lipase and phospholipase protocols

1
-
Phospholipase A2 and Phosphatidylinositol-
Specific Phospholipase C Assays by HPLC
and TLC with Fluorescent Substrate
H. Stewart Hendrickson
1. Introduction
Llpolytic enzymes have traditlonally been assayed by radlometrlc and t&-l-
metric methods (I). RadIometrIc methods are quite sensitive but require ex-
pensive radlolabeled substrates and tedious separation of labeled substrate and
products. In addition, the safe use of radioactive materials 1s of mcreasmg con-
cern. Tltrlmetrtc assays are contmuous and quite straightforward and use natu-
ral substrates but suffer from low sensltlvlty and are subject to condltlons that
may alter the amount of free hydrogen Ions released Fluorescence-based assays
have sensltlvltles that approach those of radtometrlc methods; although they
require synthetic fluorescent-labeled substrates, they are often more conve-
nient and rapid. For a recent review, see Hendrickson (2).
We first used dansyl-labeled glycerol ether analogs of phosphatidylcholme
as substrates for the assay of enzymes of the platelet-actlvatmg factor (PAF)
cycle m peritoneal polymorphonuclear leukocytes (3). This became a general
method for the assay of phosphollpase A2 (PLA,) (see Fig. 1; refs. 4 and 5).
This method can be modified to assay other enzymes of the PAF cycle such as
lyso-PAF acyltransferase, lyso-PAF acetyltransferase, and PAF acetylhydro-
lase (3) Smce the probe remains attached to the glycerol backbone, simulta-
neous assay of all of these enzymes IS possible.
The method described here for the assay of PLA2 uses thin-layer chroma-
tography (TLC) to separate products from substrate, and quantltatlon by fluo-
rescent scanning. The use of high-pressure hquld chromatography (HPLC) with
fluorescence detection IS included as an alternative to TLC. The assay IS spe-
From Methods m Molecular &o/ogy, Vol 109 Lipase and Phospholipase Protocols
Edlted by M H Doohttle and K Reue 0 Humana Press Inc , Totowa, NJ

I
Hendrickson
H20POjCH2CH2N(CH3)3
+
S02NHH
0 CH2
1 HO -H
+
H2OP03CH$IH2N(CH3)3
+
Fig
1
Reactron scheme for the assay of PLA, with dansyl-PC.
cific for PLA2 since the substrate, with an ether lmkage at the sn-1 position of
glycerol, ts not hydrolyzed by PLA,. Hydrolysis of the substrate by phosphoh-
pases C or D present m crude enzyme preparattons will be apparent as addi-
tional products that will be seen by TLC or HPLC
Phosphattdylmosttol-specific phospholipase C (PI-PLC) from
Bacdlus
cerexs catalyzes the hydrolysis of PI to a diglycertde and 1 o-myo-inositol- 1,2-
(cychc)phosphate (6) The latter is subsequently slowly hydrolyzed by the same
enzyme to 1 o-myo-mosttol- l-phosphate. This enzyme also catalyzes the release
of a number of enzymes linked to glycosylphosphatidylmosttol membrane an-
chors (7)
Several years ago we synthestzed 4-(l-pyreno)butylphosphoryl-l-myo-
mosttol (pyrene-PI) as a substrate for the assay of PI-PLC from B. cereu~ (see
Fig. 2; refs. 8 and 9) The method described here uses reverse-phase HPLC
wtth fluorescence detectton to separate and quantitate the product released.
The methods described here are well suited to the assay of crude enzyme
preparattons since the presence of other phospholtpase acttvmes will be appar-

ent. The methods are independent of the specific condtttons for the enzyme
reaction, so a variety of detergents can be used. These assays can also be auto-
mated by the use of an autosampler for HPLC and larger plates wtth multiple
Fluorescence-Based Phospholipase Assays
,
PI-PLC
I
H,CH2CH&H20H
H&
po3=
Fig 2 Reactlon scheme for the assay of PI-PLC wrth pyrene-PI
lanes for TLC; they can thus be used to screen many enzyme samples and
potential mhibltors. The TLC-based assays can done m a qualitative manner
by simply vlsuahzing the plates under an ultraviolet (UV) lamp
2. Materials
2.1. PL A2 Assay
1 PLA, buffer. 0.395 M NaCl, 66 nuW Tns, 13.2 mi!4 CaCl,, pH 7 0 (adjust with
HCl)
2 PLA, substrate dansyl-PC, 1 n-J4 In CHCl, Dissolve 1 mg of dansyl-PC (cat
no D-3765, Molecular Probes, Inc , Eugene, OR) m 1 23 mL of chloroform (see
Note 1) Store m a brown bottle at -20°C (see Note 2)
3 Trlton X-100 solutlon, 10 mM Triton X-100 (cat no. T9284, Sigma, St. LOUIS,
MO) m water
4 PLA, stock assay solution. place 100 pL of PLA, substrate (1 mMdansyl-PC) m
a small test tube (10 x 75 mm) and dry under a stream of mtrogen and then under
high vacuum for 10-15 mm to remove all traces of solvent Add 20 pL of Trlton
X-100 solution (see Note 3) and 380 pL of PLAz buffer Vortex and somcate
(bath-type somcator) repeatedly until the lipid 1s completely dissolved and the
solution is optlcally clear.
4 TLC solvent* CHC13/CH30H/conc ammoma/water (90.54 5.5:2 [v/v])

5 TLC plates. 10 x 10 cm, HPTLC plates, (cat. no. 60077, Analtech, Newark, DE).
6. Fluorescence scanner: densitometer (model CS9000, Shlmadzu) with fluorescence
accessory, or other sultable Instrument for fluorescence scanning of TLC plates.
7 Quenching solvent* hexane/lsopropanol/acetlc acid (6:8: 1.6)
8. PLA, HPLC solvent hexane/lsopropanol/water (6 8.1.6)
4
Hendrickson
2.2. PI-PLC Assay
1. PI-PLC buffer. 50 mM2-(N-morpholmo)ethane sulfomc acid (MES, Sigma), pH
7 0 (adjust with NaOH)
2. PI-PLC substrate 1 tipyrene-PI m CHCls/CH,OH (2.1). Dissolve 0 5 mg of
racemic 4-( 1 -pyreno)butylphosphoryl- 1 -myo-mositol (cat no P-3764, Molecu-
lar Probes) m 1 25 mL of solvent Store m a brown bottle at -20°C (see Note 2)
3. PI-PLC stock assay solution place 250 pL of PI-PLC substrate (1 mM pyrene-
PI) m a small test tube (10 x 75 mm) and dry under a stream of nitrogen and then
under high vacuum for 10-15 min to remove all traces of solvent Add 200 yL of
PI-PLC buffer. Vortex and sonicate (bath-type somcator) repeatedly until the
lipid is completely dissolved and the solution is clear
4. PI-PLC HPLC solvent 5 mM tetrabutylammonmm dihydrogenphosphate (cat
no 26,8 10-0, Aldrich, Milwaukee, WI) in acetomtrile/methanol/water (70 10.20)
2.3. HPLC Analysis
1 HPLC column for PLA, assay 15 cm x 4.6 mm, 5 pm spherical silica gel (cat
no 85774, Waters, Milford, MAtprotect with a guard column.
2. HPLC column for PI-PLC assay’ 25 cm x 4.6 mm 5 pm Spherisorb ODS (cat. no.
583 12, Supelco, Bellefonte, PA) protect with a guard column (see Note 4).
3. Fluorescence detector Kratos model 980 or other suitable detector
4. HPLC mstrument with autosampler (optional) and mtegrator/recorder
3. Methods
3.1. PLAl Assay
1 MIX 40 pL of PLA, stock assay solution with 60 pL of enzyme (containing the

