Abstract
Nitroalkene fatty acids are formed in vivo and exert protective and anti-inflammatory effects via reversible Michael addition to thiol-containing proteins in key signaling pathways. Nitro-conjugated linoleic acid (NO2-CLA) is preferentially formed, constitutes the most abundant nitrated fatty acid in humans, and contains two carbons that could potentially react with thiols, modulating signaling actions and levels. In this work, we examined the reactions of NO2-CLA with low molecular weight thiols (glutathione, cysteine, homocysteine, cysteinylglycine, and β-mercaptoethanol) and human serum albumin. Reactions followed reversible biphasic kinetics, consistent with the presence of two electrophilic centers in NO2-CLA located on the β- and δ-carbons with respect to the nitro group. The differential reactivity was confirmed by computational modeling of the electronic structure. The rates (kon and koff) and equilibrium constants for both reactions were determined for different thiols. LC-UV-Visible and LC-MS analyses showed that the fast reaction corresponds to β-adduct formation (the kinetic product), while the slow reaction corresponds to the formation of the δ-adduct (the thermodynamic product). The pH dependence of the rate constants, the correlation between intrinsic reactivity and thiol pKa, and the absence of deuterium solvent kinetic isotope effects suggested stepwise mechanisms with thiolate attack on NO2-CLA as rate-controlling step. Computational modeling supported the mechanism and revealed additional features of the transition states, anionic intermediates, and final neutral products. Importantly, the detection of cysteine-δ-adducts in human urine provided evidence for the biological relevance of this reaction. Finally, human serum albumin was found to bind NO2-CLA both non-covalently and to form covalent adducts at Cys-34, suggesting potential modes for systemic distribution. These results provide new insights into the chemical basis of NO2-CLA signaling actions.
Highlights
Nitroalkene fatty acids are formed in vivo and exert protective and anti-inflammatory effects via reversible Michael addition to thiol-containing proteins in key signaling pathways
We examined the reactions of Nitro-conjugated linoleic acid (NO2-Conjugated linoleic acid (CLA)) with low molecular weight thiols and human serum albumin
NO2-CLA Reacts Biphasically and Reversibly with Low Molecular Weight Thiols—Stopped-flow mixing of a solution consisting of 9- and 12-NO2-CLA (Fig. 1A) with excess GSH led to a reduction in absorbance at 330 nm, consistent with loss of double bond conjugation
Summary
NO2-CLA Reacts Biphasically and Reversibly with Low Molecular Weight Thiols—Stopped-flow mixing of a solution consisting of 9- and 12-NO2-CLA (Fig. 1A) with excess GSH led to a reduction in absorbance at 330 nm, consistent with loss of double bond conjugation. The kinetic results are consistent with two parallel and reversible processes involving two non-equivalent electrophilic centers in NO2-CLA These centers react with thiols forming two products that we hypothesize are the - and the ␦-adducts as shown in to Scheme 1, where kon and kon␦ are second-order rate constants at pH 7.4 for the forward addition reaction of the fast and slow processes, respectively, and koff and koff␦ correspond to first-order rate constants for the reverse elimination reactions.. From fits to this equation, the kon␦ for the reaction with GSH was determined to be 3.5 Ϯ 0.5 MϪ1 sϪ1 whereas Keq was (3.0 Ϯ 0.2) ϫ 10Ϫ3 M, in excellent agreement with the value determined from the fast phase. The coloring scheme spans from Ϫ6.0 ϫ 10Ϫ7 au in red to 1.1 ϫ 10Ϫ4 au in blue
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