A facile assay to monitor secretory phospholipase A

2
using 8-anilino-1-naphthalenesulfonic acid
Hamse K. Vivek
a
, Supritha G. Swamy
a
, Babu S. Priya
b
, Gautam Sethi
c
, Kanchugarakoppal S. Rangappa
b
,
S. Nanjunda Swamy
a,
a
Department of Biotechnology, Sri Jayachamarajendra College of Engineering, Mysore 570006, India
b
Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India
c
Department of Pharmacology, National University of Singapore, Singapore 117597, Republic of Singapore
a r t i c l e i n f o
Article history:
Received 6 February 2014
Received in revised form 22 May 2014
Accepted 27 May 2014
Available online 7 June 2014
Keywords:
Fluorescence property of ANS in response to
sPLA
2
Triton X-100
LPC
DMPC
a b s t r a c t
Secretory phospholipases A
2
(sPLA
2
s) are present in snake venoms, serum, and biological uids of
patients with various inammatory, autoimmune and allergic disorders. Lipid mediators in the inam-
matory processes have potential value for controlling phospholipid metabolism through sPLA
2
inhibition.
Thus, it demands the need for screening of potential leads for sPLA
2
inhibition. To date, sPLA
2
activity has
been assayed using expensive radioactive or chromogenic substrates, thereby limiting a large number of
assays. In this study, a simple and sensitive NanoDrop assay was developed using non-uorogenic and
non-chromogenic phospholipid substrate 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 8-
anilino-1-naphthalenesulfonic acid (ANS) as interfacial hydrophobic probe. The modied assay required
a 10ng concentration of sPLA
2
. ANS, as a strong anion, binds predominantly to cationic group of choline
head of DMPC through ion pair formation, imparting hydrophobicity and lipophilicity and resulting in an
increase in uorescence. Triton X-100 imparts correct geometrical space during sPLA
2
catalyzing DMPC,
releasing lysophospholipid and acidic myristoyl acid, which in turn alters the hydrophobic environment
prevailing around ANSDMPC, which leads to weakening of the electrostatic ion pair interaction between
DMPC and ANS ensuing decrease in uorescence. These characteristic uorescence changes between
DMPC and ANS in response to sPLA
2
catalysis are well documented and validated in this study.
2014 Elsevier Inc. All rights reserved.
Inammation is a complex immunological cascade associated
with various factors, out of which secretory phospholipase A
2
(sPLA
2
)
1
is found to be the primary mediator, followed by cyclooxy-
genase-2 (COX-2) or lipooxygenase (LOX) [1]. sPLA
2
represents a
ubiquitous group of enzymes serving a number of functions, from
toxicity to phospholipid metabolism, digestion, cellular signaling
and antimicrobial activities. sPLA
2
s are esterolytic enzymes that
cleave the sn-2 acyl chain of glycerophospholipids to yield lysophos-
pholipids and free fatty acids (FFAs), thereby increasing cellular ara-
chidonic acid and lysophospholipid levels, which are further
metabolized by the COX-2 or LOX enzymatic pathway. These
products produce diverse families of proinammatory eicosanoids,
platelet-activating factor (PAF), prostaglandins, and leukotrienes
that are involved in inammation and related processes [2,3]. sPLA
2
enzymes are abundantly found in nature and commonly found in
snake venoms from Viperidae, Hydrophidae, and Elaphidae families,
have been studied extensively due to their potent pharmacological
and physiopathological effects in living organisms [4]. sPLA
2
cata-
lytic mechanisms and structures are conserved with a high degree
of sequence homology between different snake species [5]. sPLA
2
s
are low-molecular-mass enzymes (1319 kDa) with a rigid tertiary
structure. So far, 11 sPLA
2
s have been described in mammals belong-
ing to groups IB, IIA, IIC, IID, IIE, IIF, III, V, X, XIIA, and XIIB. Snake ven-
oms are predominantly good sources of group I and group II sPLA
2
enzymes and are similar in their primary and secondary structures
to mammalian enzymes, but they induce various pharmacological
effects in victims [6]. In general, mammalian enzymes are nontoxic
and do not induce potent pharmacological effects. However, some
of them indeed play a crucial role in numerous diseases, such as
chronic inammation, rheumatism, osteoarthritis, tumor
http://dx.doi.org/10.1016/j.ab.2014.05.024
0003-2697/ 2014 Elsevier Inc. All rights reserved.

Corresponding author. Fax: +91 821 2548290.

E-mail address: nanju_chem@yahoo.com (S. Nanjunda Swamy).
1
Abbreviations used: sPLA
2
, secretory phospholipase A
2
; COX-2, cyclooxygenase 2;
LOX, lipooxygenase; PG, prostaglandin; HPLC, high-performance liquid chromatog-
raphy; ANS, 8-anilino-1-naphthalenesulfonic acid; DMPC, 1,2-dimyristoyl-sn-glycero-
3-phosphocholine; RFU, relative uorescence units; DCA, deoxycholic acid; LPC, 1-
myristoyl-2-hydroxy-sn-glycero-3-phosphocholine; BSA, bovine serum albumin; RT,
room temperature; 4PL, four parametric logical; S, substrate; I, inhibitor; E, enzyme.