equivalent of about 2 ng of pure snake venom PLA2 (cat no 525 150, Calbiochem,
La Jolla, CA) (see Note 5) m a 500~PL microcentrifuge tube (vortex) Incubate at
room temperature. The final concentrations are 0.15 MNaCI, 0.1 &substrate,
0 2 rnA4 Triton X-100, 5 mMCaC12, 25 mMTris-HCl, pH 7 0
2 At various times over a period of 30-60 mm, remove 5-pL aliquots Spot directly
on a TLC plate for TLC analysis, or add to 90 pL of quenchmg solvent m a 500~pL
microcentrifuge tube for HPLC analysis (vortex)
3 For TLC analysis. dry the spots and develop the TLC plates m TLC solvent After
the solvent has evaporated, scan the plates with a fluorescence densitometer (set
excitation at 256 nm; measure emission above 400 nm [cutoff filter]). The Rr val-
ues for dansyl-PAF and lyso-dansyl-PAF are 0 35 and 0 15, respectively
4 For HPLC analysis centrifuge the quenched samples at top speed m a rmcrocenmfuge for
several minutes to remove any precipitated protein Equilibrate the s&a gel HPLC col-
umn in PLAZ HPLC solvent at a rate of 1 ml/r&n Set the fluorescence detector at excna-
hOn
256 nrn and emission >4 18 nm (cutoff filter). InJect 20 pL of sample onto the column,
Elution times for dansyl-PC and lyso-dansyl-PC are about 6 and 19 min, respectively
5 Calculation of acttvtty. the mol fraction of product released is determined by
dividmg the area of the lyso-dansyl-PC peak by the sum of the areas of the dansyl-
Fluorescence-Based Phospholipase Assays
5
PC and lyso-dansyl-PC peaks Thts value times the mmal amount of substrate
present in the assay (0 01 pmol) equals the amount of product released Plot the
pmol of product released vs time to determine the mitral lmear rate (acttvtty,
umol/min)
3.2. Pi-PLC Assay
1 Add 5 pL of PI-PLC (contammg the equivalent of 0 2-4 ng of pure enzyme (cat
no P-6466, Molecular Probes) (see Notes 5 and 6) to 20 pL of PI-PLC stock
assay solutton m a 500~uL microcentrifuge tube (vortex). Incubate at room tem-
perature Fmal concentratton of substrate, 1 mM

2 At vartous times over a period of l&30 mm remove 5-uL altquots and dilute
with 95 uL of PI-PLC HPLC solvent in 500~pL microcentrifuge tubes (vortex)
Centrifuge these samples at top speed in a microcentrtfuge for several minutes to
remove any prectpttated protein (see Note 7)
3 Equilibrate the ODS reverse-phase HPLC column m HPLC solvent at a rate of
1 mL/mm (see Note 4) Set the fluorescence detector at excitation 343 nm and
emission >370 nm (cutoff filter) Inject 20 PL of sample onto the column. Elu-
tion times for pyrene-PI and pyrenebutanol are 2 4 and 6.2 mm, respectively
4 Calculatton of activity the mol fraction of product released is determmed by
dtvtdmg the area of the pyrenebutanol peak by the sum of the areas of the pyrene-
PI and pyrenebutanol peaks. This value ttmes the untial amount of substrate
present m the assay (0 025 pmol) equals the amount of product released Plot the
pmol of product released vs time to determme the mttial linear rate (activity,
pmol/min)
4. Notes
Dertvatives of dansyl-PC are useful in the assay of other enzymes of lipid
metaboltsm Lyso-dansyl-PC and dansyl-PAF (cat. no D-3766 and D-3767,
respectively, Molecular Probes) can be used as substrates m assays of lyso-PAF
acyltransferase, lyso-PAF acetyltransferase, and PAF acetylhydrolase (3)
This solution 1s stable for a year or more tf protected from moisture and stored m
a brown bottle at -20°C Before use, warm to room temperature and make sure
the lipid ts completely dissolved
Snake venom PLA, IS quite active in the presence of Trtton X- 100, but other
PLA2s (parttcularly pancreattc PLA2) may be less active with this detergent. The
pancreatic enzyme is best assayed using sodium cholate as a detergent Hexa-
decylphosphocholme (cat no H6722, Stgma) IS also a good detergent for other
phosphohpases. The concentration of substrate and the ratto of substrate to deter-
gent may be varied as destred The spectfic activity of pure snake venom PLA, in
this assay with dansyl-PC is about 13 ymol/mm/mg
Do not leave the reverse-phase ODS column in PI-PLC HPLC solvent (wtth

tetrabutylammonmm dthydrogen phosphate) for any length of time without sol-
vent running through; if so, the column will become plugged After use, tmmedt-
ately wash the column with CHsOH/H,O (80.20)
6
5
6
7
Hendrickson
Phospholipases readily adsorb onto glass or plastic surfaces Dilute soluttons of
PLA, and PI-PLC (<I mg/mL) should be stabthzed by the presence of 1% (w/v)
bovme serum albumm
With pure B cereus PI-PLC, the specific activity m this assay wtth pyrene-PI is
about 60 pmol/mm/mg, about 4 % hydrolysis IS observed m 1 mm wtth 4 ng of
pure enzyme
R, values for TLC of pyrenebutanol and pyrene-PI on silica gel plates 0.75 and
0 0, respectively, m CHCl,/CH,OH (95 5), 1 0 and 0 25, respectively m CHCl,/
CH,OHIH,O (65 35 3)
References
1. Reynolds, L J., Washburn, W N., Deems, R. A., and Dennis, E A (199 1) Assay
strategies and methods for phospholtpases Methods Enzymol 197,3-23
2 Hendrickson, H S (1994) Fluorescence-based assays of hpases, phospholtpases,
and other ltpolytx enzymes Anal Blochem 219, l-8
3. Schmdler, P W , Walter, R., and Hendrickson, H S (1988) Fluorophore-labeled
ether lipids Substrates for enzymes of the PAF cycle m peritoneal polymorpho-
nuclear leukocytes Anal Blochem 174,477-484
4 Hendrickson, H S , Kotz, K J , and Hendrtckson, E K (1990) Evaluation of
fluorescent and colored phosphatidylcholme analogs as substrates for the assay of
phosphohpase A, Anal Bzochem 185,80-83
5 Hendrickson, H. S. (1991) Phosphohpase A, assays with fluorophore-labeled lipid
substrates Methods Enzymol 197,9&94

6 Bruzik, K S and Tsar, M -D (1994) Toward the mechamsm of phosphomosttide-
spectfic phospholtpase C Bloorg Med Chem 2,49-72
7 Low, M G and Saltiel, A R (1988) Structural and functional roles for
glycosylphosphattdylmositol m membranes. Sczence 239,268-275
8 Hendrickson, E K , Johnson, J L., and Hendrickson, H S (1991) A fluorescent
substrate for the assay of phosphattdylmosnol-specific phosphohpase C 4-( l-
pyreno)butylphosphoryl- 1-myo-inositol Bloorg Med Chem Lett 1,6 19-622
9 Hendrickson, H S , Hendrickson, E K , Johnson, J L , Khan, T H , and Chlal, H
J (1992) Kinetics ofB cereus phosphatldylmositol-specific phospholtpase C with
thiophosphate and fluorescent analogs of phosphatidylmosnol Bzochemlstry 3 1,
12,169-12,172
2
Fluorometric Phospholipase Assays Based
on Polymerized Liposome Substrates
Wonhwa Cho, Shih-Kwang Wu, Edward Yoon.
and Lenka Lichtenbergova
1. Introduction
Phospholtpases are a drverse group of ltpolyttc enzymes wrth specrflcr-
ties for dtfferent sites on the phosphohptd molecule (2) Based on the site
of then hydrolytrc attack, they can be classrfted into several groups, among
whtch phospholtpase A, (PLA,), phospholipase C (PLC), and phosphoh-
pase D (PLD) have been the most extensrvely studied. Because of then
crltlcal mvolvement n-r many extra- and intracellular processes, a senstttve
contmuous kmetrc assay for phospholrpase IS an essentral tool for many
blologtcal studtes
Among the vartous assays used for the different classes of phospholt-
pases (2), those based on fluorometry (3-7) serve as the most sensttrve type
of contmuous assay. However, a maJority of current fluorometrtc methods
rely on the use of synthetic phospholiptds containmg a fluorophore at the
~2-2 posrtion of the phospholtptd that restricts then use to the assay of

