Nitrated Proteins and Allergies

Nitration by Polluted Air Enhances the Allergenicity of Proteins

  • Fig. 1: Determination of average nitration degrees with HPLC-DAD (a) and of individual nitration efficiencies of tyrosine residues sites with HPLC-MS/MS (b).Fig. 1: Determination of average nitration degrees with HPLC-DAD (a) and of individual nitration efficiencies of tyrosine residues sites with HPLC-MS/MS (b).
  • Fig. 1: Determination of average nitration degrees with HPLC-DAD (a) and of individual nitration efficiencies of tyrosine residues sites with HPLC-MS/MS (b).
  • Fig. 2: Liquid phase nitration with TNM (a) and gas phase nitration with NO2 and O3 (b). In b) the reactive oxygen intermediate-protein (ROI-protein) consists most likely of phenoxy radical derivates of tyrosine.
  • Fig. 3: 3D-structure of OVA (PDB ID: 1UHG) with highlighted tyrosine residue 107 based on  crystal structure analysis of Yamasaki et al. [9].
  • Fig. 4: Illustration of the atmospheric and physiological sources, coupling and effects of ROS  (reactive oxygen species) [10]. These are defined broadly to include ROIs as well as RNS (reactive  nitrogen species).

Nitration of proteins on tyrosine residues occur upon exposure to ozone and nitrogen oxides in polluted air. This posttranslational modification of proteins can trigger immune reactions and provides a molecular rationale for the promotion of allergies by traffic-related air pollution [1].

Laboratory and field experiments show that proteins are efficiently nitrated upon exposure to gas mixtures of nitrogen oxides and ozone or polluted urban air (summer smog). The nitration reaction leads to the addition of nitro-groups to the aromatic rings of tyrosine residues in the polypeptide chain, and this posttranslational modification can enhance the allergenic potential of proteins like the birch pollen allergen Bet v 1 [2, 3]. Additionally, food allergens like the egg allergen ovalbumin (OVA) showed enhanced allergenicity after nitration [4].

However, the kinetics of protein nitration and the dose-response relationship for nitrated proteins and allergies are not well known, and their investigation requires the development and application of suitable analytical methods.

Analytical Techniques
High-performance liquid chromatography coupled to a diode array detector of ultraviolet-visible light absorption (HPLC-DAD) can be used for the quantification of nitrotyrosine residues in protein molecules [5]. This method enables efficient determination of average nitration degrees in kinetic investigations of protein nitration, but additional site-specific information is required to elucidate reaction mechanisms and structure information.

For site-specific quantification of nitration degrees of individual tyrosine residues a microfluidic chip system with electrospray ionization and tandem mass spectrometry (HPLC-MS/MS) can be used after tryptic digestion of nitrated proteins [6]. The nitration degrees determined for individual tyrosine residues provide information about site selectivity of the nitration reaction. Both analytical methods are presented in figure 1.

Protein Nitration and Site Selectivity
Recent studies investigated the site selectivity of nitration and its dependence on the nitrating agent and protein structure for bovine serum albumin (BSA) and OVA.

In these studies two different types of nitration reactions were investigated (fig 2): the reaction of protein in aqueous solution with liquid tetranitromethane (TNM), and the reaction of solid protein with a gas mixture of O3 and NO2 in synthetic air. The nitration degrees of individual tyrosine residues (NDy) determined by HPLC-MS/MS exhibited positive correlations with the total protein nitration degree (ND) determined by HPLC-DAD, which confirms the applicability and robustness of both techniques. The ratio of NDY vs. ND can be interpreted as a proxy for the relative rate or reaction probability of individual tyrosine residues [6].
In the liquid phase reaction of BSA with TNM, the tyrosine residues at position 161, 173 and 424 were more reactive than others (NDY/ND > 2). In the heterogeneous reaction of BSA with O3 and NO2, the tyrosine residue at position 161 was the most reactive one. In the food-allergen ovalbumin (OVA), the tyrosine residue most efficiently nitrated by TNM (Tyr 107, fig. 3) is part of an epitope recognized after oral sensitization [4].

Nitrated Protein Structure and
Reaction Mechanism

The online software tools PredictProtein ( [7] and PDB Protein Workshop 3.9 [8] were used to illustrate the 3D structure of OVA, highlighting the most nitrated tyrosine residues (fig. 3). Tyrosine residues 107 and 282 are located at β-sheet-structure units while tyrosine residue 112 is located on a loop. By comparison with human serum albumin (HSA), the most nitrated tyrosine residues in BSA could be assigned to loop and helix structure units [6].

Recent experiments showed that the reaction of proteins with O3 and NO2 proceeds through long-lived reactive oxygen intermediates (ROIs) - most likely phenoxy derivatives of tyrosine [10]. The ROIs can persist over extended periods of time (up to 10 min) and react with NO2 to form nitrotyrosine (Ffig. 2b). ROIs play an important role not only in protein nitration but also in other atmospheric and physiological processes (fig. 4).


Determining nitration sites of allergenic proteins may lead to a better understanding of the interactions between the immune system and allergens. For example, the nitration of IgE and T cell epitopes in nitrated egg-allergen ovalbumin appears to have a strong influence on allergenicity [4]. Thus, we suggest and intend to pursue further investigations of the reaction kinetics and mechanism of protein nitration using the methods outlined above.


[1] Pöschl U.: Angew Chem Int Ed 44:7520-7540 (2005)
[2] Franze T. et al.: Environ Sci Technol 39:1673-1678 (2005)
[3] Gruijjthuisen YK. et al.: Int Arch Allergy Immunol 141:265275 (2006)
[4] Untersmayr E. et al.: PLoS ONE 5(12):e14210 (2010)
[5] Yang H. et al.: Anal Bioanal Chem 397:879-886 (2010)
[6] Zhang Y. et al.: Anal Bioanal Chem 399:459-471 (2011)
[7] Rost B. et al.: The PredictProtein server. Nucleic Acids Res 32:W321-W326 (2004)
[8] Moreland J.L. et al.: BMC Bioinformatics (2005)
[9] Yamasaki M. et al.: J Biol Chem 278:35524-35530 (2003)
[10] Shiraiwa M. et al.: Nature Chemistry 3(4): 291-295 (2011)

Kathrin Selzle, Yingyi Zhang, Hong Yang, Manabu Shiraiwa and Ulrich Pöschl
Max Planck Institute for Chemistry, Biogeochemistry Department, Mainz, Germany


Max-Planck-Institut für Chemie
Becherweg 27 /29
55128 Mainz

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