Analytical Biochemistry 461 (2014) 2735
Contents lists available at ScienceDirect
Analytical Biochemistry
j our nal homepage: www. el sevi er . com/ l ocat e/ yabi o
progression, asthma, psoriasis, septic shock, and adult respiratory
distress syndrome [1,79]. Inhibitors of sPLA
2
have several therapeu-
tic applications; hence, an economically feasible, short-time assay
method is needed for screening a library of compounds to identify
lead molecules showing specic sPLA
2
inhibition. In addition,
nonspecic phospholipase inhibition leads to impairment of physio-
logically important prostaglandin (PG) synthesis. The intracellular
PLA
2
G4 and PLA
2
G6 enzymes are specically important in the
regulation of PG biosynthesis because their sites of action are the
perinuclear membranes where downstream arachidonic acid metab-
olizing enzymes (cyclooxygenases and PG synthases) reside [1,10].
Most of the sPLA
2
assays developed to date are expensive, time-
consuming and tedious, involving separation of reaction mixture
by thin-layer chromatography (TLC), solvent extraction to recover
the product [11], use of pH-sensitive dyes [12], chromogenic
reagents [13], and high-performance liquid chromatography (HPLC)
coupled with a uorescence detector to monitor the product [14].
Spectrouorometric-based assays have been proved to be an alterna-
tive method for sPLA
2
assays [15,16], but they employ expensive
uorescence-labeled phospholipid that also quenches the enzyme
activity at higher concentrations. Radiometric-based sPLA
2
assays
using radiolabeled Escherichia coli cells are sensitive, but they are
tedious, time-consuming, and expensive [17]. As reported
previously, sPLA
2
activity was evaluated using scattering mode in a
spectrouorometer, which involves a simple protocol but fails to
explain enzyme inhibition and its kinetic parameters associated in
the assay [16,18].
Among various sPLA
2
assays reported, an assay using a
non-uorescence molecule having uorescence characteristics by
virtue of its chemical nature that do not interfere with reducing
enzymatic activity is favorable. The current article explains the
detailed principle behind the sPLA
2
assay by virtue of uorescent
property associated with 8-anilino-1-naphthalenesulfonic acid
(ANS) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC).
ANS is an interfacial probe molecule; when it binds to a hydropho-
bic core of substrate, it acts as a uorescent molecule [19]. The
ANS-based uorescence assay follows sPLA
2
hydrolysis by direct
substrate depletion and change to a hydrophilic environment,
causing a decrease in relative uorescence units (RFU). As reported
previously [20], the work fails to explain the characteristic features
of ANSDMPC in relation to its uorescence property and sPLA
2
catalysis. Thus, using the information from previous reports
[19,2123] and based on our results, we have made an attempt
to explain the mechanism of action of sPLA
2
catalysis. Based on
the uorescence property of ANSDMPC adduct.
As reported previously for continuous sPLA
2
assay [20], only 6%
of product formation was observed, which could be difcult to
determine the kinetic parameters. In addition, it fails to explain
the mechanism underlying the assay. This prompted us to under-
stand and explain the uorescence property behind ANSDMPC.
We modied the reported continuous sPLA
2
assay [20] protocol
into a discontinuous mode that is simple and sensitive, requires
only 10ng of sPLA
2
enzyme, and uses neutral detergent (Triton X-
100), leading to increased product formation. This work sheds light
on explaining the kinetic parameters and characteristic features of
ANSDMPC in relation to sPLA2 catalysis in relation to its uores-
cence property associated with sPLA
2
catalysis. This assay can be
employed for screening sPLA
2
activity from various sources in
either puried or crude form.
Materials and methods
Lyophilized powders of Vipera russellii and Naja naja (Indian
cobra) snake venom were purchased from Irula Cooperative Soci-
ety (Chennai, India) and Haffkine Institute for Training, Research,
and Testing (Mumbai, India), respectively. Fresh samples of pleural
and ascitic uids were collected from JSS Hospital (Mysore, India).
ANS, DMPC, deoxycholic acid (DCA), Sephadex G-75, Sephadex G-
50, and CM-Sephadex C-25 were purchased from SigmaAldrich.
1-Myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC) was
purchased from Avanti Polar Lipids. HPLC-grade methanol,
dimethyl sulfoxide (DMSO), and analytical-grade HCl were pur-
chased from Fisher Scientic. Tris(hydroxymethyl) aminomethane,
sodium azide, fatty acid free bovine serum albumin (BSA) fraction
V, sodiumhydroxide, calciumchloride dihydrate, and sodiumchlo-
ride were purchased from Merck. Quercetin and Triton X-100 were
purchased from Himedia Laboratories. Sodium acetate, glycine,
and ethyleneglycol-bis(b-aminoethyl ether)-N,N
1
-tetraacetic acid
were purchased from SRL Chemicals. Milli-Q water was used
throughout the experiments. All chemicals were of analytical grade
and all solvents were of HPLC grade. NanoDrop 3300 uorospec-
trometer from Thermo Scientic was used to record the
uorescence.
DMPC substrate preparation
Briey, a working solution of 1 mM DMPC substrate in metha-
nol containing 2 mM Triton X-100 in Milli-Q water was prepared.
