Enzymic hydrolysis of various lecithin monolayers employing surface pressure and potential technique

Abstract

Enzymic hydrolysis of dipalmitoyl, egg, soybean, and dioleoyl lecithin monolayers was studied by measuring the changes in surface pressure (π) and surface potential (Δ V) upon injection of snake venom ( Naja Naja) into the subsolution. The presence of Ca ++, which could not be substituted by Mg ++, Sr ++, or Ba ++, was essential for hydrolysis. Thin layer chromatography of monolayers removed after injection of venom into the subsolution showed that the snake venom indeed hydrolyzed lecithin monolayers into lysolecithin and free fatty acid. The rates of hydrolysis of different lecithins were in the order: dioleoyl lecithin > soybean lecithin > egg lecithin > dipalmitoyl lecithin, which is also the order of their molecular areas and hence their intermolecular spacings in monolayers. The solubility of lysolecithin molecules in the subsolution depends upon the chain length and unsaturation of the fatty acyl chains, which affects the changes in Δ V and π upon hydrolysis. The presence of citrate buffer or chelating agents with excess of Ca ++ in the subsolution increased the rate of hydrolysis by decreasing the binding of Ca ++ to lecithin monolayers. The hydrolysis of egg lecithin monolayers on different buffers was in the order, tris > veronal > collidine; which is the reverse of that found in the bulk reaction. Curves of π -area and Δ V - area of egg lecithin on these buffers indicated penetration of veronal and collidine ions into monolayers; this prevents the formation of the enzyme-substrate complex at the surface. In bulk reactions, these buffer ions act as lipid dispersants increasing the lipid/water interfacial area and consequently increase the rate of hydrolysis. Anionic, cationic, and neutral spacer molecules (15 mole %) were introduced in egg lecithin monolayers. The rate of hydrolysis increased in the presence of eicosanyl trimethylammonium, decreased in the presence of dicetyl phosphate or dipalmitin, and remained unchanged in the presence of cholesterol, indicating the importance of surface charge and state of the monolayer.