The Differences of Electrophoretic Profile and Snake Venom

Phospholipase A2 (svPLA

) Activity from the Venom of Javan

Spitting Cobra, Naja sputatrix, based on Body Scales Color and

Storage Condition

Nia Kurniawan

*, Dea Jolie Chrestella

, Dinda Sherlyndra Hapsari

, Fatchiyah

Biology Department, Faculty of Mathematics and Natural Sciences, University of Brawijaya Jalan Veteran Malang, East

Java, Indonesia

Keywords: Naja sputatrix, acidimetric assay, dorsal color, snake venom phospholipase A

, venom storage.

Abstract: Naja sputatrix (Javan spitting cobra) is one of medically important snake species in Indonesia which have

various dorsal scales color. This research purposes to examine the differences of venom general profile and

its phospholipase A

(svPLA

) activity from some N. sputatrix with different dorsal scales colors, and to

examine the activity of N. sputatrix svPLA

in different storage conditions. A total of 6 N. sputatrix from

East Java with various dorsal scales color were milked. Venom storage was performed at -80, 4 and 37°C in

a maximum period of 14 days. Venom profile and phospholipase A

activity were examined through 15%

SDS-PAGE and acidimetric method using egg yolk substrate respectively. Statistical analyses were

performed to evaluate svPLA

activity in every dorsal color and storage condition. Few protein bands range

in 16 – 22 kDa are only found in the venom of the certain dorsal color snake. Protein bands at 37°C were

found to have the lightest intensity among other groups. The svPLA

activity of brown dorsal N. sputatrix is

found as the highest activity. An interaction between the storage temperature factor and period factor has

effects on post-storage svPLA

activity. Storage in 37°C effects on svPLA

activity declining compared to

the control group and other experimental groups.

1 INTRODUCTION

Elapidae, Viperidae, and Colubridae snakes can

produce venom as their secreted product which is

useful in foraging activity and survival mechanism

(Vitt & Caldwell, 2009; Warrel, 2010). Venomous

snakes are present around the world. Thus, the

conflict between human and venomous snake

becomes a global health problem. The total of the

conflicts around the world, in 2008, reaches the

number of 421.000 – 1.841.000 envenomation cases

per year (Kasturiratne et al., 2008). Snakebite

problem does not get enough attention and is

included in Neglected Tropical Disease (NTD) since

2009 (Gutierrez et al., 2013; Williams et al., 2019).

Tropical and subtropical area, including Indonesia,

are susceptible to snakebite problem (Hijaz et al.,

2018; Megawati, 2014; Safitrih et al., 2016; Pratama

& Oktafany, 2017).

Naja sputatrix is only one of various venomous

snakes in Indonesia that is considered medically

important. N. sputatrix is classified to Catagory I

venomous snake because of its high venom and its

habitat preference that is near to human (Warrel,

2010). N. sputatrix in Indonesia can naturally be

found in Java, one of the most populated islands in

Indonesia, also in Lombok, Sumbawa, Padar, Rinca,

Komodo, Flores, Adonara, Lomblen and Alor

Islands. This snake is a terrestrial organism that

often found in rice fields and swamps near

residential areas (Iskandar, et al., 2012). N. sputatrix

has a total body length of 1,5 meters with a wide

head and an elongated hood. The dorsal scales color

of this species is varied. N. sputatrix in West Java

have blackish gray dorsal scales color, while those

from East Java and Islands of Southeast Nusa (Nusa

Tenggara) have silver to brown color (Das, 2010).

The scales color of this snake is possibly a result of

an adaptation process. The snakes with darker scales

live in rain forest with high relative humidity, while

the snakes with lighter scales color live in dry soil

habitat (Kurniawan, et al., 2017).

Kurniawan, N., Chrestella, D., Hapsari, D. and , F.

The Differences of Electrophoretic Proﬁle and Snake Venom Phospholipase A2 (svPLA2) Activity from the Venom of Javan Spitting Cobra, Naja sputatrix, based on Body Scales Color and

Storage Condition.