The resulting substrate solution was spun at 1000g for 5 min to
form uniform mixed micelles.
Purication of sPLA
2
from V. russellii venom (VRV-PL-VIIIa)
VRV-PL-VIIIa from V. russellii venom was puried to homogene-
ity as reported previously [24]. Protein was estimated by Lowrys
method [25]. Briey, V. russellii venom (100 mg) was fractionated
on Sephadex G-75 column (1.5 140 cm) using 0.05 M phosphate
buffer (pH 7.0). The protein was resolved into three fractions. The
second fraction, constituting 30% of the total protein, showed sPLA
2
activity. This sPLA
2
fraction was lyophilized and further subjected
to CM Sephadex C-25 column (1.5 40 cm) chromatography. The
fractions were eluted stepwise using phosphate buffers of varying
ionic strengths (0.050.2 M) and pHs (7.08.0). They were resolved
into two fractions labeled as V and VIII. The above eluted two frac-
tions were similar to the V and VIII protein prole as reported pre-
viously [24]. The lyophilized fraction VIII was next subjected to
Sephadex G-50 column (1.0 40 cm) chromatography and eluted
using 0.05 M phosphate buffer (pH 7.0), and the obtained peak
was checked for sPLA
2
activity. Homogeneity was checked by
sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS
PAGE) and reverse phase (RP)HPLC, which is concordant as
reported previously (data not shown) [24].
Comparison of continuous assay and improved discontinuous sPLA
2
assay
The continuous assay was performed as described previously
[20]. The difference in activity was determined using DCA or Triton
X-100 as detergent. Briey, 0.2 ml of reaction buffer (50 mM Tris
HCl [pH 7.5] containing 100 mM NaCl, 2 mM NaN
3
, 5 lg/ml BSA,
and 10 lM ANS), 20 ll of 1 mM DMPC containing 1 mM DCA/
2 mM Triton X-100, and 10 ll of CaCl
2
stock solution (100 mM)
were taken and incubated at 25 C for 10 min. Reaction was started
by adding 5 ll of sPLA
2
stock solution (3.2 lg/ml, VRV-PL-VIIIa)
and monitored for 20 min.
In our improved discontinuous assay, briey, a total reaction
mixture of 0.1 ml containing activity buffer (50 mM Tris buffer
[pH 7.5] and 10 mM CaCl
2
) was used. Then 10 ll of substrate
(1 mM DMPC containing 1 mM DCA/2 mM Triton X-100) was
added and preincubated for 5 min at 37 C. Activity was initiated
by adding 5 ll of sPLA
2
from stock solution of 146.6 nM. Reaction
28 A Facile assay to monitor secretory phospholipase A2/ H.K. Vivek et al. / Anal. Biochem. 461 (2014) 2735
mixture was incubated for 30 min at 37 C. Then 50 ll of quench-
ing solution (containing 50 lM ANS, 2 mM NaN
3
, and 50 mM eth-
ylene glycol tetra acetic acid [EGTA]) was added, vortexed for 30 s,
and incubated for 5 min at room temperature (RT). Then 2 ll of
this solution was pipetted to measure RFU in a NanoDrop
ND3300 (version 2.8) using excitation with ultraviolet (UV) LED
(370 10 nm) and emission at 480 nm in dark conditions. Enzyme
activity was calculated by Eq. (1), where DRFU is the change in RFU
of test with respect to control and the resultant RFU is compared
with standard curve of LPC to determine the sPLA
2
activity. RFU
of DMPC with enzyme and without enzyme are RFU
(t)
and RFU
(c)
,
respectively (see Fig. 3C in Results):
DRFU RFU
c
RFU
t
: 1
LPC standard curve
The LPC standard curve was constructed using different LPC
concentrations (0300 lM). Briey, a total reaction mixture of
0.1 ml containing activity buffer (50 mM Tris buffer [pH 7.5] and
10 mM CaCl
2
) and varied concentrations of LPC was used. Triton
X-100 in the ratio of 1:2 was added and incubated for 5 min at
37 C. Quenching solution was added, vortexed for 30 s, and incu-
bated for 5 min at RT. Then 2 ll of reaction mixture was pipetted to
measure RFU as described earlier.
Effect of ANS on sPLA
2
activity
Effect of ANS on sPLA
2
enzyme, inhibitor (quercetin), and sub-
strate were determined by taking respective controls to validate
the efciency of the assay. Briey, a total reaction mixture of
0.1 ml of activity buffer (50 mM Tris buffer [pH 7.5] and 10 mM
CaCl
2
) was preincubated for 5 min at 37 C. Activity and inhibition
tubes (containing 10 ll of 1 mM DMPC and 2 mM Triton X-100)
were incubated for 30 min at 37 C, after the addition of sPLA2.
Reactions were terminated on the addition of quenching solution,
and RFU values were recorded as described earlier. Furthermore,
the effect of quercetin on uorescence of S + ANS was evaluated
by titrating different concentrations of quercetin (010 lM)
containing 100 lM DMPC and 50 lM ANS.
