An Analysis of Age and Diet Related Changes in The Venom Composition of Sistrurus Iliarius Barbouri

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AN ANALYSIS OF AGE AND DIET RELATED CHANGES IN THE VENOM COMPOSITION OF SISTRURUS MILIARIUS BARBOURI BY ANDREW CAROTHERS A Thesis Submitted to the Division of Natural Sciences New College of Florida In partial fulfillment of for the degree Ch emistry / Biology Bachelor of Arts Under the sponsorship of Katherine Walstrom Sarasota, Florida April, 2012

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i Acknowledgement The author would very much like to thank Thomas Earle Plat e IV whose assistance with first deve loping and testing the assays used in this work is greatly appreciated

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ii Table of Contents List of Figures and Tables i ii Abstract iv Introduction 1 Ex perimental 1 3 Results 17 Discussion and Conclusions 23 Appendix I 25 Appendix II 29 Appendix III 30 Appendix IV 31 References 32

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iii List of Figures and Tables Figure 1 Neuromuscular Junction 5 Figure 2 S. M. Barbouri 6 Figure 3, Taxonomy 7 Figure 4 Lipid Cleavage 8 Figure 5 P LA 2 Mechanism 10 Figure 6 P ur ine Strategy 11 Figure 7 Diesterase Pathway 13 Figure 8 PLA 2 Substrate 15 Figure 9, PNPP 16 Table 1 Protein Concentration Data 18 Figure 10 Standard Curve 1 8 Table 2 Protein Mass 19 Table 3 Phospholipase A bsorbance 20 Table 4 Phosp h olipase Activity 20 Table 5 Phosphodiesterase Absorbance 21 Table 6 Phosphodiesterase Activity 21 Figure 1 1 Zymogram Gel 22

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iv AN ANALYSIS OF AGE AND DIET RELATED CHANGES IN THE VENOM COMPOSITION OF SISTRURUS MILIARIUS BARBOURI Andrew Carothers New College of Florida, 2012 ABSTRACT Envenomation by sna kebite is a serious concer n in many third world countries due to the high costs of developing and manufacturing successful antivenoms A potential method to alleviate this concern is to develop novel polyvalent antivenoms capable of treating the venoms of many different snake species. Producing a single serum would be far less c ostly than producing several capable of addressing the same range of species. The synthesis of such a serum requires detailed information of both interspecies and intraspecies div ersity in terms of the composition of the different venoms in question. This work sought to explore intraspecies venom diversity in the pygmy rattlesnake Sistrurus miliarius barbouri by observing changes in venom composition on both an ontogenetic and a dietary basis. Specifically, the activity of two key venom enzyme types, phospholipase and phosphodiesterase, were monitored across several venom samples from two juveniles which were provided specific prey. The results of this work suggest that a p r o l o n g e d diet o f warm blooded prey may induce secretion of both phospholipase and phosphodiesterase activity, although alternative

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v explanations of the data are possible and more experiments would need to be conducted to establish statistical significance. Katherine Walstrom Natural Sciences Division

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1 Introduction Ophidian envenomation is an issue which has been somewhat forgotten by the public and scientific communities of first world nations This neglect is largely due to the advent of successful antivenoms and the increasing scarcity of cases of envenomation in these countries bec ause of increasingly urban lifestyles, which has removed a sense of urgency about the problem. Unfortunately, the threat of envenomation remains a constan t problem in third world countries, and has the potential to resurface again as a significant problem in developed nations due to the cost prohibitive nature of manufacturing niche antivenoms [1] Envenoming by snakebites represents a very important pu blic health issue in many countries of Asia, Africa and Latin America Available data on the incidence of snakebites estimate more than 2.5 million accidents per year resulting in a total number of fatalities ranging from 20,000 to 125,000 [1] in additio n to a much higher rate of permanent disability resulting from both neurological damage and necrotic effects which required of life [ 2 ] These data are largely hospital ba sed and the refore underestimate the true scale of the problem, since a majority of snakebite v ictims will seek traditional treatment and may die at home unrecorded [ 2 ] Further more, children in these countries are at particular risk, since the very young may not rec ognize the danger a snake poses while older children are often called upon for agricul tural work, putting them at particular risk [2]

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2 The current crisis in the development and production of effective and safe antivenoms is a complex problem. Antivenom s are very expensive for fledging health services to afford, causing many to decrease or stop their purchase in favor of less expensive and less effective treatments. This causes biotechnology industries to either increase their prices for other purchaser s, or stop production of that particular unprofitable antivenom. This in turn results in the disappearance of medical personnel trained to use antivenoms and a subsequent fall in the confidence in Western medicine by local populations [1] It is the op inion of Dr. Juan Calvete [1] however, that the problem could be at least partially alleviated by the synthesis of a novel antivenom capable of counteracting the venoms of several different snake species across a wide geographic area. While polyvalent an tivenoms already exist, they are each currently applicable only to a small range of closely related species. The manufacture of an improved polyvalent antivenom would be very cost effective compared to the manufacture of the several different serums curre ntly needed to cover the same range of snake species. Antivenoms The principle of antivenom is based on that of vacci nes. H owever, instead of inducing immunity in an individual patient immunity is instead induced in a host animal (usually a horse or sheep) using a diluted venom sample [ 3 ] The hyperimmunized serum is then transferred from the animal, purified and transfused to a snakebite victim. The

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3 first antivenom designed against snakes, then called an anti ophidic serum, was developed by Albert Calmette in 1895 against the Indian Cobra [ 3 ] Antivenoms consist of antibodies that bind to and neutralize the venom, preventing further damage, but can not reverse the damage which has already occurred Antivenoms should thus be administered as soon as possible after the venom has been injected. Since the advent of antivenoms, some bites which were previously always fatal have become only rarely fatal provided that the antivenom is administered soon enough (often within one or two hours of the bite) [ 3 ] Since the original antivenoms were introduced, technologies for antivenom production have been greatly improved as has knowledge of what the serums actually contain and how they function Current antivenoms consist of purified immunoglobulins or an tibody fragments [3] Over time, the incidence and severity of antivenom treatment induced serum sickness and anaphylactic shock has been greatly reduced. Antivenoms are purified by several processes but will still contain other serum proteins that can act as antigens. Some individuals may react to the antivenom with an immediate hypersensitivity reaction (anaphylaxis) or a delayed hypersensitivity (serum sickness) reaction [4] Antivenom is typically the only effective treatment for life threaten ing envenomations, however, and once precautions for managing possible reactions is in place, treatment with antivenom will continue even if an allergic reaction occurs If an

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4 antivenom is not available when needed, then treatment is centered around addre ssing individual symptoms as best as possible [4] In the U.S ., the only approved antivenom for a rattlesnake bite is based on a purified product made in sheep known as CroFab. It was approved by the FDA in October, 2000 [ 3 ] U.S. coral snake antivenom is no longer manufactured b e cause it became unprofitable due to lack of sales and remaining stocks of in date antivenom for coral snakebite expired in the Fall of 2009, leaving the U.S. without a Coral snake antivenom [1] The first step in the prepara tion of a novel polyvalent antivenom is the selection of venoms to be included in the immunizing mixture This requires knowledge o f the evolutionary history and current genetic relatedness of all species of interest in the selected region in order to ma ke a broad comparison of the similarity of their venoms [1] Venom composition has both a genetic and an environmental basis, both of which must be investigated if a successful antivenom is to be produced. Snake venom genomics is a complex, fascinating, and relatively new field of study, but will not be discuss ed further here. It is entirely possible that an entire species or genera of snake must be excluded from the venom pool because the snakes produce venom containing a particular toxin or toxins whic h have a physiological effect unlike any of the other toxins in the antivenom pool. This is a potential problem because the combined venom could incapable of adapting to them at any significant dosage [2] It is also possible that the

