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EXONUCLEASE (PHOSPHODIESTERASE) FROM SNAKE VENOMS: ISOLATION, PURIFICATION, BIOCHEMICAL
CHARACTERIZATION AND SOME BIOLOGICAL STUDIES
A Thesis Submitted to
THE FACULTY OF SCIENCE
in fulfillment of the requirement for the degree of
DOCTOR OF PHILOSOPHY
in Chemistry (Biochemistry)
SAMIULLAH KHAN
Department of Chemistry
The Islamia University of Bahawalpur
Pakistan
July 2008
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APPROVAL SHEET
Certified that this thesis entitled, EXONUCLEASE (PHOSPHODIESTERASE) FROM SNAKE VENOMS:
ISOLATION, PURIFICATION, BIOCHEMICAL CHARACTERIZATION AND SOME BIOLOGICAL STUDIES, submitted
by SAMIULLAH KHAN is accepted in its present form by the Department of Chemistry, The Islamia
University of Bahawalpur, Bahawalpur, Pakistan, as satisfying the thesis requirements for the Degree
of Doctor of Philosophy in Chemistry (Biochemistry). It has been examined and approved for the
award of Ph.D. Degree.
Internal Examiner ________________________________
Chairman, Department of Chemistry ________________________________
Dean, Faculty of Science ________________________________
External Examiners 1 ________________________________
2 ________________________________
Dated:_____________.
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CERTIFICATE
It is hereby certified that this thesis entitled EXONUCLEASE (PHOSPHODIESTERASE) FROM SNAKE
VENOMS: ISOLATION, PURIFICATION, BIOCHEMICAL CHARACTERIZATION AND SOME BIOLOGICAL STUDIES,
submitted by SAMIULLAH KHAN is based upon the results of experiments carried out under my
supervision. No portion of this work has previously been presented for higher degree in this
university or any other institute of learning and to the best of the author’s knowledge, no material
has been used in this thesis which is not his own work except where due acknowledgement has been
made. He has fulfilled all the requirements and is qualified to submit this Thesis for the Degree of
Doctor of Philosophy in Chemistry (Biochemistry).
Dr. Muhammad Ashraf MSc,PhD Professor Department of Pharmacy The Islamia University of Bahawalpur.
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ACKNOWLEDGMENT
“He who taught by pen, taught man, what he knew not.”
The glorious Koran, chapter 96, verses 4 and 5.
The source of all knowledge is Allah, the knowledgeable, the wise and it is He who grants the
men capacity to Endeavour, the faculty to comprehend and the ability to use knowledge
wisely. It is therefore, befitting that all acknowledgements and thanks be directed to Allah
first.
I, therefore, thank Allah, the Praiseworthy and the Almighty for granting me the opportunity,
the resources and the energy for initiating and completing this work.
At the human level I would like to express my deep gratitude to my research supervisor,
Prof. Dr. Mohammad Ashraf, Department of Pharmacy, Faculty of Pharmacy and
Alternative Medicine for his invaluable help and advice in all aspects of my research.
I am also indebted to Prof. Dr. Saad S.M. Al-Saleh, Dean, College of Applied Medical
Sciences, King Saud University, Riyadh, Saudi Arabia, for his extra ordinary care, advice and
encouragement for my research. Thanks are due to the Dean Faculty of Science and
Chairman Department of Chemistry for the encouragement and registration with the
Department for Ph.D. program.
Finally, I would like my heartfelt appreciation to my parents and elder brother for their
encouragement and prayers, and to the members of my family for their cooperation, patience
and understanding.
SAMIULLAH KHAN
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ABBREVIATIONS
PDE-I Phosphodiesterase I
SDS Sodium dodecyle sulfate
PAGE Polyacrylamide gel electrophoresis
ALP Alkaline phosphatase
PLA2 Phospholipase A2
Ab Agistrodon bilineatus
Cv Cerastes vipera
Oh Ophiophagus hannah
Ea Energy of activation
ATP Adenosine triphosphate
cAMP 3’,5’- Cyclic adenosine monophosphate
ADP Adenosine diphosphate
IEF Isoelectric focusing
pNPP p-Nitrophenyl phosphate
TEMED N, N, N, N- tetra methyl ethylenediamine
Tris-HCl Tris (hydroxymethyl) methylamine
EDTA Ethylenediamine tetraacetic acid
pI Isoelectric point
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Contents Page
Bismillah ................................................................................................................................................ ii Approval Sheet ...................................................................................................................................... iii Certificate .............................................................................................................................................. iv Acknowledgement ................................................................................................................................. v List of Abbreviations ............................................................................................................................ vi Contents ................................................................................................................................................ vii List of Tables ......................................................................................................................................... ix List of Figures ....................................................................................................................................... xi Summary ............................................................................................................................................. xiii
1. INTRODUCTION ........................................................................................................................... 1-42
1.1. Venom and venomous snakes ................................................................................................. 1 1.2. Antivenom ............................................................................................................................... 3 1.3. Envenomation strategies ........................................................................................................ 3 1.4. Mechanisms ............................................................................................................................. 4 1.5. Composition of snake venoms ................................................................................................ 4 1.6. Toxicology of venom components .......................................................................................... 5 1.7. Potential factors of venom compositional discrepancy .......................................................... 6 1.8. Discovery of enzyme in snake venoms ................................................................................... 7 1.9. Basis of enzymes in snake venoms ......................................................................................... 7 1.10. Snake venoms as a spring of enzymes .................................................................................... 8 1.11. Distribution of enzymes in snake venoms ............................................................................ 10 1.12. Uses of snake venom enzymes in diagnostic, research and therapeutics ............................. 11 1.12.1. Snake venom enzymes as tool in biochemical research ....................................................... 12 1.12.2. Application of snake venom enzymes in clinical field ......................................................... 18 1.12.3. Medicinal use of snake venom enzymes ............................................................................... 23 1.12.4. Preparative uses of snake venom enzymes ........................................................... ……… . .26 1.13. Nucleases and their application in research .......................................................................... 29 1.14. Occurrence of phosphodiesterase in snake venoms ............................................................. 38 1.15. Isolation and purification of venom phosphodiesterase ....................................................... 39 1.16. Aims and objectives of the study .......................................................................................... 40
2. MATERIALS AND METHODS .................................................................................................. 41-60
2.1. Venoms, Chemicals and Reagents ........................................................................................ 41 2.2. Gel electrophoresis ................................................................................................................ 41 2.2.1. Analytical native polyacrylamide gel electrophoresis .......................................................... 44 2.2.2. Purification of enzyme from crude venom by preparative native PAGE ............................ 45 2.2.3. Analytical SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ..................................... 48 2.3. Recovery of proteins by electroelution ................................................................................. 51 2.4. Determination of protein concentration ................................................................................ 51 2.5. Isoelectric focusing (IEF) ...................................................................................................... 52 2.5.1. Preparation of gel .................................................................................................................. 52 2.5.2. Sample application ................................................................................................................ 53 2.5.3. Determination of pH gradient ............................................................................................... 53 2.5.4. Detection Method .................................................................................................................. 53 2.6. Assays of Phosphodiesterase activity ................................................................................... 55 2.6.1. Assay I ................................................................................................................................... 55 2.6.2. Assay II .................................................................................................................................. 55 2.6.3. Assay III ................................................................................................................................ 56 2.7. Assay for 5’-nucleotidase activity ........................................................................................ 57 2.8. Assay for alkaline phosphatase activity ................................................................................ 57 2.9. Carbohydrate contents ........................................................................................................... 57 2.10. Effect of pH ........................................................................................................................... 58 2.11. Effect of temperature ............................................................................................................. 58 2.12. Effect of metal ions ............................................................................................................... 59
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2.13. Measurement of kinetic constants ......................................................................................... 59 2.14. Estimation of IC50 .................................................................................................................. 59 2.15. Lethality Test ......................................................................................................................... 59 2.16. Determination of Re-calcified Plasma Coagulation Times .................................................. 60
3. RESULTS ..................................................................................................................................... 61-105
3.1. Purification of PDE-I enzyme by native preparative PAGE ................................................ 61 3.2. Determination of molecular weight by SDS-PAGE ............................................................. 61 3.3. Physiochemical properties .................................................................................................... 71 3.4. Enzymatic properties ............................................................................................................. 81 3.5. Effect of metal ions ............................................................................................................. 100 3.6. Substrate specificity of snake venom Phosphodiesterases (PDE-I) .................................. 102 3.7. Biological activity of snake venom Phosphodiesterases .................................................... 104
4. DISCUSSION.............................................................................................................................. 106-113
CONCLUSION ...................................................................................................................................... 114
5. REFERENCES .................................................................................................................................. 115
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LIST OF TABLES Table No. Title Page No.
1.1 Snake classification 2
1.2 Enzymes found in snake venoms 16
1.3 Applications of snake venom enzymes 17
1.4 Use of snake venom enzymes in disciplines others than hemostaseology
17
1.5 In vitro diagnostic uses of snake venom enzymes 19
1.6 Preparative uses of snake venom enzymes 20
1.7 Proposed indications for thrombin-like snake venom enzymes
27
1.8 Properties and actions of thrombocytin and batroxobin 28
1.9 The PDE superfamily 33
2.1 10% separating gel solution 46
2.2 4% Stacking gel solution 46
2.3 30% Acrylamide mix solution 47
2.4 Reservoir Buffer (0.05M Tris-glycine buffer pH9.5) 47
2.5 Sample buffer without SDS 47
2.6 Staining solution 47
2.7 De-staining solution 49
2.8 10% separating gel with SDS 49
2.9 4% Stacking gel with SDS 49
2.10 Reservoir buffer with SDS 50
2.11 Sample buffer with SDS 50
2.12 Gelling Solution for IEF 54
2.13 Electrode solution for IEF (pH 3.5-9.5) 54
2.14 Fixing solution for IEF 54
2.15 De-staining solution for IEF 55
2.16 Staining solution for IEF 55
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Table No. Title Page No.
3.1 Purification of phosphodiesterase I from different snake venoms.
69
3.2 Activities of contaminant enzymes in different crude venoms and purified PDE-I enzymes.
70
3.3 Mol. wts, pIs, activation energy (Ea) and carbohydrate content of PDE-I isolated from snake venoms.
70
3.4 Kinetic properties of PDE-I enzymes isolated from different snake venoms.
85
3.5 Effect of different compounds on the activity of PDE-I enzymes
89
3.6 Effect of different concentrations of inhibitors on the activity of PDE-I isolated from snake venoms
99
3.7 The effects of divalent metal ions on snake venom PDE-I activity
101
3.8 Hydrolysis of substrates by snake venom PDE-I 103
3.9 Effect of crude snake venoms and purified PDE-I
enzymes on coagulation of normal human plasma
105
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LIST OF FIGURES
Fig. No. Title Page No.
1.1 Hydrolysis of DNA by venom phosphodiesterase. 34
1.2 A basic structural element to which venom PDE binds for the hydrolysis of phosphodiester bond.
35
1.3 Cleavage sites of poly (ADP-ribose) with venom phosphodiesterase (C. adamanteus).
36
1.4 Hydrolysis of 3’, 5’-cAMP by venom phosphodiesterase. 37
3.1 A Analytical native PAGE of purified PDE-I from A. bilineatus venom along with crude venom.
62
3.1 B Analytical native PAGE of purified PDE-I from C. vipera venom along with crude venom.
63
3.1 C Analytical native PAGE of purified PDE-I from O. Hannah venom along with crude venom.
64
3.2 A SDS-PAGE of purified PDE from A. bilineatus venom along with crude venom.
65
3.2 B SDS-PAGE of purified PDE from C. vipera venom along with crude venom.
66
3.2 C SDS-PAGE of purified PDE from O. hannah venom along with crude venom.
67
3.3 Calibration of the SDS-PAGE system using standard proteins 68
3.4 A Effect of temperature on A. bilineatus PDE at 2mM BpNPP and pH 9.0.
72
3.4 B Effect of temperature on C. vipera PDE at 2mM BpNPP and pH 9.0. 73
3.4 C Effect of temperature on O. hannah PDE at 2mM BpNPP and pH 9.0. 74
3.5 A Arrhenius plot of A. bilineatus PDE-I. 75
3.5 B Arrhenius plot of C. Vipera PDE-I. 76
3.5 C Arrhenius plot of O. hannah PDE-I. 77
3.6 A Effect of pH on A. bilineatus PDE activity at 370C. 78
3.6 B Effect of pH on A. bilineatus PDE activity at 370C. 79
3.6 C Effect of pH on A. bilineatus PDE activity at 370C. 80
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Fig. No. Title Page No.
3.7 A Lineweaver-Burk plot for A. bilineatus PDE-I. 82
3.7 B Lineweaver-Burk plot for C. vipera PDE-I. 83
3.7 C Lineweaver-Burk plot for O. hannah PDE-I. 84
3.8 A Determination of Ki for cystein according Dixon (non-competitive inhibition) for Ab venom PDE-I
86
3.8 B Determination of Ki for cystein according Dixon (non-competitive inhibition) for Cv venom PDE-I
87
3.8 C Determination of Ki for cystein according Dixon (non-competitive inhibition) for Oh venom PDE-I
88
3.9 A Hill plot for Ab venom PDE-I inhibition by cysteine 90
3.9 B Hill plot for Cv venom PDE-I inhibition by cysteine 91
3.9 C Hill plot for Oh venom PDE-I inhibition by cysteine 92
3.10 A Determination of Ki for ADP according Dixon (competitive inhibition) for Ab venom PDE-I
93
3.10 B Determination of Ki for ADP according Dixon (competitive inhibition) for Cv venom PDE-I
94
3.10 C Determination of Ki for ADP according Dixon (competitive inhibition) for Oh venom PDE-I
95
3.11 A Hill plot for Ab venom PDE-I inhibition by ADP 96
3.11 B Hill plot for Cv venom PDE-I inhibition byADP 97
3.11 C Hill plot for Oh venom PDE-I inhibition by ADP 98
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SUMMARY
The venom Phosphodiesterase I (PDE-I, EC 3.1.15.1) is useful in elucidation of the structure
and nucleotide sequence of nucleic acids. PDE-I has been purified from three snake venoms
belonging to three different families; Agistrodon bilineatus (Ab, Crotilidae), Cerastes vipera
(Cv, Viperidae) and Ophiophagus hannah (Oh, Elapidae) by preparative native
polyacrylamide gel electrophoresis (PAGE). A single protein band was observed when
enzymes were run on analytical native PAGE. The enzymes gave a single band in SDS-
PAGE. The molecular masses of Ab, Cv and Oh PDE-I as determined by SDS-PAGE were
140, 126 and 148 kDa, respectively. The position of the band was not altered in the presence
of β-mercaptoethanol, which means the protein did not contain subunits.
The enzymes were free from 5’-nucleotidase (EC 3.1.3.5) and alkaline phosphatase (EC
3.1.3.1) activities. The maximum activity of purified Ab, Cv and Oh venom PDE-I in Tris
buffer was obtained over the pH 10.0, 9.0 and 10.0, respectively. The optimum temperature
was found to be 600C for Ab and Cv venom PDE-I, 500C for Oh venom PDE-I, with activity
decreasing at >650C. Energy of activation (Ea) was calculated to be 28.3, 128 and 135,
respectively. All the three PDE enzymes are glycoprotein in nature. The Ab and Oh venom
PDE-I exhibited basic pIs while Cv venom PDE-I showed an acidic pI. The Vmax and Km of
Ab, Cv and Oh venom PDE-I calculated were 3.85, 1.3 and 1.53 μM/min/mg and 8.3 × 10-3,
3.4 × 10-3 and 2.5 × 10-3 M, respectively. The Kcat and Ksp values for Ab, Cv and Oh PDE-I
were 23 s-1, 7.8 s-1, 9.2 s-1 and 46.4, 41.3 and 58.8 M-1 Min-1, respectively. Cysteine caused a
non-competitive inhibition, the Ki calculated for Ab, Cv and Oh venom PDE-I were 6.3 ×10
−3, 7.0×10 −3 and 8.2×10 −3 M, respectively while the IC50 determined to be 1.6, 3.0 and 3.9
mM, respectively.
ADP caused a competitive inhibition, the Ki calculated for Ab, Cv and Oh venom PDE-I were
0.8×10−3, 0.6× 10 −3 and 1.0× 10 −3 M, respectively; the IC50 determined were 5.4, 5.0 and
12.0 mM, respectively. o-Phenanthroline (10 mM) and EDTA (5 mM) inhibited the enzymes
activity significantly whereas the Mg (15 mM) greatly potentiated the activity. Zn (10 mM)
also showed significant inhibitory effect. The enzymes hydrolyzed thymidine 5’-
monophosphate p-nitrophenyl ester most readily (10 fold) while cyclic 3’,5’-cAMP (0.01
mM) was least readily hydrolyzed substrate.
