Dominican University of California
 

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Presentation or Panel Title

Purification of Lactate Dehydrogenase

Location

Guzman 114

Start Date

4-15-2016 1:30 PM

End Date

4-15-2016 2:00 PM

Department

Natural Sciences and Mathematics

Student Type

Undergraduate - Honors

Faculty Mentor

Kristylea Ojeda, Ph.D.

Presentation Format

Oral Presentation

Abstract/Description

Lactate dehydrogenase (LDH), sometimes referred to as lactic acid dehydrogenase, is an enzyme that is found in the cytoplasm of almost all living cells. It converts lactate and NAD+ (nicotinamide adenine dinucleotide) to pyruvate and NADH (nicotinamide adenine dinucleotide hydrogen), and it can also catalyze the reverse reaction. Heart muscle is highly oxygenated at all times; therefore, LDH catalyzes the reaction in the direction to form NADH and pyruvate, which is used in the Citric Acid Cycle to make energy for the cell via aerobic cellular respiration. When little or no oxygen (O2) is available to the cell, such as the case for muscle cells during exercise and other strenuous activities, LDH catalyzes the reaction in the direction to form lactate and NAD+ via lactic acid fermentation. In this way, LDH plays an important role in delaying the onset of muscle fatigue by keeping glycolysis running and providing energy to the cell until oxygen is available again. The goal of our experiment was to purify LDH from a beef heart, which is rich in LDH. The techniques of homogenization, centrifugation, ammonium sulfate precipitation, and gel-filtration chromatography were used to purify LDH. Following purification, a colorimetric assay was used to determine the protein concentration of the fractions, and the protein-containing fractions were assayed for LDH-enzyme activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was then used to separate the proteins by molecular weight, and a Coomassie-stained gel and Western blot verified the presence of LDH and the purity of our samples.

Comments

Keywords: lactate dehydrogenase, LDH, beef heart, SDS-PAGE, Western blot, lactate, pyruvate, NADH, NAD+

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Apr 15th, 1:30 PM Apr 15th, 2:00 PM

Purification of Lactate Dehydrogenase

Guzman 114

Lactate dehydrogenase (LDH), sometimes referred to as lactic acid dehydrogenase, is an enzyme that is found in the cytoplasm of almost all living cells. It converts lactate and NAD+ (nicotinamide adenine dinucleotide) to pyruvate and NADH (nicotinamide adenine dinucleotide hydrogen), and it can also catalyze the reverse reaction. Heart muscle is highly oxygenated at all times; therefore, LDH catalyzes the reaction in the direction to form NADH and pyruvate, which is used in the Citric Acid Cycle to make energy for the cell via aerobic cellular respiration. When little or no oxygen (O2) is available to the cell, such as the case for muscle cells during exercise and other strenuous activities, LDH catalyzes the reaction in the direction to form lactate and NAD+ via lactic acid fermentation. In this way, LDH plays an important role in delaying the onset of muscle fatigue by keeping glycolysis running and providing energy to the cell until oxygen is available again. The goal of our experiment was to purify LDH from a beef heart, which is rich in LDH. The techniques of homogenization, centrifugation, ammonium sulfate precipitation, and gel-filtration chromatography were used to purify LDH. Following purification, a colorimetric assay was used to determine the protein concentration of the fractions, and the protein-containing fractions were assayed for LDH-enzyme activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was then used to separate the proteins by molecular weight, and a Coomassie-stained gel and Western blot verified the presence of LDH and the purity of our samples.