Protein Protocols Protein Protocols

Carboxymethylation of Cysteine 455
59
Carboxymethylation of Cysteine Using Iodoacetamide/
Iodoacetic Acid
Alastair Aitken and Michèle Learmonth
1. Introduction
If cysteine or cystine is identified in a protein it requires modification in order to be
quantified. Thiol groups may be blocked by a variety of reagents including iodoacetic
acid and iodoacetamide. Iodoacetate produces the S-carboxymethyl derivative of cysteine,
effectively introducing new negative charges into the protein. Where such a
charge difference is undesirable, iodoacetamide may be used to derivatize cysteine to
S-carboxyamidomethylcysteine (on acid hydrolysis, as for amino acid analysis, this
yields S-carboxymethylcysteine). The charge difference between these two derivatives
has been utilized in a method to quantify the number of cysteine residues in a protein
([1], see Chapter 89).
Carboxymethylation may be carried out without prior reduction to modify only those
cysteine residues that are not involved in disulfide bridges.
If the protein is to be analyzed using gas-phase protein microsequencing, the
derivatizing agent of choice is commonly vinylpyridine as this produces a well separated
phenylthiohydantoin (PTH) derivative (PTH-S-pyridylethylcysteine), see Chapter 62.
2. Materials
1. Denaturing buffer: 6 M guanidinium hydrochloride (or 8 M deionized urea, see Note 1) in
0.6 M Tris-HCl, pH 8.6.
2. 4 mM Dithiothreitol (DTT), freshly prepared in distilled water.
3. β-Mercaptoethanol.
4. Sodium dodecyl sulfate (SDS).
5. Oxygen-free nitrogen source.
6. Iodoacetic acid: 500 mM in distilled water, pH adjusted to 8.5 with NaOH. This is light
sensitive and although the solution may be stored in the dark at –20°C, it is better to use it
freshly prepared.
7. Iodoacetamide: 500 mM, freshly prepared in distilled water.
8. Ammonium bicarbonate: 5 mM and 50 mM in distilled water.
9. 0.1% (v/v) Trifluoracetic acid (TFA), high-performance liquid chromatography (HPLC) grade.
10. Acetonitrile, far UV HPLC grade (e.g. Romil, Cambridge, UK).
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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