RE: What is an easy way to determine the active fraction of a recombinantly expressed enyzme prep?
If the Enzyme you isolated catalyses the formation of a product from the substrate that has a different absorption maximum wavelength than the substrate you should be able use your UV/VIS in single wavelength mode for the assay. Prepare a serial dilution of enzyme in a reaction buffer with catalyst if needed etc, then add substrate, then monitor the formation over time. Been almost 14 years since I have done this, but thats what comes to mind at the moment. If you do not know the absorption max for for S or P, then do full spectrum scans on them in serial dilutions. Also, the S or P could have cause allosteric interference that affect the reaction rate. I believe good Enzyme Kinetics book could help with that part. Trying to remember my favorite Author for this......its somewhere on me book shelf.
You can apply the same method if either S or P are fluorescent, and you have a flourometer. You may need to use cut off or band pass filters depending on how close the absorption or emission maximums are to the emission max of the light source, which you can find info on from the manufacturer. I think the bulbs are either Xenon or Xenon Hg
If none of those will work...try HPLC.
I use LC-MS to quantitate product formation directly for most enzymes I work on. We have a high throughput system, that with a little bit of assay development ... allows for running a 96 well plate of reaction samples in as little as an hour.
Sounds like a awesome system. Make? Always wanted a LC-MS, some day perhaps. After I have a well oiled EM lab I may get one. However, it will have to pay for itself like the EMs.
Well the only important part is the autosampler which enables the throughout. That is an Apricot ADDA.
I used to love doing full spectrum scans of substances with a UV VIS or FLourometer. Watching the different peaks form at different concentrations...
This just answers a question of how I might quantitate product. However that is not the question I am asking!
The question I am asking is, what sort of reaction would I need to set up to determine the active fraction of the enzyme. A serial dilution of the enzyme can tell me whether or not my enzyme behaves normally (IE we should see a linear relationship between enzyme concentration and product formation rate). It also potentially allows me to calculate specific activity (nmol product/min/mg protein) and that should be consistent no matter how much enzyme I include. However, none of that tells me how much of that enzyme is actually turning over, it just tells me whether the same amount relative to how much there is total, is turning over, and there are no concentration based effects (oligomerization of enzyme changing activity etc..).
You're right a good Enzyme Kinetics book could help with that part, a particular type of kinetic experiment could answer this question well (hint, look at the plot).
Ah...this is some kind of quiz. I thought you needed help. I am not so interested now, hahahaha. However, the plot appears to fit Michaelis Menten kinetics.
I don't need help with enzyme kinetics ;)
This is a StemQ question, I am hoping someone will answer it. It's not a quiz. I know the answer, but I'm not making a post to provide it.
The plot is not of the appropriate relationship for MM kinetics. :)
It's not steady state (at least not exclusively.)