A CONTINUOUS FLUOROMETRIC ASSAY FOR PHOSPHOLIPASES USING POLYMERIZED MIXED LIPOSOMES

A versatile continuous fluorometric assay for phospholipases A(2) C, a nd D has been developed utilizing polymerized mixed liposomes made of pyrene-containing phospholipids (5 mol%) uniformly inserted in the pol ymerized liposomes of 1,2-bis[12-(lipoyloxy) dodecanoyl]-sn-glycero-3- phosphoglycerol (BLPG) and its derivatives. l-2-(1-pyrenedecanoyl)-sn- glycero-3-phosphocholine was used for phospholipase A(2) and N-(1-pyre nesulfonyl)-egg phosphatidyl ethanolamine for phospholipases C and D. Fluorescence emission of pyrene moieties in polymerized mixed liposome s was strongly quenched by BLPG molecules and, thus, the hydrolysis of pyrene-containing phospholipids and the subsequent displacement of py rene moieties from the liposomes resulted in a large increase in fluor escence intensity. All the phospholipases tested selectively and rapid ly hydrolyzed the inserted pyrene-containing phospholipids, which were readily monitored by measuring an increase in fluorescence emission a t 380 nm. Assay conditions for individual phospholipases were optimize d by altering interfacial properties of polymerized liposomes, such as surface charge, and subsequently by changing the chemical structure o f hydrolyzable phospholipids. Phospholipase activities were linearly p roportional to enzyme concentrations in the range from 0.1 to 50 ng. S pecific activity determined for phospholipases from a wide variety of sources ranged from 0.5 to 100 mu mol/min/mg. Polymerized mixed liposo mes are exceptionally stable against chemical and physical degradation and the assay requires only a small amount of pyrene-containing phosp holipids. In addition, the polymerized matrix of BLPG (and its derivat ives), due to its inertness to the phospholipase hydrolysis, allows th e direct measurement of the equilibrium dissociation constant for a pr otein-liposome complex. (C) 1994 Academic Press, Inc.