Purification of protein by ion exchange chromatography

[mousumi.biotech]

Dear everybody

i am trying to purify a protein by anion exchange chromatography. After elution by changing the salt gradient, i was checking the presence of my interested protein by doing protein specific assay. The end product of the enzyme assay gives absorption maxima at 420 nm which is a yellow colour product. But after the reaction of about 2 hrs, i stored the same enzyme mixture at -20 C. I didnt get any absorbance at 420 nm after 2 hrs of reaction, but after storing the same at -20 C, it gave the yellow colour product after about 18-20hrs. what may be the reason behind that??

Mousumi

[tgd]

Hello,

This seems a very interesting problem, but I think we need a few more details. Is your assay a continuous assay, done is a spectrophotometer for example, or is it discontinuous?

Am I correct in thinking that your enzyme fraction from the DEAE-cellulose column lost activity on storage at -20 degrees C? I am not clear as what happens after this. Did the activity come back? If so, did ALL of the activity come back?

As you probably know, it would not be unusual for a enzyme to be inactivated by freezing. On strategy for avoiding this is to include glycerol (up to 20 percent) or other cryoprotectant, but this may not be your problem?

It would also help to know what the enzyme is, and the basis for the assay, but you may not will to reveal this.

As regards your assay, is the plot of velocity versus enzyme concentration linear? That is, if you double the amount of enzyme do you double the initial rate, and if you half the amount of enzyme do you half the initial rate, etc?

Is there a blank rate and has this been subtracted?

If it is a discontinuous assay ** is product formation linear with time?**. For example, is the amount of product formed after 2 hours exactly double the amount of product formed after 1 hour? In other words, are you measuring initial rates?

It seems you have an interesting problem, but I cannot fully grasp under what conditions the enzyme is reactivated.

tgd

[tgd]

Hello,

I tried to reply previously, but the post seems to have got lost!

It seems that you have an interesting problem, but we need more information. I am not clear when the enzyme became inactivated and, in particular, when the activity returned.

Am I correct in thinking that storage at -20 degrees C caused inactivation? But what happens then? Under what conditions does the activity return? How much of the activity returns? All of it or just some?

As regards your assay, is it continuous (measured in a spectrophotometer for example) or discontinuous? Is the plot of velocity versus enzyme concentration linear? If you double the amount of enzyme do you exactly double the initial rate, and if you half the amount of enzyme do you exactly half the initial rate, for example?

If the assay is discontinuous, is the amount product formed after 2 hours exactly double that formed after 1 hour. In other words, are you certain you are measuring initial rates?

What does the progress curve look like? Is there a lag? Is it sigmoid by any chance?

It would also help to know the identity of the enzyme and the basis of the assay, but you may prefer not to divulge this information.

It would seem you have a very interesting problem if apparently inactivated enzyme reactivates, but as I said above a more precise description of the problem is needed.

Good luck,

tgd