A continuous spectrophotometric assay that distinguishes between phospholipase A1 and A2 activities

Abstract

A new spectrophotometric assay was developed to measure, continuously and specifically, phospholipase A1 (PLA1) or phospholipase A2 (PLA2) activities using synthetic glycerophosphatidylcholines (PCs) containing α-eleostearic acid, either at the sn-1 position [1-α-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine (EOPC)] or at the sn-2 position [1-octadecyl-2-α-eleostearoyl-rac-glycero-3-phosphocholine (OEPC)]. The substrates were coated onto the wells of microtiter plates. A nonhydrolyzable ether bond, with a non-UV-absorbing alkyl chain, was introduced at the other sn position to prevent acyl chain migration during lipolysis. Upon enzyme action, α-eleostearic acid is liberated and then solubilized into the micellar phase. The PLA1 or PLA2 activity was measured by the increase in absorbance at 272 nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. EOPC and OEPC differentiate, with excellent accuracy, between PLA1 and PLA2 activity. Lecitase(®), guinea pig pancreatic lipase-related protein 2 (known to be a PLA1 enzyme), bee venom PLA2, and porcine pancreatic PLA2 were all used to validate the assay. Compared with current assays used for continuously measuring PLA1 or PLA2 activities and/or their inhibitors, the development of this sensitive enzymatic method, using coated PC substrate analogs to natural lipids and based on the UV spectroscopic properties of α-eleostearic acid, is a significant improvement.

Keywords: 1-octadecyl-2-α-eleostearoyl-rac-glycero-3-phosphocholine; 1-α-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine; tung oil; α-eleostearic acid; β-cyclodextrin.

Fig. 1.

Chemical structure and UV absorption spectra of EOPC and OEPC. A: Chemical structure of EOPC and OEPC. B: UV absorption spectra of α-eleostearic acid (62.5 μg ml⁻¹), EOPC (125 μg ml⁻¹) and OEPC (125 μg ml⁻¹) dissolved in ethanol containing BHT 0.001%.

Fig. 2.

Principle of the coated PC assay and time course of the enzymatic hydrolysis of OEPC or EOPC coated on microtiter plate wells. A: Schematic representation of the assay reaction showing the hydrolysis of coated EOPC or OEPC in a microwell. E, enzyme in solution; E*, enzyme at the interface; S, substrate (EOPC or OEPC); P, lipolysis products (α-eleostearic acid and non-UV-absorbing lyso-PC). β-CD (3 g·l⁻¹, final concentration) was used to solubilize the long-chain lipolysis products into the aqueous buffer. B, C: Kinetic recordings of coated EOPC (B) and OEPC (A) lipolysis by GPL-RP₂ or ppPLA₂. Variations, with time, of the absorbance at 272 nm were recorded for 10 min, for stabilization, and then for 50 min after GPL-RP₂ or ppPLA₂ (0.5 μg per well). The kinetic recordings shown here are typical of those obtained in three independent experiments.

Fig. 3.

Analysis by TLC of coated EOPC (A) and OEPC (B) hydrolysis revealed with UV light at 254 nm (left panels) and with 10% copper sulfate and 10% phosphoric acid in water followed by charring at 150°C for 15 min (right-hand panels). The standards (Std) used (10 μg of each compound) were α-eleostearic acid, EOPC, and OEPC. Coated PCs were hydrolyzed by GPL-RP₂ or ppPLA₂ (0.5 μg/well). The chromatographic solvent system was chloroform/methanol/water (65/35/4, v/v/v) and hexane/diethylether/acetic acid (84/16/1, v/v/v) containing 0.001% (w/v) BHT.

Fig. 4.

Effects of the initial PC concentration (A, B) and the amount of enzyme (C, D) on the steady-state reaction rates. Variable amounts of coated EOPC (A) or OEPC (B) were subjected to hydrolysis by GPL-RP₂ [0.5 μg (A)] or ppPLA₂ [0.5 μg (B)]. Variable amounts of GPL-RP₂ (C) or ppPLA₂ (D) were injected into the microplate well containing the coated EOPC (C) or OEPC (D) in 200 μl of standard buffer. The increase in the absorbance at 272 nm was recorded for 20–40 min after the enzyme injection, and the initial velocity (ΔmA_272nm·min⁻¹) was used for reaction rate determination. Results are given as mean ± SD for three independent experiments.

Fig. 5.

Validation of the assay to study PLA₁ or PLA₂ inhibition. Either GPL-RP₂ or ppPLA₂ was incubated with THL (enzyme:inhibitor, molar ratio of 1:70) or with MI (enzyme:inhibitor, molar ratio of 1:100) in the absence of the substrate. The residual activities of GPL-RP₂ or ppPLA₂ were then measured using coated EOPC or OEPC, respectively, as described in the Materials and Methods, and expressed as a percentage relative to the activity measured in the absence of inhibitor. Values ± SD are the mean of three independent experiments. **P < 0.01 (vs. DMSO).