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5 enzymes in the individual venoms included in the raw venom pool could begin degrading each other before research with this pool begins [1]. Unfortunately, the components capable of causing these di fficulties cannot be filtered from the pool and discarded as this would undermine the effectiveness of the antivenom. The solution to these problems is learning as much as possible about the content of the different venoms in question to avert major confl icts. Individual, geographic, and ontogenetic venom diversity reflects different levels of adaptations which can provide evolutionary advantages to a snake population, and also at least partially account for differences in the symptoms observed in human snakebite victims of different specimens of the same species [ 2 ] This further highlights the need for pooled venoms for antivenom production, even if only a single species is involved. It is also critically important to be aware of all toxic components present in composition between individuals of the same species. Despite being subjected to accelerated evolution, toxins from the same protein family but present in venoms from s nakes belonging to different genera often share antigenic determinants. Variability in venom composition can be conveniently analyzed using a combination of proteomic tools and toxicological and biochemical functional assays [ 2 ]

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6 Biology and Ve nom Composition In the case of snakes, venom has two main functions; the immobilization and digestion of prey [5] In many other organisms it is also used to fend off potential predators W hile snakes are clearly capable of using venom defense they are also capable of biting an attacker and electing not to envenomate them, thus saving valuable venom for prey while still defending themselves [6] All snake venoms contain hundreds of different compounds, m ainly enzymes, proteins, and other polypeptides Smaller components (below 1.5 KDa) include metals, peptides, lipids, nucleosides, carbohydrates, amines, and oligopeptides [5] Venom is sometimes described as a type of highly modified saliva [7] and indeed, the glands which house venom in snakes are evolutionarily derived from the parotid saliva glands of other vertebrates. These glands are usually situated on both sides of the head and behind the eye, and are wrapped in a muscular sheath [8] The venom is stored within alveoli in the glands before being conveyed by a duct to the base of the hollow fang [8] During biting, the fang is erected and venom is discharged through a duct and out of the distal orifice. As the snake bites, the jaw closes and the muscles surrounding the gland contract, causi ng venom to be ejected. It is interesting that the size of the venom fangs is not related to the virulence of the venom [6] The question of whether individual snakes are immune to their own venom is not yet definitely settled. There is a known exampl e of a cobra which self envenomated, resulting in a large abscess requiring surgical intervention but showing

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7 none of the other effects that would have proven rapidly lethal in prey species [7] Several North American species of Rat snakes as well as King snakes have proven to be immune or highly resistant to the venom of Rattle snake species. The hedgehog, mongoose, honey badger, garden dormouse, and a number of birds are all immune or partially immune to a dose of snake venom [5] There are four main ca tegories of venom effects P roteolytic toxins tend to dismantle the tissue around the site of envenomation. H emotox ins act on the heart and cardiovascular system, and are particularly prevalent in the venom of the species discussed later in this work. N e urotoxi n s act on the nervous system and brain, and can often kill a victim by paralyzing the diaphragm, rendering them unable to breathe. Cytotox ins are more difficult to classify but have a specific localized action at the site of the bite [9] Rattlen sakes are vipers, which make up the genera of Crotalus and S istrurus [10] Viper venom tends to have an especially pronounced hemotoxic effect, bringing about coagulation of the blood and clotting of the pulmonary arteries. Its action on the nervous syst em is not great, as no group of nerve cells is specifically targeted [11] The pain in the bite wound is typically severe, and is quickly followed by swelling and discoloration. The overall toxicity of viper venom varies widely. Some venoms are consiste ntly fatal unless an anitvenom or other sophisticated treatment is applied within 1 or 2 hours of envenomation. Such is the case with the venom of the Eastern Diamondback Rattlesnake, Crotalus adamanteus the largest rattlesnake in the

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8 Americas. Because of its large size, it has a particularly high venom yield of 400 1000mg, where the estimated human lethal dose is 100 150mg [10]. Other venoms seldom cause death in humans, and can be no worse than the sting of a hornet in some instances [10] In contrast to the Eastern Diamondback, the Western Diamondback Rattlesnake, Crotalus atrox has a much lower rate of mortality (10 20% for untreated victims), even though bites from this species are much more common [10]. Enzymes make up 80 90% of viper id venom. Key enzyme ty pes include phospholipases, serine proteases phosphodiesterases, thrombin like pro coagulants, and metalloproteinases [5] Polypeptide toxins include cytotoxins, cardiotoxins, and, perhaps the most well known, post synaptic neurotoxins. Th e neurotoxins bind to acetylcholine receptors at neuromuscular junction s. Amino acid oxidases and proteases are used in digestion. Amino acid oxidase triggers other enzymes and is responsible for th e yellow color of the venom of the species studied in th is work and that of many others. Hyaluronidase increases tissue permeability to accelerate absorption of other enzymes into tissues. Some snake venoms carry fasciculins, which inhibit the acetycl cholinesterase enzyme to cause the prey to lose muscle cont rol because the acetylcholine signaling cannot be stopped [12] Normal synapse function is illustrated below in Fig 1

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9 Figure 1 : Representation of a neuromuscular junction, where acetylcholine (ACh) is broken down by acetylcholinesterase after being r eceived by the postsynaptic cell. Venom fasciculins inhibit this enzyme, causing a buildup of ACh in the synapse and eventual d epletion in the axon terminal Image is public domain. Many venom components behave synergistically. Hemorrhagic toxins can cause much more significant damage in concert with anicoagulent and antiplatelet venom components [13] Another example is dendrotoxin, which induces the release of acetylcholine by its action on potassium channels, while fasciculins inhibit acetylcholin esterase. Both toxins thus enhance acetylcholine concentration at the synapse, causing paralysis through continuous muscle contraction [13] Snake venoms contain a handful of superfamilies of proteins that are mostly recruited from the body proteins. S uch proteins would mutate over time, while other mutations could cause their expression to occur in the saliva ry glands, where these new proteins would begin to provide an evolutionary advantage as the first venom enzymes. The toxin genes have evolved at an accelerated rate through gene duplication [13]

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10 Accessing Intraspecies Diversity Snake venom proteins show high levels of variation at the level of the individual but the environmental and molecular mechanisms that generate this variation are not a lways clear There is indirect evidence that some of this variation is under genetic control, possibly as a result of amino acid altering substitutions in venom genes or the presence or absence of alleles that code for specific venom proteins [14] Howev er, there is also evidence that individual venom composition can be plastic through time likely due to the effects of gene regulation. Recently, HPLC generated venom profiles ha ve shown large differences in the amount of specific proteins that are express ed by individual snakes in the same populations [14] There is evidence that the diets of mice for example, can have direct effects on the composition of salivary secretions through the autonomic nervous system, and mass spectrometric based proteomic ana lyses have recently been used to quantify shifts in specific salivary enzymes [ 30 ] Since venom glands in snakes are known to be evolutionarily derived from salivary glands, it seems possible that a feed back loop via autonomic nerves may result in chang es in the expression of specific proteins, especially if an individual consistently consumes a particular prey over long periods of time. Exploration of this hypothesis was the goal of an experiment carried out by Gibbs et al. in 2011 [14]. They used Si strurus m. barbouri common name Dusky Pigmy Rattlesnake, a small North American rattlesnake (average length 53.5 cm) found in southern Georgia, all of Florida, west though so uthern Alabama, and in Mississippi. Its

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11 natural diet consists of mainly ectothe rmic prey such as lizards and frogs although it occasionally also consumes small mammals. HPLC profiling was employed to isolate changes in venom composition. For their research, this group captured a number of pregnant female specimens of S. barbouri from northern Florida and brought them into captivity. There, each female was assigned a specific diet of either lizards, frogs, or mice. Once the females gave birth, individual juveniles were then separated into different enclosures after a short time a nd assigned a similar diet of specific prey [14] An example of the species in question is shown below in Fig. 2 followed by its taxonomic tree in Fig. 3 Figure 2 : A typical adult specimen of Sistrurus m. barbouri, gender unspecified. Image is publi c domain.

