Covalent Binding of Acetaldehyde to Proteins: Participation of Lysine Residues

Abstract

The results of this study demonstrate that lysine is the major amino acid participating in the binding of acetaldehyde to proteins. The formation of both stable and unstable acetaldehyde-albumin adducts was shown to occur via the reaction of acetaldehyde with lysine residues. This conclusion was based on the following experimental evidence: (a) the ratio of stable to unstable adducts of bovine serum albumin was similar to that observed for polylysine; (b) acetylation of albumin markedly reduced acetaldehyde binding; (c) the radioactivity profiles (obtained by high-performance liquid chromatographic analysis) of [¹⁴C]acetaldehyde modified amino acids hydrolyzed from total and stable adducts of albumin were nearly identical to those of polylysine or α-t-boc-lysine. Analysis of stable adducts of albumin indicated two major modified lysine residues; one residue was much more acidic and the other more basic than unmodified lysine. Unstable adducts were shown to be Schiff bases since NaBH₄ treatment resulted in the formation of N-ethyllysine residues. The reducing agents, NaCNBH₃ and ascorbic acid, both increased stable adduct formation via increased binding to lysine residues; however, a different elution profile of modified lysine residues was observed for these reducing agents. NaCNBH₃ increased the formation of N-ethyllysine residues exclusively, whereas ascorbate increased the formation of the acidic adduct of lysine and also caused the formation of an additional modified lysine residue which was present only in the ascorbate-treated polypeptides. In addition to their detection by radioactivity measurements, the acetaldehyde-lysine adducts could also be detected by the fluorescence of their ophthalaldehyde derivatives. Therefore, the potential exists for the detection of acetalde-hyde-protein adducts in chronically ethanol-fed animals and in alcoholic patients without the use of radioisotopes.