Modification of proteins in conditions of oxidative stress can contribute to protein dysfunction or tissue damage and disease progression. Bifunctional, most often alpha,beta-unsaturated carbonyl compounds such as 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal (ONE), and acrolein, generated from oxidation of polyunsaturated fatty acids (PUFAs), readily bind to protein nucleophiles. Modification by bifunctional aldehydes can also lead to intramolecular or intermolecular protein crosslinking. Model studies are revealing the structure of adducts that can then be more readily identified in mass spectrometric studies on proteins exposed to the various pure aldehydes or to peroxidized PUFAs. Although simple Michael and Schiff base adducts are often formed initially, only some of these adducts, such as the HNE- and ONE-derived Michael adducts on Cys and His residues, are found to survive the conditions of proteolysis and HPLC-MS analysis. Reversibly formed adducts, such as the HNE-Lys Michael adduct, can be found on proteolytic peptides only if a NaBH4-reduction step is used prior to proteolysis. Initial adducts can evolve by tautomerization, oxidation, cyclization, dehydration, and sometimes condensation with a second aldehyde molecule (the same or different), to give stable advanced lipoxidation end products (ALEs) that can be found by mass spectrometry. These include the HNE-Lys-derived 2-pentylpyrrole, the ONE-Lys-derived 4-ketoamide, the ONE-derived His-Lys pyrrole crosslink, and a Lys-derived 3-formyl-4-pentylpyrrole that results from combined action of ONE and acrolein. Michael adducts of alpha,beta-unsaturated aldehydes such as HNE and ONE can be derivatized by 2,4-dinitrophenylhydrazine (DNPH) and can thus constitute significant DNPH-detectable protein-bound carbonyl activity that serves as a key indicator of oxidative stress in tissues. It appears that lipid oxidation is a more important contributor to such activity than metal-catalyzed oxidation of protein side-chains.

trans-4-Oxo-2-nonenal (ONE) has recently been demonstrated to be a direct product of lipid peroxidation. In earlier studies to elucidate the structure of the trans-4-hydroxy-2-nonenal (HNE)-derived fluorescent Lys-Lys cross-link, we showed that ONE was capable of both oxidative and nonoxidative cross-linking of amines. A more comprehensive study on nonoxidative modification of protein nucleophiles by ONE is described here, focusing on the initial Michael addition of imidazole, thiol, and amine groups to C2 or C3 to give 4-keto aldehydes that can then condense with amines to form nucleophile-substituted pyrroles. 2,3-Substituted pyrroles (major) and 2,4-substituted pyrroles (minor) were distinguished by 2D NMR techniques, and N(tau)-substitution is preferred over N(pi)-substitution in the Michael addition of histidine. Mechanisms of both nonoxidative and oxidative side chain reactions of ONE are discussed, as is the relative propensity (ONE > HNE) to induce cross-linking of the model proteins ribonuclease A and beta-lactoglobulin.

This study provides support for better protection of menisci in ACL injury-induced conditions such as OA and indicates that menisci should be considered in the development of clinical pharmacological interventions.

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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.