Nitro-oleic acid, a novel and irreversible inhibitor of xanthine oxidoreductase

Abstract

Xanthine oxidoreductase (XOR) generates proinflammatory oxidants and secondary nitrating species, with inhibition of XOR proving beneficial in a variety of disorders. Electrophilic nitrated fatty acid derivatives, such as nitro-oleic acid (OA-NO2), display anti-inflammatory effects with pleiotropic properties. Nitro-oleic acid inhibits XOR activity in a concentration-dependent manner with an IC50 of 0.6 microM, limiting both purine oxidation and formation of superoxide (O2.). Enzyme inhibition by OA-NO2 is not reversed by thiol reagents, including glutathione, beta-mercaptoethanol, and dithiothreitol. Structure-function studies indicate that the carboxylic acid moiety, nitration at the 9 or 10 olefinic carbon, and unsaturation is required for XOR inhibition. Enzyme turnover and competitive reactivation studies reveal inhibition of electron transfer reactions at the molybdenum cofactor accounts for OA-NO2-induced inhibition. Importantly, OA-NO2 more potently inhibits cell-associated XOR-dependent O2. production than does allopurinol. Combined, these data establish a novel role for OA-NO2 in the inhibition of XOR-derived oxidant formation.

FIGURE 1.

Nitro-oleic acid inhibits XOR activity. A, following exposure of XOR (10 milliunits/ml) to a 0–10 μm concentration of either OA-NO₂ (•) or allopurinol (▪) in 50 mm phosphate buffer (pH 7.4, 25 °C), enzymatic activity was determined by the production of uric acid (λ = 292 nm). Reactions were initiated by the addition of xanthine (100 μm). Control analyses were conducted with 10 μm native oleic acid (18:1). The concentration range from 0 to 1 μm for OA-NO₂ is expanded (inset). Dashed lines, IC₅₀ values (IC₅₀ = 0.6 and 2.4 μm for OA-NO₂ and allopurinol, respectively). B, following the same protocol as in A, formation was determined by the reduction of cytochrome c (λ = 550 nm) upon OA-NO₂ (•) and allopurinol (▪) addition. Dashed lines demonstrate IC₅₀ values (IC₅₀ = 0.75 and 2.25 μm for OA-NO₂ and allopurinol, respectively). C, the effect of pH on XOR inhibition by OA-NO₂ (0.5 μm) was determined by normalizing the activity (uric acid production upon the addition of 100 μm xanthine) in the presence of OA-NO₂ to XOR alone. A nonlinear pH activity (y = (a₀ + a₁·10^{(x – a2)})/(1 + 10^{(x – a2)}) curve fit produced r² = 0.92. D, shown is the Dixon plot (1/V₀ versus [inhibitor]) for OA-NO₂ (0.25, 0.5, 1.25, and 2.5 μm) at varying concentrations (12.5 (▴), 25 (▪), and 100 μm (♦)) of xanthine. Data represent the mean ± S.E. of at least three independent determinations; *, p < 0.05.

FIGURE 2.

Nitro-oleic acid inhibition of XOR is not reversible by reducing agents. A, XOR (10 milliunits/ml) was exposed to OA-NO₂ (10μm) for 10 min at 25 °C in phosphate buffer (50 mm, pH 7.4). Next, GSH (20 mm), β-mercaptoethanol (BME; 20 mm), or dithiothreitol (DTT; 20 mm) was added for 15 min, and then enzymatic activity was determined by the production of uric acid (λ = 292 nm). B, XOR was incubated with 4-hydroxy-2-nonenol (4-HNE) (100 μm) and 15-deoxyprostaglandin J₂ (15dPGJ₂) (100 μm) or ethyl pyruvate (EP) (100 μm) for 10 min, and then enzymatic activity was determined as in A. Data points represent the mean ± S.E. of at least three independent determinations; *, p < 0.05.

FIGURE 3.

The FAD cofactor of XOR is not affected by OA-NO₂. A, XOR (10 milliunits/ml) enzymatic activity was assayed as in Fig. 1 with additions made in the order listed: XOR + xanthine (line 1), XOR + DPI + DCPIP + xanthine (line 2), XOR + DPI + OA-NO₂ + DCPIP + xanthine (line 3), XOR + DPI + xanthine (line 4). Concentrations were as follows: xanthine (100 μm), DPI (50 μm), DCPIP (15 μm), and OA-NO₂ (10 μm). B, anaerobic absorption spectra of FAD were assessed under the following conditions: 1) XOR (30 milliunits/ml) + dithionite (420 μm), 2) XOR + OA-NO₂ (50 μm), 3) XOR + OA-NO₂ + dithionite (300 μm), 4) XOR + OA-NO₂ + dithionite (420 μm), 5) XOR + OA-NO₂ + dithionite (420 μm) and then 30 min exposure to room air. C, XOR (30 milliunits/ml) was placed in a closed system O₂ monitor, and O₂ levels were monitored over time. At 4 min, NADH (500 μm) was added, and then OA-NO₂ (50 μm) and finally DPI (100 μm) were added (solid line). The dashed line represents O₂ consumption in the absence of enzyme.

FIGURE 4.

Nitro-oleic acid affects the molybdenum cofactor of XOR. A, anaerobic absorption spectra of FAD: 1) XOR, 2) XOR + xanthine, 3) XOR + OA-NO₂, 4) XOR + OA-NO₂ + xanthine. The data represent three replicate determinations. B(1), enzyme activity was measured as in Fig. 1. At t = 15 s after the initiation of enzyme turnover with xanthine (100 μm), 10 μm OA-NO₂ was added. The solid line represents conditions in the absence of DCPIP, and the dashed line indicates the presence of DCPIP (15 μm) added before the initiation of turnover with xanthine. B(2), rates of uric acid production before and after the addition of OA-NO₂ in B were calculated for four independent experiments; *, p < 0.05. C(1), XOR was exposed to either OA-NO₂ or allopurinol (AP) for 5 min, separated from free inhibitor by size exclusion column chromatography (G25 Sephadex), and reactivated by the addition of xanthine (100 μm), and uric acid production was determined. Allopurinol and then OA-NO₂ indicates that enzyme was sequentially exposed to allopurinol and then OA-NO₂. C(2), rates of uric acid production for the three conditions in C(1) were calculated for three independent experiments. Data points represent the mean ± S.E. of at least three independent determinations; *, p < 0.05.

FIGURE 5.

Nitro-oleic acid inhibits GAG-associated XOR production. A, free and HS6B-bound XOR were exposed to 10 μm allopurinol (AP) or OA-NO₂, and activity was determined by monitoring the production of uric acid (292 nm) following the addition of xanthine (100 μm). Data points represent the mean ± S.E. of at least three independent determinations; *, p < 0.05. B, bovine aortic endothelial cells were exposed to XOR (5 milliunits/ml) for 20 min and then harvested as described under “Experimental Procedures”. Cell suspensions (3 × 10⁶ cells/ml) were exposed to OA-NO₂ for 10 min and then analyzed by EPR for production with the spin trap DMPO (25 mm) at 25 °C. Reactions were initiated by the addition of xanthine (100 μm). Samples for all spectra contain cells plus DMPO and are identified beginning from the top as follows: control without added XOR and xanthine (Xan) and added XOR and xanthine (XOR + Xan). The remainder of spectra consist of cells, added XOR, xanthine, and the constituents indicated.