Hydrolysis of monomolecular films of long chain phosphatidylcholine by phospholipase A(2) in the presence of beta-cyclodextrin

The desorption rates of monomolecular films of oleic acid (OA) and lysophosphatidylcholine (lyse PC) at the air/water interface by water soluble beta-cyclodextrin (beta-CD) were studied. The desorption of OA and lyse PC involves the complexation of the single acyl chain into the beta-CD cavity and the solubilization of the beta-CD/OA or beta-CD/lyso PC complex into the aqueous subphase, associated with a rapid decrease in the surface pressure. In the case of monomolecular films of egg phosphatidylcholine (egg PC), 1,2-dihexadecanoyl-sn-glycero-3-phosphatidylcholine (DPPC); 1,2-di(cis-9-octadecenoyl)-sn-glycero-3-phosphatidycholine (DOPC), no detectable changes in the surface pressure occurred after beta-CD injection into the subphase. The hydrolysis of medium and long chain PC monomolecular films by phospholipase A(2) (PLA(2)) was investigated in the absence of beta-CD in the subphase. The considerable difference between the apparent kinetic constants was attributed to physical steps possibly involving molecular reorganization in the interface of long chain lipolytic products, associated with enzyme product inhibition. In the presence of beta-CD in the subphase, the enzymatic hydrolysis of monomolecular films of long chain PC was found to have kinetic constants which were comparable to those measured with medium chain lipids. Furthermore, comparisons between the desorption rates of the beta-CD/lipolytic product complexes and the enzymatic hydrolysis rates of long chain PC, in the presence of beta-CD in the aqueous subphase, showed that the rate limiting step is neither the formation of the beta-CD/lipolytic product complexes nor their desorption into the water subphase but the hydrolysis of the PC monomolecular films by PLA(2). The presence of beta-CD in the water subphase made it possible for the first time to perform kinetic measurements on the rates of hydrolysis of monomolecular films of long chain PC by PLA(2).