Lactate dehydrogenase also known as LDH is a critical enzyme that is found in all mammals. It exists as a tetramer in five isoforms with a combination of heart and muscle subunits. One of its main functions is to catalyze the reaction of pyruvate to lactate. This reaction occurs in an anaerobic environment where the body needs to make sufficient energy as fast as possible, therefore pyruvate ferments to lactate with the help of LDH to produce enough NAD+ to power glycolysis. LDH-5, an isoenzyme of LDH, has been a target for cancer therapy. By reducing LDH-5, mitochondrial respiration declines resulting in slow cell proliferation. (LDH-5 4) Furthermore, LDH can be used as a clinical marker to detect cellular damage in the blood. …show more content…
This was done in a series of purification steps. First, the tissue was disrupted mechanically to acquire a crude homogenate containing buffers that keep the enzymes from degrading. Next, knowing that LDH was present in the cytosol, the sample was centrifuged and cytosolic enzymes including LDH were recovered from the supernatant. After centrifugation, the sample was exposed to different concentrations of ammonium sulfate in which the least soluble enzymes would precipitate out early and most soluble enzymes would precipitate late. Then, using affinity chromatography, LDH was further purified based upon its hydrophobicity. Using a ligand that had high affinity for enzymes with NAD+ cofactors, LDH was filtered from other enzymes that didn’t contain this property. Lastly, size exclusion chromatography was used to separate enzymes by their molecular weight. Knowing the molecular weight of LDH and using beads that had specific exclusion limits, the location of LDH was identified while the sample ran inside the column. Thereafter, …show more content…
40% and 65% cut ammonium sulfate precipitation was performed on clarified homogenate. 40% cut – massive pellet observed; LDH present in supernatant. 65% cut – small pellet observed; LDH was present in the pellet.
Table 1 – Final volume of purification intermediates.
Clarified Homogenate (supernatant from crude homogenate) 120 ml
65% cut pellet (Re-suspended pellet) 9.9 ml
B) Affinity Chromatography Purification of LDH
In the Affinity Purified Chromatography, LDH was separated by hydrophobicity using Cibacron Blue Agarose as a ligand. Once the samples were loaded into the column, wash buffers were used to elute down proteins that didn’t bind to the ligand. Afterwards, 1M NaCl was used to disrupt the interaction between the ligand and the target protein so that LDH proteins could elute down. Through this elution process, 20 fractions were collected after 1M NaCl was added and LDH spot test was performed.
Graph 1: Plot of elution volume Vs the absorbance at 280nm.
LDH Spot Test A1-A2 – Positive and negative control respectively. B1-B10 – Collected Fractions 1-10