Immunochemical detection of oxidized proteins

Details

• Personal Author:
  Halmes NC ; Hinson JA ; Keller RJ ; Pumford NR
• Description:
  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. [Description provided by NIOSH]
• Subjects:
  Immune System Occupational Health Respiratory Tract Diseases Safety
• Keywords:
  Analytical-methods Blood-serum Carbonyls Immunochemistry NIOSH-Grant NIOSH-Publication Oxidative-processes Protein-chemistry Pulmonary-system-disorders
• ISSN:
  0893-228X
• Document Type:
  Text
• Funding:
  Grant
• Genre:
  Journal Article
• Place as Subject:
  Arkansas ; OSHA Region 6
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Pages in Document:
  430-433
• Volume:
  6
• Issue:
  4
• NIOSHTIC Number:
  nn:00216252
• Citation:
  Chem Res Toxicol 1993 Jul; 6(4):430-433
• Contact Point Address:
  Pharmacology-Toxicology Univ of Arkansas for Medical S 4301 West Markham, Slot 638 Little Rock, AR 72205
• CAS Registry Number:
  Hydrogen peroxide (CAS RN 7722-84-1) ; Vanadyl sulfate (CAS RN 27774-13-6)
• Federal Fiscal Year:
  1993
• NORA Priority Area:
  Pulmonary System Disorders
• Performing Organization:
  University of Arkansas Med Scis Ltl Rock, Little Rock, Arkansas
• Peer Reviewed:
  True
• Start Date:
  19930601
• Source Full Name:
  Chemical Research in Toxicology
• End Date:
  19960531
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:00059dde9a199bf7b037b62724fe7683436c3d0dd9920f5c7820e42af2e5c7cf6ff1f442e11719a7dfdd97215c81e22e892143b33402b65bfa795f6729c8c0a1
• Download URL:
  https://stacks.cdc.gov/view/cdc/204879/cdc_204879_DS1.pdf
• File Type:
  [PDF - 1.56 MB ]