Detection of sPLA
2
enzyme activity from different sources
Crude lyophilized powders of V. russellii, N. naja and inamma-
tory exudates pleural and ascitic uids were dissolved in 50 mM
Tris buffer (pH 7.5). Samples were centrifuged at 8000 rpm for
10 min at 4 C to remove cell debris, and clear supernatants were
checked for protein concentrations by Lowrys method [25]. Then
30 ng/5 ll of the respective proteins was used as the source of
sPLA
2
. Partially puried VRV-PL-V was taken along with different
sources, and enzyme activity was measured by Eq. (1).
Optimization of pH on sPLA
2
enzyme activity
Briey, a total reaction mixture of 0.1 ml containing different
buffers (pHs 4.013.0) to determine the pH dependence on activ-
ity50 mM sodium acetate buffer (pHs 4.06.0), Tris/HCI buffer
(pHs 7.08.0), and glycine/NaOH buffer (pHs 9.013.0)was added
to activity buffer containing 10 mM CaCl
2
. Then 10 ll of substrate
(1 mM DMPC and 2 mM Triton X-100) was added and incubated
for 5 min at 37 C. Activity was initiated by adding 10 ng of sPLA
2
.
Reaction mixture was incubated for 30 min at 37 C. Reactions
were stopped by adding quenching solution, and RFU values were
recorded as described earlier.
Optimization of Triton X-100 concentration
Briey, in a total reaction mixture of 0.1 ml, containing activity
buffer (50 mM Tris buffer [pH 7.5], 10 mM CaCl
2
), and 10 ll of sub-
strate (1 mM DMPC and 06 mM Triton X-100) were incubated for
5 min at 37 C. Activity was initiated by adding 10 ng of sPLA
2
.
Reaction mixtures were incubated for 30 min at 37 C. Quenching
solution was added, and RFU values were measured as described
earlier.
Calcium dependence on sPLA
2
activity
Calcium dependence on sPLA
2
activity was determined. Briey,
a total reaction mixture of 0.1 ml containing activity buffer (50 mM
Tris buffer [pH 7.5] and different concentrations of CaCl
2
ranging
from 0 to 10 mM) and 10 ll of substrate (1 mM DMPC and 2 mM
Triton X-100) were added and incubated for 5 min at 37 C. Activ-
ity was initiated by adding 10 ng of sPLA
2
enzyme. Reaction mix-
tures were incubated for 30 min at 37 C. Quenching solution
was added, and RFU values were measured as described earlier.
Activity was calculated by Eq. (1).
Optimization of sPLA
2
enzyme concentration
sPLA
2
concentration required for maximal activity was deter-
mined. Briey, a total reaction mixture of 0.1 ml containing activity
buffer (50 mM Tris buffer [pH 7.5] and 10 mM CaCl
2
) and 10 ll of
substrate (1 mM DMPC and 2 mM Triton X-100) was added and
incubated for 5 min at 37 C. Activity was initiated by adding dif-
ferent concentrations of sPLA
2
enzyme ranging from 0 to 50 ng.
Reaction mixtures were incubated for 30 min at 37 C. Quenching
solution was added, and RFU values were measured as described
earlier. Activity was calculated by Eq. (1).
Determination of sPLA
2
activity, K
m
, and V
max
sPLA
2
activity was determined for an optimal enzyme concen-
tration of 10 ng as described earlier. K
m
and V
max
values were
determined by adding different concentrations of substrate DMPC
ranging from 0 to 200 lM nal concentrations.
Inhibition of sPLA
2
by quercetin
Briey, a total reaction mixture of 0.1 ml containing activity
buffer (50 mM Tris buffer [pH 7.5] and 10 mM CaCl
2
) and different
concentrations of inhibitor quercetin (0100 lM) were used in
control and test. Here 10 ng of sPLA
2
was added to the test and
incubated for 2 min at 37 C. Activity was initiated by adding
10 ll of substrate (1 mM DMPC and 2 mM Triton X-100) and incu-
bated for 30 min at 37 C. Quenching solution was added to the
reaction mixture, vortexed for 30 s, and incubated for 5 min at
RT. Then 2 ll of this solution was pipetted to measure RFU. IC
50
was calculated using Eq. (2) and was veried and validated using
a sigmoidal four parametric logical (4PL) t curve:
IC
50
1 I V
i
=V
o
; 2
where V
i
is the initial velocity of substrate cleavage in the presence
of inhibitor at concentration [I] and V
o
is the initial velocity in the
absence of inhibitor [26,27].
Statistical analysis
V
max
and K
m
values were determined using nonlinear regression
plots. Sigmoidal 4PL t, one-way analysis of variance (ANOVA),
unpaired t test, and Tukeys multiple comparisons test were com-
puted using GraphPad Prism version 6.04 (GraphPad Software).
