Title

Evaluating Methods to Lessen Ischemic Reperfusion Injury by Testing the Effects in vitro of Pyrococcus Furiosus Superoxide Reductase in Reducing Oxidative Damage in Superoxide Exposed Cells

Date of Award

6-2014

Degree Type

Thesis

Degree Name

Master of Science (MS)

First Advisor

Francis E Jenney, Jr, PhD

Second Advisor

Kimberly Baker, PhD

Third Advisor

Melinda Maris, PhD

Abstract

schemic-Reperfusion (I/R) injuries are commonly associated with conditions such as heart attacks, liver disease, diabetes, and hypertension, due to the restoration of blood flow to oxygen-starved tissues. This restoration of oxygen has been known to cause oxidative damage within cells, producing an increased concentration of reactive oxygen species (ROS}, such as superoxide (02-). Cells use both chemical and enzymatic defenses to reduce the accumulation of ROS, although enzymes are faster at lowering this oxidative damage. Aerobic organisms use the enzyme superoxide dismutase (SOD) to eliminate superoxide by converting it to hydrogen peroxide and oxygen. Superoxide reductase (SOR) is an enzyme found in anaerobic microbes that has the ability to not only decrease the concentration of superoxide, but potentially, if utilized in aerobes, also decrease the concentration of reducing compounds that could create additional superoxide by blocking their transfer of electrons to oxygen. The highly thermostable SOR from the hyperthermophilic archaean Pyrococcus furiosus (Pf) will be tested, in vitro, for its ability to protect human cells against superoxide toxicity. The gene encoding Pf SOR (Pf 1281) was expressed in E. coli, and the protein was purified using a heat treatment (due to its hyperthermophilic nature), followed by an ion exchange and hydrophobic interaction chromatography. Pf SOR was analyzed by SDS-PAGE, a protein assay, absorption spectroscopy, and its activity was tested using a SOR activity assay. Human fibroblast cells were then incubated, experienced superoxide toxicity and later underwent viability tests, such as the Trypan Blue Exclusion Test and the CCK-8 Viability Test to determine SOR effectiveness against induced superoxide damage in vitro.

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