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PDE-I enzymes up to 4.0 mg/Kg i.p were not lethal in mice. These enzymes exhibited an
anticoagulant effect as they greatly increased the normal clotting time of normal citrated
human plasma, whereas the crude venoms showed strong coagulant effect. The above studies
show that all these three PDE-I enzymes very similar to that isolated from other snake
venoms.
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1. INTRODUCTION
1.1. Venom and Venomous Snakes
The word venom is usually applied to the poisonous material produced by a
plant or an animal in a well developed secretary organ or group of cells and which is
delivered in the process of biting or stinging. Venomous animals have a venom
gland or highly specialized group of cells, a venom duct (although it is not a
consistent finding), and a structure for delivering the venom. A term venom
apparatus is now used in broader context to denote the gland and duct in addition to
fang or sting. The rattle snake, stingray and black widow spider are examples of
venomous phanerotoxic animals (Russell, 1983). The herpetologists stress that only
some 1300 of world’s 32000 species of snake are dangerous to humans and that the
few will attack unless molested (Hider et al., 1991).
Snakes belong to the suborder Ophidia or Serpentes of the order Squamata of
the vertebrate class Repitlia. The venomous snakes are classified according to
morphological characteristics and comprise five families;
(a) Cortalidae (cortalids, pit vipers)
(b) Viperidae (viperids, vipers)
(c) Elapidae (elapids)
(d) Hydrophiidae (sea-snakes) and
(e) Colubridae (colubrids)
Even though venomous snakes seem to be well characterized by their highly
evolved venom apparatus, a clear distinction between venomous and non-venomous
snakes is hardly possible.
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Table 1.1: Classification of Snakes
FAMILY SUBFAMILY GENERA
Crotalidae
Crotallus, Sistrurus, Agistrodon, Lachesis, Trimesus
Viperidae Vipera atractaspeis, Bitis, Causus, Cerastes, Echis, Adinorhinos, Atheris, Eristicophis, Pseudocerastes, Azemiops.
Elapidae Acanthophis, Brachyaspis, Demansiar, Denisonia, Elapognathus, Glyphodon, Hypocephalus, Micropechis, Notechis, Oxyaranus, Parademansia, Parapistocalamus, Pseudopistocalamus, Pseudoechis, Rhinoplocephalus, Toxicolamus, Vermicella.
Hydrophiidae Laticaudinae, Hydrophiinae Laticauda, Aipysurus, Emydocephalus, Hydrelaps, Kerilia, Thalassophinia, Enhydrina, Hydrophis, Acalyptophis, Thalassophis, Kolophis, Leapamis, Astrotia, Pelamismicrocephalophis.
Colubridae Pseudoboinae, Apara allactinae, Atracttaspidinae, Bioginae, Homalapsinae, Xenodontinae,
Navicinae,
Colubrinae.
3 genera 16 genera Atactapis 27 genera 10 genera 11 Opisthoglyphuaus 1 Opisthoglyphous species Dispholidus, Thelotornis, + 9 others
Opisthoglyphus.
[Ref. Tu, 1977].
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1.2. Antivenom
Antivenom is the antitoxin dynamic against venom of a snake, spider or other
poisonous creature. Alternatively, it is the animal serum having antivenins. Its use is
as medicament for the treatment of poisoning caused by animals or insect venom.
The antivenom is formed by injecting a minute quantity of the targeted venom into
an animal such like a horse, sheep, goat or rabbit. The subject beast will undergo an
immune reaction to the venom and will produce antibodies in opposition to the
venom's dynamic fragment. This is then harvested from the blood of that animal. It
is then used to treat envenomation in other creatures. Internationally, snake venom
antitoxin ought to cautiously convene the principles of Pharmacopoeia and the
WHO.
1.3. Envenomation Strategies
The primary function of venom is to immobilize and kill prey organism
(Karlsson, 1979). Venoms concurrently commence digestion of the prey from
within, thereby augmenting digestion efficiency (Thomus and Pough, 1979;
Kardong, 1980, 1998). In order to achieve these objectives, snakes utilize a great
variety of biochemical mechanisms, which necessarily reveal both biology of the
snake and the nature of its major prey. These mechanisms may be grouped into three
fundamental envenomation strategies. Two of these are prey immobilization
strategies and may denominated ‘hypotensive’ and ‘paralytic’ strategies. Both serve
to limit prey flight, in the case of snake taxa, which strike, release and then track
their prey (most viperids), or to overcome prey resistance, in the case of snakes that
seize and bulldog their prey (many elapids and all colubrids). The third strategy is
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digestive and commences degradation of prey tissue internally, even before the prey
has been engulfed. Normally, all three strategies operate simultaneously and
individual venom constituents frequently participate in more than of them.
1.4. Mechanisms
Each of these three strategies contains interchangeable mechanisms, elements or
sub-strategies. Different venomous snake taxa employ different combinations of
mechanisms and no single species employs them all. In fact, 70 years of increasingly
intense research in snake venom chemistry, it is doubtful that we know enough about
the composition of any single venom to fully outline all of the primary interactions
provoked by its components. Nonetheless, to the extent possible, specific examples
will be provided based on current understanding.
1.5. Composition of Snake Venoms
No doubt snake venoms are exceptionally intricate mixtures of proteins,
peptides, carbohydrates, lipids, metal ions and organic compounds. The proteins and
peptides account for approximately 90% of the dry weight (Bougis et al., 1986;
Ownby and Colberg, 1987; Bieber, 1979). While the snake venoms have an apparent
role in self-defense, this is of relatively little importance with regard to venom
composition. The non-protein fraction of snake venom is composed of sodium,
potassium, phosphorous, chloride, calcium, magnesium, manganese, zinc and
copper. It also contains riboflavin, nucleosides, peptides, amino acids, amides, lipids
and some carbohydrates (Bieber, 1979). The snake venom proteins are enzymes,
toxins or nerve growth factors. The biological activities are predominantly found in
the protein fractions rather than the non-protein fractions (Tu, 1977).
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The diversity of the symptoms arising after a snake bite such as bleeding, shock,
hemorrhage, necrosis and muscular paralysis are caused by particular venom
component possessing enzymatic and/or toxic properties (Elliott, 1978). This highly
efficient mixture of toxins and enzymes serves two main purposes: to kill or to
immobilize the prey and to support the digestion of the un- triturated, swallowed
food. Snake bite and the harmful, often lethal effect of the venom in man is an
important health problem in many tropical countries (Mebs, 1978).
1.6. Toxicology of Venom Components
Now-a-days it is possible to measure venom or single toxins over time in
equally experimental animals and human being snakebite victims. Nevertheless this
has so far merely been performed for a very restricted variety of species. There is a
better considerate of the toxicodynamics of envenoming. Venom is injected
reasonably superficially in most of the cases, frequently subcutaneously. The nearby
acting toxins causing tissue damage will by now be at their objective site so will
initiate exerting their clinical sound effects at once. A considerable ratio of the
venom, in a few species possibly most venom, will not be absorbed
straightforwardly into the circulation. Alternatively it will be transported initially
through the lymphatic system, then ingoing the circulation by the thoracic duct. This
is useful in explaining the frequent clinical finding of distended or tender lymph
nodes draining the bite region and also the high concentration of venom in these
nodes at autopsy. The transportation through the lymphatic organization may be
quick or occasionally belated and there is a prospective for sequestration of venom
locally, by prolonged discharge more than hours or days even.
Once in the circulation, those components will disturb haemostasis will have
reached their objective location and will rapidly apply their effect. In the same
20
manner, the nephrotoxins will swiftly harm the kidneys. Though these toxins are
seeking extravascular targets predominantly the neurotoxins and myotoxins, these
will require exiting the circulation in adequate concentration to exert their effect
clinically. Hence these toxins are most probable to have a late commencement of
clinically obvious actions. A few venoms are promptly cleared from the circulation,
although some others stay detectable for days or even weeks exclusive of antivenom
therapy. The awareness of such variations is evidently pertinent in shaping
antivenom therapy.
1.7. Potential Factors of Venom Compositional Discrepancy
Snake venom composition is influenced by a host of factors, including
phylogeny, geographic origin, season, sex, age and prey preferences (Jimenez-
Porras, 1964; Minton, 1975; Fiero et al., 1972; Irwin et al., 1970; Gubensek et al.,
1974; Glenn and Straight, 1978; Zepeda et al., 1985; Mackessay, 1988; Aird and
Jorge da Silva, 1991). Comparative studies of venoms have generally neglected
evolutionary and ecological perspectives, although it is clear that genetic event, the
history of contact and isolation among populations and prey type must influence
venom composition. Williams et al., (1988) found that pattern variation among
populations of the Australian elapids, Notechis scutatus and Notechis ater niger, was
not dependent upon prey type or ecology. Aird (2005) accomplished that there is no
apparent correlation between the quantities of venom nucleosides and the literature
values for the three dominant enzymes that release endogenous nucleosides, 5’-
nucleotidase (5NUC), phosphodiesterase (PDE) and alkaline phosphomonoesterase
(PME). Aird and Silva (1991) concluded that phylogenetic affinities at higher
taxonomic levels could not be ascertained from coral snake venom enzymatic data
and suggested that the inter-specific variation might reflect prey preference.
21
Daltry et al., (1996) in a study of geographic venom variation in the pitviper,
Calloselasma rhodostoma, rejected contemporary gene flow and phylogenetic
relationship as explanation for the variation, concluding that only diet was
significantly correlated with venom variation. Sasa (1999 a,b) assailed some of the
statistical methodology of Daltry et al., (1996) and argued that variation in venom
composition may be of neutral adaptive value, having no effect on the fitness of
snake. He further argued that ‘enzymatic differences must be related to the natural
context in which the enzyme must function, the prey.’
1.8. Discovery of Enzymes in Snake Venoms
Enzymes are important and most noticeable components of snake venom.
Enzymatic effects had been first described by researchers such as Geoffrey and
Hunauld (1737) who observed that blood from cats and dogs bitten by vipers did not
coagulate. Fontana (1781) noted that even a small quantity of viper venom caused
blood coagulation in the vessels of rabbit.
1.9. Basis of Enzymes in Snake Venoms
It has been recommended that for the period of evolution the developing
venom glands first produced enzymes which were already secreted by the pancreas
and against which inhibitors were present in the blood (Kochva et al., 1983; Kochva,
1987). In a co-evolutionary fashion enzymes of pancreatic origin were consequently
produced also by oral glands of the snakes. In fact, there is a significant homology of
amino acids between mammalian pancreatic phospholipase A2 and different venom
phospholipases. The crotalase, a clotting enzyme from the venom of Crotalus
adamanteus shows close structural homology to porcine pancreatic kallikrein and
astonishingly has also kallikrein-like activity (Pirkle et al., 1981; Markland et al.,
22
1982). These conclusions prop up the idea that venom enzymes have their familial
complement in the pancreas. The occurrence of inhibitors may perhaps also clarify
the resistance of snakes to their own venom.
1.10. Snake Venoms as a Spring of Enzymes
Snake venoms are one of the most concentrated enzyme sources superior than
pancreatic juice or similar secretions. More than 26 -39 different enzyme activities
have been detected in snake venoms (Iwanaga and Suzuki, 1979; Stocker, 1990b). It
is suggested that snake venom enzyme act in the following ways (Suzuki and
Iwanaga, 1970);
(I) Affect local capillary damage and tissue necrosis by proteinases, phospholipases,
arginine ester hydrolases, and hyaluronidase.
(II) Cause diverse coagulant and anticoagulant actions by various proteinases and
phospholipase A.
(III) Induce acute hypotension and pain due to release of vasoactive peptides by
kinin-releasing enzyme. The other enzymes regarded as toxic components of snake
venom include 5’-nucleotidase, phosphodiesterase and related enzymes,
cholinesterase and L-amino acid oxidase. The enzymes 5NUC, PDE and PME
release endogenous adenosine. Adenosine suppresses cardiac function (Olsson and
Pearson, 1990; Monopoli et al., 1998) and promotes vasodilation in many vascular
beds (Sollevi and Fredholm, 1980; Gawlowski and Duran, 1986; Collis, 1989;
Cheng et al, 1996).
Many studies and the enzyme activities of the separate fraction assessed, have
demonstrated that none of these enzymes are responsible for acute toxicity of snake
venoms. However, one particular enzyme which is present in almost all snake
venoms, phospholipase A2, has undergone substantial evolutionary changes, e.g.,
23
from a non-toxic enzyme to a greatly potent neurotoxin such as notexin from the
venom of Notchis scutatus, β-bungarotoxin from the venom of Bungarus
multicinctus and taipoxin from the venom of Oxyurans scutellatus (Hagwood and
Bon, 1991). These toxins show high specificity for presynaptic membrane of motor
nerve terminals intrusive with transmitter release. Whereas, venom proteases,
principally those interfering with blood coagulation or causing hemorrhage and
bleeding, may also be considered to be toxins, ever since there in vivo effects may
cause the death of experimental animals or human beings. These examples may
reveal that an apparent distinction between “real” toxins and enzymes is vague in
snake venoms. In addition of their effects in killing and immobilizing the prey as
rapidly as possible, enzymes in snake venoms have an important function in
sustaining prey digestion (Greene, 1992).
One has to think that snake ingest their prey, sometimes large animals, whole.
To circumvent putrefaction and bacterial infections, these enzymes must make
certain that venom has spread from the site of bite such as by preventing clotting of
blood, by lowering the blood pressure and causing hemorrhage into body cavities.
By destroying membrane barriers, cells and tissue, they may also initiate and
promote autodigestion of the prey organism. The massive injection of the digestive
enzymes – from milligrams to two grams of venom such as in the case of large
specimen of Bitis gabonica which contain mostly hydrolases, must contribute to
digestion on an “inside-out” basis (Iwanaga and Suzuki, 1979; Stocker, 1990b;
Mebs, 1998).
In the course of analyzing enzymatic composition patterns relative to prey
preference among coral snake taxa (Silva and Aird, 2001) it was difficult to explain
the ubiquitous nature of some relatively non-toxic venom constituents. The nearly
24
universal occurrence of some enzymes suggested a central role in envenomation, but
no satisfactory explanation has ever been given for their presence in venoms.
Recently, Cousin and Bon (1999) noted that ‘snake venoms are rich in protein and
enzymes whose functions are unknown’, citing nerve growth factor (NGF), L-amino
acid oxidase (LAO) and phosphodiesterase (PDE) as examples. Numerous other
enzymes and non-enzymatic toxins could also have been added to this short list. It is
suggested that in addition to a digestive function, these enzyme could contribute to
envenomation through their ability to release or form purines from the endogenous
sources in the envenomed animals (Aird, 2002).
Recently, Aird (2005), argued that both exogenous (venom) purines and
endogenous purines released from prey tissues act as multifunctional toxins, exerting
synchronous effects upon virtually all cell types. It was hypothesized that purine
nucleosides constitute the central element in snake envenomation strategies,
simultaneously immobilizing the prey by hypotension and paralysis, and
contributing to prey digestion via apoptosis and fast necrotic cell death. Aird (2002)
further concluded that many nontoxic venom enzymes exist in venoms specifically
to liberate endogenous purines.
1.11. Distribution of Enzymes in Snake Venoms
Out of many enzymes, 12 are found in all snake venoms, although their
contents differ significantly. Some of these enzymes are characteristic constituents
of venoms from certain snake families or genera. Proteases including kallikreins,
hemorrhagins, enzyme acting the blood clotting cascade and hydrolase of arginine
esters are predominantly found in Viperidae and crotalid venoms; phospholipase B
and acetylcholineesterase are mainly found in Elapidae venoms. Others such as
25
phospholipase A2, L-amino acid oxidase, 5’-nucleotidase, several PDE’s and
hyaluronidases are found in most venom (Table 1.2).
Ever since immunologic studies of venoms generally show an absence of
common antigens in venoms of distantly associated species such as cobras and
rattlesnake, the extensively distributed enzymes obviously differ between these taxa
in molecular properties. The variety of enzymes and their high activity, e.g. the high
protein concentration, make snake venoms ideal source for the isolation of a
particular enzyme.
An interesting feature of enzyme in snake venom is their high stability when
stored in dry state e.g., rattle snake venom when stored for ~ 50 years has
approximately the same biologic activity as freshly extracted venom. Other venoms
have also been shown to retain lethality and enzyme activities of phosphodiesterase,
5’-nucleotidase, phosphomonoesterase, phospholipase A2, and cholinesterase, except
for L-amino acid oxidase, during storage period of 17-39 years at room temperature.
Hence, most of the properties of snake venoms seem to be well retained in promptly
lyophilized material (Iwanaga and Suzuki, 1979; Mebs, 1998).
