IR spectroscopy analysis of pancreatic lipase-related protein 2 interaction with phospholipids: 3. Monitoring DPPC lipolysis in mixed micelles.

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 D2 O buffer at pD 6 and 8. After hydrogen/deuterium exchange, DPPC hydrolysis by GPLRP2 (100nM) 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).