What is an easy way to determine the active fraction of a recombinantly expressed enyzme prep?

in #stemq5 years ago (edited)

I have recombinantly expressed an enzyme in E. coli and purified it through standard affinity and size exclusion columns on an AKTA FPLC system. I have established that this protein is purified to at least 98% purity through examination on an agarose gel. I have determined the concentration of my protein based on UV/Visible spectroscopy to be 40 uM.

I want to do some mechanistic enzyme kinetics experiments with this preparation, how can I determine what fraction of my 40 uM prep is actually catalytically active?

To assist you, I will provide you an image which may be of some help:


The above image was released for use into the public domain.

Please explain to me what type of assay I should do, how I should set it up, and how I should analyze the data to extract the information I want. I am so very clueless when it comes to enzymology, I really need your help!

StemQ Notice: This post was originally submitted on StemQ.io, a Q&A application for STEM subjects powered by the Steem blockchain.

Sort:  

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.)

To listen to the audio version of this article click on the play image.

Brought to you by @tts. If you find it useful please consider upvoting this reply.

Hi @justtryme90!

Your post was upvoted by @steem-ua, new Steem dApp, using UserAuthority for algorithmic post curation!
Your UA account score is currently 6.360 which ranks you at #193 across all Steem accounts.
Your rank has not changed in the last three days.

In our last Algorithmic Curation Round, consisting of 255 contributions, your post is ranked at #174.

Evaluation of your UA score:
  • You've built up a nice network.
  • The readers appreciate your great work!
  • Try to work on user engagement: the more people that interact with you via the comments, the higher your UA score!

Feel free to join our @steem-ua Discord server

Congratulations @justtryme90! You have completed the following achievement on the Steem blockchain and have been rewarded with new badge(s) :

You got more than 9000 replies. Your next target is to reach 9250 replies.

Click here to view your Board
If you no longer want to receive notifications, reply to this comment with the word STOP

Support SteemitBoard's project! Vote for its witness and get one more award!

Coin Marketplace

STEEM 0.39
TRX 0.12
JST 0.040
BTC 70118.22
ETH 3546.28
USDT 1.00
SBD 4.89