Quantitation of lysolipids, fatty acids, and phospholipase A2 activity and correlation with membrane polarity

Jasmeet Singh,Radha Ranganathan

doi:10.1194/jlr.d028746

Abstract

Acrylodan-labeled rat-intestinal fatty acid binding protein, ADIFAB, binds both of lysophosphatidylcholines (LPC) and FA. Binding displaces Acrylodan and its fluorescence peak shifts from 432 to 505 nm. A fluorescence assay that relies on this shift is presented for quantitating LPC, FA, and phospholipase A(2) (PLA(2)) activity in phospholipid bilayers in absolute units of μM/min/mg of enzyme. This is a development over an earlier assay that took into account only FA binding. Activities of bee venom PLA(2) on dipalmitoylphosphatidylcholine (DPPC) and dioleylphosphatidylcholine (DOPC) bilayers were measured. Standard pH-Stat assays validated the present assay. Products increase linearly with time for about one minute in DOPC and five minutes in DPPC corresponding to completion of 5 to 8% hydrolysis in DOPC and 20% in DPPC. Membrane polarity and microviscosity measured using electron spin resonance (ESR) exhibited discontinuities at compositions that mimicked similar percentages of hydrolysis products in the respective bilayers. The observed hydrolysis rate decrease following the initial linear period thus correlates to changes in membrane polarity. The ability of the assay to yield actual product concentrations, reveal structure in the reaction progress curves, and interpretation in light of the ESR data bring insight into the shape of the reaction curve.

Highlights

Acrylodan-labeled rat-intestinal fatty acid binding protein, ADIFAB, binds both of lysophosphatidylcholines (LPC) and FA
The phospholipids investigated in this work were DOPC and DPPC and the products resulting from their hydrolysis lysopalmitoylphosphatdylcholine (LPPC), palmitic acid (PA), oleic acid (OA), and lysooleylphosphatdylcholine (LOPC)
The binding of lysolipids to ADIFAB results in weaker generalized polarization (GP) response than that of FA but it is not insignificant in general. It is truer for the products, LOPC and OA, of mM)+OA(2.5 mM)+DOPC(22.5 mM) multilamellar vesicle (MLV) bilayers at 37°C; (C) LPPC (2.5 mM)+PA(2.5 mM)+DPPC(22.5

Summary

Introduction

Acrylodan-labeled rat-intestinal fatty acid binding protein, ADIFAB, binds both of lysophosphatidylcholines (LPC) and FA. A fluorescence assay that relies on this shift is presented for quantitating LPC, FA, and phospholipase A2 (PLA2) activity in phospholipid bilayers in absolute units of ␮M/min/mg of enzyme. This is a development over an earlier assay that took into account only FA binding. The goals of this work were to establish the quantitative and linear nature of ADIFAB fluorescence response to FA and LPC, develop quantitative assays for detecting both FA and LPC in aqueous solutions as well as for continuous real time monitoring of PLA2 hydrolysis at membrane interfaces expressing activity in absolute units of ␮M of product/minute/mg of enzyme, and extend the applicability of the ADIFAB PLA2 assay to more lipids and lipid bilayers. The ADIFAB fluorescence assay, first reported by Richieri and Kleinfeld, relies on the shift in fluorescence

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