An analysis of oxidative damage to lactate dehydrogenase in context of neurodegeneration and catechol-based phenolic antioxidant chemistry
Date Thesis Awarded
Honors Thesis -- Access Restricted On-Campus Only
Bachelors of Science (BS)
Lactate dehydrogenase, a redox-active enzyme with five reactive cysteine residues, reversibly converts pyruvate to lactate during anaerobic glycolysis. In the case of certain disease states, when oxygen levels are low or increased oxidative stress damages mitochondrial respiration, cells must rely heavily on anaerobic glycolysis for ATP production. This is true of Alzheimer’s disease, Parkinson’s disease, ALS, and many cancers. Diseased cells upregulate anaerobic glycolytic enzymes - particularly LDH – to produce enough energy to survive. Upregulated LDH plays a key role because as it produces lactate, it also replenishes NAD+, allowing for high levels of glycolysis to continue uninterrupted. Despite research into the function of LDH as a glycolytic enzyme, little focus has been allocated to its role outside of glycolysis and its interaction with reactive oxygen species and other cysteine reactive compounds – knowledge of which could illuminate further disease mechanics.
For this project, we first investigated the effects of bleach (HOCl) – a common reactive oxygen species found elevated in Alzheimer’s disease – on LDH structure and function. We then examined the effects of oxidized plant-derived phenolic antioxidants on LDH activity. Research shows that catechol-based antioxidants are readily oxidized by radicals to quinones. These quinones can further react with protein thiols, demonstrating the potential for beneficial antioxidants to become damaging pro-oxidants, and thus prompting our investigation into their reactivity with LDH cysteine residues. We compared our results with LDH to those obtained using alcohol dehydrogenase (ADH), a model protein that also contains readily oxidized cysteine residues. In our work we utilized UV/Vis spectroscopy, fluorescence spectroscopy, and SDS-PAGE techniques to examine how LDH and ADH structure and function changed when respective available protein thiols were reacted with HOCl or oxidized plant-derived phenolic antioxidants. Resulting data confirms these compounds inhibit LDH and ADH activity, strongly suggesting that modification of key cysteine residues occurs, changing the conformation of the protein.
Boike, Lydia, "An analysis of oxidative damage to lactate dehydrogenase in context of neurodegeneration and catechol-based phenolic antioxidant chemistry" (2017). Undergraduate Honors Theses. William & Mary. Paper 1144.
On-Campus Access Only