Development of a High-Throughput Assay for Measuring

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Development of a High-Throughput Assay for Measuring Phospholipase A Activity Using Synthetic 1,2-α-Eleostearoyl-snglycero-3-phosphocholine Coated on Microtiter Plates Meddy El Alaoui,†,‡ Alexandre Noiriel,† Laurent Soulère,‡ Lucie Grand,‡ Yves Queneau,‡ and Abdelkarim Abousalham*,† †

Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires (ICBMS) UMR 5246 CNRS, Organisation et Dynamique des Membranes Biologiques, Université Lyon 1, Bâtiment Raulin, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France ‡ Université de Lyon, INSA Lyon, ICBMS, Chimie Organique et Bioorganique, Bâtiment Jules Verne, 20 Avenue Albert Einstein, 69621 Villeurbanne Cedex, France ABSTRACT: To date, several sensitive methods, based on radiolabeled elements or sterically hindered fluorochrome groups, are usually employed to screen phospholipase A (PLA) activities. With the aim of developing a convenient, specific, sensitive, and continuous new ultraviolet (UV) spectrophotometric assay for PLA, we have synthesized a specific glycerophosphatidylcholine (PC) esterified at the sn-1 and sn-2 positions, with α-eleostearic acid (9Z, 11E, 13Eoctadecatrienoic acid) purified from Aleurites fordii seed oil. The conjugated triene present in α-eleostearic acid constitutes an intrinsic chromophore and, consequently, confers the strong UV absorption properties of this free fatty acid as well as of the glycerophospholipids harboring it. This coated PC film cannot be desorbed by the various buffers used during PLA assays. Following the action of PLA at the oil−water interface, α-eleostearic acid is freed and desorbed from the film and then solubilized with β-cyclodextrin. The UV absorbance of the α-eleostearic acid is considerably enhanced due to the transformation from an adsorbed to a water-soluble state. The PLA activity can be measured continuously by recording the variations with time of the UV absorption spectra. The rate of lipolysis was monitored by measuring the increase of absorption at 272 nm, which was found to be linear with time and proportional to the amount of added PLA. This continuous high-throughput PLA assay could be used to screen new PLA and/or PLA inhibitors present in various biological samples.

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monolayers. The two-dimensional nature of the PLA-catalyzed reactions does not follow Michaelis−Menten kinetics and it depends strongly on the quality of the interface.9−11 Obtaining accurate (i.e., substrate-specific) measurements of PLA activity, as well as developing reliable PLA assay systems, requires taking these unique features into account. Numerous assays using chromogenic,12,13 radiolabeled,14 or fluorogenic15 substrates have been developed to measure PLA activity.16 In the reported results, the different assays have provided the option of simpler and faster protocols. However, the substrates used were either expensive or unsuitable for performing large numbers of assays.17,18 Progress has been made, over the past decade, in the development of highthroughput screening methods to screen PLA activity. Some assays are able to screen the hydrolysis of the ester bond in which glycerol has been replaced by a chromogenic19 or a fluorescent group.20 More relevant substrates are usually

here are two families of phospholipase A (PLA), PLA1 (EC 3.1.1.32), and PLA2 (EC 3.1.1.4) that catalyze hydrolysis of the ester bond of the acyl group attached to the sn-1 or the sn-2 position of glycerophospholipids, respectively. They play opposite roles in organisms, either assuming vital functions for key metabolism pathways or as a powerful poison in snake and scorpion venoms.1 PLAs (essentially PLA2 and, to a lesser degree, PLA1) are also involved in different pathologies (obesity, diabetes, cardiovascular pathologies),2−4 inflammatory processes,5 and cell signaling.6 Metabolic diseases increase each year, for example, 34% of the USA adult population shows a prevalence to metabolite syndrome,7 partly due to a fat-rich diet. The study of lipid metabolism, through the actions of phospholipases A, may lead to a better understanding of their mechanisms and this knowledge is essential for the design of a new generation of drug inhibitors. Because PLA are watersoluble enzymes, hydrolyzing water-insoluble long-chain glycerophospholipids substrates, the cleavage reaction has to occur at the lipid−water interface.8,9 The mechanism involved in the enzymatic lipolysis depends strongly on the particular organization of lipid substrates in the interfacial structures, such as oil-in-water emulsions, micelles, liposomal dispersion, or © 2014 American Chemical Society

