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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, 8) 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 (³ 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 (9).

From: The Protein Protocols Handbook, Third EditionEdited by: J.M. Walker © Humana Press, a Part of Springer Science + Business Media, LLC 2009

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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.

3. Method 1. Dissolve the protein (1–2 mg/mL) in 0.1 M borate buffer pH 9.5; check the pH and

if necessary adjust it back to 9.5 using 0.1 M NaOH. 2. Dissolve 110 mg of methyl acetimidate hydrochloride in approx 1.1 mL of 1 M

NaOH (approximately 0.9 M); check the pH and if necessary adjust it to approx 10 with 1 M NaOH (see Note 1).

2. Add an aliquot of the methyl acetimidate solution to the protein solution and check the pH again (see Note 1).

3. Incubate for 40 min. 4. Dialyze the sample against an appropriate buffer (pH < 8.5) or filter on a Sephadex

G-25 column. 5. Determine the activity of the protein.

4. Notes

1. The amidination reaction proceeds with almost no side products only at pH > 9.5; at lower pH, the side reactions proceed very rapidly. Hence it is important to add the methyl acetimidine solution to the buffered protein solution without causing a change of pH (6). The methyl acetimidine is purchased as the hydrochloride salt, which is neutralized by dissolving it in 1 M NaOH (see Subheading 3., step 2). The pH of the resulting solution should be approx 10; if necessary the pH may be adjusted before the addi-tion to the protein solution with 1 M NaOH or 1 M HCl. As acetimidates are rapidly hydrolyzed at basic pH, the entire process should be performed very rapidly. It is therefore recommended, in a preliminary experiment, to dissolve the methyl acetimidine hydrochloride and determine the exact amount of 1 M NaOH that yields a solution of pH 10. The same process is then repeated with a freshly prepared methyl acetimidine solution which is rapidly added to the protein.

2. The acetimidyl group may be removed by treatment of the modified pro-tein with an ammonium acetate buffer prepared by adding concentrated ammonium hydroxide solution to acetic acid to a pH of 11.3 (Caution! Preparation of this buffer must be done carefully and in a well-venti-lated chemical hood).

3. Amidination is obviously unsuitable for the modification of proteins that are sensitive to basic pH. Reductive alkylation, using formaldehyde and sodium cyanoborohydride (see ref. 7), can be performed at neutral pH and is recommended for the modification of such proteins.

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References 1. Means, G. E. and Feeney, R. E. (1971) Chemical Modifications of Proteins.

Holden-Day, San Francisco. 2. Hirs, C. H. N. and Timasheff, S. N. (eds.) (1972) Enzyme structure B. Meth.

Enzymol. 25. 3. Lundblad, R. L. and Noyes, C. M. (1984) Chemical Reagents for Protein Modifi-

cations, Vols. 1 and 2. CRC Press, Boca Raton, FL. 4. Feeney, R. E. (1987) Chemical modification of proteins: comments and perspectives.

Int. J. Pept. Protein Res. 27, 145–161. 5. Hunter, M. J. and Ludwig, M. L. (1962) The reaction of imidoesters with small

proteins and related small molecules. J. Amer. Chem. Soc. 84, 3491–3504. 6. Wallace, C. J. A. and Harris, D. E. (1984) The preparation of fully N-e-acetimidylated

cytochrome c. Biochem. J. 217, 589–594. 7. Jentoft, N. and Dearborn, D. G. (1979) Labeling of proteins by reductive methylation

using sodium cyanoborohydride. J. Biol. Chem. 254, 4359–4365. 8. Walter, T. S. et al (2006) Lysine methylation as a routine rescue strategy for protein

crystallization Structure 14, 1617–1622. 9. Fields, R. (1972) The rapid determination of amino groups with TNBS. Meth.

Enzymol. 25, 464–468.