Quantitative evaluation of tryptophan oxidation in actin and troponin I from skeletal muscles using a rat model of acute oxidative stress.
Increased levels of "ROS" cause oxidative stress and are believed to play a key role in the development of age-related diseases and mammalian aging, e.g. through the oxidation of residues, at or close to, the protein surface. In this study, we have investigated the effects of ROS on tryptophan residues in alpha skeletal actin and troponin I (fast skeletal muscle isoform) using an established rat model of acute oxidative stress induced by X-ray irradiation. In the control samples (no oxidative stress), the single Trp residue of troponin I (position 161) and the four tryptophan residues present in actin (positions 79, 86, 340, and 356) were only oxidized at very low levels. Post-irradiation, the level of oxidized versions increased for most positions within 3 h. Tryptophan residues located inside the proteins, however, required longer time periods. Based on the increment masses of the tryptophan positions calculated from the b- and y-ion series of the tandem mass spectra, the following oxidation products of tryptophan were detected: kynurenine; oxolactone; hydroxytryptophan or oxindolylalanine (isobaric); hydroxykynurenine; dioxindolylalanine, N-formylkynurenine or dihydroxytryptophan (all three isobaric); and hydroxyl-N-formylkynurenine, with mass gains relative to tryptophan of 4, 14, 16, 20, 32, and 48 u, respectively. Despite a partial recovery after 24 h, the degree of oxidation of all oxidized versions was still higher than in the control samples.
Fedorova
Maria
M
Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Faculty of Chemistry and Mineralogy, Universität Leipzig, Leipzig, Germany.
Todorovsky
Toni
T
Kuleva
Nadezda
N
Hoffmann
Ralf
R
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000208:Acute Disease; D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D013058:Mass Spectrometry; D023421:Models, Animal; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D021241:Spectrometry, Mass, Electrospray Ionization; D014020:Tissue Extracts / Q000378:metabolism; D019210:Troponin I / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*
D000199:Actins; D010455:Peptides; D020033:Protein Isoforms; D014020:Tissue Extracts; D019210:Troponin I; D014364:Tryptophan