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12 Kingdom Animalia Phylum Chordata Subphylum Vertebrata Class Reptilia Order Squamata Suborder Serpentes Infraorder Alethinophidia Family Viperidae Subfamily Crotalinae Genus Sistrurus Species S. m iliarius Subspecies S. m b arbouri Figure 3 : Taxonomy of the Dusky Pigmy Rattlesnake, Sistrurus Miliarius Barbouri [ 28 ]. The research group found that while juveniles raised on different prey in captivity exhibited distinct c ompositional change rates, at 26 months old, their venoms show ed similar patterns of protein composition suggesting little effect of diet on the overall make up of venom in snakes this age or younger [14] In contrast, adult females raised on different prey over a 26 month period were found to have differences in t he relative abundance of major protein families from initial to final samples. From HPLC analysis mouse fed females showed substantial increases in the relative abundance of total phospholipase A 2 s and serine proteases of 95% and 100%, respectively, where as the percent increase for lizard ( 42% and 22%) and frog fed females (2% and 11%) were smaller in magnitude. These changes represent percent difference from a snake of the same species with a natural diet of lizards and frogs The team identified p rev iously characterized proteins by N terminal sequencing, mass fingerprinting, and ESI MS, after being initially recognized by retention time and SDS PAGE analysis [14]

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13 When averaged across values for all toxin classes HPLC analysis showed that frog fed individuals showed the highest absolute value for % change in composition (47%) followed by the mouse (36%) and lizard fed snakes (30%) compared to snakes of the same age and gender but with a natural diet In summary the ir results demonstrate d that an y ontogenetic changes in the venom of this species did not involve substantial changes in the broad classes of toxins but instead involve d small shifts in the relative proportion of specific venom compon ents [14]. The lack of impact of the type of pre y on the juvenile venom composition could have be en due to the use of inappropriate prey; the mouse species used was not native to Florida and some prey items were frozen and then reheated rather than presented to the snakes while alive. It is also possib le that such a limited diet caused a nutritional deficiency, which in turn altered normal venom development. Overall, the most extreme changes in venom composition were seen in mouse fed juvenile and adult females [14] This is consistent with the idea that when snakes are fed a diet of prey that was likely rarely consumed in nature, this in turn induces greater changes in venom composition. Rodents may be a rare source of nutrition for this particular species because of its small size. Phospholipa ses Phospholipases, or PLA 2 s, are hydrolytic enzymes common to all snake venoms in some form [15] PLA 2 enzymes are unique hydrolytic enzymes in that they are highly soluble in water but hydrolyze water insoluble phospholipids. They hydrolyze

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14 phospholip ids in monomeric, micellar or lipid bilayer phases. PLA 2 s can sometimes exert their full effect apart from the other venom components, but some require additional protein factors in order to form a complex. Some subunits covalently bond to each other wh ile others act like chaperones and aid in specific binding of the PLA 2 enzyme to the target site [12] As a venom component, these enzymes are capable of carrying out more than one critical role, as they are largely responsible for both the hemotoxic and neurotoxic effects of venom s [16] An example of a lipid molecule known to be a target of PLA 2 s is shown below in Fig. 4 Figure 4 : Phosphatidylcholine as an example lipid molecule showing the cleavage site of PLA 2 enzymes. Image is public domain, edi ted by the author to show the cleavage site. PLA 2 s occur naturally in many animal tissues as well. Mammalian PLA 2 enzymes play important roles in fertilization, cell proliferation, smooth muscle contraction, and chronic inflammatory disease They are also important in cellular functions such as the biosynthesis of prostaglandins and leukotrienes, and membrane homeostasis including the maintenance of the cellular phospholipid pools and membrane repair through deacylation/re acylation. However, the mamm alian enzyme s are generally nontoxic and do not produce potent pharmacological effects [17]

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15 PLA 2 enzymes require Ca 2+ for their activity, so chelators such as EDTA inhibit enzymatic activity. Ca 2+ can also be replaced with other metal ions such as Ba 2 + and Sr 2+ In some cases, Sr 2+ can replace calcium and support the normal func tions of the system, but not enzymatic activity [15] So far, the amino acid sequences of over 280 PLA enzymes have been determined. Despite differences in their pharmacolog ical properties, they share 40 99% identity in their amino acid sequences and therefore have significant similarity in their three dimensional folding [15] Calcium and substrate binding loops are a common motif in almost all known examples [18] His 48 is also conserved in PLA 2 enzymes and it plays a significant role in phospholipid hydrolysis. Alkylation of His 48 has been shown to lead to complete loss of enzymatic activity. Thus, His modified PLA 2 enzymes are suitable for both in vitro and in vivo systems for studying enzymatic activity and how histidine plays such a crucial role This alkylation does not affect the conformation of the enzyme nor its ability to bind the phospholipids [18] PLA 2 enzymes bind to target phospholipids through s pecific cleavage sites. The presence of pharmacological sites is supported by chemical modification studies, polyclonal and monoclonal antibodies and interaction of inhibitors [18] The ability of PLA 2 enzymes to induce pharmacological effects also depen ds on their penetrability. Phospholipids in plasma membranes are packed at higher density compared to phos pholipid vesicles and liposomes, and so PLA 2 s are able to hydrolyze phospholipids in the vesicles and liposomes more effectively than those found in the plasma

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16 membrane [18] A PLA 2 enzyme may fail to exhibit its pharmacological effect, despite binding to the specific target protein, if it lacks penetrability. As a venom component in rattle snakes and other vipers PLA 2 s causes hemolysis by lysing t he plasma membranes of red blood cells [15] In particular, PLA 2 s release fatty acids from the second hydroxyl group of glycerol [18] PLA 2 specifically recognizes the S N 2 acyl bond of phospholipids and hydrolyzes the bond, releasing arachidonic acid an d lysophospholipids. Upon downstream modification, arachidonic acid is modified into eicosanoids, which are inflammatory mediators. In animal tissues, PLA 2 s are normally found in low concentrations, but when administered in a venomous bite, they cause a disproportionate amount of arachidonic acid to be released, causing pain and swelling in the victim [15] The catalytic mechanisms of PLA 2 s have been studied best with sample s of endogenous pancreatic enzyme. The suggested mechanism is initiated by a H is 48/Asp 99/calcium complex within the active site. The calcium ion polarizes the S N 2 carbonyl oxygen while also coordinating with a catalytic water molecule, w5. His 48 improves the nucleophilicity of the catalytic water via a bridging second water mol ecule, w6. It has been suggested that these two water molecules are necessary to traverse the distance between the catalytic histidine and the ester. The basicity of His 48 is thought to be enhanced through hydrogen bonding with Asp 99. An asparginine s ubstitution for Hi 48 maintains activity, because the amide functional group on the asparginine can also function to lower the pKa of the bridging water molecule. The rate limiting state is the

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17 degradation of the tetrahedral intermediate which is a calci um coordinated oxyanion. PLA 2 s also possess a channel featuring a hydrophobic wall in which hydrophobic amino acid residues such as Phe, Leu, and Tyr serve to bind the substrate. Another component of PLA 2 are the seven disulfide bridges that are importa nt in regulation and stable protein folding [18] The mechanism of action of PLA 2 enzymes is displayed below in Fig. 5 Figure 5 : Mechanism of hydrolysis of a lipid catalyzed by PLA 2 Image is public domain.

