Publication Date

2013

Document Type

Thesis

Committee Members

David Cool (Advisor), Khalid Elased (Committee Member), Sharath Krishna (Committee Member)

Degree Name

Master of Science (MS)

Abstract

Snake venom is composed of many different toxins and protein components such as metalloproteases and phospholipase A2. Together, these proteins cause an up regulation in cell death pathways and disrupt the overall homeostasis of a living cell. The Red Diamondback Rattlesnake is abundantly available and well-known specie and has been used as a source for development of new drugs. It has been mainly used for the purposes of making anti-venom but it has also been therapeutically used for surgical purposes to act as an anti-aggregate. Integrins derived from RDB venom has been used for cancer cell treatment as well. Specifically a hemotoxic venom, RDB venom contains high concentrations of the toxin Ruberlysin; also known as Hemorrhagic Toxin II. An animal or human bitten by a RDB snake will experience localized tissue swelling, pain, bruising, as well as necrosis at the bit site. Systematically, the venom will cause excess internal bleeding, nausea, vomiting, and due to being hemotoxic venom, it will cause hemolysis. Due to its rich protein content as well as possible therapeutic purposes, RDB venom proves to hold the potential for studying cancer. The purpose of this research is to test the effects of Red Diamond Back rattlesnake venom protein components on different neuronal and lung cell lines, identify specific protein components and determine the venom's effects on specific cellular functions, i.e., intracellular prohormone trafficking and surfactant lipid secretion. To understand how the venom components work, the venom was fractionated by Fast Protein Liquid Chromatography and desalted through membrane dialysis using ddH2O. Neuro-2A and lung cells were incubated with individual snake venom fractions at different concentrations and over a 6 hour time.

After analysis by specific cell death assays, i.e., MTT, LDH, propidium iodide, annexin and Hoechst, several specific fractions were observed to cause cell death, while other fractions appeared to cause an increase in proliferation. Furthermore, chromogreanin A trafficking to punctate granules characteristic of the regulated secretory pathway was disupted in the Neuro-2a cells. Likewise, secretion of surfactant lipids appeared to be enhanced for several lipids from the lung cells exposed to snake venom fractions.

Specific RDB venom protein fractions were tryptically digested, spotted to a MALDItarget and sequenced using a Bruker Autoflex III MALDI TOF/TOF mass spectrometer. Ion data was collected and sent to an in-house MASCOT server database, which identified Hemorrhagic toxin (II), myotoxin, and apoxin I. The results from our study suggest that FPLC-cation exchange is a good first pass scheme for separating snake venom proteins, and that these separated proteins appear to have unique characteristics for cell death, proliferation and functionality.

Page Count

63

Department or Program

Department of Pharmacology and Toxicology

Year Degree Awarded

2013

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