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Detection of carbonyl groups on oxidized proteins using an immunochemical technique.

Authors
Halmes-NC; Keller-RJ; Hinson-JA; Pumford-NR
Source
Toxicologist 1993 Mar; 13(1):117
NIOSHTIC No.
20040980
Abstract
Chemical toxicants may produce active oxygen species that react with amino acids on proteins forming carbonyl groups and other oxidative products. An immunochemical assay was developed to detect carbonyl moieties that result from oxidative damage to proteins. Bovine serum albumin was reacted with hydroxyl radicals generated via a Fenton-like mechanism using hydrogen peroxide and vanadyl as the metal catalyst. The resulting albumin-derived carbonyls were reacted with 2,4-dinitrophenylhydrazine, giving the corresponding hydrazone. The carbonyl-dinitrophenyl groups were detected by Western blot using antidinitrophenyl antisera. Gels stained with Coomassie blue show a decrease in albumin intensity with increasing vanadyl concentration. The immunoblot demonstrated a concentration-dependent increase in carbonyl formation, as well as fragmentation of the albumin into two distinct bands with molecular weights of 51 kDa and 45 kDa. This indicates albumin has two sites that are highly susceptible to oxidative cleavage. In a parallel experiment, dinitrophenylhydrazone adducts were detected using an established spectrophotometric method. The spectrophotometric results were consistent with those of the Western blot, showing more carbonyl groups formed with increasing vanadyl concentration. However, this immunochemical assay was greater than three orders of magnitude more sensitive than the spectrophotometric method in the detection of oxidative damage.
Keywords
Analytical-methods; Immunochemistry; Carbonyls; Protein-chemistry; Blood-serum; Oxidative-processes; Analytical-processes
Publication Date
19930301
Document Type
Abstract
Funding Amount
64628
Funding Type
Grant
Fiscal Year
1993
NTIS Accession No.
NTIS Price
Identifying No.
Grant-Number-R03-OH-03061
Issue of Publication
1
ISSN
0731-9193
Priority Area
Pulmonary System Disorders
Source Name
The Toxicologist. Society of Toxicology 32nd Annual Meeting, March 14-18,1993, New Orleans, Louisiana
State
AR
Performing Organization
University of Arkansas Med Scis Ltl Rock, Little Rock, Arkansas
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