Protein Protocols Protein Protocols

Nitration of Tyrosines 469
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Nitration of Tyrosines
Dan S. Tawfik
1. Introduction
Tetranitromethane (TNM) reacts with the phenolic side chain of tyrosine under
relatively mild conditions to give 3-nitrotyrosine (1). The protocol described in this
chapter was developed for an anti-DNP antibody, but can be used with any other
antibody or protein. The major side reaction is oxidation of thiols although under more
extreme conditions tryptophans and methionines might be oxidized as well. As the
reactive species is the phenolate ion, the main factor controlling the reactivity of
protein tyrosyl side chains toward nitration by TNM is pH. Increasing the pH will
usually enhance the rate of modification.
The number of nitrated tyrosines can be determined spectrophotometrically during the
reaction. In addition, the stability of nitrotyrosine allows the specific site of modification
to be determined by amino acid analysis of cleaved fragments of the protein. Finally,
3-nitrotyrosine has a much lower pK a than tyrosine. Thus, examining the pH activity
profile of the nitrated protein, for example, the binding of the protein to its ligand can
be readily exploited not only to demonstrate the specificity of the modification but also
to engineer the binding properties of the protein (see Subheading 3.2. and refs. 2–5).
2. Materials
1. Tris-buffered saline (TBS) 8.0: 0.05 M Tris-HCl, pH 8.0, 0.15 M NaCl.
2. 1–2 mg/mL monoclonal or polyclonal antibody preparation (purified by protein A affinity
chromatography).
3. Acetonitrile.
4. TNM. Store in aliquots at –20°C.
Note: TNM should be handled with care. Preparation of aliquots and of stock solutions
should be done in a ventilated hood and with suitable gloves!
5. 2-Mercaptoethanol.
6. Phosphate buffered saline (PBS): 0.01 M phosphate buffer, pH 7.4, 0.15 M NaCl.
7. Dialysis tubes: 10,000 molecular weight cutoff.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
469