Sk. Wu et Ww. Cho, A CONTINUOUS FLUOROMETRIC ASSAY FOR PHOSPHOLIPASES USING POLYMERIZED MIXED LIPOSOMES, Analytical biochemistry, 221(1), 1994, pp. 152-159
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.