IMMUNOCHEMICAL DETECTION OF 4-HYDROXYNONENAL PROTEIN ADDUCTS IN OXIDIZED HEPATOCYTES

We report here the development of an immunochemical procedure that use s an antibody specific to the 4-hydroxynonenal (HNE) moiety for the de tection 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 preparation s of glyceraldehyde-3-phosphate dehydrogenase containing 0-7.0 equival ent of HNE-histidine residues per subunit were obtained by incubating samples of glyceraldehyde-3-phosphate dehydrogenase with increased amo unts of HNE and subjected to immunoblotting with the HNE-specific anti body, the intensities of the blots were directly proportional to the n umber of HNE-histidine adducts as measured directly by amino acid anal ysis. Binding of the HNE-conjugated glyceraldehyde-3-phosphate dehydro genase to the HNE-specific antibody could be completely inhibited by H NE-N-acetylhistidine, HNE-N-acetyllysine, or HNE-glutathione, suggesti ng that the antigenic determinant recognized by the antibody is the HN E moiety, not the HNE-amino add conjugates, such as HNE-histidine, HNE -lysine, and HNE-cysteine. The utility of the HNE-specific antibody wa s demonstrated by its ability to react selectively with a number of HN E-protein adducts in immunoblot analyses of crude homogenates of rat l iver hepatocytes that had been exposed to HNE or oxidative stresses wi th tert-butylhydroperoxide or metal-ion-catalyzed oxidation systems.