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Determination of nitration degrees for the birch pollen allergen Bet v 1


Nitration of tyrosine residues in the major birch pollen allergen Bet v 1 may alter the allergenic potential of the protein. The kinetics and mechanism of the nitration reaction, however, have not yet been well characterized. To facilitate further investigations, an efficient method to quantify the nitration degree (ND) of small samples of Bet v 1 is required. Here, we present a suitable method of high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) that can be photometrically calibrated using the amino acids tyrosine (Tyr) and nitrotyrosine (NTyr) without the need for nitrated protein standards. The new method is efficient and in agreement with alternative methods based on hydrolysis and amino acid analysis of tetranitromethane (TNM)-nitrated Bet v 1 standards as well as samples from nitration experiments with peroxynitrite. The results confirm the applicability of the new method for the investigation of the reaction kinetics and mechanism of protein nitration.

Illustration of the photometry of tyrosine and nitrotyrosine

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This work was funded by the Max Planck Society (MPG) and the Austrian Science Fund (FWF): P22236-B13. KS is supported by the Max Planck Graduate Center–Johannes Gutenberg University Mainz (MPGC-JOGU). The authors gratefully acknowledge T. Pooya and G. Gadermaier for the technical support, M. Reinmuth for the fruitful discussions, and M.O. Andreae for the support. We appreciate the support by S. Kofler, F. Ferreira, and H. Brandstetter for providing the Bet v 1.0101.

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Correspondence to Gertie J. Oostingh or Ulrich Pöschl.

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Kathrin Selzle and Chloé Ackaert contributed equally to this work.

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Selzle, K., Ackaert, C., Kampf, C.J. et al. Determination of nitration degrees for the birch pollen allergen Bet v 1. Anal Bioanal Chem 405, 8945–8949 (2013).

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