A Facile assay to monitor secretory phospholipase A2/ H.K. Vivek et al. / Anal. Biochem. 461 (2014) 2735 29
Results
Comparison of continuous assay and discontinuous sPLA
2
assay in
presence of DCA and Triton X-100
As the sPLA
2
s act on lipidwater micelles, the optimal con-
centration of detergent to form critical micelle concentration
(CMC) plays an important role during catalysis. As reported pre-
viously in the continuous mode of assay [20] using DCA, an anio-
nic detergent, only 6% product formation was observed during
catalysis. With the same assay measuring the RFU using a Nano-
Drop uorospectrometer, an increase in product formation from
6 to 26% was observed (Fig. 1A), and after replacing DCA with
Triton X-100 there was 74% product formation (Fig. 1A). Thus,
comparing the sPLA
2
activity in the presence of both DCA and
Triton X-100, it is noteworthy to mention that improved sPLA
2
activity was observed, and it is also evident from our data that
DCA interferes with DMPCANS uorescence, with a high stan-
dard deviation compared with that of Triton X-100 (Fig. 1A). In
our discontinuous assay mode, using Triton X-100 (neutral deter-
gent), 95% product formation was observed during catalysis. Eq.
(1) aids in quantifying the product formed, which can be corre-
lated with standard LPC linear t. This simple and improved dis-
continuous mode of assay accurately quanties the substrate
depletion by sPLA
2
(Fig. 1B).
LPC standard curve
ANS binds to LPC in a similar manner as that of DMPC. As LPC
concentration increases at a xed concentration of ANS, RFU
increases linearly, as shown in Fig. 2 (R
2
= 0.988, n = 3). This is
the rst report of an LPC standard curve with ANS, used to deter-
mine sPLA
2
assay kinetics. FFAs do not confer a correct binding
site with ANS; hence, the uorescence peaks get distorted and
give false results in evaluating product formation (data not
shown).
Effect of ANS on assay
The current assay principle is based on uorescence due to
ANS bound to a hydrophobic patch of substrate DMPC. Although
it is true that the assay components are hydrophobic, the mode
of ANS binding to the hydrophobic core of DMPC is specic.
The active site of sPLA
2
contains hydrophobic amino acids where
a hydrophobic substrate binds. ANS, when bound to sPLA
2
, elicits
very weak uorescence with an emission at 532 nm (Fig. 3C). Sta-
tistical analysis revealed that, comparing the different groups
using Tukeys multiple comparisons, ANS, ANSenzyme (sPLA
2
),
and ANSinhibitor (quercetin)/enzyme showed a P value > 0.05
(Fig. 3A) and remaining showed a very signicant P value
(<0.0001) when compared within groups (Table 1). sPLA
2
activity
and inhibition (5 lM quercetin) is measured as a release of LPC,
with P value < 0.0005 (Fig. 3B). When ANS + S was titrated with
increased concentrations of the inhibitor quercetin, we found
that a linear decrease in uorescence (R
2
= 0.998, n = 3), suggest-
ing that quercetin is competing with ANS binding to the sub-
strate DMPC. Thus, it can be concluded that uorescence of
ANS + DMPC is inversely proportional to concentration of inhibi-
tor (quercetin) (Fig. 3D). If the inhibitor acts on sPLA
2
, the envi-
ronment around the ANS (ANS + DMPC + I) probe will remain
hydrophobic because there is no catalysis, and it is evident that
with the increase in the inhibitor quercetin concentration with-
out sPLA
2
, quercetin competes with ANS binding to DMPC,
impairing electrostatic ion pair and charge neutralization, leading
to decreased uorescence (Fig. 3D) . As reported previously for
the continuous assay [20], the kinetic parameters are dependent
on DCA and ANS concentrations, whereas in our discontinuous
assay the kinetic parameters are independent of concentration
of ANS.
Fig.1. Comparison between DCA and Triton X-100 employed in continuous and
discontinuous assays. (A) For continuous assay, 1 mM LPC standard was prepared in
the presence of DCA and Triton X-100, and activity was correlated in terms of LPC
released in the graph labeled as DCA (test) and Triton X-100 (test). (B) For
discontinuous assay (F), 1 mM LPC standard was prepared in the presence of DCA
and Triton X-100, and activity was correlated in terms of LPC released in the graph
labeled as DCA (test) and Triton X-100 (test). Values represent arithmetical means
and standard deviations (n = 3).
Fig.2. LPC standard curve exhibiting a linear relationship between RFU and
increasing concentrations of LPC bound to ANS with linear regression (R
2
= 0.988).
Values represent arithmetical means and standard deviations (n = 3).
30 A Facile assay to monitor secretory phospholipase A2/ H.K. Vivek et al. / Anal. Biochem. 461 (2014) 2735
Fig.3. Effect of ANS on sPLA
2
assay. (A) Comparison of RFU of ANS with enzyme/substrate/inhibitor. The reaction mixture containing respective controls (i.e., sPLA
2
, inhibitor,
and substrate) was kept to determine enzyme activity and inhibition in the activity buffer containing 10 mMCaCl
2
preincubated for 5 min at 37 C. Activity and inhibition sets
were incubated for 30 min at 37 C. The reaction was terminated by adding quenching solution. Values represent the arithmetical means and standard deviations (n = 4). One-
way analysis of variance and Tukeys multiple comparison were performed. (B) Comparison of sPLA
2
activity and inhibition. sPLA
2
activity and inhibition were determined in
terms of product released per minute. Unpaired t-test value showed that P < 0.0005. (C) Determination of emission spectrum. Emission spectrum of ANSDMPC in the
presence and absence of sPLA
2
(Em = 480 nm) was recorded. Emission spectra of ANSsPLA
2
enzyme was observed at Em = 532 nm. (D) Binding of quercetin to ANSDMPC. A
titration curve with different concentrations of quercetin (010 lM) with 1 mM DMPC and 2 mM Triton X-100 is shown. Values represent arithmetical means and standard
deviations (n = 3).
