Ethoxyformylation of Histidine

  • Dan S. Tawfik
Part of the Springer Protocols Handbooks book series (SPH)


Diethylpyrocarbonate (DEP) reacts with various nucleophiles (amines, alcohols, thiols, imidazoles, or guanido groups) to yield the respective ethoxyformyl derivatives. At low pH (generally<6.0) the reaction is quite selective for histidine, as the main side reaction with the ε-amino group of lysine proceeds very slowly (see e.g., ref. 1). Still, side reactions even with hydroxyl groups (e.g., of serine or tyrosine) were observed (2). The fact that the ethoxyformyl group can be removed from the imidazole side chain by mild treatment with hydroxylamine can be exploited to ascribe the modification to a histidine residue. In addition, ethoxyformylation of histidines is characterized by an increase in absorbance at 242 nm, which is also used to determine the number of modified histidines (1-3).


Acetate Buffer Sodium Acetate Side Reaction Sodium Acetate Buffer Histidine Residue 
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  1. 1.
    Dominicini, P., Tancini, B., and Voltattorni, C. B. (1985) Chemical modification of pig kidney 3, 4-dihydroxy-phenylalanine decarboxylase with diethyl pyrocarbonate. J. Biol. Chem. 260, 10,583–10,589.Google Scholar
  2. 2.
    Melchior, W. B.,Jr. and Fahrney, D. (1970) Ethoxyformylation of proteins. Reaction of ethoxyformic anhydride with a-chymotrypsin, pepsin and pancreatic ribonuclease at pH 4. Biochemistry 9, 251–258.PubMedCrossRefGoogle Scholar
  3. 3.
    Sheflyan, G. Y., Duewel, H. S., Chen, G., and Woodard, R. W. (1999) Identification of essential histidine residues in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase: analysis by chemical modification with diethyl pyrocarbonate and site-directed mutagenesis. Biochemistry 38, 14,320–14,329.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Dan S. Tawfik
    • 1
  1. 1.Department of Biological Chemistrythe Weizman Institute of ScienceRehovotIsrael

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