(Naja naja) Venom - National University of Singapore

Comparative Proteomic Analysis Involving Captive Pakistan Cobra
(Naja naja) Venom
Doley R 1 , Kini RM 2
Modahl CM 3
Department of Biological Sciences, Faculty of Science, National University of
Singapore, 10 Kent Ridge Road, Singapore 117546
College of Agricultural, University of Kentucky, Lexington, Kentucky, 40506
ABSTRACT
Venom production facilities keep established colonies of captive snakes to obtain
venom for research and antiserum purposes. These captive colonies alleviate collection
pressures put on wild populations. Since some venomous snakes, including the Pakistan
cobra, Naja naja, have strict regulations involving importation, only a certain amount of
initial individuals are used to construct these colonies. This arrangement allows for a
decrease in genetic diversity and a closer relation between snakes.
With the use of gradient SDS-PAGE gels and reverse phase liquid chromatography
mass spectrometry (LC/MS), venom from fifteen N. naja originating from two separate
venom production colonies were analyzed. Venom variation has been well documented in
snakes and has been found to be the result of many factors, including diet, geographic
range, and individual differences. However, the affect of long-term captivity on venom
variation has not been well studied. By identifying intraspecific venom variation known
to exist, such as ontogenetic, population isolation, and sibling changes in venom
composition, a better understanding of the venom from captive snakes is developed.
1 Post Doctoral Fellow
2 Senior Lecturer
3 Undergraduate Student

This study has practical applications in the field of venom research and antiserum
production since it is from these long-term captive colonies that venom is collected for
these purposes.
INTRODUCTION
In this study, the venom from a total of fifteen N. naja housed at two different venom
production facilities is investigated. The cobras maintained at the Kentucky Reptile Zoo
and Latoxan are at least second generation captive animals, and there have not been any
studies done on the venom from snakes that have been raised in captive conditions for
that length of time.
Venom is very variable in composition and expression. Intraspecific venom variation
has been documented to be the result of many factors, including diet (Daltry et al.,
1996b), isolation (Williams et al, 1988), geographic range (Glen et al, 1983), and even
differences among siblings (Menezes et al., 2006). In fact, the venom from the same
individual can be subject to ontogenetic changes (Daltry et al., 1999).
However, the effect of long-term captivity and inbreeding on venom variation has not
been studied. The experimental objective of this study is to identify intraspecific variation
experienced by N. naja kept in a long-term captive situation.
METHODS
N. naja venom was obtained from two sources and each venom sample was collected
from a single cobra (no pooled samples were used). Collected venom was lyophilized
(10-18 mg dry weight) and reconstituted with lx SDS/Glycine Running Buffer (1 mg/ml).

Premade SDS-PAGE 4-20% Tris-HCl gradient gel was used to separate and document
the protein content of the venom from N. naja originating from the Kentucky Reptile
Zoo.
Reverse phase liquid chromatography (RPLC) was done with 150 µl of venom sample
(l mg/ml) injected into an RP-Jupiter C18 (10µ, 300 Ǻ, 10 mm X 250 mm) column
equilibrated with 0.1% trifluoroacetic acid (TFA). RPLC results were than connected to
an electrospray ionization mass spectrum to indentify the protein composition of all N.
naja individuals originating from Latoxan.
RESULTS AND DISCUSSION
The overall goal of this project was to identify venom variation observed among
different populations, age groups (ontogenetic), and siblings. All of these levels of
variation have been identified in other studies (see Introduction section), but the
composition of venom obtained from long-term captive snakes has not been reviewed.
Of the three levels of variation considered, only one is able to be distinguished, and
that is at the level of sibling and individual variation.
Since different methods were used to analyze the venom composition from cobras at
the Kentucky Reptile Zoo and cobras from Latoxan, the two can’t be directly compared.
Therefore, population variation can’t be shown in this study.
The cobras from Latoxan were all siblings from the same parents, just two separate
litters. Cobras A, B, and D were born on 01/09/01 and cobras C, E, and F were born on
01/11/02. Venom from each cobra in the different litters was examined in hopes of
finding ontogenetic venom variation trends, but none were found. It is possible that only
a year difference between the offspring is insignificant.

The only clear variation observed was between siblings. There were variations
between all the Latoxan cobras in the study, none of the venom profiles were identical to
another, but some variations were more prominent than others.
ACKNOWNLEDGEMENTS
I will forever be indebted to Prof RM Kini for allowing me the chance to spend a little
over four months working in his lab. I also couldn’t have completed this project without
the guidance of lab member Robin Doley, my advisor Bruce Webb at the University of
Kentucky, and Jim Harrison and Kristen Wiley from the Kentucky Reptile Zoo who
donated N. naja venom for my research.
REFERANCES
Daltry JC, Wüster W, Thorpe RS. 1996b. Diet and snake venom evolution. Nature. 8:
379(6565):537-40.
Daltry JC, Ponnudurai G, Shin CK, Tan NH, Thorpe RS, Wüster W. 1999.
Electrophoretic profiles and biological activities: intraspecific variation in the venom of
the Malayan pit viper (Calloselasma rhodostoma). Toxicon. 34:1. 67-79.
Glen JL and Straight R. 1978. Mojave rattlesnake Crotalus scutulatus scutulatus
venom: variation in toxicity with geographical origin. Toxicon. 16: 81-84.
Menezes M. C, Furtado M. F, Travaglia-Cardoso S. R, Camargo A C.M, and Serrano
S. M.T. 2006. Sex-based individual variation of snake venom proteome among eighteen
Bothrops jararaca siblings. Toxicon 47: 304-312.
Williams V, White J, Schwaner T.D, and Sparrow A. 1988. Variation in venom
proteins from isolated populations of tiger snakes (Notechis ater niger, N. scutatus) in
south Australia. Toxicon 26: 1067-1075.