Structural Analysis of Amino Acids, Oxidized by Reactive Oxygen Species and an Antibody against N-Formylkynurenine

Abstract

Summary Amino acid oxidation, resulting from oxidative stress, is related to certain diseases and aging. To correlate reactive oxygen species with oxidized products of individual amino acids, we oxidized Met, Trp, His, and Tyr by treatment with singlet oxygen, superoxide, or hydroxyl radicals. The structures of oxidized amino acids were determined by nuclear magnetic resonance spectroscopy, infra-red spectroscopy, and fast atom bombardment-mass spectrometry. Among the compounds identified, N-formylkynurenine (NFK), a well-known oxidation product of Trp in vivo via enzymatic and non-enzymatic reactions, and its hydroxylated form were found to be major products of the reaction with singlet oxygen. Since NFK is a pivotal oxidation product of Trp, we raised antiserum against NFK by immunizing a rabbit with NFK-conjugated bovine serum albumin. The resulting antiserum was then used to detect photooxidized trypsin and kynurenine-conjuated ovalbumin as well as NFK-albumin. An antiserum such as this would be useful tool for detecting NFK and kynurenine in situ.

Effects of CBRN Decontaminants in Common use by First Responders on the Recovery of Latent Fingerprints—Assessment of the Loss of Ridge Detail on Glass

“…GDS 2000 ® is likely to negatively affect fingerprint material due to its strong alkaline properties, while BDS 2000 ® , bleach, and SDF ® are able to oxidize components from both the eccrine and sebaceous secretions. A number of publications report the oxidation of the structural group and various side chains of amino acids . Unsaturated fatty acids are also susceptible toward oxidation .…”

Immunological detection of N-formylkynurenine in oxidized proteins

“…Not surprisingly, neither could detect NFK in photooxidized myoglobin (data not shown). A recent paper [28] describes an antibody produced by blocking the α-amine group of tryptophan, oxidizing it with 1 O 2 and then conjugating the resulting product to a carrier protein. This antiserum recognized photooxidized protein, but was used at a dilution of 1:50, a concentration likely to prohibit its wide-spread application due to non-specific binding in complex protein mixtures.…”

Photosensitizers Form in Histidine Buffer and Mediate the Photodegradation of a Monoclonal Antibody

“…[4][5][6][7][8] Visible light interaction with photosensitizers primarily produces singlet oxygen. [4][5][6][7][8] One study on the sensitizer-mediated photooxidation of free His suggested that multiple degradants of His form in the presence of singlet oxygen including oxygenated and crosslinked His arising from unstable His peroxide intermediates. 8 The authors identified the major free His photodegradant using nuclear magnetic resonance and electron paramagnetic resonance spectroscopy as a cyclized two-ring structure, 6a-hydroxy-2-oxooctahydro-pyrollo [2,3-d]imidazole-5-carboxylic acid, with mass (M + 1) of 188 Da.…”