Isolation and analysis of dityrosine from enzyme-catalyzed oxidation of tyrosine and X-irradiated peptide and proteins.
Dityrosine (DT) was isolated in a single-step by reversed-phase HPLC in 25% yield from enzyme-catalyzed oxidation of N-acetyl tyrosine followed by deacetylation. The isolated product was characterized by 1H NMR. A three-step chromatographic procedure was reported to facilitate the preparation of DT from the enzyme-catalyzed oxidation of tyrosine in 26% yield of theoretical maximum. Upon irradiation at 284 nm in acidic and 315 nm alkaline conditions, DT exhibits strong fluorescence at 400 nm-range. However, when excited at 300 nm-range, contribution of similar fluorescence by Trp oxidation and other protein modifications cannot be overruled. In order to identify the formation of DT unequivocally, Tyr was subjected to X-irradiation under nitrogen at pH 4 and labeled with dansyl chloride. HPLC conditions were devised to resolve dansylated DT from dansylated standard amino acids. Radiation-induced DT was identified by cochromatography with a dansylated, authentic sample of DT isolated and characterized from enzyme-catalyzed oxidation of Tyr. The formation of DT in the irradiated samples, determined by the integrated peak area, increased with dose (0-600 Gy). HPLC analysis of dansylated hydrolysate of the major product from an irradiated tripeptide (Tyr Gly Gly) detected Gly and DT (2:0.5). Extension of the model study to irradiated BSA and RNase A also showed DT as the major oxidation product of Tyr under the experimental conditions. Fluorescence signal of dansylated DT was linear from 0.5 pmol to 1.5 nmol (correlation coefficient 0.999, n = 3). The detection limit 0.5 pmol per 5 microliters injection hydrolysate corresponds to one molecule of DT per 300 molecules of BSA (BSA at 1 mg/ml). DT can be used as a marker for assessing oxidative damage of proteins. Most standard amino acid analysis techniques are limited to detect normal residues of proteins. The assay reported in the present study has potential for low-level detection of DT unequivocally and may be useful for monitoring oxidative stress-related physiological and pathological processes.
Sharma
M
M
Department of Biophysics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Jain
R
R
D002851:Chromatography, High Pressure Liquid; D003619:Dansyl Compounds; D005453:Fluorescence; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D009842:Oligopeptides / Q000378:metabolism / Q000528:radiation effects*; D010084:Oxidation-Reduction; D012259:Ribonuclease, Pancreatic / Q000378:metabolism / Q000528:radiation effects*; D012710:Serum Albumin, Bovine / Q000378:metabolism / Q000528:radiation effects*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism
D003619:Dansyl Compounds; D009842:Oligopeptides; D012710:Serum Albumin, Bovine; D014443:Tyrosine; C007543:dityrosine; C025787:N-acetyltyrosine; D012259:Ribonuclease, Pancreatic; C029038:dansyl chloride