DOI: 10.5220/0009587600190028

In Proceedings of the 6th International Conference on Advanced Molecular Bioscience and Biomedical Engineering (ICAMBBE 2019) - Bio-Prospecting Natural Biological Compounds for

Seeds Vaccine and Drug Discovery, pages 19-28

ISBN: 978-989-758-483-1

The venom of N. sputatrix is a dangerous

mixture solution for humans containing many

protein and non-protein components. It generally

contains major and minor components: three-finger

toxins, cytotoxin, short-chain-α-neurotoxin, long-

chain-α-neurotoxin, muscarinic toxin-like protein,

snake venom metalloproteinase, snake venom serine

protease, phospholipase A

, Kunitz-type serine

protease inhibitor, cobra venom factor,

phosphodiesterase, nucleotidase, L-amino acid

oxidase, nerve growth factor, acetylcholinesterase,

and many more (Tan et al., 2017). Phospholipase A

(PLA

), a major component of the venom, is an

enzyme that hydrolyzes glycerophospholipid

(Sunagar et al., 2015). This enzyme has been

extensively explored because of the stable structure

(Vija et al., 2009; Kang et al., 2011). Many

physiological and pathological effects are caused by

svPLA

: presynaptic neurotoxin, edema, necrosis

also hemolysis (Sunagar et al., 2015; Doley & Kini,

2009; Asad et al., 2014). Some PLA

antidote

potentials are also estimated useful to handle or

alleviate snakebite effects (Xiao et al., 2017).

The abundance of each component in venom is

varied in every single snake, even in the same

species with differences in the locality. The

composition of the venom can be affected by

geographic condition, habitat, season variation, diet

preference, the age of the snake, also sexual

dimorphism (Tan et al., 2015; Sarhan et al., 2017).

Other than the factors described in these studies, the

profile of venom from different dorsal scale colors

was not researched well yet. This makes the factor

of dorsal scales color is needed to be considered. It

would be very important in research to use fresh-

milked snake venom in wildlife to get a holistic

description of snake venom. Considering the

importance, the storage of the venom before it

arrives in the research center is very important to

note. The previous study put forward the results that

svPLA

and the venom of Crotalus molossus

molossus were generally stable in various

temperature storage for 7 days. The study however

told that the results might be generalized for other

snakes, but further researches are still required to

conduct (Munekiyo & Mackessy, 1998). This

research purposes to examine whether the

differences of venom profile and enzymatic activity,

which is represented by svPLA

as a major

component of the venom, of some N. sputatrix with

different dorsal scales colors are present or not.

Besides, this research also purposes to evaluate the

activity of N. sputatrix svPLA

in different storage

conditions. The activity of svPLA

was evaluated

after the crude venom was stored at various

temperatures for 14 days.

2 MATERIALS AND METHODS

2.1 Sample Preparation

The examination of Naja sputatrix venom profile

and svPLA

activity were carried out to examine the

difference or similarity among different dorsal scales

color snakes, also different storage condition. The

samples used in this research comprises in total of 6

East Javan N. sputatrix individuals. Black dorsal

scales color snakes were collected from Malang, the

brown dorsal scales snakes from Jombang, and

yellow dorsal scales snakes from Bangil. The snakes

used in this research have a total length of 1,2 – 1,5

meters. Venom milking was conducted after the

snakes 3 days fasting. Venom milking was done in a

beaker glass which covered with parafilm. N.

sputatrix venom from each dorsal color scales was

pooled separately. These samples were used to

examine whether dorsal scales color would affect the

venom profile and svPLA

activity or not.

Meanwhile, snake venom solution from black dorsal

scales snakes was pooled together and aliquoted into

some different storage condition groups to evaluate

the effect of storage temperature in 7, 9 and 14 days.

All fresh-milked venom were centrifuged at 4000

rpm for 5 minutes at 4°C. The supernatant was

stored for further analysis. Storage temperature used

to evaluate the effect of dorsal scales color was -

80°C, while 37, 4 and -80 °C were used to store

snake venom sample that would be evaluated under

different storage condition. Three times replication

was used at any data collection.

2.2 Protein Concentration

The whole crude protein and the svPLA

examinations were done in Molecular Biology

Laboratory of Life Science Central Laboratory and

Institut Biosains, University of Brawijaya,

Indonesia. The protein concentration of the venom

solution was measured by spectrophotometry

principle, based on the absorbance value of the

sample in wavelength 280 nm by using the

NanoDrop instrument. The protein concentration

data were used to equalize sample for further assays,

both visualization by SDS-PAGE or svPLA

activity

assay.