1.12. Uses of Snake Venom Enzymes in Diagnostic, Research and
Therapeutics
The venom snake is a concoction of hundreds to thousands of organically
dynamic proteins and enzymes which are being used in both assault and defense.
Enzymes in snake venoms are a fascinating phenomenon both from a chemical and
biological point of view. Since these proteins are competent of modulating the
physiological reaction of envenomated animals, therefore they demonstrate promise
as prospective pharmacological tools and as drug leads.
26
A number of snake venom enzymes are extensively used as tool in
biochemical research or as diagnostic or therapeutic agents. Purified snake venom
enzymes with a known mode of action and a narrow substrate specificity, resistant to
inhibitor systems present in blood and tissues, have found multiple uses in
therapeutics, diagnostics or preparative procedures in hemostaseology (Table 1.3),
while only few practical applications of snake venom enzymes are known from other
biochemical or biomedical disciplines (Table 1.4).
Snake Venom Enzymes as Tool in Biochemical Research
L-amino acid oxidase has been widely used for identifying optical isomers of
amino acids and utilized to form a number of α-keto acids from corresponding alpha
amino acids (Meister, 1952; Meister, 1956). Phospholipases A2 have been employed
to study the structural function of phospholipids as mediators of biological processes
or in membrane proteinases for the amino acid sequence studies of peptides and
proteins.
In a study, it has been demonstrated that the methanol - esterification reactions
are catalyzed by the snake venom SVP (5'-nucleotide phosphodiesterase) as
mentined by García-Díaz et al., (1991). The ability of SVP to catalyze AMP transfer
from ATP to propanol, ethanol, methanol, ethylene glycol, glycerol, 2-chloroethanol
or 2,2-dichloroethanol had been demonstrated. The HPLC, enzyme analysis,
ultraviolet and NMR spectroscopy were used to identify the AMP-O-alkyl ester
products. The potential of SVP as a tool to prepare 5'-nucleotide esters is illustrated
by these results. This also agrees with the production of a covalent 5'-nucleotidyl-
SVP intermediate susceptible to nucleophilic attack by short-chain (poly) alcohols as
acceptors substitute to water. In order to investigate the kinetic influence of the
solvent nucleophile in SVP mechanisms, initial rates of ATP solvolysis were
27
determined in diverse water/alcohol mixtures. The SVP was inactivated
comparatively by the high alcohol concentrations however lower concentrations
gave relative rates of alcoholysis.
An efficiency factor (EA), is the ratio of the mole fraction of AMP-O-alkyl
ester as a product to that of alcohol as an acceptor in water/alcohol mixtures, made
promising the evaluation of alcohols and water as AMP acceptors at low
concentrations, since it might be EA and VH rational that EA = 1 for water. The
rates of hydrolysis (VH) of different leaving groups and of substrates yielding AMP
were also assayed. The elevated values were found to correspond to those acceptors
and leaving-group conjugate acids with lower pKa and higher polar-substituent
constants respectively.
The incidence of general acid-base catalysis in the active center of SVP and
the identification of rate-limiting steps is supported by the results. For the
mechanisms of SVP-catalyzed hydrolysis and alcoholysis a model has been
proposed which accounts for the authority of the acid-base properties of alcohols on
the kinetic sketch of SVP reaction sequences (García-Díaz et al., 1993). The
alcoholysis of ATP by primary R-CH2OH alcohols with uncharged R residues,
yielding AMP-O-CH2R esterification products is catalyzed by snake venom
phosphodiesterase (SVP). The alcohols struggle with water for an SVP-bound
adenylyl intermediate. The reactions of glycerol 2–phosphate and sn-glycerol 3–
phosphate with ATP to yield AMP-O-glycerophosphoryl esters has been shown to
be catalyzed by SVP also. The HPLC was used to identify products, the reliance of
the reactions on glycerol phosphates, ultraviolet spectroscopy, and conversion to
AMP by phosphodiesterase, or to AMP-O-glyceryl esters by alkaline phosphatase.
28
The fact that the R-CH2OH alcohols with negatively charged R residues, as
well as secondary alcohols, act as adenylyl acceptors in SVP reactions revealed by
the results, thereby, extending the efficacy of SVP as a tool to produce 5'-nucleotide
derivatives.
The efficiencies (EA) of glycerol phosphates as adenylyl acceptors were found
to be very high at low, milli-molar concentrations. However they decreased
suddenly when the acceptor concentration was increased and when Pi or NaCl was
present, for glycerol 2-phosphate while the glycerol EA was not dependent on its
own concentration of Pi and NaCl. It is indicated by the responses of glycerol
phosphates that they act as adenylyl acceptors through a mechanism different from
uncharged R-CH2OH alcohols. The incidence of an acceptor-binding enzyme site
which is specific for negatively charged R residues, and its prospective significance
to the in vivo role of 5'-nucleotide phosphodiesterases as 5'-nucleotidyl transferases
are described (Vergeles et al., 1995).
In addition to its diagnostic applications, Protac® has been used in research
work on missense mutations of human PC (Miyata et al., 1994,1995; Vasse et al.,
1989) and for structure-function studies on human recombinant PC and APC,
including site directed mutants of this protein (Christiansen et al., 1995). Moreover,
Protac® has been used to study the inhibition by various inhibitors of APC in pure
systems and in plasma (Taby et al., 1990; Van der Meer et al., 1989; Heeb et al.,
1989).
Proteinases that degrade serine proteinase inhibitors (serpines) and even α2-
macroglobulin (α2-M) are present in the venoms of several snake species. In
principal, such enzymes may be used in diagnostic and preparative procedures to
abolish the inhibition of serine proteinase activities generated in plasma or plasma
29
fractions. Thus, Contant et al., (1992) described a method for the determination of
plasminogen activator inhibitor (PAI) in which the interference by α2-antiplasmin
and α2-macroglobulin is abolished by Bitis arietans venom in presence of
tranexamic acid.
Two types of proteinases which mimic part of multiple actions of thrombin
have been found in snake venoms.
1. Enzymes like thrombocytin (Niewiarowski et al., 1977) and crotalocytin
(Schmaier and Colman, 1980) which primarily act on thrombin substrate other than
fibrinogen, and
2. Enzymes like ancrod, batroxobin and venzyme which clot fibrinogen. Both types
of enzymes are used in hemostatis research; fibrinogen coagulant enzymes are also
used in diagnostic procedures. The best documented examples of the two types of
thrombin-like enzymes are thrombocytin and batroxobin, both isolated from B. atrox
venom (Table 1.5).
30
Table 1.2: Enzymes found in snake venoms
Enzymes found in all snake venoms
Phospholipase A2, L-Amino acid oxidase, Phosphodiesterase, 5’-Nucleotidase, Phosphomonoesterase, Deoxyribonuclease, Ribonuclease, Adenosine triphosphatase, Hyaluronidase, NAD-nucleosidase, Peptidase.
Enzymes in crotalid and viperid venoms
Endopeptidase, Arginine ester hydrolase, Kininogenase, Thrombin-like enzyme, Factor X activator,
Prothombin activator
Enzymes in elapid venoms
Acetylcholinesterase, Phospholipase B, Glycerophosphatase,
Enzymes found in some venoms
Glutamic-pyruvic transaminase, Catalase, Amylase, β-Glucosaminidase, Lactate dehydrogenase, Heparinase- like enzyme
31
Table 1.3: Applications of snake venom enzymes
Application Venom enzymes
Hemostatic agents Activators of factor II,X, thrombin-like enzymes
Anti-thrombotic agents
Thrombin-like enzymes, fibrinogenases
Tissue adhesives Thrombin-like enzymes
Plasma processing Thrombin-like enzymes
Hemostasis testing Activators of factor II, V, X, protein C; thrombin-like proteinases enzymes, fibrinogenases, serpinases, phospholipases, heparinase.
Table 1.4: Use of snake venom enzymes in disciplines others than emostaseology
Application Snake venom enzymes References
Neurobiology Phospholipase A2 Mebs (1990)
Compliment system research
Proteinases Vogt (1990)
DNA/RNA sequencing
Phosphodiesterases Elliot (1978)
Phospholipid analysis Phospholipase A2 Elliot (1978)
32
1.12.2. Application of Snake Venom Enzymes in Clinical Field
Snake venom proteinases that within the hemostatic system affect specific
substrates can be used to elucidate and to asses interactions of components of this
system (Table 1.6). Some of the snake venom enzymes, e.g., thrombin like enzymes
(reptilase, ancrode and batroxobin) and procoagulant (stypven), have certain
application in clinical field (Rosenberg, 1987).
The most significant groups of enzymes present in snake venom having
prospective for clinical therapeutic use are thrombin-like enzymes, which catalyze
the conversion of fibrinogen into fibrin, frequently similar, but not alike, to
thrombin. Making use of the information of their substrate specificity and catalytic
mechanism the scientists developed the reagents to carry out coagulation tests in
specimens of blood that contain heparin, and also the studies about their use as de-
fibrinogenating agents in chronic and acute diseases, e.g. stroke, deep-vein
thrombosis, myocardial infarction, and peripheral arterial thrombosis (Castro and
Rudrigues, 2006).
Good number of abnormalities in hereditary thrombophilia (hypercoagulable
states) and the disturbances within the protein C pathway due to coagulation factor
V Leiden mutation and deficiency of protein C or protein S. Moreover the acquired
dysfunctions of the protein C system could incline an individual to an amplified
thrombotic risk. The abnormalities of the protein C natural anticoagulant
organization are based on measuring protein C, protein S, and activated protein C
resistance, for diagnostic or research purposes. Nearly all of the assays include the
protein C activator Protac® from Agkistrodon contortrix contortrix (southern
copperhead).
33
Table 1.5: In vitro diagnostic uses of snake venom enzymes
Enzyme Test Reference
Ancrod Fibrin formation Haverkate et al., 1985
Batroxobin Fibrin formation, platelet functions
Stocker, 1990
RVV-X Factor-X assay,
LA testing
Bachmann et al., 1958
Svendsen et al., 1984
Thiagarajan et al., 1986
RVV-V Factor-V assay Van Dam-Mieras et al., 1984
Ecarin Assay for F-II, acarboxy F-II hirudin
Kornalik, 1988
Nowak et al., 1993
Textarin LA testing Triplett et al., 1993
Protac PC assay
PS assay
PC system
Stocker et al., 1986
Suzuki and Nishioka, 1988
Kraus et al., 1995
Taipan venom Factor-II assay
Factor-V assay
LA testing
Denson et al., 1971
Hagglund and Blomback, 1986
Rooney et al., 1994
Venzyme Fibrin research Kay et al., 1974
Thrombocytin Platelet functions Niewiarowski et al., 1979
CR-Serpinase ATIII depletion Janssen et al., 1992
Phosphodiesterase DNA research Ho and Gilham, 1973
B.arietans venom AP depletion Contant et al., 1992
34
Table 1.6: Preparative uses of snake venom enzymes
Enzyme Origin Substrate Product Reference
Batroxobin B. atrox Fibrinogen Fibrin monomer
Ranby et al., 1982
RVV-V V. russellii Plasma F-V depleted plasma
Conrad, 1988
Protac® A. controtrix Protein C Activated PC Christiansen
et al., 1995
F-II activator
O. scutalatus F-VII F-VIIa Nakagaki
et al., 1992
Ecarin E. carinatus Prothrombin Meizothrombin Nowak and Bucha, 1993
35
The Protac® straightforwardly converts the zymogen protein C into the
catalytically dynamic variety that can simply be determined by means of coagulation
or chromogenic substrate techniques, which is not like the physiological, thrombin-
catalyzed protein C activation reaction that needs thrombomodulin as a cofactor.
Numerous snake venoms contain Protein C activators. The majority of them have
been purified from venom of snake species that belongs to the Agkistrodon or
Gloydius genus. Because of its fast-acting property the protein C activator Protac®
has found a wide use in assays for research and in clinical practice to recognize
patients having the defects of the protein C system (Gempeler-Messina and Müller,
2006).
Our considerate of some molecular aspects of the hemostatic procedure has
become understandable with the help of the snake venom proteins that posses or do
not posses enzymatic activity but can bind to blood coagulation factors and
demonstrate anticoagulant effects. If an added thorough analysis of the structure-
function relationship of these molecules will be available, would definitely result in
novel medical and pharmacological applications. Moreover, the venom components
present striking templates for the progress of realistically planned curative agents
(Zingali, 2007).
The family of proteins found in nature, with low molecular weights and
extremely preserved sequences, together in their cystein arrangements and adhesive
Arg─Gly─Asp (RGD) motifs was denoted by the expression “disintegrin” first time
in 1990. An inhibitory potential in interacting with cell surface integrin receptors
was also exhibited as a general feature by these proteins. has becom a The trademark
assay for comparing the actions of members of this progressively more dissimilar
36
family revealed in the precedent two decades is the determination of the
consequence by disintegrins on the interaction amid the platelet receptor α IIbβ3 and
its ligand, fibrinogen (McLan et al., 2007).
Rhodostomin from snake venom that binds to mammalian integrins by a high
affinity through its Arg─Gly─Asp motif belongs to disintegrin family unit. The role
of soluble rhodostomin is as an integrin inhibitor by contending with the integrin-
ligand interaction. On the other hand, the immobilized rhodostomin acted as an
integrin activator through cross-linking the integrin on cell surface. Depending on
these properties the rhodostomin was utilised either as an anti-thrombosis agent or,
as an integrin agonist to trigger cell activation. The rhodostomin has been used to
learn integrin functions in numerous aspects comprising cell signaling, integrin-
mediated endcytosis, and the anti-thrombosis effects in contagious ailment models,
in recent times. The fact is revealed by the outcome of these studies that the
rhodostomin is a potent device to slice up the function of integrins (Chang and Lo,
2007).
The Elapidae and Hydrophyidae snake venoms have a cocktail of toxin
proteins with marked pharmacological properties. A few of them are neurotoxins,
while the others are cardiotoxins. The neuritoxins obstruct synaptic nerve
transmission by binding distinctively to the nicotinic acetylcholine receptor. There is
sequence homology among the short and long neurotoxins and also share a general
global fold. All of them composed of three fingers like loops coming out from a
globular center region cross-linked via disulfide bonds.
The isolation and characterization of nicotinic acetylcholine receptor (nAChR)
have been carried out by using the α-neurotoxins. The binding process of toxin to
the nAChR frequently comprises the residues from loop I and II, with a smaller
37
input from loop III. The neurotoxins are believed to unite by the nAChR through
charge-charge interaction between the base residues of the neurotoxins and acidic
residues of nAChR. The innermost loop of the toxin offers a significant character in
its binding capacity toward the receptor. The acetylcholine-binding protein
(AChBP), a natural homologue of the extra-cellular domain of the nAChR has been
discovered recently. Now it is achievable to place the toxin molecule on AchBP and
reveal an experimentally based three dimensional replica of toxin receptor complex.
There is a noteworthy difference between the modes of binding of short and long
neurotoxins to the acetylcholine receptor nevertheless both toxins contain a common
binding position on the α-7 subunit of the acetylcholine receptor (AChR), spanning
residues 182-206.
A number of groups have worked to explicate the structure and functions
relationships of cobrotoxin, a neurotoxin from Formason cobra, by means of
chemical modification studies. The groups of workers have paying attention on this
area of research because of the immense pharmaceutical appliance of cobrotoxin.
The cobrotoxin has been cloned effectively and over-expressed in high yields in
soluble state. The structure-activity relations were observed by HSQC
(Heteronuclear Single Quantum Coherence) spectrum of recombinant cobrotoxin
titrated with a peptide resulting from Torpedo nAChR. These studies have defined
The nAChR binding sites on cobrotoxin have been defined and the clues have been
specified to design the medicines that aim the nicotinic pharmacopoeia (Mohan and
Yu, 2007).
Medicinal Use of Snake Venom Enzymes
Wherever venomous snake occur, their venom has been used in occult and
folk medicine. Snake venom has been used to treat pathological condition such as
38
hemophilia, hypertension, rheumatic diseases, neuralgia, hay fever, cancer, lepra,
yellow fever, epilepsy and rabies (Reichert et al., 1944). Far before the biochemistry
of snake venom and the hemostatic system was understood, empirically diluted, un-
fractionated venom of various species, including Agistrodon piscivorus, Notechis
scutatus, Cerastes vipera, Naja naja and bothrops atrox, were tried as local and
systemic hemostatic in humans (Klobusitzky, 1951) and some observations made in
the course of such therapeutic trails stimulated systemic research. Blood clotting in
vitro upon the addition of snake venom, while patient bitten by the same snake
species had unclottable blood, was a fascinating mystery requiring clarification. This
paradoxal phenomenon stimulated scientists to elucidate the composition, properties
and action of snake venoms and to try venoms or venom fractions for the therapy of
hemorrhagic and thrombo-embolic diseases.
