Protein Protocols Protein Protocols

Pyridylethylation 461
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Pyridylethylation of Cysteine Residues
Malcolm Ward
1. Introduction
To help maintain their three-dimensional structure, many proteins contain disulfide
bridges between cysteine residues. Cysteine residues can cause problems during Edman
sequence analysis, and quantification of cysteine and cystine by amino acid analysis is
difficult since these residues are unstable during acid hydrolysis.
Chemical modification of cysteine residues can enhance the solubility of the protein,
enable more effective enzymatic digestion with proteases, such as trypsin, and
facilitate quantification by amino acid analysis.
Oxidation with performic acid can be used to convert cysteine and cystine to cysteic
acid (1) (and see Chapter 60). This, however, can lead to other nondesirable side reactions,
such as oxidation of methionine residues and destruction of tryptophan residues.
Alkylation with iodoacetic acid has been used extensively, since this enables the addition
of negative charges to the protein. The use of iodoacetic acid containing 14 C provides
a means of incorporating a radioactive label into the polypeptide chain (see
Chapter 59) (2).
The method described in this chapter is an effective alternative, where reduction and
alkylation can be achieved in one step. 4-Vinylpyridine is used to convert cysteine residues
to S-pyridylethyl derivatives. The S-pyridylethyl group is a strong chromophore at
λ254 nm, which facilitates the detection of cysteine containing peptides as well as aiding
the identification of cysteine residues during Edman sequencing (see Note 1).
2. Materials (see Note 2)
1. Denaturing buffer: 0.1 M Tris-HCl, pH 8.5, 6 M guanidine hydrochloride.
2. 4-Vinylpyridine: Store at –20°C.
3. 2-Mercaptoethanol.
3. Method
1. Dissolve 10–50 µg protein/peptide in the denaturing buffer (1 mL).
2. Add 2-mercaptoethanol (5 µL) and 4-vinyl-pyridine (2 µL) to the sample tube and shake.
Since both reagents are extremely volatile and toxic, all experimental work should be
carried out in a fumehood wearing appropriate safety clothing.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
461