Received: June 6, 2014 Accepted: September 29, 2014 Published: September 30, 2014 10576

dx.doi.org/10.1021/ac502096v | Anal. Chem. 2014, 86, 10576−10583

Analytical Chemistry

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methanol/water, 65:35:5 (v/v/v) containing 0.001% (w/v) BHT as an antioxidant. The various lipids were revealed with UV light at 254 nm. Preparation of Purified α-Eleostearic Acid from Tung Oil. A solution of 20 g of crude tung oil was hydrolyzed with 500 mg of Candida rugosa lipase in 14 mL of water and the reaction was stirred, for 3 h, at 40 °C. Total lipid was extracted into a decantation vial with 100 mL of 3 M HCl and 100 mL of diethyl ether containing 0.01% BHT (w/v). The organic layer was recovered, dried by adding anhydrous MgSO4, filtrated, and concentrated under reduced pressure. The total lipid extract (5 g), containing mainly free fatty acids, was further purified by recrystallization in 5.5 mL of acetone, at 60 °C for 20 min, and then cooled on ice. The heterogeneous mixture was filtrated and the crystalline solid obtained was treated with dry acetone. The crystals were then collected by filtration and dried in vacuo (2.2 g, 40% yield from the lipolysis extract). The specific UV spectrum obtained showed three majors peaks located at λmax (ethanol 95%) = 260, 270, and 280 nm. 1 H NMR (400 MHz CDCl3) δ 0.89 (t, 3H, J = 7 Hz, C18CH3), 1.33 (s, 12H C(4−7)−CH2, C (16,17)−CH2), 1.63 (s, 2H, C3-CH2), 2.09−2.18 (m, 4H, C(8, 15)−CH2), 2.35 (t, 2H, J = 7 Hz, C2−H2), 5.34−6.37 (m, 6H, C(9−14)-H2). 13C NMR (100 MHz CDCl3) δ 14.1 (C18), 22.4 (C17), 24.8 (C3), 28.0, 29.1, 29.16, 29.23 (C4−7), 29.8 (C8), 31.7 (C16), 32.7 (C15), 34.2 (C2), 126.1 (C12), 128.9 (C10), 130.7 (C13), 131.7 (C9), 133.0 (C11), 135.4 (C14), 180.1 (C1). HR-MS (ESI, negative mode): calculated for C18H29O2, 277.2173; found [M − H]−, 277.2167. Synthesis of 1,2-α-Eleostearoyl-sn-glycero-3-phosphocholine (EEPC). To a solution of GPC, (0.05 g, 0.2 mmol), pure α-eleostearic acid (0.278 g, 1 mmol) was added, in alcohol-free and anhydrous CHCl3 (2 mL), under stirring. A solution of freshly recrystallized PPyr32 (0.148 g, 1 mmol) and DCC (0.194 g, 1 mmol), in 1 mL of CHCl3, was then added dropwise. After 20 h, the reaction mixture was concentrated and the product was purified, by flash chromatography, on silica gel (eluent CHCl3/MeOH/H2O, 65:35:4, v/v/v). Fractions containing the product were further purified by ion exchange chromatography using Amberlyst resin (eluent CHCl3/MeOH/H2O, 65:35:4, v/v/v). EEPC (120 mg) was obtained with a 77% yield. 1 H NMR (400 MHz CDCl3) δ 0.88 (t, 6H, J = 7.1, C18CH3, C36-CH3), 1.24−1.41 (m, 24H, C(4−7, 16, 17, 22−25, 34, 35)−CH2), 1.57 (m, 4H, C(3, 21)−CH2), 2.08−2.16 (m, 8H, C(8, 15, 26, 33)−CH2), 2.26−2.30 (m, 4H C(2, 20)− CH2), 3.35 (s, 9H, N-(CH3)3), 3.79 (m, 2H, CH2−N), 3.89− 3.97 (m, 2H, CH2 sn-3), 4.11 (dd, 1H; J = 7.2 and 12.0 Hz, CH sn-1), 4.29 (m, 2H, PO−CH2) 4.38 (dd, 1H, J = 2.4 and 12.0, CH sn-1), 5.16−5.21 (m, 1H,CH sn-2) 5.33−5.41 (m, 2H C(9, 27)−CH), 5.66−5.71 (m, 2H, C14, C32-CH), 5.93−6.19 (m, 6H, C10, C11, C13, C28, C29, C31-CH), 6.31−6.40 (m, 2H, C12, C30-CH). 13C NMR (100 MHz CDCl3) δ 14.1 (C18, 36), 22.4 (C17, 35), 25.0 (C15, 33), 27.4 (C8, 26), 29.19, 29.22, 29.27, 29.33, 29.36, and 29.81 (C 4−7, 22−25), 31.6 (C16,34), 32.6 (C3, 21), 34.2 and 34.4 (C2, 20), 54.6 (N(CH3)3), 59.4 (C−N, JC−P = 5.1), 63.1 (C sn-1), 63.5 (PO-C, JC−P = 5.1), 66.5 (C sn-3, JC−P = 6.6), 70.7 (C sn-2, JC−P = 7.3), 126.0 (C12, 30), 128.9 (C10, 28), 130.7 (C13, 31), 131.9 (C9, 27), 133.0 (C11, 29), 135.4 (C14, 32), 173.3 and 173.6 (C1, 19). 31P NMR (120 MHz CDCl3) δ −0.66; HR-MS (ESI, positive mode): calculated for C44H77NO8P, 778.5381; found [M + H]+, 778.5360.