Numerous pharmaceutical preparations made from snake venoms have
become available following the pioneering work of Klobusitzky and Konig (1936)
who separated virtually a non-toxic fraction called “haemocoagulase” from Bothrops
jararaca venom and who suggested its potential use as hemostatic drug. The
currently available hemostatic agents, Reptilase® and Botropase® (Verstraete, 1987),
consists of batroxobin from B. atrox venom and of the thrombin like enzyme from B.
jararaca venom respectively. The thrombin-like enzymes ancrod from Calloselasma
(Agistrodon rhodostorma) and batroxobin from Bothrops atox moojeni act with a
narrow substrate specificity on the Aα-chain of the human fibrinogen, and are used
as antithrombotics.
In addition to ancrod and batroxobin, drugs prepared from venom of
Agistrodon acutus and Agistrodon halys Pallas are clinically used as antithrombotics
in the People’s Republic of China. They are often misnamed “Reptilase” (Liao and
39
Zou, 1992) and “Defibrase” (Haung et al., 1992) which are proprietary registered
trademarks. A novel plasminogen activator discovered in the venom of Trimeresurus
stejnegeri, TSV-PA, has been characterized as serine proteinase which cleaves the
peptide bond Arg561-Val562 of plasminogen to yield plasmin (Zhang et al., 1995).
The amino acid sequence of TSV-PA could be deduced from the cloned cDNA
encoding for TSV-PA. Plasminogen activator inhibitor type 1 (PAI-1) does not
inhibit TSV-PA, a feature which qualifies TSV-PA as a model for the design of
novel plasminogen activators to be produced by genetic engineering.
A dramatic improvement of fibrinogen-based tissue adhesives was achieved
with the development of a completely autologous system, Vivostat® (Edwardson et
al., 1995, Blomback et al., 1995). This sealant system uses a concentrate of fibrin I
monomer prepared from patient’s own plasma by incubation with biotinylated
batroxobin to form a clot of fibrin I, which is subsequently dissolved and
depolymerized at low pH and from which biotinylated batroxobin is then removed
by treatment with immobilized avidin (Burtan et al., 1995). The batroxobin-free
fibrin monomer solution is then mixed with a neutral buffer and simultaneously
applied to wound where it polymerizes immediately. The properties and actions of
batroxobin are therapeutically used to prevent thrombus formation and propagation,
to increase capillary circulation, to improve tissue oxygenation and to support
physiological or medicinal fibrinolysis (Table 1.7).
The ancrod cause defibrinogenation directly, which consequences in quite a
few secondary effects that distinguish ancrod from classic thrombolytics such as rt-
PA. The joint events of reduced fibrin deposition, indirect fibrinolysis, and decrease
in blood viscosity symbolize a collection of events that should be helpful in a range
of thrombotic conditions.
40
The ancrod has been explored most comprehensively in recent years for its
keen use for ischemic stroke. One of the two experimental studies (with a 6 h time-
to-treatment window) has established noteworthy advantage in increasing the
percentage of patients alive and free of disability at 3 months. Although elevated
doses of the preparation show to augment the threat of symptomatic intracranial
hemorrhage and diminish efficacy. The proof suggests although not conclusively,
that with customized dosing, the ancrod might capitulate a complimentary risk-
benefit ratio in stroke by restraining the danger of symptomatic intracranial blood
loss (Levy et al., 2006). Recently, Matsuda (2011) has used the snake venom
phosphodiesterase in the development of highly nuclease-resistant chemically-
modified therapeutic oligonucleotide.
Preparative Uses of Snake Venom Enzymes
Enzymes which activate the precursor forms of human and animal plasma clotting
factors are used to prepare the respective active proteins (Table 1.8). The substrate
treatment with the snake venom enzyme can in general be performed batch-wise, in
solution, or in a continuous process using immobilized venom enzymes. Active
center blocked snake venom enzymes with still intact substrate binding sites can be
used as affinity agents to purify the respective substrates, e.g. factor V, protein C,
prothrombin etc., or to deplete plasma such proteins. The thrombin-like enzyme
batroxobin, from Bothrops atrox moojeni venom, has been used to remove
fibrinogen from therapeutic human factor VIII concentrates (Lopaciuk and Latallo,
1973).
41
Table 1.7: Proposed indications for thrombin-like snake venom enzymes
Indications References
Deep vein thrombosis Blomback and Egberg, 1975
Central retinal vein thrombosis
Guadric et al., 1982
Sudden loss of hearing Shiraishi et al., 1991, 1993
Vibration disease Matsunaga et al., 1986
Thrombosis prophylaxes in vascular surgery
Furukawa and Ishimaru, 1990
Cerebral infarction Iwabuchi et al., 1991, Tanahasi et al., 1995
Myocardial infarction Apprill et al., 1985
Thrombosis prevention in hip surgery
Belch et al., 1982
Pulmonary embolism Charbonnier et al., 1983
42
Table 1.8: Properties and actions of thrombocytin and batroxobin
Property/action Thrombocytin Batroxobin
Fibrinopeptide A release − +
Inhibition by AT III/heparin
+ −
Inhibition by hirudin _ _
Activation of Factor-V + _
Activation of Factor-VIII + _
Activation of Factor-XIII + _
Activation of platelet aggregation
+ _
Activation of thrombostenin
+ _
Release of endothelial Tpa + +
Release of endothelial PG12
+ _
Activation of endothelial relaxation
n. d. +
43
1.13. Nucleases and Their Application in Research
Nucleases are enzymes which hydrolyze nucleic acids and are found in many
different venoms from front - fanged snakes. Among the myriad of enzymes present
in animal venoms, nucleotidases and nucleases are poorly investigated. Little is
known about the contribution of these enzymes to sequelae of envenomation, in
vitro activity, amino acid or cDNA sequence, degree of homology of enzymes from
various species of snakes and absolute relation of exonuclease and endonuclease
activities. Even for venom phosphodiesterase, the best studied venom nuclease, here
are noteworthy gaps in the considerate of fundamental biochemical properties of
these enzymes.
Many snake venoms also catalyze the hydrolysis of RNA molecules of
varying length, and venom RNases have been considered endonucleases. However,
experimental evidence demonstrating the presence of a unique is limited to venom of
a single species, Naja naja axiana (central Asian cobra; Babkina, 1965; Vassilinko
and Ryte, 1975).
Snake venoms usually contain one or more components capable of catalyzing
the hydrolysis of native or denatured DNA of various lengths, and internal
hydrolysis of a DNA molecule or oligonucleotide, would indicate endonuclease
activity of venom DNase. Recently, Sales and Santoro (2008) studied such enzymes
in 28 crude venoms of animals found in Brazil. Higher levels of ATPase, 5'-
nucleotidase, ADPase, phosphodiesterase and DNase activities were observed in
snake venoms belonging to Bothrops, Crotalus and Lachesis genera than to Micrurus
genus. The venom of Bothrops brazili snake showed the highest nucleotidase and
DNase activities, whereas that of Micrurus frontalis snake the highest alkaline
phosphatase activity. On the other hand, the venoms of the snake Philodryas olfersii
44
and the spider Loxosceles gaucho were devoid of most nucleotidase and DNase
activities.
Species that exhibited similar nucleotidase activities by colorimetric assays
showed different banding pattern by zymography, suggesting the occurrence of
structural differences among them. Hydrolysis of nucleotides showed that 1 mol of
ATP is cleaved in 1 mol of pyrophosphate and 1 mol of orthophosphate, whereas 1
mol of ADP is cleaved exclusively in 2 moles of orthophosphates. Pyrophosphate is
barely hydrolyzed by snake venoms.
Phosphodiesterase activity was better correlated with 5'-nucleotidase, ADPase
and ATPase activities than with DNase activity, evidencing that phosphodiesterases
are not the main agent of DNA hydrolysis in animal venoms. The omnipresence of
nucleotidase and DNase activities in viperid venoms implies a role for them within
the repertoire of enzymes involved in immobilization and death of preys.
Phosphodiesterases be a varied family of enzymes that hydrolyse cyclic nucleotides
and therefore take part in a key character in regulating intracellular levels of the
second messenger cAMP and cGMP, and thus cell function.
The phosphodiesterase (PDE) story begins with the work of Salter in 1886. An
asthmatic he noted that when he drank a strong cup of coffee on an empty stomach,
his breathing eased, an effect attributed to bronchodilator properties of caffeine.
Although the mechanism of action at the time was unknown, it has since been shown
that caffeine was acting as a non-selective, albeit weak, PDE inhibitor.
Phosphodiesterases exist both intracellulary and extracellularly in a wide variety of
tissues and organisms (Beavo, 1988). Intacellular phosphodiesterases (PDE’s) play a
role in signal transduction by regulating the cellular concentration of cyclic
nucleotides.
45
Extracellular PDE’s known as exonuclease, exist in venoms and their route in
envenomation is mostly by attacking nucleic acids through removal of
mononucleotide units from polynucleotide chain in stepwise fashion (Adams et al.,
1986). From a very early period, it was hypothesised that there were a number of
different isoforms of PDE distinguished primarily by their substrate specificity and
sensitivity to calcium-calmodulin (CaM) and these isoenzymes were numbered
according to their elucidation order (Victoria et al., 2006). They were first
differentiated in the early 1970s in rat and bovine tissue (Beavo et al., 1970).
Initially, three enzymes were identified and known as CaM-PDE, cAMP-PDE and
cGMP-PDE, which were further characterised by the use of selective inhibitors for
these enzymes (Hidaka & Endo, 1984; Nicholson et al., 1991). With the advent of
the molecular age, the number of PDE isoforms identified increased and so in 1995,
the nomenclature for the PDE family was standardised (Beavo, 1995). Today 11
isoenzyme groups, encompassing over 50 isoforms, have been identified including
the recently characterised PDE4A11 (Wallace et al., 2005) as given in Table 1.9.
There are at least four enzymes in snake venoms which act on the hydrolysis
of phosphate esters. Venom Phosphodiesterase I (Oligonucleate 5’-nucleotidehydro-
lase; EC. 3.1.15.1) catalyze the hydrolysis of phosphodiester bonds in a progressive
fashion, beginning at 3’ end (Mackessy, 1998). Although venom phosphosdiesterase
shows a broad specificity toward a number of nucleotide derivatives but its most
characteristic mode of action is in the stepwise degradation from the end of the
oligonucleotide chain bearing a 3’ hydroxyl group, resulting in the successive
release of nucleoside-5’-phosphate units (Figure 1.1). Therefore the enzyme is very
useful for determining the sequences of both ribo and deoxyribo oligo-nucleotides
46
and identifying α (alpha) and щ (gamma) terminal nucleotides (Figures 1.2, 1.3 and
1.4). Therefore this study is devoted for Phosphodiesterase I.
Numerous names exist in the literature for the same activity, often from the
same venom. The following names, when used in relation to venom derived-
products, are here considered synonymous with venom phosphodiesterase:
exonuclease, 3’-exonuclease, 5’-exonuclease, 5’-phosphodiesterase, 5’- nucleotide
phosphodiesterase, phosphodiesterase I and 2-5A phosphodiesterase. The term
DNase and ADPase has also been applied to proteins which appear to be venom
phosphodiesterases. The biochemical, diagnostic and therapeutic uses of snake
venom enzymes have led to intensive attempts to study the occurrence, purification
and characterization of the enzymes (Tu, 1977).
47
Table 1.9: The PDE superfamily
PDE isoenzyme
No. of isoforms
Substrate Km (μM) cAMP
Km (μM) GMP
Tissue expression Specific inhibitors
1 8 Ca2+/calmodulin-stimulated
1–30 3 Heart, brain, lung, smooth muscle
KS-505a
2 cGMP-stimulated 50 50 Adrenal gland, heart, lung, liver, platelets
EHNA (MEP-1)
3 4 cGMP-inhibited, cAMP-selective
0.2 0.3 Heart, lung, liver, platelets, adipose tissue, inflammatory cells
Cilostamide Enoxamone Milrinone Siguazodan
4 20 cAMP-specific 4 Sertoli cells, kidney, brain, liver, lung, inflammatory cells
Rolipram, Roflumilast Cilomilast
5 3 cGMP-specific 150 1 Lung, platelets, vascular smooth muscle
Sildenafil, Zaprinast
6
cGMP-specific 60 Photoreceptor Dipyridamole
7 3 cAMP-specific, high-affinity
0.2 Skeletal muscle, heart, kidney, brain, pancreas, T lymphocytes
BRL-50481
8 cAMP-selective, 0.06 Testes, eye, liver, skeletal muscle, heart, kidney, ovary, brain, T lymphocytes
none
9 4 cGMP-specific, 0.17 Kidney, liver, lung, brain
BAY 73-6691
10 2 cGMP-sensitive, cAMP-selective
0.05 3.0 Testes, brain None
11 4 cGMP-sensitive, dual specificity
0.7 0.6 Skeletal muscle, prostate, kidney, liver, pituitary and salivary glands, testes
None
Br J Pharmacol. 2006 January; 147(S1): S252–S257. Published online 2006 January 9. doi: 10.1038/sj.bjp.0706495.2006, Nature Publishing Group.
48
Figure 1.1: Hydrolysis of DNA by venom phosphodiesterase. Enzyme action (arrowheads) on native substrates is exonucleolytic, catalyzing hydrolysis stepwise from the 3’ end and liberating 5’-nucleotides (Mackessy, 1998).
49
Figure 1.2: A basic structural element to which venom phosphodiesterase binds for the hydrolysis of phosphodiester bond (Razzel and Khorana, 1959).
50
Figure 1.3: Cleavage sites of poly (ADP-ribose) with venom phosphodiesterase (C. adamanteus). The arrows indicate cleavage at pyrophosphate. Dotted lines indicate release of terminal phosphate by alkaline phosphomonoesterase. Ade, adenine: Rib, ribose: P, phosphate: Ado, adenosine (Matsubara et al., 1970)
51
Figure 1.4: Hydrolysis of 3’, 5’-cAMP by venom phosphodiesterase. Enzyme action (arrow) typically liberates 5’-AMP (Mackessy, 1998).
52
1.14. Occurrence of Phosphodiesterase in Snake Venoms
Delezenne and Morel (1919) were the first to witness the disintegration of
nucleic acids and production of nucleosides in the of snake venoms. Uzawa (1932)
first found the enzyme in the venom of T. flavoviridis and A. halys blomhoffii. The
first systematic study on the occurrence of phosphodiesterase and 5’-nucleotidase in
venom was made by Gulland and Jackson (1938). Venoms from most front-fanged
snakes which have been analyzed thus far show at least low levels of venom
phosphodiesterase activity. Most sea snake (family Hydrophiidae) showed no
phosphodi- esterase activity (Mackessy and Tu, 1993; Tan and Ponnudurai, 1991a).
The phosphodiesterase activity was prevalent in snake venoms of family Crotalidae
(Kuch et al., 1996). Venoms from the snakes of the family Elapidae typically
showed levels of phosphodiesterase activity which were 1-2 order of magnitude
lower than venoms from cortalid snakes (Tan and Ponnudurai, 1990a). Whereas
venoms from viperid snakes showed Intermediatelevels (Tan and Ponnudurai,
1990b). Venoms from the snakes in the family Colubridae (rear-fanged or
opisthoglyphous) are poorly characterized, but at least 7 species produced venoms
with detectable levels of phosphodiesterase activity (Weinstein and Kardong, 1994;
Hill and Mackessy, 2000).
For the intention of isolating a particular enzyme, venom must be found with a
elevated concentration of that enzyme and low concentration of the others. The
differing quantities of four enzymes (endonuclease, phosphodiesterase, 5’-
nucleotidase and nonspecific monophosphoesterase) present in venom must be
considered when isolating a particular enzyme, e.g. phosphodiesterase, an enzyme
widely used for the structural studies of nucleic acids and its related substances.
53
1.15. Isolation and Purification of Venom Phosphodiesterase
Venom phosphodiesterase has been isolated by many investigators, in
particular Laskowski and co-workers (Laskowski, 1966). The foremost goal of
purification has been directed toward removing the contaminating 5’-nucleotidase
and nonspecific monophosphoesterase. Older methods for the isolation and
purification of venom phosphodiesterases included one to several solvent
precipitation steps followed by several chromatographic steps (Williams et al., 1961;
Bjork, 1963).
More recent methods (Marcelo et al. 2009) relied entirely on chromatographic
steps, usually involving size exclusion, anion exchangers (frequently DEAE
functionalities) and cation (such as carboxymethyl resins) exchangers. One or more
method designed to eradicate all other nuclease activities, involved acetone and
ammonium sulfate precipitation and several affinity ligand chromatography steps.
