Vol.3, No.1, 44-48 (2012) Journ al of Biophysical Chemistry
http://dx.doi.org/10.4236/jbpc.2012.31006
Electroporation and electropermeabilization of lipid
bilayer membranes in the course of snakes’ venom
intoxication
Naira A yvazyan*, Narine Ghazaryan, Naira Zaqaryan
Institute of Physiology, Yerevan, Armenia; *Corresponding Author:
Received 8 November 2011; revised 21 December 2011; accepted 5 January 2012
ABSTRACT
The present study was undertaken to elucidate
how the plastic properties of model membranes
from native lipids of different tissues of rats
change in the course of Macrovipera lebetina
obtuse (MLO), Montivipera raddei (MR) and Naja
kaouthia (NK) venoms processing. T he presence
of viper venom in organism lead to increasing of
the electrical resistance of BLMs from liver and
muscle lipids approximately on a sequence,
while the BLMs from brain lipids has not show n
a noticeable differences of plastic properties
compare the control. The same concentration of
cobra venom leads to decreasing of electrical
resistance of BLMs from 1011 Ohm till 108 Ohm.
The low concentration of venom leads to ap-
pearance of channel activity. Especially it is no-
ticeable in liver lipids in media of bivalen ions.
Keywords: Lipid Bilayer; Electroporation; Viper
Venom; Breaking-Potenti al; Electropermeabilization
1. INTRODUCTION
As Vipers and pit vipers (snakes of the family Viperi-
dae) produce venoms, which contain proteins that are
normally part of the coagulation cascade, the normal
haemostatic system and can cause tissue repair. Never-
theless human envenomations are very often accompa-
nied by clotting disorders, hypofibrinogenemia and local
tissue necrosis. Although viperid venoms may contain
well over 100 protein components, venom proteins be-
long to only a few major protein families, including en-
zymes (serine proteinases, Zn2+-metalloproteinases, L-
amino acid oxidase, gro up II PLA2) and proteins without
enzymatic activity (disintegrins, C-type lectins, natri-
uretic peptides, myotoxins, CRISP toxins, nerve and
vascular endothelium growth factors, cystatin and Ku-
nitz-type proteinase inhibitors) [1,2].
The medicinal value of snake venoms has been known
from ancient times [3-5]. Snake venoms are medicinally
effective at low doses, while their therapeutic properties
are achieved by mechanisms which are different from
those of known therapies. Angiogenesis, the process by
which new blood vessels are formed, is a fundamental
event required for a number of physiological and patho-
logical conditions and the crucial role of cell extracellu-
lar matrix communication in angiogenesis is well estab-
lished [6,7]. Because of their unique biological effect,
many types of snak e’s venom have been u tilized as valu-
able pharmacological reagents for studies on the interact-
tion of their content and organized lipid interfaces, like
as BLMs, LUVs, SUVs MLVs etc. [8,9]. But usually be-
cause of their particular characteristics (size and lamel-
larity) these model membrane systems are not necessarily
accurate descriptions of cell membranes. The binding of
proteins to lipid interfaces depends on the physico-
chemical and structural properties of the membrane sur-
face. It is generally accepted that secreted (integral) en-
zymes are particularly active in the presence of transient
“membrane defects” which have been identified as the
borders between coexisting lipid phases [10 ,11].
After obtaining a stable planar bilayer lipid membrane
(BLM) from brain phospholipids in 1962 by Mueller &
Rudin [12], the electrical properties of membranes have
been intensely studied, but mainly in terms of the mem-
brane pathology related to genetic disorders, disturbance
of cholesterol-phospholipid balance in atherosclerosis,
cancer-related changes in the lipid composition of mem-
branes and etc [13-15]. Currently, there is no doubt that
BLMs formed from natural lipids isolated from tissues of
animals represent an adequate model of the lipid moiety
of biomembranes, which makes it possible to reconstruct
some features characteristic of natural bilayer mem-
branes [16].