Table 1
Statistical analysis of ANS binding.
Raw data (RFU) Tukeys multiple comparisons test P Value Raw data (RFU) Tukeys multiple comparisons test P Value
ANS vs. E + ANS ns ANS + S vs. E + S + ANS <0.0001
S + ANS <0.0001 I + ANS <0.0001
E + S + ANS <0.0001 E + I + ANS <0.0001
I + ANS ns 1012.7 S + I + ANS <0.0001
E + I + ANS ns 980.9 E + S + I + ANS <0.0001
15.2 997.4
13.7 S + I + ANS <0.0001 E + S + ANS vs. I + ANS <0.0001
12.3 E + S + I + ANS <0.0001 225.7 E + I + ANS <0.0001
E + ANS RFU at 532 nm vs. S + ANS <0.0001 246.7 S + I + ANS <0.0001
E + S + ANS <0.0001 233.9 E + S + I + ANS 0.0017
I + ANS ns I + ANS vs. E + I + ANS ns
7.2 E + I + ANS ns 15.6 S + I + ANS <0.0001
8.4 S + I + ANS <0.0001 19.3 E + S + I + ANS < 0.0001
8.0 19.6
E + S + I + ANS <0.0001 E + I + ANS
RFU at 532 nm
vs. S + I + ANS <0.0001
S + I + ANS vs. E + S + I + ANS <0.0001
639.4 19.2 E + S + I + ANS <0.0001
635.4 17.2
657.0 18.0
Note. Signicant differences with raw data comparing ANS/enzyme/inhibition/substrate groups with their respective controls are shown. The data were obtained by one-way
analysis of variance followed by Tukeys multiple comparison. Raw data are represented in RFU. ns: not signicant.
A Facile assay to monitor secretory phospholipase A2/ H.K. Vivek et al. / Anal. Biochem. 461 (2014) 2735 31
Screening for sPLA
2
activity from different sources
Crude samples pleural uid, ascitic uid, lyophilized crude
powder of V. russellii and N. naja, and partial puried VRV-PL-V
were evaluated to mark the sensitivity of our assay in detecting
sPLA
2
. The presence of sPLA
2
was observed in all of the crude sam-
ples (Fig. 4). The optimal pH for sPLA
2
from pleural uid was
observed at 10.0.
Optimization of pH for sPLA
2
activity
In the presence of different pH buffers such as 50 mM sodium
acetate (pHs 4.06.0), 50 mM Tris buffer (pHs 7.08.0), and
50 mM glycine buffer (pHs 9.013.0), it was found that with an
increase in pH, enzyme activity increased exponentially. A bell-
shaped curve was observed at the optimal pH 7.5 (Fig. 5).
Optimization of Triton X-100 concentration for critical micellar
concentration of DMPC
Triton X-100 is a non-ionic detergent that in aqueous solution
forms mixed micelles of 100 to 160 monomers. It induces a specic
geometrical surface for maximal enzymatic activity required for
sPLA
2
action. The maximal molar ratio of phospholipid/Triton X-
100 is in the range of 1:2; this molar ratio does not cause further
change in the physical state of the phospholipid and does not sol-
ubilize the enzyme [28]. With 1 mM DMPC and 2 mM Triton X-
100, an increase in the sPLA
2
activity was observed. Further
increase in Triton X-100 concentration above 2 mM, the sPLA
2
activity decreases progressively, as shown in Fig. 6. Thus, maximal
enzyme activity was observed at the ratio of 1:2 (DMPC and Triton
X-100, respectively).
Effect of calcium concentration
Phospholipids contain electron-rich oxygen on the phosphate
group at physiological pH. Enhancement of the uorescence of
lipid-bound ANS may be brought about by the addition of divalent
cationic species Ca
2+
, which is capable of binding to the electron-
rich oxygen. At a xed concentration of DMPC, an increased con-
centration of Ca
2+
increases the RFU and attains a constant, as
shown in Fig. 7B. It is well established from several studies that
the electron-rich oxygen on the phosphate group of the lipid has
a binding site for Ca
2+
. An increase in the uorescence of DMPC-
bound ANS suggests that these ions interact with the electron-rich
oxygen on the phosphate group and provides a more hydrophobic
environment in the vicinity of the ANS molecule [21]. For optimal
sPLA
2
action, 10 mM Ca
2+
was required (Fig. 7A).
Optimization sPLA
2
concentration for maximal enzyme activity
As the concentration of sPLA
2
increases with a xed DMPC sub-
strate concentration, the enzyme activity increases exponentially
and gets saturated. Our results showed that the optimal concentra-
tion of sPLA
2
was found to be 10 ng, as shown in Fig. 8, which
explains the sensitivity of the assay at very low enzyme
concentrations.
