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IR spectroscopy analysis of pancreatic lipase-related protein 2 interaction with phospholipids: 3. Monitoring DPPC lipolysis in mixed micelles
Chemistry and Physics of Lipids ( IF 3.4 ) Pub Date : 2017-11-11 , DOI: 10.1016/j.chemphyslip.2017.11.009
Eduardo Mateos-Diaz , Priscila Sutto-Ortiz , Moulay Sahaka , Jorge A. Rodriguez , Frédéric Carrière

Usual methods for the continuous assay of lipolytic enzyme activities are mainly based on the titration of free fatty acids, surface pressure monitoring or spectrophotometry using substrates labeled with specific probes. These approaches only give a partial information on the chemistry of the lipolysis reaction and additional end-point analyses are often required to quantify both residual substrate and lipolysis products. We used transmission infrared (IR) spectroscopy to monitor simultaneously the hydrolysis of phospholipids by guinea pig pancreatic lipase-related protein 2 (GPLRP2) and the release of lipolysis products. The substrate (DPPC, 1,2-Dipalmitoyl phosphatidylcholine) was mixed with sodium taurodeoxycholate (NaTDC) to form mixed micelles in D2O buffer at pD 6 and 8. After hydrogen/deuterium exchange, DPPC hydrolysis by GPLRP2 (100 nM) was monitored at 35 °C in a liquid cell by recording IR spectra and time-course variations in the CO stretching region. These changes were correlated to variations in the concentrations of DPPC, lysophospholipids (lysoPC) and palmitic acid (Pam) using calibration curves established with these compounds individually mixed with NaTDC. We were thus able to quantify each compound and its time-course variations during the phospholipolysis reaction and to estimate the enzyme activity. To validate the IR analysis, variations in residual DPPC, lysoPC and Pam were also quantified by thin-layer chromatography coupled to densitometry and similar hydrolysis profiles were obtained using both methods. IR spectroscopy can therefore be used to monitor the enzymatic hydrolysis of phospholipids and obtain simultaneously chemical and physicochemical information on substrate and all reaction products (H-bonding, hydration, acyl chain mobility).



中文翻译:

胰脂肪酶相关蛋白2与磷脂相互作用的红外光谱分析:3.监测混合胶束中的DPPC脂解

连续测定脂肪分解酶活性的常用方法主要基于游离脂肪酸的滴定,表面压力监测或使用标记有特定探针的底物进行分光光度法。这些方法仅提供有关脂解反应化学性质的部分信息,通常需要进行额外的终点分析以定量残留底物和脂解产物。我们使用透射红外(IR)光谱法来同时监测豚鼠胰脂肪酶相关蛋白2(GPLRP2)水解的磷脂和脂解产物的释放。将底物(DPPC,1,2-二棕榈酰磷脂酰胆碱)与牛磺脱氧胆酸钠(NaTDC)混合,在D 2中形成混合胶束O缓冲液位于pD 6和8。在氢/氘交换后,通过记录IR光谱和CO拉伸区域中的时程变化,在35°C的液池中监测了GPLRP2(100 nM)对DPPC的水解作用。使用将这些化合物分别与NaTDC混合建立的校准曲线,将这些变化与DPPC,溶血磷脂(lysoPC)和棕榈酸(Pam)浓度的变化相关。因此,我们能够量化磷脂脂解反应期间的每种化合物及其时程变化,并估计酶的活性。为了验证IR分析,还通过与光密度法耦合的薄层色谱法对残留DPPC,lysoPC和Pam中的变化进行了定量,并且使用这两种方法均获得了相似的水解曲线。

更新日期:2017-11-11
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