The present study was in part prompted by an interest
in the changes of the lipid condition, which take place in
native membranes under the influence of Macrovipera
lebetina obtuse (MLO) and Montivipera raddei (MR)

N. Ayvazyan et al. / Journal of Biophysical Chemistry 3 (2012) 44-48 45
venoms. The modeling of plastic properties of lipid bi-
layers in the interaction with venom could provide im-
portant information [17,18]. And as we thought, it was
necessary to compare the results of these studies with
analogous data from experiments with rats, processed by
venom of Elapidae (Naja kaouthia, NK), containing the
postsynapti c n eurotoxin
2. EXPERIMENTAL PROCEDURE
2.1. Tissue Processing
First, The venom of the Macrovipera lebetina obtusa,
Montivipera raddei and Naja kaouthia was tested for its
ability to induce supramolecular changes in rats after
short-term (10 min) intramuscular injection of the venom
(0.35 mg/kg approx. 0.5 LD50), by modeling of artificial
membranes from native lipid content from some organs
(liver, heart, brain and muscle). We tried to compare the
data of in vitro and in vivo experiments. For in vitro ex-
periments dried lyophilized toxin of MLO was dissolved
in Tris-HCl buffer (pH 7.4) with a concentration of 3 ×
10–5 M.
2.2. Phospholipid Processing
Lipids fractions were isolated from marked tissues of
rats, according to the original Kates method [19]. Then a
vacuum pump was used to remove the chloroform-
methanol mixture. For in vitro experiments there were
incubated with venom solution and held at a constant
temperature of 37˚C for 10 min. Then lipid sediments
were dissolved in nonane (3% solution).
2.3. BLMs Formation and Measurements
The lipid bilayer membranes were formed from the
lipid fractions of rat tissues on a teflon ap ertu re by means
of the Muller method [20]. A teflon cylindrical cup
having a 0.8 mm hole is coupled to a glass chamber; so
the cup separated two compartments filled with 5 ml
electrolyte each. Electrical access to the baths was through
a pair of Ag/AgCl electrodes.
Optical reflectance, electrical resistance and capaci-
tance indicated the formation of planar lipid bilayers.
The electrical parameters of the BLMs was determined
on a device equipped with a Keithley 301 differential
feedback amplifier (United States) in a voltage-fixation
mode (current-clamp mode), which let to keep up a
membrane potential on any level, independent from ionic
streams. The potential setting on exit of generator com-
pletely falls on membrane, the resistance of which is
much higher than that of electrodes, electrolyte and
effective resistance of current’s gauge. Electrometric
device can measure a current through membrane, under
fixed value of transmembrane difference of potentials.
0.1 M KCl, NaCl, LiCl and KJ, KBr was served as ionic
media.
The breaking-potential of membrane recorded in the
experiments in shielded camera is taking as the threshold
value of the voltage applied. Potential of membrane rap-
ture is a criterion for valuation of natural defects of
model membranes. Under electrical potential, radius of
these holes is increasing. There is a critical value of ra-
dius (ro), an d wh en rφ > ro membrane is destroying.
2.4. Statistical Analysis
For quantitative analysis of electrical parameters of
BLMs, a Student’s test was used to compare differences
at each time point, considering P < 0.05 as significant.
All data were presented as mean ± SEM (n = number of
experiments).
3. RESULTS
3.1. Electrical Current Measurements in
Planar Bilayer Lipid Membranes (BLMs)
Electrical measurements of planar bilayers provide a
means of measuring the changes in conductance, due to
the formation of channels, pores or defects on the mem-
branes, caused by the binding, insertion and translocation
of peptides, at lowest concentrations, at which transient
events can be detected. It also enables the determination
of other features as: open probability, ionic selectivity of
the pores and an estimative of pore’s size. Table 1 shows
the results obtained with the in vitro action of three types
of snake venom on the electrical conductance of BLMs
from brain lipids of rats in media of K+ ions.
For the first series of experiment aliquots of 0.05 ml
venom stock solution (concentration 10 mg/ml in Tris/
HCl buffer, pH 7.4) w as added to the both sides of mem-
Table 1. Snake venoms in vitro action on the electrical properties of BLMs (resistance Rm, conductivity gm and breaking-potential Ur)
formed from brain lipids in media with K+.
