TY - JOUR A1 - Backes, A. T. A1 - Reinmuth-Selzle, K. A1 - Leifke, A. L. A1 - Ziegler, K. A1 - Krevert, C. S. A1 - Tscheuschner, Georg A1 - Lucas, K. A1 - Weller, Michael G. A1 - Berkemeier, T. A1 - Pöschl, U. A1 - Fröhlich-Nowoisky, J. T1 - Oligomerization and Nitration of the Grass Pollen Allergen Phl p 5 by Ozone, Nitrogen Dioxide, and Peroxynitrite: Reaction Products, Kinetics, and Health Effects JF - International Journal of Molecular Sciences N2 - The allergenic and inflammatory potential of proteins can be enhanced by chemical modification upon exposure to atmospheric or physiological oxidants. The molecular mechanisms and kinetics of such modifications, however, have not yet been fully resolved. We investigated the oligomerization and nitration of the grass pollen allergen Phl p 5 by ozone (O3), nitrogen dioxide (NO2), and peroxynitrite (ONOO–). Within several hours of exposure to atmospherically relevant concentration levels of O3 and NO2, up to 50% of Phl p 5 were converted into protein oligomers, likely by formation of dityrosine cross-links. Assuming that tyrosine residues are the preferential site of nitration, up to 10% of the 12 tyrosine residues per protein monomer were nitrated. For the reaction with peroxynitrite, the largest oligomer mass fractions (up to 50%) were found for equimolar concentrations of peroxynitrite over tyrosine residues. With excess peroxynitrite, the nitration degrees increased up to 40% whereas the oligomer mass fractions decreased to 20%. Our results suggest that protein oligomerization and nitration are competing processes, which is consistent with a two-step mechanism involving a reactive oxygen intermediate (ROI), as observed for other proteins. The modified proteins can promote pro-inflammatory cellular signaling that may contribute to chronic inflammation and allergies in response to air pollution. KW - Protein KW - Nitrotyrosine KW - Dityrosine KW - Allergy KW - Hay fever KW - Diesel exhaust KW - Combustion KW - Exhaust PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-529729 DO - https://doi.org/10.3390/ijms22147616 VL - 22 IS - 14 PB - MDPI CY - Basel AN - OPUS4-52972 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Liu, F. A1 - Reinmuth-Selzle, K. A1 - Lai, S. A1 - Weller, Michael G. A1 - Pöschl, U. A1 - Kampf, C. J. T1 - Simultaneous determination of nitrated and oligomerized proteins by size exclusion high-performance liquid chromatography coupled to photodiode array detection JF - Journal of Chromatography A N2 - Chemical modifications such as nitration and cross-linking may enhance the allergenic potential of proteins. The kinetics and mechanisms of the underlying chemical processes, however, are not yet well understood. Here, we present a size-exclusion chromatography/spectrophotometry method (SEC-HPLC-DAD) that allows a simultaneous detection of mono-, di-, tri-, and higher protein oligomers, as well as their individual nitration degrees (NDs). The ND results of proteins from this new method agree well with the results from an alternative well-established method, for the analysis of tetranitromethane (TNM)- and nitrogen dioxide and ozone (NO2/O3)-nitrated protein samples. Importantly, the NDs for individual oligomer fractions can be obtained from the new method, and also, we provide a proof of principle for the calculation of the concentrations for individual protein oligomer fractions by their determined NDs, which will facilitate the investigation of the kinetics and mechanism for protein tyrosine nitration and cross-linking. KW - Size exclusion chromatography KW - HPLC-DAD KW - Protein nitration degree KW - Protein oligomer analysis KW - Nitrotyrosine KW - Dimer KW - Trimer KW - Oligomer KW - Protein KW - Tetranitromethane KW - BSA KW - Albumin KW - Air pollution KW - Nitrogen oxides PY - 2017 UR - http://www.sciencedirect.com/science/article/pii/S0021967317303795 DO - https://doi.org/10.1016/j.chroma.2017.03.015 SN - 0021-9673 VL - 1495 SP - 76 EP - 82 PB - Elsevier B.V. CY - Amsterdam AN - OPUS4-40304 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Reinmuth-Selzle, K. A1 - Bellinghausen, I. A1 - Leifke, A. L. A1 - Backes, A. T. A1 - Bothen, N. A1 - Ziegler, K. A1 - Weller, Michael G. A1 - Saloga, J. A1 - Schuppan, D. A1 - Lucas, K. A1 - Pöschl, U. A1 - Fröhlich-Nowoisky, J. T1 - Chemical modification by peroxynitrite enhances TLR4 activation of the grass pollen allergen Phl p 5 JF - Frontiers in Allergy N2 - The chemical modification of aeroallergens by reactive oxygen and nitrogen species (ROS/RNS) may contribute to the growing prevalence of respiratory allergies in industrialized