ICAMBBE 2019 - 6th ICAMBBE (International Conference on Advance Molecular Bioscience Biomedical Engineering) 2019

2.3 Snake Venom Electrophoresis

(SDS-PAGE)

Naja sputatrix crude venom solutions were subjected

to Sodium Dodecyl Sulfate Polyacrylamide Gel

Electrophoresis. The preparation of the venom

solution comprises the addition of reducing buffer

followed by incubation at 100°C for 15 minutes. 10-

20 µl venom solution containing 23 µg protein was

loaded to each gel well. The standard marker used

was from Jena Bioscience BlueEye Prestained

Marker 10-245 kDa. Electrophoresis was conducted

at separating gel 15% and stacking gel 3% with a

constant volt of 120 V. CBB R-250 staining process

was used to visualize the protein separation results of

the venom solution.

2.4 Phospholipase A

Assay

Phospholipase A

assay was conducted based on the

acidimetric method. Phospholipid substrate

suspension was prepared from chicken egg yolk,

CaCl2 18 mM and sodium deoxycholate 8,1 mM

1:1:1 (v/v/v). The material used was mixed well and

adjusted to pH 8,0 by using NaOH 1 M. One

hundred microlitres venom solution containing 50

µg protein was mixed into 15 mL substrate

suspension. The decrease of 1 pH unit between 5 –

65 s was considered equal to the release of 133 µmol

fatty acids (Tan & Tan, 1998).

2.5 Statistical Analysis

The results of the Naja sputatrix svPLA

activity

were analyzed statistically. We performed a

Kolmogorov-Smirnov test, Lavene statistic, One

Way ANOVA, and Games-Howell test to evaluate

the svPLA

activity in each dorsal scales color

snake. To evaluate whether interactions among

storage factors were happening or not, we performed

univariate analysis and the test of between factors

interactions. Further investigation was done by

Tukey test to compare the results between the

control group and other experimental groups.

3 RESULT AND DISCUSSION

3.1 N. sputatrix Venom Electrophoretic

Profile

The evaluation of N. sputatrix venom electrophoretic

profile based on the dorsal scales color shows that

the venom proteins range 16 – 134 kDa. There are

seven protein bands, labeled as P1 until P7. The P1

bands have similar molecular weights and

characteristics among the three kinds of venom. The

same conditions are also found on P2 until P7.

Those bands have molecular weight on 26, 54, 60,

65, 80, 92, 134 kDa (Figure 1, P1 – P7). However,

the venom from yellow dorsal scales N. sputatrix has

a higher intensity in the 2nd, 5th, 6th, and 7th bands.

Other than that, the 8th until 11th bands from all

samples used show various molecular weights and

intensity. Venom from black dorsal scales N.

sputatrix does not show both 8th and 9th protein

bands as the other samples show. Venom from

brown dorsal scales snakes observed to have 20 and

22 kDa proteins (Figure 1, PC8 and PC9). Yellow

dorsal scales snakes observed to have 19 and 21 kDa

proteins (Figure 1, PK8 and PK9). Differences are

also observed in the ≤ 20 kDa protein bands. Three

thick protein bands (16 – 17 kDa) are observed in

the venom of black dorsal scales snakes (Figure 1,

PH10, PH11, and PH12); a thick protein band (16 –

18 kDa) in the venom of brown dorsal scales snakes

(Figure 1, PC10); two protein bands (16 – 17 kDa)

in the venom of yellow dorsal scales snakes (Figure

1, PK10, PK11).

M: marker

H: black dorsal scales

C: brown dorsal scales

K: yellow dorsal scales, P1 – P8: protein bands appearing

in three samples

PH: protein band only appearing in black dorsal snakes

PC: protein band only appearing in brown dorsal snakes

PK: protein band only appearing in yellow dorsal snakes

Figure 1: Molecular weight profile of N. sputatrix protein

venom on SDS-PAGE 15%.

The Differences of Electrophoretic Proﬁle and Snake Venom Phospholipase A2 (svPLA2) Activity from the Venom of Javan Spitting Cobra,

Naja sputatrix, based on Body Scales Color and Storage Condition

Meanwhile, N. sputatrix venom samples in

which the profile are evaluated under different

storage conditions are mostly composed of proteins

that have a molecular weight of ≤ 35 kDa, which can

be seen as the thick band showed in the crude venom

separation. Separation by SDS-PAGE of N. sputatrix

crude venom does not show a well-separated band

(Figure 2). However, the analysis of control group

results in a better separation of the venom protein.

Few protein bands (19, 27, 30, 34, 39, 44, 64 and

125 kDa) are visualized well in the control group.

