Date of Award

2020

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Area of Concentration

Biochemistry

Abstract

Lactate Dehydrogenase (LDH) is an essential metabolic protein ubiquitous to most organisms. LDH catalyzes the interconversion of pyruvate to lactate. During the catalysis, NAD+/NADH are involved as a cofactor to the reaction, being reduced or oxidized. Human LDH is a tetrameric enzyme with two genes encoding two variations of subunit, subunit M and subunit H. There exist five isozymes of differing subunit composition. LDH-H4 is comprised of four H subunits, and is found mainly in blood cells, heart tissue, and brain tissue. Caenorhabditis elegans (C. elegans) was chosen as the model organism for the characterization of LDH-H4 as LDH-1 is native to the species and is orthologous to human LDH subunit H. This study aims to report on the purification of a soluble LDH-1 enzyme in addition to a kinetic characterization and optimization of reaction conditions. Previous studies on LDH-1 have reported unsuccessful attempts in purifying a soluble enzyme. The LDH-1 gene was first inserted into a pET303-His vector, transformed and overexpressed using E. coli, and purified using a Ni-affinity column. The resultant protein was found to be soluble. After purifying, the enzyme was subjected to kinetic assays. After baseline activity was established, optimum pH and temperature were determined. The optimum pH was found to be 8 and the optimum temperature was 40 oC. Finally, Michaelis-Menten kinetics curves were developed for varying pyruvate and NADH concentrations. After optimum conditions were determined, a kinetic curve following the Michaelis-Menten equation was determined for both pyruvate and NADH. The Vmax was found to be 0.134 ± 0.014 A340/min for pyruvate, and 0.196 ± 0.058 A340/min for NADH. The Km was determined to be 0.620 ± 0.164 mM for pyruvate and 62.1 ± 36.09303 µM for NADH. The specific activity of LDH-1 was determined to be 9.54 U/mg.

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