PLA*. In order to measure the activity of many types of phospholtpases, we
have recently developed a novel fluorometrrc assay utlhzmg polymertzed
mixed lrposomes (7).
Polymerized mixed hposomes are composed of a pyrene-labeled phospho-
hptd monomer Inserted mto a polymertzed phospholtptd matrix, the pyrene-
labeled phospholiprd constitutes only a small mole percentage of the total
polymerized mixed hposome substrate. In this system, the polymerized matrix
is essentially inert, and only the monomer (unpolymerrzed) inserts are selec-
tively hydrolyzed by the phosphohpase (8). For example, the PLA2 actrvtty
From Methods m Molecular Biology, Vol 109 Lfpase and Phospholfpase Protocols
Edlted by M H Dooltttle and K Reue D Humana Press Inc , Totowa, NJ
7
Cho et a/.
Fig 1 SchematIc lllustratlons of fluolometrlc assays usmg polymerized mixed
hposomes for PLA, (A) and PLC and PLD (B). F indicates a fluorescent label, such as
pyrene, attached to either the acyl cham (A) or the polar head group (B)
assay utlhzes a polymerized mixed hposome substrate that IS composed of l-
hexadecanoyl-2-( I-pyrenedecanoyl)-srt-glycero-3-phosphoglycerol (pyrene-PG)
inserted into a polymerized matrix of 1,2-bls[ 12-(hpoyloxy) dodecanoyll-sn-
glycero-3-phosphoglycerol (BLPG). The pyrene-PG Insert contams a
pyrenedecanoyl fatty acid at the sn-2 posltlon, with the fluorescent pyrene
molecule located at the methyl-termmal end of the fatty acid (see Fig. 1A);
the fluorescence emtsslon Intensity of the pyrene moiety ts largely
quenched by the lipolc acid moieties of the BLPG molecules. Using this
substrate, PLA2 hydrolysis can be contmuously monitored by measuring an
increase m pyrene fluorescence, since the hydrolyzed pyrene labeled-fatty
acids are no longer quenched as they are removed from the llposome sur-
face by serum albumin m the reaction mixture (see Fig. 1A). Alternatlvely,
for measurement of PLC and PLD hydrolysis, a new Insert IS used where
the phosphollpld monomer contams a pyrene-labeled head group; agam,

quenchmg of the pyrene moiety by the polymerized phosphollpld matrix IS
lost as the hydrolyzed pyrene moiety readily diffuses away from the llpo-
some surfaces (see Fig. 1B). Thus, polymerized mixed llposomes serve as
an extremely versatile assay system for different phosphollpases, since one
can readily modify the structure of the polymerized matrix, and the pyrene-
labeled phospholipid inserts, to create an ideal combmatlon of llposome
surfaces and hydrolyzable substrates for a specific phosphollpase. Herein,
we describe the synthesis of polymerlzable phosphollplds and pyrene-
labeled phospholiplds, the preparation of polymerized mixed llposomes
from these synthesized products, and the use of the prepared llposomes as
substrates to fluorometrlcally monitor phosphollpase hydrolysrs
Polymerized Liposome Substrates
9
2. Materials
2.1. Synthesis of the Polymerized Phospholipid Matrix,
1,2-bis[lZ-(lipoyloxy)-dodecanoy/l-sn-Glycero-
3-Phosphoglyceroi (BL PG)
1 12-hydroxydodecanotc acid, 3,4-dihydro-2H-pyran, p-toluenesulfonic actd
monohydrate, dtcyclohexylcarbodnmtde (DCC), 4-(dtmethylammo)pyrtdme
(DMAP), glycerol (Aldrtch, Mtlwaukee, WI)
2 L-a-glyceropl~ospl~orylchohne, 1. I cadmmm chloride adduct (Sigma, St Louts, MO)
3 Cabbage PLD IS prepared as follows The Inner ltght-green leaves of a Savoy
cabbage (1 Kg) IS homogemzed wtth 200 mL of cold water for 5 mm The homo-
genate IS filtered and the filtrate IS centrifuged at 20,OOOg for 30 mm The super-
natant IS kept at 55 “C for 5 mm and placed on tee for 5 mm The prectpttate 1s
removed by centrtfugatton (20,OOOg for 30 min), the supernatant 1s cooled to 0°C
and 2 vol of acetone cooled to -15°C is added to the supernatant After 10 mm,
the prectpttate containmg the active PLD IS collected by centrifugatton (20,OOOg
for 30 mm) and used for transphosphattdylatton. The acetone power can be stored
at -20°C for several months

4. PLD reaction buffer 0 1 M sodium acetate buffer, pH 5 6, 0 1 A4CaC12
2.2. Synthesis of Pyrene-Labeled Phospholipid Monomers (In-
serts) Specific for the Measurement of Cytosolic PLA2 Activity
1 lo-(1-Pyreno)decanotc actd (a k a l-pyrenedecanotc actd) (Molecular Probes,
Eugene, OR)
2 Ltthtum alummum hydrtde, pyridme, mesyl chloride, sodtum hydrtde, 2,3-
tsopropyltdene-sn-glycerol, phosphorus oxychlortde, trtethylamme (Aldrrch,
Mtlwaukee, WI)
3 Cholme tosylate, arachtdomc actd (Sigma, St Louis, MO).
2.3. Preparation of the Polymerized Mixed Liposome Substrate
1 Macro extruder Ltposofast (Avestm, Ottawa, Canada) with 0.1 -mm polycarbon-
ate filter (M&pore, Bedford, MA).
2 Polymenzatton buffer, 10 mMTris-HCI, pH 8.4,O 16 M KC1
3 Sephadex G-SO (Pharmacta, Uppsala, Sweden).
4. Mobile phase for Sephadex G-50, 10 mM HEPES buffer, pH 7.4 , 0.16 KC1
2.4. Kinetic Measurements
1 Spectrofluorometer equtpped with a thermostated cell holder and a magnettc stnrer.
2. 1-Hexadecanoyl-2-( 1 -pyrenedecanoyl)-sn-glycero-3-phosphocholme (pyrene-PC),
-phosphoethanolamme (pyrene-PE),-phosphoglycerol (pyrene-PG) (Molecular
Probes)
3. N-( I-pyrenesulfonyl)-egg phosphattdyl ethanolamme (N-pyrene-PE) (Avanti,
Alabaster, AL)
4 Fatty acid-free bovine serum albumin (BSA) (Bayer, Kankakee, IL)
70
Cho et al
5 Secretory PLA2 buffer 10 mMHEPES, pH 7 4,0 16 M KCI, 10 mMCaCl,, assay
and 10 mMBSA
6 Cytosohc PLAz buffer 10 mMTns-HCI, pH 8.0,1 mA4CaCl,, and 10 mMBSA assay
7. PLC buffer 10 mMMES buffer, pH 6 0,O 1 n&‘ZrCl,, and 10 mA4CaClz assay
8 PLD buffer 10 mA4HEPES, pH 7 4,0 16 M KCl, and 10 mM CaClz assay

3. Methods
3.1. Synthesis of the Polymerized Phospholipici Matrix, 1,2-bis[12-
(lipoyloxy)=doctecanoyl;l-sn -Glycero-5 Phosphoglycerol(6 L PG)
BLPG IS prepared from L-u-glycerophosphorylchohne by a four-step syn-
thesis according to the procedure by Sadowmk et al. (9) with slight modlfica-
tions (7,s).
3.7.1. Synthews of 12-(Tetrahydropvranyloxy)Dodecanoic Acid (TCA)
1 Suspend 10 mmol (2 16 g) of 12-hydroxydodecanolc acid m 20 mL of dry
tetrahydrofuran
2 Add 17 mm01 (1 39 g) of 3,4-dihydro-2H-pyran and stir the mixture for 10 mm at
room temperature
3 Add 0 1 mm01 (20 mg) of crystallme p-toluenesulfomc acid monohydrate and
stir the mixture for an addItIona 2 h at room temperature
4 Evaporate the solvent and purify the product by slllca gel column chromatogra-
phy usmg chloroform/methanol (20 1, v/v) as eluent. Check the product by thm-
layer chromatography (TLC) (R,= 0 5 with the same solvent) Completely remove
the solvent zn vacua
3.72. Synthesis of 1,2-Bis(lZ-Hydroxydodecanoyl)-sn-
Glycero-3-Phosphocholine
1 Dissolve the purified 12-(tetrahydropyranyloxy)dodecanolc acid (TCA) m dry
dtchloromethane (15 mL) and add DCC (2.6 mmoU4 mm01 of TCA) dissolved m
dry duzhloromethane
2 Stir the reactlon mixture at room temperature until it gets cloudy (approx 5 mm),
then add L-a-glycero-phosphorylcholme (1 mmol/4 mmol of TCA) and DMAP
(2 6 mmol/4 mm01 of TCA) Stir the mixture at room temperature overnlght
3 Filter the reactlon mixture to remove dlcyclohexylurea, evaporate the solvent
and purify the product by slhca gel column chromatography using chloroform/
methanol (5 1) first and then chloroform/methanol/water (65 25.4) Check the
product by TLC Rf= 0 35 with chloroform/ methanol/water (65 25:4)
4 Dtvlde the product mto about 200 mg fractions, dissolve each fraction m metha-

nol (2 mL) and add equlmolecular crystallme p-toluenesulfomc acid monohy-
drate Stir the mixture at room temperature for approx 1 5 h while checking the
progress of the reactlon every 15 mm by TLC usmg chloroform/methanol/water
(4 5 1) as developmg solvent (Rf = 0 3 for the product) The reaction must be
completed within 2-2 5 h, otherwlse the hydrolysis of acyl groups of the phos-
Polymerized 1 iposome Substrates
Ii
phohptd ~111 occur to a constderable degree. Use the crude product lmmedrately
for the next step
3.1.3. Synthesis of 1,2-Bls[lZ-(lipoyloxy)Dodecanoyl]-sn-Glycero-3-
Phosphocholine (BL PC)
1. Dtssolve 6,8-drthrooctanotc acid (5 mmol/l mmol of TCA) m dry dtchloro-
methane and add DCC dtssolved in dry dichloromethane (3 mmoli5 mmol of 6,8-
dtthtooctanorc acid)
2 Stir the reactton mtxture at room temperature untrl tt gets cloudy (approx 5 mm),
then add the crude product of the above reactton drssolved m dry dtchloromethane
and DMAP (2 mmol/l mmol of TCA) Star the mixture overmght m the dark at
room temperature
3 Filter the reaction mixture to remove dtcyclohexylurea, evaporate the solvent
and purify the product by silica gel column chromatography usmg chloroform/
methanol (5.1, [v/v]) first to completely remove DMAP and unreacted 6,8-
dlthlooctanotc acid and then chloroform/methanol/water (65 25 4) Check the
product by TLC R, = 0 45 wrth chloroform/methanol/water (65 25.4). The purt-
fied product (yellow sohd) should be dissolved m dtchloromethane and stored at
-20°C m the dark
3.7.4. Synthesis of BLPG
1 Suspend 50-100 mg of BLPC m ethyl ether (1 5 mL) m a small glass vial
2 Add 0 6 g of glycerol to 3 mL of 0 1 M sodium acetate buffer, pH 5 6, 0 1 A4
CaCl,, combme tt with the above BLPC solutton and vortex the mixture brrefly
3 Add approx 100 mL of cabbage PLD (approx 1 mg/ml) to the reactton mtxture

and stir the mixture vrgorously for 1 5 h at room temperature
4. Evaporate ether from the reaction mixture and extract the aqueous layer wtth
chloroform Dry the orgamc layer over anhydrous sodmm sulfate and evaporate
the solvent
In vacua
S Purify the product by sthca gel column chromatography using CHCl,/CH,OH
(3 1) first and then chloroform/methanol/water (40 10.1)
6 1,2-bts[ 12-(lipoyloxy)-dodecanoyl]-sn-glycero-3-phosphatldlc acid (BLPA) can
be synthesized by performing the PLD reaction m the absence of glycerol
3.2. Synthesis of Pyrene-Labeled Phospholipid Monomers
(Inserts) Specific for the Measurement of Cytosolic PLA2 Activity
Cytosolrc PLA, (cPLA,) has htgh specificity for the arachidonyl motety m
the
m-2
posrtron
of phospholtptds (20, II). Furthermore, cPLA, has both PLA,
acttvtty and lysophosphohpase acttvrty and, as a result, tt hydrolyzes both sn- 1
and
m-2
acyl esters tf 1,2-dtacyl-sn-glycero-3-phosphohptds are used as a sub-
strate. Thus, a pyrene-labeled substrate for cPLA, must contain ~12-2 arachtdonyl
ester and a nonhydrolyzable group at
sn-
1 posttton. l-O-( 1 -pyrenedecyl)-2-
arachrdonyl-sn-glycero-3-phosphocholine (PAPC) IS a synthetic phospholtptd
12 Cho et al.
that meets this reqmrement. PAPC IS prepared from lo-(I-pyreno)decanolc
acid by a five-step synthesis.
3.2.1. Synthesis of IO-( 1 -Pyreno)Decanol
Convert IO-( 1-pyreno)decanotc acid to an ethyl ester by refluxmg tts ethanol

solutton for 4 h wtth 1 drop of concentrated HCI
Dilute the mtxture with chloroform and wash the organic layer with 1 M solution
of sodmm bicarbonate
Dry the organic layer over anhydrous sodmm sulfate and evaporate the solvent
Dtssolve the product m dry tetrahydrofuran, place the mixture on ice,
add llthlum
alummum hydride and stir the mtxture at 0°C for 30 mm and then at room tem-
perature for 2 h
Stop the reaction by coolmg the mrxture on Ice and adding water to the mtxture
Extract the mixture wnh chloroform and purtfy IO-( 1 -pyreno)decanol by stltca
gel column chromatography using hexane/ethyl acetate (4.1, [v/v])
3 2 2. Synthesis of 1 O-( I- Pyreno) Decanyl Mesyiate
1. Drssolve IO-( I-pyreno)decanol m 5 mL of dry pyrtdme and 5 ml of hexane Stn
the mixture m a 25°C water bath and slowly add mesyl chloride (2 mmol/l mm01
of lo-( 1 -pyreno)decanol)
2 Warm the water bath to 40°C stn the mixture for 1 h and check the progress of
the reaction by TLC, R1 = 0.4 with hexane/ethyl acetate (2.1, v/v)
3 After the reaction 1s complete, dilute the mixture with 30 ml of hexane-benzene
(1 1, v/v) and wash successtvely wtth water, 0 5 N HCl, water, 5% sodmm btcar-
bonate, and water (2X).
4 Dry the organic phase over anhydrous sodmm sulfate and evaporate the solvent
Purify the product by sthca gel column chromatography usmg hexaneiethyl
acetate (2.1, v/v)
3.2.3. Synthesis of l-O-[iO-(I-Pyreno)Decanyl]-sn-Glycerol
Prepare a solutton of 0 12 g of sodmm hydride m 5 mL of dry benzene and slowly
add 2 mmol of 2,3-tsopropyltdene-sn-glycerol
Whtle sttrrmg at room temperature, add 1 5 mmol of lo-( I-pyreno)decanyl
mesylate and heat the mixture to reflux for 4 h
Cool the mixture m an Ice bath, add water to remove excess sodmm hydrtde and
neutralize the mixture wtth 1 N HCl

Dilute the mixture with 20 mL benzene and wash tt twice wtth 20 mL of metha-
nol/water (1 1, [v/v]).
Dry the orgamc phase over anhydrous magnesium sulfate, evaporate the solvent
and drssolve the solid m 5 mL of methanol
Add 20 mL of 10% HCl m methanol and heat the mtxture at 60°C for 30 min.
Evaporate the solvent m vacua, apply the crude product dissolved m acetone/
hexane (1 9, [v/v]) onto a sthca gel column and elute stepwtse with 10, 20, and
30% acetone in hexane (v/v) Check the product by TLC, Rl = 0 4 wtth hexanel
Polymerized Liposome Substrates
acetone (7 3, [v/v])
3.2.4. Synthesis of l-O-(l-Pyrenedecyl)-Z-Hydroxy-
sn
-Glycero-3-Phosphocholine
13
Combme 1 mmol of l-O-[ IO-( I-pyreno)decanyl]-sn-glycerol with 1 8 mmol of
phosphorus oxychlorlde and 3 0 mmol trlethylamme m 5 ml of dry chloroform
and stir the mixture at 0°C for 30 mm
Add choline tosylate (1 8 mmol) and stir the mixture at 0°C for another 30 mm
Check the progress of reaction by TLC, R, = 0 5 for the product with chloroform/
methanol/water (40 10 1, [v/v/v])
Quench the reaction by adding water and stirring the mixture at room tempera-
ture for IO mm
Evaporate the solvent and purify the product on a sdlca gel column Elute first
with chloroform/methanol (5. I, [v/v]) and then with chloroform/methanol/water
(40 10 1) [v/v/v])
3 2.5. Synthesis of PAPC
1 Dissolve arachldomc acid m dry dlchloromethane (2 mmol of actd/l mmol of the
above product) and add DCC dissolved m dry dlchloromethane (3 mmoli2 mmol
of arachldomc acid)
2 Stir the mixture at room temperature until it gets cloudy (approx 5 mm), then add

the above product dissolved m dnzhloromethane and DMAP (2 mmol of DMAPi
1 mmol of the above product) Stir the mixture at loom temperature overnight
3 Filter the reaction mixture to remove dlcyclohexylurea, evaporate the solvent
and purify the product by slltca gel column chromatography Elute first with chlo-
roform/methanol (5. I, [v/v]) to completely remove DMAP and unreacted fatty
acid and then chloroform/methanol/water (65 25.4, [v/v/v]) Check the final prod-
uct by TLC; R, = 0 5 with chloroform/methanol/water (65 25 4, [v/v/v]) The
purified product IS dissolved m dlchloromethane and stored at -20°C
3.3. Preparation of the Polymerized Mixed Liposome Substrate
Polymerrzed mixed hposomes are prepared by polymerlzmg pre-formed
mixed llposomes of BLPG (or its denvatlves) and a pyrene-labeled phospho-
llpld. Although the composition of pyrene-labeled phosphollpld m polymerized
mlxed hposomes can vary from 1 to 10% without affectmg the kinetic propertles
of phosphollpases, a mmlmal composltlon (1 “A) 1s maintained for all polymer-
ized mixed hposomes for kinetic consistency and economical reasons
1 To prepare 2 mL of 1 mMpolymerized mixed Iiposomes, mix 1 98 pmol of BLPG
and 0 02 pmol of pyrene-labeled phosphohpld in a glass vial, evaporate the
organic solvent with the stream of nitrogen gas and add 2 mL of IO mMTns-HCI
buffer, pH 8 4, to the hptd film and vortex the dispersion for about 5 mln
2 Prepare large umlamellar hposomes by multiple extrusion (15X) of phospholipld
dlsperslon through 0 1 -mm polycarbonate filter m a micro extruder Liposofast
14 Cho et al
3 Prepare polymerized mlxed hposomes by Incubating the mixed hposomes at 37°C
fol24 h m the presence of 10 mM dlthlothreltol The degree of polymeflzatlon can be
monitored by TLC and by the loss of the UV absorption at 333 nm (9) (see Note 1)
4 Separate the polymerized mixed hposomes from the reaction mixture on a short
Sephadex G-SO column (1 x 10 cm) eqtuhbrated with 10 mMHEPES buffer, pH 7 4
contammg 0 16 KC1 Elute the hposomes with the same buffer, monitor the absor-
bance at 280 nm and collect the first peak elutmg at the void volume of the column
5 Determine the total phosphohpld concentration of hposomes by phosphate analy-

SIS (2.5) (see Note 2)
3.4.
Kinetic Measurements
All kmetlc measurements are performed at 37°C. The change m fluores-
cence mtenslty of pyrene-labeled phosphohplds m polymerized mixed hpo-
somes IS measured as a function of time using a fluorescence spectrometer
The excltatlon wavelength 1s set at 345 nm and the emlsslon wavelength at 380 nm.
Spectral band width IS set at 5 nm for both excltatlon and emlsslon. The sample
IS contained m a stirred, thermostated 1 -cm path length quartz cuvet.
3.4.1 Secretory PLA,
1 Add 2 mL of 10 mMHEPES buffel, pH 7 4, contammg 10 pMpolymerlzed mixed
hposomes (e g , pyrene-PGiBLPG), 10 FM BSA, 0 16 M KCl, 0 1 mM EDTA to
a quartz cuvet
2. Place the cuvette m the spectrometer and monitor the fluorescence intensity at
380 nm until the baseline 1s stabilized (approx 2 mm)
3. Add an ahquot of enzyme solution and incubate the mixture for a few minutes
4 Initiate the reaction by adding 10 FL of 2 MCaCl, solution to the final concentra-
tionof lOmA
3 4.2 Cytosok PLA2
In contrast to secretory PLA,s, cPLA,s shows extremely low actlvlty toward
polymerized mixed hposomes, such as PAPUBLPG polymerized mixed hpo-
somes. This 1s presumably owing to the fact that cPLA2 must penetrate mto the
core of phosphohpld bilayer to pull a substrate molecule mto its active site, which
IS largely prohibited m the polymenzed mixed hposome system On the other hand,
cPLA, shows high activity toward PAPC m unpolymerized mixed hposomes of
PAPUBLPG (see Chapter 4). Thus, PAPUBLPG ( 1:99, mol/mol) mixed hposomes
are used as a substrate for cPLA,. As IS the case with the polymerized mixed hpo-
some system, PAPC 1s selectively hydrolyzed m this system because cPLA2, owing
to its high sn-2 specificity, shows essentially no detectable activity toward
unpolymerized BLPG. To ensure the completely selective hydrolysis of PAPC,

one can employ PAPC/D-BLPG mixed hposomes. D-BLPG can be synthesized
from D-a-glycerophosphocholme as described for BLPG (see Note 3).
Polymerized Liposome Substrates
15
1 Add 2 mL of 10 mM Tns-HCI buffer, pH 8 0, contammg 1 mM CaCI,, 10 @I
PAPUBLPG mixed hposomes and 10 @I BSA to a quartz cuvet
2 Place the cuvette m the spectrometer and momtor the fluorescence mtenslty at
380 nm until the baselme IS stablhzed (approx 2
mm)
3 Initiate the reactlon by addmg an ahquot of enzyme solution
3.4.3 PLC and PLD
With commercially available N-pyrene-PE in polymerized mixed liposomes,
one can measure the actlvlty of phosphatldylcholme-specific PLC and PLD.
For PLC, N-pyrene-PEIBLPG polymerized mlxed hposomes are used as a sub-
strate m which N-pyrene-PE 1s selectively hydrolyzed. For PLD reactions, N-
pyrene-PE/BLPA polymerized mixed liposomes are used as a substrate owing
to relatively high activity of PLD toward polymerized BLPG molecules. For
both assays, BSA IS not included.
1 For a PLC assay, add 10 pMN-pyrene-PEIBLPG polymerized mixed hposomes
m 2 mL of 10 mM MES buffer, pH 6.0,O 1 nuI4 ZnCl,, 10 ti CaCl, to a quartz
cuvet For PLD assays, use 10 @IN-pyrene-PEIBLPA polymerized mlxed llpo-
somes m 10 mA4 HEPES, pH 7 4,0 16 MKCl, 10 mM CaCl,
2 Place the cuvet m the spectrometer and monitor the fluorescence mtenslty at 380 nm
until the baseline is stabdlzed (approx 2 mm)
3 Initiate the reactlon by addmg an allquot of enzyme solution
3.4.4. Determination of Rate Constants
1 The actlvlty (pmol/mm) of PLA2 for polymerized mixed hposomes IS calculated
according to the followmg equation (7)
Actlvlty = [pyrene-PL], x (AF- A.FO)
AF,nax

where [pyrene-PL], mdlcates the total concentration of pyrene-labeled phospho-
hpld (in pmol); AF, and AF, the fluorescence change per minute for nonenzy-
matic and enzymatic hydrolysis, respectively, AF,,,,, the maxlmal fluorescence
change that IS measured by addmg an excess amount of enzyme (see Note 4)
2. The specific activity (pmol/mm/mg) IS determmed by divldmg the enzyme actlv-
Ity by the amount of protein (mg)
3 Owing to the umque structural property of polymerized mixed hposomes m which
only a small percentage of phospholipid m the hposome IS hydrolyzed, the kinetic
pattern
of hydrolysis of these hposomes IS, m general, simpler than that of the
hydrolysis of conventlonal hposomes (13) Yet various factors, such as enzyme-
to-phosphohpld ratio, can slgmficantly affect the kinetic pattern for different
phospholipases. Therefore, It 1s recommended that the range of the enzyme con-
centration wlthm which the activity IS directly proportlonal to the enzyme con-
centration be estabhshed first for each phosphohpase and Its best polymerized
mlxed hposome substrate at a given concentration (1 e., 10 @4).
16 Cho et a/.
4. Notes
1 BLPG (and BLPC) molecules are prone to polymerlzatlon even m the absence of
dlthlothreltol and at low temperature For this reason, a fresh BLPG solution IS
prepared m every three months
2 Owmg to their chemical and mechanical stablhty, polymerized mlxed llposomes
can be stored at room temperature for several weeks under argon and in the pres-
ence of 0 0 1% sodium azlde It has been notlced, however, that some phosphoh-
pases show considerably altered activity toward these hposomes after elongated
storage It 1s therefore recommended that polymerized mlxed hposomes are used
within several days of preparation for the reproduclblllty of kinetic data
3 To design an assay system using polymerized mlxed hposomes for a newly dls-
covered phosphohpase, one must first find a polymerized matrix that 1s reslstant
to the hydrolysis by the enzyme and a pyrene-labeled insert that 1s rapidly hydro-

lyzed by the enzyme The derivatives of BLPG and other pyrene-labeled phospho-
lipids (I e , pyrene-PA and pyrene-PS) (24) can be prepared by the PLD-catalyzed
transphosphatldylatlon Specific actlvlty of phosphohpases for (polymerized)
mlxed hposomes ranges from 0 5 to 1,000 mmol/mm/mg depending on the na-
ture of enzyme (7)
4 Some phosphohpases show a short lag period (approx 10 s) before they 1 each the
steady state For these enzymes, the sequence of adding enzyme and cofactors
can be varied to mmlmlze the lag If the lag persists, the mltlal velocity 1s mea-
sured after the reactlon reaches a steady state
References
Waite, M (1987) The Phospholzpases, Plenum, New York
Reynolds, L J , Washburn, W N , Deems, R A and Dennis, E A (199 I ) Assay
strategies and methods for phosphohpases Methods Enzymol
197, 3-23
Radvanyl, F , Jordan, L , Russo-Mane, F , and Bon, C (1989) A sensltlve and
contmuous fluorometrlc assay for phosphohpase A, using pyrene-labeled phos-
phohplds m the presence of serum albumm Anal Bzochem
177, 103-I 09
Thuren, T , Vlrtanen, J A , Vamlo, P , and Kmnunen, P K J (1983) Hydrolysis
of 1 -tnacontanyl-2-pyrene-l-yl)hexanoyl-sn-glycero-3-phosphochollne by human
pancreatic phosphohpase A, Chem Phy Llplds 33,283-292
Hendnkson, H S and Rauk, P N (198 1) Contmous fluorometrlc assay for phos-
phollpase A2 with pyrene-labeled leclthms as a substrate Anal Blochem
116,
553-558
Kmkald, A R and Wllton, D C (1993) A continuous fluorescence dlplacement
assay for phosphohpase A, using albumm and medium chain phosphollpld sub-
strates Anal Blochem
212, 65-70
Wu, S -K and Cho, W (1994) A contmuous fluorometric assay for phosphoh-

pases using polymerized mlxed hposomes Anal Blochem
221, 152-l 59.
Wu, S -K and Cho, W (1993) Use of polymerized llposomes to study mterac-
tlons of phosphollpase A2 with blologlcal membranes Bzochemzstry 32,
13,902-13,908
Polymerized Liposome Substrates 17
9 Sadowmk, A , Stefely, J , and Regen, S L (1986) Polymerized hposomes formed
under extremely mild condltlons. J Amer Ckem Sot 108,7789-7792
10 Clark, J. D , Lm, L -L., Knz, R W., Ramesha, C S., Sultzman, L A., Lm, A Y ,
Mllona, N., and Knopf, J. L. (1991) A novel arachldomc acid-selective cytosohc
PLA, contains a Ca2’-dependent translocatlon domam with homology to PKC
and GAP Cell 65, 1043-105 1
11 Sharp, J D , White, D L, Chlou, X G., Gooden, T, Gamboa, G C , McClure, D ,
Burgett, S , Hoskm, J., Skatrud, P L., Sportsman, J R , Becker, G W , Kang, L H ,
Roberts, E F , and Kramer, R M (199 1) Molecular clonmg and expresslon of human
Ca*+-sensltlve cytosohc phosphohpase A, J Blol Ckem 266, 14,85@-14,853
12 Kates, M (1986) Techmques of Llpldology, Elsevler, Amsterdam
13 Jam, M K and Berg, 0 G. (1989) The kinetics of mterfaclal catalysis by phos-
phohpase A, and regulation of mterfaclal actlvatlon hopping versus scootmg
Blocklm Bzopkys Actu 1002, 127-156
14 Smtko, Y , Yoon, E T , and Cho, W (1997) High Speclficlty of Human Secre-
tory Class II Phosphohpase A, for Phosphatldlc Acid Blockem J 321,737-74 1
3
Triglyceride Lipase Assays Based on a Novel
Fluorogenic Alkyldiacyl Glycerol Substrate
Albin Herrnetter
1. Introduction
Lipases are responsible for extracellular degradation and mtracellular
metabolism of lipids m animals, plants, and microorganisms The conformational

motif of all lipases whose three-dimensional structure has been determined IS
characterized by an alp hydrolase fold (1). Most hpases utilize the catalytic triad
Ser-His-Asp, with the serme residue actmg as the nucleophile in the mmal step
of carboxyl ester hydrolysis, this active site serme is contamed withm the Gly-
Xaa-Ser-Xaa-Gly consensus sequence common to most hpases (2,2)
In humans and animals, triacylglycerols (and to a lesser extent diacyl- and
monoacylglycerols) are the biological substrates of lipases Commensurate
with their function, different triglyceride lipases are found m a variety of extra-
cellular and intracellular locations. For example, lingual hpase, gastric hpase,
pancreatic lipase, and bile salt-activated lipase are present m the gastromtesti-
nal tract. Lipoprotein lipase and hepatic lipase are bound to the capillary endot-
helium and hydrolyze triacylglycerols m cnulatmg hpoprotems. Lipoprotem
lipase and bile salt-activated hpase are also present m milk. Finally, lysosomal
lipase and hormone-sensitive lipase are intracellular enzymes.
Importantly, the different lrpases exhibit specific preferences for then
triacylglycerol substrates, depending on chain length, optical and positional
isomers, and the characteristics of the lipid/water interface (3,4) For instance,
lipoprotem hpase and hepatic lipase express maximum activity only against
certain lipoprotem classes ($6). Pancreatic hpase acts on mixed micelles con-
taming bile salt, although only in the presence of colipase (7,s). Moreover,
enzyme activity may be enhanced by specific cofactors such as apohpoprotem
CII for lipoprotein lipase (9) or colipase for pancreatic lipase.
From Methods fn Molecular L?/o/ogy, Vol 109 Llpase and Phosphohpase Protocols
Edited by M H Doohttle and K Reue 0 Humana Press Inc , Totowa, NJ
19
20 Herrnetter
Impaument of hpase functton due, for example, to the absence of enzyme
activity as a consequence of mutattons m the genes coding for the enzyme or its
protein cofactor(s), may be associated with severe pathological phenotypes For
instance, elevated plasma triglycerides associated wtth metabolic disorders such

