Carbamylation of protein (no enzymatic reaction with urea) is very likely to occur!

6 months at room temperature is a long time. I think that the presence of urea is not the reason. As you know, urea provokes denaturation, but it is possible that even at those high concentrations, bacterial proteases are not totally inactive, and I suppoe that they are slowly degreded your protein.

Thank you for the reply!

Some proteases remain active in urea solution. Therefore, try to add a protease inhibitor such as phenylmethylsulfonyl fluoride (PMSF) with the protein for long storage.

Yes, I've observed this before for much shorter storage periods and lower temperatures. 8 M urea is no guarantee against some E. coli proteases -to which your protein is served completely denatured, to boot.

By the way, long-term storage in 8 M urea is never a good idea. Urea will inevitably form some cyanate which in turn will carbamylate the amines of basic residues. Better use GuHCl, if compatible with your downstream processing.

I concur with the previous comments - and I would like to add, that storing at room temperature, may expose the sample to light, including UV light from fluorescent bulbs. such light can break disulphide bridges, and thus provide a possible explanation to the observed degradation. Our studies have shown that very low levels of UV-B (280-315 nm) (significantly less than the solar output in UV-B) is sufficient to induce such changes.

@Steffen,

Nice! I have often advised students, etc. to avoid exposing their materials to light (especially intense solar light entering through a window pane due to a blind left raised). Is there a publication I can point them to?

Carbamylation of protein (no enzymatic reaction with urea) is very likely to occur!

Thank you very much for all!

I'm strongly agree with prof. Delanghe assert certainly after a period of time after denaturation would be probable the occurrence of breaksures of peptide bonds due to nucleophilic attack of urea to amide linkage between residues

Thank you for advice!

8M Urea denatures the proteins this could happen if the protein is multimeric.

Yes agrree urea will disrupt the noncovalent bonds within the protein molecules, unfold the protein and make these more susceptible to various chemical ,physical and possibly enzymatic attacks and reaction s

I always sored my proteins in phosphate buffer for short term at minus 20, but usually at minus 80. Important is that the freezing occurs fast as well as that the thawing is done as fast as possible by slightly shaking under about handwarm water. These later precautions are for enzymes which are cold labile and thus by slow freezing / thawing would get denaturated. There are --although not that frequent--- enzymes which are cold labile e.g. the a-isopropylmalate synthase (leucine biosynthesis enzyme) from Alcaligens eutropus H16.

I stayed for mesophilic enzyme away from storing in urea, since there are proteases which are not totally inhibited by 6M urea, beside as elegantly pointed out above, causing denaturation especially at RT.

Proteinases are most likely present and the urea exposes peptide bonds that are not available for clevage in the native state. I have noted this issue in the presence of SDS and was able to prevent the protein degradation by heating at 100C for 30 sec. to inactivate the preteinase. This approach may be useful in your situation if you want to recover active protein. Other possibility, freeze or maintain at O C will probably minimize degredation or remove the proteinase contamination by further chromatoraphy.

Other storage options for your protein could include keeping it as an ammonium sulfate precipitate at -20C or in a high glycerol solution (ex. 50%) at -20C if for some reason flash-freezing and -80C storage are issues.

Any protein having even a tiny molecular weight can be denatured by urea through the peptide bonds. Urea works in similar way as HCL does, as their hydrogen bonds polarizes peptide groups. This normally leads to weakening the tertiary structure. Then the result is a gradual break-up action that takes place in the inner part of the structure. consequently water and urea can access more easily into the protein structure whereas urea does more unfolding processes on the proteins molecules, finally the hydrophobicity increases which results in denaturation process.

PS. you would better keep your protein in glycerol 1:1 at - 35C. Why do you need to keep protein at room temperature in Urea!!!!

I tend to think it is residual proteases which are not denature at this urea concentrations. It is well known as was pointed out that many proteases are not affected by this condition (look at many of the partial proteolysis papers where they needed not only to denature the protein but simultaneously add protease inhibitors and your unfolded protein looks like a great substrate). On the other had, if you really have to denature the protein, its hard to see a reason but I am not in your shoes, you could use 6M guanidinium Hydrochloride which does not carbamoylate the protein. Depending upon the number of pieces you see, its is also possible that you are seeing cleavage at Asp-Pro bonds. This is sometimes seen with heat treated SDS denatured SDS-PAGE gel samples. The heating accelerates the process, which in your case it would be due to the long incubation at RT. However, unless your sequence is pretty weird it is not likely to happen at more than one or two sites at most. Finally, the comment about peptide bond breakdown in urea, while technically correct, the time frame for this is much much longer, at least according to the older literature where people tested this directly.

Thank you very much for the answers!

I came late to this discussion. I wonder whether you already commented that if some bacteria are there degradation may occur due to slow but continuous growing of such bacteria. When I wonrked with purified proteins we kept them frozen but also with sodium azide. Sodium azide is "neutral" except for few assay types.

Storing 8M urea with whatever dissolved in it at room temperature is always a very bad idea as urea turns spontaneously to ammonia (if you have stored this sample for 6 months at RT smell it and I bet you will detect the characteristic odor of ammonia). Even keeping urea buffers at room temperature for more than a few days should be avoided. I would suggest to keep your proteins stored always at -20oC with some kind of cryoprotectant like 10% glycerol.

It looks like partial proteolytic clivages. We have experimented proteases stil active when boiling samples at 100°C in Western blot SDS loading buffer. 8M urea is an even weaker denaturating agent.

I have previously worked with nuclear proteins which were purified in 8 M Urea. I was always adding Bacitracin to the lysis buffer. The proteins could be dialyzed extensely, freeze dryed and resolved before separatation on 2D gels and I did not notice degradation. See the methods in our article in H.H.Rasmussen, J. van Damme M. Puype, B. Gesser, J.E. Celis and J. Vandekerckhove (1991) Electrophoresis 12, 873-882

I just want to know why did you store your protein in urea buffer?! it's a denaturant agent and i dialyse my samples after using it in purification procedurer