employed to screen PLA activity with synthetic glycerophospholipids containing a fluorescent group21 attached to the fatty acid chain or by using a fluorochrome as an interfacial probe for hydrophobic environment detection.22 Nevertheless, these substrates present sterically hindered fluorochrome groups that may interfere with PLA activity. Wolf et al.23 used glycerophosphatidylcholine (PC) containing parinaric acid (9Z, 11E, 13E, 15E-octadecatetraenoic acid), a naturally fluorescent fatty acid bearing four conjugated double bonds, to monitor PLA2 activity. Following hydrolysis, the binding between albumin and the free parinaric acid released corresponded to an increase in the total fluorescence intensity. The drawback of this method is, on the one hand, the susceptibility of parinaric acid to oxidation by atmospheric oxygen and, on the other hand, the need for the selected detergent to solubilize the released parinaric acid into mixed micelles.24 Here we have developed a specific and continuous assay using the ultraviolet (UV) spectrophotometric properties of natural α-eleostearic acid, incorporated into PC at positions sn1 and sn-2, to measure PLA activity. α-Eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) was purified from Aleurites fordii seed oil (tung oil). Crude tung oil contains up to 70% αeleostearic acid,25,26 esterified into triglycerides, and it is one of the main primary products used in the lacquer industry, making it an ideal probe for use in large amounts at a low cost. Although the conjugated triene present in α-eleostearic acid does not confer strong fluorescent properties to this fatty acid, it is an intrinsic chromophore which confers strong UV absorption properties to fatty acids,27 esterified triglycerides28−30 and PC (this study). This continuous PLA assay is compatible with high-throughput screenings, and hence, it can be applied to the screening of PLA activities and/or inhibitors of PLA.

