title: Covalent adduction of nucleophilic amino acids by 4-hydroxynonenal and 4-oxononenal.
abstract: Peroxidation of polyunsaturated fatty acids yields the lipid aldehydes 4-hydroxynonenal (4HNE) and 4-oxononenal (4ONE). Adduction of proteins by 4HNE is thought to be involved in the pathogenesis of several diseases. At the present time, the reactivity of 4ONE toward proteins is unknown. The purpose of this study was to identify amino acids that react with 4HNE and 4ONE, characterize the chemical structure of the adduct, and determine the preference for amino acid modification. Model peptides containing one or more nucleophilic residues (i.e. Arg, Cys, His, Met and Lys) were reacted with 4HNE and 4ONE at pH 7.4, 37 degrees C and analyzed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS). Post-source decay (PSD) analysis was used to confirm peptide modification. The bimolecular rate constant for adduction of amino acids and peptides by 4HNE and 4ONE was measured. Results of this work indicate that Cys, His and Lys are modified by 4HNE. In contrast, 4ONE was found to react with Arg, Cys, His and Lys. The predominant adduct resulting from modification of peptides by 4HNE or 4ONE had a mass of 156 or 154 Da (respectively), indicating that both lipid aldehydes react primarily via Michael addition with peptide nucleophiles to yield a covalent adduct. Reactivity of amino acids toward 4HNE was found to have the following order of potency: Cys>>His>Lys. Preference for the reaction of amino acid nucleophiles with 4ONE was determined to have the following order: Cys>>His>Lys>Arg. The presence of an Arg on a Cys-containing peptide increased the reaction rate with 4HNE and 4ONE by a factor of approximately 5-6 compared to the Cys nucleophile alone. Rate constants for the modification of Cys by 4HNE and 4ONE were determined to be 1.21 and 186 M(-1) s(-1) (respectively), indicating a >150-fold difference in reactivity between the lipid aldehydes toward Cys. Spontaneous conjugation of glutathione (GSH) with the lipid aldehydes was found to occur with rate constants of 1.33 and 145 M(-1) s(-1) for 4HNE and 4ONE (respectively), demonstrating a 110-fold difference in the rate of GSH modification between the two compounds. Results of the present study indicate that both 4HNE and 4ONE react with amino acid nucleophiles via Michael addition with the following order of potency: Cys>>His>Lys. However, the reactivity of these lipid aldehydes toward amino acid nucleophiles differs qualitatively with Arg being a target for 4ONE but not 4HNE and quantitatively by a remarkable >100-fold difference in the rate of Cys modification between 4HNE and 4ONE.
authors.affiliation: Department of Pharmacology, School of Medicine, The University of Colorado Health Sciences Center, Denver, CO 80262, USA.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Doorn
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Petersen
mesh_terms: D000447:Aldehydes / Q000737:chemistry*; D000596:Amino Acids / Q000737:chemistry*; D007700:Kinetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: C403894:4-oxo-2-nonenal; D000447:Aldehydes; D000596:Amino Acids; C027576:4-hydroxy-2-nonenal