Chemical Nature of Stochastic Generation of Protein-based Carbonyls:  Metal-catalyzed Oxidation versus Modification by Products of Lipid Oxidation

Abstract

An assessment of 2,4-dinitrophenylhydrazine (DNPH)-detectable protein-based carbonyls is one of the most common assays used to quantify oxidative stress in vitro and in vivo. In this study, we compared, for the lipid-binding protein beta-lactoglobulin, the extent to which carbonyl reactivity could be introduced by adventitious metal-catalyzed oxidation (MCO) in the absence and presence of a polyunsaturated lipid or by treatment with various individual bifunctional lipid oxidation products capable of introducing carbonyls into proteins by adduction to nucleophilic side chains. With metal ions and either O2/reductant or H2O2 as the terminal oxidant, the maximal level of DNPH-detectable carbonyl generation obtainable in several hours was 0.1-0.2 mol carbonyl per mol protein monomer, with Cu(II) being more effective than Fe(II). Exposure instead to bifunctional lipoxidation-derived aldehydes (1-2 mM) generated in some cases in excess of 1 mol carbonyl per mol protein. The rank order of carbonyl incorporation reactivity was acrolein > 4-oxo-2-nonenal > 4-hydroxy-2-nonenal > 2,4-decadienal > malondialdehyde. Protein cross-linking ability followed a somewhat different rank order. Parallel studies on reductively methylated beta-lactoglobulin revealed that His and Cys residues are intrinsically more responsible than Lys residues for carbonyl appearance and that the availability of Lys residues accounts for the reduction of carbonyl content at later time (presumably reflecting cross-linking chemistry) that occurs for acrolein and 4-oxo-2-nonenal. Overall, these results suggest that DNPH reactivity observed physiologically on nonmetalloproteins may arise more from the attachment of lipid-derived products of oxidative stress than from adventitious MCO of side chains. Additional studies carried out to clarify the potential use of DNPH derivatization to tag peptide-based carbonyls for mass spectrometric analysis revealed that DNPH derivatization can reverse under the conditions used for proteolysis.

Protein Carbonylation and Adipocyte Mitochondrial Function

“…Methods are in development to isolate and analyze such alkylated peptides to determine the site(s) and stoichiometry of carbonylation. Additionally, although both amino acid side chain oxidation and carbonylation with lipid aldehydes occurs, studies indicate that side chain alkylation is ϳ10-fold more prevalent than direct side chain oxidation (41).…”

Oxidative Stress and Covalent Modification of Protein with Bioactive Aldehydes

“…Lipid peroxidation products can diffuse across membranes, allowing the reactive aldehyde-containing lipids to covalently modify proteins localized throughout the cell and relatively far away from the initial site of ROS formation. Recent studies have suggested that protein carbonylation formed from lipid-derived aldehydes is more prevalent than that formed via direct amino acid side chain oxidation (9). The cellular metabolism of lipid peroxidation products and the fates of their protein targets are diagramed in Fig.…”

Advances in purification and separation of posttranslationally modified proteins

“…Indirect protein carbonylation is more prevalent and involves a reaction with oxidized AGE (advanced glycation end) products (described in glycoproteomics, section 3.4), or hydroxyl radical-mediated oxidation of lipids. Lipid-derived aldehydes can form Michael adducts with cysteine, histidine, lysine and arginine, and in some cases can further undergo Schiff base formation with amines of adjacent lysines, producing cross-linked amino acids [120,123,124]. Analysis of the resulting PTM diversity is hindered by their instability because carbonyl groups readily undergo Schiff base formation with proximate lysine residues, even when stored at -80 °C [122].…”