Phospholipase A 2 domain formation in hydrolyzed asymmetric phospholipid monolayers at the air/water interface

Abstract

Phospholipase A 2 (PLA 2) catalyzed hydrolysis of asymmetric 1-caproyl-2-palmitoyl-phosphatidylcholine (6,16-PC) and 1-palmitoyl-2-caproyl-phosphatidylcholine (16,6-PC) lipid monolayers at the air/water interface was investigated. Surface pressure isotherms, surface potential and fluorescence microscopy at the air/water interface were used to characterize the asymmetric monolayer systems. Cobra ( N. naja naja) and bee venom PLA 2 exhibit hydrolytic activity towards 6,16-PC and 16,6-PC monolayers at all surface pressures up to monolayer collapse (37 mN m −1). Pancreatic PLA 2 hydrolytic activity, however, was observed to be blocked at a lateral surface pressure of approx. 18 mN m −1 for both 6,16-PC and 16,6-PC monolayers. For 6,16-PC monolayers, fluorescence microscopy revealed that monolayer hydrolysis by PLA 2 from cobra, bee, and bovine pancreatic sources all produced monolayer microstructuring. Fluorescence microscopy also showed that PLA 2 is bound to these monolayer microstructures. Very little PLA 2-induced microstructuring was observed to occur in 16,6-PC monolayer systems where caproic acid (C6) hydrolysis products were readily solubilized in the aqueous monolayer subphase. Surface potential measurements for 16,6-PC monolayer hydrolysis indicate dissolution of caproic acid reaction products into the monolayer subphase. Monolayer molecular area as a function of 6,16-PC monolayer hydrolysis time indicates the presence of monolayer-resident palmitic acid reaction products. With bovine serum albumin present in the monolayer subphase, PLA 2 domain formation was observed only in hydrolyzed 6,16-PC monolayers. These results are consistent with laterally phase separated monolayer regions containing phospholipid and insoluble fatty acid reaction products from PLA 2 monolayer hydrolysis electrostatically driving PLA 2 adsorption to and enzyme domain formation at the heterogeneous, hydrolyzed lipid monolayer interface.