Determination K
m
and V
max
The dependence of sPLA
2
activity on substrate concentration
was determined. The enzyme activity exhibited classical Michae-
lisMenten kinetics. Fluorescence of DMPC bound with ANS is
brought about by electron-rich oxygen of the phosphate group of
DMPC at physiological pH. sPLA
2
acts on its substrate and releases
LPC and hydrophilic FFA (myristic acid) ensuing decrease in uo-
rescence relates to a corresponding increase in LPC release, as
shown in Fig. 9. This is brought about by two factors: hydrophobic-
Fig.4. Comparison of sPLA
2
activity in crude inammatory exudates. sPLA
2
activity
were screened from inammatory exudates like Ascitic, Pleural uid, Snake
venoms, viz., Naja naja (Acidic sPLA
2
), and VRV-PL-V (Basic sPLA
2
) by using
discontinuous method. sPLA
2
sample from pleural uid is active at pH 10. Activity
was initiated by addition of 30ng of protein sample in the activity buffer. Reaction
was terminated by addition of quenching solution. Values represent the arithmetic
mean and SD (n = 3).
Fig.5. Effect of pH on sPLA
2
activity. The pH-dependent sPLA
2
(VRV-PL-VIIIa)
activity curve is shown, where activity was measured in the presence of different
buffers ranging from pH 4.0 to pH 13.0 added into the activity buffer. The reaction
was terminated by the addition of quenching solution. Values represent arithmet-
ical means and standard deviations (n = 3).
Fig.6. Effect of Triton X-100 on sPLA
2
activity. The dependence of Triton X-100 on
sPLA
2
(VRV-PL-VIIIa) activity is shown. The reaction mixture contained 10 mM
CaCl
2
, 50 mM Tris buffer (pH 7.5), and 10 ll of 1 mM DMPC with varied
concentrations of Triton X-100 (06 mM). The reaction was initiated by adding
10 ng of sPLA
2
and was incubated at 37 C for 30 min. The reaction was terminated
by adding quenching solution. Values represent arithmetical means and standard
deviations (n = 3).
32 A Facile assay to monitor secretory phospholipase A2/ H.K. Vivek et al. / Anal. Biochem. 461 (2014) 2735
ity of the interfacial environment and relative change in pH
(V
max
= 10.38 lmol/min and K
m
= 63.24 lM, as determined by our
studies).
sPLA
2
inhibition by quercetin
A small increase in RFU was observed, which can be attributed
to inhibitor competing at the active site of the enzyme and affects
binding equilibrium between Ca
2+
and substrate, thereby prevent-
ing DMPC cleavage. Quercetin binds to sPLA
2
at an active site, mak-
ing no room for substrate binding (Fig. 10A). The IC
50
value of
quercetin for VRV-PL-VIIIa was found to be 2.044 lM, and using
sigmoidal 4PL t (y = Bottom + (Top Bottom)/(1 + 10^((LogIC
50
-
x)HillSlope))) it was further validated (Fig. 10B) using GraphPad
Prism 6.04. Quercetin showed an IC
50
value of 2.01 0.2 lM for V.
russellii sPLA
2
, as shown in Fig. 10A and B, which is concordant
with the previously reported IC
50
value [29].
Fig.7. Effect of CaCl
2
on sPLA
2
activity. (A) The activity buffer contained 50 mM Tris
buffer (pH 7.5) and 10 ll of 1 mM DMPC with 2 mM Triton X-100, and varied
concentrations of CaCl
2
were added into the activity buffer. The reaction was
initiated by adding 10 ng of sPLA
2
. (B) The activity buffer contained 50 mM Tris
buffer (pH 7.5) and 10 ll of 1 mM DMPC with 2 mM Triton X-100, and varied
concentrations of CaCl
2
were added into the activity buffer without sPLA
2
. Values
represent arithmetical means and standard deviations (n = 3).
Fig.8. Optimization of sPLA
2
concentration for maximal enzymatic activity. Linear
regression was performed with the steady-state time points. Values represent
arithmetical means and standard deviations (n = 3).
Fig.9. Dependence of sPLA
2
activity on DMPC concentration. The reaction mixture
contained 50 mM Tris buffer (pH 7.5) and DMPC with Triton X-100 in the ratio 1:2,
and activity was initiated by adding 10 ng of sPLA
2
. The reaction was terminated by
adding quenching solution. Values represent arithmetical means and standard
deviations (n = 4).
Fig.10. sPLA
2
inhibition by quercetin. (A) Inhibitor concentration-dependent
activity plot. The activity buffer contained 50 mM Tris (pH 7.5), 10 ll of 1 mM
DMPC with 2 mM Triton X-100, and 10 mM CaCl
2
, and varied concentrations of
quercetin were added (0100 lM). The reaction was initiated by adding 10 ng of
enzyme. The reaction was terminated by adding quenching solution. Values
represent arithmetical means and standard deviations (n = 3). (B) Sigmoidal 4PL
t of quercetin (0100 lM). Values represent arithmetical means and standard
deviations (n = 3).
