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

  • K. Selzle
  • , C. Ackaert
  • , C.J. Kampf
  • , A.T. Kunert
  • , A. Duschl
  • , G.J. Oostingh
  • , U. P̈öschl
  • Multiphase Chemistry Department, Max Planck Institute for Chemistry
  • Department of Molecular Biology, University of Salzburg

Research output: Contribution to journalArticlepeer-review

Abstract

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 efficientmethod to quantify the nitration degree (ND) of small samples of Bet v 1 is required. Here, we present a suitable method of highperformance 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. © Springer-Verlag Berlin Heidelberg 2013.
Original languageEnglish
Pages (from-to)8945-8949
Number of pages5
JournalAnalytical and Bioanalytical Chemistry
Volume405
Issue number27
DOIs
Publication statusPublished - 2013

Keywords

  • Bet v 1.0101
  • HPLC-DAD
  • Nitration degree
  • Protein tyrosine nitration
  • Allergens
  • Amino acids
  • Liquid chromatography
  • Proteins
  • Reaction kinetics
  • Amino acid analysis
  • Diode array detectors
  • Kinetics and mechanism
  • Nitration reactions
  • Protein tyrosine nitrations
  • Tyrosine residues
  • Nitration
  • 3 nitrotyrosine
  • 3-nitrotyrosine
  • Bet v 1 allergen, Betula
  • drug derivative
  • peroxynitrous acid
  • plant antigen
  • recombinant protein
  • tyrosine
  • vegetable protein
  • article
  • birch
  • calibration
  • chemistry
  • high performance liquid chromatography
  • immunology
  • pollen
  • protein processing
  • Antigens, Plant
  • Betula
  • Calibration
  • Chromatography, High Pressure Liquid
  • Peroxynitrous Acid
  • Plant Proteins
  • Pollen
  • Protein Processing, Post-Translational
  • Recombinant Proteins
  • Tyrosine

Classification according to Österreichische Systematik der Wissenschaftszweige (ÖFOS 2012)

  • 303012 Health sciences

Applied Research Level (ARL)

  • Not applicable

Research focus/foci

  • Applied Health Innovation

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