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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.
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