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18 Phosphodiesterases Phosphodiesterases are a broad, variable category of enzymes that all break phosphodiester bonds of some type [19] In snake venom, phosphodiesterases mainly pressure. Venoms from most front fanged snakes show at least low levels of venom pho s phodiesterase activity, but absolute amounts of phosphodiesterase activity relative to other nucleases vary considerably. Phosphodiesterases from snake venoms appear to be able to catalyze the hydrolysis of numerous native substances including double and single stranded DNA, ribosomal and transfer RNA, oligonucle otides and cyclic nucleotides [19] Before more intense studies were conducted, phosphodiesterases in venom were thought to aid in digestion o nly. It is now known that t hey naturally liberate purines, which act as a multitoxin [19] The identification of free purines as a constituent of venoms has further supported the role of purinergic signaling in envenomation. Purines are known to cause venom induce d hypotension and paralysis via purine receptors, which are commonly found in various or ganisms envenomed by snakes [19] In humans, there are four types of purine receptors, where the two primary types, A 1 and A 2A ; both play important roles in the heart in regulating coronary blood flow. Stimulation of these receptors in the heart leads to the hypotension and eventual immobilization [ 29 ]. An overview of this and the other events leading to immobilization are represented below in Fig. 6

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19 F igure 6 : Overview of venom immobilization strategy, with emphasis on the role of both liberated and venom purines. Purines from both sources play a role in triggering hypotension in the prey/victim through stimulation of the A 1 A 2A and A 2B adenosine r eceptors [ 29 ]. Few studies since have attempted to purify and characterize diesterase from snake venom. They are mellaoenzymes in general; the existence of a zinc active site was demonstrated by flame absorption spectrometry [19] There is no informa tion on the

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20 amino acid sequence of full length venom diesterases as of 2010 Enzymes of this type have been shown to inhibit platelet aggregation when induced by ADP, collagen, and sodium arachidonate in platelet rich plasma, and when induced by thrombin in platelet poor plasma [19] It is possible that during envenomation, diesterases synergistically act with hemorrhagic proteases and fibrinogens to affect normal hemostatic functions, leading to blood loss and circulatory collapse in the prey/victim. Adenosine generation is pharmacologically important as it induces several snake envenomation related symptoms. Generation of adenosine by venom enzymes can occur in many pathways. Enzymes like nucleotidase and PDE act immediately upon envenomation on av ailable ATP molecules to release adenosine [19] ATPases were shown to be M g 2+ dependent. In addition, a few reports also suggest that some of these enzymes act synergistically with other toxins, contributing to the overall lethal effects of the venoms It is unclear whether there exist specific nucleotidases in venom or if single enzymes are capable of acting on multiple nucleotide types. On incubation, venom containing phosphodiesterase is capable of hydrol yzing ATP into either AMP or ADP and the ac companying phosphate depending on experimental conditions [19] It appears possible that immobilization of prey could be partially achieved by depletion of ATP by the action of ATPases, but this has not yet been experimentally verified, and such an effect would undoubtedly have to act in concert with neurotoxic paralysis to be effective Phosphodiesterases also liberate nucleotides from the cell genome [19] The liberation is preceded by cell death caused by venom proteases/hemorrhagins,

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21 phospholipase s, myotoxins, cardio toxins, and cytolytic peptides. Once the cell is ruptured by these agents the venom diesterase acts on the DNA and RNA. After liberation, there is a possibility that the adenosine will be deaminated into inosine, which is an importa nt metabolite capable of inducing many pharmacological actions including potential antiviral and neuroprotective abilities [19] Inosine and can be oxidized to xanthosine monophosphate, an important precursor in purine metabolism Liberated adenosine ha s many functions once in the prey. It can help in th e through vasodilation. Adenosine is also known to cause paralysis by inhibiting neurotransmitter release at both cent ral and peripheral ne rve termini [19] Adenosine is also naturally released in mammals as a response to stress or injury, further elevating levels after an envenomation. The main pathways for adenosine liberation are shown below in Fig. 7 Figure 7 : Sc hematic of various pathways of adenosine generation by phosphodiesterase and related enzymes. Image acquired from [ 19 ].

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22 Present Work This work sought to explore intraspecies venom diversity in S. barbouri by observing changes in venom composition on both an ontogenetic and a dietary basis, in an experiment similar to that conducted by Gibbs et. al. [14]. Specifically, the activity of two key venom enzyme types, phospholipase and phosphodiesterase, were monitored across several venom samples from two juveniles that were provided a strict diet of specific prey. This work differs from that done previously in this area because of the inclusion of the phosphodiesterase activity assay; potential changes in phosphodiesterase activity with diet and age have not been studied before. In addition to these assays, the protein concentration in each collected venom sample was analyzed, and gelatin zymography was performed as a qualitative assay of protease activity.

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23 Experimental Section Venom samples u sed in these experiments were obtained from Medtoxin Venom Laboratories in DeLand, FL, who in turn captured female specimens of S. m. barbouri and their offspring. The pregnant females were captured from Seminole, Columbia, and Volusia counties in Florida Individual juveniles were fed specific prey in much the same way they were by Gibbs et al. [ 14 ], but in this case there existed only two diet options; warm blooded prey, consisting of rodents, and cold blooded prey, consisting of both lizards and frogs. Individuals were milked for their venom in 2 week intervals for a 6 month period, and after each milking, the venom was lyophilized for long term storage. Mackessy et. al. [ 8 ] found that crude venom is highly refractive to a wide array of storage condit ions, and suggested that most venom activities should remain stable even if the venom is stored under conditions that would normally be adverse for biological samples. They speculated that endogenous mechanisms present in venom must inhibit autolysis duri ng it s long term storage in vivo in the gland [ 8 ] Of the numerous samples available from several individuals, venom samples were exclusively assayed from two individual snakes; one with a warm blooded and one with a cold blooded diet. These two individu als were specifically chosen because they had the largest number of dated samples available, making an ontogenetic analysis possible. Many samples from these two juveniles and the others had been previously used in research in another laboratory making t he selection of samples and the construction of a timeline for their milkings and date of birth somewhat difficult. The

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24 two individuals were labeled 2VCR and 4VCLF these individuals was captured in Volusia County, blooded diet of blooded diet of lizards and frogs. A large sample of lyophilized venom from an adult of this species was also acquired from Medtoxin and used as a control to test the following assays before data collection began. Protein Concentration Assay Before assaying phospholipase or phosphodiesterase activity, the protein concentration in each milking sample was determined. The assay performed was colorimetric in nature and based on the well documented Lowry assay. Each assay was carried out using a Bio Rad Protein Assay Reagent Kit and a Varian Cary 300 UV VIS spectrophotometer. The assay is based on the reaction of protein with an alkaline copper tartrate solution and a Folin reagent. T he Folin or Folin Ciocalteu reagent is a mixture of phosphomolybdate and phosphotungstate, which measure the total reducing ability of the sample. This reagent is often used to detect the presence of phenolic compounds but it is also reactive towards thi ols, several vitamins, guanine, and some inorganic ions [ 20 ] To focus the reaction onto phenols specifically, the phenols are complexed with copper, to increase their reactivity towards the reagent [ 21 ] Thus i n the Lowry assay, there are two steps which lead to color development: t he reaction between protein and copper in an alkaline medium, and the subsequent reduction of Folin reagent by the

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25 copper treated protein. Final c olor development is primarily due to the amino acids tyrosine and tryptophan a s they are phenolic compounds an d to a lesser extent, cysteine and histidine. Proteins cause a reduction of the Folin reagent by loss of 1, 2, or 3 oxygen atoms, thereby producing one or more of several possible reduced species which have a characteristi c blue color with maximum absorbance at 750nm [ 20 ] Dilutions of bovine serum alb umin, obtained from Sigma Aldri ch, were assayed as standards to produce a standard curve to determine protein concentration. All standards and samples were assayed twice, a nd the results of each assay were averaged together to generate the standard curve. A new standard curve was prepared for each venom sample. Phospholipase Assay Phospholipase activity was also assayed by colorimetric means using the same spectrophotom eter The substrate used in this assay was 4 Nitro 3 (octanoyloxy)benzoic Acid, first synthesized by Cho et. al. [ 2 2 ] for use in this type of assay. The release of the nitrophenol group from the hydrophobic chain simulate s the cleavage of the acyl bond i n phospholipids. The resulting product, para nitrobenzoic acid, is the chromophore in this assay [2 3 ] The structure of the substrate is shown below in Fig. 8.