countries. Post-translational modifications can alter the immunological properties of proteins, but the underlying mechanisms and effects are not well understood. In this study, we investigate the Toll-like receptor 4 (TLR4) activation of the major birch and grass pollen allergens Bet v 1 and Phl p 5, and how the physiological oxidant peroxynitrite (ONOO–) changes the TLR4 activation through protein nitration and the formation of protein dimers and higher oligomers. Of the two allergens, Bet v 1 exhibited no TLR4 activation, but we found TLR4 activation of Phl p 5, which increased after modification with ONOO– and may play a role in the sensitization against this grass pollen allergen. We attribute the TLR4 activation mainly to the two-domain structure of Phl p 5 which may promote TLR4 dimerization and activation. The enhanced TLR4 signaling of the modified allergen indicates that the ONOO–-induced modifications affect relevant protein-receptor interactions. This may lead to increased sensitization to the grass pollen allergen and thus contribute to the increasing prevalence of allergies in the Anthropocene, the present era of globally pervasive anthropogenic influence on the environment. KW - Bet v 1 KW - Birch pollen allergen KW - Phl p 5 KW - Grass pollen KW - Phleum pratense KW - Betula pendula KW - Nitration KW - Nitrotyrosine KW - Protein nitration KW - Toll-like receptor 4 KW - Allergy KW - Enhancement KW - Oligomerization KW - Dimerization KW - TLR4 activation KW - Air pollution KW - Nitrogen oxides KW - Inflammation PY - 2023 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-570298 DO - https://doi.org/10.3389/falgy.2023.1066392 VL - 4 SP - 1 EP - 7 PB - Frontiers Media SA CY - Lausanne, Switzerland AN - OPUS4-57029 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Reinmuth-Selzle, K. A1 - Tchipilov, Teodor A1 - Backes, A. T. A1 - Tscheuschner, Georg A1 - Tang, K. A1 - Ziegler, K. A1 - Lucas, K. A1 - Pöschl, U. A1 - Fröhlich-Nowoisky, J. A1 - Weller, Michael G. T1 - Determination of the protein content of complex samples by aromatic amino acid analysis, liquid chromatography-UV absorbance, and colorimetry JF - Analytical and Bioanalytical Chemistry N2 - Fast and accurate determination of the protein content of a sample is an important and non-trivial task of many biochemical, biomedical, food chemical, pharmaceutical, and environmental research activities. Different methods of total protein determination are used for a wide range of proteins with highly variable properties in complex matrices. These methods usually work reasonably well for proteins under controlled conditions, but the results for non-standard and complex samples are often questionable. Here, we compare new and well-established methods, including traditional amino acid analysis (AAA), aromatic amino acid analysis (AAAA) based on the amino acids phenylalanine and tyrosine, reversed-phase liquid chromatography of intact proteins with UV absorbance measurements at 220 and 280 nm (LC-220, LC-280), and colorimetric assays like Coomassie Blue G-250 dye-binding assay (Bradford) and bicinchoninic acid (BCA) assay. We investigated different samples, including proteins with challenging properties, chemical modifications, mixtures, and complex matrices like air particulate matter and pollen extracts. All methods yielded accurate and precise results for the protein and matrix used for calibration. AAA, AAAA with fluorescence detection, and the LC-220 method yielded robust results even under more challenging conditions (variable analytes and matrices). These methods turned out to be well-suited for reliable determination of the protein content in a wide range of samples, such as air particulate matter and pollen. KW - Air particulate matter KW - Aromatic amino acid analysis KW - Atmospheric aerosol KW - Chemical protein modification KW - Derivatization KW - Nitration KW - Nitrotyrosine KW - LC-UV absorbance KW - Pollen extract KW - Protein quantification KW - Protein test KW - Kjeldahl KW - Tyrosine KW - Phenylalanine KW - Hydrolysis KW - Bradford KW - BCA test KW - 280 nm KW - Air filter samples KW - Fluorescence KW - HPLC KW - Chromatography KW - Protein content KW - 150th anniversary of BAM KW - Topical collection: Analytical Methods and Applications in the Materials and Life Sciences PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-545924 UR - https://pubmed.ncbi.nlm.nih.gov/35320366/ DO - https://doi.org/10.1007/s00216-022-03910-1 SP - 1 EP - 14 PB - Springer Nature Limited CY - New York, Heidelberg AN - OPUS4-54592 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -