hyperpolarization. The mechanism of the hyperpolarization is discussed. The application of the method to assay phospholipases, allows detection ofanactivity.
BIOCHEMICAL
Vol. 99, No. 1,198l March
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages 275-283
16,198l
A SENSITIVE
ASSAY OF, PHOSPHOLIPASE USING
THE FLUORESCENT PROBE 2-PARINAROYLLECITHIN CLAUDE WOLF, LUCIEN SAGAERT, GILBERT BEREZIAT Laboratoire Saint-Antoine,
C.H.U. Received
January
de Biochimie 27 rue Chaligny,
75012-Paris
5, 1981
Summary A,-phospholipase from Crotalus Adamanteus venom has been assayed using the fluorescent probe 2-all trans-parinoyllecithin. The hydrolysis of the lecithin in an "albumin-rich" incubation medium is paralleled with a fluorescence hyperpolarization. The mechanism of the hyperpolarization is discussed. The application of the method to assay phospholipases, allows detection ofanactivity under 1 nanomole/min and continuous monitoring of the hydrolysis. Introduction The assay procedures
with
determination
of crude
methods
bonds
(time
of IOC-ICOO
nmoles/min.
has been requested phospholipases of protein.
-consuming
acyl-thioester by the Ellman and of their
The availability
assay)
described
of phospholipids.
reagent,
the different
thioesterases
substrate of the
The assay for
assay
are overcome
by the
and co11
(4)
of the
of acylthioester
and the disturbance discussed
(4).
by A successful
dansylphosphatidylethano-
published
phospholipid
275
the range
of radiochemical
hydrolysis
a fluorescent
has been recently
with
of acti-
Disturbances
have been already
fluorescent
2) allows
by Aarsman
oxyesters,
using
(1,
preparationsin
analogs
of lipoprotein-lipase
lamine-labeled
assay
corresponding
by various
The disadvantages
and fixed-time
spectrophotometric
monospecific assay
Titrimetry
intracellular
nmoles/min/mg
continuous
method
range
has been described
sensitivity.
phospholipids(3)
of 0.5-10
using
a variable in the
radiolabeled vities
of phospholipase
by J.D.Johnson
analog
(5).
2-parinoyl-
0006-291X/%1/050275-09$01.00/0 Cop.vrigh f 0 1981 by Academic Press, Inc. All rights of reproducrion in any form reserved.
Vol. 99, No. 1,198l
8lOCHEMlCAL
phosphatidylcholine parinaric
acid
fluorescence perform
described with
polarization,
a simple,
Material Synthesis
respect
AND
by Sklar
et al
(6)
RESEARCH
and the
to the environnement, have been
continuous
BIOPHYSICAL
combined
and sensitive
as it in the
assay
COMMUNICATIONS
sensitiveness
of
is measured
present
work
by to
of AZ-phospholipase.
and methods of 2-all
trans-parinoylphosphatidylcholine.
All-trans parinaric acid (9, 11, 13, 15-octadecatetraeneoic acidin the all-trans configuration) is purchased from Molecular Probes [Texas, USA] or prepared from the seed of Parinari glaberrimum [kindly provided by Dr. M.Lambert, WesternSamoa]. Lyso-1-acyl-phosphatidylcholine was prepared from egg yolk lecithin by the action of Crotalus Adamanteus venom [Sigma] and purified by repeated washing with anhydrous ether (7). The acylation conditions differ in 2 aspects from the conditions described by Sklar (6) : 1) Pure parinaric anhydride was made by the Lspidot procedure (8) 2) The conditions described by Robles (9) were substituted by the efficient procedure of Gupta (10) using the N, N-dimethyl-4-aminopyridine catalyst [Merck]. The ratio lysocompoundlcatalyst is 1.5. The yield is 40 % determinated by phosphate analysis after 24 h. The absorption spectra were consistent with those described by Sklar (6) and were repeated with minor changes during storage of the fluorescent phospholipid in chloroform at -18°C. Incubation
conditions
for
phospholipase
assay.
AZ-phospholipase [from Crotalus Adamanteus (Sigma)] is assayed at 37°C in an albumin-rich (10 mg/ml) buffer (4 mM CaC12, 250 mM NaCl, 10 mM Tris HCl (pH 8.5)). The high ionic strenght achieved by NaCl prevents the segregation of released fatty acids by Ca++ and hence favours the binding onto albumin. [Sigma ; "essentialy fatty acids free" grade]. The substrate is added as phosphatidylcholine vesicles prepared by sonication until optical clarity of the following mixture : ovolecithin (1 mg), 2-all trans-parinoyllecithin (1 ug) and butylhydroxytoluene as antioxydant (10 nmoles) for 2 ml of carefully deoxygenated buffer. Fluorescence
measurement
Fluorescence polarization is continuously monitored in the spectrofluorometer JY3 [Jobin Yvonl fitted with 2 linear polarizers [PolaroTd HPN'BI. The temperature is measured by a thermistance immersed in the cuvette. The incubation medium is continuously stirred by a micromagnet. The fluorescence polarization (P) is calculated according to the formula p _ (111 -111 (In -111
Albumin) Albumin)
- (IA-ILAlbumin) + (IL-II Albumin)
X G where X'G
;
III and 11 are the intensities of fluorescence at 432 nm polarized oarallel and perpendicular to the polarized exitation light at 320 nm, 111 Albumin and I&Albumin are the fluorescence intensities in both directions from the incubation medium without the fluorescent phospholipid vesicles and G is the correction factor for instrumental anisotropy (II).
276
BIOCHEMICAL
Vol. 99, No. 1,19Bl
PN*,mn-rlu-
0.17..
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
V*drlrI~) 1 AlbumIn
Experimental acid bound
diagram of onto albumin
fluorescence or intercalated
polarization variations for in phospholipid vesicles
free parinaric at 38OC.
3 nmoles of parinaric acid (PNA) in 1 ~1 tetrahydrofuran are added under continuous stirring in 2.5 ml aqueous buffer containing either 400 nmoles of fatty acid-free albumin (1) or 40 nmoles of non-fluorescent lecithin vesicles (2). Fluorescence polarization ( A excitation = 320 nm ; X emission = 432 nm) is recorded and then.either non-fluorescent lecithin vesicles (1) or fatty acid-free albumin (2) is added and the fluorescence polarization recorded again.
Results The summarized bound to
in
to
fig.1
at
tional
diffusion.
38°C
increasing
phospholipid of
a high
the
hydrolysis
fluorescence of
the
amount
Hence
of
the
acid
parinaric to
acid
albumin.
0.31.
to
It bound
onto
the fluidity
in
will
277
even
the
fluo-
fast
rota-
AZ-phospholipase off
the
hydrophobic is
noticeable albumin
move
tight
parinoyllecithin is
bound
of this by
by
the
is
when parinaric
2-parinoyllecithin
of
and
depolarization
uptaken
hydrolysis
acid
hindered
acid
is
alternatively
parinaric
On the contrary,
parinaric be
been
strongly
vesicles,
of
polarization
has
When
is
released
is
fluorescence
fluorescence
hyperpolarization. parinaric
by
in phospholipid
environnement
albumin.
assay
diffusion
allows
During an
or
reaches
intercalated
medium
free
rotational
polarization
is
the
vesicles
its
rescence
of where
lecithin
albumin
acid
principles
in
figure
in
the
fluid pockets
paralleled 1 that presence
with
a
most of
vesicles
Vol. 99, No. 1,198l
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
-5oj
.Tima.min.
0
Fig. 2 Albumin-dependence hydrolysis of
10
then
20
of fluorescence 2-parinoyllecithin
The fluorescence of 2-parinoyllecithin 320 nm and analysed continuously polarizers. Experiment is started addition of the enzyme. At time hydrolysis is stopped by addition
and
COMMUNICATIONS
the polarization
is
hyperpolarization by A,-phospholipase.
recorded
during
the
is excitated by a polarized beam at at 432 nm through parallel and perpendicular at time 0 in a albumin-free buffer by 7.5 min, the albumin powder is added. The of 100 mM EDTA.
close
to its
maximum value
even
in the
presence
of vesicles. The dependence is
seen
lipase albumin.
albumin
in is
figure not
parinaric
a low
a burst
depolarization
in
has been ended
by addition
of EDTA since
(2)
slight
the Ca++ -segregated In
and the
the
paralled
sented.
The
dependence
in the observed.
accounts
the vesicles.
The
But
for
experiment
Crotalus
as
of
early
as
the released shown
Adamanteus
depolarization
absence
in
2
figure
A,-phospholipase
due to the release
of
PNA.
figure
through
is
of hyperpolarization
accumulated
Ca ++ dependent
hyperpolarization by the phospho-
polarization
acid
is
fluorescence
of Z-parinoyllecithin
by an increased
contrary
added,
of the
The hydrolysis
paralleled
On the
is
2.
on albumin
3 the (I
experimental
) and on
pH of
the the
recordings perpendicular snake
278
of (
phospholipase
I )
the
fluorescence
polarizer is
seen
are (optimum
prepH
BIOCHEMICAL
Vol. 99, No. 1,198l
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Ilnl..rnh.
Fiq. 3 Phospholipase activity in function Crotalus Adamenteus venom is added with 10 mM Tris-HCl at pH 7, 6 or continuously monitored through the (see Method).
around
9) in agreement
remains
stable
and I
polarization.
This
polarization
throughout
in figure
medium
throughout
method
as reported
remaining
given change
the
fluorescence
In this
with
its
acid
release
as well
( Itot ).
This yield
evidences this
in polarization
as with increase is from
experiment
with
the
acids
aliquots
variation
by the
(12)
and the fluorescence First,
it
is
for
(not
of the
seen that
is
with
correlated
parinaric
acid
lifetimes
an increase
time
if
bound
onto
In figure is
total
explained
measurements
published).
279
the
in the fluorescence
is easily
incubation
the
of the fluorescent Secondarily
of Itot
venom is
by the Dole-
by the venom.
an increase
of
of the incubation
have been extracted
albumin
Since
with the
process
fluorescence
the
with
(2).
intensity
simultaneously
with the disappearance
assumed
assumption
total
of fatty
hydrolysis onto
of M. A. Wells
correlated
has been recorded.
of parinaric
to support
the
by S. A. Ibrahim
i.e.
quantic
of Itot
is correlated
polarization
Further
results
hydrolysis
phospholipid
phospholipid,
creased
4. the
hyperpolarization
intensity
revious
increases,
variation
(p)
displayed
in
the
+ 2 I ) is assumed to increase
I tot(ltot=l
binding
with
of the pH of incubation medium. at time 0 in an albumin-rich medium buffered 9. The fluorescence of parinaric acid is parallel and perpendicular polarizers
displayed
an inalbumin. will
be
5 the
linear
for
various
I
Vol. 99, No. 1,1981
Fig. 4 Relationships &?!h'
8lOCHEMlCAL
AND
between hyperpolarization L 1 and release of ."-11-; 2-
in I:;
BIOPHYSICAL
(p), acids
fatty
RESEARCH
total from
COMMUNICATIONS
fluorescence the fluorescent
intensity Z-parinoyl-
Incubation is carried out under the standard conditions (final volume = 4ml) in the cuvette and III and 11 are continuously monitored. At fixed-times 0.4 ml aliquots are sampled and extracted according to the Dole procedure. Release of FA was calculated -from the fluorescence of the remaining substrate.
quantities
of
substrate.
From
tivity
of
the
substrate of
enzyme
in
the
the
lowest
assay
has
acids
been
(6
a non-limiting ul
calculated
It
after
of
activity
hydrolysedlmin.
fatty
presence
is
a 5 min
to
of and
say
incubation
venom)
reaches
that
the
will
concentration added, 0.3
release
be
the
sensi-
nanomoles
of
of
easily
of
1.5
nanomoles
detected.
Discussion Thanks parinoyllecithin, to
the
availability
previously
monitor
Crotalus
to
the
hydrolysis
Adamenteus
cence
emitted
ratio
(10m3)
assay
an
activity
by or
of
venom. the bound of
described
probe onto 0.3
lecithin The
the
fluorescent
by
Sklar
(6),
by
analysis
incorporated albumin
of
after
nanomoles/min.
the
280
we
have
the
polarization
in
the
substrate
hydrolysis, sensitivity
2been
A,-phospholipase
of
This
probe
from of at
is
able
a very
efficient is
fluoreslow to
very
close
BlOCHEMlCAL
Vol.99,No.1,1981
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
L. c . t
80
60
40
Fig. 5 Rate of hyperpolarization concentration.
in
function
of
Crotaleus
Adamanteus
An-phospholipase
Various concentrations of crude venom ( (w--m ) 6 VI, (O-0 ) 12 ul I (&--A ) 25 ~1) are added to 10 ug of lecithin vesicles (ovolecithin/ Z-parinoyllecithin : 1 000/l) suspended in the albumin-rich buffer (300 mM INaCl. 4mM CaC12 , 10 mM Tris-HCl, 1 % albumin ["free fatty-acid" grade], (pH 8.5)). Incubation is carried out in the temperature-controled cuvette (37°C) undercontinuousstirring and fluorescence polarization is recorded in function of time.
to the sensitivity method
of the radiochemical
has the advantages
reaction
products)
structural is probably nolamine
closer (5)
with
the
to the
ideal
and than
by spectrometry
following
to oxygen tricks
1) a careful and addition 2) fluorescence
the
and the
time-consuming
continuous natural
With
regard
of the to its
the 2-parinoyllecithin
than
analogs
fluorescence
(no separation
assays.
lecithin,
formula
thioester
the enzyme The 2 main
sensibility
to be less
and to allow
similarity
methods
the dansylphosphatidyletha(4)
used previously
to monitor
lipolysis.
disadvantages
of the parinoyl-substrate,
and to photobleaching,have
i.e.
been overcome
by the
: deoxygenation
of the buffer
of butylhydroxytoluene is
excitated
used to sonicate
lecithin
asantioxydant
underalow
illumination
281
by the use of
a
BIOCHEMICAL
Vol. 99, No. 1,198l
Polaroid (at
polarizer
AND
preferably
X excitation
to the optically
for
the particularity the
Then
reaction
the
triglyceride
products
monitoring from
the
monitor
in the
tely
it
used
from
is
AI-and
Parinari
Nichols
parinaric
acid
COMMUNICATIONS
polarizer
activity.
noticeable "inner
throughout
the that
filter" the
of albumin
this
present
as well
be
energy
to parinaric parameter
acid
usable
is
to
of parinoyl-substrate.
energy
effect
The natural
the fluorescence
hydrolysis
site
as lysolecithin).
seed could
fluorescence
system
as well
A*-phospholipase.
Finally,
residues
binding
as a low specificity
acid
glaberrimum
interresting
"albumin"
masked by the
buffer
clear
as well
parinaric
to assay
tryptophyl
to be another
affinity
binds
a lipase
assumed
Nevertheless
(it
is able
extracted
transfert
as "hindered"
of a high
procedure
used for
RESEARCH
= 320 nm).
The use of albumin offers
BIOPHYSICAL
transfert
has been
in the standard
comple-
"albumin-rich"
work.
Acknowlegment Grants this
from
DRET (79137)
and DGRST (7970791)
have generously
supported
work.
References 1. de Haas, G.H., (1971) Biochim.
Bonsen, P.P.M., Biophys. Acta,
2. Wells,
M.A.
(1972)
Biochem.,
3. Zieve,
F.J.
(1973)
Anal.
11,
Van Deenen, A.J., Chem. 5, 241-253.
5. Johnson, J. Biol.
J.D., Taskinen, M-R., Chem. 255, 3461-3465.
7. Hanahan, 6. Salinger,
L.A., D.J.
Hudson, (1952)
B.S.
Z. and Lapidot,
9. Robles, E.C. 187, 520-526.
and Van den Bosch,H.
Matsuoka,
and Simoni,
R.D.
(1977)
R.L.
Biochem.
(1980)
l6-,
199-210.
J. Lipid
D. (1969)
282
(1976)
N. and Jackson,
Chem. -195,
Y. (1966)
and Van Den Berg,
L.L.M.
-51, 436-441.
L.L.M.
J. Biol.
and Van Deenen,
1030-1036.
Biochem.
4. Aarsman, Bioorgan.
6. Sklar,
Pieterson, W.A. -239, 252-262.
Res. I,
Biochim.
174-175.
Biophys.
Acta,
819-828.
Vol. 99, No. 1,198l
BIOCHEMICAL
AND
BIOPHYSICAL
10. Gupta, C.Ll., Radhakrishman, R. and Khorana, Acad. Sci. USA 2, 4315-4319. 11. Azumi, 12.
Ibrahim,
T. and MC Glynn, S.A.
(1967)
S.P.
Biochim.
(1962) Biophys.
283
J.
RESEARCH
H.G.
Chem. Phys. Acta,
137,
COMMUNICATIONS
(1977) 37, -
Proc.
Natl.
2413-2416.
413-419.