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26 Figure 8 : Structure of the substrate, 4 Nitro (octanoyloxy)benzoic Acid, shown with the P LA 2 cleavage site. Image was drawn by the author The substrate was obtained from Enzo life sciences, and an appropriate mass was dissolved in acetonitrile for each daily use. Each assay was again performed in duplicate. The entire reaction mixture w as as follows: 1 mL buffer (10 mM tris HCl, 10 mM CaCl 2 100mM NaCl; pH 8.0), 100 uL 4.0 mg/mL venom, 100 uL substrate (4 nitro 3 (octanoyloxy)benzoic acid, 3.0 mM in acetonitrile). Once complete, the mixtures were allowed to incubate for 20 minutes in a 37 C water bath to simulate physiological conditions for enzyme activity Afterwards, the enzyme activity was stopped with 100 uL of 2.5% Triton X 100. The samples were held at room temperature for 10 15 minutes before spectrophotometric analysis at 4 25 nm. Blanks consisted of 1mL buffer, 100 uL deionized water, and 100 uL acetonitrile. The molar extinction coefficient of the enzyme products is 5039 M 1 cm 1 [2 3 ] Since venoms are a mixture of proteins, specific activity for phospholipase could not be determined.

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27 Phosphodiesterase Assay Phosphodiesterase activity was a lso assayed by colorimetric means. The substrate used was PNPP (Bis(4 nitrophenyl) phosphate). When attacked by a phosphatase, PNPP decays to para nitrophenol, the chromophore i n this assay. Most spectrophotometric assays of venom phosphodiesterase are based on the quantification of the nitrophenol chromophore released at hydrolysis. PNPP was chosen in particular because it is widely available commercially, is inexpensive and pr oduces reliable results for most typical assays. PNPP was obtained from Sigma Aldrich [2 4 ] The structure of the substrate is shown below in Fig. 9. Figure 9 : Structure of the substrate, Bis(4 nitrophenyl) phosphate, shown with the phosphodiesterase cl eavage site. Image is public domain, edited by the author to show the cleavage site. After attempting the assay at a variety of protein concentrations with adult venom, a concentration of 2mg/ml finally selected Concentrations above this value were to o potent to produce results comparable to those of the phospholipase assay. Each assay was performed in duplicate. A fresh solution of 1mM PNPP was prepared for

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28 each venom sample. The entire reaction mixture was as follows: 0.2 mL 0.1M Tris HCl (pH 8.9) 0.24 mL 1mM PNPP, 6 uL 3M Acetate, 114 uL Di Water, and 40 uL venom dilution, with an overall working pH of 7.4. Once complete, the mixtures were allowed to incubate for 25 minutes in a 37 C water bath. After 25 minutes, the activity was stopped with 600 uL of 50 mM NaOH. The samples were then analyzed at 400 nm with the same spectrophotometer. Blanks consisted of 0.2 mL of 0.1 M Tris HCl, 6 uL 3M Mg Acetate, and 394 uL of DI water. P ara nitrophenol has a molar extinction coefficient of 18,300 M 1 cm 1 [2 5 ] Again, since the venom is a mixture of proteins, a specific activity for phosphodiesterase could not be determined. Gelatin Zymography Zymography is an electrophoretic technique used to identify proteolytic activity in enzymes separated in poly acrylamide gels under nonreducing conditions [2 6 ] It has been used extensively in the qualitative evaluation of proteases present in tumors and cell culture conditioned media. In a zymogram, e vidence of enzymatic activity is demonstrated by the absence of staining in areas where the large protein substrate embedded in the gel has been degraded. The initial rate of digestion is proportional to the enzyme loading. Zymography is useful because it can determine if any proteins being analyzed possess proteo lytic activity, provide a qualitative measure of this activity, and provide and estimation of the molecular weight of the protease(s) [ 27 ]

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29 Zymography was performed according to the method outlined by Renata [ 28 ]. Gelatin was copolymerized with the acryl amide during casting of the running gel. T he running gel was non reducing, in order to preserve enzyme activity Three lanes were loaded with venom from the juvenile 2VCR, at 1 mg/ml, 0.5 mg/ml, and 0.1 mg/ml concentrations in nanopure water The total volume of each loaded sample was 3 l. Three adjacent lanes were loaded with venom samples from an adult rattlesnake at the same concentrations. This allowed for a qualitative examination of the strength of the A 0.5 mg/ml solution of papain, an enzyme found in papaya, was prepared in water and loaded onto the gel as a positive control. Papain was obtained from Sigma Aldrich. A lane prepared with 3 l of load buffer only was used as a negative control. Two sta ndard protein ladders were also r u n in this zymogram. Once electrolysis was complete, t he enzymes were allowed to refold in Triton X 100 and were then incubated in a buffer containing 20 mM Tris HCl, 150 mM NaCl, and 5 mM CaCl 2 final pH 7.4 The gel was then stained with Coomassie brilliant blue, and allowed to de stain overnight before being imaged.

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30 Results Section Protein Concentration In total, 12 samples from the 2 selected juveniles were assayed, 7 from the 2VCR individual, and 5 from the 4VCLF individual. Equal numbers of samples from the two individuals would have been preferred, but there were insufficient samples from the 4VCLF individual to achieve this. Listing the age of the individual at the time of each venom collection rather than date of the collection would have also been preferred, but the lack of a recorded date of birth for these individuals makes this impossible. The averaged absorbances reported from the protein concentration assays are reported below for each sa mple. These averages are obtained from combining the absorbance data from two replicates. The data is displayed below in Table 1. Sample Abs or bance (AU) Standard Deviation 2VCR 9/20/04 0.4252 0.0059 2VCR 1/23/05 0.4039 0.0004 2VCR 3/19/05 0.3293 0.000 5 2VCR 4/3/05 0.6552 0.0037 2 VCR 5/1/05 0.6913 0.0039 2VCR 5/15/05 0.4082 0.0031 2VCR 7/10/05 0.5028 0.0022 4VCLF 2/20/05 0.2118 0.0023 4VCLF 3/19/05 0.3288 0.0054 4VCLF 4/3/05 0.4765 0.0049 4VCLF 5/15/05 0.4906 0.0016 4VLF 7/10/05 0.5143 0.0 135 Table 1 : Absorbances of the protein assays at 750nm of dated venom samples reported in absorbance units. Absorbances were obtained by averaging together two separate readings from separate replicates during the protein concentration assay in identica l conditions.

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31 The absorbance data was then interpreted using standard curves constructed for each sample with BSA as a standard. This allowed for an estimation of the mass of protei n present in each venom sample. A sample of one these standard curves is shown below in Fig. 10. Figure 10 : Sample standard curve produced for the analysis of sample2VCR 4/3/05 Solutions of BSA were prepared at concentration of 0.2, 0.5, 0.8, 1.2, and 1.5 mg/ml and then measured at 750 nm to produce the data points of the curve. The curve was then fit with a linear trend line to allow for interp olation of the venom sample absorbance. Once all standard curves were complete, the linear fit of each curve was used to calculate the approximate mass of protein present in each sample based on the absorbance of that sample. These calculated masses are shown below in Table 2.