The majority of methods required several ion exchange steps with different
functionalities in order to remove contaminating nucleases from phosphodiesterases
(Perron et al., 1993; Mackessy, 1989).
So there is a potential to develop a methodology for the isolation and
purification of phosphodiesterase enzyme from snake venoms which should involve
single step and / or minimum steps with reasonable good results, time saving and
economical. Since there are considerable gaps in the understanding of basic
biochemical, biological properties of venom phosphodiesterases which should be
filled by doing such studies extensively on purified preparations.
54
1.16. Aims and Objectives of the Study
The study conducted to achieve the following aims and objectives.
1. To develop a simple, short, effective and economical procedure for the isolation
and purification of phosphodiesterase I from snake venoms belonging to
different families.
2. To carry out biochemical characterization of the purified enzymes.
3. To carry out some biological studies on purified enzymes
55
2. MATERIALS AND METHODS
2.1. Venoms, Chemicals and Reagents
Agistrodon bilineatus (Ab) and Ophiophagus hannah (Oh) crude venoms were
purchased from Venom Supplies Pty Ltd, Tanunda, South Australia. Cerastes vipera
(Cv) crude venom was obtained from Latoxana, Rosans, France. Bis-p-nitrophenyl
phosphate, p-nitrophenyl thymidine-5’-phosphate, cyclic 3’,5’-AMP, ATP, 1,10-
phenanthroline and L- glutathione were from Sigma-Aldrich Co. St. Louis, MO.
USA. Acrylamide was obtained from Merck, Munchen, Germany. N,N-methylene
bisacrylamide and Coomassie Brilliant blue R250 were purchased from Fluka
Chemie AG, Buchs, Switzerland. Ammonium persulphate, sodium dodecyl
sulphate (SDS) and Tris (hydroxymethyl) methylamine were from BDH Chemicals,
Poole, UK.
Standard proteins (BlueRanger® Pre-stained Protein Molecular Weight Marker
Mix) were from Pierce Biotechnology, Inc. Illinois USA. Ampholine (pH 3.5–10.0)
was from Pharmacia Uppsala, Sweden. Glycerol from Winlab, Ltd., Maidenhead,
UK and glycine and N,N,N,N-Tetramethyl ethylenediamine were from Riedel-
Dekhen AG, Wunstrofer StraBe, Germany. p-Nitrophenyl phosphate (pNPP-
substrate) was purchased from Amresco Solon, Ohio, USA. 2-Mercaptoethanol was
from British Drug House Chemicals Poole, UK. All other chemicals used were of
analytical grade.
2.2. Gel Electrophoresis
It is a technique so as to separate macromolecules both nucleic acids and
proteins according to electric charge, size and other substantial properties. A number
of vital biological molecules like proteins, amino acids, peptides, nucleic acids and
56
nucleotides having ionisable groups and hence, exit in solution as electrically
charged species whichever as positive (cation) or negative (anions), at any certain
pH. The charged particles will travel either to anode or to cathode depending on the
character of remaining charge.The separation of big (macro) molecules is based on
two forces i.e charge and mass. An organic sample like proteins or DNA when
mixed in a buffer solution and loaded onto a gel, the two forces work jointly. The
molecules are repeled from electrical current of one electrode, whereas the other
electrode concurrently attracts the molecules. The role of frictional power of the
geling substance is as a “molecular sieve”, sorting out the molecules according to
size. The molecules are enforced to travel through the pores while the electrical
current is applied in electrophoresis.
The polyacrylamide gel electrophoresis (PAGE) may be used effectively as a
powerful preparative device to get a pure protein sample and also as an analytical
device to give information on the mass, charge, purity or occurrence of a protein.
Quite a few forms of PAGE be present and be capable of providing diverse types of
information concerning the protein(s). The native PAGE, separates the proteins
according to their mass: charge ratio, whereas the SDS-PAGE, separates proteins
principally by mass. The 2-D PAGE separates proteins according to isoelectric point
in the first dimension and according to mass in the second dimension. The
polyacrylamide gel could be primed so as to offer an extensive range of
electrophoretic conditions. In order to make diverse molecular sieving properties for
separating proteins of diverse sizes the pore size of the gel might be altered.
Acrylamide is considered the material of preference for preparation of
electrophoretic gels to separate proteins by size. A cross-linked polymer system of
acrylamide mixed with bisacrylamide is obtained while the polymerizing agent,
57
ammonium persulfate, is added (Figure 1). The ammonium persulfate generates free
radicals quicker in the company of TEMED (N,N,N,N'-tetramethylenediamine). The
dimension of the pores fashioned in the gel is inversely correlated to the quantity of
acrylamide used. For instance, a 8% polyacrylamide gel would have bigger pores in
the gel as compared to a 13% polyacrylamide gel. The gels having a high fraction of
acrlyamide are used to separate small proteins, whereas gels with a low proportion
of acrylamide are characteristically used to separate bulky proteins.
Two methods of polyacrylamide gel electrophoresis were used in our work:
(a) Non-denaturing or Native polyacrylamide gel electrophoresis
(b) SDS polyacrylamide gel electrophoresis
Both of these systems involved the use of double-stack polyacrylamide gels,
with a large-pore upper gel acting as a stacking gel and concentrating the protein
into a fine band separation in the small pore lower gel.
58
2.2.1. Analytical Native Polyacrylamide Gel Electrophoresis
Preparation of Gel
In order to establish the acrylamide monomer concentration that gives the best
separation of venom proteins. Three slabs 18 cm long, 16 cm wide and 0.3 cm thick
gel containing 8%, 10% and 12% acrylamide respectively were prepared as
described by Laemmli et al., (1970) with minor modifications using gel casting
system. The stock solutions for the lower small pore gel were mixed in proportions
given in Table 2.1 to give desired gel concentration. The first three components of
gel solution were mixed well followed by the addition of ammonium per-sulfate and
N,N,N,N- tetramethylenediamine (TEMED). The solution was then poured in
cassette, and a thin layer of distilled water was pipetted over the top to achieve a flat
surface after polymerization. The gel solution took about 30 minutes to polymerize.
After the completion of polymerization of lower gel, the solutions for the upper,
large-pore gel were mixed in the proportion shown in Table 2.2. The layer of water
was removed from the top of the lower gel and solution for upper gel was poured
over the separating gel in the presence of a plastic comb to make 10 wells. The
capacity of each well is 100-200 μl. The polymerization took about 15 minutes; the
wells were washed with distilled water to remove un-polymerized gel materials.
Preparation of Samples for Electrophoresis
The crude snake venom (freeze dried powder) was dissolved in distilled water
(30mg/ml) and centrifuged at 5,000 g for 15 minutes to remove any turbidity. The
crude venom (200 μg) samples were dissolved in sample buffer solution as given in
Table 2.5.
59
Electrophoresis
The plate containing a slab gel was clamped to a vertical slab gel apparatus
(Model SE600-15-1.5, Hoefer Pharmasia Biotech Inc. CA, USA,). The upper and
lower chambers contained the same reservoir buffer as described in Table 2.4. The
pre-electrophoresis was carried out for 30 minutes. A sample volume up to 200 μl
containing up to 200 μg of protein of three different snake venoms was then applied
to three separate wells. Sucrose in the sample buffer solution gives the sample
sufficient density to displace the upper reservoir buffer. The slab was run at 80C with
a constant voltage of 100 V for 6 hours until the dye front reached the lower end of
gel. The current was then turned off and the gel was removed from the plates,
stained with Coomassie Brilliant Blue R-250 (Table 2.6) for 30 minutes and then
destained with the de-staining solution consisting of water: glacial acetic acid :
methanol in the ratio of 68: 7: 25, respectively as given in Table 2.7.
2.2.2. Purification of Enzyme from Crude Venom by Preparative Native PAGE
Preparation of Gel
According to the result of analytical native PAGE section (2.2.1), the 10%
acrylamide monomer gel showed the best separation of different proteins present in
the three different snake venoms used. So a slab 18 cm long, 16 cm wide and 0.6 cm
thick gel containing 10% acrylamide was prepared as described table 2.1using the
apparatus and procedure as described in section 2.2.1. After the completion of
polymerization of lower gel, the solutions for the upper, large-pore gel were mixed
in the proportion shown in Table 2.2. The layer of water was removed from the top
of the lower gel and solution for upper gel was poured over the separating gel in the
presence of a plastic comb to make single trough. The polymerization took about 15
minutes; the trough was washed with distilled water to remove un-polymerized gel
60
materials. Then crude venom (10 mg of protein) was dissolved in sample buffer
solution of Table 2.5.
A sample volume up to 1ml containing up to 10 mg of protein was then
applied to the trough. The slab was run at 8 0C with a constant voltage of 100 V for 8
hours until the dye front reached the lower end of gel (Al-Saleh et al., 2002). The
current was then turned off. Two guide strips were cut from both sides of the slab
gel and stained with Coomassie Brilliant Blue R-250 (Table 2.6) for 30 minutes and
then de-stained with the de-staining solutions consisting of water: glacial acetic acid
: methanol in the ratio of 68: 7: 25, respectively (Table 2.7). The locations of the
protein bands on the unstained gel were then carefully marked and removed.
Table 2.1: 10% separating gel solution
Distilled water 48.8 ml
30% Acrylamide Mix 40 ml
3M Tris-HCl buffer, pH 9.5 30 ml
10% Ammonium persulfate 1.2 ml
TEMED 0.050 ml
Table 2.2: 4% Stacking gel solution
Distilled water 13.9 ml
30% Acrylamide Mix 3.4 ml
0.5M Tris-H3PO4 buffer, pH 9.0 2.5 ml
10% ammonium persulfate 0.2 ml
TEMED 0.025 ml
61
Table 2.3: 30% Acrylamide mix solution
Acrylamide 29.2 gm
N,N-methylene bis-acrylamide 0.8 gm
Distilled water up to 100 ml
Table 2.4: Reservoir Buffer (0.05M Tris-glycine buffer pH 9.5)
Tris-HCl (base) 6.06 gm
Glycine 3.75 gm
Distilled water 1000 ml
Table 2.5: Sample buffer without SDS
Reservoir buffer pH 9.5 4.0 ml
20% Sucrose 0.8 ml
0.5% Bromophenol blue 0.2 ml
Table 2.6: Staining solution
Coomassie Brilliant blue R250 1.25 gm
Methanol 125 ml
Glacial acetic acid 25 ml
Distilled water 350 ml
62
2.2.3. Analytical SDS-Polyacrylamide Gel Electrophoresis
(SDS-PAGE)
A slab 18 cm long, 16 cm wide and 0.3 cm thick gel containing 10%
acrylamide was prepared from solution given in Table 2.8 using the apparatus and
procedure as described in section 2.2.1. After the completion of polymerization of
lower gel, the solutions for the upper, large-pore gel were mixed in the proportion
shown in Table 2.9. The layer of water was removed from the top of the lower gel
and solution for upper gel was poured over the separating gel in the presence of a
plastic comb to make 10 wells. The capacity of each well is 100-200 μl. The crude
venom (250 μg) and purified enzyme (40 μg) samples were boiled with sample
buffer mentioned in Table 2.11.
The pre-electrophoresis was carried out for 30 minutes. Purified venom
fraction (40 μg) along with crude venom (250 μg) and standard proteins were
electrophoresed on SDS-PAGE at 10% acrylamide concentration according to
method of Laemmli et al., (1970). The standard molecular weight proteins used were
Myosin (215 kDa), phosphorylase b (120 kDa), bovine serum albumin (84 kDa),
ovalbumin (60 kDa), carbonic anhydrase (39.2 kDa), trypsin inhibitor (28 kDa), and
lsozyme (18.3 kDa). The slab was run at 80C with a constant voltage of 100 V for 6
hours until the dye front reached the lower end of gel. The current was then turned
off and the gel was removed from the plates, stained with Coomassie Brilliant Blue
R-250 (Table 2.6) for 30 min and then destained with the destaining solution given
in Table 2.7. The reference value (Rf) for each known and unknown protein was
determined. Then a standard curve of log of molecular weight of known proteins
against their reference values (Rf) was drawn. Finally the molecular weight of
unknown protein (enzyme) was determined from the standard curve. It is noted that
63
the covalently bound dye alters the apparent molecular weight (M.W.) of the
proteins relative to unstained proteins and tends to produce broader bands (Pierce
Biotechnology, Inc.).
Table 2.7: De-staining solution
Methanol 250 ml
Glacial acetic acid 70 ml
Distilled water 680 ml
Table 2.8: 10% separating gel with SDS
Distilled water 24.4 ml
Acrylamide mix 20 ml
3M Tris-HCl buffer pH 9.5 15 ml
10% SDS (w/v) 0.6 ml
10% Ammonium per-sulfate 0.6 ml
TEMED 0.025 ml
Table 2.9: 4% Stacking gel with SDS
Distilled water 13.6 ml
Acrylamide mix 3.4 ml
0.5M Tris-H3PO4 buffer, pH 9.0 2.5 ml
10% SDS (w/v) 0.2 ml
10% Ammonium persulfate 0.2 ml
TEMED 0.025 ml
64
Table 2.10: Reservoir buffer with SDS (0.05M Tris-glycine buffer pH 9.5)
Tris-HCl (base) 6.06 gm
Glycine 3.75 gm
SDS 5 gm
Distilled water 1000 ml
Table 2.11: Sample buffer with SDS
Reservoir buffer pH 9.5 4.0 ml
20% Sucrose 0.8 ml
0.5% Bromophenol blue 0.2 ml
10% SDS (w/v) 1.6 ml
65
2.3. Recovery of Proteins by Electroelution
The separated venom fractions were eluted from gel using a procedure
described by Walker et al., (1982). The area of preparative gel that contained the
venom fractions was sliced to small pieces. The gel slices were then put in a column
(10 ml), whose lower end was connected to a dialysis bag (4-5 cm length). A small
piece of cotton wool was placed at the bottom and top of the gel. After ensuring that
Laemmli reservoir buffer filled the column and dialysis bag, the upper chamber was
filled with buffer, while the end of the column was immersed in the tank which
contained the same buffer. The power was turned on and after 12 hours at 40 mA the
power was turned off. The eluted protein fractions were dialyzed for 30 minutes
against distilled water, lyophilized and stored at 4oC. The fraction obtained was
analyzed for purity on analytical native (non-SDS) PAGE as described under section
2.2.1.
2.4. Determination of Protein Concentration
The concentration of protein in the fraction thus obtained was determined by
using the method of Lowry et al., (1951). Lowry reagent was prepared from stock
solution of 2% (w/v) sodium carbonate in 0.1 M sodium hydroxide, 1% copper
sulfate and 2% (w/v) sodium potassium tartarate in a volume ratio of 1: 0.1: 0.1
respectively. A (5-25µl volume) of protein fraction was incubated with 1ml of
Lowry reagent at room temperature for 10 minutes, followed by addition of diluted
(1:1) Folin Ciocaltuae reagent (0.1 ml). The mixture was then incubated for 30
minutes at room temperature. The absorbance of fraction and standard was read at
730 nm and the protein content was determined by comparison with a standard (200
µg) of bovine serum albumin.
66
2.5. Isoelectric Focusing (IEF)
Electrofocusing is a very special electrophoretic technique by which protein
are separated according to their isoelectric points in a stable pH-gradient. Proteins
differing by only a few hundredths of a pH unit in their isoelectric points may be
resolved by this technique. Electrofocusing differs from conventional
electrophoresis in a manner that the pH is not set steady all over the entire system,
alternatively the sample components travel electrophoretically in a fixed pH
gradient. Thus a steady state will eventually be reached at which all the sample
components are concentrated or focused as sharp bands at their respective isoelectric
points (pI). Besides giving detailed information on the composition of a protein
mixture, electrofocusing permits estimation of isoelectric points (pIs) in a very
simple way.
2.5.1. Preparation of Gel
A thin layer of polyacryamide gel was moulded on a thin glass plate with good
heat conducting properties. The mould used for making gels that fit the LKB2117
Multiphor system consisted of a 1mm thick supporting glass plate, a 2mm thick
rubber gasket and two 3mm thick glass plates. All were clamped together. The
rubber gasket was lubricated with silicon grease to ensure a tight seal between glass
plates. After mixing the reagent as given in Table 2.12 , the solution was made up to
60 ml with distilled water (30.2 ml), degassed in a 250 ml vacuum flask for 10 min.
Then 1.5 ml of 1% ammonium persulfate solution was added and the complete gel
solution was mixed by swirling the flask. Care was taken to ovoid the re-uptake of
air because of the inhibitory effect of dissolved oxygen on polymerization reaction.
67
The solution was loaded onto the mould from the open end. The mould was sealed
and made air tight by applying the remaining clamps. Polymerization reaction was
completed in an hour at room temperature. The thick glass plate and rubber gasket
were removed. The template was placed on the cooling plate of a multiphore with a
thin film of insulating fluid. Electrode strips were soaked in the electrode solution
(Table 2.13).
