Monitoring of phospholipid monolayer hydrolysis by phospholipase A2 by use of polarization-modulated Fourier transform infrared spectroscopy

Abstract

Polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) was used to follow the hydrolysis of phospholipid monolayers at the air–water interface by phospholipase A2 (PLA2). The decrease in the intensity of the υCO ester band of dipalmitoylphosphatidylcholine at 1733 cm −1 and the appearance of two new infrared bands in the 1530-1580 cm −1 region allowed to monitor phospholipid hydrolysis by PLA2. Indeed, the decrease in the intensity of the band at 1733 cm −1 was attributed to the enzymatic hydrolysis of the acyl ester linkage of the sn-2 fatty acid on the glycerol backbone whereas the doublet appearing at 1537 and 1575 cm −1 was attributed to the υ a COO − vibration of the newly formed calcium-palmitate. The presence of this band as a doublet indicates the formation of a crystalline-like calcium-palmitate monolayer. This observation supports our previously postulated mechanism for the formation of PLA2 domains at the air–water interface. Definitive assignment of the infrared bands has been possible by measuring PM-IRRAS spectra of the individual hydrolysis products (palmitic acid and lysopalmitoylphosphatidylcholine) as well as of 1-caproyl-2-palmitoyl-phosphatidylcholine and 1-palmitoyl-2-caproyl-phosphatidylcholine monolayers before and after hydrolysis by PLA2.