Bhat M K, Mueller-Harvey I, Sumner I G, Goodenough P W
AFRC Institute of Food Research, Department of Protein Engineering, Reading Laboratory, UK.
Biochim Biophys Acta. 1993 Feb 24;1166(2-3):244-50. doi: 10.1016/0005-2760(93)90104-h.
A simple and straight forward method was developed for the synthesis of 2-hexadecanoylthio-1-ethyl phosphorylcholine (HEPC). The new procedure, which used p-toulenesulfonate instead of 2-bromoethyl phosphorylcholine, not only reduced the reaction time but also allowed the reaction to proceed under mild conditions. Using HEPC as a substrate, we have also developed a microplate assay for measuring phospholipase A2 activity which is rapid and will be useful for analyzing a large number of samples in a very short time. The applicability of this assay method for assessing phospholipases A2 from two different sources and determining their kinetic constants is also demonstrated. This method can also be extended for measuring lipases and lysophospholipases using a suitable thioester. Thus, both synthesis and assay methods will be useful in basic and applied research on phospholipases and related enzymes.
开发了一种简单直接的方法来合成2-十六烷酰硫基-1-乙基磷酰胆碱（HEPC）。新方法使用对甲苯磺酸盐代替2-溴乙基磷酰胆碱，不仅缩短了反应时间，还使反应能在温和条件下进行。以HEPC为底物，我们还开发了一种用于测量磷脂酶A2活性的微孔板检测方法，该方法快速，对于在极短时间内分析大量样品很有用。还证明了该检测方法在评估来自两种不同来源的磷脂酶A2及其动力学常数方面的适用性。该方法还可以扩展用于使用合适的硫酯测量脂肪酶和溶血磷脂酶。因此，合成和检测方法在磷脂酶及相关酶的基础研究和应用研究中都将很有用。