2.5.2. Sample Application
Small plastic frames were placed on the gel surface. Crude venom and purified
enzyme were applied directly to the gel surface as droplets about 10-25µl (25µg) in
size. Cytochrome C, a red color protein a basic pI(10.0) was also applied as standard
and visual guide for monitering the progress of experiment. The experiment was
performed at 25 watt constant power. It took about 1.5 hours until the voltage
became around 1000V and the current was at 25 mA. The power was turned off.
2.5.3. Determination of pH Gradient
Immediately after the process, the lower part of the gel was cut into pieces at
0.5cm intervals, suspended in 10 mL distilled water and the pH gradient was
determined from cathode to anode.
2.5.4. Detection Method
The gel was immersed in fixing solution (Table 2.14) for 0.5-1.0 hour. This
will irreversibly precipitate the proteins and wash out most of the ampholine. The
gel was removed from the fixing solution and placed in destaining solution (Table
2.15) for 15-30 minutes. This will wash remainder ampholine and adjust the pH of
the gel to match that of staining solution. The gel was then stained in a staining
68
solution of 0.4% Coomassie Brilliant Blue R-250 (Table 2.16) for 10 min at room
temperature, with the lid in place. The gel was then immersed in destaining solution
over night at room temperature. The pH gradient was then compared with each band
to the isoelectric point of purified enzyme and crude venom.
Table 2.12: Gelling Solution for IEF
Acrylamide solution 29.1% 10 ml
Bisacrylamide solution 0.9% 10 ml
Glycerol 87% (v/v) 7 ml
1809 Ampholine pH 3.5-10 2.8 ml
Distilled water 30.2 ml
Table 2.13: Electrode solution for IEF (pH 3.5-9.5)
1M H3PO4 (Anode) 28.82 ml
1M NaOH 10gm
Distilled water 250 ml
Table 2.14: Fixing solution for IEF
Sulfosalicylic acid 17.3 gm
Trichloroacetic acid 57.5 gm
Distilled water 500 ml
69
Table 2.15: De-staining solution for IEF
Ethanol 250 ml
Acetic acid 80 ml
Distilled water 670 ml
Table 2.16: Staining solution for IEF
Coomassie brilliant blue 0.46 gm
Destaining solution 400 ml
2.6. Assays of Phosphodiesterase Activity
2.6.1. Assay I
The PDE-I activity was determined according to spectrophotometeric method
of Sulkowski and Laskowski (1971). The assay mixture contained in 1 ml: 5 μmoles
bis-p-nitrophenyl phosphate, 10 μmoles MgCl2, 100 μmoles Tris-HCl, pH 9.0. Pre-
incubation at 37 0C lasted for 5 minutes; enzyme (10µg) was added and incubated
for the next 5 min. The reaction was stopped by 2 ml 0.1N NaOH; the absorbence
was read at 400 nm, using 17,600 as molar extinction coefficient; activity was
expressed in μmoles × min-1 × ml-1.
2.6.2. Assay II
The method is fundamentally that of Razell and Khorana (1959) wherever the
reaction velocity is estimated through an augment in optical density at 400 nm
ensuing from the breakdown of p-nitrophenyl thymidine-5’-phosphate. One unit of
enzyme hydrolyzes 1 μmole of p-nitrophenyl thymidine-5’-phosphate per min at pH
70
8.9, and 37 0C under specified conditions. The reaction mixture contained in 1 ml:
0.9 ml of 0.11M Tris-HCl buffer, pH 8.9 containing 0.11M NaCl and 15 mM MgCl2
and 0.1 ml of 5 mM p-nitrophenyl thymidine-5’-phosphate. Pre-incubation at 370C
lasted for 5 minutes, 10 μl (10 µg) enzyme was added and incubated for the next 5
minutes.
2.6.3. Assay III
The standard assay of Butcher and Sutherland (1961) was used which
consisted of measuring the release of inorganic phosphate with use of an excess of
5’-nucleotidase. This reaction mixture contained 0.01 mM of cAMP or 2mM ATP
(as the case may be) 2 μmoles of MgSO4, and 36 μmoles of Tris-HCl buffer, pH 7.5
with a suitable dilution (50µg) of the enzyme sample being tested in a total volume
of 0.9 ml. This mixture was incubated at 370C for 3 hours in case of cAMP and 30
min in case of ATP. After the first 170 minutes of incubation in case of cAMP and
20 min in case of ATP, 0.1 ml of a Crotalus atrox venom solution was added
containing 0.1mg of venom in 0.001 M Tris-HCl, pH 7.5. The entire reaction was
terminated by the addition of 0.1 ml of cold 55% trichloroacetic acid. After addition
of trichloroacetic acid, the precipitate was removed by centrifugation, and aliquots
of the supernatant fluids were analyzed for inorganic phosphate by the method of
Fiske and SubbaRow (1925).
The Crotalus atrox venom contained a potent 5’-nucleotidase that hydrolyzed
the 5’-AMP formed in the phosphodiesterase reaction to adenosine and phosphate.
The venom, when used in this concentration, was without effect on cAMP. One unit
of enzyme was defined as the amount that caused the release of inorganic phosphate
per μmole of cAMP or ATP as the case may be.
71
2.7. Assay for 5’-Nucleotidase Activity
The activity of 5’-Nucleotidase was determined by the modified method of
Sinsheimer and Koerner (1952). The reaction mixture contained: 0.1 ml of 1M
glycine buffer, pH 9.0, 0.1 ml of 0.1M MgCl2, 0.3ml of 0.01M AMP, 0.1 ml (10 µg)
of enzyme solution and water to make total volume of 1ml. The mixture was
incubated for 15 min at 370C. The liberated phosphate was determined according to
Fiske and SubbaRow (1925). One unit was defined as the amount of enzyme
liberating 1 μmole of inorganic phosphate per min at 370C.
2.8. Assay for Alkaline Phosphatase Activity
The alkaline phosphatase (ALP) activity was determined according to the
spectrophotometric method of Hausamen et al., (1967) at 25oC using p-nitrophenyl
phosphate as substrate. The assay mixture (1.2 ml) contained 0.9 M diethanolamine
(DEA) at pH 9.8, 0.5 mM magnesium sulfate and (10 µg) purified PDE-I as the
source of enzyme. The blank was also run under the same conditions and with the
same components except for that the enzyme was omitted. One unit of enzyme
activity is defined as the amount of ALP that catalyzes the hydrolysis of 1 µmole of
pNPP per min per mg of protein under the given experimental conditions.
2.9. Carbohydrate Contents
Carbohydrate contents of the purified sample were measured according to the
method of Dubois et al., (1956). Galactose was used as standard. Two milliliters of
sugar solution containing between 10 and 70 µg of sugar is pipetted into a
colorimetric tube and 0.05 ml of 80% phenol. Then 5ml of concentrated sulfuric acid
is constituted quickly, the flow of acid being aimed in opposition to the liquid
surface instead against the sides of test tube so as to get excellent mixing. These
72
tubes are permitted to stand 10 minutes, and then they are shaken and placed for 20
minutes in water bath at 30oC, before readings are taken. The color is stable for
several hours and reading may be made later if necessary. The absorbance of
characteristic yellow-orange color is measured at 490nm. Blanks are set by replacing
aqua distilla for the carbohydrate solution. The quantity of sugar may then be
estimated by reference to a typical curve. All solutions are prepared in triplicate to
minimize errors resulting from accidental contamination with cellulose lint.
2.10. Effect of pH
The outcome of pH on enzyme action was tested in the range between 6.5 and
10.5 using 0.11M Tris-HCl buffer. A substrate control was also included at every pH
tested. For kinetic studies the substrate thymidine 5’-monophosphate p-nitrophenyl
ester was used.
2.11. Effect of Temperature
Enzyme activity was tested at different temperatures in the range between 200
and 90 0C. A substrate control was included with each test to correct for auto
substrate destruction at different temperatures. The activation energy (Ea) for the
hydrolysis of bis-p-nitrophenyl phosphate by Agistrodon bilineatus (Ab) venom
PDE-I was estimated by scheming the log of PDE-I activity Vs 1/T (Arrhenius plot).
The worth of the negative slope of this plot was put in to the following equation to
give the activation energy, Ea
Slope = Ea ___ (Segel, 1975)
2.3R
73
Whereas R is the Boltzmann constant and its value is equal to 1.987
calories/deg/moles.
2.12. Effect of Metal Ions
Enzyme assays were carried out in the absence and presence of Mg++, Ca++ or
Zn++. The concentrations of 5.0, 10.0, 15.0 and 20.0 mM of Mg++ were used whereas
the concentrations of 5.0, 7.0 and 10.0 mM for Zn++ or Ca++ were used.
2.13. Measurement of Kinetic Constants
The effect of varying substrate concentration on enzyme activity was tested
and Km for the enzyme was determined from Lineweaver-Burk plot (Segel, 1975).
The Km was calculated using the Grafit software package, version 3.0
(Leatherbarrow, 1992). It was repeated in the presence of different inhibitors and the
type of inhibition and Ki were determined. For competitive and non-competitive
inhibitors the Ki were determined by plotting 1/v against i (inhibitor’s concentration)
according to the graphical method of Dixon (1953).
2.14. Estimation of IC50
The altered data log V/ (Vo-V) (where Vo = Velocity in the absence of
inhibitor and V in the presence of inhibitor) vs log inhibitor’s concentration were
plotted for the estirmation of IC50 for each type of inhibitor.
2.15. Lethality Test
Swiss albino male mice (15-20 gm) obtained from the animal house of the College.
The animals were fed on a commercial pellet diet and water ad libitum. The purified
venom enzyme was injected intraperitoneally (0.25, 0.5, 1.0, 2.0 and 4.0mg/kg body
weight) in a fixed volume of 200 µl to determine their lethal effect and the LD50 was
74
calculated according to the arithmetic method of Karber. Eight mice were used per
group. Control mice were injected with 0.9% NaCl. The observation time limit used
was 24 h, post-injection. These experiments were approved by an institutional ethics
committee for animal care and use.
2.16. Determination of Re-calcified Plasma Coagulation Times
The effect of PDE-I on the plasma recalcification time was determined using a
Biomeriux apparatus (Option 2), according to the manufacturer’s instructions. Blood
samples from healthy volunteers were collected into sodium citrate (3.2%) and
citrated human plasma (100 µl) was incubated with venom (400 µg) or PDE-I (50 µg)
(each in 100 µl) at 37oC for 4 min after which 100 µl of 25 mM CaCl2 was added and
the clotting time was recorded. Control clotting times were obtained by adding 100 µl
of 0.9% NaCl without venom or PDE-I. This blood collection and use was approved
by an institutional ethics committee for research in humans.
75
3. RESULTS
3.1. Purification of PDE-I Enzyme by Native Preparative PAGE
Three crude venoms viz; A. bilineatus, C. vipera and O. hannah were
fractionated by native preparative PAGE into many fractions. Fraction No.1 showed
PDE-I activity only. Fraction No.1 appeared as single band on native analytical
PAGE (Figure 3.1 A, B & C) and on SDS-PAGE (Figure 3.2 A, B & C) which
means the enzyme is homogenous. The Ab, Cv and Oh venom PDE-I, enzymes were
purified 3.0, 5.6 and 2.0 fold over the crude venom with a specific activity of 2.0,
2.8 and 4.1 U/mg protein, respectively. Table 3.1 shows that the recovery of Ab, Cv
and Oh PDE-I activity was 38%, 68% and 33% whereas the protein yield of
enzymes was 4.5%, 6.1% and 5%, respectively. All three purified PDE-I enzymes
were free from 5’-nucleotidase activity. The non-specific alkaline phosphatase
activity was also negligible (Table 3.2).
3.2. Determination of Molecular Weight by SDS-PAGE
The purified enzymes from the three venoms yielded a single band on SDS-
PAGE with an estimated molecular mass of 140, 126 and 148 KDa respectively
following Coomassie Brilliant Blue staining (Figure 3.2 A, B &C and Figure 3.3).
The purified enzymes when treated with β-mercaptoethanol did not alter the
positions and still showed single band on SDS-PAGE (result not shown), indicating
that the enzymes were a single chain polypeptide. Ab and Oh venom PDE-I enzymes
exhibited basic isoelectric points (7.4 and 7.5) while the Cv venom PDE-I showed
an acidic pI; 5.8 (Table 3.3).
76
Figure 3.1A: Analytical native PAGE of purified PDE-I from A. bilineatus venom along with crude venom. (1) Crude venom (200 μg); (2) Purified PDE-I (30 μg).
77
Figure 3.1B: Analytical native PAGE of purified PDE-I from C. vipera venom along with crude venom. (1) Crude venom (200 μg); (2) Purified PDE-I (30 μg).
78
Figure 3.1C: Analytical native PAGE of purified PDE-I from O. hannah venom along with crude venom. (1) Purified PDE-I (30 μg) ; (2) Crude venom (200 μg)
79
Figure 3.2 A: SDS-PAGE of purified PDE from A. bilineatus venom along with crude venom. (1) Crude venom (300 μg); (2) Purified PDE (40 μg); (3) Pre-stained proteins molecular weight marker mix.
80
Figure 3.2 B: SDS-PAGE of purified PDE from C. vipera venom along with crude venom. (1) Crude venom (300 μg); (2) Purified PDE (40 μg); (3) Pre-stained proteins molecular weight marker mix.
81
Figure 3.2 C: SDS-PAGE of purified PDE from O. hannah venom along with crude venom. (1) Crude venom (300 μg); (2) Purified PDE (40 μg); (3) Pre-stained proteins molecular weight marker mix.
82
0.0 0.2 0.4 0.6 0.8 1.01.2
1.4
1.6
1.8
2.0
2.2
2.4
X
BSA(84 KDa)
Phosphodiestease I
Lysozyme(18.3 KDa)
Trypsin inhibitor(28 KDa)Carbonic anhydrase(39.2KDa)
Ovalbumin(60 KDa)
Phosphorylase b(120 KDa)
Myosine(215 KDa)
Lo
g M
ol W
t
Rf
Figure 3.3: Calibration of the SDS-PAGE system using standard proteins and the estimation of molecular weight of PDE-I.
83
Table: 3.1. Purification of PDE-I from different snake venoms
Crude venom / PDE-I
Total activity (Units)
Specific activity (Units/mg)
Recovery of protein (%)
Recovery of activity (%)
Purification (fold)
A. bilineatus 45± 2 0.67±0.03
A. bilineatus
PDE-I
17±0.7 2±0.06 4.5±0.2 38±1.5 3.0±0.07
C. vipera 34±1.4 0.5±.02
C. vipera
PDE-I
23±1.0 2.8±0.07 6.1±0.25 68±2.2 5.6±0.2
O. hannah 102±4.0 2±0.06
O. hannah
PDE-I
34±1.2 4.1±0.15 5±0.15 33±1.2 2.0±0.06
84
Table 3.2: Activities of contaminant enzymes in different crude venoms and purified PDE-I enzymes from the venoms.
Crude venom/PDE-I 5’-nucleotidase (U/L) ALP (U/L)
A. bilineatus 5.9± 0.3 732±10
A. bilineatus PDE-I 0.00 0.00
C. vipera 6.7±0.3 26±1.2
C. vipera PDE-I 0.00 2±0.05
O. Hannah 4.7±0.1 121±4
O. hannah PDE-I 0.00 1±0.1
Table 3.3: Molecular weights, pIs, activation energy (Ea) and carbohydrate content of PDE-I isolated from different snake venoms.
PDE-I source Activation energy (Ea)
M.W. (KDa)
pI Carbohydrate Contents (mg/gm)
A. bilineatus 28±1.5 140±5 7.4±0.2 14±0.3
C. vipera 128±5 126±5 5.8±015 21±0.4
O. hannah 135±6 148±4.5 7.5±0.2 24±0.5
85
3.3. Physiochemical Properties
The optimum temperature for Ab, Cv venom PDE-I activity was found to be
60oC whereas for Oh venom PDE-I activity was 50oC, with activity decrease at >
65oC (Figure 3.4 A, B & C). Energy of activation (Ea) for Ab, Cv and Oh venom
PDE-I calculated from an Arrhenius plot was 28.3, 128 and 135 respectively (Figure
3.5 A, B & C). The maximum activity of purified Ab and Cv, Oh venom PDE-I in
Tris buffer was obtained over the pH 10.0, 9.0 and 10.0 respectively (Figure 3.6 A,
B & C). As shown in Table 3.3, Ab, Cv and Oh venom PDE-I enzymes are
glycoproteins having 14%, 21% and 24% of carbohydrate contents, respectively.
