Protein Modification by Lipid Peroxidation Products: Formation of Malondialdehyde-DerivedNϵ-(2-Propenal)lysine in Proteins

Malondialdehyde (MDA), a naturally occurring dialdehyde produced in the membrane lipid peroxidation, is known to react with lysine residues of proteins, but the MDA–lysine adducts generated in the proteins have not been characterized adequately. In the present study, we provide evidence that the enaminal-type MDA–lysine adduct,Nϵ-(2-propenal)lysine, is formed in human low-density lipoprotein (LDL) upon reaction with MDA or Cu2+. We found that the incubation ofNα-acetyllysine with MDA generatedNα-acetyl-Nϵ-(2-propenal)lysine as the predominant product. In addition, a polyclonal antiserum raised against the MDA-modified protein was found to contain antibody populations that could be purified by affinity gel prepared by covalent attachment ofNα-acetyl-Nϵ-(2-propenal)lysine. It was concluded that the affinity-purified anti-Nϵ-(2-propenal) lysine antibody was highly specific to the enaminal derivative of both lysine residues and phosphatidylethanolamine, based on the observations that (i) MDA was the only aldehyde which generated immunoreactive materials in proteins; (ii) among structurally defined MDA-lysine adducts tested, the antibody recognized the enaminal adduct only; and (iii) immunoreactivity toN-(2-propenal)serine was still significant but much weaker than its reactivity toN-(2-propenal)ethanolamine. Furthermore, analysis of antibody recognition sites with a variety ofN-(2-propenal)alkylamines revealed that the mono-specific antibody recognized theN-2-propenal-N-ethyl moiety [—(CH2)2—NH—:CH=CH—CHO] of enaminal adducts. Determination by a competitive enzyme-linked immunosorbent assay demonstrated thatNϵ-(2-propenal)lysine accounted for 33.7 and 3.1% of the lysine residues that disappeared duringin vitroincubation of LDL with MDA and Cu2+, respectively. These results suggest thatNϵ-(2-propenal)lysine represents a major form of MDA covalently attached to proteins.