Date of Award

2018

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Area of Concentration

Chemistry

Abstract

Lactate dehydrogenase (LDH) is a metabolic, cytoplasmic protein that is found in nearly all living organisms. LDH catalyzes the interconversion of pyruvate to lactate, while generating nicotinamide adenine dinucleotide (NADH) from NAD+ (and vice versa). In humans, there are two predominant LDH genes that give rise two subunits, subunit M and subunit H, that produce five isozymes. The LDH-H4 isozyme is made from four of the H subunits and is found in heart muscle. LDH-H4 is referred to as LDH-1 in this study. This study uses Caenorhabditis elegans (C. elegans) as a model organism to further characterize LDH-1, as C. elegans LDH-1 is orthologous to human LDH-1. There is little previous published work on kinetic characterization of LDH-1 from C. elegans. This study reports progress on the optimization of purification and kinetic analysis of LDH-1. Previously, LDH-1 has been found to be insoluble. The expression vector used here contained a C-terminal 6X Histidine tag, and the resultant protein was more soluble than in past research. After purification under native conditions, enzyme assays were taken using the Cary 300 spectrophotometer. A baseline Michaelis-Menten kinetics plot was created by varying pyruvate concentrations. A KM of 0.50 +/- 0.27 mM and a Vmax of 0.0022 +/- 0.00028 mM/sec for pyruvate were calculated using the Michaelis-Menten equation. The optimal pH of LDH-1 was 8.0. Oxalic acid had inhibitory effects on the enzyme, increasing the Km to 1.0 +/- 0.69 and decreasing the Vmax to 0.00014 +/- 2.7E- 05. Future research should be done to find conditions to increase the stability of the enzyme as well as to test a wider array of concentrations of oxalic acid to further understand inhibition of LDH-1.

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