86
0
0.5
1
1.5
2
2.5
3
0 20 40 60 80 100Temperature (C)
Spe
cific
act
ivity
(µ
M/M
in/m
g)
Figure 3.4 A: Effect of temperature on A. bilineatus PDE at 2mM BpNPP and pH 9.0. Enzyme activity was tested at different temperatures in the range between 20 0C and 90 0C. Each point represents the mean of five values.
87
0
0.02
0.04
0.06
0.08
0.1
0.12
0 20 40 60 80 100Temperature (C)
Spe
cific
act
ivity
(µ
M/M
in/m
g)
Figure 3.4B: Effect of temperature on C. vipera PDE at 2mM BpNPP and pH 9.0. Enzyme activity was tested at different temperatures in the range between 20 0C and 90 0C. Each point represents the mean of five values.
88
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 20 40 60 80 100
Temperature (C)
Spe
cific
act
ivity
(µ
M/M
in/m
g)
Figure 3.4 C: Effect of temperature on O. hannah PDE at 2mM BpNPP and pH 9.0. Enzyme activity was tested at different temperatures in the range between 20 0C and 90 0C. Each point represents the mean of five values.
89
y = -6.2077x + 0.4277
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04
Figure 3.5 A: Arrhenius plot of A. bilineatus PDE-I. Each point represents the mean ± SD of four independent experiments.
90
y = -28.172x - 0.5391
-1.6
-1.4
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
0 0.005 0.01 0.015 0.02 0.025 0.03 0.0351/T
Lo
g V
ma
x
Figure 3.5 B: Arrhenius plot of C. vipera PDE-I. Each point represents the mean ± SD of four independent experiments.
91
y = -29.547x + 0.0468
-1.6
-1.4
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
0 0.01 0.02 0.03 0.04 0.05 0.061/T
Lo
g V
max
Figure 3.5 C: Arrhenius plot of O. hannah PDE-I. Each point represents the mean ± SD of four independent experiments.
92
0
0.5
1
1.5
2
2.5
3
3.5
4
0 2 4 6 8 10 12
pH
Sp
ecif
ic a
cti
vity
Figure 3.6 A: Effect of pH on A. bilineatus PDE-I activity at 370C. The effect of pH on enzyme activity was tested in the range between 6.5 and 10.5 using 0.11M Tris-HCl buffer. A substrate control was included at every pH tested. Each point represents the mean of three independent experiments.
93
0
0.5
1
1.5
2
2.5
3
3.5
0 2 4 6 8 10 12
pH
Sp
eci
fic
act
ivit
y
Figure 3.6 B: Effect of pH on C. vipera PDE-I activity at 37oC. The effect of pH on enzyme activity was tested in the range between 6.5 and 10.5 using 0.11M Tris-HCl buffer. A substrate control was included at every pH tested. Each point represents the mean of three independent experiments.
94
0
0.5
1
1.5
2
2.5
3
3.5
4
0 2 4 6 8 10 12
pH
Sp
eci
fic
act
ivit
y
Figure 3.6 C: Effect of pH on O. hannah PDE activity at 37oC. The effect of pH on enzyme activity was tested in the range between 6.5 and 10.5 using 0.11M Tris-HCl buffer. A substrate control was included at every pH tested. Each point represents the mean of three independent experiments.
95
3.4. Enzymatic properties
The V max and Km for Ab, Cv and Oh PDE-I calculated from Lineweaver-Burk
plot were 3.85, 1.3 and 1.5 μM/min/mg protein and 8.3×10-3, 3.4×10-3 and 2.5×10-3
M respectively (Figure 3.7 A, B & C). Table 3.4 shows K cat and K sp values for Ab,
Cv and Oh PDE-I which are 23 s-1, 7.8 s-1 and 9.2 s-1 and 46.4, 41.3 and 58.8 M-1
Min-1 respectively.
Cysteine caused a non-competitive inhibition, the Ki calculated for Ab, Cv
and Oh venom PDE-I were 6.3×10−3, 7.0×10−3 and 8.2×10−3 M respectively (Figure
3.8 A, B & C) while the IC50 values were 1.6, 3.0 and 3.9 mM respectively (Figure
3.9 A, B & C). Whereas ADP exhibited a competitive inhibition, the Ki calculated
for Ab, Cv and Oh venom PDE-I were 0.8×10−3, 0.6×10−3 and 1.0×10−3 M
respectively (Figure 3.10 A, B & C). The IC50 values were 5.4, 5.0 and 12.0 mM
respectively (Figure 3.11 A, B & C and Table 3.5).
ADP, cysteine, Glutathione, and o-phenanthroline at a concentration of 2.5-10
mM inhibited the Ab venom PDE activity (25-60%), (47-75%), (60-83%) and (87-
98.3%) respectively. In case of Cv venom PDE-I activity inhibition by these
compounds was (30-83%), (87-90%), (23-47%) and (98-100%) respectively while
the Oh venom PDE-I activity inhibition was (18-35%), (40-62%), (2-50%) and (92-
100%) respectively. The EDTA at a concentration of 0.5-10mM inhibited the Ab,
Cv and Oh venom PDE-I activities by (96.6-100%), (98-100%) and (50-88%)
respectively (Table 3.6). The addition of 10 mM of Ca+2 reactivated the PDE activity
by only 8%.
96
1.110.90.80.70.60.50.40.30.20.10-0.1-0.2
2.62.42.2
21.81.61.41.2
10.80.60.40.2
0
1/[S] mM
1/v
uM
/Min
/mg
Figure 3.7 A: Lineweaver-Burk plot for A. bilineatus PDE-I. The effect of varying substrate concentration on enzyme activity was tested and Km for the enzyme was determined from Lineweaver-Burk plot. The Km was calculated using the GraFit software package, version 3.0. The points represent the mean ± SD of 4-5 determinations, each in duplicate.
97
0.60.50.40.30.20.10-0.1-0.2-0.3-0.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
1/[S] mM
1/v
uM
/Min
/mg
Figure 3.7 B: Lineweaver-Burk plot for C. vipera PDE-I. The effect of varying substrate concentration on enzyme activity was tested and Km for the enzyme was determined from Lineweaver-Burk plot. The Km was calculated using the GraFit software package, version 3.0.The points represents the mean ± SD of 4-5 determination, each in duplicate.
98
1.110.90.80.70.60.50.40.30.20.10-0.1-0.2-0.3-0.4-0.5
2.42.2
21.81.61.41.2
10.80.60.40.2
0
1/[S] mM
1/v
uM/M
in/m
g
Figure 3.7 C: Lineweaver-Burk plot for O. hannah PDE-I. The effect of varying substrate concentration on enzyme activity was tested and Km for the enzyme was determined from Lineweaver-Burk plot. The Km was calculated using the GraFit software package, version 3.0.The points represents the mean ± SD of 4-5 determination, each in duplicate.
99
Table:3.4: Kinetic properties of PDE-I enzymes isolated from different snake venoms.
PDE-I source Vmax μM/min/mg)
Km (M) Kcat (S-1)
Ksp (M-1)
A. bilineatus 3.85±0.15 8.3×10-3 ±0.25 23±1.0 46.4±1.8
C. vipera 1.30±0.05 3.4×10-3 ±0.15 7.8±0.2 41.3±1.6
O. hannah 1.53±0.05 2.5×10-3 ±0.1 9.2±0.3 58.8±2.0
100
Figure 3.8 A: Determination of Ki for cystein according to Dixon (non-competitive inhibition). Dixon plot for Ab PDE-I at two different concentrations of substrate. (S1=5.0 mM and S2=1.0 mM).
101
Figure 3.8 B: Determination of Ki for cystein according to Dixon (non-competitive inhibition). Dixon plot for Cv PDE-I at two different concentrations of substrate. (S1=5.0 mM and S2=1.0 mM)
102
Figure 3.8 C: Determination of Ki for cystein according to Dixon (non-competitive inhibition). Dixon plot for Oh PDE-I at two different concentrations of substrate. (S1=5.0 mM and S2=1.0 mM).
103
Table: 3.5: Effect of different compounds on the activity of PDE-I enzymes isolated from snake venoms and the type of inhibition.
PDE-I source
Compound Type of inhibition Ki (M) IC50 (mM)
A. bilineatus
ADP Cystein
Competitive Non-competitive
0.8×10−3 ±0.02 6.3 ×10−3 ± 0.2
5.4±0.25 1.6± 0.04
C. vipera
ADP Cystein
Competitive Non-competitive
0.6 ×10−3 ±0.02 7.0 ×10−3±0.2
5.0±0.25 3.0±0.08
O. hannah ADP Cystein
Competitive Non-competitive
1.0 ×10−3±0.03 8.2 ×10−3±0.25
12.0 ±0.5 3.9 ±0.1
104
1.61.41.210.80.60.40.20-0.2-0.4
0.05
0
-0.05
-0.1
-0.15
-0.2
-0.25
Log[I]
Log[
v/(v
0-v)
]
Figure 3.9A: A. bilineatus PDE-I inhibition by cystein. The transformed data is represented in the form of a Hill plot, where V and V0 are the reaction rates for experimental and control system respectively. The Hill coefficient is 0.04 and slope was -0.2. Each point is the mean ±D of three independent determinations.
105
1.61.41.210.80.60.40.20-0.2-0.4
0.4
0.2
0
-0.2
-0.4
-0.6
-0.8
-1
-1.2
-1.4
Log[I]
Log[
v/(v
0-v)
]
Figure 3.9 B: C. vipera PDE-I inhibition by cystein. The transformed data is represented in the form of a hill plot, where V and Vo are the reaction rates for experimental and control system respectively. The Hill coefficient is 0.6 and slope was -1.39. Each point is the mean ±D of three independent determinations.
106
1.61.41.210.80.60.40.20-0.2-0.4
0.4
0.2
0
-0.2
-0.4
-0.6
Log[I]
Log[
v/(v
0-v)
]
Figure 3.9 C: O. hannah PDE-I inhibition by cystein. The transformed data is represented in the form of a Hill plot, where V and Vo are the reaction rates for experimental and control system respectively. The Hill coefficient is 0.33 and slope was -0.56. Each point is the mean ±D of three independent determinations.
107
Figure 3.10:A. Determination of Ki for ADP according to Dixon (competitive inhibition). Dixon plot for Ab PDE-I at two different concentrations of substrate. (S1=1.0 mM and S2=5.0 mM).
108
Figure 3.10 B: Determination of Ki for ADP according to Dixon (competitive inhibition). Dixon plot for Cv PDE-I at two different concentrations of substrate. (S1=1.0 mM and S2=5.0 mM).
109
Figure 3.10 C: Determination of Ki for ADP according to Dixon (competitive inhibition). Dixon plot for Oh PDE-I at two different concentrations of substrate. (S1=1.0 mM and S2=5.0 mM).
110
1.61.41.210.80.60.40.20-0.2-0.4
0.80.60.40.2
0-0.2-0.4-0.6-0.8
-1-1.2-1.4-1.6
Log[I]
Log[
v/(v
0-v)
]
Figure 3.11 A: A. bilineatus PDE-I inhibition by ADP. The transformed data is represented in the form of a Hill plot, where V and Vo are the reaction rates for experimental and control system respectively. The Hill coefficient is 1.05 and slope was -1.42. Each point is the mean ±D of three independent determinations.
111
1.61.41.210.80.60.40.20-0.2-0.4
1.61.41.2
10.80.60.40.2
0-0.2-0.4-0.6-0.8
-1-1.2-1.4-1.6-1.8
Log[I]
Log[
v/(v
0-v)
]
Figure 3.11 B: C. vipera PDE-I inhibition by ADP. The transformed data is represented in the form of a Hill plot, where V and Vo are the reaction rates for experimental and control system respectively. The Hill coefficient is 1.36 and slope was -1.99. Each point is the mean ±D of three independent determinations.
112
1.61.41.210.80.60.40.20-0.2-0.4
1.41.2
10.80.60.40.2
0-0.2-0.4-0.6-0.8
-1-1.2
Log[I]
Log[
v/(v
0-v)
]
Figure 3.11 C: O. hannah PDE-I inhibition by ADP. The transformed data is represented in the form of a Hill plot, where V and Vo are the reaction rates for experimental and control system respectively. The Hill coefficient is 1.53 and slope was -1.46. Each point is the mean ±D of three independent determinations.
113
Table: 3.6: Effect of different concentrations of inhibitors on the activity of PDE-I isolated from different snake venoms. (Percent activity)*
Inhibitor PDE-I source Specific activity (μM/min/mg protein) at different concentrations (mM) 0.5 2.5 5.0 10.0
ADP A. bilineatus
C. vipera
O. hannah
--- 81±3.0 70 ±2.5 40±1.5
--- 70 ±2.5 42±1.5 17±0.5
--- 82±3.0 71±2.5 65±1.5
Cysteine
A. bilineatus
C. vipera
O. hannah
--- 53±2.0 35±1.5 25±1.0
--- 17±0.5 14±0.4 10±0.3
--- 60±2.0 57±2.0 42±1.0
EDTA A. bilineatus
C. vipera
O. hannah
3.4±0.2 1.5±0.1 0.0 0.0
2.0±0.15 1.0±0.05 0.0 0.0
50±1.5 23±0.75 15±0.5 12±0.5
Glutathione
A. bilineatus
C. vipera
O. Hannah
---- 40±1.5 25±1.0 17±0.6
---- 77±2.8.0 64±2.5 53±2.0
---- 98±3.0 79±2.5 50±1.5
o-Phenanthroline
A. bilineatus
C. vipera
O. hannah
---- 13±0.5 6.0±0.25 1.7±0.1
---- 2.0±0.1 1.0±0.05 0.0
---- 8.0±0.25 2.0 ±0.1 0.0
*Enzyme activity in the presence of Ca+2 or Mg+2 in the assay mixture was taken as 100%. All assays were done in triplicate and only mean ±D values are given.
114
3.5. Effect of Metal Ions
The studies with chelating agents indicate the requirement of divalent metal
ions for activity of PDE-I. The Ca+2 did not affect the activity of all three snake
venom PDE-I enzymes. 5mM Mg+2 caused a 56% increase in activity of Ab venom
PDE-I, 5 mM Mg+2 increased the Cv venom PDE-I activity by 28% while a 70%
increase was caused by 15 mM of Mg+2 in Oh venom PDE-I activity. Zn+2 showed
an inhibitory effect, as a 19% decrease was caused in Ab venom PDE-I activity by
5mM, a 36% decreased was observed in Cv venom PDE-I activity by 10 mM of
Zn+2. Whereas a 23% decrease was produced in Oh venom PDE-I activity by 10mM
of Zn+2 (Table 3.7).
115
Table: 3.7: Effects of divalent metal ions on snake venom PDE-I activity.
Addition PDE-I source Specific activity (μM/min/mg protein) at different concentrations (mM) None 3 5 7 1 0 15 20
Ca+2 A. bilineatus
C. vipera
O. hannah
5.3±0.2 5.3±0.2 5.3±0.2 --- 5.3±0.2 --- ----
5.1±0.2 5.1±0.2 5.1±0.2 5.2±0.2 5.3±0.2 --- ----
5.3±0.2 5.3±0.2 5.3±0.2 5.3±0.2 5.3±0.2 --- ---
Mg+2 A. bilineatus
C. vipera
O. hannah
2.4±0.1 ---- 3.8±0.2 ---- 3.8±0.2 2.6±0.2 2.6±0.2
0.6±0.03 --- 0.9±0.04 -- 0.9±0.04 0.9±0.040 0.9±0.04
0.88±0.04 --- 0.94±0.04 --- 1.1±0.05 1.4±0.06 0.94±0.04
Zn+2 A. bilineatus
C. vipera
O. hannah
0.31±0.02 -- 0.25±0.01 0.25±0.01 0.25±0.01 --- ---
0.63±0.04 --- 0.63±0.04 0.50±0.03 0.4±0.03 --- ---
0.88±0.04 ---- 0.88±0.04 0.7±0.03 0.68±0.03 --- ---
Note: All assays were done in triplicate; only mean ±SD data is given.
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3.6. Substrate Specificity of Snake Venom Phosphodiesterases (PDE-I)
Snake venom PDE-I shows negligible activity towards p-nitrophenyl
phosphate or mono-phosphate nucleotides. Di- and tri-phosphate nucleotides, DNA,
RNA and many derivatives of these native molecules can serve as substrate for
phosphodiesterases. Ab, Cv and Oh venom PDE-I enzymes hydrolyzed bis-p-
nitrophenyl phosphate, ATP, cAMP and thymidine 5’-monophosphate p-nitrophenyl
ester. Ab, Cv and Oh venom PDE-I enzymes hydrolyzed thymidine 5’-
monophosphate p-nitrophenyl ester most readily, the specific activity was 15.0, 8.0
and 11.0 Units/mg protein (10-fold) while cAMP was the least readily hydrolyzed
substrate with a specific activity of 0.1, 0.06 and 0.13 Units/mg protein, respectively,
(Table 3.8).
