Apocynin: chemical and biophysical properties of a NADPH oxidase inhibitor

Abstract

Apocynin is the most employed inhibitor of NADPH oxidase (NOX), a multienzymatic complex capable of catalyzing the one-electron reduction of molecular oxygen to the superoxide anion. Despite controversies about its selectivity, apocynin has been used as one of the most promising drugs in experimental models of inflammatory and neurodegenerative diseases. Here, we aimed to study the chemical and biophysical properties of apocynin. The oxidation potential was determined by cyclic voltammetry (Epa = 0.76V), the hydrophobicity index was calculated (logP = 0.83) and the molar absorption coefficient was determined (e275nm = 1.1 × 104 M-1 cm-1). Apocynin was a weak free radical scavenger (as measured using the DPPH, peroxyl radical and nitric oxide assays) when compared to protocatechuic acid, used here as a reference antioxidant. On the other hand, apocynin was more effective than protocatechuic acid as scavenger of the non-radical species hypochlorous acid. Apocynin reacted promptly with the non-radical reactive species H2O2 only in the presence of peroxidase. This finding is relevant, since it represents a new pathway for depleting H2O2 in cellular experimental models, besides the direct inhibition of NADPH oxidase. This could be relevant for its application as an inhibitor of NOX4, since this isoform produces H2O2 and not superoxide anion. The binding parameters calculated by fluorescence quenching showed that apocynin binds to human serum albumin (HSA) with a binding affinity of 2.19 × 104 M-1. The association did not alter the secondary and tertiary structure of HSA, as verified by synchronous fluorescence and circular dichroism. The displacement of fluorescent probes suggested that apocynin binds to site I and site II of HSA. Considering the current biomedical applications of this phytochemical, the dissemination of these chemical and biophysical properties can be very helpful for scientists and physicians interested in the use of apocynin.

Figure 1

UV-Vis spectrum of apocynin and molar absorption coefficients. (a) UV-Vis spectra of apocynin dissolved in water (6.25–100 μM); (b) Linear regression at 275 nm, r² = 0.9994; (c) Linear regression at 303 nm, r² = 0.9989.

Figure 2

Molecular structures of apocynin and protocatechuic acid and their hydrophobicity indexes (log P) and acidity constants (pKa).

Figure 3

Cyclic voltammograms for apocynin (a) and protocatechuic acid (b) (0.1 mM) obtained in 0.2 M sodium phosphate buffer at pH 7.0. The scan rate was 5 mV s.

Figure 4

Reactivity of apocynin (APO) and protocatechuic acid (PA) with peroxyl radical (ROO•). (a,c) Bleaching of the triene (eleostearic acid) by ROO• and the lag phase provoked by the addition of the tested substances. (b,d) AUC versus concentration.

Figure 5

Reactivity of apocynin (APO) and protocatechuic acid (PA) with Nitric oxide. The reaction mixtures were constituted of 10 mM sodium nitroprusside and various concentration of the tested substances in 10 mM PBS, pH 7.4. The production of NO was measured by the Griess method.

Figure 6

Reactivity of apocynin and protocatechuic acid with hydrogen peroxide. The reaction mixtures were constituted of 1 mM tested substances, 1 mM H₂O₂ and 100 nM HRP in 0.1 M PBS, pH 7.4, at 25 °C.

Figure 7

Reactivity of apocynin and protocatechuic acid with hypochlorous acid. The reaction mixtures were constituted of 100 μM HOCl and various concentrations of the tested substances in 0.01 M PBS, pH 7.4, at 25 °C. The consumed HOCl was measured using the taurine-chloramine/TMB assay.

Figure 8

Fluorescence quenching of HSA by apocynin. (a) Emission spectra of HSA (1 μM) (λ_ex = 295 nm) in the presence of apocynin (0–40 μM). The arrow indicates the addition of apocynin; (b) Stern-Volmer plot; (c) Plot for apparent binding constant determination.

Figure 9

Effect of apocynin on HSA synchronous fluorescence. (a) Quencher of synchronous fluorescence (scanning at fixed Δλ = 15 nm). (b) Quencher of synchronous fluorescence (scanning at fixed Δλ = 60 nm). The mixtures consisted of 1.0 μM HSA and apocynin (0–30 μM) in 10 mM PBS, pH 7.2 at 25 °C. The arrow indicates the addition of apocynin.

Figure 10

Displacement of fluorophores from HSA by apocynin. (a) Displacement of dansylamide (DNSA); (b) Displacement of dansylglycine (DG). The mixtures consisted of 10.0 μM HSA, 5.0 μM fluorophores and apocynin (0, 5, 10, 20, 30, 40 and 50 μM) in 10 mM PBS, pH 7.2 at 25 °C.

Figure 11

Effect of apocynin on circular dichroism spectra of HSA. (a) Far-UV-CD: The mixtures consisted of 1.0 μM HSA and 30 μM apocynin in 10 mM PBS, pH 7.2 at 25 °C. The inset is a zoom of graph “a”; (b) Near-UV-CD: The mixtures consisted of 30 μM HSA and 60 μM apocynin in 10 mM PBS, pH 7.2 at 25 °C.