Hydrolysis of milk phospholipid and phospholipid–protein monolayers by pancreatic phospholipase A2

Abstract

Recently, phospholipids have been used for the liposomal encapsulation of drugs and bioactive compounds. Pancreatic phospholipase A2 is responsible for the hydrolysis of phospholipids in the intestines. Using fluorescence microscopy, Langmuir monolayers at the air–water interface and atomic force microscopy, the aim of this work was to obtain preliminary data on the effect of the liquid-ordered domains and proteins on the hydrolysis of phospholipids by pancreatic phospholipase A2. Increasing the enzyme:substrate ratio (1:40 to 1:2) and the temperature (30°C to 37°C) increased phospholipid hydrolysis as seen by a decrease in surface pressure and change in the surface area corresponding to the formation and desorption of hydrolytic products. Hydrolysis occurred rapidly in the phase coexistence region and led to the clustering of the liquid-ordered domains likely through reduction of electrostatic forces. Atomic force microscopic images of the monolayers under simulated intestinal conditions revealed the presence of packing defects in the liquid-ordered domains where phospholipase A2 could adsorb. Hydrolysis took place starting in the center of the liquid-ordered domains and proceeded towards their edges. It did not lead to any measurable height changes by atomic force microscopy within the domains but it was evident that mechanical changes took place during hydrolysis. The presence of β-casein appeared to increase the rate of hydrolysis, possibly through interaction with phospholipase A2 at the interface.