Amino Acids, Peptides and Proteins - Dévényi T., Gergely J. 1976

Hydrolysis of Proteins and Peptides
Preparation of Samples for Hydrolysis
Reduction and Carboxymethylation of Proteins and Peptides

The sample is dissolved in 6 M urea at pH 7.2, and mercaptoethanol is added in a 10-fold molar excess relative to the Cysteine content. The mixture is stirred at room Temperature for 3 h, then iodo- or bromoacetate is added in a 50- to 100-fold molar excess relative to the cystine content in the sample. The mixture is left for 2 h at room temperature and for 24 h at 4°C. The solution is then dialyzed against distilled Water to remove urea.

NOTES

1. Since the free SH groups of reduced Proteins are highly reactive, they should be carboxymethylated with iodo- or bromoacetate to prevent reoxidation.

2. Reduced and carboxymethylated proteins are usually poorly soluble in water. If they precipitate as the urea concentration decreases, dialysis is no longer necessary. In this case, the precipitated protein can be washed directly to remove urea with 0.01 N HCl, which also removes the excess alkylating reagent.

3. After carboxymethylation and acid washing, or after dialysis, it is recommended to suspend the sample in distilled water and lyophilize it.



Last update: 06/08/2026

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