Those bands, in contrast, are not visualized well in

the other experimental group, except for the S1 and

S2 bands. Electrophoretic visualization of the stored

N. sputatrix venom solutions shows a lower

intensity, with the lowest intensity is showed in the

experimental groups under 37°C storage

temperature.

NSC: control group

D7: 7 days storage

D9: 9 days storage

D14: 14 days storage.

Figure 2: N. sputatrix venom visualization on 15% SDS-

PAGE in variation of storage condition.

Black outline squares show protein with molecular

weight range on 13 – 16 kDa. N. sputatrix svPLA2

is estimated to be in the range.

Figure 3: Dissulfide bonds in the three-dimentional

structure of N. sputatrix svPLA2 (Q92084).

Dissulfide bonds are represented by yellow parts. A)

Front side, B) Back side, C) Right side, D) Left side,

E) Upper side, F) Under side.

3.2 The Activity of N sputatrix svPLA

N. sputatrix svPLA2 from the black dorsal scale

snake performs the lowest activity (144,08

µmoles/minutes/mg) compared to the brown and

yellow dorsal scale color snakes (Figure 4). Statistic

analysis with a confidence level of 95% shows that a

significant difference is present between the svPLA2

activity of the black dorsal scales snakes and the

brown dorsal scales snakes. On the other hand, the

difference of svPLA2 activity in yellow dorsal scales

snakes with both black and brown dorsal color

snakes are not significant (Figure 4). This condition

indicates that the ability to hydrolyze biological cell

phospholipid membrane in brown dorsal snakes

venom is higher than the other venom, and is found

significantly different from the venom of black

dorsal snakes, and not significantly different with

the venom of yellow dorsal snakes. Along with it,

the venom from brown dorsal color might be riskier

to raise various pathophysiological effects in

following the envenomation compared to the two

other snakes.

ICAMBBE 2019 - 6th ICAMBBE (International Conference on Advance Molecular Bioscience Biomedical Engineering) 2019

Figure 4: The snake venom phospholipase A2 activity rate

of Javan spitting cobra (N. sputatrix) with different dorsal

scales color. Letter a and b define the statistic notation

among sample groups.

To confirm the stable electrophoretic results of

svPLA2 under different storage conditions, we

performed the svPLA2 activity assay. In this

research, N. sputatrix svPLA2 activity is affected by

the interaction between storage duration and

temperature factors. However, svPLA2 in our

research are estimated stable during the storage at

both 4 and -80°C for two weeks long. There are no

significant differences between svPLA2 activity

from the control group and from the venom

solutions which are kept at 4 and -80°C for 7 - 14.

On the other hand, the activity of svPLA2 that

had been stored at 37°C does not remain stable since

the first 7 days of storage. The N. sputatrix svPLA2

activities in 37°C storage temperature are found

similar in 7, 9 and 14 days storage duration, where

only reach about ¾ of svPLA2 activity in the control

group sample (Figure 5). This may indicate the

damage of svPLA2 native form, which affects its

performance.

Figure 5: Effect of interaction between temperature and

period of time storage on svPLA2 activity rate of N.

sputatrix venom. Letter a, b, c define notation among

sample groups based on statistical data.

All protein bands from the venom of each dorsal

scales color snake (Figure 1) are estimated as four

venom protein families, those are Phospholipase A2

(svPLA

), Cysteine-Rich Secretory Protein (CRiSP),

Snake Venom Metalloproteinase (SVMP), and

Nerve Growth Factor (NGF) (Table 1). PLA

estimated as the protein family from protein bands

16 – 19 kDa. Even though PLA

is known as a major

component of venom, the toxic effects caused by

this enzyme are varied. This enzyme also

synergistically works with the other venom

components to support the toxicity potential of cobra

venom (Wong et al., 2017; Tan et al., 2017).

Protein bands 16, 18, 20, 21 and 26 kDa in the

SDS-PAGE results are estimated as CRiSP. This

protein family possesses the inhibition the smooth

muscle contraction through the blockade of cyclic

nucleotide-gated (CNG) and L-type calcium

channels. Protein bands with molecular weight on 22

and 26 kDa can also be estimated as NGF family, a

non-enzyme protein that effects on apoptosis

induction and cytotoxic activity (Tan et al., 2017;

Wong et al., 2017). Few higher molecular weight

venom proteins (80, 65, 60 and 54 kDa) are

estimated as SVMP, which effects on local and

systemic bleeding induction, hemostatic disruption

through the properties of procoagulant or

anticoagulant, inflammation and tissue necrosis

(Sanhajariya et al., 2018).