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32 Sample Protein Mass (mg) 2VCR 9/20/04 0.47 2VCR 1/23/05 0. 13 2VCR 3/19/05 0. 12 2VCR 4/3/05 0.61 2 VCR 5/1/05 0.7 2 2VCR 5/15/05 0.2 7 2VCR 7/10 /05 0.1 6 4VCLF 2/20/05 No Data 4VCLF 3/19/05 0. 12 4VCLF 4/3/05 0.33 4VCLF 5/15/05 0.32 4VLF 7/10/05 0.41 Table 2 : Approximate mass of protein present in analyzed venom samples, reported in mg. Total volume in each assayed sample was 1 ml. Value s were calculated b y entering the average d absorbances of the two replicates into the linear fit equations generated for each standard curve. Sample 4VCLF has no protein mass data because the average absorbance of this sample was lower than the y intercep t of its standard curve. Phospholipase Activity Phospholipase and phosphodiesterase activity were both analyzed immediately after the completion of the protein concentration assay. The average absorbance s from two individual assays are reported here in Table 3 below

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33 Sample Absorbance (AU) Standard Deviation 2VCR 9/20/04 0.1231* No Data 2VCR 1/23/05 0.2101 0.0553 2VCR 3/19/05 0.0936 0.0050 2VCR 4/3/05 0.5991 0.2449 2VCR 5/1/05 No Data No Data 2VCR 5/15/05 0.2032 0.0105 2VCR 7/10/05 0.3422 0.0326 4VCLF 2/20/05 0.0692 0.0146 4VCLF 3/19/05 0.1177 0.1108 4VCLF 4/3/05 0.2241 0.1563 4VCLF 5/15/05 0.2112 0.0504 4VLF 7/10/05 0.4055 0.1001 Table 3 : Absorbances of dated venom samples reported in absorbance units. Absorbances were obtained by averaging together two separate readings from separate replicates recorded during the phospho lipase activity assay in identical conditions at 425 nm. The al iquoted sample for 2VCR 5/1/05 was ruined during the assay. The value stated for sample 2VCR 9 /20/04 is not an average but a single reading, due its duplicate reading being an outlier. In an attempt to normalize the lipase activity data based on the amount of protein present in the corresponding sample, each average absorbance value was divided b y the protein mass in the sample to yield the relative enzyme activities found in Table 4 Sample Relative Activity 2VCR 9/20/04 0.26 2VCR 1/23/05 1.60 2VCR 3/19/05 0.79 2VCR 4/3/05 0.9 8 2VCR 5/1/05 No Data 2VCR 5/15/05 0.76 2VCR 7/10/05 2.14 4VCLF 2/20/05 No Data 4VCLF 3/19/05 0.12 4VCLF 4/3/05 0.6 8 4VCLF 5/15/05 0.6 6 4VLF 7/10/05 0.98 Table 4 : Relative phospholipase enzyme activity obtained by dividing assay average absorbance by total protein mass. The allotted sample for 2VCR 5/1 /05 was ruined during the assay, while mass data for s a m p l e 4VCLF 2 / 2 0 / 5 i s u n a v a i l a b l e p r e c l u d i n g a n a c t i v i t y c a l c u l a t i o n

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34 Phosphodiesterase Activity The phosphodiesterase assay for each sample was carried out concurrently with the phospholipase assay. The average absorbance from two individual assays is reported here i n Table 5 Sample Absorbance (AU) Standard Deviation 2VCR 9/20/04 0.0440 0.0004 2VCR 1/23/05 0.0305 0.0055 2VCR 3/19/05 0.0220 0.0019 2VCR 4/3/05 0.0989 0.0074 2VCR 5/1/05 0.0535 0.0039 2VCR 5/15/05 0.0859 0.0416 2VCR 7/10/05 0.0393 0.0013 4V CLF 2/20/05 0.0688 0.0012 4VCLF 3/19/05 0.0340 0.0029 4VCLF 4/3/05 0.0538 0.0083 4VCLF 5/15/05 0.0579 0.0252 4VLF 7/10/05 0.0103 0.0023 Table 5 : Absorbances of dated venom samples reported in absorbance units. Absorbances were obtained by averaging to gether two separate readings from separate samples during the phospho diesterase activity assay in identical conditions at 400 nm. The same normalizing procedure performed on the phospholipase data was also conducted on the phosphodiesterase absorbances. This normalized data is presented in Table 6 below.

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35 Sample Relative Activity 2VCR 9/20/04 0.09 2VCR 1/23/05 0 2 3 2VCR 3/19/05 0. 1 8 2VCR 4/3/05 0.16 2VCR 5/1/05 0.07 2VCR 5/15/05 0.32 2VCR 7/10/05 0.2 5 4VCLF 2/20/05 N o D a t a 4VCLF 3/19/05 0. 2 9 4VCLF 4/3/05 0.16 4VCLF 5/15/05 0.18 4VLF 7/10/05 0.0 3 Table 6 : Relative phospho diesterase enzyme activity obtained by dividing assay absorbance by total protein mass. M ass data for s a m p l e 4VCLF 2 / 2 0 / 5 i s u n a v a i l a b l e p r e c l u d i n g a n a c t i v i t y c a l c u l a t i o n Zymography A number of zymograms were performed prior to beginning the colori metric assays. The image presented here is of the final zymogram that was performed, as all previous attempts required refinement while this gel imaged clearly. The venoms used in this zymogram were from an undated sample from 2VCR and from an adult snak e of the same species, for comparison. Dark bands in the gel indicate the presence of proteins of a particular molecular mass, just as in typical SDS PAGE, while lightened areas are evidence of proteolytic enzymes acting on the gelatin embedded within the gel. A contrast enhanced photograph of the zymogram is shown below in Fig. 11.

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36 Figure 11 : Stained and imaged zymogram gel. Lanes L1 and L2 contain standard protein ladder. L2 was cut away from the gel prior to staining for fear that the ladder would stain too darkly to recognize. Lane C was a negative control and contained load buffer only. Lane P was a positive control and contained the known proteolytic enzyme papain at 0.5 mg/ml Lanes 1, 2, and 3 contained 0.1, 0.5, and 1.0 mg/mL concentration s of adult venom, respectively. Lanes A, B, and C contained 0.1, 0.5, and 1.0 mg/ml of venom from the juvenile 2VCR, respectively. Total volume loaded into each lane was 3 ul. The arrow indicates the position of the proteolytic band.

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37 Discu ssion and Conclusions Although the concentration assa y was only intended to serve as a means to interpret the results of the enzyme activity assays, the data seemed to exhibit a trend in the case of the 4VCLF as protein concentrations steadily increased with the age of this individual. This trend could also potentially be an artifact, however, as it was not mirrored in the concentration data from the 2VCR venom samples. Overall the protein concentration data appears to be reliable based on low standar d deviations and errors, though again larger sample sizes would be much preferred. It would also be easier to acquire larger amounts of crude venom from larger snake species. The zymogram gel seems to indicate that venom from a juvenile individual has a higher protein concentration than a n adult individual, evidenced by the darker protein bands in lanes A, B, and C. Since venoms are not entirely composed of proteins, there is a possibility that the juvenile crude venom sample contained more protein by m ass than that of the adult sample. Clearly, proteolytic enzymes represent only a small portion of the total enzymes within the venom of this species, as 0.5 mg/ml papain shows much higher gelatinase activity than any concentration of rattlesnake venom. N o significant ontogenetic pattern was observed in the phospholipase activity of 2VCR venom and a weak pattern of increasing activity was demonstrated by the venom of 4VCLF. This partially agrees with the results obtained in a report by [ 14 ] as this group demonstrated that juvenile snakes did not demonstrate significant changes in venom composition. In terms of overall phospholipase activity, ind ividual 2VCR had an average

