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-Monograph 0005 

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Carbamylation of Proteins and Peptides

(the dangers of using urea in protein characterization)

Urea is a common chaotrope used in the solublization and denaturation of proteins. Urea is sometimes used as a protein denaturant in S-carboxymethylation reactions .  In this reaction a reducing agent is used to break disulfide bonds as a prelude to cysteine modification.  Urea helps to make the buried disulfides accessible to reduction and modification.  This process goes by the common abbreviation "RCM."   After the reduction and S-carboxymethylation (RCM) process the next step is often enzymatic digestion of the modified protein in the course of protein characterization.

Urea in solution is in equilibrium with ammonium cyanate.  The form that reacts with protein amino groups is isocyanic acid.  Carbamylatoin by isocyanic acid is detrimental to further protein characterization because isocyanic acid notably reacts with the amino terminus of proteins blocking the peptide or protein to N-terminal sequencing.  Isocyanic acid also attacks the side chains of lysine and arginine residues rendering a protein unsuitable for many enzymatic digests.  In addition even if the carbamylation does not prevent the enzymatic digest it will often confound the results with peptides that have unexpected retention times and masses.  It does not seem to make a difference what grade of urea is used because, urea + heat + protein = carbamylation.  The bottom line is that a urea solution will always degrade to isocyanic acid no matter what the grade.  Urea solutions should always be used fresh and should be treated to remove isocyanic acid.  Urea solutions can be acidified (100mM HCl) to drive the equilibrium to favor urea over the isocyanic acid, or a urea solution can be bulk deionized by using an ion exchange resin such as AG 501-X8 Resin ^TM.  It is not always necessary to use urea in an RCM procedure and the use of urea should be investigated on a case by case basis.  If a chaotrope is required one should investigate whether guanidine hydrochloride will work just as well. 

Remove Urea Before a Proteolytic Digest

Even though some enzymes will tolerate small amounts of urea it is important to remove urea from the digest mixture because normally enzyme digests are preformed at elevated temperature over a relatively long period of time.  Remember, urea + heat + protein = carbamylation.  Urea can be removed after the reduction and S-carboxymethylation reaction by fast reversed phase chromatography or by using spin columns (small SEC columns), or dialysis prior to the enzyme digest.

Interestingly, there is ample evidence of the carbamylation of proteins in vivo which have been observed in several disease states, references are provided below.  In addition researchers have found evidence for cysteine carbamylation. As always it is important to remember that careful sample preparation is needed to insure that the modification observed in an analysis is natural and not an artifact of the sample work-up.

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Last updated:  Saturday, February 17, 2007 10:20:33 PM