Control Vipera lebetina Montivipera raddei Naja kaoutia
Rm (Ohm) (1  0.7) × 1011 (6  0.8) × 108 (1.9  0.3) × 109 (3.3  0.2) × 108
gm (Ohm–1 ) (2  0.2) × 10–11 3.3 × 10–9 4 × 10–10 3 × 10–9
Ur (mV) 448  12 270  8 580  10 280  9
*Average value (±SEM) of mi ni mum 7 different experiments.

N. Ayvazyan et al. / Journal of Biophysical Chemistry 3 (2012) 44-48
46
brane. After a few minutes of equilibration, –100 mV
potential was applied in order to monitor changes in
electrical properties of BLMs. Starting at 0.2 ml, venoms
of MLO and NK showed dramatic changes of the mem-
brane conductance (macroscopic integral conductance)
and venom of MR showed significant activity (Tabl e 1).
An excess venom added in both sides of BLM raised
sharply the membrane conductance and witnessed about
cumulative mode of venom-membrane interaction. For
the next series of experiment, the lipid fraction, after
removing the chloroform-methanol mixture was incu-
bated with venom stock solution and held at a constant
temperature of 37˚C for 10 min. Then venom solution
was washed out and BLMs were formed from lipid mix-
ture in nonane. This procedure didn’t lead to any signifi-
cant changes in the means of conductance of bilayers
induced by MLO and MR venoms, but BLMs from brain
lipids of rats incubated with cobra venom showed a de-
pendence of electrical resistance of bilayers from proc-
essing time (Figure 1) and venom concentration (Figure
2).
Collectively these results indicate that components of
MLO and MR venom demonstrate rather surface activity
during membrane-peptide interaction, while at least one
or more components of cobra venom is definitely pene-
trate the bilayer. Indeed, single-channel current events
induced by NK venom have been recorded in BLMs at
lowest concentrations (<8 × 10–4 mg/ml).
3.2. Planar Lipid Bilayer Preparation for in
Vivo Experiments and Ionic
Permeability of BLMs
Planar lipid bilayers for these series of experiments
were formed from solutions of native lipid mixtures from
different tissues of rats (liver, heart, brain and muscle)
after short-term (10 min) intramuscular injection of the
venom (0.35 mg/kg approx. 0.5 LD 50).
The presence of viper venom in organism lead to the
increase of the electrical resistance of BLMs from liver
and muscle lipids approximately on a sequence, while
the BLMs from brain lipids have not shown noticeable
(Tables 2, 3). The same concentration of cobra venom
leads to the decrease of electrical resistance of BLMs
from 1011 Ohm till 108 Ohm. The low concentration of
venom leads to the app earance of chann el activity (Table
4). It is especially noticeable in liver lipids in media of
bivalen ions.
4. CONCLUSIONS
As we can see from the experiments with planar lipid
layers, that compare cobra venom with viper venom,
peptides affinity is dominated by the electrostatic term.
The hydrophobic effect is thus not sufficient to maintain
a deep embedding of the peptides within the lipids. So
the components of venom are very probably adsorbed at
such a lipid interface with a few non-polar residues at the
contact with lipid chain. This is in agreement with the
data concerned the interaction of different peptides from
snake venoms [21,22]. In such cases the binding to lipids
Figure 1. A dependence of electrical resistance of
rat’s brain lipi ds BLMs (incubate d with cobra ven om)
on the processing time.
Figure 2. A dependence of electrical resistance
of rat’s brain lipids BLMs (incubated with cobra
venom) on the venom concentration.
Table 2. Snake venoms’ in vivo effect on the electrical properties of BLMs (resistance Rm, conductivity gm and breaking-potential Ur)
formed from different tissue’s lipids in K+ medium.
