Quantitation of lysolipids, fatty acids, and phospholipase A 2 activity and correlation with membrane polarity

摘要

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.