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EXPERIMENTAL SECTION Reagents and Materials. β-Cyclodextrin (β-CD), butylhydroxytoluene (BHT), N,N′-dicyclohexylcarbodiimide (DCC), 4-pyrrolidinopyridine (PPyr), Amberlyst 15, and crude tung oil were purchased from Sigma-Aldrich-Fluka Chimie (St-QuentinFallavier, France). sn-Glycero-3-phosphocholine (GPC) was purchased from Bachem (Budendorf, Switzerland). Methyl Indoxam was kindly provided by Dr. D. Charmot (Ardelyx, Frement). Microtiter plates (Costar UV-Plate) were purchased from Corning Inc., Corning, NY. Thin-layer chromatography (TLC) aluminum sheets, coated with 0.2 mm silica gel 60 F254, were from Merck. All other chemicals and solvents, of the highest quality, were obtained from local suppliers. Proteins. Bovine serum albumin (BSA), porcine pancreatic PLA2 (ppPLA2), honey bee venom (Apis mellifera) PLA2 (hbPLA2), and PLA1 from Thermomyces lanuginosus (known as Lecitase Ultra) (tlPLA1) were all purchased from SigmaAldrich-Fluka Chimie (St-Quentin-Fallavier, France). Horse pancreatic PLA2 (hpPLA2) was kindly provided by Dr. R. Verger (Marseille, France). Candida rugosa lipase AY30 was purchased from Amano Pharmaceuticals Ltd. (Nogoya, Japan). The protein concentrations were determined using Bradford’s procedure,31 with Bio-Rad Dye Reagent and BSA as the standard. Thin-Layer Chromatography (TLC). Glycerophospholipids were separated by performing analytic TLC on aluminum sheets coated with 0.2 mm silica gel 60. The following chromatographic solvent system was used: chloroform/ 10577



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Figure 1. (A) Synthesis of EEPC from GPC; (B) 1H NMR (400 MHz) spectrum of pure EEPC in deuterated chloroform. Chemical shifts are given in δ-values in ppm downfield from the chloroform (δCHCl3 = 7.26).

Coating Microtiter Plates with EEPC. Microtiter plates were coated with EEPC as described previously.28,30 EEPC solution (0.5 mg mL−1) was prepared in ethanol, containing 0.01% BHT as an antioxidant, and the wells of a UV-microtiter plate were filled with EEPC (100 μL/well). The microtiter plate was first partially dried under a fume hood and then left in a vacuum desiccator until the solvent had completely evaporated (about 30 min). After ethanol evaporation, the coated EEPC plates were found to be stable in the dark for at least 1 week, at 4 °C. UV Spectrophotometric PLA Assay Using Coated EEPC. PLA activity was assayed spectrophotometrically by measuring the amount of α-eleostearic acid continuously released from EEPC (Figure 1). The PLA activity measurement was performed with 10 mM Tris-HCl buffer (pH 8.0) containing 3 g L−1 β-CD, 150 mM NaCl, 6 mM CaCl2, and 1 mM EDTA. The substrate was dissolved in ethanol to obtain the desired final concentration and the wells of a 96-well, flatbottomed microtiter plate were then coated with the lipids, as described above. The substrate-coated wells were then washed with the assay buffer and left to equilibrate for 10 min, at 37 °C. The assays were run in a 200 μL final volume at 37 °C. The PLA solutions (2−10 μL) were injected into the microtiter plate wells, and the absorbance, at 272 nm, was recorded continuously, at regular 1 min intervals, against the buffer alone. A microtiter plate scanning spectrophotometer (Tecan Infinite M200 Pro) was used. The plate was shook for 5 s before each reading.

The specific activity of the various PLAs used was calculated from the steady-state reaction rate, expressed as the change in absorbance per minute using an apparent molar extinction of 5320 M−1 cm−1 (see the Results and Discussion). The PLA specific activity was expressed as international units per mg of pure enzyme. One international unit corresponds to 1 μmol of fatty acid released per minute, under the assay conditions. Alternatively, after different periods of time, the hydrolytic reaction was stopped by acidification, using 20 μL of 0.1 M HCl, and the total aqueous phase (220 μL) was transferred to a glass vial. In order to maximize total lipid recovery, each well was washed with 100 μL of ethanol and added to the aqueous phase. Lipids were then extracted with 1 mL of chloroform/ methanol (2:1; v/v), shaken vigorously, and centrifuged for 1 min at 1000g. The lower organic phase was collected and transferred to a new glass tube in which it was dried with anhydrous MgSO4. MgSO4 was removed by centrifugation, for 1 min at 1000g, and the clear lipid extract was transferred to a 2 mL vial. The remaining solvent was evaporated under nitrogen flow, to prevent oxidation, and the lipids were dissolved in 20 μL of a mixture of chloroform methanol (2:1 v/v) containing 0.001% BHT. A TLC analysis of lipids was carried out using TLC precoated plates 60 F254, as described above. Microtiter Plate Assay to Study PLA Inhibition. The enzyme−inhibitor incubation method33 was used to test, in aqueous medium and in the absence of the substrate, whether any direct interactions occur between the PLA and the inhibitor. ppPLA2 (0.5 μg, 0.14 nM) was incubated, at 20 °C, with methyl indoxam (MI)34 solution (14 nM in dimethyl 10578



