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

Polarization-modulated infrared reflection absorption spectroscopy (PM-IRRA S) was used to follow the hydrolysis of phospholipid monolayers at the air- water interface by phospholipase A2 (PLA2). The decrease in the intensity o f the vC=O ester band of dipalmitoylphosphatidylcholine at 1733 cm(-1) and the appearance of two new infrared bands in the 1530-1580 cm(-1) region all owed to monitor phospholipid hydrolysis by PLA2. Indeed, the decrease in th e intensity of the band at 1733 cm(-1) was attributed to the enzymatic hydr olysis of the acyl ester linkage of the sn-2 fatty acid on the glycerol bac kbone whereas the doublet appearing at 1537 and 1575 cm(-1) was attributed to the v(a) COO- vibration of the newly formed calcium-palmitate. The prese nce of this band as a doublet indicates the formation of a crystalline-like calcium-palmitate monolayer. This observation supports our previously post ulated mechanism for the formation of PLA2 domains at the air-water interfa ce. Definitive assignment of the infrared bands has been possible by measur ing PM-IRRAS spectra of the individual hydrolysis products (palmitic acid a nd lysopalmitoylphosphatidylcholine) as well as of l-caproyl-2-palmitoyl-ph osphatidylcholin and 1-palmitoyl-2-caproylphosphatidylcholine monolayers be fore and after hydrolysis by PLA2. (C) 2000 Elsevier Science B.V. All right s reserved.