Department of Physics, California State University Northridge, Northridge, CA 91330-8268, USA.
J Lipid Res. 2012 Sep;53(9):1993-2001. doi: 10.1194/jlr.D028746. Epub 2012 Jul 5.
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.
丙烯酰胺标记的大鼠肠脂肪酸结合蛋白（ADIFAB）结合了溶血磷脂酰胆碱（LPC）和脂肪酸（FA）。结合会导致丙烯酰胺的置换，其荧光峰从 432nm 位移至 505nm。基于这种位移的荧光测定法用于定量磷脂双层中的 LPC、FA 和磷脂酶 A2（PLA2）活性，单位为μM/min/mg 酶。这是对早期仅考虑 FA 结合的测定法的发展。蜂毒 PLA2 在二棕榈酰磷脂酰胆碱（DPPC）和二油酰基磷脂酰胆碱（DOPC）双层上的活性进行了测量。标准 pH-Stat 测定法验证了本测定法。在 DOPC 中，产物在线性增加约一分钟，在 DPPC 中增加约五分钟，与 DOPC 中 5%至 8%的水解和 DPPC 中 20%的水解完成相对应。使用电子自旋共振（ESR）测量的膜极性和微粘度在模拟相应双层中类似百分比水解产物的组成处表现出不连续性。因此，初始线性期后观察到的水解速率下降与膜极性的变化相关。该测定法能够产生实际的产物浓度、揭示反应进度曲线中的结构，以及根据 ESR 数据进行解释，为反应曲线的形状提供了深入的了解。
