Immunochemical detection of 4-hydroxynonenal protein adducts in oxidized hepatocytes.
We report here the development of an immunochemical procedure that uses an antibody specific to the 4-hydroxynonenal (HNE) moiety for the detection of HNE-protein adducts. The HNE-specific antibody was prepared by immunizing rabbits with a HNE-keyhole limpet hemocyanin conjugate and purifying the rabbit serum on an affinity gel prepared by covalent attachment of a HNE-conjugated heptapeptide. When various preparations of glyceraldehyde-3-phosphate dehydrogenase containing 0-7.0 equivalent of HNE-histidine residues per subunit were obtained by incubating samples of glyceraldehyde-3-phosphate dehydrogenase with increased amounts of HNE and subjected to immunoblotting with the HNE-specific antibody, the intensities of the blots were directly proportional to the number of HNE-histidine adducts as measured directly by amino acid analysis. Binding of the HNE-conjugated glyceraldehyde-3-phosphate dehydrogenase to the HNE-specific antibody could be completely inhibited by HNE-N-acetylhistidine, HNE-N-acetyllysine, or HNE-glutathione, suggesting that the antigenic determinant recognized by the antibody is the HNE moiety, not the HNE-amino acid conjugates, such as HNE-histidine, HNE-lysine, and HNE-cysteine. The utility of the HNE-specific antibody was demonstrated by its ability to react selectively with a number of HNE-protein adducts in immunoblot analyses of crude homogenates of rat liver hepatocytes that had been exposed to HNE or oxidative stresses with tert-butylhydroperoxide or metal-ion-catalyzed oxidation systems.
Uchida
K
K
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Szweda
L I
LI
Chae
H Z
HZ
Stadtman
E R
ER
D000447:Aldehydes / Q000032:analysis / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006639:Histidine / Q000378:metabolism; D015151:Immunoblotting; D007150:Immunohistochemistry / Q000379:methods; D007700:Kinetics; D015227:Lipid Peroxidation; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D011914:Rats, Inbred BN; D011916:Rats, Inbred F344
D000447:Aldehydes; D009842:Oligopeptides; D011506:Proteins; D006639:Histidine; C027576:4-hydroxy-2-nonenal