Effect of curcuminoids on cell-free 5-lipoxygenase activity

Human semi-purified or purified 5-lipoxygenase (5-LOX) enzyme was pre-treated with DMSO or curcuminoids, 5-LOX product formation was initiated by arachidonic acid and 5-LOX products (et-LTB4, t-LTB4 and 5-H(p)ETE) were analyzed by RP-UV-HPLC.
Raw data including the absolute values (ng) and normalized data (% of control) of all individual experiments was uploaded.
The methods and results were published in Rao et al., Biochem Pharmacol. 2022 Sep:203:115202. doi: 10.1016/j.bcp.2022.115202 
To determine 5-LOX activity, semi-purified 5-LOX (0.5 µg) was pre-treated with vehicle (DMSO), test compounds or the selective 5-LOX inhibitor BWA4C (0.1 μM, Merck) in 1 mL PBS pH 7.4 plus EDTA (1 mM) and ATP (1 mM) for 10 min at 4 °C. The mixture was pre-warmed for 30 s at 37 °C before 5-LOX product formation was initiated by the addition of AA (20 μM or as indicated, Cayman Chemical) and CaCl2 (2 mM). After 10 min incubation at 37 °C, the reaction was stopped with an equal volume of ice-cold methanol containing the internal standard PGB1 (2 ng, Cayman Chemical). Lipid mediators formed were extracted by solid phase extraction using Sep-Pak C18 35 cc Vac Cartridges (Waters, Milford, MA). Major 5-LOX products (all-trans isomers of LTB4 and 5-H(p)ETE) were analyzed by RP-UV-HPLC using a Nova-Pak C18 Radial-Pak Column (4 μm, 5 × 100 mm, Waters) under isocratic conditions (73 % methanol/27 % water/0.007 % trifluoroacetic acid) at a flow rate of 1.2 mL/min and detected at 235 nm (for 5-H(p)ETE) or 280 nm (all-trans isomers of LTB4). The 5-LOX reference inhibitor BWA4C (0.1 μM, Merck) inhibited 5-LOX product formation by 87.6 ± 1.7 %.
For the AA competition studies shown in Fig. 1E-G, human recombinant 5-LOX (Cayman Chemical, 25–50 units in 1 mL PBS pH 7.4 with 1 mM EDTA and 1 mM ATP) was pre-incubated with vehicle (DMSO), compound 1a, 3b, or 2f for 10 min on ice. Product formation was initiated by the addition of AA (10 µM, 20 µM or 40 µM, Cayman Chemical) and CaCl2 (2 mM) followed by incubation at 37 °C. After 10 min, the reaction was stopped by the addition of ice-cold methanol (1 mL) containing PGB1 (2 ng, Cayman Chemical) as internal standard. PBS pH 7.4 (498.2 µL) acidified with HCl (1 M, 31.8 µL) was added to the samples, which were centrifuged (750 × g, 10 min, 4 °C) and subjected to solid phase extraction. Clean-Up C-18 Endcapped SPE cartridges (100 mg, 10 mL, UCT, Bristol, PA) were washed twice with methanol and pre-conditioned with water (1 mL each). Supernatants were loaded onto the cartridges, which were washed with 1 mL water and 1 mL water/methanol (75/25, v/v) prior to elution of 5-LOX products with 300 µL methanol. After addition of 120 µL water, samples were centrifuged (21,000xg, 10 min, 4 °C) and supernatants subjected to UPLC-photodiode array detector (PDA) analysis.
Chromatographic separation of 5-LOX products (all-trans isomers of LTB4 and 5-H(p)ETE) was performed at 40 °C on a Kinetex C-18 LC column (100 Å, 1.3 μm, 2.1 × 50 mm, Phenomenex, Torrance, CA) using a Nexera X2 UHPLC system (Shimadzu, Kyoto, Japan) that was coupled to a PDA (SPD-M20A, Shimadzu). The UHPLC system was operated at a flow rate of 0.45 mL/min using buffer A (50 % methanol/50 % water/0.05 % trifluoroacetic acid) and buffer B (100 % methanol/0.05 % trifluoroacetic acid). Following sample injection (10 µL), the initial mobile phase composition (A/B = 86/14) was kept constant for 2 min before it was stepwise decreased to A/B = 54/46 (2 min) and then to A/B = 10/90 (another 2 min).
For 5-H(p)ETE analysis, the wavelength was set to 235 nm, whereas PGB1 (internal standard, Cayman Chemical) and the all-trans isomers of LTB4 were detected at 280 nm. Chromatograms were acquired and processed using LabSolutions (version 5.97, Shimadzu).