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Figure 2. Principle of the PLA assay using coated EEPC: (A) A microtiter plate well containing EEPC in ethanol, and UV absorption spectrum of purified EEPC in ethanol with three major peaks located at 260, 270, and 280 nm. (B) After solvent evaporation, the coated EEPC exhibited a UV spectrum with low absorbance. (C) Kinetic UV absorption spectra following EEPC lipolysis. PLA2 (2.5 μg/mL, final concentration) was added to coated EEPC in the standard buffer containing β-CD (3 mg/mL, final concentration). The UV spectra were determined every 3 min, for 30 min, in the 250−300 nm range. A blank was run at t = 0. E, PLA in solution; E*, PLA at the interface; S, substrate; P, lipolysis products.

was obtained with a 77% yield. The product was stored under argon, in the dark at −20 °C to avoid any oxidation. The mass spectrometry analysis showed that the product was also stable for more than 2 years in ethanol. The UV absorption spectrum (220−320 nm) of EEPC, dissolved in ethanol, displayed three major peaks located at around 260, 270, and 280 nm. This profile spectrum is similar to that of pure α-eleostearic acid (this study and refs 28−30) as well as to the spectrum of pure tung oil triglycerides29 and synthetic triglycerides containing α-eleostearic acid.28 The1H NMR spectrum of EEPC (Figure 1B) showed signals of the vinylic system, between δ 6.4 and 5.4 ppm, as a complex set of multiplets. A single signal at δ 5.19 ppm is characteristic of the sn-2 single proton. At δ 4.4 to 3.8 ppm, multiplets are referenced to protons close to the glycerol chain group (sn-1 and sn-3 positions and the phosphatidyl group). The methyl groups of the quaternary ammonium give a specific signal at δ 3.3 ppm. Saturated protons from the carbonyl chain are located from δ 2.3 ppm (α carbon) to δ 0.9 ppm (ω carbon). Principle of the PLA Assay. The EEPC was coated onto the wells of microtiter plates, as indicated in the Experimental Section. The principle of the PLA assay is schematically shown in Figure 2. EEPC was dissolved in ethanol, and the UV absorption spectrum was recorded from 250 to 300 nm (Figure 2A). The sensitivity of α-eleostearic acid to oxidation was prevented by using BHT in all the EEPC ethanolic solutions. After ethanol evaporation, under reduced pressure, the thin EEPC films coated on the 96-well microtiter plate exhibited a UV spectrum with very low absorbance (Figure 2B). The hydrolysis of EEPC was performed by adding ppPLA2 in the buffered phase containing the nontensioactive β-CD in order to solubilize the long-chain lipolytic products38 (α-eleostearic acid and 1-α-eleostearoyl-sn-glycero-3-phosphocholine (lyso-PC)). β-CD has been previously used as an acceptor for the long-

sulfoxide (DMSO)) at an enzyme/inhibitor molar ratio of 1:100 (final DMSO concentration 2.5%). The residual ppPLA2 activity was then measured at various incubation times using EEPC coated on the surface of the microtiter plate wells. Alternatively, inhibition during the lipolysis method33 was also used to test whether any inhibition reaction occurred in the presence of a water insoluble substrate during hydrolysis. ppPLA2 (0.14 nM, final concentration) was injected into the reaction medium containing EEPC coated on the surface of the microtiter plate wells. MI (14 nM, final concentration) was injected a few minutes after ppPLA2 addition, and the activity was then continuously recorded.