Table 1: Protein family estimation of the protein bands

appearing in N. sputatrix venom solution 15% SDS-

PAGE.

Protein

family

prediction

Band Molecular

weight

(kDa)

References

SVMP P3 80 Lauridsen et al.,

2017; Shan et al.,

2016; Xu et al.,

2017

P4 65

P5 60

P6 54

NGF P7 26 Xu et al., 2017

PC8 22

CRiSP P7 26 Xu et al., 2017;

Sanhajariya et al.,

2018; Shan et al.,

2016

PK8 21

PK9 20

PH10,

PC10,

PK10,

PH11,

PK11,

PH12,

16 - 18

PLA

PK9,

PH10,

PC10,

PK10,

PH11,

PK11,

PH12

16-19 Shan et al. 2016;

Xu et al., 2017

The Differences of Electrophoretic Proﬁle and Snake Venom Phospholipase A2 (svPLA2) Activity from the Venom of Javan Spitting Cobra,

Naja sputatrix, based on Body Scales Color and Storage Condition

Venom protein visualization in this research is in

accordance to the previous studies. Liu et al. (2018)

conducted research which found that few protein

families are identified on the whole venom of Naja

atra, those are SVMP, Venom Complement C3,

CRiSP, PLA

, NGF, and 3FT. Xiao et al. (2017)

found that the venom of Naja naja, Naja

melanoleuca, Naja nigricollis, and Micrurus fluvicus

consist of acetylcholinesterase, SVMP, a serine

protease, CRiSP, PLA

, and 3FT. The protein

families found in previous studies are estimated

appearing in the sample used. Even few differences

are found in the separation profile, the differences

are in the same protein families among the three

kinds of venom used. This indicates that the color of

Javan spitting cobra dorsal scales might have effects

in the abundance or characteristics of venom protein

bands. The unclear results lead to the need for

further research with more supportive methods that a

holistic analysis of cobra venom in the consideration

of dorsal color could be done better.

Under different storage condition, the protein

separation through SDS-PAGE of Crotalus molussus

molussus venom showed few variations. The storage

conditions under 4, -20, and -80ºC, in general, did

not affect the visualized protein band. The similar

results also found in previous researches. The

visualization of venom protein through SDS-PAGE

appears to be not affected by the storage temperature

(-80, -20, 4 and so 20ºC) for 1-7 days long (Egen &

Russell, 1984; Munekiyo & Mackessy, 1998). Other

than cold storage, the proteins of snake venom are

found stable in dried storage even until more than 50

years. Few degradations in the protein may happen

but limited to the functionally unimportant peptides

(Jesupret et al., 2014). In contrast, the venom

solution storage under 37ºC caused some changes in

the protein bands. The decrease in intensity,

absence, and appearance of some bands in the

visualization may indicate an autolytic degradation

of some proteins in the venom solution (Munekiyo

& Mackessy, 1998). This also possibly happens in

the N. sputatrix venom solution stored at 37ºC,

which can be observed from the decrease of

intensity in the separation results.

The differences in svPLA

activity of N.

sputatrix from Jombang, as the highest activity

among other groups, are possibly an effect of their

habitat and so prey availability. Variation in snake

venom (Casewell et al., 2014) component is an

adaptation to choose prey. These phenomena exist in

both interspecific and intraspecific levels. The

venom system is an important adaptation that

evolved independently in every animal lineage. The

toxins in snake venom are encoded by a few gene

families, in which each gene family can produce

related isoform that had been produced from gene

duplication during the evolution process. Birth and

death model of toxin-gene evolution is often used as

a mechanism that brings out toxin gene paralogue,

with the evidence that natural selection does

facilitate the encoded protein subfunctionalization or

neofunctionalization. This process produces a toxin

complex that synergistically works to cause death in

prey. Venom evolution in the advanced level enables

the changes in prey capture from mechanic

(constricting) to chemical (venom). It plays an

important role in snakes diversification. The

diversity in snake venom is caused by the new toxin

gene recruitment, or the diversification of existing

toxin genes, that happened before and during the

evolution (Xu et al., 2017). The svPLA

enzyme is

coded by the ancestor’s physiological gene that

experiences convergent and divergent evolution

several times. PLA2 in snake venom is a single

chain polypeptide consists of 115 – 125 amino acid

residues with a molecular weight of 13 – 15 kDa and

has a high homolog sequence in many cobra species.