Brain Heart Liver Muscle
Rm
Ohm
gm
Ohm–1 Ur mV
Rm
Ohm gm Ohm–1
Ur
mV Rm Ohm gm Ohm–1
Ur
mV
Rm
Ohm gm Ohm–1 Ur mV
Control 1.3 × 1010 0.7 × 10–11 355 1.3 × 10916 × 10–10 2002 × 1095 × 10–10 219 2 × 109 5 × 10–10180
Vipera lebetina 5.6 × 1010 1.8 × 10–11 309 6 × 1010 1.6 × 10–11 2285.6 × 1010 1.7 × 10–11 291 4.7 × 1010 2 × 10–11 225
Vipera raddei 7.3 × 1 010 1.3 × 10–11 313 - - - 5.3 × 1010 1.9 × 10–11 239 4.6 × 1010 2.1 × 10–11304
Naja kaoutia 7.4 × 1010 1.3 × 10–11 407 2.3 × 1010 4.3 × 10–11 2524.2 × 1010 2.4 × 1 0–11 249 2.9 × 1010 4.2 × 10–11232
Each group contained 20 BLMs from four tissues. P > 0.01 by Stu dent’s t-test relative to the corre sponding control.

N. Ayvazyan et al. / Journal of Biophysical Chemistry 3 (2012) 44-48 47
Table 3. Montivipera raddei venom in vivo effect on the electrical properties of BLMs (resistance Rm, conductivity gm and breaking-
potential Ur) formed from different tissue’s lipids in univalent ions media.
Brain Liver Muscle
Rm
Ohm gm Ohm–1 Ur mV
Rm
Ohm gm Ohm–1
Ur
mV
Rm
Ohm gm Ohm–1 Ur mV
K+ 7.25 × 1010 1.3 × 10–11 313 5.26 × 1010 1.9 × 10–11 239 4.62 × 1010 2.1 × 10–11 304
Na+ 5 × 1010 2 × 10–11 234 1.17 × 109 8.5 × 10–10 162 1.94 × 1010 5.1 × 10–11 227
Li+ 16 × 1010 6 × 10–12 309 2.54 × 1010 3.9 × 10–11 173 1.9 4 × 109 5.1 × 10–10 200
J– 1.33 × 109 7.5 × 10–10 355 5.98 × 108 1.6 × 10–9 180 2.71 × 109 3. 6 × 1 0–10 348
Cl– 7.25 × 1 010 1.3 × 10–11 313 5.26 × 1010 1.9 × 10–11 239 4.62 × 1010 2.1 × 10–11 304
Br– 10 × 1010 1 × 10–11 312 2.58 × 108 3.8 × 10–9 162 3.3 × 105 3 × 10–6 327
Each group contained 2 0 BLMs from th ree tissues. P > 0.01 by S tudent’s t-test re l ative to the c orr esponding control.
Table 4. Naja kaoutia venom in vivo effect on the electrical
properties of BLMs (resistance Rm, conductivity gm and break-
ing-potential Ur) formed from different tissue’s lipids in univa-
lent ions medium.
Brain Liver
Rm
Ohm gm Ohm–1 Ur mV Rm Ohm gm Ohm–1
Ur
mV
K+ 7.4 × 1010 1.4 × 10–11 4074.2 × 1010 2.4 × 10–11 249
Na+ 52 × 1010 1.9 × 10–11 3592.1 × 1010 4.8 × 10–11 233
Li+ - - 4482.15 × 1010 4.8 × 10–11 170
J– - - 4273.7 × 109 2.7 × 10–10 180
Cl– 7.4 × 1010 1.4 × 10–11 4074.2 × 1010 2.4 × 10–11 249
Br– 1.02 × 1010 1 × 10–10 3541.49 × 108 6.7 × 10–9 157
was also strictly curvature-dependent.
Studies following the changes in the plastic properties
of membranes of different tissues during snake venom
envenomation are scarce. The analysis of the results ob-
tained in the experiments with whole venom is difficult
because of the complexity of ongoing processes, each of
which may induce a various cascades of tissue damages.
At the same time an important task of su ch research is to
provide not only opportunities to improv e tissue recov ery
and clinical outcome of patients, but also to adopt novel
therapeutic strategies to treat a number of diseases, such
as different types of aggressive cancers [23] or neuro-
logical damages [24,25].
5. ACKNOWLEDGEMENTS
This work was supported by Grant # 1923-EN from the Armenian
National Science and Education Fund (ANSEF) based in New York,
USA.
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