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RESULTS AND DISCUSSION

Synthesis of EEPC and Spectroscopic Properties. The α-eleostearic acid was obtained from tung oil enzymatic hydrolysis, and it was recrystallized from the total lipid extract. The α-eleostearic acid crystals were collected by filtration and dried in vacuo (2.2 g, 40% yield from the crude lipolysis extract). The purified α-eleostearic acid was further characterized by 1H NMR, 13C NMR, and mass spectrometry. The vinylic protons carried by the trienic system resonate as a complex set of multiplets between δ 5.3 and δ 6.4 ppm. The αcarboxylic acid methylene protons and the C-omega position resonate as characteristic triplets at δ 2.35 ppm and δ 0.9 ppm, respectively. Chemical shifts, coupling constants, and integrations were in agreement with the values reported in the literature28,35 confirming the purity of the recrystallized αeleostearic acid. Synthesis of EEPC was performed as indicated in Figure 1A. DCC was used as a chemical coupling agent, in the presence of freshly recrystallized32 PPyr, to esterify the α-eleostearic acid at both the sn-1 and sn-2 positions of PC.36−38 EEPC (120 mg) 10579



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chain free fatty acids as well as for lysolecithin resulting from the PLA2 hydrolysis of lecithin at the argon−water interface.39−41 β-CD (seven glucopyranoside units) is able to form inclusion complexes with long-chain free fatty acids, lyso-PC, and cholesterol.40,42,43 The desorption rate of the waterinsoluble reaction products (α-eleostearic acid and lyso PC) probably involves the complexation of the single acyl chain into the β-CD cavity together with the desorption, into the aqueous phase, of either soluble free fatty acid/β-CD or lyso-PC/β-CD complexes. The UV absorbance of α-eleostearic acid was considerably enhanced due to its transition from the adsorbed state to the soluble state. Consequently, the ppPLA2 activity can be followed continuously by recording the variations of the UV absorption spectra with time. The UV absorption spectrum was recorded every 3 min for 30 min (Figure 2C). An increase in the UV absorption peaks was observed in the 250−300 nm range. It is worth noting that a 2 nm redshift of the UV absorption spectrum occurred in aqueous buffer, as compared with the UV absorption spectrum in ethanol where the major absorption peak was shifted from 270 to 272 nm. This solvatochromic effect observed in the EEPC spectrum has already been highlighted,44 and it could provide some information about the local polarity surrounding the conjugated triene carried by both acyl chains. Stability of Coated EEPC and Enzyme Kinetic Recording. Using EEPC coated on the microtiter plate (50 μg/well), the stability of the lipid film was tested by recording the variations in absorbance at 272 nm in the presence of buffer alone (without enzyme). As shown in Figure 3A, a constant baseline was recorded for at least 60 min, indicating that the coated EEPC was not perturbed by any interaction due to the buffer components (Figure 3A, curve 1). Following an injection of ppPLA2 (0.5 μg/well), an increase in the absorbance at 272 nm was observed showing a biphasic kinetic pattern: a slow accelerating phase for around 10 min (the lag time) followed by a second phase reaching a steady state (Figure 3A, curve 2). The lag time may reflect a rate-limiting step due to the slow penetration of the enzyme into the water/oil interface.45 In these heterogeneous conditions, the kinetic values were measured during the steady state phase. Similar kinetic behavior was observed using hpPLA2 and hbPLA2, with a lag time of around 10 and 6 min, respectively (data not shown). In the literature,45 the lag time for ppPLA2 is clearly described and shown to be due to the adsorption state of the enzyme. Nevertheless, for hbPLA2, no lag time has been described previously using a monolayer assay.46 The difference in the substrate organization (coated versus monolayer) and the enzymatic concentration (30-fold less in our experiment) in these assays may explain the differences in the resulting kinetics. The ability of these PLA2 to efficiently hydrolyze coated EEPC was further investigated by performing TLC on the lipolysis products. The qualitative decrease in the EEPC spots and the appearance of α-eleostearic acid and lyso-PC can be seen clearly in Figure 3B (lines 3, 4, and 5 for hpPLA2, ppPLA2, and hbPLA2, respectively). The hydrolysis of coated EEPC was also tested using tlPLA1, known as Lecitase. This commercially available enzyme is obtained from the fusion genes of lipase from Thermomyces lanuginosus and phospholipase from Fusarium oxysporum, and it has been shown to hydrolyze specifically the sn-1 position of PC.47 In contrast to ppPLA2, the hydrolysis rate of EEPC with tlPLA1 was very low and no lag time was apparent from the kinetics (data not shown).