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38 normalized phospholipase act ivity across all dates of 0 8 5 0 6 7 while individual 4VCLF h ad an average activity of 0 6 1 0. 3 6 The rodent fed individual having higher phospholipase activity is in accordance with the results of [ 14 ]. The results of the phospholipase assays had somewhat higher standard deviations and errors than either the protein concentration or phosphodiesterase assays, however, casting doubt on the r eliability of any conclusions drawn from this data. No o ntogenetic pattern was recognizable in either the absorbance or activity data from phosphodiesterase assays. Individual 2VCR had an average normalized phosphodiesterase act ivity across all dates of 0. 1 9 0. 0 9 while individual 4VCLF had an activity of 0. 1 7 0. 1 1 The averages show that phosphodiesterase activity may be higher in the individual with the diet of w a r m blooded prey, which is fascinating since the activity of this enzyme class has not been examined in an experiment of this type before a l t h o u g h t h e d i f f e r e n c e i s q u i t e s m a l l It would be very interesting for another group to continue this research and perform similar phosphodiesterase assa ys, since the influence of diet and age on phosphodiesterase activity has not been analyzed before. Th e phosphodiesterase data appear to be fairly reliable based on standard deviations and errors between those of concentration and phospholipase assays. O verall, this study produced interesting results but has a number of weaknesses. Foremost, small sample sizes prevent ed any significant statistical interpretation of the results, l i k e l y negating their potential u tility in future studies. T h e t e c h n i q u e s u s e d i n t h i s w o r k h o w e v e r h a v e g r e a t p o t e n t i a l u t i l i t y i n f u t u r e e x p e r i m e n t s o f t h i s n a t u r e

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39 In addition, several t echnical problems relating to the source of the venom samples also affected the interpretation of the data. The specimens were captured, raised and milked by another research group, which also labeled all samples and appeared to use several of them in the ir own research before passing the remainder on. This limited the quality of the ontogenetic analyses as gaps in the collection sequence of variable time length are present between the available samples Further, the previous group did not specify in th e transcript they provided as to what species of rodents, frogs and mice composed the diets of the snakes. It is unclear whether a single or a number of different species were used for each prey category, and it is also unclear as to what ratio of frogs v ersus lizards were presented to the snake s with the cold blooded diet s These uncertainties limit the reliability of any comparison between the results presented here and the results obtained by Gibbs et al. in [ 14 ], as this group clearly listed the choic e of prey for their work. Finally, this study suffers an uncertainty that is more difficult to address and that w as faced by the researchers in [ 14 ] as well. I t is possible that any venom variation observed in this experiment could be caused by variation s in the individuals studied which are unrelated to the experimental conditions, such as i ntraspecies genetic differences In any case, confirming the results of this work will require larger sample sizes capable of accounting for individual variation. F uture work of this type could potentially examine individuals for a considerably longer duration, and if a diet induced change in venom composition is believed to be induced during this time, the diet could then be altered to see if this observed change is reversible. It would also be interesting

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40 to attempt to determine if any prey related changes in venom composition are in fact advantageous when feeding on this particular prey, or if the observed changes are only the result of a physiological necessity i n response to a limited diet.

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41 Appendix I Concentration Assay Data 2 VCR 1 23 05 Trial 1 BSA Concentration (mg/ml) Absorbance (at 750nm) 0.2 0.4448 0.5 0.7092 0.8 0.8807 1.2 1.0574 1.5 1.1492 Venom Sample 0.4036 2 VCR 1 23 05 Tr ial 2 0.2 0.4485 0.5 0.6976 0.8 0.9235 1.2 1.0640 1.5 1.1675 Venom Sample 0.4042 4VCLF 7 10 05 Trial 1 BSA Concentration (mg/ml) Absorbance (at 750nm) 0.2 0.3316 0.5 0.5875 0.8 0.7589 1.2 0.9080 1.5 1.0002 Venom Sample 0.5047 4VCLF 7 10 05 T rial 2 0.2 0.3507 0.5 0.6113 0.8 0.7800 1.2 0.9276 1.5 1.0175 Venom Sample 0.5238 2VCR 7 10 05 Trial 1 BSA Concentration (mg/ml) Absorbance (at 750nm) 0.2 0.4387 0.5 0.6996 0.8 0.8949 1.2 1.1009 1.5 1.1785 Venom Sample 0.5012 2VCR 7 10 05 Tr ial 2 0.2 0. 4359 0.5 0. 7600 0.8 0. 8961

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42 1.2 1.1107 1.5 1. 1807 Venom Sample 0.5 043 2VCR 4 3 05 Trial 1 BSA Concentration (mg/ml) Absorbance (at 750nm) 0.2 0.3601 0.5 0.6120 0.8 0.8128 1.2 1.0261 1.5 1.1310 Venom Sample 0.6526 2VCR 4 3 05 Trial 2 0.2 0.3477 0.5 0.6168 0.8 0.8206 1.2 1.0280 1.5 1.1371 Venom Sample 0.6578 4VCLF 4 3 05 Trial 1 BSA Concentration (mg/ml) Absorbance (at 750nm) 0.2 0.3525 0.5 0.5914 0.8 0.7547 1.2 0.9963 1.5 1.0391 Venom Sample 0.4730 4VCLF 4 3 05 Trial 2 0.2 0.3581 0.5 0.5993 0.8 0.7644 1.2 1.0024 1.5 1.0478 Venom Sample 0.4799 2VCR 3 19 05 Trial 1 BSA Concentration (mg/ml) Absorbance (at 750nm) 0.2 0.3524 0.5 0.5852 0.8 0.7 673 1.2 0. 9757 1.5 1.0889 Venom Sample 0.3197 2VCR 3 19 05 Trial 2 0.2 0. 3484 0.5 0. 5774 0.8 0.7 560 1.2 1.00 43

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43 1.5 1.0 873 Venom Sample 0. 3186 2VCR 5 15 05 Trial 1 BSA Concentration (mg/ml) Absorbance (at 750nm) 0.2 0.2937 0.5 0.5421 0.8 0.7840 1.2 0.8422 1.5 0.9490 Venom Sample 0.4052 2VCR 5 15 05 Trial 2 0.2 0.2970 0.5 0.5482 0.8 0.7851 1.2 0.8474 1.5 0.9534 Venom Sample 0.4112 4VCLF 5 15 05 Trial 1 BSA Concentration (mg/ml) Absorbance (at 750nm) 0.2 0.3702 0.5 0.6256 0.8 0.8361 1.2 1.0978 1.5 1.2134 Venom Sample 0.4917 4VCLF 5 15 05 Trial 2 0.2 0.3705 0.5 0.6225 0.8 0.8339 1.2 1.0977 1.5 1.2110 Venom Sample 0.4895 4VCLF 3 19 05 Trial 1 BSA Concentration (mg/ml) Absorbance (at 750nm) 0.2 0.3684 0.5 0.6474 0.8 0.8318 1.2 0.9938 1.5 1.0744 Venom Sample 0.3249 4VCLF 3 19 05 Trial 2 0.2 0.3724 0.5 0.6311 0.8 0.83 97 1.2 0.9961 1.5 1. 0779

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44 Venom Sample 0. 3326 2VCR 9 20 04 Trial 1 BSA Concentration (mg/ml) Absorbance (at 750nm) 0.2 0.2546 0.5 0.4806 0.8 0.5999 1.2 0.7362 1.5 0.9939 Venom Sample 0.4210 2VCR 9 20 04 Trial 2 0.2 0.2673 0.5 0.4921 0.8 0.6048 1.2 0.7422 1.5 1.0001 Venom Sample 0.4293 2VCR 5 1 05 Trial 1 BSA Concentration (mg/ml) Absorbance (at 750nm) 0.2 0.3903 0.5 0.7059 0.8 0.7365 1.2 0.9100 1.5 0.9934 Venom Sample 0.6940 2VCR 5 1 05 Trial 2 0 .2 0.3830 0.5 0.6991 0.8 0.7259 1.2 0.9001 1.5 0.9825 Venom Sample 0.6885 4VCLF 2 20 05 Trial 1 BSA Concentration (mg/ml) Absorbance (at 750nm) 0.2 0.4279 0.5 0.6542 0.8 0.8765 1.2 1.0487 1.5 1.1766 Venom Sample 0.2101 4VCLF 2 20 05 Trial 2 0.2 0.4204 0.5 0.6438 0.8 0.8723 1.2 1.0442 1.5 1.1747 Venom Sample 0.2134

