Abstract
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 (NO2-OA) and its ability to bind to the fatty acid transporter human serum albumin (HSA). The nitro group of NO2-OA underwent electrochemical reduction at −0.75 V at pH 7.4 in an aqueous milieu. Based on observations of the R–NO2 reduction process, the stability and reactivity of NO2-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-NO2 and C10-NO2 positional isomers of NO2-OA occurred in two conformers with different internal angles (69° and 110°) between the methyl- and carboxylate termini. Both NO2-OA positional isomers have LUMO energies of around −0.7 eV, affirming the electrophilic properties of fatty acid nitroalkenes. In addition, the binding of NO2-OA and OA with HSA revealed a molar ratio of ~7:1 [NO2-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 NO2-OA resulted in the formation of nitric oxide radical. Finally, the interaction of NO2-OA with HSA was monitored via Tyr and Trp residue electro-oxidation. The results indicate that not only non-covalent binding but also NO2-OA-HSA adduction mechanisms should be taken into consideration. This study of the redox properties of NO2-OA is applicable to the characterization of other electrophilic mediators of biological and pharmacological relevance.
Highlights
Inflammatory and metabolic reactions increase rates of production and levels of reactive oxygen and nitrogen species (RONS), including nitric oxide and nitrogen dioxide radicals, hydroxyl radicals, hydrogen peroxide, superoxide, and their downstream reaction products [1]
The electrophilic character of nitro-oleic acid (NO2-oleic acid (OA)) corresponds to a reduction process that can be observed around the potential of −0.75 vs. Ag|AgCl|3 M KCl at pH 7.4 using cyclic voltammetry (CV), see Fig. 1B
We focused on evaluating the redox behavior of NO2OA and its ability to bind to human serum albumin (HSA)
Summary
Inflammatory and metabolic reactions increase rates of production and levels of reactive oxygen and nitrogen species (RONS), including nitric oxide and nitrogen dioxide radicals, hydroxyl radicals, hydrogen peroxide, superoxide, and their downstream reaction products [1]. NO2-FAs induce multiple pleiotropic signaling responses, resulting in anti-inflammatory and antioxidant effects [9] These actions have been attributed to the electrophilic capacity of the β-carbon of the reactive nitroalkenyl substituent of NO2-FAs, which undergo a kinetically rapid and reversible Michael addition [15]. This reaction, termed nitroalkylation, occurs primarily with functionally-significant Cys residues of transcription factors and enzymes [15,16]. (d) apply EPR techniques to evaluate the potential for nitro-moiety release (nitric oxide radical formation) and binding affinity to HSA This experimental and theoretical work utilizes oleic acid (OA) as a negative control
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