Figure 3. Enzymatic hydrolysis of coated EEPC (50 μg) by PLA2s. (A). After EEPC coating, variation, with time, of the absorbance at 272 nm was recorded for 10 min for stabilization and then for 60 min after an enzyme injection with buffer only (curve 1) or with 0.5 μg of ppPLA2 (curve 2). (B) TLC analysis of lipolytic products (αeleostearic acid and lyso-PC) after extraction, revealed under UV light at 254 nm. Lane 1, purified α-eleostearic acid (10 μg); lane 2, purified EEPC (10 μg); lane 3, EEPC hydrolyzed by hpPLA2 (0.5 μg, 35 pmol); lane 4, EEPC hydrolyzed by ppPLA2 (0.5 μg, 33 pmol); lane 5, EEPC hydrolyzed by hbPLA2 (0.5 μg, 31 pmol). The chromatographic solvent system was chloroform/methanol/water 65:35:5 (v/v/v) containing 0.001% (w/v) BHT.

Influence of β-CD and the Initial Coated EEPC Concentrations on the Reaction Rate. The optimal concentration of β-CD in the reaction buffer was determined using 50 μg of coated EEPC and 1 μg of ppPLA2. Under these conditions, the ppPLA2 reaction rate increases with the β-CD concentration, reaching a maximal value at about 17 mA272 nm/ min with 0.6 mg/well of β-CD (3 g L−1 final concentration) and then slightly decreasing with higher concentrations of βCD (Figure 4). A final amount of 0.6 mg/well of β-CD in the reaction buffer was selected for further kinetic experiments. Various amounts of EEPC were coated onto the microtiter plate wells, ranging from 0 to 80 μg/well, and their lipolysis rates by ppPLA2 (0.5 μg, 143 pM) were then compared. Reaction rates were measured at a steady state (slope of the variations, with time, in the absorbance at 272 nm) and plotted as a function of the amounts of coated EEPC (Figure 5). The reaction rate increased quasi-linearly with the EEPC concentration, up to 50 μg/well. A slight increase in the reaction rate was observed with higher amounts of coated EEPC (Figure 5). On the basis of these results, a final amount of 50 μg of EEPC (64 nmol) per well was selected for further kinetic assays. Influence of the Amount of ppPLA2 on the SteadyState Reaction Rates. Using coated EEPC (50 μg/well), the increase, with time, of the absorbance at 272 nm was recorded after injecting variable amounts of ppPLA2. The steady-state reaction rates were plotted as a function of the amounts of ppPLA2 and were found to be linear with time and proportional to the amount of added enzyme (Figure 6). It is possible to 10580



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coating, performed under controlled conditions of nitrogen flow and temperature, may improve the reproducibility of the results. The detection sensitivity of this coated method is, however, around 50 times lower compared to that obtained with the fluorescent assays. Beisson et al.24 set up a lipase assay using parinaric acid-containing triacylglycerols, purified from Parinari glaberrimum seed oil, and quantities as low as 0.1 ng of pure pancreatic lipase could be detected under standard conditions. Unlike the fluorescence method, which is solely based on the micellar solubilization of the free fatty acid released, the UV spectrophotometric method is based on the coated phospholipid-containing α-eleostearic acid and it can be performed without detergent, which would be convenient when assaying phospholipases sensitive to detergents. Estimation of the Specific Activity Using Various PLAs. The apparent molar extinction coefficient (εapp) of the αeleostearic acid was determined using 50 μg of coated EEPC per well. Various amounts of pure α-eleostearic acid were injected and the OD at 272 nm was recorded. εapp of pure αeleostearic acid was found to be 5320 ± 160 M−1 cm−1. Under these conditions, the specific activities of tlPLA1, hpPLA2, ppPLA2, and hbPLA2 were calculated to be 0.06, 0.13, 0.94, and 5.84 U/mg, respectively. For the sake of comparison, the specific activity of ppPLA2 was found to be 937 U/mg, using the pH-stat technique and with egg yolk PC as the substrate.48 This difference was probably due to the vigorous stirring of the reaction mixture and the emulsification conditions in the pHstat vessel, which no doubt resulted in a more efficient dispersion of the substrate than that which occurred in the UV spectrophotometric microtiter plate. The heterogeneous character of the lipolytic reaction makes it difficult to accurately quantify both the interface (specific surface) and the interfacial parameters (such as the interfacial tension, surface viscosity, surface potential, etc.) responsible for the “interfacial quality” of the substrate8−11 on which the lipolytic process largely depends. Consequently, the specific activity depends, to a great extent, on the nature of the substrate, its organization at the interface, and the technique used for the assay. Microtiter Plate PLA Assay to Study PLA2 Inhibition. One of the main goals of this work is its application to the screening of potential inhibitors for different PLAs. MI, a known pancreatic PLA2 inhibitor, was used in this study in two different ways. First, coated EEPC (50 μg, 64 nmol) was hydrolyzed by ppPLA2 (0.5 μg, 143 pM) and injected alone or after its preincubation (1 h) with a 50 or 100 molar excess of the inhibitor. The steady state residual ppPLA2 activity reached 31% and 21% with an inhibitor to a ppPLA2 molar excess of 50 and 100, respectively (data not shown). Second, ppPLA2 inhibition was tested during lipolysis by successively injecting the DMSO alone (Figure 7, curve 1) or the inhibitor (14 nM in DMSO) (Figure 7, curve 2), at 30 min and at 50 min. A slight increase in absorbance was observed immediately after the injection due to the DMSO used to solubilize the inhibitor. The steady state residual ppPLA2 activity reached 58% and 18%, measured at 40−50 min and 60−70 min, respectively (Figure 7).