A Facile assay to monitor secretory phospholipase A2/ H.K. Vivek et al. / Anal. Biochem. 461 (2014) 2735 33
Discussion
Phospholipase A
2
requires a micellar surface, which acts on sub-
strates that are part of a lipid/water interface [28]. Triton X-100, a
non-ionic detergent, does not interfere with SO
3

and N
+
of ANS-
DMPC adduct because the uorescence of DMPCANS depends
on charge neutralization, which is well documented from our
results. The addition of cationic detergent (hexadecyltrimethylam-
monium bromide) decreases the uorescence (data not shown),
and anionic detergent DCA increases the uorescence of DMPC
ANS, whereas Triton X-100 does not interfere with the change in
RFU of DMPCANS.
VRV-PL-VIIIa showed increased activity in the presence of Tri-
ton X-100 in our discontinuous assay, as compared with the use
of DCA in continuous assay. Following the previously reported con-
tinuous assay using DCA as detergent, the measurement of RFU
using NanoDrop employed in our assay improved the quantica-
tion of product formation from 6 to 26%. It has been reported that
the use of sodium deoxycholic acid as detergent in the assay will
inhibit the sPLA
2
enzyme [30]. In several assays, BSA and NaCl
are used to improve signal linearity and reduce background noise,
respectively [19,22]. It is noteworthy that with higher concentra-
tions of NaCl, Cl

anions may compete with ANS binding to BSA

and result in false interpretation of result. In our discontinuous
assay, the use of these was eliminated.
The mechanism involved in sPLA
2
catalysis is that sPLA
2
hydro-
lyzes the phospholipid substrate, which is a highly specic reaction
among the active site histidine, the Ca
2+
cofactor, conserved water,
and the glycerophospholipid substrate. During catalysis, Ca
2+
binds
to the enzyme at the active site, the conserved Ca
2+
binding loop,
that lies between residues 25 and 33 with a consensus sequence
(YGCYCGXGG) in VRV-PL-VIIIa [24,31]. All Ca
2+
-dependent sPLA
2
s
possess a Ca
2+
binding loop at the active site. Our results also reveal
the role of Ca
2+
as an important cofactor for sPLA
2
catalysis. As the
substrate DMPC is bound to Ca
2+
ion, a relativity small increase in
RFU is observed (Fig. 7B) because it creates a hydrophobic environ-
ment in the close vicinity of ANS. Ca
2+
is masked within the active
site as the sPLA
2
s cleave DMPC at the sn-2 position [31]. sPLA
2
acts
on the substrate DMPC, releasing LPC and hydrophilic acidic FFA
(i.e., myristic acid), which changes the hydrophobic environment
to hydrophilic, leading to weakening of ion pairing between sulfo-
nate anion of ANS with the nitrogen atom of choline (Fig. 3C)
[9,20]. As the concentration of LPC increases, RFU increases linearly
(R
2
= 0.988) (Fig. 2). This reported improved assay method is sim-
ple and accurate when compared with the existing sPLA
2
assays
[13,16,18,20], and it gains an advantage of using non-radiolabeled
substrate, neutral detergent, simple measurements, and short anal-
ysis time for determining enzymatic activity. The results obtained
were consistent by this assay, as is evident from the statistical
analysis. Fundamentally, this assay is based on hydrophobicity;
hence it is important to maintain proper control. ANSenzyme/
inhibitor elicits weak uorescence. ANSenzyme shows emission
at 532 nm, but ANSDMPC and ANSLPC show emission at
480 nm (Fig. 3C). The values of ANSsubstrate/inhibitor (control),
ANSsubstrate/substrateenzyme (activity), and ANSsubstrate
inhibitor/substrateenzyme (inhibition) complexes were found to
be statistically signicant with a P value < 0.0001 (Table 1 and
Fig. 3A).
The core principle behind evaluating sPLA
2
activity using ANS in
our assay lies in the presence (high RFU) and absence (low RFU) of
FFA present at the sn-2 position of DMPC (Fig. 3C). This assay can
be employed in screening of library of compounds targeting inhibi-
tion of sPLA
2
from various sources. In our study, we have deter-
mined the IC
50
value of quercetin against sPLA
2
enzyme VRV-PL-
VIIIa. The IC
50
value is concordant with the value obtained from
radiometric assay [29]; thus, our study overcomes the use of
expensive radioactive substrates. Reagents used in this assay are
stable up to 6 months at 4 C. Overall, this improved discontinuous
assay is simple and accurate and employs a portable NanoDrop u-
orospectrometer for measurements. The assay is cheaper and can
be used to screen a library of compounds in a short time.
Acknowledgments
S.N.S. thanks University Grants Commission (UGC), New Delhi
[UGCMRP vide no. F, no. 38-220/2009 (SR) dated 24 December
2009] and Visvesvaraya Technological University (VTU), Belgaum
[VTU Research Grants vide no. VTU/Aca/2011-12/A-9/739] for
nancial assistance. H.K.V. thanks Council of Scientic and Indus-
trial Research (CSIR), Government of India, for CSIRSenior
Research Fellowship (08/584 (0001)/2012-EMR-I). We gratefully
acknowledge Prof. Cletus J.M. DSouza, Dr. Satish Srinivasan, Dr.
M. Guruswamy, Dr. J.R. Kumar, Prof. K. Kemparaju and Dr. K.S. Gir-
ish for their help with this work.
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