Protein Protocols Protein Protocols

Nitration of Tyrosines 471
1. Nitrate the antibody as described in Subheading 3.1., steps 1–5).
2. Perform a series of dilutions of the nitrated antibody (1:50 – 1:50,000; see Note 5) in 50 mM
2-morpholinoethane sulfonic acid (MES) saline buffer pH 5.8, and in TBS, pH 9.0.
3. Determine the binding activity of the diluted antibody at pH 5.8 and 9.0 by ELISA on
microtiter plates coated with DNP–bovine serum albumin (BSA) (see Note 6).
4. Notes
1. In the first modification experiment of a protein a wide range of TNM concentrations of
should be applied, for example, 0–10,000 molar excess. If loss of activity is not observed
it is recommended to try again at higher pH (e.g., at pH 9.0) and with longer incubations at
room temperature.
2. Although proteins hardly absorb at this 428 nm, a sample of the same concentration of the
unmodified protein should be used as blank. Relatively high quantities of a protein
are required for the determination of low modification ratios; for example, a single
3-nitrotyrosine per site (i.e., two per antibody molecule) would give an OD 428nm of approx
0.11 at antibody concentration of 2 mg/mL (13.3 mM).
3. Demonstrating specificity by modifying the protein in the presence of an active site
specific ligand should be done under the mildest conditions that cause full loss of activity;
these conditions (e.g., excess of TNM and pH) should be determined in a preliminary
experiment. In some cases dialysis with 6 M urea (or a similar reagent) is required to
release the ligand from the protein to allow the determination of its activity. In any case,
control samples (without the addition of TNM) containing the protein alone and the
protein incubated with the ligand should be included for comparison of the remaining
activity after modification.
4. Nitrated proteins (and other chemically modified proteins as well) are often unstable.
Therefore, measure the residual activity soon after modification and avoid freezing and
defrosting of the samples. In those cases in which the activity assay can be performed at
low protein concentrations, for example, measuring the binding activity of an antibody by
ELISA, dialysis that follows the quenching can be avoided. Protein stability can be
improved by adding an equal volume of a 10 mg/mL BSA solution after the addition of
the 2-mercaptoethanol.
5. To be able to observe pH-dependent binding the nitration should be performed under mild
modification conditions (e.g., 200 molar excess of TNM at pH 8.3); under more extreme
conditions, e.g., 1000 molar excess of TNM, the antibody is irreversibly inactivated, that
is, hapten-binding is not recovered at pH 5.8 (2).
6. Conditions of the binding assay (e.g., ELISA) should be optimized to clearly identify the
pH dependency of binding; in particular, the concentration of the immobilized ligand
(DNP in this example) must be low enough so that the differences in binding affinities at
pH 5.8 vs pH 9.0 may be observed. Thus, antigen carrying low ratios of DNP (3–10 DNPs
per molecule of BSA) should be used and its concentration for the coating of the ELISA
microtiter plates should be determined.
References
1. Riordan, J. F., Sokolovsky, M., and Valee, B. L. (1966) Tetranitromethane: a reagent for
the nitration of tyrosine and tyrosyl residues in proteins. J. Am. Chem. Soc. 88, 4104–4105.
2. Tawfik, D. S., Chap, R., Eshhar, Z., and Green, B. S. (1994) pH ‘on-off’ switching of
antibody-hapten binding obtained by site-specific chemical modification of tyrosine. Protein
Eng. 7, 431–434.
3. Resmini, M., Vigna, R., Simms, C., Barber, N. J., HagiPavli, E. P., Watts, A. B., et al.
(1997) Characterisation of the hydrolytic activity of a polyclonal catalytic antibody