Figure 4. Effect of β-CD concentration on the steady state reaction rate. Results are given as means ± SD for three independent experiments performed with ppPLA2 (1 μg per well).

Figure 5. Effect of different amounts of coated EEPC on the steady state reaction rate, calculated from the increase in absorbance at 272 nm. Results are given as means ± SD for three independent experiments performed with ppPLA2 (0.5 μg per well).

Figure 6. Effect of varying amounts of enzyme on the steady state reaction rate. Variable amounts of ppPLA2 were injected into microtiter plate wells containing coated EEPC (50 μg/well) in standard buffer. The kinetics of the enzyme rate were measured by the increase of absorbance at 272 nm for 30−70 min after enzyme injection. Results are given as means ± SD for three independent experiments.

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estimate that 5 ng is the lowest quantity of ppPLA2 detectable using coated EEPC on a microtiter plate, corresponding to 3 times the background observed with buffer alone. A slight difference was seen in the reaction rate measured during these different assays, in the same experimental conditions (Figures 4−6), partly due to the manual coating and to solvent evaporation under the fume hood. A spotted

CONCLUSIONS We describe here a new phospholipid esterified with a naturally conjugated polyene fatty acid which does not contain any chemical substituent likely to create a steric hindrance in measuring PLA activity. Moreover, this phospholipid, contain10581



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Figure 7. Validation of the PLA assay to study ppPLA2 inhibition. EEPC (50 μg/well) was coated on microtiter plate wells, as described in the Experimental Section. After ppPLA2 (0.5 μg) injection, DMSO (curve 1) or MI in DMSO (14 nM, final concentration, curve 2) was injected into the reaction medium during lipolysis, successively, at 30 and 50 min. The steady state residual activity was measured at 40−50 min and at 60−70 min. Curves are representative of three independent experiments.

ing α-eleostearic acid, shows excellent stability and can be stored for at least 2 years. The assay is continuous, sensitive, and easy to perform and could be used for high-throughput screening of new PLA and/or PLA inhibitors present in various biological samples. We have established that as little as 5 ng of pure ppPLA2 and 50 ng of pure tlPLA1 can be detected using this UV assay in microtiter plates.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +33 4 72 44 81 02. E-mail: [email protected] Author Contributions

The manuscript was written from contributions by all the authors. All the authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Prof. Alain Doutheau for his support and constructive discussions. We gratefully acknowledge support from the “Région Rhône-Alpes” Programme Grant CIBLE-2012-1201080801. Meddy El Alaoui was supported by a Ph.D grant from the “Région Rhône-Alpes” (€32,116/year). English revision by Valerie James is also acknowledged.

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