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45 Appendix II Phospholipase Assay Data 2VCR 1 23 05 Trial 1 Trial 2 0.2492 0.1710 4VCLF 7 10 05 Trial 1 Trial 2 0.3347 0.4752 2VCR 7 10 05 Trial 1 Trial 2 0.3652 0.31 91 2VCR 4 3 05 Trial 1 Trial 2 0.4259 0.7722 4VCLF 4 3 05 Trial 1 Trial 2 0.3346 0.1135 2VCR 3 19 05 Trial 1 Trial 2 0.0971 0.900 2VCR 5 15 05 Trial 1 Trial 2 0.1958 0.2106 4VCLF 5 15 05 Trial 1 Trial 2 0.2468 0.1755 4VCLF 3 19 05 Trial 1 Trial 2 0.1960 0.0393 2VCR 9 20 04 Trial 1 Trial 2 0.1231 0.0247 2VCR 5 1 05 Trial 1 Trial 2 No Data No Data 4VCLF 2 20 05 Trial 1 Trial 2 0.0795 0.0588

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46 Appendix III Phosphodiesterase Assay Data 2VCR 1 23 05 Trial 1 Trial 2 0.0344 0.02 66 4VCLF 7 10 05 Trial 1 Trial 2 0.0119 0.0086 2VCR 7 10 05 Trial 1 Trial 2 0.0383 0.0402 2VCR 4 3 05 Trial 1 Trial 2 0.0936 0.1041 4VCLF 4 3 05 Trial 1 Trial 2 0.0597 0.0479 2VCR 3 19 05 Trial 1 Trial 2 0.0206 0.0233 2VCR 5 15 05 Trial 1 Trial 2 0.0565 0.1153 4VCLF 5 15 05 Trial 1 Trial 2 0.0401 0.0757 4VCLF 3 19 05 Trial 1 Trial 2 0.0319 0.0 360 2VCR 9 20 04 Trial 1 Trial 2 0. 0436 0.0 443 2VCR 5 1 05 Trial 1 Trial 2 0.0497 0.0552 4VCLF 2 20 05 Trial 1 Trial 2 0.0 679 0.0 696

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47 Appendix IV Standard Curve Samples

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48 References 1) J. Calvete. Antivenomics and venom phenotyping: A marriage of convenience to address the performance and range of clinical use of antivenoms Toxincon 56 (7) (2010) pp. 128 4 1291 2) Guitierrez, J. M., et. al. Confronting the Neglected Problem of Snake Bite Envenoming: The Need for a Global Partnership PLoS Medicine, June 2006 3 ) World Health Organization (1981). Progress in the characterization of venoms and standardization of antivenoms. Geneva: WHO Offset Publications. pp. 5 4) James Forster, MD, MS (2006 03 14). "Snake Envenomations, Sea" eMedicine Emergency Medicine (environmental). Antivenom precautions section 5) (Edited by) Halliday; Adler, Tim; Kraig (2002). Firefly Encyclopedia of Reptil es and Amphibians. Toronto, Canada: Firefly Books Ltd. 6) Mattison, Chris (2007 (first published in 1995)). The New Encyclopedia of Snakes. New Jersey, USA (first published in the UK): Princeton University Press (Princeton and Oxford) pp. 117. 7) M.D. Mc Cue (1 Aug 2007). "Prey envenomation does not improve digestive performance in western diamondback rattlesnakes (Crotalus atrox)" J. Exp. Zool. A 307a pp. 568 77 8) Mackessy, S. Comp. Biochem. Physiol. Vol. 119B No. 1, pp. 119 127, 1998 9) Bottrall, Joshua L.; Frank Madaras, Christopher D Biven, Michael G Venning, and Peter J Mirtschin (30). "Proteolytic activity of Elapid and Viperid Snake venoms and its implication to digestion". Journal of Venom Research 1 (3): 18 28. 10) Price, Andrew H. (2009). Venomous Snakes of Texas: A Field Guide. University of Texas Press. pp. 38 39. 11) Kini, R. Manjunatha et al., ed. (2011). Toxins and Hemo stasis. Springer 2011. p. 99. 12) Rodrguez Ithurralde, D.; R. Silveira, L. Barbeito, F. Dajas (1983). "Fasciculin, a powerful anticholinesterase polypeptide from Dendroaspis angusticeps venom". Neurochemistry International 5 (3): 267 274. 13) Doley, R., K ini, R. 66 (17) (2009) 2851 2871 14) Gibbs et. al. related changes in venom composition of juvenile and adult Dusky Pigmy rattlesnakes (S Journal of Proteomics 74 (2011) pp. 2169 2179

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49 15) Dennis EA (May 1994). "Diversity of group types, regulation, and function of phospholipase A2" J. Biol. Chem. 269 (18): 13057 60. 16) Nicolas JP, Lin Y, Lambeau G, Ghomashchi F, Lazdunski M, Gelb MH (March 1997). "Localization of structural elements of bee venom phospholipase A2 involved in N type receptor binding and neurotoxicity". J. Biol. Chem. 272 (11): 7173 81. 17) Six DA, Dennis EA (2000). "The expanding superfamily o f phospholipase A(2) enzymes: classification and characterization" Biochim. Biophys. Acta 1488 (1 2): 1 19. 18) Berg OG, Gelb MH, Tsai MD, Jain MK (September 2001). "Interfacial enzymology: the secreted phospholipase A(2) paradigm". Chem. Rev. 101 (9): 26 13 54. 19) Cell Biochemistry and Function. 28 (3) (2010) 171 177 20) Oliver H. Lowry,Nira J. Rosebrough,A. Lewis Farr, and Rose J. Randall (1951). "Protein Measurement with the Folin Phenol Reagent". J. Biol. Chem. 193 (1): 265 275. 21) Everette, Jace D.; Bryant, Quinton M.; Green, Ashlee M.; Abbey, Yvonne A.; Wangila, Grant W.; Walker, Richard B. (2010). "Thorough Study of Reactivity of Various Compound Classes toward J. Agric. Food Chem. 58 (14): 8139. 22) Cho et. al., 2 Substrates: Kinetics of the Phospohlipase A 2 Catalyzed Hydrolysis of 3 (Acyloxy) 4 J. Am. Chem. Soc. 1988 5166 5171 2 3) An aqueous endpoint assay of snake venom phospholipase A 2 Toxicon 34 (10) (1996) pp. 1149 1155 24) Mackessy, S., 1998 pp. 361 404 25) Aird, S., et al. ractionation and characterization of the venom of the great basin Herpetologica 44 (1) 1988 71 85 26) D.E. Kleiner, W.G. Stetlerstevenson Quantities of Gelatinases 329 27) Terra, R. et. al. Toxicon 54 (6) (2009) 836 844 28) Collins, J. T. Standard Common and Current Sc ientific names for North American Amphibians and Reptiles Fourth Edition, (1997) pp ii + 40 29) Aird, S. Ophidian envenomation strategies and the role of purines Toxicon 40 (2002) 335 393

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50 30) Da Costa, G., et. al. Salivary amylase induction by tannin enriched diets as possible countermeasure against tannins. J Chem Ecol 2008; 34 pp 376 87

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