Immunochemical detection of oxidized proteins.
Keller RJ; Halmes NC; Hinson JA; Pumford NR
Chem Res Toxicol 1993 Jul; 6(4):430-433
An immunochemical technique for detecting oxidative protein damage was developed. The procedure was developed using bovine-serum- albumin (BSA). Samples of oxidized BSA formed by a Fenton type reaction involving vanadyl-sulfate (27774136) and hydrogen-peroxide (7722841) or by gamma ray induced radiolysis were incubated with an equal volume of 0.05 millimolar 2,4-dinitrophenylhydrazine (DNPH) in 0.1 molar phosphate buffer, pH 6.3, for 1 hour at 37 degrees-C. Carbonyl groups formed in the proteins were converted by DNPH to the corresponding diphenylhydrazones. The derivatized proteins were separated electrophoretically under reducing conditions by sodium- dodecyl-sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). The proteins were then transferred electrophoretically to nitrocellulose membranes, which were incubated with antidinitrophenyl sera for 2 hours. This was followed by a 5 hour incubation with mouse antirabbit immunoglobulin-G conjugated to alkaline-phosphatase. After development with 5-bromo-4-chloro-3-indolyl-phosphate, the extent of carbonyl group formation in the nitrocellulose membranes was determined by computed laser densitometry. Carbonyl formation in BSA induced by the Fenton reaction or by gamma radiolysis was linear over the range 0 to 900 micromolar vanadyl-sulfate and 0 to 80 gray. The detection limits were in the picogram range. When compared to the standard spectrophotometric method for determining carbonyl groups, the detection limit for the immunochemical procedure was approximately three orders of magnitude lower. Examination of the SDS/PAGE data indicated that carbonyl formation induced by the Fenton reaction was associated with protein bands having apparent molecular weights of 51 and 45 kilodaltons (kDa). Radiolysis produced protein bands having molecular weights of 62 and 46kDa. The authors conclude that the immunochemical method can determine carbonyl groups in oxidized proteins with much greater sensitivity than the conventional spectrophotometric procedure, which can also determine the site of damage.
NIOSH-Publication; NIOSH-Grant; Pulmonary-system-disorders; Analytical-methods; Immunochemistry; Carbonyls; Protein-chemistry; Blood-serum; Oxidative-processes
Pharmacology-Toxicology Univ of Arkansas for Medical S 4301 West Markham, Slot 638 Little Rock, AR 72205
Pulmonary System Disorders
Chemical Research in Toxicology
University of Arkansas Med Scis Ltl Rock, Little Rock, Arkansas