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Table: 3.8: The hydrolysis of different substrates by snake venom PDE-I enzymes.
Substrate Assay PDE-I Source
Activity (U/L)
Sp. activity (Units/mg)
ATP Butcher and
Sutherland
A. bilineatus
C. vipera
O. hannah
129±4
103±4
103±4
6.45±0.3
5.15±0.2
5.15±0.2
cAMP Butcher and
Sutherland
A. bilineatus
C. vipera
O. hannah
4.5±0.2
3.1±0.15
6.5±0.25
0.1±0.01
0.06±0.002
0.13±0.01
BpNPP Sulkowski and
Laskowski
A. bilineatus
C. vipera
O. hannah
17±0.75
23±1
34±1
2.0±0.1
2.8±0.1
4.1±0.15
T5’PpNP
Ester
Razell and
Khorana
A. bilineatus
C. vipera
O. hannah
150±5
78±4
113±5
15±0.7
8.0±0.2
11±0.3
Note: All assays were done in triplicate; only mean ±SD data is given.
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3.7. Biological Activity of Snake Venom Phosphodiesterase I
Snake venom PDE-I is studied extensively but few have investigated the
biological activity of this unique venom component. The Ab, Cv and Oh venom
PDE-I enzymes up to 4.0 mg/Kg i.p were not lethal in mice. The animals showed
some symptoms of intoxication like hypoactivity and extension of hind limbs during
first hour of a 24 hours experiment.
As Table 3.9 shows the Ab, Cv and Oh venom PDE-I enzymes exhibited an
anticoagulant effect as all the enzymes significantly increased the normal clotting
time of normal citrated human plasma, whereas the Ab and Oh crude venoms
showed coagulant effect. But the Cv crude venom exhibited anticoagulant effect.
119
Table 3.9: Effect of different crude snake venoms and purified phosphodiesterases on coagulation of normal human plasma *
Coagulation parameters Clotting time (s)
Plasma + CaCl2 175 ± 10
A. bilineatus crude venom
Ab venom PDE
15 ± 3
530 ± 5
C. vipera crude venom
Cv venom PDE
500 ± 6
375 ± 5
O. hannah crude venom
Oh venom PDE
85 ± 3
360 ± 5
* Note: All assays were done in triplicate; only mean ±SD data is given.
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4. DISCUSSION
The snake venom PDE-I has been isolated from a number of species,
including C. adamenteus (Razzell, 1963; Razzell and Khorana, 1959; Sinsheimer
and Koerner, 1952 (missing); Privat de Garilhe, and Laskowski, 1955; Richards,
1967; Phillips, 1975; Laskowski, 1980); A. acutus (Sugihara et al., 1984); A. halys
blomhoffii (Tatsuki et al., 1975); A. pisivorous (Butler, 1955); B. atrox (Phillips,
1976; Bjork, 1963; Frischauf and Eckstein, 1973); C. durissus terrificus (Phillips,
1975); C. ruber rubber (Mori et al., 1987); T. mucrosquamatus (Sugihara et al.,
1986); V. palestinae (Levy and Bdolah, 1976); V. aspis (Ballario et al., 1977); C.
cerastes (Halim et al., 1987); Vipera russellii formosensis (Win et al., 1998) and B.
alternatus (Valerio et al., 2002). The purity of PDE-I obtained by the methods
described in the above mentioned reports varied considerably particularly in the
early attempts at purification, the main reason is the difficulty in removing the
contaminating 5’-nucleotidase and alkaline phosphatase.
In contrast to other current methods for obtaining PDE-I from snake venoms,
our procedure is single step. Our procedure apparently provided pure PDE-I
enzymes as shown by the single protein on native analytical PAGE and SDS-PAGE.
The PDE-I enzymes were free of these contaminants, as evident by the absence of
5’-nucleotidase and alkaline phosphatase activity in the enzyme preparation. The
PDE-I enzymes appeared at same position on native PAGE from all the three snake
venoms, though the venoms were from three different families. So our method is
equally useful for isolating and purifying PDE-I enzyme from the three families i.e.
crotalidae, viperidae and elapidae.
We isolated and purified the PDE-I enzymes from the above mentioned three
families. More over our procedure requires slab gel to be run for 8-10 hours only
121
whereas the preparative gel procedure of Ballario et al., required 22 hours to get rid
of 5’-nucleotidase and two other unknown proteins. The yield of protein for Ab, Cv
and Oh venom PDE-I (4.5, 6.1 & 5.0% respectively) was also fair enough as
reported for many other venoms PDE-I purified by a variety of protocols (Phillips,
1976; Williams et al., 1961; Bjork, 1963; Phillips, 1975; Laskowski, 1980; Frischauf
and Eckstein, 1973; Mori et al., 1987; Sugihara et al., 1986; Ballario et al., 1977;
Valerio et al., 2002; Felix et al., 1960).
The only drawback of the purification procedure described here is the limited
amount (10 mg) of venom protein which can be applied to the slab gel but is still
useful for the small-scale preparation of a highly purified phosphodiesterase I from
snake venom.
The snake venom PDE are generally high molecular weight (90-150 KDa)
glycoprotein comprising of a single chain polypeptide with iso-electric points
ranging from 7.4-10.5 (Razzell and Khorana, 1959; Phillips, 1976; Phillips, 1975;
Laskowski, 1980; Mori et al., 1987; Sugihara et al., 1986; Ballario et al., 1977;
Valerio et al., 2002; Stoynov et al., 1997). The molecular masses of PDE-I from Ab,
Cv and Oh venom (140,126 &148 KDa respectively) are close to the other snake
venom PDE’s masses. The molecular masses being not affected by β-
mercaptoethanol indicates that the proteins are a single chain polypeptide, as is
generally the case for venom PDE-I.
Although there are reports that PDE-I from C. rubber rubber venom exist as a
homo-dimer with subunits of 49 KDa (Mori et al., 1987), PDE from C. viridis
oreganus venom exists as a homo-dimer with subunits of 57 KDa (Mackessy, 1989)
and PDE from C. mitchelli pyrrhus venom exists as a homodimer with subunits of
55 KDa (Perron et al., 1993). Throughout the whole purification procedure and in
122
SDS-PAGE, only one fraction of activity and one protein band were obtained
respectively for all three venom phosphodiesterases which indicates that this enzyme
probably does not occur in iso-forms as reported about PDE of V. palestinae (Levy
and Bdolah, 1976) and PDE of T. flavoviridis (Kini and Gowda, 1984). The pIs for
Ab and Oh venom PDE-I are basic while Cv venom PDE-I exhibited an acidic pI
(5.8). The pIs for phosphodieaterases so far reported are basic but Sittenfeld et al.,
(1991), mentioned that DNase with acidic pIs were present in B. godmani and L.
muta venoms.
Halim et al., 1987 reported an energy of activation (Ea) 0.913 for C. cerastes
PDE which is lower as compared to the energy of activation of PDE-I enzymes
isolated by us. Many of these enzymes rapidly loose activity (within 1-4 minutes) at
temperatures > 65-70oC as reported by many workers (Phillips, 1976; Bjork, 1963;
Phillips, 1975; Mori et al., 1987; Sugihara et al., 1986; Ballario et al., 1977; Halim
et al., 1987). This property was also found to be the same for the PDE-I enzymes
from Ab, Cv and Oh venoms.
The pH optima of the PDE-I enzymes isolated from Ab, Cv and Oh venoms is
in agreement as reported by others e.g. pH 9.2, 9.0, 8.5 and 8.5 for PDE-I enzymes
from B. atrox, C. cerastes C. adamentus and C. mitchelli pyrrhue, respectively
(Razzell and Khorana, 1959; Phillips, 1976; Bjork, 1963; Levy and Bdolah, 1976;
Valerio et al., 2002; Frischauf and Eckstien, 1973; Halim et al., 1987; Phillips,
1975; Stoynov et al., 1997; Perron et al., 1993).
The three PDE-I enzymes were found to be glycoprotein. The PDE-I from T.
flavoviridis contained about 24% carbohydrate (Kini, and Gowda., 1984); C.
adamentus PDE contains 9.2% neutral and 1.9% amino sugars (Dolapchiev et al.,
123
1980). A PDE from B. atrox (Frischauf and Eckstein, 1973) and C. ruber rubber
(Mori et al., 1987) reported to contain carbohydrate.
The Km values are in the same order as reported by others e. g 5.6 × 10-3 M
and 8.3 × 10-3 M (Frischauf and Eckstein, 1973; Mori et al., 1987) by using same
substrate but recently Marcelo et al. 2009 reported 8.5× 10-4 M for same substrate
which is lower than the values reported to date. The Kcat values (23 s-1, 7.8 s-1 &
9.2 s-1) for the Ab, Cv and Oh venom PDE-I enzymes are in reported range.
Dolapchiev et al., (1980) reported Kcat values 1.9-40 s-1 for PDE-I from C.
adamentus using ATP as substrate while Pollack and Auld (1982) reported the
values in the range 200-600 s-1 for snake venom PDE using nucleotide analog as
substrate. Recently, Marcelo et al. (2009) reported Kcat values 9.6×105 s-1 and
4.4×104 s-1 for thymidine 5'-monophosphate p-nitrophenyl ester and bis (p-
nitrophenyl) phosphate, respectively. The only report about K sp is by Pollack and
Auld, (1982) which mentioned a value in the range 18-66 μM -1 s-1 for nucleotide
analog. The values for Kcat and Ksp mentioned above are quite different from PDE-I
enzymes isolated by us, this is may be due to using different substrates.
Halim et al., (1987) reported a non-competitive inhibition by cysteine, Ki
calculated was 3.346 × 10-3 M whereas they reported a competitive inhibition for
ADP and the Ki was calculated to be 0.47 × 10-3 M. An IC50 of 7.5 mM has been
reported for cystein by Razzell and a competitive type of inhibition was also
observed with ADP and AMP (Razzell, 1963). Lysophosphatidic acid (LPA) and its
cyclic form (cLPA) were found to inhibit snake venom PDE in non-competitive
(LPA) and competitive (cLPA) manner (Mamillapalli et al., 1998). Mahroof-Tahir et
al., (2005) observed a non-competitive inhibition by mononuclear oxovanadium
(IV) complexes.
124
Choudhary et al., (2006) have reported a Ki up to 1.15 × 10-3 M and IC50 up to
1.0 mM for biscoumarin derivatives. Mostafa et al., (2006) reported that PDE-I
enzyme activity was significantly inhibited by quinovic acid-3-O-alpha-L-
rhamnopyranoside and quinovic acid-3-O-beta-D-glucopyranosyl (1→4)-beta-D-
fucopyranoside. Recently Khalid et al., (2009) reported that PDE-I activity was also
inhibited by 1, 3, 4-oxadiazole-2(3H)-thione and its analogues and the inhibition was
of pure non-competitive type. Fatima et al., (2002) mentioned 50% inhibition by
0.748 mM cystein, 0.274 mM EDTA, 0.166 mM quinovic acid and 0.374 mM
quinovic acid 3-O-(beta-D-glucopyranoside).
The PDE-I enzymes from Ab, Cv and Oh venoms exhibited similar type of
behavior towards the inhibitors as reported above by other workers. The PDE-I
enzymes from Ab, Cv and Oh venom were inhibited by EDTA etc. These findings
are in agreement with the observations that metal chelators such as EDTA, EGTA
and o-phenanthroline generally inhibit PDE activity and indicate that these proteins
are metalloenzymes. In addition, the inhibition of PDE activity by cysteine,
glutathione and ADP suggest that S-S bonds or –SH residue are essential for
enzymatic activity and the active site of PDE contains cysteine residue with free –
SH group (Razzell and Khorana, 1959; Phillips, 1976; Mori et al., 1987; Sugihara et
al., 1986; Halim et al., 1987; Valerio et al., 2002; Kini and Gowda, 1984).
The enzymes were active in the absence of any addition of divalent ions and in
fact were stimulated by high concentration of Mg+2. It is suggested that the
necessary cation is already present on enzyme (Razzell and Khorana, 1959).
Although zinc has been shown to be metal cofactor for several venom
phodphodiesterases but Ab, Cv and Oh venom PDE-I enzymes were inhibited by
125
Zn+2 significantly. Mori et al., (1987) reported that Zn+2 were inhibitory to Crr
venom PDE-I.
Activity levels of various nucleases in crude venom are usually distinguished
by the ability to catalyze the hydrolysis of specific synthetic substrate. The three
PDE-I enzymes hydrolyzed thymidine 5’-mono-phosphate p- nitro-phenyl ester most
readily (10 fold) while cyclic 3’-5’-AMP was least readily hydrolyzed substrate.
Many workers reported that the p-nitrophenyl esters are most readily hydrolyzed
chromogenic substrates (Mackessy, 1998). According to Suzuki et al., (1960), the
cyclic 3’-5’-AMP is also readily hydrolyzed by PDE-I isolated from N.n. atra, T.
flavoviridis and A. halys blomhoffii venom and was better substrate based on
reaction rate. Whereas the cyclic 3’-5’-AMP was the least hydrolyzed substrate by
the PDE-I enzymes isolated from Ab, Cv and Oh venoms. The abilities of three 3'-
and 5'-exonuclease enzymes to hydrolyze the DNA past this linkage are examined
and it is found that the linkage causes significant pauses at the sulfur linkage for T4
DNA polymerase and calf spleen phosphodiesterase, but not for snake venom
phosphodiesterase (Xu and Kool, 1998).
The biological activities of snake venom PDE have not been extensively
studied, although a role for these enzymes in envenoming has been suggested.
Purines make up the ideal multifunctional toxins, because they are endogenous
regulatory and homeostatic compounds in all vertebrates, it is impossible for any
prey organism to develop resistance to them. Purine generation from endogenous
precursors also explains the presence of many hitherto unexplained enzymes
activities in venoms, comprising 5'-nucleotidase, endonucleases, phosphodiesterase,
ATPase, alkaline and acid phosphomonoesterases and NADase, all of which are
involved in endogenous purine release. The widespread distribution of PDE in snake
126
venom again suggests a central role for PDE in envenomation strategies as described
by Aird (2002). Although the PDE was not lethal, the mice were hypoactive and
showed hind limb extension within the first hour after enzyme injection.
The Ab, Cv and Oh venom PDE enzymes did not show lethality while Russell
et al., (1963), demonstrated an LD50 of 3.08-4.65mg/Kg i.v. in mice, which is fairly
toxic. The lethality may be due to the enzyme or may be due to the presence of some
other proteins in the enzyme preparation of Russell et al., (1963), as several bands
were observed on disc gel electrophoresis.
The Ab. Cv and Oh venom PDE-I enzymes showed strong anticoagulant
effect. Ouyang and Huang (1986), reported platelet aggregation inhibiting activity of
venom PDE-I isolated from A. acutus but a specific biological role was not assigned.
Sakura et al., (1998) reported that the platelet aggregation by ADP was inhibited by
the addition of the PDEase (from fetal serum) in the platelet-rich plasma. It is
possible that a synergistic interaction with hemorrhagic proteases and fibrinogenases
found in the same venom occurs during envenomation, interfering with normal
haemostatic mechanisms and promoting blood loss leading to circulatory collapse.
Adenosine exercise anti-aggregatory effects on human platelets in vitro by
escalating intraplatelet levels of cAMP. In whole blood the half life of adenosine is
merely about 10-15 s, indicative of that the effects of adenosine in vivo on blood
platelets are normally transient and highly localized. However, in the event of large
scale release of adenosine, as take place in envenomation by many species of snakes,
adenosine could possibly have a considerable anticoagulant effect. This could be the
possible explanation in case of our venom PDEs as venom PDE it can release
adenosine as it hydrolyze nucleotides, which would have possibly exhibited
anticoagulant effect.
127
Species specific variation in relative amounts of various nucleases has been
reported by Richards et al., (1965). Biochemical characterization and comparative
analysis of these enzymes reveal that the differences in physical parameter of these
enzymes from various species sources are likely. The comparative analysis of
various biochemical and biological properties of phosphodiesterases isolated from
different snake venoms reveal the fact that the phylogenetic affinities at higher
taxonomic levels could not be ascertained by enzymatic data, as also concluded by
Aird and Silva (2001).
128
CONCLUSIONS
In conclusion, the properties of Ab, Cv and Oh venom PDE-I isolated in this study
were similar to those of other snake venoms PDE-I. The purification procedure
described here is simple, rapid and reproducible, should prove beneficial in
providing pure protein, for investigations into the contribution of this enzyme to the
biological activities of A. bilineatus, C. vipera and O. hannah venoms and of venom
PDE-I in general.
129
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