as type I hyperhpoprotememta results from the deficiency of hpoprotem hpase
activity (10,12) The consequence of a deficiency m lysosomal actd ltpase IS
cholesterol ester storage disease (12). Moreover, under pathological condmons,
hpases may be found m an envnonment m which they usually do not occur and,
as such, may represent markers of destructive organ processes For instance, as a
consequence of pancreatttts, high levels of pancreatic hpases are often present n-t
the blood (13).
Determmatton of hpase acttvlty 1s routmely used for monitormg enzyme pro-
tem durmg tsolatton, purtficatlon, detectton, and characterization of natural and
recombinant proteins, for studies of biologtcal enzyme functton, and for the
screenmg of biologtcal samples for diagnosis m medtcme. A number of methods
are available for the determmation of hpase acttvmes (#J&16), and include a
variety of different substrate types (natural or synthetic triacylglycerols, radtoac-
ttve or chromogenic llptds), as well as methods of substrate preparation (mono-
disperse and polydisperse substrates, phospholtptd vesicles or detergent
micelles) Both substrate type and its preparation are cructal wtth respect to
reproducibility and relevance of the measured data
A htgh-performance lipase assay 1s expected to meet several criteria that are
important for facility, quality, and selecttvity of the procedure (see Table 1)
We
have developed new fluorogemc trtacylglycerol analogs as substrates for a con-
tmuous llpase assay that fulfills these requirements (1618). These substrates
can be solubthzed m water containing fatty acid-free albumin Alternatively,
stable lyophihsates of substrate/albumm complexes can be obtained commerctally
and represent ready-to-use hpase substrates after dlssolutton m an appropnate aque-
ous buffer (17) In addmon, these
fluorogernc substrates can be substituted for
triacylglycerol m many types of standard substrate preparations, mcludmg deter-
gent mtcelles, phosphohptd vesicles, emulsions, and orgamc solvents (18)
The structure of these novel fluorogemc lipase substrates 1s an alkyldtacyl-

glycerol m an enanttomerically pure form, as shown m Fig. 1. In posttions sn-1
or ~2-3, a hexadecyl residue is linked to glycerol by an ether bond, whtch pre-
vents esterolysts of this bond by the hpase In posttton 92-2, glycerol is estertfied
with the fluorescent molecule pyrenedecanotc acid. Finally, a dodecanoyl chain,
substituted at its omega-end with a fluorescence quencher (a trlmtrophenylammo
residue), IS bound by an ester linkage to posttion sn-3 or sn- 1 of the glycerolipld
In then Intact form, these substrates show only low fluorescence intensity due to
mtramolecular resonance energy transfer. In the presence of a hpase, the fatty
acid ester containing the quencher 1s hydrolyzed and the fluorescent product, an
Lipase Assays Usmg Fluorogemc Substrates
21
Table1
Requirements for a High-Performance Lipase Assay
Category I facllltatlon of the assay
Fast results
High throughput of samples
Contmuous measurement
Water-soluble substrates
No radloactlvlty
Category II quahty and selectlvlty of the assay
High sensltlvlty
High accuracy
High reproduclblllty
No detergent
General apphcablhty to different reaction media
Determmatlon of enzyme actlvlty under native condltlons
No interference with nonspeclfic esterases
Fig 1 Chemical structure of the fluorogemc hpase substrate l-tnmtrophenyl-
amlnododecanoyl-2-pyrenedecanoyl-3-0-hexadecyl-sn-glycerol
alkylacylglycerol, IS formed. Thus, from the contmuous mcrease m fluores-

cence mtenstty, substrate hydrolysts can be monitored as a measure of ltpase
activity, using a calibratton curve obtamed by plotting fluorescence mtenst-
ties vs concentratton of an unquenched standard (pyrenedecanotc acid) (see
Figs.
2 and 3) Under condtttons of nonltmttmg substrate concentrattons
whtch depend on the reaction medium, enzyme actrvrty IS lmearly propor-
tional to enzyme concentratton. The detetectton limit for pure lipases IS >5 ng
depending on the lipase type, the stabthty of the hpase, and the quality of the
enzyme preparation.
For all animal hpases so far mvesttgated, there IS a very high stereopref-
erence for the sn-1 posttton of the fluorogemc alkyldtacylglycerols, espe-
cially tf solubthzed m the presence of albumin (2618) Therefore, only the
preferred enantromer, namely, 1 -trinitrophenyl-ammo-dodecanoyl-2-pyren-
edecanoyl- 3-O- hexadecyl-sn-glycerol, will be described as the hpase sub-
22
Herme tter
800
600
0
0 40 80 120 160
Standard concentration [pmollml]
Fig 2 Caltbration plot for an unquenched standard fluorophore. Fluorescence
mtensities at 400 nm are plotted agamst concentrations of pyrenedecanoic acid as a
complex with albumm m PBS
600
400
200
0
0
1

2 3
Time I min
4 6
Fig 3 Plot of the change m fluorescence intensity due to the addition of lipase
activity Fluorescence mtensity of substrate (lipid-albumm complex) was recorded at
400 nm and 37”C, followed by measurement of the time-dependent Increase of fluo-
rescence mtensity (AI) at 37% after addition of 15 pL post-heparm serum
strate m this chapter. Importantly, this long-chain trtacylglycerol analog 1s
ltpase specific, we have tested esterases from porcme pancreas and various
mrcroorgamsms and have found no hydrolytrc actrvtty using this substrate
Lipase Assays Using Fluorogenic Substrates
23
The features of this novel fluorogemc substrate that provide advantages com-
pared with existmg techmques may be summarized as follows*
1 The substrate can be prepared m detergent-free form, or at submtcellar detergent
concentration, and wtth a defined particle size m solutton (approxtmately 40 A) that
does not disrupt membranes or affect hpoprotem structure Thus, hpase acttvmes can
be measured under native condmons
2. The fluorogemc substrate/albumm complex IS avarlable as a water-soluble
lyophtbsate that represents a ready-to-use hpase substrate after reconstttutlon m
aqueous buffer The lyophlltsate IS very stable and easily stored
3 The use of radioactive substrates is avoided
4 The assay IS continuous and much more sensitive than the continuous tttrtmetrtc
methods using ahphatic short-cham triglycerides as substrates
5 The fluorogemc substrate is lipase specific Other commonly used short-chain
trtglycertdes (e g,. trtbutyrm, or carboxyhc acldp-mtrophenol esters) are not spe-
cific smce they are also hydrolyzed by esterases
6 A large number of samples can be analyzed in a short ttme when a fluorescence
plate reader IS used
In this chapter, detatled protocols are provided for the preparation of the

