Title

The Potential Effect of Superoxide Reductase in Reducing ISchemia-Reperfusion Injury

Date of Award

2010

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

Department

Basic Sciences

First Advisor

Francis E. Jenney, Jr, PhD

Second Advisor

Harold Komiskey, PhD

Third Advisor

Brian M Matayoshi, PhD

Abstract

Ischemia reperfusion (I/R) injury, associated with such conditions as myocardial infarctions, hypertension, and diabetes occurs mainly due to reactive oxygen species production in affected areas upon restoration of blood flow to oxygen-starved tissues. Aerobes use the enzyme superoxide dismutase (SOD) to convert superoxide to hydrogen peroxide, in order to limit potential cell damage. However, it is incapable of decreasing reducing compounds and limiting further superoxide production. Superoxide reductase (SOR), an antioxidant enzyme found in anaerobic microbes, has been shown to utilize reducing equivalents from its in vivo partner rubredoxin, as well as mammalian cytochrome c, in vitro, to reduce superoxide directly to hydrogen peroxide. The SOR enzyme from an hyperthermophilic archaeon, Pyrococcus furiosus (Pf), has been shown to be highly thermostable, and, remarkably, functional even at 25oC, which is 75oC below its optimal growth temperature. Therefore, in this study, superoxide induced oxidative damage was stimulated in Human Jurkat T lymphocytes to test the protective effect of Pf SOR, with and without rubredoxin. Variations in cell viability were assessed using the Trypan Blue Exclusion Test and the Lactate Dehydrogenase (LDH) Assay. Lipid damage accumulation was quantified using the Thiobarbituric Acid (TBARS) Assay. Analysis of post incubated samples showed that SOR reduced cytotoxicity by 15%, as measured by the LDH Assay. The Trypan Blue Exclusion test concluded that SOR treated Jurkats experienced a 40% increase in cell viability. The Thiobarbituric Acid Assay showed that SOR, when supplemented with rubredoxin, resulted in a 14% reduction in superoxide induced lipid damage.

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