Redox properties and human serum albumin binding of nitro-oleic acid

Nitro-fatty acids modulate inflammatory and metabolic stress responses, thus displaying potential as new drug candidates. Herein, we evaluate the redox behavior of nitro-oleic acid (NO(2)-OA) and its ability to bind to the fatty acid transporter human serum albumin (HSA). The nitro group of NO(2)-OA underwent electrochemical reduction at −0.75 V at pH 7.4 in an aqueous milieu. Based on observations of the R–NO(2) reduction process, the stability and reactivity of NO(2)-OA was measured in comparison to oleic acid (OA) as the negative control. These electrochemically-based results were reinforced by computational quantum mechanical modeling. DFT calculations indicated that both the C9-NO(2) and C10-NO(2) positional isomers of NO(2)-OA occurred in two conformers with different internal angles (69° and 110°) between the methyl- and carboxylate termini. Both NO(2)-OA positional isomers have LUMO energies of around −0.7 eV, affirming the electrophilic properties of fatty acid nitroalkenes. In addition, the binding of NO(2)-OA and OA with HSA revealed a molar ratio of ~7:1 [NO(2)-OA]:[HSA]. These binding experiments were performed using both an electrocatalytic approach and electron paramagnetic resonance (EPR) spectroscopy using 16-doxyl stearic acid. Using a Fe(DTCS)(2) spin-trap, EPR studies also showed that the release of the nitro moiety of NO(2)-OA resulted in the formation of nitric oxide radical. Finally, the interaction of NO(2)-OA with HSA was monitored via Tyr and Trp residue electro-oxidation. The results indicate that not only non-covalent binding but also NO(2)-OA-HSA adduction mechanisms should be taken into consideration. This study of the redox properties of NO(2)-OA is applicable to the characterization of other electrophilic mediators of biological and pharmacological relevance.