However, the pharmacology of svPLA

envenomation cases are contributed in various way

even the sequence was generally homolog. Naja

melanoleuca, an African cobra, has a very high

PLA

activity that reaches 2120,66

µmoles/minutes/mg. Meanwhile, svPLA

activity of

Asian cobras range from 864,04 – 1157,56

µmoles/minutes/mg. Asian cobras used in the

research are Naja sputatrix, Naja naja, Naja

kaouthia, Naja atra, Naja sumatrana. Some African

cobras, for example, Naja katiensis, Naja nigricollis,

Naja pallida, Naja mossambica and Naja nubiae

have svPLA

activities that do not show many

differences with those in Asian cobras venom. Some

other African cobras, Naja senegalensis, Naja haje,

Naja annulifera, and Naja nivea have very low

svPLA

activity (Tan et al., 2019).

A slight difference in venom molecular weight

profile, also with the significant differences in the

svPLA

activity which is found in our research could

indicate that some different protein kinds and/or

abundance might exist in different dorsal scales

color snakes, and are related to the habitat of the

snakes. However, the differences are not studied

further because of data limitations. Further studies

with more supportive methods were needed to

confirm these results.

Under different storage conditions, we evidence

that svPLA

activity of N. sputatrix venom is

influenced by the interaction of temperature and

ICAMBBE 2019 - 6th ICAMBBE (International Conference on Advance Molecular Bioscience Biomedical Engineering) 2019

storage time. The activity of svPLA

is observed

decreasing significantly under the temperature of

37ºC. These results are not in accordance to the

previous study by Munekiyo and Mackessy (1998).

Munekiyo and Mackessy did research that results in

the stable activity of some enzymes, including

svPLA

that had been stored in various

temperatures: -80, -20, 4 and 37°C for 7 days long.

The svPLA

enzyme activity, specifically, are

maintained under the storage of 37°C . PLA

enzyme is considered as a stable enzyme in various

temperature conditions even in the presence of

proteolysis enzymes because of its small size and

molecular structure (Vija et al., 2009; Kang et al.,

2011). Our examination on a member of N. sputatrix

svPLA

family shows that there are 7 dissulfide

bonds in the structure of N. sputatrix svPLA2.

Dissulfide bonds play an important role in

maintaining this molecule stability through

decreasing the protein entropy in the unfolding

condition (Xiao et al., 2017; Fass, 2012).

With the exception in the research conducted by

Munekiyo and Mackessy (1998), Naja naja venom

PLA

shows an optimum temperature at 45 – 55°C

after its incubation at 37°C for 60 minutes

(Shashidharamurthy & Kemparaju, 2006). A similar

condition also found in the venom of Ecis ocellatus

and Crotalis durissus terificus (Sallau et al., 2008;

Toyama et al., 2003). In addition, Bothrops asper

svPLA

have an optimum temperature at 52°C after

its incubation in the temperature range 6 – 92°C for

30 minutes (Avila et al., 2004). These indicate that

the stable feature of PLA

at various temperature are

phenomena performed by svPLA

after its

incubation at a various temperature in a relatively

short time, which are not intended to storage

condition.

The crude venom solutions which we stored at

37°C show a visual difference compared to the other

storage condition groups. The solutions are more

turbid and contained some precipitation. The

presence of precipitation is an indication of protein

changes. Wrong storage could lead to the instability

and formation of precipitation. The precipitation

usually is the non-native form of protein which is

irreversible. The aggregate formation that leads to

protein precipitation could occur in some conditions:

changes of pH, freeze-thawing cycles and high-

temperature exposure (including the temperature of

37°C). The aggregate appearance could decrease the

amount of native-form svPLA

protein molecule in

the N. sputatrix venom solution, which can be

observed from the decrease of N. sputatrix svPLA

rate activity which had been stored in 37°C. The

svPLA

that have already aggregated with another

protein would undergo conformation changes that it

could not function on the substrate (Carpenter et al..

2002; Calamai et al., 2005).

Protease activity might be a factor that leads to

the svPLA2 degradation at the 37°C venom solution.

