, however it looses tons of activity (The overall yield after AS precipitation is 1.8% of the original cell-free extract). I don't know what I should do at this point. My gut tells me that it's not the overnight dialysis (post-precipitation) that is causing loss of activity, because I have dialyzed cell-free extract overnight and loose only 50% of the activity compared to just leaving the extract in the fridge overnight.

 

Is it worth trying to optimize this step, or should I try to use another approach entirely?

Is it "normal" for an enzyme to loose this much activity during AS precipitation? I had always thought that most enzymes were quite stable in an AS pellet...

Thanks!

[Jens]

I would try out to start with something else (e.g. gel filtration).

Since your essay is sensitive, you can try mutliple different things with small amounts.

If purifaction turns out to be very difficult and you can invest more time you might think about your activity test can give you hints to what your protein might bind more specifically.

 

 

 

[CharonY]

Assuming everything else was ok and you really have sufficient enzyme (i.e. normalized activity against concentration, for example), then is possible that e.g. pH shifts due to the ammonium sulphate (or other treatment) may have denatured part of the proteins. Sometimes high salts also lead to irreversible activity losses, but especially with ammonium sulphate it is rather rare.

[BabcockHall]

I would desalt a small portion of the ammonium sulfate precipitate quickly, and run an activity assay on that. Sometimes just centrifuging then removing the supernatant removes most of the ammonium sulfate, and sometimes a Penefsky column works well. I also think starting with a different purification method might be a good idea, as others have said. You might want to pick up Scopes' book and Clarence Suelter's book.