Protein Protocols Protein Protocols

Amidination of Lysine 479
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Amidination of Lysine Side Chains
Dan S. Tawfik
1. Introduction
Perhaps the largest variety of modifications available is that for ε-amino group of
lysine (1–4). The amino side chain can be acylated (using e.g., acetic anhydride) or
alkylated by trinitrobenzenesulfonic acid (TNBS); these reactions alter both the size
and the charge of the amino group. Other modifications, using anhydrides of dicarboxylic
acids (e.g., succinic anhydride), replace the positively charged amino group
with a negatively charged carboxyl group. Amidinations (5,6) and reductive alkylations
(see ref. 7) offer an opportunity to modify the structure of the ε-amino group of
lysines, while maintaining the positive charge. Modifications that usually do not
disrupt the overall structure of the protein are preferred, particularly in those cases
when one wishes to identify the specific role of lysine in the active site of the protein
being studied.
Amidination is performed by reacting the protein with imidoesters such as methyl or
ethyl acetimidate at basic pH. The reaction proceeds solely with amino groups to give
mainly the positively charged acetimidine derivative, which is stable under acidic and
mildly basic pH. Side products can be avoided by maintaining the pH above 9.5
throughout the reaction (see Note 1 and 2, ref. 6). The modification can be removed at
a higher pH (≥ 11.0) and in the presence of amine nucleophiles (e.g., ammonia) (5,6,
and see Note 2).
A major drawback of this modification is that the number of amidinated lysines
cannot be readily determined. However, it is possible to take advantage of the fact that
the amidine group is not reactive with amine modifying reagents such as TNBS and
thereby to indirectly determine the number of the remaining unmodified lysine
residues after the reaction (8).
2. Materials
1. Methyl acetimidate hydrochloride.
2. 0.1 M and 1 M NaOH.
3. Protein for modification.
4. 0.1 M borate buffer, pH 9.5.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
479