PHOSPHOLIPASE A(2) DOMAIN FORMATION IN HYDROLYZED ASYMMETRIC PHOSPHOLIPID MONOLAYERS AT THE AIR/WATER INTERFACE

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, 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, 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.