substrate and the appropriate standards used for quantitation of the hydrolyzed
product. Also mcluded are methods for the determmation of hpase activtty
from purified enzyme preparations or from biological samples (e g , serum or
post-heparm serum.) Selective conditions are discussed for the detection of
hpoprotem lipase and hepattc lipase based on measurements of enzyme active-
ties m buffers of different iomc strengths. A buffer system for the selective
determmatton of pancreas ltpase activity m serum has recently been established
m our laboratory (see Note 1).
2. Materials
2.1. Fluorescence Standards
Tetrahydrofuran (analytical grade).
Pyrenedecanorc acid, availatble from Molecular Probes (Eugene, OR, USA, or
2333 AA Leaden, The Netherlands), or from Lambda Probes (Krrchbach/
Stelermark, Austria).
Bovine serum albumm, fatty-acid-free, fraction V (Boehrmger-Mannhelm,
Mannhelm, Germany, or Sigma, Munchen, Germany)
Buffer for hpase assay (see Subheading 2.3.)
Trtton X-100, especially purified for membrane research (Boehrmger-Mannhelm)
Alternatively, fluorescence standards can be purchased from PROGEN
Biotechntk (Heidelberg, Germany) as stable lyophtltzed preparations of
pyrenedecanoic acid/albumin complexes containing 0, 0.6, 1.2, and 2 4 nmol
pyrenedecanorc acid, respectively, with 16 mg of fatty actd-free albumin each,
2 4 nmol Trtton X- 100, and 300 mg starch as a stabilizer.
24
Hen-netter
2.2. Lipase Substrate
1 Buffer for hpase assay (see Subheading 2.3.)
2 Bovine serum albumm, Trtton X- 100, and tetrahydrofuran (see Subheading 2.1.)
3 Ltpase substrate (fluorogemc alkyldtacylglycerol) 1 -truutrophenylammodo-decanoyl-
2-pyrenedecanoyl-3-0-hexadecyl-sn-glycerol (mol wt 1078) IS eastly soluble tn meth-

ylene chloride and tetrahydrofuran and soluble in ethanol, its absorptton spectrum IS the
sum of the mdtvtdual absorptton spectra of pyren-edecanotc acid (&,,, = 345 nm, E =
50,000) and tnnttrophenylammododecanotc acid (h,,, = 415 nm, E = 7000, E at 345 nm
= 16,000), solvent methylene chloride) Accurate substrate concentratrons m organic
soluttons are determmed from the absorbance at 415 nm (whtch momtors trmt-
trophenylamme absorptron) Purity of the alkyldtacylglycerol substrate can be deter-
mmed by thin-layer chromatography (I&= 0 7), using sthca gel plates and chloroform/
acetone/acetrc acrd (96 4 1, by vol ) as a solvent The alkyldracylglycerol substrate m
methylene chloride solutton or m solvent-tree form IS stored at -20°C The chemtcal
syntheses of the hptd 1s described In ref. 16
4 Alternattvely, the alkyldtacylglycerol substrate can be purchased from PROGEN
Btotechmk as a lyophthzed substrate/albumin complex A typical preparatton con-
tams 60 nmol fluorogeruc alkyldracylglycerol, 60 nmol (approximately 4 mg) fatty
actd-free albumm, 2 4 nmol Tnton X- 100, and 300 mg starch as a stabrhzer (accord-
mg to Zulkowsky, from Merck, Darmstadt, Germany), store m refrigerator (4°C)
2.3. Lipase Activity Assay
1 A fluorometer, equipped wrth a cell holder (preferably with four cuvet posmons)
that 1s water Jacketed for temperature control Suitable mstruments mclude the
Perkm-Elmer LSSOB, Perkm Elmer, Uberlmgen, Germany, Shtmadzu RF-5301
PC, Shtmadzu, Kyoko, Japan, and Hitachi F-2000, Ntsset Sangyo, Toyko, Japan
2 Cuvets for fluorescence spectroscopy Suitable cuvets Include quartz cuvettes, 4 mL,
d = 1 cm (Hellma, Mullhelm, Germany), or polyacrylate cuvets, 4 mL,
d =
1 cm (cat
no 2,3893, Merck)
3 Ltpase assay buffer Ltsted are recommended buffers for determmmg the enzymatic
actrvrty of some hpases For purtfied preparattons of hpoprotem hpase, hepatrc hpase,
and pancreattc hpase, use 0 1 MTns-HCI, pH 7 4, or phosphate-buffered sahne (PBS),
pH 7 4,0 2 g KCVL, 0 2 g KH,PO,/L, 8 g NaCl/‘L, 1 15 g Na2HP04/L These buffers
also work well to assay total hpase acttvtty m serum or post-hepann serum or tn cell

culture supematants (see Note 2) Addttton of 1 0 A4 NaCl to these buffers ~111 rnhrbrt
hpoprotetn hpase acttvrty but ~111 not reduce hepatrc hpase actrvtty A buffer suttable for
the specific assay of pancreatic hpase acttvtty has recently been developed (see Note 1)
3. Methods
3.1. Preparation of Fluorescence Standards and Lipase Substrate
3 1 1. Preparation of the Fluorescence (Pyrenedecanoic Aad) Standards
The followmg procedure can be used to prepare 30 standard samples con-
taming defined concentrations of unquenched fluorophore. These standards are
Lipase Assays Using Fluorogenic Substrates 25
used to create a cahbratlon plot to quantltate the amount of substrate hydro-
lyzed during the hpase assay (see
Fig. 2).
1 Dissolve 480 nmol fatty acid-free bovine serum albumin (approx 32 mg) m 240
mL PBS or the same buffer used for the hpase assay (see Subheading 2.3. for
examples of different hpase buffers)
2 Prepare four concentrations of pyrenedecanotc acid by dlssolvmg 0,O 6, 1 2 and
2 4 nmol of pyrenedecanotc acid m 200 uL tetrahydrofuran With constant stu-
rmg at 37°C add each concentration of pyrenedecanolc acid to 60 mL of the
albumm solutton prepared m step 1, The final concentration of pyrenedecanotc
acid m these standards will be 0, 10, 20,40 pmol/mL; 2 mL of each standard 1s
used to construct the calibration plot (see Subheading 3.3. and Fig. 2) These
standards can be stored for 1 wk at 4°C
3 If detergents are present m the hpase assay, prepare the standards as descrtbed m
step 2, except either supplement or replace the albumm solutton wtth the appro-
priate detergent (e.g , Trtton X- 100, alkylsulfobetam, octylglucostde, sodmm
cholate, or deoxycholate). For supplementation, add 200 uL of the pyrene-
decanolc actd/tetrahydrofuran solution to 60 mL of an albumm solution as pre-
pared m step 1 that contains 0 04 mMTriton X-l 00 (see Note 4) For replacement,
add 200 uL of the pyrenedecanolc actdftetrahydrofuran solutton to 60 mL of
0 2 mM Triton X- 100 m PBS

4 Alternattvely, lyophthzed pyrenedecanotc actd/albumm standards can be pur-
chased commercially (see Subheading 2.1.) These preparations represent ready-
to-use standards after reconstitution in 2 mL of PBS or the same buffer used for
the ltpase assay The final concentrattons of these standards are 0, 10, 20, and
40 pmol/mL or higher
3.1.2. Preparatron of the Lipase (Alkykiiacylglycerol) Substrate
The followmg procedure can be used to prepare enough llpase substrate for
30 assays Once prepared, the substrate can be stored overmght at 4°C or 3 h
at
room temperature
1. Dissolve 120 nmol fatty acid-free bovme serum albumin (approx 8 mg) m 60 mL
PBS or the same buffer used for the lipase assay (see Subheading 2.3. for
examples of dtfferent hpase buffers).
2. To prepare a detergent-free substrate, dissolve 120 nmol of the fluorogemc
alkyldtacylglycerol substrate rn 200 pL of tetrahydrofuran wtth constant sttrrmg
at 37°C Add the substrate/tetrahydrofuran solution to the 60 mL albumm solu-
tion prepared in step 1 to gave a final concentration of 2 nmol/mL alkyl-
dtacylglycerol Each hpase assay utilizes 2 mL of this substrate solutton
3 To prepare substrate supplemented with detergent and albumm, follow the same
procedure descrtbed m step 2 except dissolve 240 nmol fluorogemc alkyl-
dtacylglycerol substrate m 200 uL of tetrahydrofuran and add this solution to
60-mL albumm solution contammg 0 04 pA4 Trtton X- 100 The assay using this
detergent supplement substrate is much more sensmve.