Radiation-induced formation of 3,4-dihydroxyphenylalanine in tyrosine-containing peptides and proteins as a function of X-irradiation dose.
Radiation-induced formation of 3,4-dihydroxyphenylalanine (DOPA) in Tyr and Tyr-containing peptides and proteins was investigated as a function of X-irradiation dose. Irradiated Tyr (0-30 Gy) and the acid hydrolysates of irradiated peptide and protein (0-240 Gy) were conjugated with dansyl chloride. The dansylated amino acids were analyzed by reversed-phase HPLC using fluorescence detection. Formation of DOPA, determined by integrated peak area, increased with dose. Analysis of the major product from irradiated tripeptide Tyr-Gly-Gly detected Gly and DOPA (2:1). Extension of the model study to irradiated BSA and RNase A showed correlation of DOPA formation with Tyr modification up to 120 Gy. Higher dose induced further transformation of DOPA. The fluorescence signal of dansylated DOPA was linear from 1.5 nmol to 0.5 pmol (correlation coefficient of 0.999, n = 3). The detection limit allows the detection of 1 molecule of DOPA/300 molecules of BSA in 5 micrograms of dansylated hydrolysate. Most standard amino acid analysis techniques are limited to detect normal residues of protein. Protein-bound DOPA has been suggested to have a role in the replenishment of reduced transition metal ion involved free-radical-generating system in vivo. Sensitive analysis of protein-bound DOPA will be useful to study amplification of the radical-damaging event.
Jain
R
R
Department of Biophysics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Freund
H G
HG
Budzinsky
E
E
Sharma
M
M
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003619:Dansyl Compounds; D004295:Dihydroxyphenylalanine / Q000737:chemistry* / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D005609:Free Radicals / Q000737:chemistry / Q000528:radiation effects; D066298:In Vitro Techniques; D009842:Oligopeptides / Q000737:chemistry / Q000528:radiation effects; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry* / Q000528:radiation effects*; D011506:Proteins / Q000737:chemistry* / Q000528:radiation effects*; D017382:Reactive Oxygen Species; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000528:radiation effects; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000737:chemistry* / Q000528:radiation effects*
D003619:Dansyl Compounds; D005609:Free Radicals; D009842:Oligopeptides; D010455:Peptides; D011506:Proteins; D017382:Reactive Oxygen Species; C044757:tyrosyl-glycyl-glycine; D012710:Serum Albumin, Bovine; D014443:Tyrosine; D004295:Dihydroxyphenylalanine; C029038:dansyl chloride