Protease could degrade endogenous inhibitor and/or

svPLA2, which impacts to the decrease of svPLA2

activity. Munekiyo & Mackessy in their study

detected the degradation of endogenous protein

inhibitor in the venom stored for 7 days at 37°C

(Munekyo & Mackessy, 1998). Endogenous protein

inhibitor has a crucial role in maintaining the whole

venom quality. Venom protein in the whole venom

solution form might undergo proteolysis caused by

protease, that impacts on the venom impotent.

Besides, the protease in the venom solution could

also damage the cells that make up the venom gland.

An endogenous inhibitor is found inside the venom

solution and functions to inhibit the protease,

including its activity to damage the svPLA2 (Francis

et al., 1992; Francis & Ivan, 1993).

Another factor that might influence the svPLA2

activity inside the venom solutions which had been

stored at 37ºC is a growth of microbes. Snpake

venom has been known for its antibacterial potential,

however, there is association between bacterial

infection and venomous snakebite cases. Ten years

of research (2001 – 2010) conducted in North

Taiwan pointed that Morganella morganii and

Enterococcus are the most abundant bacteria

identified in the victim's wound culture (Chen et al.,

2011). The snakebite victims in KwaZulu Natal,

South Africa, have an infection of a few kinds of

bacteria, for example, Morganella morganii,

Enterococcus faecalis, Proteus sp., and Salmonella

enterica. The bacteria were collected from necrosis

tissue samples from the victim (Wagener et al.,

2017). The microbes mentioned above can be found

in the intestinal track of human and other warm-

blooded animals (Lee et al., 2009; Dubin & Eric,

2014; Drzewiecka, 2016; ); which is associated to

the storage temperature we performed.

Microbial analysis at oral swab and venom

samples in recent research pointed that microbial

diversity of venomous snake oral cavity is dependent

on the food type and water resource as result from

faeces that might enter the oral cavity or venom

gland. Bacteria from the oral cavity (including

fangs) and venom solution are found to be appearing

in two different clusters, indicates that the venom

gland might be a different ecological niche. The

bacteria community in both the oral cavity and

venom gland was different. The bacteria in the

The Differences of Electrophoretic Proﬁle and Snake Venom Phospholipase A2 (svPLA2) Activity from the Venom of Javan Spitting Cobra,

Naja sputatrix, based on Body Scales Color and Storage Condition

venom solution also show that those are viable even

though the venom are air-dried or lyophilized. The

research also pointed out that two new strains for

Enterococcus faecalis appeared as a result of

adaptation to the venom. (Esmaeilishirazifard et al.,

2018).

Protein cold storage at -80°C until 4°C is an easy

method to store protein solution, specifically for a

stable protein in a short time of period range to 4

weeks. The storage at 4°C usually accompanied by

the addition of stabilizer solutions like sucrose,

glycine or glycerol to reduce the protein

concentration. This is important to decrease the

degradation of protein risk as an effect of the kinetic

process inside the protein solution (Carpenter et al.,

2002). Snake venom solution, however, does not

need a stabilizer solution due to its stable profile in

4°C storage. Venom storage at -80°C (or lower)

could also decrease the degradation risk to one year.

A factor that needs to be considered is a freeze-

thawing cycle that might denaturate protein on the

ice surface during freezing or thawing (Cao et al.,

2003), however, research studies also pointed its

stability during freeze-thawing cycles (Egen &

Russell, 1984; Munekiyo & Mackessy, 1998).

4 CONCLUSIONS

To conclude, we evidence differences in protein

bands' abundance and characteristics. Different

dorsal color N. sputatrix. The differences we found

are estimated as a similar protein family. The

svPLA2 activities of these snake venom solutions

also show a significant difference between black and

brown dorsal color. Yet the yellow dorsal color

snakes do not show a significant difference of

svPLA2 activity with both black or brown dorsal

color N. sputatrix. Considering the variation in

storage condition, svPLA2 visualization of Javan

spitting cobra venom in this research were remain

similar, accompanied by the decrease in band

intensity in the 37°C condition. The svPLA2 activity

of N. sputatrix based on the storage condition is

influenced by the factor of storage temperature and

time with a significant different results in the 37°C

storage temperature condition. We suggest the

storage of venom solution in a relatively short time

until 14 days would be performed at 4 or -80°C.

ACKNOWLEDGEMENTS

The present study was funded by Kemenristekdikti -

the Government of the Republic of Indonesia

through the scheme of PDUPT 2019 to Nia

Kurniawan with contract number

330.13/UN10.C10/PN/2019.

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